Professor Roberto La Ragione


Professor of Veterinary Microbiology and Pathology, Head of the School of Biosciences
BSc (Hons) MSc PhD FRSB CBiol FIBMS CSci AECVM FRCPath HonAssocRCVS
+44 (0)1483 686920
14 AX 01
Please contact Lara Haswell for an appointment
Personal Assistant: Lara Haswell
+44 (0)1483 686920

Academic and research departments

School of Veterinary Medicine, School of Biosciences.

About

University roles and responsibilities

  • Module coordinator for MSc Vet Micro research projects
  • Head of the School of Biosciences and Medicine
  • Professor of Veterinary Microbiology and Pathology
  • Member of the Senior Leadership Team
  • Deputy chair of AWERB committee
  • Member of the NASPA committee

    News

    Research

    Research interests

    Supervision

    Postgraduate research supervision

    Teaching

    Publications

    T. Rai, I. Papanikolaou, N. Dave, A. Morisi, B. Bacci, S. A. Thomas, R. La Ragione, K. Wells (2023)Investigating the potential of untrained convolutional layers and pruning in computational pathology, In: J E Tomaszewski, A D Ward (eds.), MEDICAL IMAGING 2023124711247119pp. 1247119-1247119-10 Spie-Int Soc Optical Engineering

    The effectiveness of untrained convolutional layers for feature extraction in a computational pathology task using real-world data from a necrosis detection dataset is investigated. The study aims to determine whether ImageNet pretrained layers from deep CNNs combined with frozen untrained weights are sufficient for effective necrosis detection in canine Perivascular Wall Tumour (cPWT) whole slide images. Additionally, the authors investigate the impact of pruning CNNs, and whether it can be effective for necrosis detection as this technique can contribute towards reducing memory requirements and improve inference speed in diagnostic settings. The study found that fine-tuning the last (deepest) layers of a pretrained ImageNet model for necrosis detection in cPWT produces the highest test F1-score (0.715) when compared to alternative set ups. This score is further improved to 0.754 when the results are optimised using an optimal threshold predetermined on maximising the validation set F1-score. Resetting weights (untrained) and freezing the last few convolutional layers in the last dense block also demonstrated some capability in necrosis detection with an optimised F1-score of 0.747, still outperforming models trained from scratch as well as an ImageNet pretrained feature extraction model. Pruning the fine-tuned model using lower thresholds also showed the potential to improve performance, however thresholds higher than 0.40 negatively impacted performance.

    Tara Davis, Dagmara Bialy, Joy Leng, Roberto La Ragione, Holly Shelton, Klaudia Chrzastek (2023)Alteration of the Chicken Upper Respiratory Microbiota, Following H9N2 Avian Influenza Virus Infection, In: Pathogens (Basel)12(9)1168 Mdpi

    Several studies have highlighted the importance of the gut microbiota in developing immunity against viral infections in chickens. We have previously shown that H9N2 avian influenza A virus (AIV) infection retards the diversity of the natural colon-associated microbiota, which may further influence chicken health following recovery from infection. The effects of influenza infection on the upper respiratory tract (URT) microbiota are largely unknown. Here, we showed that H9N2 AIV infection lowers alpha diversity indices in the acute phase of infection in the URT, largely due to the family Lactobacillaceae being highly enriched during this time in the respiratory microbiota. Interestingly, microbiota diversity did not return to levels similar to control chickens in the recovery phase after viral shedding had ceased. Beta diversity followed a similar trend following the challenge. Lactobacillus associate statistically with the disturbed microbiota of infected chickens at the acute and recovery phases of infection. Additionally, we studied age-related changes in the respiratory microbiota during maturation in chickens. From 7 to 28 days of age, species richness and evenness were observed to advance over time as the microbial composition evolved. Maintaining microbiota homeostasis might be considered as a potential therapeutic target to prevent or aid recovery from H9N2 AIV infection.

    María Getino, María López-Díaz, Nicholas Ellaby, John Clark, Matthew J. Ellington, Roberto Marcello La Ragione (2022)A Broad-Host-Range Plasmid Outbreak: Dynamics of IncL/M Plasmids Transferring Carbapenemase Genes, In: Antibiotics11(11)1641 MDPI

    IncL/M broad-host-range conjugative plasmids are involved in the global spread of blaOXA-48 and the emergence of blaNDM-1. The aim of this study was to evaluate the transmission potential of plasmids encoding the emergent NDM-1 carbapenemase compared to the pandemic OXA-48. The conjugation rate and fitness cost of IncM2 and IncL plasmids encoding these carbapenemase genes were tested using a variety of host bacteria. Genomic analysis of uropathogenic Escherichia coli SAP1756 revealed that blaNDM-1 was encoded on an IncM2 plasmid, which also harboured blaTEM-1, bleMBL and sul1 and was highly similar to plasmids isolated from the same geographical area. Conjugation experiments demonstrated that NDM-1 and OXA-48-carrying plasmids transfer successfully between different Enterobacterales species, both in vitro and in vivo. Interestingly, E. coli isolates tested as recipients belonging to phylogroups A, B1, D and F were able to receive IncM2 plasmid pSAP1756, while phylogroups B2, C, E and G were not permissive to its acquisition. In general, the IncL OXA-48-carrying plasmids tested transferred at higher rates than IncM2 harbouring NDM-1 and imposed a lower burden to their host, possibly due to the inactivation of the tir fertility inhibition gene and reflecting their worldwide dissemination. IncM2 plasmids carrying blaNDM-1 are considered emergent threats that need continuous monitoring. In addition to sequencing efforts, phenotypic analysis of conjugation rates and fitness cost are effective methods for estimating the pandemic potential of antimicrobial resistance plasmids.

    Myron Christodoulides, Daysiane de Oliveira, David W. Cleary, Maria Victoria Humbert, Ricardo A. Machado-de-Avila, Roberto M. La Ragione (2022)An in silico reverse vaccinology study of Brachyspira pilosicoli, the causative organism of intestinal spirochaetosis, to identify putative vaccine candidates, In: Process biochemistry (1991)122(Part 1)pp. 128-148 Elsevier

    Brachyspira pilosicoli is a zoonotic bacterium that can cause intestinal spirochaetosis (IS) in avian species (AIS), pigs (PIS) and humans (HIS). In the absence of vaccines to prevent infections, we used genome-based reverse vaccinology (RV) to identify putative B. pilosicoli vaccine candidates. Genome sequence of B. pilosicoli strain B2904, an AIS isolate, was analysed with PSORTb3, CELLO, SOSUIGramN, LipoP, SignalP-5.0, TMHMM, BLAST 2.12.0 +, PDB database, SEED Viewer, eggNOG-mapper, UniProt, VaxiJen and Vaxign2, and Tblastn to generate a RV list of putative vaccine candidates. We also generated a linear B-cell chimera antigen using Blast-p, Emini Surface Accessibility Prediction, ABCpred, Expasy ProtParam and PepCalc programs. RV defined a list of 162 proteins containing 48 Outer Membrane (OM), 27 OM/Extracellular, 27 Extracellular, 4 Periplasm, 2 Surface, 2 Cytoplasm and 52 Unknown proteins. The list was characterised by an abundance of SPII lipoproteins. We found that genes encoding amino acid sequences of 146/162 (90%) proteins were present in 19 other B. pilosicoli genomes. A linear B-cell chimera antigen was generated from the amino acid sequences of 18 OM and Extra-cellular proteins. Our contemporary RV study represents a starting point for a comprehensive vaccine development strategy for preventing intestinal spirochaetosis.

    Owen Higgins, Alexandra Chueiri, Louise O'Connor, Sinéad Lahiff, Liam Burke, Dearbháile Morris, Nicola Maria Pfeifer, Belén González Santamarina, Christian Berens, Christian Menge, Manuela Caniça, Vera Manageiro, Veljo Kisand, Marwa M. Hassan, Brian Gardner, Arnoud H.M. van Vliet, Roberto M. La Ragione, Bruno Gonzalez-Zorn, Terry J. Smith (2022)Portable Differential Detection of CTX-M ESBL Gene Variants, bla(CTX-M-1) and bla(CTX-M-15), from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification, In: Microbiology spectrum11(1) Amer Soc Microbiology

    CTX-M ESBL-producing E. coli is an increasing AMR public health issue with the transmission between animals and humans via zoonotic pathogens now a major area of interest. Accurate and timely identification of ESBL-expressing E. coli CTX-M variants is essential for disease monitoring, targeted antibiotic treatment and infection control. Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamase (ESBL) enzymes produced by Enterobacteriaceae confer resistance to clinically relevant third-generation cephalosporins. CTX-M group 1 variants, CTX-M-1 and CTX-M-15, are the leading ESBL-producing Enterobacteriaceae associated with animal and human infection, respectively, and are an increasing antimicrobial resistance (AMR) global health concern. The bla(CTX-M-1) and bla(CTX-M-15) genes encoding these variants have an approximate nucleotide sequence similarity of 98.7%, making effective differential diagnostic monitoring difficult. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) enables rapid real-time multiplex pathogen detection with single-base specificity and portable on-site testing. We have developed an internally controlled multiplex CTX-M-1/15 LEC-LAMP assay for the differential detection of bla(CTX-M-1) and bla(CTX-M-15). Assay analytical specificity was established using a panel of human, animal, and environmental Escherichia coli isolates positive for bla(CTX-M-1) (n = 18), bla(CTX-M-15) (n = 35), and other closely related bla(CTX-Ms) (n = 38) from Ireland, Germany, and Portugal, with analytical sensitivity determined using probit regression analysis. Animal fecal sample testing using the CTX-M-1/15 LEC-LAMP assay in combination with a rapid DNA extraction protocol was carried out on porcine fecal samples previously confirmed to be PCR-positive for E. coli bla(CTX-M). Portable instrumentation was used to further analyze each fecal sample and demonstrate the on-site testing capabilities of the LEC-LAMP assay with the rapid DNA extraction protocol. The CTX-M-1/15 LEC-LAMP assay demonstrated complete analytical specificity for the differential detection of both variants with sensitive low-level detection of 8.5 and 9.8 copies per reaction for bla(CTX-M-1) and bla(CTX-M-15), respectively, and E. coli bla(CTX-M-1) was identified in all bla(CTX-M) positive porcine fecal samples tested.IMPORTANCE CTX-M ESBL-producing E. coli is an increasing AMR public health issue with the transmission between animals and humans via zoonotic pathogens now a major area of interest. Accurate and timely identification of ESBL-expressing E. coli CTX-M variants is essential for disease monitoring, targeted antibiotic treatment and infection control. This study details the first report of portable diagnostics technology for the rapid differential detection of CTX-M AMR markers bla(CTX-M-1) and bla(CTX-M-15), facilitating improved identification and surveillance of these closely related variants. Further application of this portable internally controlled multiplex CTX-M-1/15 LEC-LAMP assay will provide new information on the transmission and prevalence of these CTX-M ESBL alleles. Furthermore, this transferable diagnostic technology can be applied to other new and emerging relevant AMR markers of interest providing more efficient and specific portable pathogen detection for improved epidemiological surveillance.

    Charlotte Bain, Getika Rathor, Ben P. Jones, Marwa M. Hassan, Przemyslaw Papke, Ben South, Mark Elliott, Ian Jones, Roberto M. La Ragione, Martha Elizabeth Betson (2022)beta-Lactam resistance genes present in UK pheasants and red-legged partridges, In: Veterinary record195(5)pp. 185-224 Wiley

    BackgroundDespite considerable recent reductions in antimicrobial use, the UK gamebird industry continues to struggle with production diseases during the rearing season, necessitating significant antibiotic use. This observational study investigated the presence of genes conferring resistance to beta-lactam antibiotics within industry-reared pheasants and red-legged partridges in the UK. MethodsDNA was extracted from 60 pooled caecal samples collected from gamebirds at routine postmortem examinations during the rearing season. Genes encoding extended-spectrum beta-lactamases (ESBL) were detected by PCR and the corresponding alleles were determined. ResultsOver half (53%) of the samples harboured genes encoding bla(TEM) resistance, with bla(SHV) identified in 20% of samples. The bla(TEM) gene was more common on sites with higher antibiotic use, whereas bla(SHV) was predominantly found in birds younger than 5 weeks. Genotyping of the identified resistance genes revealed the presence of bla(TEM-1), bla(SHV-1) and bla(SHV-11) alleles. LimitationsThis was a small-scale study conducted at four sites in southern England. ConclusionThis is the first report of the presence of ESBL genes in gamebirds, highlighting the need for further research into antimicrobial resistance in UK gamebirds.

    Brian Gardner, Martha Betson, Mark A. Chambers, Francesca M. Contadini, Laura C. Gonzalez Villeta, Marwa M. Hassan, Roberto M. La Ragione, Joaquin M. Prada, Lorenzo A. Santorelli, Nick Selemetas, Mukunthan Tharmakulasingam, Arnoud H. M. Van Vliet, Inaki Deza-Cruz, Giovanni Lo Iacono (2023)Mapping the evidence of the effects of environmental factors on the prevalence of antibiotic resistance in the non-built environment: Protocol for a systematic evidence map, In: Environment International171107707 Elsevier

    Background Human, animal, and environmental health are increasingly threatened by the emergence and spread of antibiotic resistance. Inappropriate use of antibiotic treatments commonly contributes to this threat, but it is also becoming apparent that multiple, interconnected environmental factors can play a significant role. Thus, a One Health approach is required for a comprehensive understanding of the environmental dimensions of antibiotic resistance and inform science-based decisions and actions. The broad and multidisciplinary nature of the problem poses several open questions drawing upon a wide heterogeneous range of studies. Objective This study seeks to collect and catalogue the evidence of the potential effects of environmental factors on the abundance or detection of antibiotic resistance determinants in the outdoor environment, i.e., antibiotic resistant bacteria and mobile genetic elements carrying antibiotic resistance genes, and the effect on those caused by local environmental conditions of either natural or anthropogenic origin. Methods Here, we describe the protocol for a systematic evidence map to address this, which will be performed in adherence to best practice guidelines. We will search the literature from 1990 to present, using the following electronic databases: MEDLINE, Embase, and the Web of Science Core Collection as well as the grey literature. We shall include full-text, scientific articles published in English. Reviewers will work in pairs to screen title, abstract and keywords first and then full-text documents. Data extraction will adhere to a code book purposely designed. Risk of bias assessment will not be conducted as part of this SEM. We will combine tables, graphs, and other suitable visualisation techniques to compile a database i) of studies investigating the factors associated with the prevalence of antibiotic resistance in the environment and ii) map the distribution, network, cross-disciplinarity, impact and trends in the literature.

    Florent Kempf, Guido Cordoni, Anne-Marie Chaussé, Rosanna Drumo, Helen Brown, Daniel L Horton, Frédéric Paboeuf, Martine Denis, Philippe Velge, Roberto La Ragione, Annaëlle Kerouanton (2023)Inflammatory Responses Induced by the Monophasic Variant of Salmonella Typhimurium in Pigs Play a Role in the High Shedder Phenotype and Fecal Microbiota Composition, In: mSystems8(1)e0085222

    Pigs infected with Salmonella may excrete large amounts of Salmonella, increasing the risk of spread of this pathogen in the food chain. Identifying Salmonella high shedder pigs is therefore required to mitigate this risk. We analyzed immune-associated markers and composition of the gut microbiota in specific-pathogen-free pigs presenting different shedding levels after an oral infection with Salmonella. Immune response was studied through total blood cell counts, production of anti-Salmonella antibodies and cytokines, and gene expression quantification. Total Salmonella shedding for each pig was estimated and hierarchical clustering was used to cluster pigs into high, intermediate, and low shedders. Gut microbiota compositions were assessed using 16S rRNA microbial community profiling. Comparisons were made between control and inoculated pigs, then between high and low shedders pigs. Prior to infection, high shedders had similar immunological profiles compared to low shedders. As soon as 1 day postinoculation (dpi), significant differences on the cytokine production level and on the expression level of several host genes related to a proinflammatory response were observed between high and low shedders. Infection with Salmonella induced an early and profound remodeling of the immune response in all pigs, but the intensity of the response was stronger in high shedders. In contrast, low shedders seroconverted earlier than high shedders. Just after induction of the proinflammatory response (at 2 dpi), some taxa of the fecal microbiota were specific to the shedding phenotypes. This was related to the enrichment of several functional pathways related to anaerobic respiration in high shedders. In conclusion, our data show that the immune response to Salmonella modifies the fecal microbiota and subsequently could be responsible for shedding phenotypes. Influencing the gut microbiota and reducing intestinal inflammation could be a strategy for preventing Salmonella high shedding in livestock. Salmonellosis remains the most frequent human foodborne zoonosis after campylobacteriosis and pork meat is considered one of the major sources of human foodborne infections. At the farm, host heterogeneity in pig infection is problematic. High Salmonella shedders contribute more significantly to the spread of this foodborne pathogen in the food chain. The identification of predictive biomarkers for high shedders could help to control Salmonella in pigs. The purpose of the present study was to investigate why some pigs become super shedders and others low shedders. We thus investigated the differences in the fecal microbial composition and the immune response in orally infected pigs presenting different Salmonella shedding patterns. Our data show that the proinflammatory response induced by Typhimurium at 1 dpi could be responsible for the modification of the fecal microbiota composition and functions observed mainly at 2 and 3 dpi and to the low and super shedder phenotypes.

    Mukunthan Tharmakulasingam, Brian Gardner, Roberto La Ragione, Anil Fernando (2022)Explainable Deep Learning Approach for Multilabel Classification of Antimicrobial Resistance with Missing Labels, In: IEEE Access10pp. 113073-113085 Institute of Electrical and Electronics Engineers (IEEE)

    Predicting Antimicrobial Resistance (AMR) from genomic sequence data has become a significant component of overcoming the AMR challenge, especially given its potential for facilitating more rapid diagnostics and personalised antibiotic treatments. With the recent advances in sequencing technologies and computing power, deep learning models for genomic sequence data have been widely adopted to predict AMR more reliably and error-free. There are more than 30 different types of AMR; therefore, any practical AMR prediction system must be able to identify multiple AMRs present in a genomic sequence. Unfortunately, most genomic sequence datasets do not have all the labels marked, thereby making a deep learning modelling approach challenging owing to its reliance on labels for reliability and accuracy. This paper addresses this issue by presenting an effective deep learning solution, Mask-Loss 1D convolution neural network (ML-ConvNet), for AMR prediction on datasets with many missing labels. The core component of ML-ConvNet utilises a masked loss function that overcomes the effect of missing labels in predicting AMR. The proposed ML-ConvNet is demonstrated to outperform state-of-the-art methods in the literature by 10.5%, according to the F1 score. The proposed model's performance is evaluated using different degrees of the missing label and is found to outperform the conventional approach by 76% in the F1 score when 86.68% of labels are missing. Furthermore, the proposed ML-ConvNet is established with an explainable artificial intelligence (XAI) pipeline, thereby making it ideally suited for hospitals and healthcare settings where model interpretability is an essential requirement.

    Ambra Morisi, Taran Rai, Nicholas J. Bacon, Spencer A. Thomas, Miroslaw Bober, Kevin Wells, Michael J. Dark, Tawfik Aboellail, Barbara Bacci, Roberto M. La Ragione (2023)Detection of Necrosis in Digitised Whole-Slide Images for Better Grading of Canine Soft-Tissue Sarcomas Using Machine-Learning, In: Veterinary sciences10(1)45 MDPI

    The definitive diagnosis of canine soft-tissue sarcomas (STSs) is based on histological assessment of formalin-fixed tissues. Assessment of parameters, such as degree of differentiation, necrosis score and mitotic score, give rise to a final tumour grade, which is important in determining prognosis and subsequent treatment modalities. However, grading discrepancies are reported to occur in human and canine STSs, which can result in complications regarding treatment plans. The introduction of digital pathology has the potential to help improve STS grading via automated determination of the presence and extent of necrosis. The detected necrotic regions can be factored in the grading scheme or excluded before analysing the remaining tissue. Here we describe a method to detect tumour necrosis in histopathological whole-slide images (WSIs) of STSs using machine learning. Annotated areas of necrosis were extracted from WSIs and the patches containing necrotic tissue fed into a pre-trained DenseNet161 convolutional neural network (CNN) for training, testing and validation. The proposed CNN architecture reported favourable results, with an overall validation accuracy of 92.7% for necrosis detection which represents the number of correctly classified data instances over the total number of data instances. The proposed method, when vigorously validated represents a promising tool to assist pathologists in evaluating necrosis in canine STS tumours, by increasing efficiency, accuracy and reducing inter-rater variation.

    Owen Higgins, MARWA M. HASSAN, Arnoud Van Vliet, Liam P. Burke, Alexandra Chueiri, Louise O'Connor, Dearbháile Morris, Terry J. Smith, Roberto Marcello La Ragione (2023)Rapid culture‐independent loop‐mediated isothermal amplification detection of antimicrobial resistance markers from environmental water samples, In: Microbial Biotechnology16(5)pp. 977-989 Wiley

    Environmental water is considered one of the main vehicles for the transmission of antimicrobial resistance (AMR), posing an increasing threat to humans and animals health. Continuous efforts are being made to eliminate AMR; however, the detection of AMR pathogens from water samples often requires at least one culture step, which is time-consuming and can limit sensitivity. In this study, we employed comparative genomics to identify the prevalence of AMR genes within among: Escherichia coli, Klebsiella, Salmonella enterica and Acinetobacter, using publicly available genomes. The mcr-1, blaKPC (KPC-1 to KPC-4 alleles), blaOXA-48, blaOXA-23 and blaVIM (VIM-1 and VIM-2 alleles) genes are of great medical and veterinary significance, thus were selected as targets for the development of isothermal loop-mediated amplification (LAMP) detection assays. We also developed a rapid and sensitive sample preparation method for an integrated culture-independent LAMP-based detection from water samples. The developed assays successfully detected the five AMR gene markers from pond water within 1 h and were 100% sensitive and specific with a detection limit of 0.0625 μg/mL and 10 cfu/mL for genomic DNA and spiked bacterial cells, respectively. The integrated detection can be easily implemented in resource-limited areas to enhance One Health AMR surveillances and improve diagnostics.

    Sumeet K. Tiwari, Boas C. L. van der Putten, Thilo M. Fuchs, Trung N. Vinh, Martin Bootsma, Rik Oldenkamp, Roberto La Ragione, Sebastien Matamoros, Ngo T. Hoa, Christian Berens, Joy Leng, Julio Alvarez, Marta Ferrandis-Vila, Jenny M. Ritchie, Angelika Fruth, Stefan Schwarz, Lucas Dominguez, Maria Ugarte-Ruiz, Astrid Bethe, Charlotte Huber, Vanessa Johanns, Ivonne Stamm, Lothar H. Wieler, Christa Ewers, Amanda Fivian-Hughes, Herbert Schmidt, Christian Menge, Torsten Semmler, Constance Schultsz (2023)Genome-wide association reveals host-specific genomic traits in Escherichia coli, In: BMC biology2176 BMC

    Background Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. Results We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. Conclusions This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

    Jonathan W. Betts, Shaun Cawthraw, Joan A. Smyth, Robert K. Poole, Patrick Roth, Ulrich Schatzschneider, Roberto M. La Ragione (2023)The manganese carbonyl complex [Mn(CO)3(tqa-κ3N)]Br: A novel antimicrobial agent with the potential to treat avian pathogenic Escherichia coli (APEC) infections, In: Veterinary microbiology284109819 Elsevier

    The development of alternatives to antibiotics is essential for the treatment of animal infections and as a measure to reduce the selective pressure on antibiotics that are critical for human medicine. Metal complexes have been highlighted for their antimicrobial activity against several bacterial pathogens. In particular, manganese carbonyl complexes have shown efficacy against multidrug-resistant Gram-negative pathogens, and relatively low cytotoxicity against avian macrophages and in wax moth larval models. They are thus potential candidates for deployment against Avian Pathogenic Escherichia coli (APEC), the aetiological agent of avian colibacillosis, which results in severe animal welfare issues and financial losses worldwide. This study aimed to determine the efficacy of [Mn(CO)3(tqa-κ3N)]Br in Galleria mellonella and chick models of infection against APEC. The results demonstrated in vitro and in vivo antibacterial activity against all antibiotic-resistant APEC test isolates screened in the study. •In vitro [Mn(CO)3(tqa-κ3N)]Br exposure results in bacterial killing at 4-hours.•[Mn(CO)3(tqa-κ3N)]Br reduces mortality in G. mellonella post APEC infection.•[Mn(CO)3(tqa-κ3N)]Br significantly reduces bacterial shedding in chicks.•Bacterial colonisation is reduced in chicks after [Mn(CO)3(tqa-κ3N)]Br treatment.

    Lucy Coleman, James R. G. Adams, Will Buchanan, Tao Chen, Roberto M. M. La Ragione, Lian X. X. Liu (2023)Non-Antibiotic Compounds Synergistically Kill Chronic Wound-Associated Bacteria and Disrupt Their Biofilms, In: Pharmaceutics15(6)1633 Mdpi

    Chronic wounds and their treatment present a significant burden to patients and healthcare systems alike, with their management further complicated by bacterial infection. Historically, antibiotics have been deployed to prevent and treat infections, but the emergence of bacterial antimicrobial resistance and the frequent development of biofilms within the wound area necessitates the identification of novel treatment strategies for use within infected chronic wounds. Here, several non-antibiotic compounds, polyhexamethylene biguanide (PHMB), curcumin, retinol, polysorbate 40, ethanol, and D-& alpha;-tocopheryl polyethylene glycol succinate 1000 (TPGS) were screened for their antibacterial and antibiofilm capabilities. The minimum inhibitory concentration (MIC) and crystal violet (CV) biofilm clearance against two bacteria frequently associated with infected chronic wounds, Staphylococcus aureus and Pseudomonas aeruginosa, were determined. PHMB was observed to have highly effective antibacterial activity against both bacteria, but its ability to disperse biofilms at MIC levels was variable. Meanwhile, TPGS had limited inhibitory activity but demonstrated potent antibiofilm properties. The subsequent combination of these two compounds in a formulation resulted in a synergistic enhancement of their capability to kill both S. aureus and P. aeruginosa and disperse their biofilms. Collectively, this work highlights the utility of combinatory approaches to the treatment of infected chronic wounds where bacterial colonization and biofilm formation remains significant issues.

    Frederico Alves, Karin Artursson, Juliette Bloch, Anne Brisabois, Hein Imberechts, Pikka Jokelainen, Roberto La Ragione, Mats Lindblad, Rebecca Litzell Forss, Denise A. Marston, Omid Parvizi, Lena Tuominen, Anna Omazic (2023)A multi-country One Health foodborne outbreak simulation exercise: cross-sectoral cooperation, data sharing and communication, In: Frontiers in public health111121522 Frontiers Media S.A
    Mukunthan Tharmakulasingam, Wenwu Wang, Michael Kerby, Roberto La Ragione, Anil Fernando (2023)TransAMR: An Interpretable Transformer Model for Accurate Prediction of Antimicrobial Resistance Using Antibiotic Administration Data, In: IEEE access11pp. 75337-75350 IEEE

    Antimicrobial Resistance (AMR) is a growing public and veterinary health concern, and the ability to accurately predict AMR from antibiotics administration data is crucial for effectively treating and managing infections. While genomics-based approaches can provide better results, sequencing, assembling, and applying Machine Learning (ML) methods can take several hours. Therefore, alternative approaches are required. This study focused on using ML for antimicrobial stewardship by utilising data extracted from hospital electronic health records, which can be done in real-time, and developing an interpretable 1D-Transformer model for predicting AMR. A multi-baseline Integrated Gradient pipeline was also incorporated to interpret the model, and quantitative validation metrics were introduced to validate the model. The performance of the proposed 1D-Transformer model was evaluated using a dataset of urinary tract infection (UTI) patients with four antibiotics. The proposed 1D-Transformer model achieved 10% higher area under curve (AUC) in predicting AMR and outperformed traditional ML models. The Explainable Artificial Intelligence (XAI) pipeline also provided interpretable results, identifying the signatures contributing to the predictions. This could be used as a decision support tool for personalised treatment, introducing AMR-aware food and management of AMR, and it could also be used to identify signatures for targeted interventions.

    Henry Nicholls, Bobby Kainth, Paul Cook, Rick Mumford, Charles Keevil, Roberto La Ragione, Eric Threlfall, David McDowell, Kathryn Callaghan (2023)Foodborne antimicrobial resistance (AMR) research and surveillance in the UK: priorities identified through the Food Standards Agency’s AMR programme review (2023), In: Journal of medical microbiology72(10) Microbiology Society

    The significance and burden of antimicrobial resistance (AMR) has been well documented. The 2016 O’Neill report highlighted the enormous risk that AMR poses to human health and made recommendations to address this global challenge [1]. The report prompted the UK’s 20 year vision for AMR [2], highlighting how the UK will contain and control antimicrobial resistance by 2040. Alongside this vision, a 5 year National Action Plan (NAP) [3], 2019–2024, was created; laying out how government departments would deliver co-ordinated action to mitigate the impact of AMR.

    Aurore C. Poirier, Ruben D. Riaño Moreno, Leona Takaindisa, Jessie Carpenter, Jai W. Mehat, Abi Haddon, Mohammed A. Rohaim, Craig Williams, Peter Burkhart, Chris Conlon, Matthew Wilson, Matthew McClumpha, Anna Stedman, Guido Cordoni, Manoharanehru Branavan, Mukunthan Tharmakulasingam, Nouman S. Chaudhry, Nicolas Locker, Anil Fernando, Wamadeva Balachandran, Mark Bullen, Nadine Collins, David Rimer, Daniel L. Horton, Muhammad Munir, Roberto M. La Ragione (2023)VIDIIA Hunter diagnostic platform: a low-cost, smartphone connected, artificial intelligence-assisted COVID-19 rapid diagnostics approved for medical use in the UK, In: Frontiers in molecular biosciences101144001 Frontiers Media S.A

    Introduction: Accurate and rapid diagnostics paired with effective tracking and tracing systems are key to halting the spread of infectious diseases, limiting the emergence of new variants and to monitor vaccine efficacy. The current gold standard test (RT-qPCR) for COVID-19 is highly accurate and sensitive, but is time-consuming, and requires expensive specialised, lab-based equipment. Methods: Herein, we report on the development of a SARS-CoV-2 (COVID-19) rapid and inexpensive diagnostic platform that relies on a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay and a portable smart diagnostic device. Automated image acquisition and an Artificial Intelligence (AI) deep learning model embedded in the Virus Hunter 6 (VH6) device allow to remove any subjectivity in the interpretation of results. The VH6 device is also linked to a smartphone companion application that registers patients for swab collection and manages the entire process, thus ensuring tests are traced and data securely stored. Results: Our designed AI-implemented diagnostic platform recognises the nucleocapsid protein gene of SARS-CoV-2 with high analytical sensitivity and specificity. A total of 752 NHS patient samples, 367 confirmed positives for coronavirus disease (COVID-19) and 385 negatives, were used for the development and validation of the test and the AI-assisted platform. The smart diagnostic platform was then used to test 150 positive clinical samples covering a dynamic range of clinically meaningful viral loads and 250 negative samples. When compared to RT-qPCR, our AI-assisted diagnostics platform was shown to be reliable, highly specific (100%) and sensitive (98–100% depending on viral load) with a limit of detection of 1.4 copies of RNA per µL in 30 min. Using this data, our CE-IVD and MHRA approved test and associated diagnostic platform has been approved for medical use in the United Kingdom under the UK Health Security Agency’s Medical Devices (Coronavirus Test Device Approvals, CTDA) Regulations 2022. Laboratory and in-silico data presented here also indicates that the VIDIIA diagnostic platform is able to detect the main variants of concern in the United Kingdom (September 2023). Discussion: This system could provide an efficient, time and cost-effective platform to diagnose SARS-CoV-2 and other infectious diseases in resource-limited settings.

    Joanna Urbaniec, Maria Getino, Tahnee B-D McEwan, Martina L Sanderson-Smith, Johnjoe McFadden, Faisal Hai, Roberto La Ragione, Marwa M Hassan, Suzie Hingley-Wilson (2023)Anti-persister efficacy of colistin and meropenem against uropathogenic Escherichia coli is dependent on environmental conditions, In: Microbiology169(11)001403 Microbiology Society

    Antibiotic persistence is a phenomenon observed when genetically susceptible cells survive long-term exposure to antibiotics. These 'persisters' are an intrinsic component of bacterial populations and stem from phenotypic heterogeneity. Persistence to antibiotics is a concern for public health globally, as it increases treatment duration and can contribute to treatment failure. Furthermore, there is a growing array of evidence that persistence is a 'stepping-stone' for the development of genetic antimicrobial resistance. Urinary tract infections (UTIs) are a major contributor to antibiotic consumption worldwide, and are known to be both persistent (i.e. affecting the host for a prolonged period) and recurring. Currently, in clinical settings, routine laboratory screening of pathogenic isolates does not determine the presence or the frequency of persister cells. Furthermore, the majority of research undertaken on antibiotic persistence has been done on lab-adapted bacterial strains. In the study presented here, we characterized antibiotic persisters in a panel of clinical uropathogenic Escherichia coli isolates collected from hospitals in the UK and Australia. We found that a urine-pH mimicking environment not only induces higher levels of antibiotic persistence to meropenem and colistin than standard laboratory growth conditions, but also results in rapid development of transient colistin resistance, regardless of the genetic resistance profile of the isolate. Furthermore, we provide evidence for the presence of multiple virulence factors involved in stress resistance and biofilm formation in the genomes of these isolates, whose activities have been previously shown to contribute to the formation of persister cells.

    Giovanni Lo Iacono, Alasdair Cook, Gianne Derks, Lora E. Fleming, Nigel French, Emma L. Gillingham, Laura Cristina Gonzalez Villeta, Clare Heaviside, Roberto M. La Ragione, Giovanni Leonardi, Christophe Sarran, Sotiris Vardoulakis, Francis Senyah, Arnoud H. M. van Vliet, Gordon L Nichols (2023)A mathematical, classical stratification modeling approach to disentangling the impact of weather on infectious diseases: a case study using spatio-temporally disaggregated Campylobacter surveillance data for England and Wales, In: Dataset for Campylobacter project Public Library of Science

    Disentangling the impact of the weather on transmission of infectious diseases is crucial for health protection, preparedness and prevention. Because weather factors are co-incidental and partly correlated, we have used geography to separate out the impact of individual weather parameters on other seasonal variables using campylobacteriosis as a case study. Campylobacter infections are found worldwide and are the most common bacterial food-borne disease in developed countries, where they exhibit consistent but country specific seasonality. We developed a novel conditional incidence method, based on classical stratification, exploiting the long term, high-resolution, linkage of approximately one-million campylobacteriosis cases over 20 years in England and Wales with local meteorological datasets from diagnostic laboratory locations. The predicted incidence of campylobacteriosis increased by 1 case per million people for every 5° (Celsius) increase in temperature within the range of 8° − 15°. Limited association was observed outside that range. There were strong associations with day-length. Cases tended to increase with relative humidity in the region of 75 − 80%, while the associations with rainfall and wind-speed were weaker. The approach is able to examine multiple factors and model how complex trends arise, e.g. the consistent steep increase in campylobacteriosis in England and Wales in May-June and its spatial variability. This transparent and straightforward approach leads to accurate predictions without relying on regression models and/or postulating specific parameterisations. A key output of the analysis is a thoroughly phenomenological description of the incidence of the disease conditional on specific local weather factors. The study can be crucially important to infer the elusive mechanism of transmission of campylobacteriosis; for instance, by simulating the conditional incidence for a postulated mechanism and compare it with the phenomenological patterns as benchmark. The findings challenge the assumption, commonly made in statistical models, that the transformed mean rate of infection for diseases like campylobacteriosis is a mere additive and combination of the environmental variables.

    Aurore Poirier, Jennifer Cantlay, E Campling, D Horton, W.H.M van der Poel, Roberto La Ragione (2023)D6.17 Report on the Fourth One Health EJP Final School614516 One Health European Joint Programme (OHEJP)

    The One Health EJP (OHEJP) Final “Summer” School 2022 “Sustainability in One Health – how can it be achieved?” was held from 5th to 7th December 2022. The event focused on sustainability, since it is at the core of the One Health approach to optimise the health of people, animals, plants, and ecosystems and needs to be at the centre of the training of young One Health scientists and practitioners. Sustainable solutions must be adopted that recognise the importance of animal welfare, biodiversity and ecosystem integrity for overall health and well-being, now and for future generations. This Final School was organised by the Local Organising team at University of Surrey in Guildford – UK, led by Roberto La Ragione (Professor of Veterinary Microbiology and Pathology at the School of Veterinary Medicine and Head of the School of Biosciences). The event was organised and hosted in collaboration with the OHEJP Work Package 6 (WP6) team and Communications team based at the University of Surrey, UK. The event was held online to reach a wider international audience. In total, over 200 scientists from countries across the globe participated. The event was a major success as it provided the first international training initiative entirely devoted to sustainability to a multidisciplinary audience of delegates and involved many OHEJP partners as well as OHEJP Stakeholders. The testimonials from the delegates provide telling evidence of the Final School success.

    Roberto La Ragione, Aurore Poirier, Jennifer Cantlay, Dan Horton, Wim van der Poel (2022)D6.22 Fourth periodic report on PhD projects One Health EJP
    Aurore C. Poirier, Ruben D. Riaño Moreno, Leona Takaindisa, Mukunthan Tharmakulasingam, Jessie Carpenter, Nouman S. Chaudhry, Jai W. Mehat, Anil Fernando, Abi Haddon, Daniel L. Horton, Mohammed A. Rohaim, Roberto M. La Ragione, Craig Williams, Peter Burkhart, Chris Conlon, Matthew Wilson, Matthew McClumpha, Anna Stedman, Nicolas Locker, Guido Cordoni, Manoharanehru Branavan, Wamadeva Balachandran, Mark Bullen, Nadine Collins, David Rimer, Muhammad Munir (2023)Corrigendum: VIDIIA Hunter: a low-cost, smartphone connected, artificial intelligence-assisted COVID-19 rapid diagnostic platform approved for medical use in the UK, (Front. Mol. Biosci, (2023), 10, (1144001), 10.3389/fmolb.2023.1144001), In: Frontiers in molecular biosciences10 Frontiers Media

    In the published article, there was an error in the Article title. Instead of “VIDIIA Hunter diagnostic platform: a low-cost, smartphone connected, artificial intelligence-assisted COVID-19 rapid diagnostics approved for medical use in the UK,” it should be “VIDIIA Hunter: a low-cost, smartphone connected, artificial intelligence-assisted COVID-19 rapid diagnostic platform approved for medical use in the UK”. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

    Jai Mehat, James R. G. Adams, Roberto La Ragione, Shahriar Behboudi (2023)Preventing bacterial disease in poultry in the post-antibiotic era: a case for innate immunity modulation as an alternative to antibiotic use, In: Frontiers in immunology14

    The widespread use of antibiotics in the poultry industry has led to the emergence of antibiotic-resistant bacteria, which pose a significant health risk to humans and animals. These public health concerns, which have led to legislation limiting antibiotic use in animals, drive the need to find alternative strategies for controlling and treating bacterial infections. Modulation of the avian innate immune system using immunostimulatory compounds provides a promising solution to enhance poultry immune responses to a broad range of bacterial infections without the risk of generating antibiotic resistance. An array of immunomodulatory compounds have been investigated for their impact on poultry performance and immune responses. However, further research is required to identify compounds capable of controlling bacterial infections without detrimentally affecting bird performance. It is also crucial to determine the safety and effectiveness of these compounds in conjunction with poultry vaccines. This review provides an overview of the various immune modulators known to enhance innate immunity against avian bacterial pathogens in chickens, and describes the mechanisms involved.

    Khushboo Borah Slater, Muhammad Ahmad, Aurore Poirier, Ash Stott, Bianca S. Siedler, Matthew Brownsword, Jai Mehat, Joanna Urbaniec, Nicolas Locker, Yunlong Zhao, Roberto La Ragione, S. Ravi P. Silva, Johnjoe McFadden (2023)Development of a loop-mediated isothermal amplification (LAMP)-based electrochemical test for rapid detection of SARS-CoV-2, In: iScience

    Rapid, reliable, sensitive, portable, and accurate diagnostics are required to control disease outbreaks such as COVID-19 that pose an immense burden on human health and the global economy. Here we developed a loop-mediated isothermal amplification (LAMP)-based electrochemical test for the detection of SARS-CoV-2 that causes COVID-19. The test is based on the oxidation-reduction reaction between pyrophosphates (generated from positive LAMP reaction) and molybdate that is detected by cyclic voltammetry using inexpensive and disposable carbon screen printed electrodes. Our test showed higher sensitivity (detecting as low as 5.29 RNA copies/μL) compared to the conventional fluorescent reverse transcriptase (RT)-LAMP. We validated our tests using human serum and saliva spiked with SARS-CoV-2 RNA and clinical (saliva and nasal-pharyngeal) swab samples demonstrating 100% specificity and 93.33% sensitivity. Our assay provides a rapid, specific, and sensitive test with an electrochemical readout in less than 45 min that could be adapted for point-of-care settings.

    Mohammed A Rohaim, Emily Clayton, Irem Sahin, Roberto Marcello La Ragione, Julianne Vilela, Anil Fernando, Manar E. Khalifa, Mohammad Q. Al-Natour, Mahmoud Bayoumi, Aurore Poirier, Manoharanehru Branavan, Mukunthan Tharmakulasingam, Nouman S. Chaudhry, Ravinder Sodi, Amy Brown, Peter Burkhart, Wendy Hacking, Judy Botham, Joe Boyce, Hayley Wilkinson, Craig Williams, Jayde Whittingham-Dowd, Elisabeth Shaw, Matt Hodges, Lisa Butler, M Bates, Wamadeva Balachandran, Muhammad Munir (2020)Artificial intelligence-assisted loop mediated isothermal amplification (AI-LAMP) for rapid detection of SARS-CoV-2, In: Viruses12(9)972 MDPI

    Until vaccines and effective therapeutics become available, the practical solution to transit safely out of the current coronavirus disease 19 (CoVID-19) lockdown may include the implementation of an effective testing, tracing and tracking system. However, this requires a reliable and clinically validated diagnostic platform for the sensitive and specific identification of SARS-CoV-2. Here, we report on the development of a de novo, high-resolution and comparative genomics guided reverse-transcribed loop-mediated isothermal amplification (LAMP) assay. To further enhance the assay performance and to remove any subjectivity associated with operator interpretation of results, we engineered a novel hand-held smart diagnostic device. The robust diagnostic device was further furnished with automated image acquisition and processing algorithms and the collated data was processed through artificial intelligence (AI) pipelines to further reduce the assay run time and the subjectivity of the colorimetric LAMP detection. This advanced AI algorithm-implemented LAMP (ai-LAMP) assay, targeting the RNA-dependent RNA polymerase gene, showed high analytical sensitivity and specificity for SARS-CoV-2. A total of similar to 200 coronavirus disease (CoVID-19)-suspected NHS patient samples were tested using the platform and it was shown to be reliable, highly specific and significantly more sensitive than the current gold standard qRT-PCR. Therefore, this system could provide an efficient and cost-effective platform to detect SARS-CoV-2 in resource-limited laboratories.

    Tiphany Chrun, Joy Leng, Roberto M La Ragione, Simon P Graham, Elma Tchilian (2021)Changes in the nasal microbiota of pigs following single or co-infection with porcine reproductive and respiratory syndrome and swine influenza a viruses, In: Pathogens10(10)1225 MDPI

    Host-microbiota interactions are important in shaping immune responses that have the potential to influence the outcome of pathogen infection. However, most studies have focused on the gut microbiota and its possible association with disease outcome, while the role of the nasal microbiota and respiratory pathogen infection has been less well studied. Here we examined changes in the composition of the nasal microbiota of pigs following experimental infection with porcine reproductive and respiratory syndrome virus 2 (PRRSV-2), swine influenza A H3N2 virus (H3N2) or both viruses. DNA extracted from nasal swabs were subjected to 16S rRNA sequencing to study the composition of the nasal microbiota. Bacterial richness fluctuated in all groups, with a slight reduction in pigs singly infected with PRRSV-2 and H3N2 during the first 5 days of infection compared to uninfected controls. In contrast, nasal bacterial richness remained relatively stable after PRRSV-2/H3N2 co-infection. PRRSV-2 and H3N2, alone or in combination differentially altered the abundance and distribution of bacterial families. Single and co-infection with PRRSV-2 or H3N2 was associated with the expansion of the Neisseriaceae family. A positive correlation between H3N2 viral load and the relative abundance of the Neisseriaceae was observed. However, further mechanistic studies are required to understand the significance of the changes in specific bacterial families following these viral infections.

    Giorgia Tura, Federica Savini, Laura Gallina, Roberto M. La Ragione, Andy E. Durham, Martina Mazzeschi, Mattia Lauriola, Giancarlo Avallone, Giuseppe Sarli, Barbara Brunetti, Luisa Muscatello, Cinzia Girone, Barbara Bacci (2021)Fibroblast-associated protein-α expression and BPV nucleic acid distribution in equine sarcoids, In: Veterinary pathology58(6)pp. 1044-1050 Sage

    Sarcoids are the most common cutaneous tumor of equids and are caused by bovine papillomavirus (BPV). Different clinical subtypes of sarcoids are well characterized clinically but not histologically, and it is not known whether viral activity influences the clinical or histological appearance of the tumors. The aim of this study was to verify whether the development of different clinical types of sarcoids or the presence of certain histological features were associated with BPV distribution within the tumor. The presence of BPV was assessed by polymerase chain reaction (PCR) and visualized in histological sections by chromogenic in situ hybridization (CISH) in 74 equine sarcoids. Furthermore, to better characterize the molecular features of neoplastic cells, immunohistochemistry for S100, smooth muscle actin-alpha (alpha SMA), and fibroblast-associated protein-alpha (FAP alpha) was performed. The presence of BPV was confirmed in all tissues examined by either or both PCR and CISH (72/74, 97% each). Of 70/74 CISH-positive cases, signal distribution appeared as either diffuse (61/70, 87%) or subepithelial (9/70, 13%); the latter was more frequently observed in the verrucous subtype. However, no statistically significant association was found between clinical subtypes and specific histological features or hybridization pattern. Moreover, CISH signal for BPV was not detected in the epidermis overlying sarcoids nor in the tissue surrounding the neoplasms. By immunohistochemistry, alpha SMA confirmed the myofibroblastic differentiation of neoplastic cells in 28/74 (38%) sarcoids. Using tissue microarrays, FAP alpha labelling was observed in neoplastic fibroblasts of all sarcoids, suggesting this marker as a potential candidate for the immunohistochemical diagnosis of sarcoids.

    James P.R Sorensen, Phil Aldous, Sarah Y Bunting, Susan McNally, Barry R Townsend, Megan J Barnett, Tessa Harding, Roberto M La Ragione, Marianne E Stuart, Holly J Tipper, PHILIP ALDOUS, Steve Pedley (2021)Seasonality of enteric viruses in groundwater-derived public water sources, In: Water Research207117813 Elsevier

    We investigated the seasonal prevalence of seven enteric viruses in groundwater-derived public water sources distributed across the dominant aquifers of England. Sampling targeted four periods in the hydrological cycle with typically varying microbial risks, as indicated using a decade of Escherichia coli prevalence data. Viruses were concentrated onsite by filtration of raw groundwater, and extracted nucleic acid (NA) was amplified by qPCR or RT-qPCR. Seven out of eight sources, all aquifers, and 31% of samples were positive for viral NA. The most frequently detected viral NA targets were Hepatitis A virus (17% samples, 63% sites), Norovirus GI (14% samples, 38% sites), and Hepatitis E virus (7% samples, 25% sites). Viral NA presence was episodic, being most prevalent and at its highest concentration during November and January, the main groundwater recharge season, with 89% of all positive detects occurring during a rising water table. Seasonal Norovirus NA detections matched its seasonal incidence within the population. Viral NA is arriving with groundwater recharge, as opposed to persisting for long-periods within the saturated zone. Neither total coliforms nor E. coli were significant predictors of viral NA presence-absence, and there was limited co-occurrence between viruses. Nevertheless, a source with an absence of E. coli in regularly collected historical data is unlikely to be at risk of viral contamination. To manage potential groundwater viral contamination via risk assessment, larger scale studies are required to understand key risk factors, with the evidence here suggesting viral NA is widespread across a range of typical microbial risk settings.

    Jade L Passey, Roberto M La Ragione (2022)JMM Profile: Brachyspira species: The causative agent of Avian Intestinal Spirochaetosis, In: Journal of medical microbiology71(9)

    The genus Brachyspira includes nine officially recognised species, several of which are pathogenic to mammals and birds. B. pilosicoli, B. intermedia, and B. alvinipulli are the causative agents of avian intestinal spirochaetosis (AIS), a gastrointestinal disease in poultry caused by the colonisation of the caeca and/ or colo-rectum by Brachyspira . AIS primarily affects layer hens and broiler breeders over the age of 15 weeks. The severity of symptoms can vary but typically presents as reduced growth rates, delayed onset of lay, reduced egg production, faecally stained eggs, and diarrhoea. This disease is estimated to cost the UK laying industry £18 million per annum. Brachyspira colonisation in humans is common in populations from developing countries and HIV-positive patients; however, it is rarely investigated as a human pathogen.

    Klaudia Chrzastek, Joy Leng, Mohammad Khalid Zakaria, Dagmara Bialy, Roberto La Ragione, Holly Shelton (2021)Low pathogenic avian influenza virus infection retards colon microbiota diversification in two different chicken lines, In: Animal microbiome3(1)64 BioMedCentral

    Background A commensal microbiota regulates and is in turn regulated by viruses during host infection which can influence virus infectivity. In this study, analysis of colon microbiota population changes following a low pathogenicity avian influenza virus (AIV) of the H9N2 subtype infection of two different chicken breeds was conducted. Methods Colon samples were taken from control and infected groups at various timepoints post infection. 16S rRNA sequencing on an Illumina MiSeq platform was performed on the samples and the data mapped to operational taxonomic units of bacterial using a QIIME based pipeline. Microbial community structure was then analysed in each sample by number of observed species and phylogenetic diversity of the population. Results We found reduced microbiota alpha diversity in the acute period of AIV infection (day 2–3) in both Rhode Island Red and VALO chicken lines. From day 4 post infection a gradual increase in diversity of the colon microbiota was observed, but the diversity did not reach the same level as in uninfected chickens by day 10 post infection, suggesting that AIV infection retards the natural accumulation of colon microbiota diversity, which may further influence chicken health following recovery from infection. Beta diversity analysis indicated a bacterial species diversity difference between the chicken lines during and following acute influenza infection but at phylum and bacterial order level the colon microbiota dysbiosis was similar in the two different chicken breeds. Conclusion Our data suggest that H9N2 influenza A virus impacts the chicken colon microbiota in a predictable way that could be targeted via intervention to protect or mitigate disease.

    Roberta Maria Antonello, Stefano Di Bella, Jonathan Betts, Roberto La Ragione, Raffaela Bressan, Luigi Principe, Stefano Morabito, Federica Gigliucci, Rosangela Tozzoli, Marina Busetti, Anna Knezevich, Linda Furlanis, Francesco Fontana, Francesco Luzzaro, Roberto Luzzati, Cristina Lagatolla (2021)Zidovudine in synergistic combination with fosfomycin: an in vitro and in vivo evaluation against multidrug-resistant Enterobacterales, In: International journal of antimicrobial agents58(1)106362 Elsevier Ltd

    •Fosfomycin (FOF) is an antibiotic option against multidrug-resistant (MDR) Enterobacterales.•Zidovudine (ZDV) has unexploited antibacterial properties.•Study of ZDV in combination with FOF against MDR Enterobacterales.•In vitro, ZDV restored FOF susceptibility in ~90% of resistant strains.•In vivo, G. mellonella survival was higher (20–50%) with FOF + ZDV combination. Multidrug-resistant (MDR) Enterobacterales are a priority health issue with few treatment options. Recently, fosfomycin has been reconsidered for MDR bacterial infections. Zidovudine, licensed for the treatment of human immunodeficiency virus (HIV), has unexploited antibacterial properties and has been considered for drug repurposing. The aim of this study was to assess the effect of the combination of fosfomycin plus zidovudine against clinical MDR Enterobacterales isolates. Minimum inhibitory concentration (MIC) determination and checkerboard assays for 36 MDR Enterobacterales strains were performed. In addition, fosfomycin-resistant strains were evaluated using time–kill assay and in an in vivo Galleria mellonella infection model. Zidovudine and fosfomycin MICs ranged between 0.06 to >64 mg/L and 0.125 to >512 mg/L, respectively. A synergistic effect [fractional inhibitory concentration index (FICI) ≤0.5] was observed in 25 isolates and no antagonistic effect was observed in the remaining isolates. For 7 of 8 fosfomycin-resistant strains (MIC > 32 mg/L), zidovudine combination was able to restore fosfomycin susceptibility. These results were confirmed by time–kill assays. Fosfomycin + zidovudine presented greater larval survival (20–50%) than monotherapy. Synergistic activity was observed for fosfomycin + zidovudine in 69.4% of the tested strains. In vivo experiments confirmed the enhanced effectiveness of the combination. The zidovudine concentrations tested here can be reached in human serum using the actual licensed dosage, therefore this combination deserves further clinical investigation.

    André Becker Simões Saidenberg, Arnoud H.M van Vliet, Paulo Eduardo Brandão, Lilian Rose Marques de Sá, Marcos Paulo Vieira Cunha, Roberto M La Ragione, Terezinha Knöbl (2020)Genomic characterization of enteropathogenic Escherichia coli (EPEC) of avian origin and rabbit ileal loop response; a pet macaw (Ara chloropterus) as a possible zoonotic reservoir, In: The Veterinary Quarterly40(1)pp. 331-341 Taylor & Francis

    Enteropathogenic Escherichia coli (EPEC) constitutes one of the main causes of mortality in children in low- to medium-income countries. Diverse animal species have been linked as reservoirs, including birds. The aim of this study was to describe the genomic and phylogenetic features of an EPEC recovered from a pet macaw and further characterizing the macro and microscopic lesion in a rabbit ileal loop experimental model. The isolate was whole-genome sequenced (WGS) obtaining its genotypic and phenotypic in silico characteristics and inoculated in a rabbit experimental model with subsequently evaluating the strain's pathogenicity by scanning electron microscopy (SEM) and histopathology. The isolate was characterized as O109:H21-B1-ST40 typical EPEC, harboring several virulence factors of diarrheagenic E. coli. The macaw EPEC genome was located in a monophyletic clade of human and animal ST40 EPEC sequences. In vivo inoculation demonstrated severe hemorrhage with SEM and histopathological analysis confirming these lesions to be associated with intra-epithelial lymphocytes. Therefore, the isolate not only shared several genotypic and phylogenetic similarities with EPEC that affects humans and animals, but was able to induce severe tissue injury in a mammal model. These findings highlight the underrated role of pet birds as zoonotic reservoirs and the diversity in virulence factors being unraveled by new WGS studies.

    MARWA M. HASSAN, Lucy F. Grist, AURORE POIRIER, ROBERTO MARCELLO LA RAGIONE (2022)JMM profile: Loop-mediated isothermal amplification (LAMP): For the rapid detection of nucleic acid targets in resource-limited settings, In: Journal of medical microbiology71(5)
    Mukunthan Tharmakulasingam, Nouman S. Chaudhry, Manoharanehru Branavan, Wamadeva Balachandran, Aurore C. Poirier, Mohammed A. Rohaim, Muhammad Munir, Roberto M. La Ragione, Anil Fernando (2021)An artificial intelligence-assisted portable low-cost device for the rapid detection of sars-cov-2, In: Electronics (Basel)10(17)2065 Mdpi

    An artificial intelligence-assisted low-cost portable device for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is presented here. This standalone temperature-controlled device houses tubes designed for conducting reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays. Moreover, the device utilises tubes illuminated by LEDs, an in-built camera, and a small onboard computer with automated image acquisition and processing algorithms. This intelligent device significantly reduces the normal assay run time and removes the subjectivity associated with operator interpretation of colourimetric RT-LAMP results. To further improve this device's usability, a mobile app has been integrated into the system to control the LAMP assay environment and to visually display the assay results by connecting the device to a smartphone via Bluetooth. This study was undertaken using similar to 5000 images produced from the similar to 200 LAMP amplification assays using the prototype device. Synthetic RNA and a small panel of positive and negative SARS-CoV-2 patient samples were assayed for this study. State-of-the-art image processing and artificial intelligence algorithms were applied to these images to analyse them and to select the most efficient algorithm. The template matching algorithm for image extraction and MobileNet CNN architecture for classification results provided 98.0% accuracy with an average run time of 20 min to confirm the endpoint result. Two working points were chosen based on the best compromise between sensitivity and specificity. The high sensitivity point has a sensitivity value of 99.12% and specificity value of 70.8%, while at the high specificity point, the sensitivity is 96.05% and specificity 93.59%. Furthermore, this device provides an efficient and cost-effective platform for non-health professionals to detect not only SARS-CoV-2 but also other pathogens in resource-limited laboratories, factories, airports, schools, universities, and homes.

    Taranpreet Rai, Ambra Morisi, Barbara Bacci, Nicholas J. Bacon, Michael J. Dark, Tawfik Aboellail, Spencer A. Thomas, Miroslaw Bober, Roberto La Ragione, Kevin Wells (2022)Deep learning for necrosis detection using canine perivascular wall tumour whole slide images, In: Scientific Reports1210634

    Necrosis seen in histopathology Whole Slide Images is a major criterion that contributes towards scoring tumour grade which then determines treatment options. However conventional manual assessment suffers from inter-operator reproducibility impacting grading precision. To address this, automatic necrosis detection using AI may be used to assess necrosis for final scoring that contributes towards the final clinical grade. Using deep learning AI, we describe a novel approach for automating necrosis detection in Whole Slide Images, tested on a canine Soft Tissue Sarcoma (cSTS) data set consisting of canine Perivascular Wall Tumours (cPWTs). A patch-based deep learning approach was developed where different variations of training a DenseNet-161 Convolutional Neural Network architecture were investigated as well as a stacking ensemble. An optimised DenseNet-161 with post-processing produced a hold-out test F1-score of 0.708 demonstrating state-of-the-art performance. This represents a novel first-time automated necrosis detection method in the cSTS domain as well specifically in detecting necrosis in cPWTs demonstrating a significant step forward in reproducible and reliable necrosis assessment for improving the precision of tumour grading.

    Apostolos Liakopoulos, Roberto M. La Ragione, Christoph Nagel, Ulrich Schatzschneider, Daniel E. Rozen, Jonathan W. Betts (2020)Manganese complex [Mn(CO)3(tpa-κ3N)]Br increases antibiotic sensitivity in multidrug-resistant Streptococcus pneumonia, In: Journal of Global Antimicrobial Resistance Elsevier

    Objectives The emergence of multidrug-resistance (MDR) in Streptococcus pneumoniae clones and non-vaccine serotypes necessitate the development of novel treatment strategies. This work aimed to determine the efficacy of the Mn complex [Mn(CO)³(tpa-κ³N)]Br against clinically important MDR strains of S. pneumoniae. Methods Twenty MDR clinicalS. pneumoniae strains were included in this study. Minimum inhibitory concentrations (MICs) of [Mn(CO)₃(tpa-κ³N)]Br were determined via broth microdilution alone and in combination with other antimicrobial agents using checkerboard assays and/or disc diffusion tests. In vitro efficacy was assessed by time-kill assays while in vivo efficacy was tested using the insect model Galleria mellonella. Results [Mn(CO)₃(tpa-κ³N)]Br showed moderate in vitro efficacy against S. pneumoniae coupled with bactericidal activity. Checkerboard and disc diffusion assays showed synergy between [Mn(CO)₃(tpa-κ³N)]Br and tetracycline, and the combination of both agents caused rapid kill-kinetics and reduced the MIC below the susceptibility breakpoint of 1 mg/L even for tetracycline-resistant strains of S. pneumoniae. Similar results were observed for the erythromycin- and the co-trimoxazole-Mn complex combination. In the G. mellonella infection model, mortality and morbidity rates at 96 h were significantly lower in larvae treated with [Mn(CO)₃(tpa-κ³N)]Br than phosphate buffered saline, while treatment with the tetracycline-Mn complex combination was superior to monotherapy, resulting in significantly lower mortality and morbidity rates (p ˂ 0.049). Conclusions We show that [Mn(CO)₃(tpa-κ³N)]Br has in vitro and in vivo antibacterial activity against clinically relevant strains of S. pneumoniae and has the potential to be used in combination with currently available antibiotics to increase their effectiveness against MDR S. pneumoniae.

    Joy Leng, Susan McNally, Gemma Walton, Jonathan Swann, Caroline Argo, Sue Emery, Roberto La Ragione, Robert Eustace, Christopher John Proudman (2021)Hay versus haylage: Forage type influences the equine urinary metabonome and faecal microbiota, In: Equine Veterinary Journal54(3)pp. 614-625 Wiley

    Gut microbial communities are increasingly being linked to diseases in animals and humans. Obesity and its associated diseases are a concern for horse owners and veterinarians, and there is a growing interest in the link between diet, the intestinal microbiota and metabolic disease. Assess the influence of long-term hay or haylage feeding on the microbiota and metabolomes of 20 Welsh mountain ponies. Longitudinal study. Urine, faeces and blood were collected from 20 ponies on a monthly basis over a 13-month period. Urine and faeces were analysed using proton magnetic resonance ( H NMR) spectroscopy and faecal bacterial DNA underwent 16S rRNA gene sequencing. Faecal bacterial community profiles were observed to be different for the two groups, with discriminant analysis identifying 102 bacterial groups (or operational taxonomic units, OTUs) that differed in relative abundance in accordance with forage type. Urinary metabolic profiles of the hay and haylage fed ponies were significantly different during 12 of the 13 months of the study. Notably, the urinary excretion of hippurate was greater in the hay fed ponies for the duration of the study, while ethyl-glucoside excretion was higher in the haylage fed ponies. The study was undertaken over a 13-month period and both groups of ponies had access to pasture during the summer months. The data generated from this study, suggest that the choice of forage may have implications for the intestinal microbiota and metabolism of ponies and therefore, potentially their health status. Understanding the potential implication of feeding a particular type of forage will enable horse owners to make more informed choices with regard to feed, especially if their horse or pony is prone to weight gain.

    Jono W Betts, Patrick Roth, Calum A Pattrick, Hannah M Southam, Roberto M La Ragione, Robert K Poole, Ulrich Schatzschneider (2020)Antibacterial activity of Mn(i) and Re(i) tricarbonyl complexes conjugated to a bile acid carrier molecule, In: Metallomics12(10)1563pp. 1563-1575

    A bifunctional cholic acid-bis(2-pyridylmethyl)amine (bpa) ligand featuring an amide linker was coordinated to a manganese(i) or rhenium(i) tricarbonyl moiety to give [M(bpacholamide)(CO)3] with M = Mn, Re in good yield and very high purity. Strong antibacterial activity was observed against four strains of methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus, with minimum inhibitory concentrations (MICs) in the range of 2-3.5 μM. No difference in response was observed for the MSSA vs. MRSA strains. Activity was also independent of the nature of the metal center, as the Mn and Re complexes showed essentially identical MIC values. In contrast to some other metal carbonyl complexes, the activity seems to be unrelated to the release of carbon monoxide, as photoactivation of the Mn complex reduced the potency by a factor of 2-8. Both metal complexes were non-toxic in Galleria mellonella larvae at concentrations of up to 100× the MIC value. In vivo testing in Galleria larvae infected with MRSA/MSSA demonstrated a significant increase in overall survival rates from 46% in the control to 88% in the group treated with the metal complexes. ICP-MS analysis showed that the Mn and Re cholamide complexes are efficiently internalized by E. coli cells and do not interfere with membrane integrity, as evident from a lack of release of intracellular ATP. An increased sensitivity was observed in acrB, acrD, and mdt mutants that are defective in multidrug exporters, indicating that the compounds have an intracellular mechanism of action. Furthermore, E. coli mntP mutants defective in the gene encoding an Mn exporter were more sensitive than the wildtype, while inactivation of the regulator that controls expression of the Mn uptake proteins MntP and MntH slightly increased sensitivity to the compound. Single knockout mutants defective in genes linked to bile salt and oxidative stress response (dinF, yiaH, sodA, katE, and soxS) did not show increased sensitivity relative to the wild type. Overall, neither the cholic acid moiety nor the metal-carbonyl fragment alone appear to be responsible for the biological activity observed and thus the search for the primary intracellular target continues.

    Aurore C. Poirier, Dai Kuang, Bianca S. Siedler, Khushboo Borah, Jai W. Mehat, Jialin Liu, Cui Tai, Xiaoli Wang, Arnoud H. M. van Vliet, Wei Ma, David R. Jenkins, John Clark, Roberto M. La Ragione, Jieming Qu, Johnjoe McFadden (2022)Development of Loop-Mediated Isothermal Amplification Rapid Diagnostic Assays for the Detection of Klebsiella pneumoniae and Carbapenemase Genes in Clinical Samples, In: Frontiers in molecular biosciences8794961 Frontiers Media Sa

    Klebsiella pneumoniae is an important pathogenic bacterium commonly associated with human healthcare and community-acquired infections. In recent years, K. pneumoniae has become a significant threat to global public and veterinary health, because of its high rates of antimicrobial resistance (AMR). Early diagnosis of K. pneumoniae infection and detection of any associated AMR would help to accelerate directed therapy and reduce the risk of the emergence of multidrug-resistant isolates. In this study, we identified three target genes (yhaI, epsL, and xcpW) common to K. pneumoniae isolates from both China and Europe and designed loop-mediated isothermal amplification (LAMP) assays for the detection of K. pneumoniae in clinical samples. We also designed LAMP assays for the detection of five AMR genes commonly associated with K. pneumoniae. The LAMP assays were validated on a total of 319 type reference strains and clinical isolates of diverse genetic backgrounds, in addition to 40 clinical human sputum samples, and were shown to be reliable, highly specific, and sensitive. For the K. pneumoniae-specific LAMP assay, the calculated sensitivity, specificity, and positive and negative predictive values (comparison with culture and matrix-assisted laser desorption/ionization-time of flight mass spectrometry) were all 100% on clinical isolates and, respectively, of 100%, 91%, and 90%, and 100% when tested on clinical sputum samples, while being significantly faster than the reference methods. For the bla(KPC) and other carbapenemases' LAMP assays, the concordance between the LAMP results and the references methods (susceptibility tests) was 100%, on both pure cultures (n = 125) and clinical samples (n = 18). In conclusion, we developed highly sensitive and specific LAMP assays for the clinical identification of K. pneumoniae and detection of carbapenem resistance.

    Johannes Charlier, Herman W. Barkema, Paul Becher, Paola De Benedictis, Ingrid Hansson, Isabel Hennig-Pauka, ROBERTO MARCELLO LA RAGIONE, Lars E Larsen, Evelyn Madoroba, Dominiek Maes, Clara M Marín, Franco Mutinelli, Alasdair J Nisbet, Podgórska Katarzyna , Jozef Vercruysse, Fabrizio Vitale, Diana J.L Williams, Ruth N. Zadoks (2022)Disease control tools to secure animal and public health in a densely populated world, In: The Lancet Planetary health6(10)pp. E812-E824 Elsevier

    Animal health is a prerequisite for global health, economic development, food security, food quality, and poverty reduction, while mitigating against climate change and biodiversity loss. We did a qualitative review of 53 infectious diseases in terrestrial animals with data from DISCONTOOLS, a specialist database and prioritisation model focusing on research gaps for improving infectious disease control in animals. Many diseases do not have any appropriate control tools, but the prioritisation model suggests that we should focus international efforts on Nipah virus infection, African swine fever, contagious bovine pleuropneumonia, peste des petits ruminants, sheeppox and goatpox, avian influenza, Rift Valley fever, foot and mouth disease, and bovine tuberculosis, for the greatest impact on the UN's Sustainable Development Goals. Easy to use and accurate diagnostics are available for many animal diseases. However, there is an urgent need for the development of stable and durable diagnostics that can differentiate infected animals from vaccinated animals, to exploit rapid technological advances, and to make diagnostics widely available and affordable. Veterinary vaccines are important for dealing with endemic, new, and emerging diseases. However, fundamental research is needed to improve the convenience of use and duration of immunity, and to establish performant marker vaccines. The largest gap in animal pharmaceuticals is the threat of pathogens developing resistance to available drugs, in particular for bacterial and parasitic (protozoal, helminth, and arthropod) pathogens. We propose and discuss five research priorities for animal health that will help to deliver a sustainable and healthy planet: vaccinology, antimicrobial resistance, climate mitigation and adaptation, digital health, and epidemic preparedness.

    Barbara Holzer, Pramila Rijal, Adam McNee, Basudev Paudyal, Veronica Martini, Becky Clark, Tanuja Manjegowda, Francisco J Salguero, Emily Bessell, John C Schwartz, Katy Moffat, Miriam Pedrera, Simon P Graham, Alistair Noble, Marie Bonnet-Di Placido, Roberto M La Ragione, William Mwangi, Peter Beverley, John W McCauley, Rodney S Daniels, John A Hammond, Alain R Townsend, Elma Tchilian (2021)Protective porcine influenza virus-specific monoclonal antibodies recognize similar haemagglutinin epitopes as humans, In: PLoS pathogens17(3)e1009330

    Pigs are natural hosts for the same subtypes of influenza A viruses as humans and integrally involved in virus evolution with frequent interspecies transmissions in both directions. The emergence of the 2009 pandemic H1N1 virus illustrates the importance of pigs in evolution of zoonotic strains. Here we generated pig influenza-specific monoclonal antibodies (mAbs) from H1N1pdm09 infected pigs. The mAbs recognized the same two major immunodominant haemagglutinin (HA) epitopes targeted by humans, one of which is not recognized by post-infection ferret antisera that are commonly used to monitor virus evolution. Neutralizing activity of the pig mAbs was comparable to that of potent human anti-HA mAbs. Further, prophylactic administration of a selected porcine mAb to pigs abolished lung viral load and greatly reduced lung pathology but did not eliminate nasal shedding of virus after H1N1pdm09 challenge. Hence mAbs from pigs, which target HA can significantly reduce disease severity. These results, together with the comparable sizes of pigs and humans, indicate that the pig is a valuable model for understanding how best to apply mAbs as therapy in humans and for monitoring antigenic drift of influenza viruses in humans, thereby providing information highly relevant to making influenza vaccine recommendations.

    Florent Kempf, Roberto La Ragione, Barbara Chirullo, Catherine Schouler, Philippe Velge (2022)Super Shedding in Enteric Pathogens: A Review, In: Microorganisms (Basel)10(11)2101 Mdpi

    Super shedding occurs when a small number of individuals from a given host population shed high levels of a pathogen. Beyond this general definition, various interpretations of the shedding patterns have been proposed to identify super shedders, leading to the description of the super shedding phenomenon in a wide range of pathogens, in particular enteric pathogens, which are of considerable interest. Several underlying mechanisms may explain this observation, including factors related to the environment, the gut microbiota, the pathogen itself (i.e., genetic polymorphism), and the host (including immune factors). Moreover, data suggest that the interplay of these parameters, in particular at the host-pathogen-gut microbiota interface, is of crucial importance for the determination of the super shedding phenotype in enteric pathogens. As a phenomenon playing an important role in the epidemics of enteric diseases, the evidence of super shedding has highlighted the need to develop various control strategies.

    Megan G. Taggart, William J. Snelling, Patrick J. Naughton, Roberto M. La Ragione, James S. G. Dooley, Nigel G. Ternan (2021)Biofilm regulation in Clostridioides difficile: Novel systems linked to hypervirulence, In: PLoS pathogens17(9)1009817pp. e1009817-e1009817 Public Library Science

    Clostridiodes difficile (C. difficile) was ranked an "urgent threat" by the Centers for Disease Control and Prevention (CDC) in 2019. C. difficile infection (CDI) is the most common healthcare-associated infection (HAI) in the United States of America as well as the leading cause of antibiotic-associated gastrointestinal disease. C. difficile is a gram-positive, rodshaped, spore-forming, anaerobic bacterium that causes infection of the epithelial lining of the gut. CDI occurs most commonly after disruption of the human gut microflora following the prolonged use of broad-spectrum antibiotics. However, the recurrent nature of this disease has led to the hypothesis that biofilm formation may play a role in its pathogenesis. Biofilms are sessile communities of bacteria protected from extracellular stresses by a matrix of self-produced proteins, polysaccharides, and extracellular DNA. Biofilm regulation in C. difficile is still incompletely understood, and its role in disease recurrence has yet to be fully elucidated. However, many factors have been found to influence biofilm formation in C. difficile, including motility, adhesion, and hydrophobicity of the bacterial cells. Small changes in one of these systems can greatly influence biofilm formation. Therefore, the biofilm regulatory system would need to coordinate all these systems to create optimal biofilm-forming physiology under appropriate environmental conditions. The coordination of these systems is complex and multifactorial, and any analysis must take into consideration the influences of the stress response, quorum sensing (QS), and gene regulation by second messenger molecule cyclic diguanosine monophosphate (c-di-GMP). However, the differences in biofilmforming ability between C. difficile strains such as 630 and the "hypervirulent" strain, R20291, make it difficult to assign a "one size fits all" mechanism to biofilm regulation in C. difficile. This review seeks to consolidate published data regarding the regulation of C. difficile biofilms in order to identify gaps in knowledge and propose directions for future study.

    Arnoud HM van Vliet, Siddhartha Thakur, Joaquin M Prada, Jai W Mehat, Roberto La Ragione (2021)Genomic screening of antimicrobial resistance markers in UK and US Campylobacter isolates highlights stability of resistance over an 18 year period, In: bioRxiv Cold Spring Harbor Laboratory
    Caroline I Le Roy, Luke J Mappley, Roberto M La Ragione, Martin J Woodward, Sandrine P Claus (2015)Brachyspira pilosicoli-induced avian intestinal spirochaetosis, In: Microbial ecology in health and disease26(1)pp. 28853-28853 Taylor & Francis

    Avian intestinal spirochaetosis (AIS) is a common disease occurring in poultry that can be caused by Brachyspira pilosicoli, a Gram-negative bacterium of the order Spirochaetes. During AIS, this opportunistic pathogen colonises the lower gastrointestinal (GI) tract of poultry (principally, the ileum, caeca, and colon), which can cause symptoms such as diarrhoea, reduced growth rate, and reduced egg production and quality. Due to the large increase of bacterial resistance to antibiotic treatment, the European Union banned in 2006 the prophylactic use of antibiotics as growth promoters in livestock. Consequently, the number of outbreaks of AIS has dramatically increased in the UK resulting in significant economic losses. This review summarises the current knowledge about AIS infection caused by B. pilosicoli and discusses various treatments and prevention strategies to control AIS.

    Caroline I Le Roy, Luke J Mappley, Roberto M La Ragione, Martin J Woodward, Sandrine P Claus (2015)Brachyspira pilosicoli-induced avian intestinal spirochaetosis, In: Microbial ecology in health and disease26(1)pp. 28853-28853 Taylor & Francis

    Avian intestinal spirochaetosis (AIS) is a common disease occurring in poultry that can be caused by Brachyspira pilosicoli, a Gram-negative bacterium of the order Spirochaetes. During AIS, this opportunistic pathogen colonises the lower gastrointestinal (GI) tract of poultry (principally, the ileum, caeca, and colon), which can cause symptoms such as diarrhoea, reduced growth rate, and reduced egg production and quality. Due to the large increase of bacterial resistance to antibiotic treatment, the European Union banned in 2006 the prophylactic use of antibiotics as growth promoters in livestock. Consequently, the number of outbreaks of AIS has dramatically increased in the UK resulting in significant economic losses. This review summarises the current knowledge about AIS infection caused by B. pilosicoli and discusses various treatments and prevention strategies to control AIS.

    H. R. Horvitz, M. T. Clegg, M. M. Howe, S. L. Lindquist, R. J. Rothstein, Roberto Marcello La Ragione (1989)Genetics Society of America Program and Abstracts, In: Genetics (Austin)122(2)pp. s1-s48
    Paul Güntzel, Christoph Nagel, Jeanette Weigelt, Jono W. Betts, Calum A. Pattrick, Hannah M. Southam, Roberto M. La Ragione, Robert K. Poole, Ulrich Schatzschneider (2019)Biological activity of manganese(i) tricarbonyl complexes on multidrug-resistant Gram-negative bacteria: From functional studies to in vivo activity in Galleria mellonella, In: Metallomics Royal Society of Chemistry

    Three new manganese(I) tricarbonyl complexes [Mn(bpqa-κ³N)(CO)₃]Br, [Mn(bqpa-κ³N)(CO)₃]Br, and [Mn(CO)₃(tqa-κ³N)]Br as well as the previously described compound [Mn(CO)₃(tpa-κ³N)]Br with bpqa = bis(2-pyridinylmethyl)(2-quinolinylmethyl)amine, bqpa = bis(2-quinolinylmethyl)(2-pyridinylmethyl)amine, tqa = tris(2-quinolinylmethyl)amine, and tpa = tris(2-pyridinylmethyl)amine were examined for their antibacterial activities on 14 different multidrug-resistant clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa, in recognition of the current antimicrobial resistance (AMR) concerns with these pathogens. Minimal inhibitory concentrations (MIC) of the most potent tqa compound were in the mid-micromolar range and generally lower than that of the free ligand. Activity against both bacterial species increased with the number of quinolinylmethyl groups and lipophilicity in the order of tpa < bpqa < bqpa ≈ tqa, consistent with measured increases in release of ATP, a uniquely cytoplasmic biomolecule and induced permeability to exogenous fluorescent intercalating compounds. [Mn(CO)₃(tqa-κ³N)]Br was also evaluated in the Galleria mellonella model of infection, and displayed a lack of host toxicity combined with effective bacterial clearance.

    Matthew Crossfield, Rachel Gilroy, Anuradha Ravi, David Baker, Roberto M La Ragione, Mark J Pallen (2022)Archaeal and Bacterial Metagenome-Assembled Genome Sequences Derived from Pig Feces, In: Microbiology resource announcements11(1)e0114221

    We report the recovery of metagenome-assembled genomes (MAGs) from fecal samples collected in 2018 from five healthy adult female pigs in southeast England. The resulting nonredundant catalog of 192 MAGs encompasses 102 metagenomic species, 41 of them novel, spanning 10 bacterial and 2 archaeal phyla.

    Tom R. Kupfer, Kayleigh J. Wyles, Fraje Watson, Roberto Marcello La Ragione, Mark A. Chambers, Alastair S. Macdonald (2019)Determinants of hand hygiene behaviour based on the Theory of Interpersonal Behaviour, In: Journal of Infection Prevention Sage Publications

    Background: Many investigations into the determinants of hand hygiene (HH) behaviour have explored only individual predictors or were designed according to arguably overly simplistic models of behaviour. Consequently, important influences on HH behaviour, including habit and emotion, are sometimes neglected. This study is the first to employ the Theory of Interpersonal Behaviour as a comprehensive model for understanding the determinants of HH behaviour. Method: A self-report questionnaire was conducted with staff from two large UK veterinary referral practices. Participants (n = 75) reported their HH behaviour and responded to statements rating the importance of social norms, self-protection, patient protection, time pressures, access to equipment, habit and disgust, to their HH behaviour. Results: Regression analysis showed that, overall, determinants explained 46% of variance (p 

    Alice A. K. King, Brigitta Matta-Domjan, Matthew J. Large, Csaba Matta, Sean P. Ogilvie, Niki Bardi, Hugh J. Byrne, Anvar Zakhidov, Izabela Jurewicz, Eirini Velliou, Rebecca Lewis, Roberto La Ragione, Alan Dalton (2017)Pristine carbon nanotube scaffolds for the growth of chondrocytes, In: Journal of Materials Chemistry B5(41)pp. 8178-8182 Royal Society of Chemistry

    The effective growth of chondrocytes and the formation of cartilage is demonstrated on scaffolds of aligned carbon nanotubes; as two dimensional sheets and on three dimensional textiles. Raman spectroscopy is used to confirm the presence of chondroitin sulfate, which is critical in light of the unreliability of traditional dye based assays for carbon nanomaterial substrates. The textile exhibits a very high affinity for chondrocyte growth and could present a route to implantable, flexible cartilage scaffolds with tuneable mechanical properties.

    Jai W. Mehat, Simon F. Park, Arnoud H. M. van Vliet, Roberto M. La Ragione (2018)CapC, a novel autotransporter and virulence factor of Campylobacter jejuni, In: Applied and Environmental Microbiologypp. e01032-18 American Society for Microbiology

    Campylobacter jejuni is recognized as an important causative agent of bacterial gastroenteritis in the developed world. Despite the identification of several factors contributing to infection, characterization of the virulence strategies employed by C. jejuni remains a significant challenge. Bacterial autotransporter proteins are a major class of secretory proteins in Gram-negative bacteria and notably many autotransporter proteins contribute to bacterial virulence. The aim of this study was to characterise the C. jejuni 81116 C8J_1278 gene (capC), predicted to encode an autotransporter protein, and examine the contribution of this factor to virulence of Campylobacter jejuni. The predicted CapC protein has a number of features that are consistent with autotransporters including the N-terminal signal sequence and the C-terminal β-barrel domain and was determined to localise to the outer membrane. Inactivation of the capC gene in C. jejuni 81116 and C. jejuni M1 resulted in reduced insecticidal activity in Galleria mellonella larvae. Furthermore, C. jejuni capC mutants displayed significantly reduced adherence to and invasion of non-polarized, partially differentiated Caco-2 and T84 intestinal epithelial cells. Gentamicin treatment showed that the reduced invasion of the capC mutant is primarily caused by reduced adherence to intestinal epithelial cells, not by reduced invasion capability. C. jejuni capC mutants caused reduced IL-8 secretion from intestinal epithelial cells and elicited a significantly diminished immune reaction in Galleria larvae indicating that CapC functions as an immunogen. In conclusion, CapC is a new virulence determinant of C. jejuni that contributes to the integral infection process of adhesion to human intestinal epithelial cells.

    L Roberts, R La Ragione, M Alder (2012)Starting a new vet school, In: VETERINARY RECORD171(21)pp. 523-524 BRITISH VETERINARY ASSOC
    AF Flockhart, JJ Tree, X Xu, M Karpiyevich, SP McAteer, R Rosenblum, DJ Shaw, CJ Low, A Best, V Gannon, C Laing, KC Murphy, JM Leong, T Schneiders, R La Ragione, DL Gally (2012)Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion., In: Mol Microbiol83(1)pp. 208-223

    This study has identified horizontally acquired genomic regions of enterohaemorrhagic Escherichia coli O157:H7 that regulate expression of the type III secretion (T3S) system encoded by the locus of enterocyte effacement (LEE). Deletion of O-island 51, a 14.93 kb cryptic prophage (CP-933C), resulted in a reduction in LEE expression and T3S. The deletion also had a reduced capacity to attach to epithelial cells and significantly reduced E. coli O157 excretion levels from sheep. Further characterization of O-island 51 identified a novel positive regulator of the LEE, encoded by ecs1581 in the E. coli O157:H7 strain Sakai genome and present but not annotated in the E. coli strain EDL933 sequence. Functionally important residues of ECs1581 were identified based on phenotypic variants present in sequenced E. coli strains and the regulator was termed RgdR based on a motif demonstrated to be important for stimulation of gene expression. While RgdR activated expression from the LEE1 promoter in the presence or absence of the LEE-encoded regulator (Ler), RgdR stimulation of T3S required ler and Ler autoregulation. RgdR also controlled the expression of other phenotypes, including motility, indicating that this new family of regulators may have a more global role in E. coli gene expression.

    Mukunthan Tharmakulasingam, Brian Gardner, Roberto La Ragione, Anil Fernando (2022)Rectified Classifier Chains for Prediction of Antibiotic Resistance from Multi-labelled Data with Missing Labels, In: IEEE/ACM transactions on computational biology and bioinformaticsPP(1)pp. 1-1 IEEE

    Predicting Antimicrobial Resistance (AMR) from genomic data has important implications for human and animal healthcare, and especially given its potential for more rapid diagnostics and informed treatment choices. With the recent advances in sequencing technologies, applying machine learning techniques for AMR prediction have indicated promising results. Despite this, there are shortcomings in the literature concerning methodologies suitable for multi-drug AMR prediction and especially where samples with missing labels exist. To address this shortcoming, we introduce a Rectified Classifier Chain (RCC) method for predicting multi-drug resistance. This RCC method was tested using annotated features of genomics sequences and compared with similar multi-label classification methodologies. We found that applying the eXtreme Gradient Boosting (XGBoost) base model to our RCC model outperformed the second-best model, XGBoost based binary relevance model, by 3.3% in Hamming accuracy and 7.8% in F1-score. Additionally, we note that in the literature machine learning models applied to AMR prediction typically are unsuitable for identifying biomarkers informative of their decisions; in this study, we show that biomarkers contributing to AMR prediction can also be identified using the proposed RCC method. We expect this can facilitate genome annotation and pave the path towards identifying new biomarkers indicative of AMR.

    Mark A Chambers, Simon P Graham, Roberto M La Ragione (2016)Challenges in Veterinary Vaccine Development and Immunization, In: Methods in molecular biology (Clifton, N.J.)1404pp. 3-35

    In approaching the development of a veterinary vaccine, researchers must choose from a bewildering array of options that can be combined to enhance benefit. The choice and combination of options is not just driven by efficacy, but also consideration of the cost, practicality, and challenges faced in licensing the product. In this review we set out the different choices faced by veterinary vaccine developers, highlight some issues, and propose some pressing needs to be addressed.

    M Courtenay, P Conrad, M Wilkes, R La Ragione, N Fitzpatrick (2014)Interprofessional initiatives between the human health professions and veterinary medical students: a scoping review., In: J Interprof Care

    Abstract This article presents the findings of a scoping review designed to identify the extent, nature and range of literature on interprofessional education (IPE) initiatives between the human health professions and veterinary medical students, which is particularly important to advance One Health education and research. Nine published articles were identified. The websites of six universities were searched in order to collect further information. Interventions vary widely with regards to their structure and delivery, their objectives, the participants involved, and outcome measures. Healthcare professional programmes focus upon interprofessional collaborative practice in the human healthcare setting. By contrast, postgraduate programmes focus upon topics under the One Health paradigm but make little mention of interprofessional collaboration. Evidence of the impact of interventions on team processes at the human, animal, and environmental interface is extremely limited. In order to enhance our understanding of what constitutes effective IPE between veterinary medical students and the human health professions, guide intervention development, and the development of outcome measures, there is a need to further explore, define, differentiate and validate some of the terms and concepts used to describe interprofessional interventions.

    Michael Hornsey, Jonathan W. Betts, Jai W. Mehat, David W. Wareham, Arnoud H. M. van Vliet, Martin J. Woodward, Roberto M. La Ragione (2019)Characterization of a colistin-resistant Avian Pathogenic Escherichia coli ST69 isolate recovered from a broiler chicken in Germany, In: Journal of Medical Microbiology68(1)pp. 111-114 Microbiology Society

    In recent years, several plasmids harbouring genes encoding phosphoethanolamine transferases conferring colistin resistance have been described in multiple Enterobacteriaceae species. Avian Pathogenic E. coli (APEC) causes colibacillosis and is responsible for a considerable proportion of the disease burden in commercial poultry flocks, and may be linked to zoonotic infections in humans. Here, we describe the genotypic and phenotypic characteristics of a multidrug-resistant APEC ST69 isolate (APECA2), recovered in 2016 from a diseased broiler at post-mortem examination in Germany. The isolate was resistant to several antibiotics of human and veterinary importance, including colistin. The mcr-1 gene was detected on a mobile genetic element located on an IncHI2/ST4 plasmid, which was characterized using long-read Nanopore and short-read Illumina sequencing of purified plasmid. Isolate APECA2 displayed resistance to chicken serum and harbours numerous virulence genes. This study highlights the public health importance of enhanced antimicrobial resistance surveillance and strict antimicrobial stewardship in human and veterinary healthcare.

    Stefanie Gerson, Jonathan W. Betts, Kai Lucaßen, Carolina Silva Nodari, Julia Wille, Michaele Josten, Stephan Göttig, Jennifer Nowak, Danuta Stefanik, Ignasi Roca, Jordi Vila, José M. Cisneros, Roberto M. La Ragione, Harald Seifert, Paul G. Higgins (2019)Investigation of novel pmrB and eptA mutations in isogenic Acinetobacter baumannii isolates associated with colistin resistance and increased virulence in vivo, In: Antimicrobial Agents and Chemotherapy American Society for Microbiology

    Colistin resistance in Acinetobacter baumannii is of great concern and a threat to human health. In this study we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, ΔL9-G12). Increased expression of pmrC was shown by qRT-PCR for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain-specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain-specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC-homologue eptA and a point mutation in ISAba1 upstream of eptA were associated with colistin-resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain-specific.

    O Diribe, S Thomas, M AbuOun, N Fitzpatrick, R La Ragione (2015)Genotypic relatedness and characterization of Staphylococcus pseudintermedius associated with post-operative surgical infections in dogs, In: JOURNAL OF MEDICAL MICROBIOLOGY64pp. 1074-1081 SOC GENERAL MICROBIOLOGY
    Jonathan W. Betts, Michael Hornsey, Paul G. Higgins, Kai Lucassen, Julia Wille, Francisco Salguero, Harald Seifert, Roberto M. La Ragione (2019)Restoring the activity of the antibiotic aztreonam using the polyphenol epigallocatechin gallate (EGCG) against multidrug-resistant clinical isolates of Pseudomonas aeruginosa, In: Journal of Medical Microbiology68(10) Microbiology Society

    Introduction. Pseudomonas aeruginosa is an important Gram-negative pathogen that is intrinsically multidrug-resistant (MDR) and frequently associated with healthcare-associated outbreaks. With increasing resistance to antibiotics and with very few novel drugs under development, clinicians often use combinations to treat critically ill patients. Aim. The aim of this study was to evaluate the ability of epigallocatechin (EGCG) to restore the activity of aztreonam against clinical MDR strains of P. aeruginosa. Methodology. Checkerboard and time–kill kinetic assays were performed to assess synergy in vitro and the Galleria mellonella model of infection was used to test the efficacy of the combination in vivo. Accumulation assays were performed to gain insight into the mechanism of action. Results. The results demonstrate that synergy between aztreonam and EGCG exists [fractional inhibitory concentration indices (FICIs) 0.02-0.5], with the combination affording significantly (P=˂0.05) enhanced bacterial killing, with a ˃3 log10 reduction in colony-forming units ml−1 at 24 h. EGCG was able to restore susceptibility to aztreonam to a level equal to or below the breakpoint set by the European Committee for Antimicrobial Susceptibility Testing. In G. mellonella, the combination was superior to monotherapy, with increased larval survival observed (94 % vs ≤63 %). We also demonstrated the relatively low toxicity of EGCG to human keratinocytes and G. mellonella larvae. Accumulation assay data suggest that the mechanism of synergy may be due to EGCG increasing the uptake of aztreonam. Conclusion. EGCG was able to restore the activity of aztreonam against MDR P. aeruginosa . The data presented support further evaluation of the aztreonam–EGCG combination and highlight its potential for use in clinical medicine.

    O Diribe, S North, J Sawyer, L Roberts, N Fitzpatrick, R La Ragione (2014)Design and application of a loop-mediated isothermal amplification assay for the rapid detection of Staphylococcus pseudintermedius., In: J Vet Diagn Invest

    Staphylococcus pseudintermedius is a commensal and opportunistic pathogen of dogs. It is mainly implicated in canine pyoderma, as well as other suppurative conditions of dogs. Although bacterial culture is routinely used for clinical diagnosis, molecular methods are required to accurately identify and differentiate S. pseudintermedius from other members of the Staphylococcus intermedius group. These methods, owing largely to their cost, are not easy to implement in nonspecialized laboratories or veterinary practices. In the current study, loop-mediated isothermal amplification (LAMP), a novel isothermal nucleic acid amplification procedure, was employed to develop a rapid, specific, and sensitive S. pseudintermedius assay. Different detection strategies, including the use of a lateral flow device, were evaluated. The assay was evaluated for cross-reactivity against 30 different bacterial species and validated on a panel of 108 S. pseudintermedius isolates, originating from different dog breeds and locations within the United Kingdom. The assay was specific, showing no cross-reactivity during in silico and in vitro testing. When tested using DNA extracts prepared directly from 35 clinical surgical site swabs, the assay could detect S. pseudintermedius in less than 15 min, with a diagnostic sensitivity of 94.6%, superior to that of a polymerase chain reaction method. The LAMP assay also had an analytical sensitivity in the order of 10(1) gene copies, and the amplified products were readily detected using a lateral flow device. The LAMP assay described in the present study is simple and rapid, opening up the possibility of its use as a diagnostic tool within veterinary practices.

    JJ Tree, AJ Roe, A Flockhart, SP McAteer, X Xu, D Shaw, A Mahajan, SA Beatson, A Best, S Lotz, MJ Woodward, R La Ragione, KC Murphy, JM Leong, DL Gally (2011)Transcriptional regulators of the GAD acid stress island are carried by effector protein-encoding prophages and indirectly control type III secretion in enterohemorrhagic Escherichia coli O157:H7, In: MOLECULAR MICROBIOLOGY80(5)pp. 1349-1365 WILEY-BLACKWELL
    Usman O. Adekanye, Abel Bulamu Ekiri, Erika Galipo, Abubakar Bala Muhammad, Ana Mateus, Roberto M. La Ragione (2020)Knowledge, Attitudes and Practices of Veterinarians Towards Antimicrobial Resistance and Stewardship in Nigeria, In: Antibiotics MDPI

    Antimicrobial resistance (AMR) is a global health concern and the inappropriate use of antibiotics in animals and humans are considered contributing factors. A cross-sectional survey to assess the knowledge, attitudes and practices of veterinarians regarding AMR and antimicrobial stewardship was conducted in Nigeria. A total of 241 respondents completed an online survey. Only 21% of respondents correctly defined the term antimicrobial stewardship and 59.8% were unaware of the guidelines provided by the Nigeria AMR National Action Plan. Over half (51%) of respondents indicated that prophylactic antibiotic use was appropriate when farm biosecurity was poor. Only 20% of the respondents conducted antimicrobial susceptibility testing (AST) frequently, and the unavailability of veterinary laboratory services (82%) and the owner’s inability to pay (72%) were reported as key barriers to conducting AST. The study findings suggest a focus on the following areas of potential intervention may be useful in improving appropriate antibiotic use and antimicrobial stewardship among veterinarians in Nigeria: increased awareness of responsible antimicrobial use among practicing and new graduated veterinarians, increased dissemination of 33 regularly updated antibiotic use guidelines, increased understanding of the role of good biosecurity 34 and vaccination practices in disease prevention, and increased provision of AST at affordable costs.

    Roberto La Ragione, MJ Woodward, M Kumar, J Rodenberg, H Fan, Andrew Wales, K Karaca (2013)Efficacy of a Live Attenuated Escherichia coli O78∶K80 Vaccine in Chickens and Turkeys, In: Avian Diseases57(2)pp. 273-279 American Association of Avian Pathologists

    A candidate live vaccine for avian pathogenic Escherichia coli (APEC) was constructed from a virulent field APEC O78 strain by mutation of the aroA gene. The mutant was highly similar to the parent wild-type strain in respect of colony morphology, motility, growth in suspension, hemagglutination, Congo Red binding, HEp-2 cell adhesion, and the elaboration of surface antigens type 1 fimbriae and flagella, although production of curli fimbriae was reduced marginally. The mutant proved avirulent when inoculated into 1-day-old chicks by spray application and when presented again in the drinking water at 7 days of age. Chickens and turkeys vaccinated with an O78 aroA mutant were protected against a challenge at 6 wk of age by virulent APEC strains.

    O Diribe, N Fitzpatrick, J Sawyer, R La Ragione, S North (2015)A Rapid and Simple Loop-Mediated Isothermal Amplification Assay for the Detection of Pseudomonas aeruginosa From Equine Genital Swabs, In: JOURNAL OF EQUINE VETERINARY SCIENCE35(11-12)pp. 929-934 ELSEVIER SCIENCE INC
    Camilla Pedersen, Edith Gallagher, Felicity Horton, Richard J. Ellis, Umer Z. Ijaz, Huihai Wu, Etana Joy Jaiyeola, Onyinye Diribe, Thibaut Duparc, Patrice D. Cani, Glenn R. Gibson, Paul Hinton, John Wright, Roberto La Ragione, M. Denise Robertson (2016)Host-microbiome interactions in human type 2 diabetes following prebiotic dietary fibre (galacto-oligosaccharide) intake, In: The British Journal of Nutrition: an international journal of nutritional science116(11)pp. 1869-1877 Cambridge University Press

    Aberrant microbiota composition and function have been linked to several pathologies, including type 2 diabetes. In animal models, prebiotics induce favourable changes in the intestinal microbiota, intestinal permeability (IP) and endotoxaemia which are linked to concurrent improvement in glucose tolerance. This is the first study to investigate the link between intestinal permeability, glucose tolerance, and intestinal bacteria in human type 2 diabetes. Twenty-nine males with well-controlled type 2 diabetes were randomised to a prebiotic (galactooligosaccharide mixture) or placebo (maltodextrin) supplement (5.5g/day for 12 weeks). Intestinal microbial community structure, IP, endotoxaemia, inflammatory markers and glucose tolerance were assessed at baseline and post-intervention. IP was estimated by the urinary recovery of oral 51Cr-EDTA and glucose tolerance by insulin modified IVGTT. Intestinal microbial community analysis was performed by high-throughput Next-Generation Sequencing of 16S rRNA amplicons and quantitative PCR. Prebiotic fibre supplementation had no significant effects on clinical outcomes or bacterial abundances compared with placebo; however, changes in the bacterial family Veillonellaceae correlated inversely with changes in glucose response and IL-6 levels (r = -0.90, P = 0.042 for both) following prebiotic intake. The absence of significant changes to the microbial community structure at a prebiotic dosage/length of supplementation shown to be effective in healthy individuals is an important finding, We propose that concurrent metformin treatment and the high heterogeneity of human type 2 diabetes may have played a significant role. It is also plausible that prebiotics may play a more important role in prevention rather than in the treatment of human type 2 diabetes.

    P Permpoonpattana, J Phetcharaburanin, A Mikelsone, M Dembek, S Tan, MC Brisson, R La Ragione, AR Brisson, N Fairweather, HA Hong, SM Cutting (2013)Functional Characterization of Clostridium difficile Spore Coat Proteins., In: J Bacteriol

    Spores of Clostridium difficile play a key role in the dissemination of this important human pathogen and until recently little has been known of their functional characteristics. Genes encoding six spore coat proteins (cotA, cotB, cotCB, cotD, cotE, and sodA) were disrupted by ClosTron insertional mutagenesis. Mutation of one gene, cotA, presented a major structural defect in spore assembly with a clear miss-assembly of the outermost layers of the spore coat. The CotA protein is most probably subject to post-translational modification and could play a key role in stabilising the spore coat. Surprisingly, mutation of the other spore coat genes did not affect the integrity of the spore although for the cotD, cotE and sodA mutants enzyme activity was reduced or abolished. This could imply that these enzymatic proteins are located in the exosporium or alternatively they are structurally redundant. Of the spore coat proteins predicted to carry enzymatic activity, three were confirmed as enzymes using both in vivo and in vitro methods, the latter using recombinant expressed proteins. cotD encoding a manganese catalase, sodA a superoxide dismutase (SOD) and cotE a bifunctional enzyme with peroxiredoxin and chitinase activity. These enzymes being exposed on the spore surface would play a role in coat polymerisation and detoxicification of H(2)O(2). Two additional proteins, CotF (a tyrosine rich protein and potential substrate for SodA) and CotG (a putative managanese catalase) were shown to be located to the spore surface.

    C Pedersen, U Ijaz, E Gallagher, F Horton, R Ellis, Etana Joy Jaiyeola, T Duparc, David Russell-Jones, P Hinton, P Cani, Roberto La Ragione, Margaret Robertson (2018)Faecal Enterobacteriales enrichment is associated with increased in-vivo intestinal permeability in humans, In: Physiological Reports6(7)e13649 Wiley Open Access

    Type 2 diabetes (T2D) has been linked with increased intestinal permeability, but the clinical significance of this phenomenon is unknown. The objective of this study was to investigate the potential link between glucose control, intestinal permeability, diet and intestinal microbiota in patients with T2D. Thirty-two males with well-controlled T2D and 30 age-matched male controls without diabetes were enrolled in a case-control study. Metabolic parameters, inflammatory markers, endotoxaemia and intestinal microbiota in individuals subdivided into high (HP) and normal (LP) colonic permeability groups, were the main outcomes. In T2D, the HP group had significantly higher fasting glucose (P = 40 0.034) and plasma non-esterified fatty acid levels (P = 0.05) compared with the LP group. Increased colonic permeability was also linked with altered abundances of selected microbial taxa. The microbiota of both T2D and control HP groups was enriched with Enterobacteriales. In conclusion, high intestinal permeability was associated with poorer fasting glucose control in T2D patients and changes in some microbial taxa in both T2D patients and non-diabetic controls. Therefore, enrichment in the gram- negative order Enterobacteriales may characterise impaired colonic permeability prior to/independently from a disruption in glucose tolerance.

    Jonathan Betts, Michael Hornsey, David W. Wareham, Roberto La Ragione (2017)In vitro and In vivo Activity of Theaflavin–Epicatechin Combinations versus Multidrug-Resistant Acinetobacter baumannii, In: Infectious Diseases and Therapy6(3)pp. 435-442 Adis, Springer Healthcare

    Introduction: Acinetobacter baumannii is an important human nosocomial pathogen; most clinical isolates are multidrug-resistant (MDR). Infections caused by A. baumannii often lead to high morbidity and mortality, with limited treatment options. Owing to the small number of anti-Gram-negative antibiotics in the development pipeline, researchers are looking to other natural compounds. The aim of this study was to determine the in vitro kill kinetics, in vivo efficacy and toxicity of theaflavin-epicatechin combinations against MDR A. baumannii. Methods: Kill-kinetic assays were performed in Mueller Hinton 2 broth over 24 h. Toxicity of the compound in the insect model, Galleria mellonella was investigated. The effect of theaflavin-epicatechin combinations on mortality and morbidity were assessed in Acinetobacter baumannii infected G. mellonella. Larvae were scored for morbidity (melanisation: scale; 0-4) and mortality over 96 h. Results: Kill-kinetic assays revealed that monotherapy had bacteriostatic activity over 24 h, whereas theaflavin-epicatechin combinations were bactericidal (a >3 log reduction in bacterial numbers at 24 h compared with the starting inoculum). Both polyphenols were non-toxic to G. mellonella at concentrations of up to 1000 mg/kg. In vivo treatment assays showed that the combination significantly increased (t-test; p =

    M Giles, S Cawthraw, M AbuOun, C Thomas, D Munera, M Waldor, Roberto La Ragione, Jennifer Ritchie (2018)Host-specific differences in the contribution of an extended spectrum β-lactamase (ESBL) IncI1 plasmid to intestinal colonisation by Escherichia coli O104:H4, In: Journal of Antimicrobial Chemotherapy73(6)pp. 1579-1585 Oxford University Press

    Objectives. To assess stability and contribution of a large extended spectrum β-lactamase (ESBL)-containing IncI1 plasmid to intestinal colonization by Escherichia coli O104:H4 in two different mammalian hosts. Methods. Specific-pathogen-free 3-day old New Zealand White rabbits and conventionally-reared 6-week-old weaned lambs were orally infected with wild-type E. coli O104:H4 or the ESBL-plasmid cured derivative, and the recovery of bacteria in intestinal homogenates and faeces monitored over time. Results. Carriage of the ESBL plasmid had differing impacts on E. coli O104:H4 colonisation of the two experimental hosts. The plasmid cured strain was recovered at significantly higher levels than wild type during late-stage colonization of rabbits, but at lower levels than wildtype in sheep. Regardless of the animal host, the ESBL plasmid was stably maintained in virtually all in vivo passaged bacteria that were examined. Conclusions. These findings suggest that carriage of ESBL plasmids has distinct effects on the host bacterium depending upon the animal species it encounters and demonstrates that, as for E. coli O157:H7, ruminants could represent a potential transmission reservoir.

    X Wang-Kan, JMA Blair, B Chirullo, Jonathan Betts, Roberto La Ragione, A Ivens, V Ricci, TJ Opperman, LJV Piddock (2017)Lack of AcrB efflux function confers loss of virulence to Salmonella Typhimurium, In: mBio8(4)pp. e00968-17 American Society for Microbiology

    AcrAB-TolC is the paradigm resistance-nodulation-division (RND) multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a non-functional AcrB mutant by substituting D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal media. However, compared with wild-type SL1344, the mutant had increased accumulation of the Hoechst 33342 dye, decreased efflux of ethidium bromide and was multidrug hyper-susceptible. The D408A mutant was attenuated in vivo in a mouse model and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro. A dose dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNAseq revealed down-regulation of bacterial factors necessary for infection, including those in the Salmonella Pathogenicity Islands 1, 2 and 4, quorum sensing genes and phoPQ. Several general stress response genes were up-regulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggests that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps.

    CI Le Roy, Jade Passey, MJ Woodward, Roberto La Ragione, SP Claus (2017)Metabonomics-based analysis of Brachyspira pilosicoli's response to tiamulin reveals metabolic activity despite significant growth inhibition, In: Anaerobe45pp. 71-77 Elsevier

    Pathogenic anaerobes Brachyspira spp. are responsible for an increasing number of Intestinal Spirochaetosis (IS) cases in livestock against which few approved treatments are available. Tiamulin is used to treat swine dysentery caused by Brachyspira spp. and recently has been used to handle avian intestinal spirochaetosis (AIS). The therapeutic dose used in chickens requires further evaluation since cases of bacterial resistance to tiamulin have been reported. In this study, we evaluated the impact of tiamulin at varying concentrations on the metabolism of B. pilosicoli using a 1 H-NMR-based metabonomics approach allowing the capture of the overall bacterial metabolic response to antibiotic treatment. Based on growth curve studies, tiamulin impacted bacterial growth even at very low concentration (0.008 mg/mL) although its metabolic activity was barely affected 72 h post exposure to antibiotic treatment. Only the highest dose of tiamulin tested (0.250 mg/mL) caused a major metabolic shift. Results showed that below this concentration, bacteria could maintain a normal metabolic trajectory despite significant growth inhibition by the antibiotic, which may contribute to disease reemergence post antibiotic treatment. Indeed, we confirmed that B. pilosicoli remained viable even after exposition to the highest antibiotic dose. This paper stresses the need to ensure new evaluation of bacterial viability post bacteriostatic exposure such as tiamulin to guarantee treatment efficacy and decrease antibiotic resistance development

    Joy Leng, G. Walton, J. Swann, A. Darby, Roberto La Ragione, Christopher Proudman, Charles M. Dozois (2019)“Bowel on the Bench”: Proof of Concept of a Three-Stage, In Vitro Fermentation Model of the Equine Large Intestine, In: Applied and Environmental Microbiology86(1) American Society for Microbiology

    The intestinal microbiota of the horse, an animal of huge economic and social importance worldwide, is essential to the health of the animal. Understanding the intestinal ecosystem and its dynamic interaction with diet and dietary supplements currently requires the use of experimental animals, with consequent welfare and financial constraints. Here, we describe the development and assessment, using multiple analytical platforms, of a three-vessel, continuous-flow, in vitro model of the equine hindgut. After inoculation of the model with fresh horse feces, the bacterial communities established in each vessel had a taxonomic distribution similar to that of the source animal. Short-chain fatty acid (SCFA) and branched-chain fatty acid (BCFA) production within the model at steady state was consistent with the expected bacterial function, although higher concentrations of some SCFA/BCFA relative to those in the ex vivo gut content were apparent. We demonstrate the intermodel repeatability and the ability of the model to capture some aspects of individual variation in bacterial community profiles. The findings of this proof-of-concept study, including recognition of the limitions of the model, support its future development as a tool for investigating the impact of disease, nutrition, dietary supplementation, and medication on the equine intestinal microbiota.

    OL Champion, AV Karlyshev, NJ Senior, M Woodward, R La Ragione, SL Howard, BW Wren, RW Titball (2010)Insect Infection Model for Campylobacter jejuni Reveals That O-methyl Phosphoramidate Has Insecticidal Activity, In: JOURNAL OF INFECTIOUS DISEASES201(5)pp. 776-782 UNIV CHICAGO PRESS
    T Ooka, MAM Vieira, Y Ogura, L Beutin, R La Ragione, PM van Diemen, MP Stevens, I Aktan, S Cawthraw, A Best, RT Hernandes, G Krause, TAT Gomes, T Hayashi, G Frankel (2007)Characterization of tccP2 carried by atypical enteropathogenic Escherichia coli, In: FEMS MICROBIOLOGY LETTERS271(1)pp. 126-135 BLACKWELL PUBLISHING
    Caroline Ivanne Le Roy, Martin John Woodward, Richard John Ellis, Roberto Marcello La Ragione, Sandrine Paule Claus (2019)Antibiotic treatment triggers gut dysbiosis and modulates metabolism in a chicken model of gastro-intestinal infection, In: BMC Veterinary Research15(37)pp. 1-13 BMC

    Background Infection of the digestive track by gastro-intestinal pathogens results in the development of symptoms ranging from mild diarrhea to more severe clinical signs such as dysentery, severe dehydration and potentially death. Although, antibiotics are efficient to tackle infections, they also trigger dysbiosis that has been suggested to result in variation in weight gain in animal production systems. Results Here is the first study demonstrating the metabolic impact of infection by a gastro-intestinal pathogen (Brachyspira pilosicoli) and its resolution by antibiotic treatment (tiamulin) on the host (chicken) systemic metabolism and gut microbiota composition using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy and 16S rDNA next generation sequencing (NGS). Clear systemic metabolic markers of infections such as glycerol and betaine were identified. Weight loss in untreated animals was in part explained by the observation of a modification of systemic host energy metabolism characterized by the utilization of glycerol as a glucose precursor. However, antibiotic treatment triggered an increased VLDL/HDL ratio in plasma that may contribute to reducing weight loss observed in treated birds. All metabolic responses co-occurred with significant shift of the microbiota upon infection or antibiotic treatment. Conclusion This study indicates that infection and antibiotic treatment trigger dysbiosis that may impact host systemic energy metabolism and cause phenotypic and health modifications.

    M Courtenay, J Sweeney, P Zielinska, SB Blake, R La Ragione (2015)One Health: An opportunity for an interprofessional approach to healthcare, In: JOURNAL OF INTERPROFESSIONAL CARE29(6)pp. 641-642 TAYLOR & FRANCIS INC
    Jonathan Betts, Christoph Nagel, Ulrich Schatzschneider, Robert Poole, Roberto La Ragione (2017)Antimicrobial activity of carbon monoxide-releasing 1 molecule [Mn(CO)3(tpa-k3N)]Br versus multidrug-2 resistant isolates of Avian Pathogenic Escherichia coli 3 and its synergy with colistin, In: PLoS ONE12(10) Public Library of Science

    The purpose of this study is to demonstrate the properties of novel nanocomposites, based on cycloaliphatic epoxy resin additionally reinforced with silicon-containing nanostructures (mono- or octa-functional POSS or nanosilica). The changes in properties are discussed for the varied combinations of cycloaliphatic epoxy with a curing agent (cycloaliphatic amine or anhydride) and the nanomodifier. The in uence of modification on thermal stability, curing behaviour, morphology, surface chemistry, and topography were studied with TGA, DSC, ATR-FTIR, XPS and LCM. The results show that when POSS and/or nanosilica are incorporated to the cycloaliphatic matrix they in uence curing behaviour and glass transition temperatures (Tg), where mono-POSS increases Tg and octa-POSS decreases it with respect to nanosilica. Mono-POSS produces silicon-rich surfaces but tends to agglomerate and increase surface roughness. Octa-POSS and nanosilica penetrate the polymer matrix more deeply and disperse more easily. From the selected modifiers, octa-POSS shows the highest thermal stability.

    Luca Guardabassi, Jens Peter Christensen, John Elmerdahl Olsen, J Scott Weese, Roberto Marcello La Ragione, Guillaume Lhermie (2020)Indications for the use of highest priority critically important antimicrobials in the veterinary sector, In: Journal of Antimicrobial Chemotherapy Oxford University Press (OUP)

    Background Among the measures taken to preserve the clinical efficacy of highest priority critically important antimicrobials (HP-CIAs), the WHO has recommended avoiding their use in food-producing animals. Little is known regarding the indications for which different antimicrobial classes are used in animals, even in countries where data on antimicrobial use are available. Objectives To outline, in a narrative review, the diseases for which HP-CIAs are used in veterinary medicine, highlighting incongruences with international guidelines and disease conditions where effective alternatives to HP-CIAs are missing. Methods Scientific literature, national reports and expert opinion were used to describe the indications for the use of HP-CIAs in the main food-producing (pigs, cattle and poultry) and companion (horses, dogs and cats) animal species. Results The most common indications for use of HP-CIAs are enteric and respiratory infections in pigs, cattle and poultry, urogenital infections in dogs and cats and respiratory infections in horses. In some instances, no valid and convenient alternatives to colistin and macrolides are available against certain porcine enteric and bovine respiratory pathogens. Effective, legal and convenient alternatives to HP-CIAs are also lacking for managing common infections in cats, for which oral administration is difficult, Rhodococcus equi infections in horses, some enteric and respiratory infections in poultry and MDR infections in all companion animal species. Conclusions Future research and stewardship programmes should focus on the disease conditions identified by this review to reduce the use of HP-CIAs in the veterinary sector.

    CI Le Roy, LJ Mappley, Roberto La Ragione, MJ Woodward, SP Claus (2016)NMR-based metabolic characterization of chicken tissues and biofluids: a model for avian research, In: Metabolomics12157 Springer

    Introduction Poultry is one of the most consumed meat in the world and its related industry is always looking for ways to improve animal welfare and productivity. It is therefore essential to understand the metabolic response of the chicken to new feed formulas, various supplements, infections and treatments. Objectives As a basis for future research investigating the impact of diet and infections on chicken’s metabolism, we established a high-resolution proton nuclear magnetic resonance (NMR)-based metabolic atlas of the healthy chicken (Gallus gallus). Methods Metabolic extractions were performed prior to 1 H-NMR and 2D NMR spectra acquisition on twelve biological matrices: liver, kidney, spleen, plasma, egg yolk and white, colon, caecum, faecal water, ileum, pectoral muscle and brain of 6 chickens. Metabolic profiles were then exhaustively characterized. Results Nearly 80 metabolites were identified. A crosscomparison of these matrices was performed to determine metabolic variations between and within each section and highlighted that only eight core metabolites were systematically found in every matrice. Conclusion This work constitutes a database for future NMR-based metabolomic investigations in relation to avian production and health.

    D Newell, Roberto La Ragione (2018)Enterohaemorrhagic and other Shiga toxin producing Escherichia coli (STEC); where are we now regarding diagnostics and control strategies?, In: Transboundary and Emerging Diseases65(S1)pp. 49-71 Wiley

    Escherichia coli comprises a highly diverse group of Gram-negative bacteria and is a common member of the intestinal microflora of humans and animals. Generally, such colonization is asymptomatic; however, some E. coli strains have evolved to become pathogenic and thus cause clinical disease in susceptible hosts. One pathotype, the Shiga toxigenic E. coli (STEC) comprising strains expressing a Shiga-like toxin is an important foodborne pathogen. A subset of STEC are the enterohaemorrhagic E. coli (EHEC), which can cause serious human disease, including haemolytic uraemic syndrome (HUS). The diagnosis of EHEC infections and the surveillance of STEC in the food chain and the environment require accurate, cost-effective and timely tests. In this review, we describe and evaluate tests now in routine use, as well as upcoming test technologies for pathogen detection, including loop-mediated isothermal amplification (LAMP) and whole-genome sequencing (WGS). We have considered the need for improved diagnostic tools in current strategies for the control and prevention of these pathogens in humans, the food chain and the environment. We conclude that although significant progress has been made, STEC still remains an important zoonotic issue worldwide. Substantial reductions in the public health burden due to this infection will require a multipronged approach, including ongoing surveillance with high-resolution diagnostic techniques currently being developed and integrated into the routine investigations of public health laboratories. However, additional research requirements may be needed before such high-resolution diagnostic tools can be used to enable the development of appropriate interventions, such as vaccines and decontamination strategies.

    P Wragg, RM La Ragione, A Best, R Relchel, MF Anjum, A Mafura, MJ Woodward (2009)Characterisation of Escherichia fergusonii isolates from farm animals using an Escherichia coli virulence gene array and tissue culture adherence assays, In: RESEARCH IN VETERINARY SCIENCE86(1)pp. 27-35 ELSEVIER SCI LTD
    Roberto La Ragione, A Best, MJ Woodward, Andrew Wales (2009)Escherichia coli O157:H7 colonisation in small domestic ruminants, In: FEMS Microbiology Reviews33(2)pp. 394-410 Oxford University Press

    Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 was first implicated in human disease in the early 1980s, with ruminants cited as the primary reservoirs. Preliminary studies indicated cattle to be the sole source of E. coli O157:H7 outbreaks in humans, however further epidemiological studies soon demonstrated that E. coli O157:H7 was widespread in other food sources and that a number of transmission routes existed. More recently, small domestic ruminants (sheep and goats) have emerged as important sources of E. coli O157:H7 human infection, particularly with the widespread popularity of petting farms and the increased use of sheep and goat food products, including unpasteurised cheeses. Although the colonisation and persistence characteristics of E. coli O157:H7 in the bovine host have been studied intensively, this is not the case for small ruminants. Despite many similarities with the bovine host, the pathobiology of E. coli O157:H7 in small domestic ruminants does appear to differ significantly from that described in cattle. This review aims to critically review the current knowledge regarding colonisation and persistence of E. coli O157:H7 in small domestic ruminants, including comparisons with the bovine host where appropriate.

    R Tozzoli, L Grande, V Michelacci, R Fioravanti, D Gally, X Xu, R La Ragione, M Anjum, G Wu, A Caprioli, S Morabito (2014)Identification and Characterization of a Peculiar vtx2-Converting Phage Frequently Present in Verocytotoxin-Producing Escherichia coli O157 Isolated from Human Infections, In: INFECTION AND IMMUNITY82(7)pp. 3023-3032 AMER SOC MICROBIOLOGY
    F Martelli, R Gosling, E Kennedy, A Rabie, H Reeves, F Clifton-Hadley, R Davies, R La Ragione (2014)Characterization of the invasiveness of monophasic and aphasic Salmonella Typhimurium strains in 1-day-old and point-of-lay chickens, In: AVIAN PATHOLOGY43(3)pp. 269-275 TAYLOR & FRANCIS LTD
    RM La Ragione, IM McLaren, G Foster, WA Cooley, MJ Woodward (2002)Phenotypic and genotypic characterization of avian Escherichia coli O86 : K61 isolates possessing a gamma-like intimin, In: APPLIED AND ENVIRONMENTAL MICROBIOLOGY68(10)pp. 4932-4942 AMER SOC MICROBIOLOGY
    ST Cartman, RM La Ragione, MJ Woodward (2008)Bacillus subtilis spores germinate in the chicken gastrointestinal tract, In: APPLIED AND ENVIRONMENTAL MICROBIOLOGY74(16)pp. 5254-5258 AMER SOC MICROBIOLOGY
    Jai Mehat, Roberto M. La Ragione, Arnoud van Vliet (2020)Campylobacter jejuni and Campylobacter coli autotransporter genes exhibit lineageassociated distribution and decay, In: BMC Genomics21314 BMC

    Background: Campylobacter jejuni and Campylobacter coli are major global causes of bacterial gastroenteritis. Whilst several individual colonisation and virulence factors have been identified, our understanding of their role in the transmission, pathogenesis and ecology of Campylobacter has been hampered by the genotypic and phenotypic diversity within C. jejuni and C. coli. Autotransporter proteins are a family of outer membrane or secreted proteins in Gram-negative bacteria such as Campylobacter, which are associated with virulence functions. In this study we have examined the distribution and predicted functionality of the previously described capC and the newly identified, related capD autotransporter gene families in Campylobacter. Results: Two capC-like autotransporter families, designated capC and capD, were identified by homology searches of genomes of the genus Campylobacter. Each family contained four distinct orthologs of CapC and CapD. The distribution of these autotransporter genes was determined in 5829 C. jejuni and 1347 C. coli genomes. Autotransporter genes were found as intact, complete copies and inactive formats due to premature stop codons and frameshift mutations. Presence of inactive and intact autotransporter genes was associated with C. jejuni and C. coli multi-locus sequence types, but for capC, inactivation was independent from the length of homopolymeric tracts in the region upstream of the capC gene. Inactivation of capC or capD genes appears to represent lineagespecific gene decay of autotransporter genes. Intact capC genes were predominantly associated with the C. jejuni ST-45 and C. coli ST-828 generalist lineages. The capD3 gene was only found in the environmental C. coli Clade 3 lineage. These combined data support a scenario of inter-lineage and interspecies exchange of capC and subsets of capD autotransporters. Conclusions: In this study we have identified two novel, related autotransporter gene families in the genus Campylobacter, which are not uniformly present and exhibit lineage-specific associations and gene decay. The distribution and decay of the capC and capD genes exemplifies the erosion of species barriers between certain lineages of C. jejuni and C. coli, probably arising through co-habitation. This may have implications for the phenotypic variability of these two pathogens and provide opportunity for new, hybrid genotypes to emerge.

    J-M Huang, RM La Ragione, A Nunez, SM Cutting (2008)Immunostimulatory activity of Bacillus spores, In: FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY53(2)pp. 195-203 WILEY-BLACKWELL
    Susan Knowler, C Cross, S Griffiths, AK McFadyen, J Jovanovik, A Tauro, Z Kibar, KJ Driver, Roberto La Ragione, Clare Rusbridge (2017)Use of Morphometric Mapping to Characterise Symptomatic Chiari-Like Malformation, Secondary Syringomyelia and Associated Brachycephaly in the Cavalier King Charles Spaniel, In: PLoS One12(1)e0170315 Public Library of Science (PLoS)

    Objectives To characterise the symptomatic phenotype of Chiari-like malformation (CM), secondary syringomyelia (SM) and brachycephaly in the Cavalier King Charles Spaniel using morphometric measurements on mid-sagittal Magnetic Resonance images (MRI) of the brain and craniocervical junction. Methods This retrospective study, based on a previous quantitative analysis in the Griffon Bruxellois (GB), used 24 measurements taken on 130 T1-weighted MRI of hindbrain and cervical region. Associated brachycephaly was estimated using 26 measurements, including rostral forebrain flattening and olfactory lobe rotation, on 72 T2-weighted MRI of the whole brain. Both study cohorts were divided into three groups; Control, CM pain and SM and their morphometries compared with each other. Results Fourteen significant traits were identified in the hindbrain study and nine traits in the whole brain study, six of which were similar to the GB and suggest a common aetiology. The Control cohort had the most elliptical brain (p = 0.010), least olfactory bulb rotation (p = 0.003) and a protective angle (p = 0.004) compared to the other groups. The CM pain cohort had the greatest rostral forebrain flattening (p = 0.007), shortest basioccipital (p = 0.019), but a greater distance between the atlas and basioccipital (p = 0.002) which was protective for SM. The SM cohort had two conformation anomalies depending on the severity of craniocervical junction incongruities; i) the proximity of the dens (p

    A Best, RM La Ragione, WA Cooley, CD O'Connor, P Velge, MJ Woodward (2003)Interaction with avian cells and colonisation of specific pathogen free chicks by Shiga-toxin negative Escherichia coli O157 : H7 (NCTC 12900), In: VETERINARY MICROBIOLOGY93(3)pp. 207-222 ELSEVIER SCIENCE BV
    F Girard, G Frankel, AD Phillips, W Cooley, U Weyer, AHA Dugdale, MJ Woodward, RM La Ragione (2008)Interaction of enterohemorrhagic Escherichia coli O157 : H7 with mouse intestinal mucosa, In: FEMS MICROBIOLOGY LETTERS283(2)pp. 196-202 WILEY-BLACKWELL
    HJ Metcalfe, RM La Ragione, DG Smith, D Werling (2013)Functional characterisation of bovine TLR5 indicates species-specific recognition of flagellin., In: Vet Immunol Immunopathol

    Mammalian toll-like receptor 5 (TLR5) senses flagellin of several bacterial species and has been described to activate the innate immune system. To assess the role of bovine TLR5 (boTLR5) in the cattle system, we cloned and successfully expressed boTLR5 in human embryonic kidney (HEK) 293 cells, as indicated by quantitative PCR and confocal microscopy. However, in contrast to huTLR5-transfected cells, exposure of boTLR5-transfected cells to flagellin neither activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nor CXCL8 production. Subsequent comparison of the flagellin response induced in human and bovine primary macrophages revealed that flagellin did not lead to phosphorylation of major signalling molecules. Furthermore, the CXCL8 and TNFα response of primary bovine macrophages stimulated with flagellin was very low compared to that observed in human primary macrophages. Our results indicate that cattle express a functional TLR5 albeit with different flagellin sensing qualities compared to human TLR5. However, boTLR5 seemed to play a different role in the bovine system compared to the human system in recognizing flagellin, and other potentially intracellular expressed receptors may play a more important role in the bovine system to detect flagellin.

    JW Collins, NG Coldham, FJ Salguero, WA Cooley, WR Newell, RA Rastall, GR Gibson, MJ Woodward, RM La Ragione (2010)Response of Porcine Intestinal In Vitro Organ Culture Tissues following Exposure to Lactobacillus plantarum JC1 and Salmonella enterica Serovar Typhimurium SL1344, In: APPLIED AND ENVIRONMENTAL MICROBIOLOGY76(19)pp. 6645-6657 AMER SOC MICROBIOLOGY
    Susan Knowler, A-M Kiviranta, AK McFadyen, TS Jokinen, Roberto La Ragione, Clare Rusbridge (2017)Craniometric Analysis of the Hindbrain and Craniocervical Junction of Chihuahua, Affenpinscher and Cavalier King Charles Spaniel Dogs With and Without Syringomyelia Secondary to Chiari-Like Malformation, In: PLoS One12(1)e0169898 Public Library of Science (PLoS)

    Objectives:To characterize and compare the phenotypic variables of the hindbrain and craniocervical junction associated with syringomyelia (SM) in the Chihuahua, Affenpinscher and Cavalier King Charles Spaniel (CKCS). Method Analysis of 273 T1-weighted mid-sagittal DICOM sequences of the hindbrain and craniocervical junction from 99 Chihuahuas, 42 Affenpinschers and 132 CKCSs. The study compared 22 morphometric features (11 lines, eight angles and three ratios) of dogs with and without SM using refined techniques based on previous studies of the Griffon Bruxellois (GB) using Discriminant Function Analysis and ANOVA with post-hoc corrections. Results The analysis identified 14/22 significant traits for SM in the three dog breeds, five of which were identical to those reported for the GB and suggest inclusion of a common aetiology. One ratio, caudal fossa height to the length of the skull base extended to an imaginary point of alignment between the atlas and supraoccipital bones, was common to all three breeds (p values 0.029 to

    A Carter, M Adams, Roberto La Ragione, MJ Woodward (2016)Colonisation of poultry by Salmonella Enteritidis S1400 is reduced by combined administration of Lactobacillus salivarius 59 and Enterococcus faecium PXN-33, In: Veterinary Microbiology199pp. 100-107 Elsevier

    Salmonella Enteritidis remains a significant issue within the poultry industry and one potential solution is to use probiotic bacteria to prevent Salmonella colonisation through competitive exclusion (CE). We demonstrate that combined administration of Lactobacillus salivarius 59 and Enterococcus faecium PXN33 were effective competitive excluders of Salmonella Enteritidis S1400 in poultry. Two models were developed to evaluate the efficacy of probiotic where birds received Salmonella Enteritidis S1400 by a) oral gavage and b) sentinel bird to bird transmission. A statistically significant (p < 0.001) 2 log reduction of Salmonella Enteritidis S1400 colonisation was observed in the ileum, caecum and colon at day 43 using combined administration of the two probiotic bacteria. However, no Salmonella Enteritidis S1400 colonisation reduction was observed when either probiotic was administered individually. In the sentinel bird model the combined probiotic administered at days 12 and 20 was more effective than one-off or double administrations at age 1 and 12 days. In vitro cell free culture supernatant studies suggest the mechanism of Salmonella Enteritidis S1400 inhibition was due to a reduction in pH by the probiotic bacteria. Our current study provides further evidence that probiotics can significantly reduce pathogenic bacterial colonisation in poultry and that mixed preparation of probiotics provide superior performance when compared to individual bacterial preparations.

    Brachyspira pilosicoli is a potentially zoonotic anaerobic intestinal spirochaete that is one of several species causing avian intestinal spirochaetosis. The aim of this study was to develop a reproducible model of infection in point of lay chickens and compare the virulence of two strains of B. pilosicoli in a model using experimentally challenged laying chickens. Seventeen week-old commercial laying chickens were experimentally challenged by oral gavage with either B. pilosicoli strain B2904 or CPSp1, following an oral dose of 10% sodium bicarbonate to neutralise acidity in the crop. Approximately 80% of the chickens became colonised and exhibited increased faecal moisture content, reduced weight gain and delayed onset of lay. Tissues sampled at post-mortem examination were analysed to produce a quantitative output on the number of spirochaetes present and hence, the extent of colonisation. The liver and spleen were colonised and novel histopathology was observed in these tissues. The infection model we report here has potential use in studies to improve our understanding of the mechanisms by which Brachyspira elicit disease in poultry and in testing novel intervention strategies.

    IA Naqid, JP Owen, BC Maddison, DS Gardner, N Foster, MA Tchórzewska, RM La Ragione, KC Gough (2015)Prebiotic and probiotic agents enhance antibody-based immune responses to Salmonella Typhimurium infection in pigs, In: Animal Feed Science and Technology201pp. 57-65

    © 2014 Elsevier B.V.Salmonellosis causes significant economic losses to the pig industry and contaminated pork products are an important source of Salmonella for humans. The EU ban on the use of antibiotic growth promoters in pig production, and the emergence of antibiotic resistance has meant there is a pressing need for alternative control strategies for pathogenic bacteria such as S. Typhimurium in pigs. Here, we determined the effects of prebiotic, probiotic and synbiotic diet regimes on antibody responses to oral Salmonella challenge of pigs. The data demonstrate that the inclusion of the probiotic Lactobacillus plantarum B2984 in the diet of piglets (~1×1010cfu/animal/day) enhanced serum IgM (P

    IA Naqid, JP Owen, BC Maddison, A Spiliotopoulos, RD Emes, A Warry, RJ Flynn, F Martelli, RJ Gosling, RH Davies, RM La Ragione, KC Gough (2016)Mapping B-cell responses to Salmonella enterica serovars Typhimurium and Enteritidis in chickens for the discrimination of infected from vaccinated animals, In: Scientific Reports6ARTN 31186 NATURE PUBLISHING GROUP
    I Aktan, B Carter, H Wilking, RM La Ragione, L Wieler, MJ Woodward, MF Anjum (2007)Influence of geographical origin, host animal and stx gene on the virulence characteristics of Escherichia coli O26 strains, In: JOURNAL OF MEDICAL MICROBIOLOGY56(11)pp. 1431-1439 SOC GENERAL MICROBIOLOGY
    LJ Mappley, MA Tchórzewska, A Nunez, MJ Woodward, PM Bramley, RM La Ragione (2012)Oral treatment of chickens with Lactobacillus reuteri LM1 reduces Brachyspira pilosicoli-induced pathology., In: J Med Microbiol

    Avian intestinal spirochetosis (AIS) results from the colonisation of the caeca and colon of poultry by pathogenic Brachyspira, notably B. pilosicoli. There have been increased reports in the number of cases of AIS since ban on the use of antibiotic growth promoters in the European Union in 2006 which, together with emerging antimicrobial resistance in Brachyspira, has driven renewed interest in alternative intervention strategies. Lactobacillus-based probiotics have been reported previously to protect against infection with common enteric pathogens in livestock. Our previous studies have shown that L. reuteri LM1 antagonises aspects of the pathobiology of Brachyspira in vitro. Here, we aimed to assess whether L. reuteri LM1 mitigates against the clinical symptoms of AIS in chickens experimentally challenged with B. pilosicoli. In this study, two groups of fifteen commercial laying hens were challenged experimentally by oral gavage with B. pilosicoli B2904 at 18 weeks of age; one group received un-supplemented drinking water and the other received drinking water supplemented with L. reuteri LM1 from one-week prior to challenge with Brachyspira and thereafter for the duration of the study. The group dosed with L. reuteri LM1 were protected against experimentally-induced B. pilosicoli infection and showed reduced clinical symptoms associated with AIS. Specifically, B. pilosicoli was detected by culture in fewer birds, bird weights were higher, faecal moisture contents were significantly lower (p

    Ioannis Sakaridis, R Ellis, S Cawthraw, Arnoud van Vliet, D Stekel, J Penell, Mark Chambers, Roberto La Ragione, Alasdair Cook (2018)Investigating the association between the caecal microbiomes of broilers and Campylobacter burden, In: Frontiers in Microbiology9927pp. 1-9 Frontiers Media

    One of the major transmission routes for the foodborne bacterial pathogen Campylobacter is undercooked poultry meat, contaminated from intestinal contents during processing. In broilers, Campylobacter can grow to very high densities in the caeca, and is often considered to be a commensal or an opportunistic pathogen in poultry. Reduction of caecal loads of Campylobacter may assist in lowering incidence rates of Campylobacter food poisoning. To achieve this, there needs to be a better understanding of the dynamics of Campylobacter colonisation in its natural niche, and the effect of the local microbiome on colonisation. Previous studies have shown that the microbiome differed between Campylobacter colonised and non–colonised chicken intestinal samples. To characterise the microbiome of Campylobacter-colonised broilers, caecal samples of 100 randomly selected birds from four farms were analysed using amplified 16S rRNA gene sequences. Bacterial taxonomic analysis indicated that inter-farm variation was greater than intra-farm variation. The two most common bacterial groups were Bacteroidetes and Firmicutes which were present in all samples and constituted 29.7 – 63.5% and 30.2 – 59.8% of the bacteria present, respectively. Campylobacter was cultured from all samples, ranging from 2 to 9 log10 CFU g-1. There was no clear link between Campylobacter counts and Firmicutes, Bacteroidetes or Tenericutes levels in the 16S rRNA Operational Taxonomic Unit (OTU)-based analysis of the caecal microbiome, but samples with high Campylobacter counts (> 9 log CFU g-1) contained increased levels of Enterobacteriaceae. A decrease in Lactobacillus abundance in chicken caeca was also associated with high Campylobacter loads. The reported associations with Lactobacillus and Enterobacteriaceae match changes in the intestinal microbiome of chickens and mice previously reported for Campylobacter infection, and raises the question about temporality and causation; as to whether increases in Campylobacter loads create conditions adverse to Lactobacilli and/or beneficial to Enterobacteriaceae, or that changes in Lactobacilli and Enterobacteriaceae levels created conditions beneficial for Campylobacter colonisation. If these changes can be controlled, this may open opportunities for modulation of chicken microbiota to reduce Campylobacter levels for improved food safety.

    K Stapleton, SA Cawthraw, SW Cooles, NG Coldham, RM La Ragione, DG Newell, AM Ridley (2010)Selecting for development of fluoroquinolone resistance in a Campylobacter jejuni strain 81116 in chickens using various enrofloxacin treatment protocols, In: JOURNAL OF APPLIED MICROBIOLOGY109(4)pp. 1132-1138 WILEY-BLACKWELL
    LJ Mappley, ML Black, M Abuoun, AC Darby, MJ Woodward, J Parkhill, AK Turner, MI Bellgard, T La, ND Phillips, RM La Ragione, DJ Hampson (2012)Comparative genomics of Brachyspira pilosicoli strains: genome rearrangements, reductions and correlation of genetic compliment with phenotypic diversity., In: BMC Genomics13pp. 454-?

    ABSTRACT:

    A Berto, WH Van der Poel, R Hakze-van der Honing, F Martelli, RM La Ragione, N Inglese, J Collins, S Grierson, R Johne, J Reetz, A Dastjerdi, M Banks (2012)Replication of hepatitis E virus in three-dimensional cell culture., In: J Virol Methods

    Hepatitis E is an acute, viral hepatitis epidemic in developing regions, but which is detected with increasing frequency in sporadic form in developed regions. Pigs and possibly some other mammals are considered reservoirs of zoonotic infection with hepatitis E virus (HEV). However, whilst the relative significance of potential transmission routes from pigs to people is still unclear, the consumption of raw or undercooked pig meat has been implicated as a source of HEV infection. The lack of information about HEV zoonotic transmission is due in part to the difficulties of in vitro propagation of HEV. The Rotating Wall Vessel (RVW) has been described as a useful tool for the culture of cell lines in a 3-dimensional (3D) configuration. The aim of this work was to develop a 3D cell culture system for HEV to facilitate studies into the viability of virions contaminating pig tissues. This study, demonstrated that HEV can replicate efficiently in the RWV in human hepatoblastoma PLC/PRF/5 cells for up to 5 months not only by real time RT-PCR but also by detection of complete virions via electron microscopy. Furthermore, the replication of HEV progeny was observed by detecting HEV RNA by RT-PCR. The progeny were able to infect fresh 3D cultures, showing that this method is able to produce infectious hepatitis E virions.

    I Aktan, RM La Ragione, MJ Woodward (2007)Colonization, persistence, and tissue tropism of Escherichia coli O26 in conventionally reared weaned lambs, In: APPLIED AND ENVIRONMENTAL MICROBIOLOGY73(3)pp. 691-698 AMER SOC MICROBIOLOGY
    MJ Woodward, L Mappley, C Le Roy, SP Claus, P Davies, G Thompson, RM La Ragione (2015)Drinking water application of Denagard (R) Tiamulin for control of Brachyspira pilosicoli infection of laying poultry, In: RESEARCH IN VETERINARY SCIENCE103pp. 87-95 ELSEVIER SCI LTD
    G Wu, M AbuOun, E Hackl, RM La Ragione, M Fookes, J Fenner, Z Pan, P Wenzl, MF Anjum, MJ Woodward (2010)Epidemic multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport strains contain three phage regions and a MDR resistance plasmid, In: ENVIRONMENTAL MICROBIOLOGY REPORTS2(2)pp. 228-235 WILEY-BLACKWELL PUBLISHING, INC
    S Huehn, RM La Ragione, M Anjum, M Saunders, MJ Woodward, C Bunge, R Helmuth, E Hauser, B Guerra, J Beutlich, A Brisabois, T Peters, L Svensson, G Madajczak, E Litrup, A Imre, S Herrera-Leon, D Mevius, DG Newell, B Malorny (2010)Virulotyping and Antimicrobial Resistance Typing of Salmonella enterica Serovars Relevant to Human Health in Europe, In: FOODBORNE PATHOGENS AND DISEASE7(5)pp. 523-535 MARY ANN LIEBERT INC
    T Rosser, T Dransfield, L Allison, M Hanson, N Holden, J Evans, S Naylor, R La Ragione, JC Low, DL Gally (2008)Pathogenic Potential of Emergent Sorbitol-Fermenting Escherichia coli O157:NM, In: INFECTION AND IMMUNITY76(12)pp. 5598-5607 AMER SOC MICROBIOLOGY
    HJ Metcalfe, A Best, T Kanellos, RM La Ragione, D Werling (2010)Flagellin expression enhances Salmonella accumulation in TLR5-positive macrophages, In: DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY34(8)pp. 797-804 ELSEVIER SCI LTD
    Marta Ferrandis-Vila, Sumeet K Tiwari, Svenja Mamerow, Torsten Semmler, Christian Menge, Christian Berens, ROBERTO MARCELLO LA RAGIONE, Jennifer Mary Ritchie (2022)Using unique ORFan genes as strain-specific identifiers for Escherichia coli, In: BMC microbiology22135 BioMedCentral

    Bacterial identification at the strain level is a much-needed, but arduous and challenging task. This study aimed to develop a method for identifying and differentiating individual strains among multiple strains of the same bacterial species. The set used for testing the method consisted of 17 Escherichia coli strains picked from a collection of strains isolated in Germany, Spain, the United Kingdom and Vietnam from humans, cattle, swine, wild boars, and chickens. We targeted unique or rare ORFan genes to address the problem of selective and specific strain identification. These ORFan genes, exclusive to each strain, served as templates for developing strain-specific primers. Most of the experimental strains (14 out of 17) possessed unique ORFan genes that were used to develop strain-specific primers. The remaining three strains were identified by combining a PCR for a rare gene with a selection step for isolating the experimental strains. Multiplex PCR allowed the successful identification of the strains both in vitro in spiked faecal material in addition to in vivo after experimental infections of pigs and recovery of bacteria from faecal material. In addition, primers for qPCR were also developed and quantitative readout from faecal samples after experimental infection was also possible. The method described in this manuscript using strain-specific unique genes to identify single strains in a mixture of strains proved itself efficient and reliable in detecting and following individual strains both in vitro and in vivo, representing a fast and inexpensive alternative to more costly methods.

    Siddhartha Thakur, Joaquin M Prada, Jai W Mehat, Roberto M La Ragione, Arnoud Van Vliet (2022)Genomic Screening of Antimicrobial Resistance Markers in UK and US Campylobacter Isolates Highlights Stability of Resistance over an 18-Year Period, In: Antimicrobial agents and chemotherapy66(5)e0168721

    Campylobacter jejuni and Campylobacter coli are important bacterial causes of human foodborne illness. Despite several years of reduced antibiotics usage in livestock production in the United Kingdom (UK) and United States (US), a high prevalence of antimicrobial resistance (AMR) persists in . Both countries have instigated genome sequencing-based surveillance programs for , and in this study, we have identified AMR genes in 32,256 C. jejuni and 8,776 C. coli publicly available genome sequences to compare the prevalence and trends of AMR in isolated in the UK and US between 2001 and 2018. AMR markers were detected in 68% of C. coli and 53% of C. jejuni isolates, with 15% of C. coli isolates being multidrug resistant (MDR), compared to only 2% of C. jejuni isolates. The prevalence of aminoglycoside, macrolide, quinolone, and tetracycline resistance remained fairly stable from 2001 to 2018 in both C. jejuni and C. coli, but statistically significant differences were observed between the UK and US. There was a statistically significant higher prevalence of aminoglycoside and tetracycline resistance for US C. coli and C. jejuni isolates and macrolide resistance for US C. coli isolates. In contrast, UK C. coli and C. jejuni isolates showed a significantly higher prevalence of quinolone resistance. Specific multilocus sequence type (MLST) clonal complexes (e.g., ST-353/464) showed >95% quinolone resistance. This large-scale comparison of AMR prevalence has shown that the prevalence of AMR remains stable for in the UK and the US. This suggests that antimicrobial stewardship and restricted antibiotic usage may help contain further expansion of AMR prevalence in but are unlikely to reduce it in the short term.

    Jai W Mehat, Roberto M La Ragione, Arnoud Van Vliet (2021)The Avian Pathogenic Escherichia coli (APEC) pathotype is comprised of multiple distinct, independent genotypes, In: Avian Pathology50(5)pp. 402-416 Taylor and Francis

    Avian Pathogenic E. coli (APEC) is the causative agent of avian colibacillosis, resulting in economic losses to the poultry industry through morbidity, mortality and carcass condemnation, and impacts the welfare of poultry. Colibacillosis remains a complex disease to manage, hampered by diagnostic and classification strategies for E. coli that are inadequate for defining APEC. However, increased accessibility of whole genome sequencing (WGS) technology has enabled phylogenetic approaches to be applied to the classification of E. coli and genomic characterisation of the most common APEC serotypes associated with colibacillosis O1, O2 and O78. These approaches have demonstrated that that the O78 serotype is representative of two distinct APEC lineages, ST-23 in phylogroup C and ST-117 in phylogroup G. The O1, and O2 serotypes belong to a third lineage comprised of 3 sub-populations in phylogroup B2; ST-95, ST-140 and ST-428/ST-429. The frequency with which these genotypes are associated with colibacillosis implicates them as the predominant APEC populations and distinct from those causing incidental or opportunistic infections. The fact that these are disparate clusters from multiple phylogroups suggests that these lineages may have become adapted to the poultry niche independently. WGS studies have highlighted the limitations of traditional APEC classification and can now provide a path towards a robust and more meaningful definition of the APEC pathotype. Future studies should focus on characterising individual APEC populations in detail and use this information to develop improved diagnostics and interventions.

    M Urashima, G Teoh, M Akiyama, Y Yuza, K C Anderson, K Maekawa, Roberto Marcello La Ragione (1999)Restoration of p16INK4A protein induces myogenic differentiation in RD rhabdomyosarcoma cells, In: British journal of cancer79(7-8)pp. 1032-1036 Nature Publishing Group

    p16 INK4A (p16) tumour suppressor induces growth arrest by inhibiting function of cyclin-dependent kinase (CDK)4 and CDK6. Homozygous p16 gene deletion is frequent in primary rhabdomyosarcoma (RMS) cells as well as derived cell lines. To confirm the significance of p16 gene deletion in tumour biology of RMS, a temperature-sensitive p16 mutant (E119G) gene was retrovirally transfected into the human RMS cell line RD, which has homozygous gene deletion of p16 gene. Decrease from 40°C (restrictive) to 34°C (permissive) culture temperature reduced CDK6-associated kinase activity and induced G1 growth arrest. Moreover, RD-p16 cells cultured under permissive condition demonstrated differentiated morphology coupled with expressions of myogenin and myosin light chain. These suggest that deletion of p16 gene may not only facilitate growth but also inhibit the myogenic differentiation of RD RMS cells. © 1999 Cancer Research Campaign

    CHUNHUA Yuan, THOMAS L. Selby, JUNAN LI, IN-JA L. Byeon, MING-DAW Tsai, Roberto Marcello La Ragione (2000)Tumor suppressor INK4: Refinement of p16INK4A structure and determination of p15INK4B structure by comparative modeling and NMR data, In: Protein science9(6)pp. 1120-1128 Cambridge University Press

    Within the tumor suppressor protein INK4 (inhibitor of cyclin-dependent kinase 4) family, p15INK4B is the smallest and the only one whose structure has not been determined previously, probably due to the protein's conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16INK4A as the template, followed by restrained energy minimization with NMR constraints (NOE and H-bonds). For this purpose, the solution structure of p16INK4A, whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure-based iterative NOE assignments. The nonhelical regions showed major improvement with root-mean-square deviation (RMSD) improved from 1.23 to 0.68 Å for backbone heavy atoms. The completion of p15INK4B coupled with refinement of p16INK4A made it possible to compare the structures of the four INK4 members in depth, and to compare the structures of p16INK4A in the free form and in the p16INK4A-CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each INK4 member.

    Celeste Bento, Melanie J. Percy, Betty Gardie, Tabita Magalhaes Maia, Richard van Wijk, Silverio Perrotta, Fulvio Della Ragione, Helena Almeida, Cedric Rossi, Francois Girodon, Maria Åström, Drorit Neumann, Susanne Schnittger, Britta Landin, Milen Minkov, Maria Luigia Randi, Stephane Richard, Nicole Casadevall, William Vainchenker, Susana Rives, Sylvie Hermouet, M. Leticia Ribeiro, Mary Frances McMullin, Holger Cario, Roberto Marcello La Ragione (2014)Genetic Basis of Congenital Erythrocytosis, In: Human mutation35(1)pp. 15-26

    Congenital erythrocytosis (CE), or congenital polycythemia, represents a rare and heterogeneous clinical entity. It is caused by deregulated red blood cell production where erythrocyte overproduction results in elevated hemoglobin and hematocrit levels. Primary congenital familial erythrocytosis is associated with low erythropoietin (Epo) levels and results from mutations in the Epo receptor gene (EPOR). Secondary CE arises from conditions causing tissue hypoxia and results in increased Epo production. These include hemoglobin variants with increased affinity for oxygen (HBB, HBA mutations), decreased production of 2,3-bisphosphoglycerate due to BPGM mutations, or mutations in the genes involved in the hypoxia sensing pathway (VHL, EPAS1, and EGLN1). Depending on the affected gene, CE can be inherited either in an autosomal dominant or recessive mode, with sporadic cases arising de novo. Despite recent important discoveries in the molecular pathogenesis of CE, the molecular causes remain to be identified in about 70% of the patients. With the objective of collecting all the published and unpublished cases of CE the COST action MPN&MPNr-Euronet developed a comprehensive Internet-based database focusing on the registration of clinical history, hematological, biochemical, and molecular data (http://www.erythrocytosis.org/). In addition, unreported mutations are also curated in the corresponding Leiden Open Variation Database.

    Giovambattista Capasso, Chiara Iolanda Di Gennaro, Fulvio Della Ragione, Caterina Manna, Roberto Ciarcia, Salvatore Florio, Angelica Perna, Rosa Maria Pollastro, Sara Damiano, Orazio Mazzoni, Patrizia Galletti, Vincenzo Zappia, Roberto Marcello La Ragione (2008)In vivo effect of the natural antioxidant hydroxytyrosol on cyclosporine nephrotoxicity in rats, In: Nephrology, dialysis, transplantation23(4)pp. 1186-1195 Oxford University Press

    Background. Cyclosporine A (CsA) is the first-line immunosuppressant used in transplant patients and in auto- immune diseases. Nephrotoxicity is the major limitation of CsA use. Although the mechanisms of nephrotoxicity have not been completely defined, some evidence suggests that reactive oxygen species (ROS) play a causal role. The present study was designed to investigate in vivo effects of hydroxytyrosol (DOPET), a natural olive oil antioxidant, on oxidative stress, renal histology and haemodynamic alterations induced in rats by CsA treatment. Methods. Adult Sprague–Dawley rats were treated i.p. with CsA (15 mg/kg) alone or in combination with DOPET (20 mg/kg) for 3 weeks. At the end of the treatment, superoxide concentration within the cells of the abdominal aorta and renal artery was quantified from the oxidation of dihydroethidium (DHE) using fluorescence microscopic imaging analysis. In kidney tissues, lipid peroxidation was measured by thiobarbituric acid-reacting substances (TBARS) assay, glutathione level was assessed enzymatically and the expression of haem oxygenase-1 (HO-1) gene was evaluated by semiquantitative RT-PCR. Renal morphology was studied by classical histological techniques, while the glomerular filtration rate (GFR) was estimated by inulin clearance. Systemic blood pressure was monitored by the tail method and through the catheterization of the carotid artery. Results. CsA administration increased superoxide concentration both in the aorta and in the renal artery, while DOPET completely prevented this effect. Higher levels of TBARS, a significant decrease in GSH and an upregulation of HO-1 mRNA were observed in the kidneys of CsA-treated rats. DOPET treatment reversed quantitatively these effects. However, CsA-dependent changes in renal histology were only partially reversed by DOPET. Finally, CsA induced a severe reduction in GFR and a significant increase in both systolic and diastolic blood pressure; the DOPET treatment had no significant effect on these haemodynamic alterations. Conclusion. The reported data indicate that effective DOPET protection from CsA-induced oxidative stress is associated with a mild effect on histological damages and does not affect the altered glomerular function and the hypertension, thus indicating that kidney injury by CsA is only in part dependent on oxidative stress.

    A Iolascon, L Giordani, A Borriello, R Carbone, A Izzo, G P Tonini, C Gambini, F Della Ragione, Roberto Marcello La Ragione (2000)Reduced expression of transforming growth factor-beta receptor type III in high stage neuroblastomas, In: British journal of cancer82(6)pp. 1171-1176 Nature Publishing Group

    Transforming growth factor beta (TGF-β) is a powerful inhibitor of cell proliferation and a potent inducer of differentiation. Resistance to TGF-β action is a characteristic of many malignancies and has been attributed to alterations of TGF-β receptors as well as disturbance of downstream transduction pathways. To analyse the TGF-β response in neuroblastoma, the expression of TGF-β1 and TGF-β type I, II and III receptor genes was investigated in 61 cancer samples by means of reverse transcription polymerase chain reaction. The specimens analysed belong to different stages, namely nine samples of stage 1, ten of stage 2, nine of stage 3 and 28 of stage 4. Moreover, five samples were of stage 4S, which represents a tumour form undergoing spontaneous regression. The results obtained show that TGF-β1 and TGF-β type I and II receptor genes appear to be almost equally expressed in neuroblastomas of all stages. Conversely, TGF-β type III receptor gene expression, which is required for an efficacious TGF-β binding and function, is strongly reduced exclusively in neuroblastomas of stages 3 and 4. These findings were directly confirmed by immunohistochemical analyses of ten neuroblastoma specimens. Our results suggest the occurrence of an altered TGF-β response in advanced neuroblastomas which might be an important mechanism for escaping growth control and for developing invasiveness. Moreover, our findings allow the proposal of a novel mechanism, namely down-regulation of TGF-β type III receptor gene expression, to avoid TGF-β inhibitory activity. © 2000 Cancer Research Campaign

    Mukunthan Tharmakulasingam, Cihan Topal, Anil Fernando, Roberto La Ragione (2019)Improved Pathogen Recognition using Non-Euclidean Distance Metrics andWeighted kNN, In: ICBBE 2019: 2019 6TH INTERNATIONAL CONFERENCE ON BIOMEDICAL AND BIOINFORMATICS ENGINEERINGpp. 118-124 Assoc Computing Machinery

    The timely identification of pathogens is vital in order to effectively control diseases and avoid antimicrobial resistance. Non-invasive point-of-care diagnostic tools are recently trending in identification of the pathogens and becoming a helpful tool especially for rural areas. Machine learning approaches have been widely applied on biological markers for predicting diseases and pathogens. However, there are few studies in the literature that have utilized volatile organic compounds (VOCs) as non-invasive biological markers to identify bacterial pathogens. Furthermore, there is no comprehensive study investigating the effect of different distance and similarity metrics for pathogen classification based on VOC data. In this study, we compared various non-Euclidean distance and similarity metrics with Euclidean metric to identify significantly contributing VOCs to predict pathogens. In addition, we also utilized backward feature elimination (BFE) method to accurately select the best set of features. The dataset we utilized for experiments was composed from the publications published between 1977 and 2016, and consisted of associations in between 703 VOCs and 11 pathogens. We performed extensive set of experiments with five different distance metrics in both uniform and weighted manner. Comprehensive experiments showed that it is possible to correctly predict pathogens by using 68 VOCs among 703 with 78.6% accuracy using k-nearest neighbour classifier and Sorensen distance metric.

    Karen K.W. Siu, Kyle Asmus, Allison N. Zhang, Cathy Horvatin, Sheng Li, Tong Liu, Barbara Moffatt, Virgil L. Woods, P. Lynne Howell, Roberto Marcello La Ragione (2011)Mechanism of substrate specificity in 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidases, In: Journal of structural biology173(1)pp. 86-98 Elsevier Inc

    5′-Methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase (MTAN) plays a key role in the methionine-recycling pathway of bacteria and plants. Despite extensive structural and biochemical studies, the molecular mechanism of substrate specificity for MTAN remains an outstanding question. Bacterial MTANs show comparable efficiency in hydrolyzing MTA and SAH, while the plant enzymes select preferentially for MTA, with either no or significantly reduced activity towards SAH. Bacterial and plant MTANs show significant conservation in the overall structure, and the adenine- and ribose-binding sites. The observation of a more constricted 5′-alkylthio binding site in Arabidopsis thaliana AtMTAN1 and AtMTAN2, two plant MTAN homologues, led to the hypothesis that steric hindrance may play a role in substrate selection in plant MTANs. We show using isothermal titration calorimetry that SAH binds to both Escherichia coli MTAN ( EcMTAN) and AtMTAN1 with comparable micromolar affinity. To understand why AtMTAN1 can bind but not hydrolyze SAH, we determined the structure of the protein–SAH complex at 2.2 Å resolution. The lack of catalytic activity appears to be related to the enzyme’s inability to bind the substrate in a catalytically competent manner. The role of dynamics in substrate selection was also examined by probing the amide proton exchange rates of EcMTAN and AtMTAN1 via deuterium–hydrogen exchange coupled mass spectrometry. These results correlate with the B factors of available structures and the thermodynamic parameters associated with substrate binding, and suggest a higher level of conformational flexibility in the active site of EcMTAN. Our results implicate dynamics as an important factor in substrate selection in MTAN.

    Kerstin de Verdier, Ann Nyman, Christina Greko, Björn Bengtsson, Roberto Marcello La Ragione (2012)Antimicrobial resistance and virulence factors in Escherichia coli from swedish dairy calves, In: Acta veterinaria scandinavica54(1)pp. 2-2 BioMed Central
    Molly Courtenay, Michael Wilkes, Patricia A. Conrad, Roberto M. La Ragione, Scott Reeves (2014)One Health: The importance of education and the impact of interprofessional interventions, In: The veterinary journal (1997)201(3)pp. 241-242 Elsevier Ltd
    S A Warren, K G Warren, S Greenhill, M Paterson, Roberto Marcello La Ragione (1982)How multiple sclerosis is related to animal illness, stress and diabetes, In: Canadian Medical Association journal126(4)pp. 377-385

    At the University of Alberta's multiple sclerosis research clinic 100 patients with multiple sclerosis were matched to control patients for age, sex, race and zone of residence before the age of 15 years. Case and control subjects were interviewed and information was collected by questionnaire on factors that might play a role in the development of multiple sclerosis. The only factors found to be significantly associated with the development of this disorder were a history of leisure time spent in physical activities before the onset of symptoms, exposure to animal illness -- specifically canine distemper -- and a history of severe or prolonged emotional stress. The study also confirmed a familial predisposition to multiple sclerosis and suggested a relation between the disorder and a personal or family history of diabetes mellitus.

    Alastair S Macdonald, Mark A Chambers, Roberto La Ragione, Kayleigh Wyles, Matthieu Poyade, Andrew Wales, Naomi Klepacz, Tom R Kupfer, Fraje Watson, Shona Noble (2021)Addressing Infection Risk in Veterinary Practice through the Innovative Application of Interactive 3D Animation Methods, In: The Design Journal24(1)pp. 51-72 Routledge

    Antimicrobial resistance is of growing concern in human and animal health. The aim of this study was to raise awareness and perception of risk of infection-related behaviours during routine preparation for veterinary surgery. We took a multi-disciplinary and multi-method approach to 'make visible, the invisible' by illustrating how microbial contamination can be spread during the preparation process for surgical procedures. The design-led visualization approach enhanced inter-disciplinary team and workshop participant contributions during the co-development of an innovative digital tool to support training for veterinary practitioners and students. After experiencing the intervention, 92% of 51 participants agreed to change their behaviour and stated an intention to implement an infection control behaviour that aligned with training objectives. The 3D graphics enhanced the delivery of training content by making difficult and abstract contamination concepts easy to understand. A similar approach could be taken for human health applications.

    Anish Pandey, Maria Victoria Humbert, Alexandra Jackson, Jade L Passey, David J Hampson, David W Cleary, Roberto M La Ragione, Myron Christodoulides (2020)Evidence of homologous recombination as a driver of diversity in Brachyspira pilosicoli, In: Microbial Genomics6(12)

    The enteric, pathogenic spirochaete Brachyspira pilosicoli colonizes and infects a variety of birds and mammals, including humans. However, there is a paucity of genomic data available for this organism. This study introduces 12 newly sequenced draft genome assemblies, boosting the cohort of examined isolates by fourfold and cataloguing the intraspecific genomic diversity of the organism more comprehensively. We used several in silico techniques to define a core genome of 1751 genes and qualitatively and quantitatively examined the intraspecific species boundary using phylogenetic analysis and average nucleotide identity, before contextualizing this diversity against other members of the genus Brachyspira . Our study revealed that an additional isolate that was unable to be species typed against any other Brachyspira lacked putative virulence factors present in all other isolates. Finally, we quantified that homologous recombination has as great an effect on the evolution of the core genome of the B. pilosicoli as random mutation (r/m=1.02). Comparative genomics has informed Brachyspira diversity, population structure, host specificity and virulence. The data presented here can be used to contribute to developing advanced screening methods, diagnostic assays and prophylactic vaccines against this zoonotic pathogen.

    Giorgia Tura, Barbara Brunetti, Elena Brigandì, Riccardo Rinnovati, Giuseppe Sarli, Giancarlo Avallone, Luisa Vera Muscatello, Roberto Marcello La Ragione, Andy E Durham, Barbara Bacci (2022)Expression of Cell-Cycle Regulatory Proteins pRb, Cyclin D1, and p53 Is Not Associated with Recurrence Rates of Equine Sarcoids, In: Veterinary sciences9(9)474
    Cristina Lagatolla, Jai W. Mehat, Roberto Marcello La Ragione, Roberto Luzzati, Stefano Di Bella (2022)In Vitro and In Vivo Studies of Oritavancin and Fosfomycin Synergism against Vancomycin-Resistant Enterococcus faecium, In: Antibiotics (Basel)11(10)1334 MDPI

    Therapeutic options for infections caused by vancomycin-resistant enterococci are currently suboptimal. Combination regimens where fosfomycin is used alongside existing treatments are emerging given the proven synergistic potential and PK/PD properties. In the studies presented here, we tested five vanA and five vanB clinical isolates of Enterococcus faecium using a combination of oritavancin + fosfomycin both in vitro (checkerboard, time killing) and in vivo ( Galleria mellonella ). The combination of oritavancin and fosfomycin increased drug susceptibility, showing a synergistic effect in 80% of isolates and an additive effect in the remaining isolates. The combination restored fosfomycin susceptibility in 85% of fosfomycin-resistant isolates. Time killing on four selected isolates demonstrated that the combination of oritavancin and fosfomycin provided a CFU/mL reduction > 2 log 10 compared with the most effective drug alone and prevented the bacterial regrowth seen after 8–24 h at sub-inhibitory drug concentrations. In addition, the combination was also tested in a biofilm assay with two isolates, and a strong synergistic effect was observed in one isolate and an additive effect in the other. Finally, we demonstrated in vivo ( Galleria mellonella ) a higher survival rate of the larvae treated with the combination therapy compared to monotherapy (fosfomycin or oritavancin alone). Our study provides preclinical evidence to support trials combining oritavancin and fosfomycin for VRE BSI in humans, even when biofilm is involved.

    J. Nunez-Garcia, M. AbuOun, N. Storey, M. S. Brouwer, J. F. Delgado-Blas, S. S. Mo, N. Ellaby, K. T. Veldman, M. Haenni, P. Châtre, J. Y. Madec, J. A. Hammerl, C. Serna, M. Getino, R. La Ragione, T. Naas, A. A. Telke, P. Glaser, M. Sunde, B. Gonzalez-Zorn, M. J. Ellington, M. F. Anjum (2022)Harmonisation of in-silico next-generation sequencing based methods for diagnostics and surveillance, In: Scientific reports1214372 Nature Portfolio

    Abstract Improvements in cost and speed of next generation sequencing (NGS) have provided a new pathway for delivering disease diagnosis, molecular typing, and detection of antimicrobial resistance (AMR). Numerous published methods and protocols exist, but a lack of harmonisation has hampered meaningful comparisons between results produced by different methods/protocols vital for global genomic diagnostics and surveillance. As an exemplar, this study evaluated the sensitivity and specificity of five well-established in-silico AMR detection software where the genotype results produced from running a panel of 436 Escherichia coli were compared to their AMR phenotypes, with the latter used as gold-standard. The pipelines exploited previously known genotype–phenotype associations. No significant differences in software performance were observed. As a consequence, efforts to harmonise AMR predictions from sequence data should focus on: (1) establishing universal minimum to assess performance thresholds (e.g. a control isolate panel, minimum sensitivity/specificity thresholds); (2) standardising AMR gene identifiers in reference databases and gene nomenclature; (3) producing consistent genotype/phenotype correlations. The study also revealed limitations of in-silico technology on detecting resistance to certain antimicrobials due to lack of specific fine-tuning options in bioinformatics tool or a lack of representation of resistance mechanisms in reference databases. Lastly, we noted user friendliness of tools was also an important consideration. Therefore, our recommendations are timely for widespread standardisation of bioinformatics for genomic diagnostics and surveillance globally.

    OLUYINKA DAVID OLUWOLE, LUCY COLEMAN, William Buchanan, TAO CHEN, ROBERTO MARCELLO LA RAGIONE, LIAN XIANG LIU (2022)Antibiotics-Free Compounds for Chronic Wound Healing, In: Pharmaceutics14(5)1021 MDPI
    Mary-Anne Frank, William S M Justice, Roberto La Ragione, Mark A Chambers (2021)ANTIBIOTIC RESISTANCE IN ESCHERICHIA COLI AND ENTEROCOCCUS SPP. ISOLATED FROM UNGULATES AT A ZOOLOGICAL COLLECTION IN THE UNITED KINGDOM, In: Journal of Zoo and Wildlife Medicine51(4)pp. 761-770 American Association of Zoo Veterinarians

    Increase of antimicrobial resistance (AMR) is a global threat to health. The AMR profile of bacteria isolated from domesticated animals and free-ranging wildlife has been studied, but there are relatively few studies of bacteria isolated from captive wild animals. Understanding the dynamics of AMR in different populations is key to minimizing emergence of resistance and to preserve the efficacy of antimicrobials. In this study, fecal samples were collected from 17 species of healthy ungulates from a zoological collection in southeast England, which yielded 39 and 55 spp. isolates for further analysis. Antibiotic sensitivity was investigated using agar disk diffusion. isolates were resistant to a range of antibiotics, with resistance to ampicillin being the most common (28%). All isolates were susceptible to apramycin, enrofloxacin, chloramphenicol, and florfenicol. None tested positive for extended-spectrum beta-lactamase or AmpC activity. Seven of 39 (18%) isolates were resistant to three or more antibiotic classes. The isolates were further analyzed using multilocus sequence typing, which identified four pairs of identical sequence type isolates and 27 diverse strains. The spp. isolates were resistant to a range of antibiotics, with resistance to cefpodoxime seen in 95% of isolates. All spp. isolates were susceptible to ampicillin, gentamicin, chloramphenicol, and vancomycin. This study identified multidrug-resistant phenotypes in enterobacterial isolates that were like those commonly found in domestic ungulates. There was no apparent spatial clustering of the resistance profiles within the zoo. Review of the medical records of individual animals showed no direct relation to the AMR profiles observed. Observed resistance to antibiotics rarely or never used may have been due to coselection or directly acquired from other sources.

    Rachel Gilroy, Joy Leng, Anuradha Ravi, Evelien Adriaenssens, Aharon Oren, Dave Baker, Roberto M. La Ragione, Christopher Proudman, Mark J. Pallen (2022)Metagenomic investigation of the equine faecal microbiome reveals extensive taxonomic diversity, In: PeerJ – the Journal of Life & Environmental Sciences10e13084 PeerJ

    Background The horse plays crucial roles across the globe, including in horseracing, as a working and companion animal and as a food animal. The horse hindgut microbiome makes a key contribution in turning a high fibre diet into body mass and horsepower. However, despite its importance, the horse hindgut microbiome remains largely undefined. Here, we applied culture-independent shotgun metagenomics to thoroughbred equine faecal samples to deliver novel insights into this complex microbial community. Results We performed metagenomic sequencing on five equine faecal samples to construct 123 high- or medium-quality metagenome-assembled genomes from Bacteria and Archaea. In addition, we recovered nearly 200 bacteriophage genomes. We document surprising taxonomic diversity, encompassing dozens of novel or unnamed bacterial genera and species, to which we have assigned new Candidatus names. Many of these genera are conserved across a range of mammalian gut microbiomes. Conclusions Our metagenomic analyses provide new insights into the bacterial, archaeal and bacteriophage components of the horse gut microbiome. The resulting datasets provide a key resource for future high-resolution taxonomic and functional studies on the equine gut microbiome.

    Helen L Brown, Jade L Passey, Maria Getino, Isabella Pursley, Piyali Basu, Daniel L Horton, Roberto M La Ragione (2020)The One Health European Joint Programme (OHEJP), 2018–2022: an exemplary One Health initiative, In: Journal of medical microbiology69(8)pp. 1037-1039 Microbiology Society

    Overview One Health is an increasingly popular approach used to tackle complex health problems. The One Health concept recognizes that human health is tightly connected to the health of animals and the environment. Although the related fields are now more aware of the benefits of collaborative working, the full benefits have not yet been realized as research efforts are often focussed on just one of these health domains. To address regional and global issues such as foodborne zoonoses (FBZ), antimicrobial resistance (AMR) and emerging infectious threats (ET), there must be transdisciplinary collaboration between the health domains, in addition to active dialogue between scientists and international policy makers. This editorial introduces the One Health European Joint Programme (OHEJP) as an example of a One Health initiative. Zoonoses, AMR and their global burden Zoonoses are infectious diseases that can be transmitted directly or indirectly between humans and animals. Although the severity of zoonotic infections varies, their global impact is undisputable. The World Bank estimates that just six zoonotic disease outbreaks between 1997 and 2009 led to a global economic loss of US$ 80 billion [1]. This high cost is due to medical costs, loss of individual productivity and restrictions on trade and movement during outbreaks. Despite improvements in the management and treatment of zoonotic outbreaks, high disease burdens caused by zoonotic pathogens continue to be reported globally. These problems have been amply demonstrated recently by the SARS-CoV-2 pandemic. Although it is still too soon to fully assess the total economic and societal cost of this virus, recent publications, such as Nicola et al. [2] have begun to highlight just how widespread the impact of a truly global zoonotic disease can be. Alongside zoonoses, AMR is a growing international issue. The World Health Organization (WHO) has listed AMR as one of the ten greatest global health threats in 2019 [3]. AMR is defined as the ability of microorganisms to survive the effect of antimicrobial drugs, hindering not only our ability to treat infectious diseases, but also to perform medical procedures requiring prophylactic antibiotic administration. It has been predicted that by 2050, the number of deaths due to unresponsive infections will reach 10 million annually, with the associated costs being estimated at US$ 100 trillion [4]. Increased and inappropriate use of antimicrobials has contributed to the development and spread of AMR, which can be transmitted between humans, animals and the environment. The history of the ‘One Health’ concept The origins of One Health go as far back as 1855, when Rudolf Virchow founded comparative pathology, which could be seen as the origin of the One Health concept. Building upon this, Calvin W. Schwabe argued in the twentieth century against compartmentalization in medical research, using the term ‘One Medicine’. The term One Health was then popularized in 2004 by the Wildlife Conservation Society at a conference in New York [5], and its use has continued to evolve since then, fostering the revival of comparative medicine (Fig. 1, and reviewed in Gibbs [6]). One Health has now been adopted by the WHO [7], the Food and Agriculture Organization (FAO) [8] and the World Organization for Animal Health (OIE) [9].

    Rachel Gilroy, Anuradha Ravi, Maria Getino, Isabella Pursley, Daniel L. Horton, Nabil-Fareed Alikhan, Dave Baker, Karim Gharbi, Neil Hall, Mick Watson, Evelien M. Adriaenssens, Ebenezer Foster-Nyarko, Sheikh Jarju, Arss Secka, Martin Antonio, Aharon Oren, Roy R. Chaudhuri, Roberto La Ragione, Falk Hildebrand, Mark J. Pallen (2021)Extensive microbial diversity within the chicken gut microbiome revealed by metagenomics and culture, In: PeerJ (San Francisco, CA)9e10941 Peerj Inc

    Background: The chicken is the most abundant food animal in the world. However, despite its importance, the chicken gut microbiome remains largely undefined. Here, we exploit culture-independent and culture-dependent approaches to reveal extensive taxonomic diversity within this complex microbial community. Results: We performed metagenomic sequencing of fifty chicken faecal samples from two breeds and analysed these, alongside all (n = 582) relevant publicly available chicken metagenomes, to cluster over 20 million non-redundant genes and to construct over 5,500 metagenome-assembled bacterial genomes. In addition, we recovered nearly 600 bacteriophage genomes. This represents the most comprehensive view of taxonomic diversity within the chicken gut microbiome to date, encompassing hundreds of novel candidate bacterial genera and species. To provide a stable, clear and memorable nomenclature for novel species, we devised a scalable combinatorial system for the creation of hundreds of well-formed Latin binomials. We cultured and genome-sequenced bacterial isolates from chicken faeces, documenting over forty novel species, together with three species from the genus Escherichia, including the newly named species Escherichia whittamii. Conclusions: Our metagenomic and culture-based analyses provide new insights into the bacterial, archaeal and bacteriophage components of the chicken gut microbiome. The resulting datasets expand the known diversity of the chicken gut microbiome and provide a key resource for future high-resolution taxonomic and functional studies on the chicken gut microbiome.

    Aiste Dijokaite, Maria Victoria Humbert, Emma Borkowski, Roberto M. La Ragione, Myron Christodoulides (2021)Establishing an invertebrate Galleria mellonella greater wax moth larval model of Neisseria gonorrhoeae infection, In: Virulence12(1)pp. 1900-1920 Taylor & Francis

    Neisseria gonorrhoeae (gonococcus) causes the human sexually transmitted disease gonorrhea. Studying gonococcal pathogenesis and developing new vaccines and therapies to combat the increasing prevalence of multi-antibiotic resistant bacteria has made use of many ex vivo models based on human cells and tissues, and in vivo vertebrate models, for example, rodent, pig and human. The focus of the current study was to examine the utility of the invertebrate greater wax moth Galleria mellonella as an in vivo model of gonococcal infection. We observed that a threshold of similar to 10(6) similar to 10(7) gonococci/larva was required to kill >50% of larvae (P < 0.05), and increased toxicity correlated with reduced health index scores and pronounced histopathological changes such as increases in the total lesion grade, melanized nodules, hemocyte reaction, and multifocal adipose body degeneration. Larval death was independent of the expression of pilus or Opa protein or LOS sialylation within a single gonococcal species studied, but the model could demonstrate relative toxicity of different isolates. N. meningitidis, N. lacatamica and gonococci all killed larvae equally, but were significantly less toxic (P > 0.05) than Pseudomonas aeruginosa. Larvae primed with nontoxic doses of gonococci were more susceptible to subsequent challenge with homologous and heterologous bacteria, and larval survival was significantly reduced (P < 0.05) in infected larvae after depletion of their hemocytes with clodronate-liposomes. The model was used to test the anti-gonococcal properties of antibiotics and novel antimicrobials. Ceftriaxone (P < 0.05) protected larvae from infection with different gonococcal isolates, but not azithromycin or monocaprin or ligand-coated silver nanoclusters (P > 0.05).

    Simon P. Graham, Yuen-Ki Cheong, Summer Furniss, Emma Nixon, Joseph A. Smith, Xiuyi Yang, Rieke Fruengel, Sabha Hussain, Monika A. Tchorzewska, Roberto M. La Ragione, Guogang Ren (2021)Antiviral Efficacy of Metal and Metal Oxide Nanoparticles against the Porcine Reproductive and Respiratory Syndrome Virus, In: Nanomaterials (Basel, Switzerland)11(8)2120 Mdpi

    Porcine reproductive and respiratory syndrome viruses (PRRSV) are responsible for one of the most economically important diseases affecting the global pig industry. On-farm high-efficiency particulate air (HEPA) filtration systems can effectively reduce airborne transmission of PRRSV and the incidence of PRRS, but they are costly, and their adoption is limited. Therefore, there is a need for low-cost alternatives, such as antimicrobial filters impregnated with antiviral nanoparticles (AVNP). During the past 10 years, tailored intermetallic/multi-elemental AVNP compositions have demonstrated effective performance against human viruses. In this study, a panel of five AVNP was evaluated for viricidal activity against PRRSV. Three AVNP materials: AVNP2, copper nanoparticles (CuNP), and copper oxide nanoparticles (CuONP), were shown to exert a significant reduction (>99.99%) in virus titers at 1.0% (w/v) concentration. Among the three, CuNP was the most effective at lower concentrations. Further experiments revealed that AVNP generated significant reductions in viral titers within just 1.5 min. For an optimal reduction in viral titers, direct contact between viruses and AVNP was required. This was further explained by the inert nature of these AVNP, where only negligible leaching concentrations of Ag/Cu ions (0.06-4.06 ppm) were detected in AVNP supernatants. Real-time dynamic light scatting (DLS) and transmission electron microscopic (TEM) analyses suggested that the mono-dispersive hydrodynamic behavior of AVNPs may have enhanced their antiviral activity against PRRSV. Collectively, these data support the further evaluation of these AVNP as candidate nanoparticles for incorporation into antimicrobial air-filtration systems to reduce transmission of PRRSV and other airborne pathogens.

    Andre Becker S. Saidenberg, Arnoud H.M. van Vliet, Marc Stegger, Thor Bech Johannesen, Torsten Semmler, Marcos Cunha, Alessandro C. de O. Silveira, Eleine Kuroki Anzai, Isabel C.A. Scaletsky, Anders Dalsgaard, Roberto M. La Ragione, Terezinha Knöbl (2022)Genomic analysis of the zoonotic ST73 lineage containing avian and human extraintestinal pathogenic Escherichia coli (ExPEC), In: Veterinary microbiology267109372 Elsevier B.V

    •Extraintestinal pathogenic Escherichia coli ST/CC73 are rarely reported in poultry.•Brazilian APEC ST/CC73 lack common APEC markers but harbor highly similar profiles to human ExPEC.•Phylogenetically, these APEC clustered closely with international UPEC/SEPEC human isolates. Extraintestinal pathogenic Escherichia coli (ExPEC) is a globally distributed pathogen, with uropathogenic E. coli (UPEC) and sepsis-associated E. coli (SEPEC) pathotypes particularly involved in human and companion animal disease, while avian pathogenic pathotype (APEC) severely impact poultry health and production. Similarities between APEC from poultry/meat and human ExPEC suggest that some APEC lineages may have zoonotic potential. ExPEC sequence type 73 (ST73) and its clonal complex (CC) are increasing causes of urinary tract infections and sepsis, but its role in zoonotic disease is less well understood. Here, we analyzed the genome sequences of 25 E. coli isolates from Brazil (11 APEC and 14 UPEC) from two time periods, from poultry producing areas and hospitals in the same geographical regions. Isolates were compared to 558 publicly available ST73/CC73 global sequences. Brazilian APEC harbored virulence factors associated with UPEC/SEPEC such as sfa, cnf1, vat, usp, hlyA, iron acquisition and protectins/serum resistance systems, while lacking some common APEC markers and widespread multidrug resistance. Analysis of core genome MLST and SNP phylogenetic trees indicated evolutionary relationships between subgroups of the Brazilian APEC to two contemporary Brazilian UPEC isolates from the same region, and one Brazilian UPEC available from another study. Phylogenies showed a non-host, geographical, or pathotype specificity, with APEC isolates clustering closely with international human UPEC, SEPEC. The remaining Brazilian UPEC grouped within human clusters. Collectively, this suggests a zoonotic potential for subgroups of Brazilian APEC from the ST73 lineage that could contaminate poultry products and subsequently cause human infection.

    Nicholas M. Thomson, Rachel Gilroy, Maria Getino, Ebenezer Foster-Nyarko, Arnoud H. M. van Vliet, Roberto M. La Ragione, Mark J. Pallen (2022)Remarkable genomic diversity among Escherichia isolates recovered from healthy chickens, In: PeerJ (San Francisco, CA)10e12935 Peerj Inc

    The genus Escherichia has been extensively studied and it is known to encompass a range of commensal and pathogenic bacteria that primarily inhabit the gastrointestinal tracts of warm-blooded vertebrates. However, the presence of E. coli as a model organism and potential pathogen has diverted attention away from commensal strains and other species in the genus. To investigate the diversity of Escherichia in healthy chickens, we collected fecal samples from antibiotic-free Lohmann Brown layer hens and determined the genome sequences of 100 isolates, 81 of which were indistinguishable at the HC0 level of the Hierarchical Clustering of Core Genome Multi-Locus Sequence Typing scheme. Despite initial selection on CHROMagar Orientation medium, which is considered selective for E. coli, in silico phylotyping and core genome single nucleotide polymorphism analysis revealed the presence of at least one representative of all major clades of Escherichia, except for E. albertii, Shigella, and E. coli phylogroup B2 and cryptic clade I. The most frequent phylogenomic groups were E. coli phylogroups A and B1 and E. ruysiae (clades III and IV). We compiled a collection of reference strains isolated from avian sources (predominantly chicken), representing every Escherichia phylogroup and species, and used it to confirm the phylogeny and diversity of our isolates. Overall, the isolates carried low numbers of the virulence and antibiotic resistance genes typically seen in avian pathogenic E. coli. Notably, the clades not recovered are ones that have been most strongly associated with virulence by other studies.

    Vasco T. M. Gomes, Luisa Z. Moreno, Ana Paula S. Silva, Siddhartha Thakur, Roberto M. La Ragione, Alison E. Mather, Andrea M. Moreno (2022)Characterization of Salmonella enterica Contamination in Pork and Poultry Meat from Sao Paulo/Brazil: Serotypes, Genotypes and Antimicrobial Resistance Profiles, In: Pathogens (Basel)11(3)358 Mdpi

    Salmonellosis is a zoonosis of major relevance to global public health. Here we present the assessment of Salmonella enterica contamination in pork and poultry meat sold at retail markets in Sao Paulo, Brazil. A total of 780 meat samples (386 poultry meat and 394 pork samples) were collected from 132 markets. From these, 57 samples (7.3%) were positive for S. enterica isolation, including 32 (8.3%) poultry meat and 25 (6.3%) pork samples. S. enterica isolates were further characterized for serotyping, antimicrobial resistance and genotyping by amplified fragment length polymorphism and pulsed field gel electrophoresis. Antimicrobial resistance analysis demonstrated two main profiles: pork isolates were more resistant to macrolides, beta-lactams, tetracycline, phenicols, and fluoroquinolones, and poultry meat isolates presented higher resistance to fluoroquinolones, sulfonamides, tetracycline, and beta-lactams. A total of 72.4% of poultry meat isolates were identified as S. Heidelberg, while most of pork isolates were S. Typhimurium (31.7%) and S. Give (16.7%). Genotyping resulted in most clusters consisting exclusively of pork or poultry meat, no crosscontamination was detected, and a tendency to differentiate isolates according to their serotypes and markets of origin. High resistance rates to critically important antimicrobials reinforce the importance of controlling Salmonella contamination in meat production chains.

    Norman K. Fry, Roberto M. La Ragione (2022)JMM Profiles for the Journal of Medical Microbiology: an update, In: Journal of medical microbiology71(3) Microbiology Soc
    J Szych, T Wolkowicz, R La Ragione, G Madajczak (2014)Impact of Antibiotics on the Intestinal Microbiota and on the Treatment of Shiga-toxin-Producing Escherichia coli and Salmonella Infections, In: CURRENT PHARMACEUTICAL DESIGN20(28)pp. 4535-4548 BENTHAM SCIENCE PUBL LTD
    A Caprioli, S Morabito, F Scheutz, H Chart, E Oswald, M Brigotti, L Monnens, A Aspan, R La Ragione, C Low, D Newell (2006)Pathogenesis of verocytotoxin/shiga toxin-producing Escherichia coli infection, In: Emerging Infectious Diseases12(8)
    Alistair Macdonald, Mark Chambers, Roberto La Ragione, Kayleigh Wyles, Matthieu Poyade, Andrew Wales, Naomi Klepacz, Tom Kupfer, Fraje Watson, Shona Noble (2020)Using novel visualisation methods to combat infection risk during clinical practices., In: Kirsty Christer, Claire Craig, Paul Chamberlain (eds.), Proceedings of the 6th International Conference on Design4Health
    CI Le Roy, LJ Mappley, RM La Ragione, MJ Woodward, SP Claus (2015)Brachyspira pilosicoli-induced avian intestinal spirochaetosis., In: Microb Ecol Health Dis26pp. 28853-?

    Avian intestinal spirochaetosis (AIS) is a common disease occurring in poultry that can be caused by Brachyspira pilosicoli, a Gram-negative bacterium of the order Spirochaetes. During AIS, this opportunistic pathogen colonises the lower gastrointestinal (GI) tract of poultry (principally, the ileum, caeca, and colon), which can cause symptoms such as diarrhoea, reduced growth rate, and reduced egg production and quality. Due to the large increase of bacterial resistance to antibiotic treatment, the European Union banned in 2006 the prophylactic use of antibiotics as growth promoters in livestock. Consequently, the number of outbreaks of AIS has dramatically increased in the UK resulting in significant economic losses. This review summarises the current knowledge about AIS infection caused by B. pilosicoli and discusses various treatments and prevention strategies to control AIS.

    Arnoud H M van Vliet, MARWA M. HASSAN, Owen Higgins, LA Burke, Louise O'Connor, D Morris, ROBERTO MARCELLO LA RAGIONE, TC Smith (2020)WorldCOM Deliverable 1: Prevalence of ESBL subtypes in bacterial pathogens and a sequence database of selected alleles Zenodo

    OHEJP Project: WorldCOM, Deliverable 1, Work Package 1. This dataset is connected to Work Package 1, Task1 of the WorldCOM consortium grant within the One Health EJP group. The aim was to analyse publicly available sequences for antimicrobial resistance genes associated with Salmonella, Campylobacter and E. coli. For the initial phase of this work package, we have focused on ESBL-related AMR genes. As these genes are absent from Campylobacter, we have not included this bacterium in these analyses, and have used the important pathogens Klebsiella and Acinetobacter. All types and subtypes of Extended Spectrum β-Lactamases (ESBLs) and plasmid-mediated colistin resistance genes have been analysed for frequency among reported and extracted sequences. High frequency resistant genes subtypes have been highlighted for further sequence analysis to illustrate geographic distribution and geographic-specific single nucleotide polymorphisms (SNPs). The data shown are work in progress.