Campylobacter jejuni genes Cj1492c and Cj1507c are involved in host cell adhesion and invasion

Background Campylobacter jejuni (C. jejuni) has been assigned as an important food-borne pathogen for human health but many pathogenicity factors of C. jejuni and human host cell responses related to the infection have not yet been adequately clarified. This study aimed to determine further C. jejuni pathogenicity factors and virulence genes based on a random mutagenesis approach. A transposon mutant library of C. jejuni NCTC 11168 was constructed and the ability of individual mutants to adhere to and invade human intestinal epithelial cells was evaluated compared to the wild type. We identified two mutants of C. jejuni possessing altered phenotypes with transposon insertions in the genes Cj1492c and Cj1507c. Cj1492c is annotated as a two-component sensor and Cj1507c is described as a regulatory protein. However, functions of both mutated genes are not clarified so far. Results In comparison to the wild type, Cj::1492c and Cj::1507c showed around 70–80% relative motility and Cj::1492c had around 3-times enhanced adhesion and invasion rates whereas Cj::1507c had significantly impaired adhesive and invasive capability. Moreover, Cj::1492c had a longer lag phase and slower growth rate while Cj::1507c showed similar growth compared to the wild type. Between 5 and 24 h post infection, more than 60% of the intracellular wild type C. jejuni were eliminated in HT-29/B6 cells, however, significantly fewer mutants were able to survive intracellularly. Nevertheless, no difference in host cell viability and induction of the pro-inflammatory chemokine IL-8 were determined between both mutants and the wild type. Conclusion We conclude that genes regulated by Cj1507c have an impact on efficient adhesion, invasion and intracellular survival of C. jejuni in HT-29/B6 cells. Furthermore, potential signal sensing by Cj1492c seems to lead to limiting attachment and hence internalisation of C. jejuni. However, as the intracellular survival capacities are reduced, we suggest that signal sensing by Cj1492c impacts several processes related to pathogenicity of C. jejuni

Motility related gene expression of Campylobacter jejuni NCTC 11168 derived from high viscous media

Flagellation is one of the major virulence factors of Campylobacter jejuni (C. jejuni), enabling bacterial cells to swarm in rather high viscous fluids. The aim of this study was to determine the impact of the surrounding viscosity on the expression of motility related genes of C. jejuni. Therefore, bacterial RNA was extracted from liquid cultures as well as from bacterial cells recovered from the edge and the center of a swarming halo from high viscous media. The expression pattern of selected flagellar and chemotaxis related genes was investigated by RT-PCR. Higher mRNA levels of class 1 and lower levels of class 2 and 3 flagellar assembly genes were detected in cells derived from the edge of a swarming halo than in cells from the center. This indicates different growth states at both locations within the swarming halo. Furthermore, higher mRNA levels for energy taxis and motor complex monomer genes were detected in high viscous media compared to liquid culture, indicating higher demand of energy if C. jejuni cells were cultivated in high viscous media. The impact of the surrounding viscosity should be considered in future studies regarding motility related questions

The transcriptional response of Arcobacter butzleri to cold shock

Arcobacter (A.) butzleri is an emerging zoonotic pathogen associated with gastrointestinal diseases, such as abdominal cramps and diarrhea, and is widely detected in animals, showing a high prevalence in poultry and seafood. The survival and adaptation of A. butzleri to cold temperatures remains poorly studied, although it might be of interest for food safety considerations. To address this, growth patterns of eight A. butzleri isolates were determined at 8 °C for 28 days. A. butzleri isolates showed strain‐dependent behavior: six isolates were unculturable after day 18, one exhibited declining but detectable cell counts until day 28 and one grew to the stationary phase level. Out of 13 A. butzleri cold shock‐related genes homologous to Escherichia coli, 10 were up‐regulated in response to a temperature downshift to 8 °C, as demonstrated by reverse transcription‐quantitative PCR. Additionally, we compared these data with the cold‐shock response in E. coli. Overall, we provide a deeper insight into the environmental adaptation capacities of A. butzleri, which we find shares similarities with the E. coli cold‐shock response

Editorial: Campylobacter-associated food safety

Introduction: Campylobacteriosis is an enteric bacterial zoonotic infection caused by members of the Campylobacter genus (Kirkpatrick and Tribble, 2011). C. jejuni (> 85%) and C. coli (5–10%) are the most common species associated with the disease (Patrick et al., 2018). Ingestion of as few as 500 bacteria can cause campylobacteriosis (Robinson, 1981). Although Campylobacter typically causes self-limiting human gastroenteritis, it can lead to prolonged post-infectious complications, such as Guillain-Barré syndrome, reactive arthritis, and/or post infectious-irritable bowel syndrome (Rees et al., 1995). The treatment of campylobacteriosis poses significant economic burdens worldwide, resulting in $1.56 billion in healthcare costs in the USA,$80 million in Canada, and €2.4 billion in the European Union per year (Devleesschauwer et al., 2017). The high prevalence of Campylobacter in the agri-food system is likely a major contributing factor to the incidence of campylobacteriosis. Due to its microaerobic nature, Campylobacter can colonize the intestinal tract of food-producing animals such as poultry, cattle, sheep, and swine (Hansson et al., 2018). However, it can also survive under aerobic conditions and infect humans through the food supply chain by forming biofilms or entering the viable but non-culturable state (Lv et al., 2019; Ma et al., 2022). The main route of infection has been identified as the consumption of contaminated food commodities, such as unpasteurized dairy products, undercooked poultry meat and/or contaminated water (Silva et al., 2011). Therefore, detection, characterization, and reduction of Campylobacter in the agroecosystem are of great importance. This mini-review provides an overview of the current trends in understanding Campylobacter and its interaction with the agroecosystem. We first introduce the improved methods to detect Campylobacter in various agri-food settings. Then, the prevalence of this microbe in the agri-food system as well as its characteristics are summarized. Finally, novel control strategies of Campylobacter are summarized and discussed

Lessons from a Meta-Analysis of Murine Infection Studies

Background: Only limited information is available about the immunopathogenic properties of Arcobacter infection in vivo. Therefore, we performed a meta- analysis of published data in murine infection models to compare the pathogenic potential of Arcobacter butzleri with Campylobacter jejuni and commensal Escherichia coli as pathogenic and harmless reference bacteria, respectively. Methodology / Principal Findings: Gnotobiotic IL-10-/- mice generated by broad-spectrum antibiotic compounds were perorally infected with A. butzleri (strains CCUG 30485 or C1), C. jejuni (strain 81-176) or a commensal intestinal E. coli strain. Either strain stably colonized the murine intestines upon infection. At day 6 postinfection (p.i.), C. jejuni infected mice only displayed severe clinical sequelae such as wasting bloody diarrhea. Gross disease was accompanied by increased numbers of colonic apoptotic cells and distinct immune cell populations including macrophages and monocytes, T and B cells as well as regulatory T cells upon pathogenic infection. Whereas A. butzleri and E. coli infected mice were clinically unaffected, respective colonic immune cell numbers increased in the former, but not in the latter, and more distinctly upon A. butzleri strain CCUG 30485 as compared to C1 strain infection. Both, A. butzleri and C. jejuni induced increased secretion of pro-inflammatory cytokines such as IFN-γ, TNF, IL-6 and MCP-1 in large, but also small intestines. Remarkably, even though viable bacteria did not translocate from the intestines to extra-intestinal compartments, systemic immune responses were induced in C. jejuni, but also A. butzleri infected mice as indicated by increased respective pro-inflammatory cytokine concentrations in serum samples at day 6 p.i. Conclusion / Significance: A. butzleri induce less distinct pro-inflammatory sequelae as compared to C. jejuni, but more pronounced local and systemic immune responses than commensal E. coli in a strain-dependent manner. Hence, data point towards that A. butzleri is more than a commensal in vertebrate hosts

Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

C. jejuni is an important food-borne pathogen displaying high genetic diversity, substantially based on natural transformation. The mechanism of DNA uptake from the environment depends on a type II secretion/type IV pilus system, whose components are partially known. Here, we quantified DNA uptake in C. jejuni at the single cell level and observed median transport capacities of approximately 30 kb per uptake location. The process appeared to be limited by the initialization of DNA uptake, was finite, and, finalized within 30 min of contact to DNA. Mutants lacking either the outer membrane pore PilQ or the inner membrane channel ComEC were deficient in natural transformation. The periplasmic DNA binding protein ComE was negligible for DNA uptake, which is in contrast to its proposed function. Intriguingly, a mutant lacking the unique periplasmic protein Cj0683 displayed rare but fully functional DNA uptake events. We conclude that Cj0683 was essential for the efficient initialization of DNA uptake, consistent with the putative function as a competence pilus protein. Unravelling features important in natural transformation might lead to target identification, reducing the adaptive potential of pathogens

Control of Campylobacter spp. and Yersinia enterocolitica by virulent bacteriophages

The efficacy of the Campylobacter (C.) phages NCTC12684 (group II) and CP81 (group III) and of the Yersinia (Y.) phage PY100 to reduce the numbers of Campylobacter and Y. enterocolitica in meat at 4(o)C applying different Multiplicities of Infection (MOIs) was analyzed. Initial experiments were carried out in broth at 4(o)C and 37(o)C to compare cell number reductions under chilling and optimized growth conditions, respectively. The results showed a 1 log(10) unit reduction of Campylobacter cell numbers at 37(o)C in broth. However, no reduction was observed in broth and meat at 4(o)C. In contrast, Y. enterocolitica cell numbers were reduced in broth at 4(o)C (up to 3 log(10) units after 24hr) and 37(o)C (5 log(10) units after 1.5hr) and also in meat at 4(o)C (2 log(10) units after 48hr). The highest cell number reductions were obtained at the highest MOIs

Microrna response of primary human macrophages to Arcobacter Butzleri infection

The role of microRNAs (miRNAs) in infectious diseases is becoming more and more apparent, and the use of miRNAs as a diagnostic tool and their therapeutic application has become the major focus of investigation. The aim of this study was to identify miRNAs involved in the immune signaling of macrophages in response to Arcobacter (A.) butzleri infection, an emerging foodborne pathogen causing gastroenteritis. Therefore, primary human macrophages were isolated and infected, and miRNA expression was studied by means of RNAseq. Analysis of the data revealed the expression of several miRNAs, which were previously associated with bacterial infections such as miR-155, miR-125, and miR-212. They were shown to play a key role in Toll-like receptor signaling where they act as fine-tuners to establish a balanced immune response. In addition, miRNAs which have yet not been identified during bacterial infections such as miR-3613, miR-2116, miR-671, miR-30d, and miR-629 were differentially regulated in A. butzleri-infected cells. Targets of these miRNAs accumulated in pathways such as apoptosis and endocytosis — processes that might be involved in A. butzleri pathogenesis. Our study contributes new findings about the interaction of A. butzleri with human innate immune cells helping to understand underlying regulatory mechanisms in macrophages during infection

Prevalence, antimicrobial susceptibility and virulence gene profiles of Arcobacter species isolated from human stool samples, foods of animal origin, ready-to-eat salad mixes and environmental water

Background Members of the genus Arcobacter are considered as emerging zoonotic food and waterborne pathogens that cause gastroenteritis and bacteremia in humans. However, the potential risk that Arcobacter species pose to public health remains unassessed in various countries, including Baltic states. Therefore, the aim of this study was to determine the prevalence, antimicrobial susceptibility and presence of putative virulence genes of Arcobacter isolates recovered from humans, food products and environmental water in Lithuania. Results A total of 1862 samples were collected and examined from 2018 to 2020 in the city of Kaunas. Overall, 11.2% (n = 208) of the samples were positive for the presence of Arcobacter spp. The highest prevalence was detected in chicken meat (36%), followed by environmental water (28.1%), raw cow milk (25%), ready-to-eat salad mixes (7.1%) and human stool (1.7%). A. butzleri was the most frequently isolated species (n = 192; 92.3%), followed by A. cryaerophilus (n = 16; 7.7%). Arcobacter spp. antimicrobial susceptibility testing revealed unimodally distributed aggregated minimal inhibitory concentrations (MICs) for gentamicin, tetracycline, ciprofloxacin, ampicillin and erythromycin. However, a bimodal distribution for azithromycin was found with 96.2% of determined MICs above the epidemiological cut-off value (ECOFF) defined for Campylobacter jejuni (0.25 µg/ml). Majority of the Arcobacter isolates (n = 187; 89.9%) showed high susceptibility to ciprofloxacin with MICs below or equal to the ECOFF value of 0.5 µg/ml. The putative virulence genes cadF (100%), ciaB (100%), cj1349 (99%), tlyA (99%), mviN (97.9%) and pldA (95.8%) were the predominant genes detected among A. butzleri isolates. In contrast, the mviN and ciaB genes were present in all, whereas cj1349 (12.5%), tlyA (25%) and hecA (12.5%) were only detected in few A. cryaerophilus isolates. Conclusions Our results demonstrate that food products and environmental water in Lithuania are frequently contaminated with Arcobacter spp. that carry multiple putative virulence genes. Furthermore, A. butzleri were isolated from 1.7% of inpatients. Fluoroquinolones and aminoglycosides were found to be more effective against Arcobacter in comparison to other antimicrobial agents. However, further studies are needed to determine the pathogenic mechanisms and factors that facilitate the spread of Arcobacter infections

Intestinal Barrier in Post-Campylobacter jejuni Irritable Bowel Syndrome

Background: Campylobacter jejuni (C. jejuni) is one of the most common causes of bacterial gastroenteritis worldwide. One sequela of this infection is the development of post-infectious irritable bowel syndrome (PI-IBS). It has been suggested that a dysfunctional intestinal barrier may promote IBS development. We aimed to test this hypothesis against the background of the leaky gut concept for low-grade inflammation in PI-IBS. Methods: We identified patients with persistent PI-IBS symptoms after C. jejuni infection. During sigmoidoscopy, forceps biopsies were obtained for electrophysiological measurements of epithelial transport and barrier function in miniaturized Ussing devices. C. jejuni absence was checked by PCR and cytokine production with immunohistochemistry. Results: In PI-IBS, the epithelial resistance of the colon epithelium was unaltered, reflecting an intact paracellular pathway. In contrast, temperature-dependent horseradish peroxidase (HRP, 44 kDa) permeation increased. Short-circuit current (Isc) reflecting active anion secretion and ENaC-dependent electrogenic sodium absorption was unaffected. Early endosome antigen-1 (EEA1) and IL-4 levels increased. C. jejuni is not incorporated into the resident microbiota of the colon mucosa in PI-IBS. Conclusions: In PI-IBS after C. jejuni infection, macromolecule uptake via endocytosis was enhanced, leading to low-grade inflammation with pro-inflammatory cytokine release. The findings will allow C. jejuni-induced pathomechanisms to be targeted during infection and, thereafter to reduce sequelae such as PI-IBS