Campylobacter jejuni Virulence Factors Identified by Modulating Their Synthesis on Ribosomes With Altered rRNA Methylation

Campylobacter jejuni is a major cause of food poisoning worldwide, and remains the main infective agent in gastroenteritis and related intestinal disorders in Europe and the USA. As with all bacterial infections, the stages of adhesion to host tissue, survival in the host and eliciting disease all require the synthesis of proteinaceous virulence factors on the ribosomes of the pathogen. Here, we describe how C. jejuni virulence is attenuated by altering the methylation of its ribosomes to disrupt the composition of its proteome, and how this in turn provides a means of identifying factors that are essential for infection and pathogenesis. Specifically, inactivation of the C. jejuni Cj0588/TlyA methyltransferase prevents methylation of nucleotide C1920 in the 23S rRNA of its ribosomes and reduces the pathogen’s ability to form biofilms, to attach, invade and survive in host cells, and to provoke the innate immune response. Mass spectrometric analyses of C. jejuni TlyA-minus strains revealed an array of subtle changes in the proteome composition. These included reduced amounts of the cytolethal distending toxin (CdtC) and the MlaEFD proteins connected with outer membrane vesicle (OMV) production. Inactivation of the cdtC and mlaEFD genes confirmed the importance of their encoded proteins in establishing infection. Collectively, the data identify a subset of genes required for the onset of human campylobacteriosis, and serve as a proof of principle for use of this approach in detecting proteins involved in bacterial pathogenesis.

Comparative Analysis of New Zealand Campylobacter Isolates Using MLST, PFGE and flaA PCR RFLP Genotyping

<p>Campylobacter jejuni and Campylobacter coli are the most commonly identified sources of campylobacteriosis in New Zealand, yet little is known about the distribution of genotypes within the respective population structures. Using multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and flaA genotyping, the current study identified the distribution of genotypes within New Zealand C. jejuni and C. coli isolates from an outbreak situation, as well as isolates present in the ESR Campylobacter collection. Although the most commonly identified MLST genotypes were similar to international genotypes, a number of internationally rare, or unique to New Zealand genotypes were observed. One rare dominant genotype, ST-474, arising from a point source outbreak, was found to cause a large proportion of human campylobacteriosis cases in New Zealand. A unique cluster of New Zealand genotypes were isolated only from river water, identifying a potentially water adapted C. jejuni strain. Frequent homologous recombination and horizontal gene transfer events were identified within the seven housekeeping genes characterised in the New Zealand sample and the MLST C. jejuni/C. coli database. The identified genetic instability within the current study questions the legitimacy of bacterial species boundaries, especially when examining closely related species such as C. jejuni and C. coli.</p>

Comparative Analysis of New Zealand Campylobacter Isolates Using MLST, PFGE and flaA PCR RFLP Genotyping

<p>Campylobacter jejuni and Campylobacter coli are the most commonly identified sources of campylobacteriosis in New Zealand, yet little is known about the distribution of genotypes within the respective population structures. Using multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and flaA genotyping, the current study identified the distribution of genotypes within New Zealand C. jejuni and C. coli isolates from an outbreak situation, as well as isolates present in the ESR Campylobacter collection. Although the most commonly identified MLST genotypes were similar to international genotypes, a number of internationally rare, or unique to New Zealand genotypes were observed. One rare dominant genotype, ST-474, arising from a point source outbreak, was found to cause a large proportion of human campylobacteriosis cases in New Zealand. A unique cluster of New Zealand genotypes were isolated only from river water, identifying a potentially water adapted C. jejuni strain. Frequent homologous recombination and horizontal gene transfer events were identified within the seven housekeeping genes characterised in the New Zealand sample and the MLST C. jejuni/C. coli database. The identified genetic instability within the current study questions the legitimacy of bacterial species boundaries, especially when examining closely related species such as C. jejuni and C. coli.</p>

Impact of the gut microecology on Campylobacter presence revealed by comparisons of the gut microbiota from chickens raised on litter or in individual cages

Background Poultry is the major reservoir of Campylobacter that contributes to human campylobacteriosis and threatens food safety. Litter contact has been linked to Campylobacter colonization, but the gut microecological impact underlying this link remains not fully clear. Here, we sought to investigate the impact of the gut microecology on the presence of Campylobacter by examining the microbiota in the duodenum, jejunum, ileum, ceca, and feces from chickens raised on commercial litter and in individual cages at 0–57 days of age. Results Through litter contact, the presence of Campylobacter was found to benefit from microecological competition among Lactobacillus, Helicobacter, and genera that are halotolerant and aerobic or facultatively anaerobic in the upper intestine, such as Corynebacterium and Brachybacterium. The presence was also promoted by the increased abundance in obligate anaerobic fermentation microbes, especially members of the orders Clostridiales and Bacteroidales. The longitudinal analysis supported the vertical or pseudo-vertical transmission but suggested that colonization might occur immensely at 7–28 days of age. We observed a host genetic effect on the gut microecology, which might lead to increased heterogeneity of the microecological impact on Campylobacter colonization. Conclusions The findings advance the understanding of the gut microecological impact on Campylobacter presence in the chicken gut under conditions of litter contact and suggest that manipulations of the gut microecology, as well as the microbes identified in the Campylobacter association networks, might be important for the development of intervention strategies.

Immune-Modulatory Effects upon Oral Application of Cumin-Essential-Oil to Mice Suffering from Acute Campylobacteriosis

Human campylobacteriosis, commonly caused by Campylobacter jejuni, is a food-borne infection with rising prevalence causing significant health and socioeconomic burdens worldwide. Given the threat from emerging antimicrobial resistances, the treatment of infectious diseases with antibiotics-independent natural compounds is utmost appreciated. Since the health-beneficial effects of cumin-essential-oil (EO) have been known for centuries, its potential anti-pathogenic and immune-modulatory effects during acute experimental campylobacteriosis were addressed in the present study. Therefore, C. jejuni-challenged secondary abiotic IL-10-/- mice were treated perorally with either cumin-EO or placebo starting on day 2 post-infection. On day 6 post-infection, cumin-EO treated mice harbored lower ileal pathogen numbers and exhibited a better clinical outcome when compared to placebo controls. Furthermore, cumin-EO treatment alleviated enteropathogen-induced apoptotic cell responses in colonic epithelia. Whereas, on day 6 post-infection, a dampened secretion of pro-inflammatory mediators, including nitric oxide and IFN-γ to basal levels, could be assessed in mesenteric lymph nodes of cumin-EO treated mice, systemic MCP-1 concentrations were elevated in placebo counterparts only. In conclusion, our preclinical intervention study provides first evidence for promising immune-modulatory effects of cumin-EO in the combat of human campylobacteriosis. Future studies should address antimicrobial and immune-modulatory effects of natural compounds as adjunct antibiotics-independent treatment option for infectious diseases.

Survey of Pathogen-Lowering and Immuno-Modulatory Effects Upon Treatment of Campylobacter coli-Infected Secondary Abiotic IL-10-/- Mice with the Probiotic Formulation Aviguard®

The prevalence of infections with the zoonotic enteritis pathogen Campylobacter coli is increasing. Probiotic formulations constitute promising antibiotic-independent approaches to reduce intestinal pathogen loads and modulate pathogen-induced immune responses in the infected human host, resulting in acute campylobacteriosis and post-infectious sequelae. Here, we address potential antipathogenic and immuno-modulatory effects of the commercial product Aviguard® during experimental campylobacteriosis. Secondary abiotic IL-10-/- mice were infected with a C. coli patient isolate on days 0 and 1, followed by oral Aviguard® treatment on days 2, 3 and 4. Until day 6 post-infection, Aviguard® treatment could lower the pathogen burdens within the proximal but not the distal intestinal tract. In contrast, the probiotic bacteria had sufficiently established in the intestines with lower fecal loads of obligate anaerobic species in C. coli-infected as compared to uninfected mice following Aviguard® treatment. Aviguard® application did not result in alleviated clinical signs, histopathological or apoptotic changes in the colon of infected IL-10-/- mice, whereas, however, Aviguard® treatment could dampen pathogen-induced innate and adaptive immune responses in the colon, accompanied by less distinct intestinal proinflammatory cytokine secretion. In conclusion, Aviguard® constitutes a promising probiotic compound to alleviate enteropathogen-induced proinflammatory immune responses during human campylobacteriosis.

Antimicrobial Resistance, FlaA Sequencing, and Phylogenetic Analysis of Campylobacter Isolates from Broiler Chicken Flocks in Greece

Human campylobacteriosis caused by thermophilic Campylobacter species is the most commonly reported foodborne zoonosis. Consumption of contaminated poultry meat is regarded as the main source of human infection. This study was undertaken to determine the antimicrobial susceptibility and the molecular epidemiology of 205 Campylobacter isolates derived from Greek flocks slaughtered in three different slaughterhouses over a 14-month period. A total of 98.5% of the isolates were resistant to at least one antimicrobial agent. In terms of multidrug resistance, 11.7% of isolates were resistant to three or more groups of antimicrobials. Extremely high resistance to fluoroquinolones (89%), very high resistance to tetracycline (69%), and low resistance to macrolides (7%) were detected. FlaA sequencing was performed for the subtyping of 64 C. jejuni and 58 C. coli isolates. No prevalence of a specific flaA type was observed, indicating the genetic diversity of the isolates, while some flaA types were found to share similar antimicrobial resistance patterns. Phylogenetic trees were constructed using the neighbor-joining method. Seven clusters of the C. jejuni phylogenetic tree and three clusters of the C. coli tree were considered significant with bootstrap values >75%. Some isolates clustered together were originated from the same or adjacent farms, indicating transmission via personnel or shared equipment. These results are important and help further the understanding of the molecular epidemiology and antimicrobial resistance of Campylobacter spp. derived from poultry in Greece.

Preclinical Evaluation of Oral Urolithin-A for the Treatment of Acute Campylobacteriosis in Campylobacter jejuni Infected Microbiota-Depleted IL-10−/− Mice

Human campylobacteriosis represents an infectious enteritis syndrome caused by Campylobacter species, mostly Campylobacter jejuni. Given that C. jejuni infections are rising worldwide and antibiotic treatment is usually not indicated, novel treatment options for campylobacteriosis are needed. Urolithin-A constitutes a metabolite produced by the human gut microbiota from ellagitannins and ellagic acids in berries and nuts which have been known for their health-beneficial including anti-inflammatory effects since centuries. Therefore, we investigated potential pathogen-lowering and immunomodulatory effects following oral application of synthetic urolithin-A during acute campylobacteriosis applying perorally C. jejuni infected, microbiota-depleted IL-10−/− mice as preclinical inflammation model. On day 6 post infection, urolithin-A treated mice harbored slightly lower pathogen loads in their ileum, but not colon as compared to placebo counterparts. Importantly, urolithin-A treatment resulted in an improved clinical outcome and less pronounced macroscopic and microscopic inflammatory sequelae of infection that were paralleled by less pronounced intestinal pro-inflammatory immune responses which could even be observed systemically. In conclusion, this preclinical murine intervention study provides first evidence that oral urolithin-A application is a promising treatment option for acute C. jejuni infection and paves the way for future clinical studies in human campylobacteriosis.