Reuter M, Periago PM, Mulholland F, Brown HL, van Vliet AH (2015) A PAS domain-containing regulator controls flagella-flagella interactions in Campylobacter jejuni, Frontiers in Microbiology 6 770 Frontiers Media
The bipolar flagella of the foodborne bacterial pathogen Campylobacter jejuni confer motility, which is essential for virulence. The flagella of C. jejuni are post-translationally modified, but how this process is controlled is not well understood. In this work, we have identified a novel PAS-domain containing regulatory system, which modulates flagella-flagella interactions in C. jejuni. Inactivation of the cj1387c gene, encoding a YheO-like PAS6 domain linked to a helix-turn-helix domain, resulted in the generation of a tightly associated ?cell-train? morphotype, where up to four cells were connected by their flagella. The morphotype was fully motile, resistant to vortexing, accompanied by increased autoagglutination, and was not observed in aflagellated cells. The cj1387c mutant displayed increased expression of the adjacent Cj1388 protein, which comprises of a single endoribonuclease L-PSP domain. Comparative genomics showed that cj1387c (yheO) orthologs in bacterial genomes are commonly linked to an adjacent cj1388 ortholog, with some bacteria, including C. jejuni, containing another cj1388-like gene (cj0327). Inactivation of the cj1388 and cj0327 genes resulted in decreased autoagglutination in Tween-20-supplemented media. The cj1388 and cj0327 mutants were also attenuated in a Galleria larvae-based infection model. Finally, substituting the sole cysteine in Cj1388 for serine prevented Cj1388 dimerization in non-reducing conditions, and resulted in decreased autoagglutination in the presence of Tween-20. We hypothesize that Cj1388 and Cj0327 modulate post-translational modification of the flagella through yet unidentified mechanisms, and propose naming Cj1387 the Campylobacter Flagella Interaction Regulator CfiR, and the Cj1388 and Cj0327 protein as CfiP and CfiQ, respectively.
CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated) systems are sequence-specific adaptive defenses against phages and plasmids which are widespread in prokaryotes. Here we have studied whether phylogenetic relatedness or sharing of environmental niches affects the distribution and dissemination of Type II CRISPR-Cas systems, first in 132 bacterial genomes from 15 phylogenetic classes, ranging from Proteobacteria to Actinobacteria. There was clustering of distinct Type II CRISPR-Cas systems in phylogenetically distinct genera with varying G+C%, which share environmental niches. The distribution of CRISPR-Cas within a genus was studied using a large collection of genome sequences of the closely related Campylobacter species Campylobacter jejuni (N = 3,746) and Campylobacter coli (N = 486). The Cas gene cas9 and CRISPR-repeat are almost universally present in C. jejuni genomes (98.0% positive) but relatively rare in C. coli genomes (9.6% positive). Campylobacter jejuni and agricultural C. coli isolates share the C. jejuni CRISPR-Cas system, which is closely related to, but distinct from the C. coli CRISPR-Cas system found in C. coli isolates from nonagricultural sources. Analysis of the genomic position of CRISPR-Cas insertion suggests that the C. jejuni-type CRISPR-Cas has been transferred to agricultural C. coli. Conversely, the absence of the C. coli-type CRISPR-Cas in agricultural C. coli isolates may be due to these isolates not sharing the same environmental niche, and may be affected by farm hygiene and biosecurity practices in the agricultural sector. Finally, many CRISPR spacer alleles were linked with specific multilocus sequence types, suggesting that these can assist molecular epidemiology applications for C. jejuni and C. coli.
Pearson BM, Gaskin DJ, Segers RP, Wells JM, Nuijten PJ, van Vliet AH (2007) The complete genome sequence of Campylobacter jejuni strain 81116 (NCTC11828), Journal of Bacteriology 189 (22) pp. 8402-8403 American Society for Microbiology
Campylobacter jejuni is a major human enteric pathogen that displays genetic variability via genomic reorganization and phase variation. This variability can adversely affect the outcomes and reproducibility of experiments. C. jejuni strain 81116 (NCTC11828) has been suggested to be a genetically stable strain (G. Manning, B. Duim, T. Wassenaar, J. A. Wagenaar, A. Ridley, and D. G. Newell, Appl. Environ. Microbiol. 67:1185-1189, 2001), is amenable to genetic manipulation, and is infective for chickens. Here we report the finished annotated genome sequence of C. jejuni strain 81116.
Stoof J, Kuipers EJ, van Vliet AH (2009) Characterization of NikR-responsive promoters of urease and metal transport genes of Helicobacter mustelae, Biometals 23 pp. 145-159 Springer
The NikR protein is a nickel-responsive regulator, which in the gastric pathogen Helicobacter pylori controls expression of nickel-transporters and the nickel-cofactored urease acid resistance determinant. Although NikR-DNA interaction has been well studied, the Helicobacter NikR operator site remains poorly defined. In this study we have identified the NikR operators in the promoters of two inversely nickel-regulated urease operons (ureAB and ureA2B2) in the ferret pathogen Helicobacter mustelae, and have used bioinformatic approaches for the prediction of putative NikR operators in the genomes of four urease-positive Helicobacter species. Helicobacter mustelae NikR bound to the ureA2 promoter to a sequence overlapping with the ?35 promoter region, leading to repression. In contrast, NikR binding to a site far upstream of the canonical Ã80 promoter in the H. mustelae ureA promoter resulted in transcriptional induction, similar to the situation in H. pylori. Using H. pylori NikR operators and the newly identified H. mustelae NikR operators a new consensus sequence was generated (TRWYA-N15-TRWYA), which was used to screen the genomes of four urease-positive Helicobacter species (H. mustelae, H. pylori, H. acinonychis and H. hepaticus) for putative NikR-regulated promoters. One of these novel putative NikR-regulated promoters in H. mustelae is located upstream of a putative TonB-dependent outer membrane protein designated NikH, which displayed nickel-responsive expression. Insertional inactivation of the nikH gene in H. mustelae resulted in a significant decrease in urease activity, and this phenotype was complemented by nickel-supplementation of the growth medium, suggesting a function for NikH in nickel transport accross the outer membrane. In conclusion, the H. mustelae NikR regulator directly controls nickel-responsive regulation of ureases and metal transporters. The improved consensus NikR operator sequence allows the prediction of additional NikR targets in Helicobacter genomes, as demonstrated by the identification of a new nickel-repressed outer membrane protein in H. mustelae.
Reuter M, Mallett A, Pearson BM, van Vliet AH (2010) Biofilm formation by Campylobacter jejuni is increased under aerobic conditions, Applied and Environmental Microbiology 76 (7) pp. 2122-2128 American Society for Microbiology
The microaerophilic human pathogen Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that C. jejuni NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O2) than under microaerobic conditions (5% O2, 10% CO2), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in C. jejuni. Moreover, preformed biofilms shed high numbers of viable C. jejuni cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions, C. jejuni adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of C. jejuni.
Stoof J, Breijer S, Pot RG, van der Neut D, Kuipers EJ, Kusters JG, van Vliet AH (2008) Inverse nickel-responsive regulation of two urease enzymes in the gastric pathogen Helicobacter mustelae, Environmental Microbiology 10 (10) pp. 2586-2597 Wiley
The acidic gastric environment of mammals can be chronically colonized by pathogenic Helicobacter species, which use the nickel-dependent urea-degrading enzyme urease to confer acid resistance. Nickel availability in the mammal host is low, being mostly restricted to vegetarian dietary sources, and thus Helicobacter species colonizing carnivores may be subjected to episodes of nickel deficiency and associated acid sensitivity. The aim of this study was to investigate how these Helicobacter species have adapted to the nickel-restricted diet of their carnivorous host. Three carnivore-colonizing Helicobacter species express a second functional urea-degrading urease enzyme (UreA2B2), which functions as adaptation to nickel deficiency. UreA2B2 was not detected in seven other Helicobacter species, and is in Helicobacter mustelae only expressed in nickel-restricted conditions, and its expression was higher in iron-rich conditions. In contrast to the standard urease UreAB, UreA2B2 does not require activation by urease or hydrogenase accessory proteins, which mediate nickel incorporation into these enzymes. Activity of either UreAB or UreA2B2 urease allowed survival of a severe acid shock in the presence of urea, demonstrating a functional role for UreA2B2 in acid resistance. Pathogens often express colonization factors which are adapted to their host. The UreA2B2 urease could represent an example of pathogen adaptation to the specifics of the diet of their carnivorous host, rather than to the host itself.
van Vliet AH, Ernst FD, Kusters JG (2004) NikR-mediated regulation of Helicobacter pylori acid adaptation, Trends in Microbiology 12 (11) pp. 489-494 Elsevier
Nickel is the cofactor of the Helicobacter pylori urease
enzyme, a factor essential for the chronic colonization of
the acidic hostile environment in the human stomach.
The NikR regulatory protein directly controls urease
expression and regulates the uptake of nickel, and is also
able to regulate the expression of other regulatory
proteins including the iron-responsive regulator Fur.
Through regulatory crosstalk and overlapping regulons,
the NikR protein controls the expression of many
systems important for colonization and acid adaptation.
Despite the paucity of regulatory proteins, this enables
H. pylori to optimally adapt to conditions in the
stomach, making it one of the most successful human
Handley RA, Mulholland F, Reuter M, Ramachandran VK, Musk H, Clissold L, Le Brun NE, van Vliet AH (2015) PerR controls oxidative stress defence and aerotolerance but not motility-associated phenotypes of Campylobacter jejuni, Microbiology 161 (7) pp. 1524-1536 Microbiology Society
The foodborne bacterial pathogen Campylobacter jejuni is an obligate microaerophile that is exposed to atmospheric oxygen during transmission through the food chain. Survival under aerobic conditions requires the concerted control of oxidative stress systems, which in C. jejuni are intimately connected with iron metabolism via the PerR and Fur regulatory proteins. Here, we have characterized the roles of C. jejuni PerR in oxidative stress and motility phenotypes, and its regulon at the level of transcription, protein expression and promoter interactions. Insertional inactivation of perR in the C. jejuni reference strains NCTC 11168, 81-176 and 81116 did not result in any growth deficiencies, but strongly increased survival in atmospheric oxygen conditions, and allowed growth around filter discs infused with up to 30 % H2O2 (8.8 M). Expression of catalase, alkyl hydroperoxide reductase, thioredoxin reductase and the Rrc desulforubrerythrin was increased in the perR mutant, and this was mediated at the transcriptional level as shown by electrophoretic mobility shift assays of the katA, ahpC and trxB promoters using purified PerR. Differential RNA-sequencing analysis of a fur perR mutant allowed the identification of eight previously unknown transcription start sites of genes controlled by Fur and/or PerR. Finally, inactivation of perR in C. jejuni did not result in reduced motility, and did not reduce killing of Galleria melonella wax moth larvae. In conclusion, PerR plays an important role in controlling oxidative stress resistance and aerobic survival of C. jejuni, but this role does not extend into control of motility and associated phenotypes.
Butcher J, Handley RA, van Vliet AH, Stintzi A (2015) Refined analysis of the Campylobacter jejuni iron-dependent/independent Fur- and PerR-transcriptomes, BMC Genomics 16 498 BioMed Central
The genome of Campylobacter jejuni contains two iron activated Fur-family transcriptional regulators, CjFur and CjPerR, which are primarily responsible for regulating iron homeostasis and oxidative stress respectively. Both transcriptional regulators have been previously implicated in regulating diverse functions beyond their primary roles in C. jejuni. To further characterize their regulatory networks, RNA-seq was used to define the transcriptional profiles of C. jejuni NCTC11168 wild type, fur, perR and furperR isogenic deletion mutants under both iron-replete and iron-limited conditions. Results
It was found that 202 genes were differentially expressed in at least one mutant under iron-replete conditions and 331 genes were differentially expressed in at least one mutant under iron-limited conditions. The CjFur and CjPerR transcriptomes characterized in this study were compared to those previously identified using microarray profiling and found to be more extensive than previously understood. Interestingly, our results indicate that CjFur/CjPerR appear to co-regulate the expression of flagellar biogenesis genes in an opposing and iron-independent fashion. Moreover the furperR isogenic deletion mutant revealed that CjFur and CjPerR can compensate for each other in certain cases, suggesting that both regulators may compete for binding to specific promoters. Conclusions
The CjFur and CjPerR transcriptomes are larger than previously reported. In particular, deletion of perR results in the differential expression of a large group of genes in the absence of iron, suggesting that CjPerR may also regulate genes in an iron-independent manner, similar to what has already been demonstrated with CjFur. Moreover, subsets of genes were found which are only differentially expressed when both CjFur and CjPerR are deleted and includes genes that appear to be simultaneously activated by CjFur and repressed by CjPerR. In particular the iron-independent co-regulation of flagellar biogenesis by CjFur/CjPerR represents a potentially novel regulatory function for these proteins. These findings represent additional modes of co-regulation by these two transcriptional regulators in C. jejuni.
Brown HL, Hanman K, Reuter M, Betts RP, van Vliet AH (2015) Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment, Frontiers in Microbiology 6 699 Frontiers Media
Biofilms make an important contribution to survival and transmission of bacterial pathogens in the food chain. The human pathogen Campylobacter jejuni is known to form biofilms in vitro in food chain-relevant conditions, but the exact roles and composition of the extracellular matrix are still not clear. Extracellular DNA has been found in many bacterial biofilms and can be a major component of the extracellular matrix. Here we show that extracellular DNA is also an important component of the C. jejuni biofilm when attached to stainless steel surfaces, in aerobic conditions and on conditioned surfaces. Degradation of extracellular DNA by exogenous addition of DNase I led to rapid biofilm removal, without loss of C. jejuni viability. Following treatment of a surface with DNase I, C. jejuni was unable to re-establish a biofilm population within 48 h. Similar results were obtained by digesting extracellular DNA with restriction enzymes, suggesting the need for high molecular weight DNA. Addition of C. jejuni genomic DNA containing an antibiotic resistance marker resulted in transfer of the antibiotic resistance marker to susceptible cells in the biofilm, presumably by natural transformation. Taken together, this suggest that eDNA is not only an important component of C. jejuni biofilms and subsequent food chain survival of C. jejuni, but may also contribute to the spread of antimicrobial resistance in C. jejuni. The degradation of extracellular DNA with enzymes such as DNase I is a rapid method to remove C. jejuni biofilms, and is likely to potentiate the activity of antimicrobial treatments and thus synergistically aid disinfection treatments.
Shaw FL, Mulholland F, Le Gall G, Porcelli I, Hart DJ, Pearson BM, van Vliet AH (2012) Selenium-dependent biogenesis of formate dehydrogenase in Campylobacter jejuni is controlled by the fdhTU accessory genes, Journal of Bacteriology 194 (15) pp. 3814-3823 American Society for Microbiology
The food-borne bacterial pathogen Campylobacter jejuni efficiently utilizes organic acids such as lactate and formate for energy production. Formate is rapidly metabolized via the activity of the multisubunit formate dehydrogenase (FDH) enzyme, of which the FdhA subunit is predicted to contain a selenocysteine (SeC) amino acid. In this study we investigated the function of the cj1500 and cj1501 genes of C. jejuni, demonstrate that they are involved in selenium-controlled production of FDH, and propose the names fdhT and fdhU, respectively. Insertional inactivation of fdhT or fdhU in C. jejuni resulted in the absence of FdhA and FdhB protein expression, reduced fdhABC RNA levels, the absence of FDH enzyme activity, and the lack of formate utilization, as assessed by 1H nuclear magnetic resonance. The fdhABC genes are transcribed from a single promoter located two genes upstream of fdhA, and the decrease in fdhABC RNA levels in the fdhU mutant is mediated at the posttranscriptional level. FDH activity and the ability to utilize formate were restored by genetic complementation with fdhU and by supplementation of the growth media with selenium dioxide. Disruption of SeC synthesis by inactivation of the selA and selB genes also resulted in the absence of FDH activity, which could not be restored by selenium supplementation. Comparative genomic analysis suggests a link between the presence of selA and fdhTU orthologs and the predicted presence of SeC in FdhA. The fdhTU genes encode accessory proteins required for FDH expression and activity in C. jejuni, possibly by contributing to acquisition or utilization of selenium.
Metris A, Reuter M, Gaskin DJ, Baranyi J, van Vliet AH (2011) In vivo and in silico determination of essential genes of Campylobacter jejuni, BMC Genomics 12 535 BioMed Central
In the United Kingdom, the thermophilic Campylobacter species C. jejuni and C. coli are the most frequent causes of food-borne gastroenteritis in humans. While campylobacteriosis is usually a relatively mild infection, it has a significant public health and economic impact, and possible complications include reactive arthritis and the autoimmune diseases Guillain-Barré syndrome. The rapid developments in "omics" technologies have resulted in the availability of diverse datasets allowing predictions of metabolism and physiology of pathogenic micro-organisms. When combined, these datasets may allow for the identification of potential weaknesses that can be used for development of new antimicrobials to reduce or eliminate C. jejuni and C. coli from the food chain. Results
A metabolic model of C. jejuni was constructed using the annotation of the NCTC 11168 genome sequence, a published model of the related bacterium Helicobacter pylori, and extensive literature mining. Using this model, we have used in silico Flux Balance Analysis (FBA) to determine key metabolic routes that are essential for generating energy and biomass, thus creating a list of genes potentially essential for growth under laboratory conditions. To complement this in silico approach, candidate essential genes have been determined using a whole genome transposon mutagenesis method. FBA and transposon mutagenesis (both this study and a published study) predict a similar number of essential genes (around 200). The analysis of the intersection between the three approaches highlights the shikimate pathway where genes are predicted to be essential by one or more method, and tend to be network hubs, based on a previously published Campylobacter protein-protein interaction network, and could therefore be targets for novel antimicrobial therapy.Conclusions
We have constructed the first curated metabolic model for the food-borne pathogen Campylobacter jejuni and have presented the resulting metabolic insights. We have shown that the combination of in silico and in vivo approaches could point to non-redundant, indispensable genes associated with the well characterised shikimate pathway, and also genes of unknown function specific to C. jejuni, which are all potential novel Campylobacter intervention targets.
Belzer C, Stoof J, van Vliet AH (2007) Metal-responsive gene regulation and metal transport in Helicobacter species, Biometals 20 pp. 417-429 Springer
Helicobacter species are among the most successful colonizers of the mammalian gastrointestinal and hepatobiliary tract. Colonization is usually lifelong, indicating that Helicobacter species have evolved intricate mechanisms of dealing with stresses encountered during colonization of host tissues, like restriction of essential metal ions. The recent availability of genome sequences of the human gastric pathogen Helicobacter pylori, the murine enterohepatic pathogen Helicobacter hepaticus and the unannotated genome sequence of the ferret gastric pathogen Helicobacter mustelae has allowed for comparitive genome analyses. In this review we present such analyses for metal transporters, metal-storage and metal-responsive regulators in these three Helicobacter species, and discuss possible contributions of the differences in metal metabolism in adaptation to the gastric or enterohepatic niches occupied by Helicobacter species.
Le MT, van Veldhuizen M, Porcelli I, Bongaerts RJ, Gaskin DJ, Pearson BM, van Vliet AH (2015) Conservation of sigma28-Dependent Non-Coding RNA Paralogs and Predicted sigma54-Dependent Targets in Thermophilic Campylobacter Species, PLoS One 10 (10) Public Library of Science (PLoS)
Assembly of flagella requires strict hierarchical and temporal control via flagellar sigma and anti-sigma factors, regulatory proteins and the assembly complex itself, but to date non-coding RNAs (ncRNAs) have not been described to regulate genes directly involved in flagellar assembly. In this study we have investigated the possible role of two ncRNA paralogs (CjNC1, CjNC4) in flagellar assembly and gene regulation of the diarrhoeal pathogen Campylobacter jejuni. CjNC1 and CjNC4 are 37/44 nt identical and predicted to target the 5' untranslated region (5' UTR) of genes transcribed from the flagellar sigma factor Ã54. Orthologs of the Ã54-dependent 5' UTRs and ncRNAs are present in the genomes of other thermophilic Campylobacter species, and transcription of CjNC1 and CNC4 is dependent on the flagellar sigma factor Ã28. Surprisingly, inactivation and overexpression of CjNC1 and CjNC4 did not affect growth, motility or flagella-associated phenotypes such as autoagglutination. However, CjNC1 and CjNC4 were able to mediate sequence-dependent, but Hfq-independent, partial repression of fluorescence of predicted target 5' UTRs in an Escherichia coli-based GFP reporter gene system. This hints towards a subtle role for the CjNC1 and CjNC4 ncRNAs in post-transcriptional gene regulation in thermophilic Campylobacter species, and suggests that the currently used phenotypic methodologies are insufficiently sensitive to detect such subtle phenotypes. The lack of a role of Hfq in the E. coli GFP-based system indicates that the CjNC1 and CjNC4 ncRNAs may mediate post-transcriptional gene regulation in ways that do not conform to the paradigms obtained from the Enterobacteriaceae.
Gerrits MM, van Vliet AH, Kuipers EJ, Kusters JG (2006) Helicobacter pylori and antimicrobial resistance: molecular mechanisms and clinical implications, Lancet Infectious Diseases 6 (11) pp. 699-709
Helicobacter pylori is an important human pathogen that colonises the stomach of about half of the world's population. The bacterium has now been accepted as the causative agent of several gastroduodenal disorders, ranging from chronic active gastritis and peptic ulcer disease to gastric cancer. The recognition of H pylori as a gastric pathogen has had a substantial effect on gastroenterological practice, since many untreatable gastroduodenal disorders with uncertain cause became curable infectious diseases. Treatment of H pylori infection results in ulcer healing and can reduce the risk of gastric cancer development. Although H pylori is susceptible to many antibiotics in vitro, only a few antibiotics can be used in vivo to cure the infection. The frequent indication for anti-H pylori therapy, together with the limited choice of antibiotics, has resulted in the development of antibiotic resistance in H pylori, which substantially impairs the treatment of H pylori-associated disorders. Antimicrobial resistance in H pylori is widespread, and although the prevalence of antimicrobial resistance shows regional variation per antibiotic, it can be as high as 95%. We focus on the treatment of H pylori infection and on the clinical relevance, mechanisms, and diagnosis of antimicrobial resistance.
Ernst FD, Homuth G, Stoof J, Mader U, Waidner B, Kuipers EJ, Kist M, Kusters JG, Bereswill S, van Vliet AH (2005) Iron-responsive regulation of the Helicobacter pylori iron-cofactored superoxide dismutase SodB is mediated by Fur., Journal of Bacteriology 187 (11) pp. 3687-3692 American Society for Microbiology
Maintaining iron homeostasis is a necessity for all living organisms, as free iron augments the generation of reactive oxygen species like superoxide anions, at the risk of subsequent lethal cellular damage. The iron-responsive regulator Fur controls iron metabolism in many bacteria, including the important human pathogen Helicobacter pylori, and thus is directly or indirectly involved in regulation of oxidative stress defense. Here we demonstrate that Fur is a direct regulator of the H. pylori iron-cofactored superoxide dismutase SodB, which is essential for the defense against toxic superoxide radicals. Transcription of the sodB gene was iron induced in H. pylori wild-type strain 26695, resulting in expression of the SodB protein in iron-replete conditions but an absence of expression in iron-restricted conditions. Mutation of the fur gene resulted in constitutive, iron-independent expression of SodB. Recombinant H. pylori Fur protein bound with low affinity to the sodB promoter region, but addition of the iron substitute Mn2+ abolished binding. The operator sequence of the iron-free form of Fur, as identified by DNase I footprinting, was located directly upstream of the sodB gene at positions ?5 to ?47 from the transcription start site. The direct role of Fur in regulation of the H. pylori sodB gene contrasts with the small-RNA-mediated sodB regulation observed in Escherichia coli. In conclusion, H. pylori Fur is a versatile regulator involved in many pathways essential for gastric colonization, including superoxide stress defense.
Moons LM, Kusters JG, Bultman E, Kuipers EJ, van Dekken H, Tra WM, Kleinjan A, Kwekkeboom J, van Vliet AH, Siersema PD (2005) Barrett's oesophagus is characterized by a predominantly humoral inflammatory response, Journal of Pathology 207 (3) pp. 269-276 Wiley
Barrett's oesophagus (BO) is thought to be an intermediate step in the progression from reflux oesophagitis (RO) to oesophageal adenocarcinoma. Premalignant conditions that develop in the presence of chronic inflammation are often associated with the development of a more pronounced humoral immune response during progression of the disease. The aim of this study was to determine whether BO is also associated with a more pronounced humoral immune response when compared to RO. Immunohistochemical studies were performed to quantify the mean numbers of Th2 effector cells (plasma cells and mast cells) and Th1 effector cells (macrophages and CD8+ T cells) to detect the antibody classes produced by plasma cells (IgA, IgG, IgM or IgE) and to determine the presence of isolated lymph follicles [segregated B and T cell areas, follicular dendritic cells (CD23) and expression of CXCL13] in 124 oesophageal biopsies from 20 patients with BO and 20 patients with RO. The proportion of Th2 effector cells was higher in BO than in RO, mainly due to higher numbers of plasma cells and mast cells in BO (p
van Vliet AH, Wren BW (2009) New levels of sophistication in the transcriptional landscape of bacteria, GENOME BIOLOGY 10 233 BioMed Central
An extra layer of complexity in the regulation of gene expression in bacteria is now apparent through previously unanticipated roles of noncoding and antisense RNAs. Bacteria are the great survivors on planet Earth, where they can adapt and flourish in harsh environments ranging from deep-sea vents to acidic mine shafts. A feature of many bacteria, particularly pathogenic bacteria, is their ability to adapt and thrive in multiple environments, which provides them with a competitive advantage. For example, the facultative intracellular pathogen Listeria monocytogenes happily survives in the ambient environment as a saprophyte, but on occasions it has an inherent capacity to turn nasty and cause brain and materno-fetal infections in humans . This requires the bacterium to switch genes on and off as it traverses different environments, ranging from a saprophytic lifestyle to the gut lumen after ingestion to invasion of epithelial cells and intracellular survival. The key to the survivalist success of pathogens is their ability to coordinate, redirect and fine-tune their genetic repertoire as and when required. Traditionally, transcriptional reshaping in bacteria has been considered to be controlled by a hierarchical network of interconnected global transcriptional regulators, such as sigma factors and one- and two-component regulatory systems . In the past decade it has become apparent that the various forms of noncoding regulatory RNA (previously considered as intergenic junk) play important roles in the global regulation of cellular functions, and may represent connecting links between many cellular networks [3, 4]. As such, noncoding RNA also plays a subtle but crucial role in the coordination of the expression of bacterial virulence determinants . Two recent papers from Pascale Cossart and colleagues [6, 7] present a comprehensive microarray analysis of the trans-criptome of Listeria monocytogenes in different conditions, uncovering an unsuspected variety of regulatory roles for noncoding RNAs in controlling changes in gene expression that characterize the transition from saprophytic to pathogenic lifestyle.
Gerrits MM, van der Wouden EJ, Bax DA, van Zwet AA, van Vliet AH, de Jong A, Kusters JG, Thijs JC, Kuipers EJ (2004) Role of the rdxA and frxA genes in oxygen-dependent metronidazole resistance of Helicobacter pylori, Journal of Medical Microbiology 53 pp. 1123-1128 Microbiology Society
Almost 50 % of all Helicobacter pylori isolates are resistant to metronidazole, which reduces the efficacy of metronidazole-containing regimens, but does not make them completely ineffective. This discrepancy between in vitro metronidazole resistance and treatment outcome may partially be explained by changes in oxygen pressure in the gastric environment, as metronidazole-resistant (MtzR) H. pylori isolates become metronidazole-susceptible (MtzS) under low oxygen conditions in vitro. In H. pylori the rdxA and frxA genes encode reductases which are required for the activation of metronidazole, and inactivation of these genes results in metronidazole resistance. Here the role of inactivating mutations in these genes on the reversibility of metronidazole resistance under low oxygen conditions is established. Clinical H. pylori isolates containing mutations resulting in a truncated RdxA and/or FrxA protein were selected and incubated under anaerobic conditions, and the effect of these conditions on the MICs of metronidazole, amoxycillin, clarithromycin and tetracycline, and cell viability were determined. While anaerobiosis had no effect on amoxycillin, clarithromycin and tetracycline resistance, all isolates lost their metronidazole resistance when cultured under anaerobic conditions. This loss of metronidazole resistance also occurred in the presence of the protein synthesis inhibitor chloramphenicol. Thus, factor(s) that activate metronidazole under low oxygen tension are not specifically induced by low oxygen conditions, but are already present under microaerophilic conditions. As there were no significant differences in cell viability between the clinical isolates, it is likely that neither the rdxA nor the frxA gene participates in the reversibility of metronidazole resistance.
Reuter M, van Vliet AH (2013) Signal balancing by the CetABC and CetZ chemoreceptors controls energy taxis in Campylobacter jejuni, PLoS One 8 e54390 Public Library of Science (PLoS)
The coupling of environmental sensing to flagella-mediated directed motility allows bacteria to move to optimum environments for growth and survival, either by sensing external stimuli (chemotaxis) or monitoring internal metabolic status (energy taxis). Sensing is mediated by transducer-like proteins (Tlp), either located in the membrane or in the cytoplasm, which commonly influence motility via the CheA-CheY chemotaxis pathway. In this study we have investigated the role of PAS-domain-containing intracellular Tlp-sensors in energy taxis of the food-borne pathogen Campylobacter jejuni, using plate- and tube-based assays utilising the conversion of the redox indicator dyes triphenyl tetrazolium chloride (TTC) and resazurin. Inactivation of the genes encoding the Campylobacter Energy Taxis system (CetA (Tlp9) and CetB (Aer2)) in C. jejuni strain NCTC 11168 resulted in reduced taxis. Inactivation of the cj1191c gene, encoding the CetB homolog CetC (Aer1), did not affect taxis per se, but the cetC gene complemented a cetB mutant in trans, indicating that CetC can form a functional signal transduction complex with CetA in the absence of CetB. Inactivation of both CetB and CetC resulted in greatly reduced taxis confirming the role of CetC in energy taxis. Inactivation of the cj1110c gene, encoding Tlp8 (CetZ), a cytoplasmic sensor with two PAS-domains, resulted in increased taxis, a phenotype opposite to that of CetAB. Inactivation of the cheA gene resulted in the same overall phenotype as the cetAB mutant in both wild-type and cetZ backgrounds, suggesting that both systems use the CheA system for signal transduction. Absence of both CetAB and CetZ resulted in the cetAB taxis phenotype, suggesting that CetZ is subordinate to CetAB. In conclusion, we present evidence that C. jejuni balances the input from two counteracting PAS-domain-containing sensory systems to position itself for optimal usage of energy resources.
van Vliet AH (2010) Next generation sequencing of microbial transcriptomes: challenges and opportunities, FEMS Microbiology Letters 302 pp. 1-7 Oxford University Press
Over the past 15 years, microbial functional genomics has been made possible by the combined power of genome sequencing and microarray technology. However, we are now approaching the technical limits of microarray technology, and microarrays are now being superseded by transcriptomics based on high-throughput (next generation) DNA-sequencing technologies. The term RNA-seq has been coined to represent transcriptomics by next-generation sequencing. Although pioneered on eukaryotic organisms due to the relative ease of working with eukaryotic mRNA, the RNA-seq technology is now being ported to microbial systems. This review will discuss the opportunities of RNA-seq transcriptome sequencing for microorganisms, and also aims to identify challenges and pitfalls of the use of this new technology in microorganisms
Belzer C, Kusters JG, Kuipers EJ, van Vliet AH (2006) Urease induced calcium precipitation by Helicobacter species may initiate gallstone formation, Gut 55 (11) pp. 1678-1679 BMJ Publishing Group
Helicobacter species can colonise the mammalian gastrointestinal and hepatobiliary tract which usually results in a chronic infection coupled to an inflammatory host response. It is therefore not surprising that colonisation with Helicobacter species is linked with a range of inflammation associated gastrointestinal and hepatobiliary diseases.1 Recently, this range has been expanded, with an association of infection with enterohepatic Helicobacter species and the formation of cholesterol gallstones.2 In their study, Maurer and colleagues2 demonstrated that murine infection with the enterohepatic Helicobacter species H bilis and H hepaticus accelerated the formation of cholesterol gallstones in mice fed a lithogenic diet. Although the gallbladder mucosa in mice with gallstones displayed signs of inflammation, Helicobacter species were not cultured from the inflamed gallbladder or bile. Therefore, Maurer et al hypothesised that the chronic immune stimulation caused by Helicobacter species, rather than a direct bacterial factor, led to the production of nucleating agents, thus indirectly linking &
Porcelli I, Reuter M, Pearson BM, Wilhelm T, van Vliet AH (2013) Parallel evolution of genome structure and transcriptional landscape in the Epsilonproteobacteria, BMC Genomics 14 616 BioMed Central
Gene reshuffling, point mutations and horizontal gene transfer contribute to bacterial genome variation, but require the genome to rewire its transcriptional circuitry to ensure that inserted, mutated or reshuffled genes are transcribed at appropriate levels. The genomes of Epsilonproteobacteria display very low synteny, due to high levels of reshuffling and reorganisation of gene order, but still share a significant number of gene orthologs allowing comparison. Here we present the primary transcriptome of the pathogenic Epsilonproteobacterium Campylobacter jejuni, and have used this for comparative and predictive transcriptomics in the Epsilonproteobacteria.
Differential RNA-sequencing using 454 sequencing technology was used to determine the primary transcriptome of C. jejuni NCTC 11168, which consists of 992 transcription start sites (TSS), which included 29 putative non-coding and stable RNAs, 266 intragenic (internal) TSS, and 206 antisense TSS. Several previously unknown features were identified in the C. jejuni transcriptional landscape, like leaderless mRNAs and potential leader peptides upstream of amino acid biosynthesis genes. A cross-species comparison of the primary transcriptomes of C. jejuni and the related Epsilonproteobacterium Helicobacter pylori highlighted a lack of conservation of operon organisation, position of intragenic and antisense promoters or leaderless mRNAs. Predictive comparisons using 40 other Epsilonproteobacterial genomes suggests that this lack of conservation of transcriptional features is common to all Epsilonproteobacterial genomes, and is associated with the absence of genome synteny in this subdivision of the Proteobacteria. Conclusions
Both the genomes and transcriptomes of Epsilonproteobacteria are highly variable, both at the genome level by combining and division of multicistronic operons, but also on the gene level by generation or deletion of promoter sequences and 52 untranslated regions. Regulatory features may have evolved after these species split from a common ancestor, with transcriptome rewiring compensating for changes introduced by genomic reshuffling and horizontal gene transfer.
Clostridium botulinum is a highly dangerous pathogen that forms very resistant endospores that are ubiquitous in the environment, and which, under favorable conditions germinate to produce vegetative cells that multiply and form the exceptionally potent botulinum neurotoxin. To improve the control of botulinum neurotoxin-forming clostridia, it is important to understand the mechanisms involved in spore germination. Here we present models for spore germination in C. botulinum based on comparative genomics analyses, with C. botulinum Groups I and III sharing similar pathways, which differ from those proposed for C. botulinum Groups II and IV. All spores germinate in response to amino acids interacting with a germinant receptor, with four types of germinant receptor identified [encoded by various combinations of gerA, gerB, and gerC genes (gerX)]. There are three gene clusters with an ABC-like configuration; ABC [gerX1], ABABCB [gerX2] and ACxBBB [gerX4], and a single CA-B [gerX3] gene cluster. Subtypes have been identified for most germinant receptor types, and the individual GerX subunits of each cluster show similar grouping in phylogenetic trees. C. botulinum Group I contained the largest variety of gerX subtypes, with three gerX1, three gerX2, and one gerX3 subtypes, while C. botulinum Group III contained two gerX1 types and one gerX4. C. botulinum Groups II and IV contained a single germinant receptor, gerX3 and gerX1, respectively. It is likely that all four C. botulinum Groups include a SpoVA channel involved in dipicolinic acid release. The cortex-lytic enzymes present in C. botulinum Groups I and III appear to be CwlJ and SleB, while in C. botulinum Groups II and IV, SleC appears to be important.
Kusters JG, van Vliet AH, Kuipers EJ (2006) Pathogenesis of Helicobacter pylori infection, Clinical Microbiology Reviews 19 (3) pp. 449-490 American Society for Microbiology
Helicobacter pylori is the first formally recognized bacterial carcinogen and is one of the most successful human pathogens, as over half of the world's population is colonized with this gram-negative bacterium. Unless treated, colonization usually persists lifelong. H. pylori infection represents a key factor in the etiology of various gastrointestinal diseases, ranging from chronic active gastritis without clinical symptoms to peptic ulceration, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Disease outcome is the result of the complex interplay between the host and the bacterium. Host immune gene polymorphisms and gastric acid secretion largely determine the bacterium's ability to colonize a specific gastric niche. Bacterial virulence factors such as the cytotoxin-associated gene pathogenicity island-encoded protein CagA and the vacuolating cytotoxin VacA aid in this colonization of the gastric mucosa and subsequently seem to modulate the host's immune system. This review focuses on the microbiological, clinical, immunological, and biochemical aspects of the pathogenesis of H. pylori.
van Vliet AH, Kusters JG (2015) Use of Alignment-Free Phylogenetics for Rapid Genome Sequence-Based Typing of Helicobacter pylori Virulence Markers and Antibiotic Susceptibility, Journal of Clinical Microbiology 53 (9) pp. 2877-2888 American Society for Microbiology
Whole-genome sequencing is becoming a leading technology in the typing and epidemiology of microbial pathogens, but the increase in genomic information necessitates significant investment in bioinformatic resources and expertise, and currently used methodologies struggle with genetically heterogeneous bacteria such as the human gastric pathogen Helicobacter pylori. Here we demonstrate that the alignment-free analysis method feature frequency profiling (FFP) can be used to rapidly construct phylogenetic trees of draft bacterial genome sequences on a standard desktop computer and that coupling with in silico genotyping methods gives useful information for comparative and clinical genomic and molecular epidemiology applications. FFP-based phylogenetic trees of seven gastric Helicobacter species matched those obtained by analysis of 16S rRNA genes and ribosomal proteins, and FFP- and core genome single nucleotide polymorphism-based analysis of 63 H. pylori genomes again showed comparable phylogenetic clustering, consistent with genomotypes assigned by using multilocus sequence typing (MLST). Analysis of 377 H. pylori genomes highlighted the conservation of genomotypes and linkage with phylogeographic characteristics and predicted the presence of an incomplete or nonfunctional cag pathogenicity island in 18/276 genomes. In silico analysis of antibiotic susceptibility markers suggests that most H. pylori hspAmerind and hspEAsia isolates are predicted to carry the T2812C mutation potentially conferring low-level clarithromycin resistance, while levels of metronidazole resistance were similar in all multilocus sequence types. In conclusion, the use of FFP phylogenetic clustering and in silico genotyping allows determination of genome evolution and phylogeographic clustering and can contribute to clinical microbiology by genomotyping for outbreak management and the prediction of pathogenic potential and antibiotic susceptibility.
Contamination of mussels with the human pathogen Listeria monocytogenes occurs during processing in the factory, possibly from bacteria persisting in the factory's indoor and outdoor areas. In this study, a selection of persistent (n = 8) and sporadic (n = 8) L. monocytogenes isolates associated with mussel-processing premises in New Zealand were investigated for their phenotypic and genomic characteristics. To identify traits that favour or contribute to bacterial persistence, biofilm formation, heat resistance, motility and recovery from dry surfaces were compared between persistent and sporadic isolates. All isolates exhibited low biofilm formation at 20 °C, however, at 30 °C persistent isolates showed significantly higher biofilm formation after 48 h using cell enumeration and near significant difference using the crystal violet assay. All 16 isolates were motile at 20 °C and 30 °C and motility was fractionally higher for sporadic isolates, but no significant difference was observed. We found persistent isolates tend to exhibit greater recovery after incubation on dry surfaces compared to sporadic isolates. Two of the three most heat-resistant isolates were persistent, while four of five isolates lacking heat resistance were sporadic isolates. Comparison of genome sequences of persistent and sporadic isolates showed that there was no overall clustering of persistent or sporadic isolates, and that differences in prophages and plasmids were not associated with persistence. Our results suggest a link between persistence and biofilm formation, which is most likely multifactorial, combining subtle phenotypic and genotypic differences between isolates.
Brown HL, Reuter M, Hanman K, Betts RP, van Vliet AH (2015) Prevention of biofilm formation and removal of existing biofilms by extracellular DNases of Campylobacter jejuni, PLoS One 10 e0121680 Public Library of Science (PLoS)
The fastidious nature of the foodborne bacterial pathogen Campylobacter jejuni contrasts with its ability to survive in the food chain. The formation of biofilms, or the integration into existing biofilms by C. jejuni, is thought to contribute to food chain survival. As extracellular DNA (eDNA) has previously been proposed to play a role in C. jejuni biofilms, we have investigated the role of extracellular DNases (eDNases) produced by C. jejuni in biofilm formation. A search of 2791 C. jejuni genomes highlighted that almost half of C. jejuni genomes contains at least one eDNase gene, but only a minority of isolates contains two or three of these eDNase genes, such as C. jejuni strain RM1221 which contains the cje0256, cje0566 and cje1441 eDNase genes. Strain RM1221 did not form biofilms, whereas the eDNase-negative strains NCTC 11168 and 81116 did. Incubation of pre-formed biofilms of NCTC 11168 with live C. jejuni RM1221 or with spent medium from a RM1221 culture resulted in removal of the biofilm. Inactivation of the cje1441 eDNase gene in strain RM1221 restored biofilm formation, and made the mutant unable to degrade biofilms of strain NCTC 11168. Finally, C. jejuni strain RM1221 was able to degrade genomic DNA from C. jejuni NCTC 11168, 81116 and RM1221, whereas strain NCTC 11168 and the RM1221 cje1441 mutant were unable to do so. This was mirrored by an absence of eDNA in overnight cultures of C. jejuni RM1221. This suggests that the activity of eDNases in C. jejuni affects biofilm formation and is not conducive to a biofilm lifestyle. These eDNases do however have a potential role in controlling biofilm formation by C. jejuni strains in food chain relevant environments.
Parkhill J, Wren BW, Mungall K, Ketley JM, Churcher C, Basham D, Chillingworth T, Davies RM, Feltwell T, Holroyd S, Jagels K, Karlyshev AV, Moule S, Pallen MJ, Penn CW, Quail MA, Rajandream MA, Rutherford KM, van Vliet AH, Whitehead S, Barrell BG (2000) The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences, Nature 403 pp. 665-668 Nature Publishing Group
Campylobacter jejuni, from the delta-epsilon group of proteobacteria, is a microaerophilic, Gram-negative, flagellate, spiral bacterium?properties it shares with the related gastric pathogen Helicobacter pylori. It is the leading cause of bacterial food-borne diarrhoeal disease throughout the world1. In addition, infection with C. jejuni is the most frequent antecedent to a form of neuromuscular paralysis known as Guillain?Barré syndrome2. Here we report the genome sequence of C. jejuni NCTC11168. C. jejuni has a circular chromosome of 1,641,481 base pairs (30.6% G+C) which is predicted to encode 1,654 proteins and 54 stable RNA species. The genome is unusual in that there are virtually no insertion sequences or phage-associated sequences and very few repeat sequences. One of the most striking findings in the genome was the presence of hypervariable sequences. These short homopolymeric runs of nucleotides were commonly found in genes encoding the biosynthesis or modification of surface structures, or in closely linked genes of unknown function. The apparently high rate of variation of these homopolymeric tracts may be important in the survival strategy of C. jejuni.
The human bacterial pathogen Helicobacter pylori has a highly variable genome, with significant allelic and sequence diversity between isolates and even within well-characterised strains, hampering comparative genomics of H. pylori. In this study, pan-genome analysis has been used to identify lineage-specific genes of H. pylori. A total of 346 H. pylori genomes spanning the hpAfrica1, hpAfrica2, hpAsia2, hpEurope, hspAmerind and hspEAsia multilocus sequence typing (MLST) lineages were searched for genes specifically over- or underrepresented in MLST lineages or associated with the cag pathogenicity island (PAI). The only genes overrepresented in cagpositive genomes were the cag PAI genes themselves. In contrast, a total of 125 genes were either overrepresented or underrepresented in one or more MLST-lineages. Of these 125 genes, alcohol/aldehyde-reducing enzymes linked with acid-resistance and production of toxic aldehydes were found to be overrepresented in African lineages. Conversely, the FecA2 ferric citrate receptor was missing from hspAmerind genomes, but present in all other lineages. This work shows the applicability of pan-genome analysis for identification of lineage-specific genes of H. pylori, facilitating further investigation to allow linkage of differential distribution of genes with disease outcome or virulence, and can be used with other microbial pathogens with highly variable genomes.
Campylobacter jejuni is the leading cause of bacterial foodborne diarrhoeal disease worldwide. Despite the microaerophilic nature of the bacterium, C. jejuni can survive the atmospheric oxygen conditions in the environment. Bacteria that can survive either within a host or in the environment like C. jejuni require variable responses to survive the stresses associated with exposure to different levels of reactive oxygen species. The MarR-type transcriptional regulators RrpA and RrpB have recently been shown to play a role in controlling both the C. jejuni oxidative and aerobic stress responses. Analysis of 3,746 Campylobacter jejuni and 486 Campylobacter coli genome sequences showed that whilst rrpA is present in over 99% of C. jejuni strains, the presence of rrpB is restricted and appears to correlate with specific MLST clonal complexes (predominantly ST-21 and ST-61). C. coli strains in contrast lack both rrpA and rrpB. In C. jejuni rrpB+ strains, the rrpB gene is located within a variable genomic region containing the IF subtype of the type I Restriction-Modification (hsd) system, whilst this variable genomic region in C. jejuni rrpB- strains contains the IAB subtype hsd system and not the rrpB gene. C. jejuni rrpB- strains exhibit greater resistance to peroxide and aerobic stress than C. jejuni rrpB+ strains. Inactivation of rrpA resulted in increased sensitivity to peroxide stress in rrpB+ strains, but not in rrpB- strains. Mutation of rrpA resulted in reduced killing of Galleria mellonella larvae and enhanced biofilm formation independent of rrpB status. The oxidative and aerobic stress responses of rrpB- and rrpB+ strains suggest adaptation of C. jejuni within different hosts and niches that can be linked to specific MLST clonal complexes.
Draper J, Hansen L, Bernick D, Abedrabbo S, Underwood J, Kong N, Huang B, Weis A, Welmer B, van Vliet A, Pourmand N, Solnick J, Karplus K, Ottemann K (2017) Fallacy of the unique genome: sequence diversity within single Helicobacter pylori strains, mBio 8 (1) e02321-16
American Society for Microbiology
Many bacterial genomes are highly variable but nonetheless are typically published as a single assembled genome. Experiments tracking bacterial genome evolution have not looked at the variation present at a given point in time. Here, we analyzed the mouse-passaged Helicobacter pylori strain SS1 and its parent PMSS1 to assess intra- and intergenomic variability. Using high sequence coverage depth and experimental validation, we detected extensive genome plasticity within these H. pylori isolates, including movement of the transposable element IS607, large and small inversions, multiple single nucleotide polymorphisms, and variation in cagA copy number. The cagA gene was found as 1 to 4 tandem copies located off the cag island in both SS1 and PMSS1; this copy number variation correlated with protein expression. To gain insight into the changes that occurred during mouse adaptation, we also compared SS1 and PMSS1 and observed 46 differences that were distinct from the within-genome variation. The most substantial was an insertion in cagY, which encodes a protein required for a type IV secretion system function. We detected modi?cations in genes coding for two proteins known to affect mouse colonization, the HpaA neuraminyllactose-binding protein and the FutB -1,3 lipopolysaccharide (LPS) fucosyltransferase, as well as genes predicted to modulate diverse properties. In sum, our work suggests that data from consensus genome assemblies from single colonies may be misleading by failing to represent the variability present. Furthermore, we show that high-depth genomic sequencing data of a population can be analyzed to gain insight into the normal variation within bacterial strains.
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.
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 ²-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.
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.