miR-125a-5p regulates the sialyltransferase ST3GAL1 in murine model of human intestinal campylobacteriosis.

BACKGROUND: Zoonotic microorganisms are increasingly impacting human health worldwide. Due to the development of the global population, humans and animals live in shared and progressively crowded ecosystems, which enhances the risk of zoonoses. Although Campylobacter species are among the most important bacterial zoonotic agents worldwide, the molecular mechanisms of many host and pathogen factors involved in colonisation and infection are poorly understood. Campylobacter jejuni colonises the crypts of the human colon and causes acute inflammatory processes. The mucus and associated proteins play a central host-protective role in this process. The aim of this study was to explore the regulation of specific glycosyltransferase genes relevant to differential mucin-type O-glycosylation that could influence host colonisation and infection by C. jejuni. RESULTS: Since microRNAs are known to be important regulators of the mammalian host cell response to bacterial infections, we focussed on the role of miR-125a-5p in C. jejuni infection. Combining in vitro and in vivo approaches, we show that miR-125a-5p regulates the expression of the sialyltransferase ST3GAL1 in an infection-dependent manner. The protein ST3GAL1 shows markedly increased intestinal levels in infected mice, with enhanced distribution in the mucosal epithelial layer in contrast to naïve mice. CONCLUSION: From our previous studies and the data presented here, we conclude that miR-125a-5p and the previously reported miR-615-3p are involved in regulating the glycosylation patterns of relevant host cell response proteins during C. jejuni infection. The miRNA-dependent modulation of mucin-type O-glycosylation could be part of the mucosal immune response, but also a pathogen-driven modification that allows colonisation and infection of the mammalian host.

Genomic Characterization of Arcobacter butzleri Strains Isolated from Various Sources in Lithuania.

Arcobacter (A.) butzleri, the most widespread species within the genus Arcobacter, is considered as an emerging pathogen causing gastroenteritis in humans. Here, we performed a comparative genome-wide analysis of 40 A. butzleri strains from Lithuania to determine the genetic relationship, pangenome structure, putative virulence, and potential antimicrobial- and heavy-metal-resistance genes. Core genome single nucleotide polymorphism (cgSNP) analysis revealed low within-group variability (≤4 SNPs) between three milk strains (RCM42, RCM65, RCM80) and one human strain (H19). Regardless of the type of input (i.e., cgSNPs, accessory genome, virulome, resistome), these strains showed a recurrent phylogenetic and hierarchical grouping pattern. A. butzleri demonstrated a relatively large and highly variable accessory genome (comprising of 6284 genes with around 50% of them identified as singletons) that only partially correlated to the isolation source. Downstream analysis of the genomes resulted in the detection of 115 putative antimicrobial- and heavy-metal-resistance genes and 136 potential virulence factors that are associated with the induction of infection in host (e.g., cadF, degP, iamA), survival and environmental adaptation (e.g., flagellar genes, CheA-CheY chemotaxis system, urease cluster). This study provides additional knowledge for a better A. butzleri-related risk assessment and highlights the need for further genomic epidemiology studies in Lithuania and other countries.

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.

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.

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.

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.

Challenging the "gold standard" of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses.

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the "gold standard" in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log10Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination. Campylobacter served as a model organism, challenging CFU as "gold standard" and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens.

The glycosyltransferase ST3GAL2 is regulated by miR-615-3p in the intestinal tract of Campylobacter jejuni infected mice.

BACKGROUND: Campylobacter jejuni (C. jejuni) infections are of increasing importance worldwide. As a typical mucosal pathogen, the interaction of C. jejuni with mucins is a prominent step in the colonisation of mucosal surfaces. Despite recent advances in understanding the interaction between bacterial pathogens and host mucins, the mechanisms of mucin glycosylation during intestinal C. jejuni infection remain largely unclear. This prompted us to identify relevant regulatory networks that are concerted by miRNAs and could play a role in the mucin modification and interaction. RESULTS: We firstly used a human intestinal in vitro model, in which we observed altered transcription of MUC2 and TFF3 upon C. jejuni NCTC 11168 infection. Using a combined approach consisting of in silico analysis together with in vitro expression analysis, we identified the conserved miRNAs miR-125a-5p and miR-615-3p associated with MUC2 and TFF3. Further pathway analyses showed that both miRNAs appear to regulate glycosyltransferases, which are related to the KEGG pathway 'Mucin type O-glycan biosynthesis'. To validate the proposed interactions, we applied an in vivo approach utilising a well-established secondary abiotic IL-10-/- mouse model for infection with C. jejuni 81-176. In colonic tissue samples, we confirmed infection-dependent aberrant transcription of MUC2 and TFF3. Moreover, two predicted glycosyltransferases, the sialyltransferases ST3GAL1 and ST3GAL2, exhibited inversely correlated transcriptional levels compared to the expression of the identified miRNAs miR-125a-5p and miR-615-3p, respectively. In this study, we mainly focused on the interaction between miR-615-3p and ST3GAL2 and were able to demonstrate their molecular interaction using luciferase reporter assays and RNAi. Detection of ST3GAL2 in murine colonic tissue by immunofluorescence demonstrated reduced intensity after C. jejuni 81-176 infection and was thus consistent with the observations made above. CONCLUSIONS: We report here for the first time the regulation of glycosyltransferases by miRNAs during murine infection with C. jejuni 81-176. Our data suggest that mucin type O-glycan biosynthesis is concerted by the interplay of miRNAs and glycosyltransferases, which could determine the shape of intestinal glycosylated proteins during infection.

Molecular Mechanisms of Campylobacter Biofilm Formation and Quorum Sensing.

Even though Campylobacter spp. are known to be fastidious organisms, they can survive within the natural environment. One mechanism to withstand unfavourable conditions is the formation of biofilms, a multicellular structure composed of different bacterial and other microbial species which are embedded in an extracellular matrix. High oxygen levels, low substrate concentrations and the presence of external DNA stimulate the biofilm formation by C. jejuni. These external factors trigger internal adaptation processes, e.g. via regulating the expression of genes encoding proteins required for surface structure formation, as well as motility, stress response and antimicrobial resistance. Known genes impacting biofilm formation will be summarized in this review. The formation of biofilms as well as the expression of virulence genes is often regulated in a cell density depending manner by quorum sensing, which is mediated via small signalling molecules termed autoinducers. Even though quorum sensing mechanisms of other bacteria are well understood, knowledge on the role of these mechanisms in C. jejuni biofilm formation is still scarce. The LuxS enzyme involved in generation of autoinducer-2 is present in C. jejuni, but autoinducer receptors have not been identified so far. Phenotypes of C. jejuni strains lacking a functional luxS like reduced growth, motility, oxygen stress tolerance, biofilm formation, adhesion, invasion and colonization are also summarized within this chapter. However, these phenotypes are highly variable in distinct C. jejuni strains and depend on the culture conditions applied.

Inhibition of AI-2 Quorum Sensing and Biofilm Formation in Campylobacter jejuni by Decanoic and Lauric Acids.

Campylobacter jejuni is a major bacterial cause of human diarrheal diseases worldwide. Despite its sensitivity to environmental stresses, C. jejuni ubiquitously distributes throughout poultry production chains. Biofilm formation mediated by quorum sensing is suggested to be critical to the survival of C. jejuni in agroecosystem. C. jejuni possesses LuxS, the enzyme involved in the production of autoinducer-2 (AI-2) signaling molecules. In this study, two fatty acids, namely decanoic acid and lauric acid, were identified to be effective in inhibiting AI-2 activity of C. jejuni. Both decanoic acid and lauric acid at 100 ppm inhibited ∼90% AI-2 activity (P < 0.05) of C. jejuni without bacterial inactivation. The biofilm biomass of two C. jejuni strains was reduced by 10-50% (P < 0.05) after treatment by both fatty acids, while increased biofilm formation was observed for one C. jejuni strain. In addition, both fatty acids effectively reduced the motility of all tested C. jejuni strains. These findings can aid in developing alternative C. jejuni control strategies in agri-food and clinical settings.

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.

Arcobacter Identification and Species Determination Using Raman Spectroscopy Combined with Neural Networks.

Rapid and accurate identification of Arcobacter is of great importance because it is considered an emerging food- and waterborne pathogen and potential zoonotic agent. Raman spectroscopy can differentiate bacteria based on Raman scattering spectral patterns of whole cells in a fast, reagentless, and easy-to-use manner. We aimed to detect and discriminate Arcobacter bacteria at the species level using confocal micro-Raman spectroscopy (785 nm) coupled with neural networks. A total of 82 reference and field isolates of 18 Arcobacter species from clinical, environmental, and agri-food sources were included. We determined that the bacterial cultivation time and growth temperature did not significantly influence the Raman spectral reproducibility and discrimination capability. The genus Arcobacter could be successfully differentiated from the closely related genera Campylobacter and Helicobacter using principal-component analysis. For the identification of Arcobacter to the species level, an accuracy of 97.2% was achieved for all 18 Arcobacter species using Raman spectroscopy combined with a convolutional neural network (CNN). The predictive capability of Raman-CNN was further validated using an independent data set of 12 Arcobacter strains. Furthermore, a Raman spectroscopy-based fully connected artificial neural network (ANN) was constructed to determine the actual ratio of a specific Arcobacter species in a bacterial mixture ranging from 5% to 100% by biomass (regression coefficient >0.99). The application of both CNN and fully connected ANN improved the accuracy of Raman spectroscopy for bacterial species determination compared to the conventional chemometrics. This newly developed approach enables rapid identification and species determination of Arcobacter within an hour following cultivation.IMPORTANCE Rapid identification of bacterial pathogens is critical for developing an early warning system and performing epidemiological investigation. Arcobacter is an emerging foodborne pathogen and has become more important in recent decades. The incidence of Arcobacter species in the agro-ecosystem is probably underestimated mainly due to the limitation in the available detection and characterization techniques. Raman spectroscopy combined with machine learning can accurately identify Arcobacter at the species level in a rapid and reliable manner, providing a promising tool for epidemiological surveillance of this microbe in the agri-food chain. The knowledge elicited from this study has the potential to be used for routine bacterial screening and diagnostics by the government, food industry, and clinics.

Detection of Arcobacter species in different intestinal compartments of broiler chicken during slaughter and processing.

Arcobacter spp. are commonly present on meat products. However, the source of contamination on chicken meat is under dispute. Since different studies reported contradictory results on the occurrence of Arcobacter spp. inside the intestinal tract of chicken, our study examined four intestinal compartments at four significant production steps during broiler slaughter and processing in the slaughterhouse. Altogether, 157 intestinal tracts from 19 flocks were examined qualitatively and semiquantitatively applying a selective enrichment. Further verification was performed by mPCR and rpoB sequencing. Arcobacter spp. were only detected sporadically in intestinal contents after bleeding (2/32) and in none after scalding (0/32). After defeathering, Arcobacter spp. were detected in 62% (18/29) of the intestinal contents with 28% (8/29) of the duodenal, 21% (6/29) of the jejunal, 3% (1/29) of the cecal, and 55% (16/29) of the colonic samples tested positive with loads up to 24,000 MPN/g in the colonic content. Further 88% (7/8) of colonic tissue samples were tested positive. After evisceration, the prevalences (58/64) and loads of Arcobacter spp. display comparable levels in the intestinal contents like after defeathering. In conclusion, our data point out that Arcobacter spp. are most likely detected in the colonic intestinal compartment of the chicken after defeathering and evisceration. Therefore, not only cross-contamination originating from the environment inside the slaughterhouse may cause carcass contamination with Arcobacter spp. on broiler chicken carcasses. The detection of Arcobacter spp. in duodenal and jejunal contents as well as in the colonic tissue indicates that there possibly exists an Arcobacter reservoir inside the chicken.

Differences in the Transcriptomic Response of Campylobacter coli and Campylobacter lari to Heat Stress.

Campylobacter spp. are one of the most important food-borne pathogens, which are quite susceptible to environmental or technological stressors compared to other zoonotic bacteria. This might be due to the lack of many stress response mechanisms described in other bacteria. Nevertheless, Campylobacter is able to survive in the environment and food products. Although some aspects of the heat stress response in Campylobacter jejuni are already known, information about the stress response in other Campylobacter species are still scarce. In this study, the stress response of Campylobacter coli and Campylobacter lari to elevated temperatures (46°C) was investigated by survival assays and whole transcriptome analysis. None of the strains survived at 46°C for more than 8 h and approximately 20% of the genes of C. coli RM2228 and C. lari RM2100 were differentially expressed. The transcriptomic profiles showed enhanced gene expression of several chaperones like dnaK, groES, groEL, and clpB in both strains, indicating a general involvement in the heat stress response within the Campylobacter species. However, the pronounced differences in the expression pattern between C. coli and C. lari suggest that stress response mechanisms described for one Campylobacter species might be not necessarily transferable to other Campylobacter species.

Prevalence and antimicrobial susceptibility of Arcobacter species in human stool samples derived from out- and inpatients: the prospective German Arcobacter prevalence study Arcopath.

BACKGROUND: Arcobacter species, particularly A. butzleri, but also A. cryaerophilus constitute emerging pathogens causing gastroenteritis in humans. However, isolation of Arcobacter may often fail during routine diagnostic procedures due to the lack of standard protocols. Furthermore, defined breakpoints for the interpretation of antimicrobial susceptibilities of Arcobacter are missing. Hence, reliable epidemiological data of human Arcobacter infections are scarce and lacking for Germany. We therefore performed a 13-month prospective Arcobacter prevalence study in German patients. RESULTS: A total of 4636 human stool samples was included and Arcobacter spp. were identified from 0.85% of specimens in 3884 outpatients and from 0.40% of specimens in 752 hospitalized patients. Overall, A. butzleri was the most prevalent species (n = 24; 67%), followed by A. cryaerophilus (n = 10; 28%) and A. lanthieri (n = 2; 6%). Whereas A. butzleri, A. cryaerophilus and A. lanthieri were identified in outpatients, only A. butzleri could be isolated from samples of hospitalized patients. Antimicrobial susceptibility testing of Arcobacter isolates revealed high susceptibilities to ciprofloxacin, whereas bimodal distributions of MICs were observed for azithromycin and ampicillin. CONCLUSIONS: In summary, Arcobacter including A. butzleri, A. cryaerophilus and A. lanthieri could be isolated in 0.85% of German outpatients and ciprofloxacin rather than other antibiotics might be appropriate for antibiotic treatment of infections. Further epidemiological studies are needed, however, to provide a sufficient risk assessment of Arcobacter infections in humans.

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.

Characterization of Arcobacter strains isolated from human stool samples: results from the prospective German prevalence study Arcopath.

BACKGROUND: Arcobacter constitute emerging food- and waterborne pathogens causing gastroenteritis in humans, but the underlying mechanisms are only incompletely understood. We therefore characterized Arcobacter isolates derived from human stool samples that had been collected during a prospective prevalence study in Germany in vitro. Thirty-six bacterial isolates belonging to the species A. butzleri (n = 24), A. cryaerophilus (n = 10) and A. lanthieri (n = 2) were genotyped by ERIC-PCR, the presence of 10 putative virulence genes was assessed and cytotoxic effects on the human intestinal cell line HT-29/B6 were analyzed applying the WST-assay. RESULTS: Genotyping revealed high genetic diversity within the species A. butzleri, A. cryaerophilus and A. lanthieri. Both, A. butzleri and A. lanthieri encoded for a large number of putative virulence genes, while fewer genes were detectable in A. cryaerophilus isolates. Notably, the three cytolethal distending toxin (CDT) genes cdtA, cdtB and cdtC were abundant in both A. lanthieri isolates. Furthermore, all A. butzleri and A. lanthieri, but only one of the A. cryaerophilus isolates exerted cytotoxic effects. CONCLUSIONS: Our study provides evidence for the abundance of putative virulence genes in Arcobacter isolates and prominent cytotoxic effects of A. butzleri and A. lanthieri in vitro. The presence of cdtA, cdtB, cdtC in A. lanthieri points towards CDT secretion as potential mechanism underlying cytotoxicity as opposed to A. butzleri. However, the association of the Arcobacter virulence factors detected and human morbidity should be addressed in future studies.

Characterization of Arcobacter spp. isolated from retail seafood in Germany.

Arcobacter species are considered emerging zoonotic pathogens associated with human gastroenteritis. They were already isolated from a wide range of habitats and hosts worldwide. However, information about the prevalence of Arcobacter in retail seafood products is still scarce. This study aimed to evaluate the presence of Arcobacter in retail seafood and characterize Arcobacter isolates derived from these matrices. In total, seven species of Arcobacter were isolated from 56 of 318 (17.6%) seafood samples, including bivalves (mussels, clams and razor clams), shrimps and cephalopods (squids and octopuses). The highest prevalence was detected in cephalopods (27.4%), followed by bivalves (18%) and lowest in shrimps (8.5%). PCRs of 10 putative virulence genes demonstrated higher prevalences of these genes among A. butzleri, compared to other species, such as A. cryaerophilus, A. aquimarinus and A. venerupis. Further, high genetic diversity could be determined by ERIC-PCR. Our study indicates the potential transmission of Arcobacter to humans by consuming uncooked or undercooked seafood.

Prevalence and quantitative analysis of ESBL and AmpC beta-lactamase producing Enterobacteriaceae in broiler chicken during slaughter in Germany.

Food producing animals are considered a reservoir for Extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase (AmpC) producing Enterobacteriaceae. Therefore, meat is discussed to be a potential source for the transmission of these resistant bacteria to humans. There is only limited information about the quantitative load of ESBL-/AmpC-producing Enterobacteriaceae in different sample matrices during slaughter and their distribution in the slaughterhouse environment. Therefore, the aim of this study was to determine the prevalence as well as quantitative load of ESBL-/AmpC-producing Enterobacteriaceae in caecum, skin and filet samples of different broiler chicken flocks during slaughter in Germany. In addition, environmental samples were taken during slaughter of the respective flocks. To gain insights into possible transmission routes of ESBL-/AmpC-producing Enterobacteriaceae, the corresponding phylogroup and beta-lactamase genes were determined for selected isolates. ESBL-/AmpC-producing Enterobacteriaceae were detected during slaughter of all seven investigated flocks. On average, 47% (83/175) of caecum, 55% (96/175) of skin, 28% (49/175) of filet and 28% (25/89) of environmental samples harboured ESBL-/AmpC-producing Enterobacteriaceae. Prevalence varied widely between the flocks as well as between the different sample matrices. In about half of the caecum (23/40) and skin (19/40) samples as well as 85% (17/20) of the filet samples, the number of putative ESBL-/AmpC-producing Enterobacteriaceae (cefotaxime resistant Enterobacteriaceae) was below quantification limit. The median of cefotaxime resistant Enterobacteriaceae was 2.5 × 103 cfu/g in caecum, 1.5 × 103 cfu/g in skin and 1.5 × 102 cfu/g in filet samples. The median of cefotaxime resistant Enterobacteriaceae was, depending on the sample matrix, 1-4 log units below the median of total Enterobacteriaceae. Using real-time PCR, in 82% (629/767) of the cefotaxime resistant Enterobacteriaceae at least one of the investigated beta-lactamase genes blaCTX-M, blaSHV, blaTEM, blaAmpC-CIT was detected. The respective resistance genes of 322 isolates were further sequenced. The predominant bla-gene was blaCMY-2 (48%), followed by blaSHV-12 (23%). A contamination from the broiler chicken to the slaughterhouse environment and vice versa seems probable as isolates of the same species and phylogroup, encoding the same resistance genes were detected in all matrices during slaughter of the respective flock as well as in the slaughterhouse environment.