Professor Roberto La Ragione


Professor of Veterinary Microbiology and Pathology, Head of the Department of Pathology and Infectious Diseases, Deputy Head of School
BSc (Hons) MSc PhD FRSB CBiol FIBMS CSci AECVM FRCPath HonAssocRCVS
+44 (0)1483 689265
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Academic and research departments

School of Veterinary Medicine.

Biography

University roles and responsibilities

  • Module coordinator for MSc Vet Micro research projects
  • Head of Department (Pathology and Infectious Diseases)
  • Professor of Veterinary Microbiology and Pathology
  • Vet Sch SMT member
  • Deputy chair of AWERB committee
  • Deputy Head of School

    News

    Research

    Research interests

    Supervision

    Postgraduate research supervision

    My teaching

    My publications

    Publications

    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].
    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.
    Brachyspira are the causative agent of avian intestinal spirochaetosis, a gastrointestinal disease common in layer hens and broiler breeders. This disease costs the UK laying industry approximately £18 million per annum, resulting from reduced egg production and poor egg quality. Prevalence of Brachyspira is increasing, and due to the poor understanding of this pathogen, mitigation strategies have been largely unsuccessful. Therefore, preventative measures are essential. These studies aimed to improve the understanding of Brachyspira pathobiology and investigate Lactobacillus probiotics as a suitable mitigation strategy. Brachyspira and Lactobacillus species were characterised using phenotypic and genotypic methods. Four Lactobacillus isolates were selected for their inhibition of Brachyspira in vitro and demonstrated inhibition by a number of mechanisms. Secreted metabolites in Lactobacillus cell free supernatant inhibited Brachyspira (p value < 0.05) and metabolomic studies identified the production of organic acids to be a major contributor to inhibition. Protein denaturation in cell free supernatants significantly reduced Brachyspira inhibition (p value < 0.05), suggesting the role of bacteriocins in inhibition. Furthermore, L. reuteri isolates co-aggregated with Brachyspira in vitro, reducing pathogen viability (p value < 0.05). Pro-inflammatory responses to Brachyspira in HD11 avian macrophages were dominated by upregulation of IFNg (p value < 0.01) and pre-treatment of cells with Lactobacillus significantly reduced this response (p value < 0.0001), demonstrating the ability of probiotics to alter immune responses to Brachyspira. Galleria mellonella were utilised to study Brachyspira virulence and probiotic intervention. G. mellonella exhibited a varied response to Brachyspira iv infection and Lactobacillus isolates were able to protect against the mortality associated with Brachyspira isolates (p value < 0.05). The studies here demonstrated that Lactobacillus probiotics are a suitable mitigation strategy against Brachyspira. A number of mechanisms were identified, however future studies are required to explore these mechanisms in a more relevant in vivo chicken model.
    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.
    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.
    Porcine reproductive and respiratory syndrome viruses (PRRSV) cause the most economically significant disease of swine. The emergence of highly pathogenic strains and the limitations of current vaccines pose significant challenges to PRRS control. An improved understanding of pathogenesis is needed to support the development of improved vaccines. Experimental infection with virulent PRRSV-1 subtype 3 strains is associated with enhanced immune responses, which have been attributed to the more effective viral clearance compared to ‘typical’ PRRSV-1 subtype 1 strains. Previous ex vivo data has also shown that this increased virulence correlates with a broader cell tropism for cells of the myeloid lineage. The causation of this correlation was investigated by assessing whether European PRRSV-1 strains can infect dendritic cells (DC), which are not canonically a target cell. A staining strategy was optimised to enrich and phenotype blood-derived DC (BDC) subsets. Infection experiments demonstrated that BDC were not susceptible, and that cytokine responses in enriched BDC culture were suppressed by inoculation of subtype 3 strain Lena. Further characterisations of host responses instead used monocyte-derived macrophages (MΦ) and DC. Functional assays determined that pro-inflammatory responses were increased by subtype 3 strain SU1-Bel more than subtype 1 strains, including modulation of surface activation markers, ability to prime allogeneic T cell proliferation, and secretion of inflammatory cytokines; Lena, however, did not induce pro-inflammatory cytokine responses but decreased endocytic and phagocytic capacity. This trend was validated by transcriptomic analysis of infected monocyte-derived MΦ, which identified putative genes of interest for future studies, including pattern recognition receptors. Inflammasome treatments suggested that SU1-Bel primed inflammasome activation and that subtype 3 strain replication was decreased by inflammasome inhibition, proportionately increasing MΦ viability. Finally, preliminary trials of a novel macrophage cell line demonstrated its utility for further investigation of the mechanistic basis for the enhanced virulence of PRRSV-1 subtype 3 strains.
    Varicose veins, often thought as a purely cosmetic issue, are a common symptom resulting from chronic venous insufficiency (CVI). CVI can result in serious fasciocutaneous and haematological complications. In 2013, NICE recommended that the treatment of varicose veins should preferably be carried out by endothermal ablation (ETA). The mechanism of action of ETA is still not fully understood. In 2004 it was suggested that successful treatment must involve transmural vein wall death (TMVD) in the target vein. Through the development of a novel in-vitro¬ model, using ex-vivo veins, the work carried out in this thesis has indicated that TMVD is reliant on a combination of thermal necrosis, followed by the upregulation of apoptosis. The novel model has been used to compare four different ETA techniques; endovenous laser ablation (EVLA) using an 810nm endovenous laser (EVL) with a jacketed fibre, EVLA using a 1470nm EVL with a jacketed fibre, EVLA using a 1470nm EVL with a radial fibre and Radiofrequency-induced Thermo Therapy (RFiTT). The comparisons indicate that treatment with the 810nm EVL is inferior at causing TMVD, in comparison to the 1470nm EVL. Treatment with a radial fibre, compared to a jacketed fibre, when using a 1470nm EVL, shows an improved damage profile that is much more homogenous with less overtreatment of target tissue. Comparisons between RFiTT and EVLA, indicate that treatment with RFiTT is as effective at causing TMVD as the 1470nm EVL with a radial fibre but with differences in the thermal damage profile. These results correlate well with reports from the current literature. However, this novel model has been able to show that the upregulation of apoptosis plays a pivotal role in TMVD after ETA treatment. The results also show that histology is often not sufficient to alone determine the difference between successful and inadequate treatment.
    In this work two lichen species (Cladonia portentosa and Ramalina farinacea) were investigated. Lichen have been used as antimicrobials, perfume and dye ingredients. Five known compounds were isolated from C. portentosa and seven known compounds were isolated from R. farinacea. Antibacterial activity investigation was carried out for four compounds isolated from these lichen the compounds were: (-)-usnic acid (43), olivetocarboxylic acid (45), perlatolic acid (46) and atranorin (49). Their activity was investigated against seven different bacteria, Escherichia coli (90, 91, 93, 94, 98), Salmonella Enteritidis (446, 496), Klebsiella pneumonia (97), Acinetobacter baumannii (210, 211), Pseudomonas aeruginosa (2, 92), Staphylococcus aureus (NCTC 12493) and Enterococcus faecalis (ATCC 29212). All compounds were active at high concentrations. In Larix decidua bark two active compounds (larixol and larixyl acetate) have previously been isolated and are being developed for use in the agrochemical Larixyne ®. To extract the compounds traditional solvents such as methanol were used. In this work, two environmentally friendly natural deep eutectic solvents, choline chloride / lactic acid (1:1 mol) and choline chloride / malonic acid (1:1 mol), were investigated. It was determined that the NADES successfully extract larixol and larixyl acetate from ground L. decidua bark. Antimicrobial resistance is a global threat, with some pathogens demonstrating resistance to all currently available commercial antimicrobials. With few novel antimicrobials in the pharmaceutical pipeline, treatments for bacterial infections, particularly those caused by Gram-negative bacteria, are extremely limited. In this work synergy between the natural product vanillin and the commercially available antibiotic colistin was investigated against various Gram-negative bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella Enteritidis, Salmonella Typhimurium and Esherichia coli). It was determined that the combination has a synergistic relationship against Gram-negative bacteria. Traditionally, the leaves of Sambucus ebulus are used to treat Newcastle’s Disease Virus in chicken. In this work the antiviral activity of the oil of the S. ebulus plant, its main component (isovaleric acid) and three compounds of a similar structure to isovaleric acid (menthyl isovalerate, benzyl isovalerate and levulinic acid) were investigated. Three main types of fluorometric antiviral assays were carried out: combined, therapeutic and prophylactic. The oil was found to be active against NDV therefore supporting its traditional use. Isovaleric acid and other compounds were found to have some activity against NDV.
    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.
    L Roberts, R La Ragione, M Alder (2012)Starting a new vet school, In: VETERINARY RECORD171(21)pp. 523-524 BRITISH VETERINARY ASSOC
    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 < .001) in self-reported HH behaviour, with time constraints being the strongest predictor (β = −.47, p < .001) followed by difficulty finding equipment (β = −.21, p = .05). Discussion: Time constraints may be the most important influence on HH adherence among the determinants investigated. Future researchers should consider employing theoretical models to aid a more comprehensive understanding of the psychology underlying HH adherence and HH interventions.
    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.
    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.
    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.

    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.
    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.
    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
    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: Proceedings of the 6th International Conference on Design4Health
    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.
    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
    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.
    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 = <0.05) larval survival at 96 h to 86% (±17 standard deviation percentage points [pp]) compared to monotherapy with theaflavin (52% ±14 pp), epicatechin (44% ±25 pp) or PBS (31% ±13 pp). Morbidity was also lower in larvae treated with the combination, compared to monotherapy. Conclusions: Polyphenol combinations produce effective antibacterial action against A. baumannii and show great potential for the treatment of infections caused by MDR A. baumannii.
    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
    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.
    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
    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.
    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 pneumoniae, 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.

    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
    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
    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
    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 <0.001) ii) increased airorhynchy with a smaller, more ventrally rotated olfactory bulb (p <0.001). Both generated `concertina' flexures of the brain and craniocervical junction. Conclusion Morphometric mapping provides a diagnostic tool for quantifying symptomatic CM, secondary SM and their relationship with brachycephaly. It is hypothesized that CM pain is associated with increased brachycephaly and SM can result from different combinations of abnormalities of the forebrain, caudal fossa and craniocervical junction which compromise the neural parenchyma and impede cerebrospinal fluid flow.
    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.
    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.
    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
    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
    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
    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.
    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.

    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
    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
    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.
    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
    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.
    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.
    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
    Usman O. Adekanye, Abel Bulamu Ekiri, Erika Galipo, Abubakar Bala Muhammad, Ana Mateus, Roberto M. La Ragione 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.
    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
    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.
    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
    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<0.001), IgG (P=0.001) and IgA (P=0.039) responses to S. Typhimurium infection including cross-reacting antibodies to S. Enteritidis. Similarly, inclusion of the prebiotic lactulose at 1% (w/w) of the feed on a daily basis in the diet enhanced serum IgM (P=0.010), IgG (P=0.004) and IgA (P=0.046) responses to S. Typhimurium infection and also cross-reacting antibodies to S. Enteritidis. Inclusion of both additives in the synbiotic diet also elicited an enhanced immune response with IgM (P=0.009) and IgG (P=0.046) levels being increased, however a significant interaction of the pre and probiotics was observed when considering the immune responses to S. Typhimurium (IgM P=0.004; IgG and IgA, P<0.001 for interaction). With respect to immune responses, the effects of pre or probiotic administration were the same or reduced in the synbiotic diet compared to when used in isolation. The data support the use of Lactobacillus plantarum B2984 or lactulose as strategies to contribute to the protection of weaned piglets from zoonotic bacterial pathogens, but caution must be taken when combining dietary supplements as combinations can interact.
    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
    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
    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)
    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
    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
    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:
    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
    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
    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
    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
    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.
    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<0.05) and egg production as assessed by egg weight and faecal staining score (p<0.05) were better. Also, at post-mortem examination, significantly fewer B. pilosicoli were recovered from treated birds (p<0.05), with only mild-moderate histopathological changes were observed.
    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.
    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.
    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
    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.
    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 <0.001). Associated with SM were a reduced occipital crest and two acute changes in angulation i) `sphenoid flexure' at the spheno-occipital synchondrosis ii) `cervical flexure' at the foramen magnum allied with medulla oblongata elevation. Comparing dogs with and without SM, each breed had a unique trait: Chihuahua had a smaller angle between the dens, atlas and basioccipital bone (p value < 0.001); Affenpinschers had a smaller distance from atlas to dens (p value 0.009); CKCS had a shorter distance between the sphenooccipital synchondrosis and atlas (p value 0.007). Conclusion The selected morphometries successfully characterised conformational changes in the brain and craniocervical junction that might form the basis of a diagnostic tool for all breeds. The severity of SM involved a spectrum of abnormalities, incurred by changes in both angulation and size that could alter neural parenchyma compliance and/or impede cerebrospinal fluid channels.
    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.
    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
    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.
    Chiari-like Malformation (CM) and secondary Syringomyelia (SM) is a complex, debilitating abnormality which compromises the normal cerebrospinal fluid movement of the central nervous system culminating in the development of fluid-containing cavities within the spinal cord and associated with behavioural signs of pain and neurological deficits. The prevalence of asymptomatic CM dogs suggest that cerebellar indentation and impaction may be normal anatomical variations and unsuitable as a definition of CM. Magnetic Resonance Imaging (MRI) remains the definitive means of diagnosing CM/SM and a morphometric technique of quantifying CM and SM on mid-sagittal MRI has been successfully applied and validated in previous studies to a cohort of Griffon Bruxellois (GB) dogs with and without CM and a mixed breed GB family crossed with a mesaticephalic breed (Australian Terrier). Using a refined technique which took account of recent research findings, morphometries using a triangulation of circles, lines and angles were used to ‘map’ MRIs of the whole brain and cervical region in order to quantify the severity of the CM and SM phenotype in the Cavalier King Charles (CKCS). A further morphometric analysis was undertaken to explore brachycephaly and miniaturization as risk factors for CM and SM by comparing their impact in the CKCS, Affenpinscher and Chihuahua breeds. The collective framework of lines and angles generated a unique ‘signature’ for the dog, characterised by “concertina” type flexures demonstrating the combined nature of segregated traits towards the severity in the phenotype. Compared to controls, CKCS with CM pain are characterised by increased brachycephaly and airorhynchy, while significant traits for SM in the three dog breeds included those reported for the GB, suggesting a common aetiology. The characterisation of the CM phenotype provides the possibility of a diagnostic tool for veterinarians and means to assist breeders with mate selection to reduce symptomatic prevalence of CM/SM.
    The emergence of extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae represents a major challenge to public and animal health in many countries including Lebanon. Carriage of ESBLs confers resistance to β-lactam antibiotics and more than 200 different sub-types have been identified, all of which are primarily encoded by genes located on conjugative plasmids. Despite the high prevalence of ESBLs, there is still a paucity of information regarding the impact of ESBL plasmid carriage on the host bacterium and how the spread of antibiotic resistance relates to antibiotic use in hospital environments. Therefore, the aim of this study was, firstly, to characterise the genetic and phenotypic properties of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolated from a hospital in Lebanon. Secondly, to assess the fitness of isolates harbouring different sub-types of ESBLs in order to better understand the influence of the plasmid on the host bacterium, and thirdly, to correlate hospital antibiotic consumption data calculated using ABC Calc software (in defined daily doses (DDD) per 100 bed days), with antimicrobial susceptibility profiles obtained over a three year period. A total of 140 isolates of E. coli and K. pneumoniae that were resistant to at least one 3rd- or 4th- generation cephalosporin were collected from the Centre Hospitalier Du Nord (CHN) in Lebanon, between 1st May 2011 and the 31st December 2012. ESBL-production was confirmed in all isolates using the double disk synergy assay and the E-test ESBL strip. Out of 140 isolates, 35 were also found to harbour a chromosomal AmpC β-lactamase, whereas none harboured the plasmidic gene. PCR analyses showed that all isolates harboured CTX-M type β-lactamase, with 74% also harbouring TEM and approximately 40% harbouring either SHV or OXA type β-lactamases. Most E. coli isolates belonged to phylogenetic group B2, and harboured genes important in iron uptake. PFGE analysis of both E. coli and K. pneumoniae indicated significant clonal diversity amongst the isolates. Analysis of the β-lactamase-encoding plasmids from selected isolates revealed the plasmids were conjugative and bore similarities to pEC_L8 and Inc group types. Plasmid curing studies indicated that ESBL plasmids influenced bacterial host cell fitness through altered carbon metabolism and generation time. Both positive and negative correlations were found between antibiotic consumption and the susceptibility of E. coli and K. pneumoniae to different types of antibiotics. This study highlights the diversity of ESBL-producing isolates found among members of the Enterobacteriaceae in Lebanon and supports the idea that horizontal transfer of CTX-M-15 harbouring ESBL plasmids, rather than clonal expansion of particular isolates, has occurred. Moreover, the use of β-lactam antibiotics and quinolones may promote their spread, and thus effective hospital infection control programs and more pragmatic antimicrobial usage is recommended.
    Campylobacter jejuni and Campylobacter coli are recognised as the principal causative agents of bacterial gastroenteritis in the developed world. However, despite the identification of factors integral to infection, characterisation of the virulence strategies employed by Campylobacter remains a significant challenge. Bacterial autotransporter proteins comprise the largest and most diverse class of secretory proteins in Gram-negative bacteria; notably almost all previously characterised autotransporter proteins contribute to bacterial virulence to some extent. The aim of this study was to characterise CapC, a newly identified, strain-specific gene predicted to encode an autotransporter protein, and to examine the contribution of this factor to the virulence of Campylobacter jejuni. The capC gene was initially confirmed as being encoded by approximately 60% of C. jejuni and C. coli human clinical and poultry associated isolates. Moreover, CapC was confirmed as a member of the autotransporter family through the use of bioinformatic prediction tools and the localisation site of this protein was determined as the outer membrane of C. jejuni. Targeted mutagenesis of the capC gene in C. jejuni 81116 and C. jejuni M1 and subsequent comparison of wild-type and isogenic mutant strains demonstrated that CapC contributes directly to virulence in the Galleria mellonella invertebrate model (p=0.00017; p=0.002323). Furthermore, tissue culture assays using non-polarised, partially differentiated Caco-2 and T84 intestinal epithelial cells indicate that deletion of CapC resulted in significantly decreased adhesion and invasion efficiency. Additional analyses indicated that CapC primarily contributes to adhesion to intestinal epithelial cells. Additional assays showed that deletion of the capC gene has no significant phenotypic effect on cytotoxicity in a Caco-2 cell model. A secondary aim of this study was to examine the distribution of capC amongst campylobacters and to establish any potential genetic associations of this virulence determinant. Using publically vi available genome sequences, capC was established to be highly prevalent in C. jejuni (67.9%) and C. coli (84%). Campylobacter autotransporter proteins were also shown to be present in truncated and full length forms. Interestingly, full length CapC was shown to be primarily associated with the ST-45, ST-283 and ST-573 clonal complexes in C. jejuni and the ST-828 clonal complex in C. coli. Furthermore, this study detailed the identification of a novel autotransporter in Campylobacter species, tentatively designated as CapD. This autotransporter was found to be genetically distinct from CapC and is the most prevalent autotransporter identified in Campylobacter species. The studies presented in this thesis indicate that CapC is a strain-specific virulence determinant in Campylobacter species that is associated with select lineages of C. jejuni and C. coli. CapC contributes to the integral infection process of adhesion however further studies are required to fully elucidate the exact nature of this interaction. Additionally, it can be concluded that possession of Campylobacter autotransporter proteins is dependent on genetic background.
    Campylobacter is the most common cause of bacterial gastroenteritis in the developed world, with approximately 70,000 cases reported in the UK per annum. It is well accepted that Campylobacter spp. form biofilms which aid its survival in both the environment and the host. The formation of biofilms in poultry processing plants are of particular concern, as they are potential sources of contamination between meat batches, and facilitate the transmission of the pathogen through the human food chain. However, despite the importance of biofilms, the molecular mechanisms and metabolic pathways associated with biofilm formation in Campylobacter have not been well elucidated. Here, 30 C. jejuni strains were isolated from commercial chicken meat and assayed for their motility and ability to form biofilms, using crystal violet staining at 37ᵒC and 42ᵒC. Only five of the 30 isolates were able to form biofilms, with more complex biofilm phenotypes observed at 37ᵒC. Although all isolates were motile, a weak correlation between motility and biofilm formation was observed, indicating that motility is not essential to the phenotype. Ten isolates were selected, representing the five most competent, and five poorest biofilm formers. These isolates were screened for their virulence profiles using Galleria mellonella and adhesion and invasion of Caco-2 models. No correlation between the ability to form biofilms and virulence phenotypes was observed. A competent biofilm former (isolate CJP13) was selected and a mariner transposon mutant library was constructed in this strain. Over 3,000 of the resulting transposon mutants were individually screened for their ability to form biofilms. Thirteen of the 3,000 transposon mutants showed reduced ability to form biofilms across two independent biological replicates. Of those, individual knock-out mutants of Cj0080, Cj1623 (memP), hydA and trbJ and complemented mutants were constructed in CJP13 and NCTC11168 strains. All mutants showed reduced ability to form biofilms compared to wild type strains, although the NCTC11168 ΔmemP mutant showed the most significant reduction, with almost no biofilm ability observed (p<0.001). Complemented mutants presented a mixed ability to restore biofilm formation to wild type levels. Next-Generation Sequencing (NGS) and subsequent pangenome analysis revealed genes which were differentially present/absent in competent and poor biofilm genomes, two of which are involved with sialic acid synthesis and transport. Phylogenetic analysis revealed CJP17 and CJP19 strains (competent and poor biofilm formers respectively) to be almost genetically identical, with three gene mutations in the CJP17 genome. One such mutation is predicted to cause truncation of pflA, which is suggested to be the cause of reduced motility in this strain compared to CJP19. Despite this mutation, CJP17 displayed a competent biofilm phenotype, suggesting the mechanisms involved in biofilm formation are motility independent. The panel of 10 isolates were subjected to Biolog phenotypic array analysis to study the ability of Campylobacter to metabolise 95 different carbon substrates. Competent biofilm formers were able to significantly metabolise several carbon sources more readily; D-ribose and L-lyxose when using lag phase to define utilisation, and L-lactic acid when using max utilisation and max slope to represent substrate utilisation parameters. However, varying concentrations of L-lactic acid failed to induce biofilm formation in chicken isolates when added to complex media. The studies reported here demonstrate significant differences in the metabolism and genetic composition between poor and competent biofilm isolates. Moreover, this work provides evidence that multiple C. jejuni genes are responsible for the biofilm phenotype in currently circulating C. jejuni isolates. This study suggests that the role of membrane proteins, such as memP, is key in the formation of biofilm in NCTC11168, but less so for CJP13, indicating strain-specific mechanisms for C. jejuni biofilm formation. The continuation of genomic and metabolic analysis of biofilm formation is required to facilitate the development of novel control strategies aimed at mitigating biofilm formation in poultry processing plants, to prevent subsequent human infection.
    The gut microbiota plays an important role in the development of type 2 diabetes (T2D), which is an alteration in the diversity and abundance of the gut microbiota, favouring the growth of Gram-negative bacteria. Although a lot of studies have shown this to be the case, most of this work has been done in animal models with few studies in humans. In animal models of T2D, it is known that a high-fat diet alters the gut microbiota in favour of the growth of Gram–negative bacteria. The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS) which is an endotoxin that can trigger inflammation leading to metabolic disorders such insulin resistance and T2D, hence T2D is considered a low grade inflammatory disorder. In this thesis, the effect of Galactooligosaccharide (GOS), a prebiotic, on the composition of the gut microbiota was investigated. Next generation sequencing (NGS) of the gut microbiota of T2D and healthy control subjects showed no significant difference at the phylum level between the two groups. Furthermore, T2D patients in the prebiotic group had a significant increase in the level of Firmicutes compared to the placebo group. Also, although not significant, T2D patients on metformin had increased level of Bacteroidetes, Proteobacteria and Actinobacteria compared to those not on metformin. The ability of human faecal water (FW) to distinguish between healthy and T2D patients using an in vitro model of the intestinal mucosa was studied. FW from T2D patients decreased Caco-2 cell monolayer integrity when compared to the healthy controls and in the T2D patients, FW activity in vitro correlated with biological markers of T2D severity measured in vivo. Additionally, cytokines were measured in T2D faecal samples using a human cytokine array. Finally, GOS anti-cytotoxic activity was also assessed in vitro using cell viability assays and the anti-cytotoxic effect of GOS was time and concentration dependent. Together, the thesis explored potential new ways of using faecal samples as biomarker for T2D in vitro and relating it to in vivo parameters of the patients. Also future work in this area may reveal mechanistic insight to the use of FW as a non-invasive biomarker for T2D.