Greta Gölz

Presence of virulence genes, adhesion and invasion of Arcobacter butzleri.

The pathogenic potential of Arcobacter butzleri isolates was investigated by detecting the presence of putative virulence genes and analysing the adhesive and invasive capabilities in cell cultures of human cell lines.

Relevance of Campylobacter to public health—The need for a One Health approach

Campylobacter species belong to the most important foodborne bacteria which cause gastroenteritis in humans in both developed and developing countries. With increasing reporting rates, the public awareness towards Campylobacter infections is growing continuously. This strengthens the necessity to establish intervention measures for prevention and control of thermophilic Campylobacter spp. along the food chain, as in particular poultry and poultry meat represent a major source of human infections. An interdisciplinary One Health approach and a combined effort of all stakeholders are necessary to ultimately reduce the burden of campylobacteriosis cases in humans. Numerous studies point out, however, that at present a complete elimination of Campylobacter in the food chain is not feasible. The present aim should therefore be to establish control measures and intervention strategies to minimize the occurrence of Campylobacter spp. in livestock (e.g. poultry flocks) and to reduce the quantitative Campylobacter burden in animals and foods. To this end, a combination of intervention methods at different stages of the food chain appears most promising. That has to be accompanied by targeted consumer advice and education campaigns to raise the awareness towards Campylobacter infections.

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 4oC applying different Multiplicities of Infection (MOIs) was analyzed. Initial experiments were carried out in broth at 4oC and 37oC to compare cell number reductions under chilling and optimized growth conditions, respectively. The results showed a 1 log10 unit reduction of Campylobacter cell numbers at 37oC in broth. However, no reduction was observed in broth and meat at 4oC. In contrast, Y. enterocolitica cell numbers were reduced in broth at 4oC (up to 3 log10 units after 24hr) and 37oC (5 log10 units after 1.5hr) and also in meat at 4oC (2 log10 units after 48hr). The highest cell number reductions were obtained at the highest MOIs.

Prevalence and genotypes of Campylobacter jejuni from urban environmental sources in comparison with clinical isolates from children.

This study aimed to investigate the prevalence of Campylobacter jejuni in potential contamination sources that are not regularly monitored such as free-living urban pigeons and crows, dogs, cats and urban environmental water and to assess the possible impact on the epidemiology of campylobacteriosis in children using multilocus sequence typing (MLST). Campylobacter spp. were detected in 36.2 % of faecal samples of free-living urban birds and in 40.4 % of environmental water samples. A low prevalence of Campylobacter spp. was detected in dogs and cats, with 7.9 and 9.1 %, respectively. Further identification of isolates revealed that environmental water and pet samples were mostly contaminated by other Campylobacter spp. than C. jejuni, whereas C. jejuni was the most prevalent species in faecal samples of free-living birds (35.4 %). This species was the dominant cause of campylobacteriosis in children (91.5 %). In addition, the diversity of C. jejuni MLST types in free-living birds and children was investigated. Clonal complex (CC) 179 was predominant among free-living urban birds; however, only two isolates from children were assigned to this CC. One dog and one child isolate were assigned to the same clonal complex (CC48) and sequence type (ST) 918. The dominant two clonal complexes among the child clinical isolates (CC353 and CC21) were not detected among C. jejuni strains isolated from environmental sources examined in this study. As only two CCs were shared by environmental and child C. jejuni isolates and a high number of novel alleles and STs were found in C. jejuni isolated from free-living urban birds and environmental water, there is probably only a limited link between urban environmental sources and campylobacteriosis in children, particularly in rather cold climatic conditions.

QUORUM SENSING DEPENDENT PHENOTYPES AND THEIR MOLECULAR MECHANISMS IN CAMPYLOBACTERALES

Quorum sensing comprises the mechanism of communication between numerous bacteria via small signalling molecules, termed autoinducers (AI). Using quorum sensing, bacteria can regulate the expression of multiple genes involved in virulence, toxin production, motility, chemotaxis and biofilm formation, thus contributing to adaptation as well as colonisation. The current understanding of the role of quorum sensing in the lifecycle of Campylobacterales is still incomplete. Campylobacterales belong to the class of Epsilonproteobacteria representing a physiologically and ecologically diverse group of bacteria that are rather distinct from the more commonly studied Proteobacteria, such as Escherichia and Salmonella. This review summarises the recent knowledge on distribution and production of AI molecules, as well as possible quorum sensing dependent regulation in the mostly investigated species within the Campylobacterales group: Campylobacter jejuni and Helicobacter pylori.

Arcobacter butzleri Induce Colonic, Extra-Intestinal and Systemic Inflammatory Responses in Gnotobiotic IL-10 Deficient Mice in a Strain-Dependent Manner

Background The immunopathological impact of human Arcobacter (A.) infections is under current debate. Episodes of gastroenteritis with abdominal pain and acute or prolonged watery diarrhea were reported for A. butzleri infected patients. Whereas adhesive, invasive and cytotoxic capacities have been described for A. butzleri in vitro, only limited information is available about the immunopathogenic potential and mechanisms of infection in vivo. Methodology/Principal Findings Gnotobiotic IL-10-/- mice were generated by broad-spectrum antibiotic treatment and perorally infected with the A. butzleri strains CCUG 30485 and C1 shown to be invasive in cell culture assays. Bacterial colonization capacities, clinical conditions, intestinal, extra-intestinal and systemic immune responses were monitored at day six and 16 postinfection (p.i.). Despite stable intestinal A. butzleri colonization at high loads, gnotobiotic IL-10-/- mice were virtually unaffected and did not display any overt symptoms at either time point. Notably, A. butzleri infection induced apoptosis of colonic epithelial cells which was paralleled by increased abundance of proliferating cells. Furthermore A. butzleri infection caused a significant increase of distinct immune cell populations such as T and B cells, regulatory T cells, macrophages and monocytes in the colon which was accompanied by elevated colonic TNF, IFN-γ, nitric oxide (NO), IL-6, IL-12p70 and MCP-1 concentrations. Strikingly, A. butzleri induced extra-intestinal and systemic immune responses as indicated by higher NO concentrations in kidney and increased TNF, IFN-γ, IL-12p70 and IL-6 levels in serum samples of infected as compared to naive mice. Overall, inflammatory responses could be observed earlier in the course of infection by the CCUG 30485 as compared to the C1 strain. Conclusion/Significance Peroral A. butzleri infection induced not only intestinal but also extra-intestinal and systemic immune responses in gnotobiotic IL-10-/- mice in a strain-dependent manner. These findings point towards an immunopathogenic potential of A. butzleri in vertebrate hosts.

Experimental infection of weaned piglets with Campylobacter coli--excretion and translocation in a pig colonisation trial.

Campylobacter (C.) is one of the most common food-borne pathogen causing bacterial enteric infections in humans. Consumption of meat and meat products that have been contaminated with Campylobacter are the major source of infection. Pigs are a natural reservoir of Campylobacter spp. with C. coli as the dominant species. Even though some studies focussed on transmission of C. coli in pig herds and the excretion in faeces, little is known about the colonisation and excretion dynamics of C. coli in a complex gut microbiota present in weaned piglets and the translocation to different tissues. Therefore, an experimental trial was conducted to evaluate the colonisation and translocation ability of the porcine strain C. coli 5981 in weaned pigs. Thus, ten 35 days old piglets were intragastrically inoculated with strain C. coli 5981 (7 × 10(7)CFU/animal) encoding resistances against erythromycin and neomycin. Faecal samples were taken and C. coli levels were enumerated over 28 days. All piglets were naturally colonised with C. coli before experimental inoculation, and excretion levels ranged from 10(4) to 10(7)CFU/g faeces. However, no strain showed resistances against the additional antimicrobials used. Excretion of C. coli 5981 was seen for all piglets seven days after inoculation and highest counts were detectable ten days after inoculation with 10(6)CFU/g faeces. Post-mortem, translocation and subsequent invasion of luminal C. coli was observed for gut tissues of the small intestine and for the gut associated lymphatic tissues, such as jejunal mesenteric lymph nodes and tonsils as well as for spleen and gall bladder. In conclusion, this pig colonisation trial offers the opportunity to study C. coli colonisation in weaned piglets using the porcine strain C. coli 5981 without the need for gnotobiotic or specific pathogen-free animals.

Analysis of in vitro and in vivo effects of probiotics against Campylobacter spp.

Campylobacter (C.) spp. are well recognised as the leading cause of bacterial food-borne diarrheal disease worldwide, with C. jejuni and C. coli as the most important species: C. coli is highly abundant in pigs and pork meat has often been implicated as a source for human infection. Intestinal colonisation of C. coli in pigs plays a role in carcass contamination during slaughter. Different pre-harvest intervention measures are proposed to reduce the C. coli burden in the porcine intestine. Among others, the use of probiotics to prevent or reduce the colonisation of intestinal pathogens is discussed. One aim of this study was to screen a variety of probiotics to evaluate their inhibitory activity against Campylobacter spp. in vitro. Therefore, cell-free culture supernatants of Lactobacillus spp., Bifidobacterium spp., Enterococcus (E.) faecium NCIMB 10415, and Escherichia coli Nissle 1917 were tested against C. jejuni and C. coli by a well-diffusion agar assay. Seven out of eleven Lactobacillus strains showed an inhibitory activity against at least one of the three tested Campylobacter strains. This antagonistic activity against Campylobacter spp. was caused by the production of organic acids that lowered the pH. Application with pH neutralised cell-free culture supernatants abolished this inhibitory effect. Other tested strains with probiotic properties showed no inhibitory activity against any Campylobacter spp. strain. The strain E. faecium NCIMB 10415 was chosen to test its inhibitory activity against C. coli in vivo. Twenty weaned piglets were allocated into two groups, a probiotic group and a control group.The diet of the probiotic group was supplemented with E. faecium NCIMB 10415 (10(9) cfu/kg feed, Cylactin) since weaning, whereas the control group received no probiotic treatment. All piglets were naturally colonised with C. coli. The excretion load of C. coli was monitored for 28 days. The results indicate that dietary supplementation of E. faecium NCIMB 10415 did not significantly affect C. coli excretion levels in pigs. In this study, E. faecium NCIMB 10415 showed no antagonistic activity against C. coli in vitro and in vivo and had no impact on the growth performance of weaned piglets.

Effects of meat juice on biofilm formation of Campylobacter and Salmonella

Campylobacter and Salmonella are leading causes of foodborne illnesses worldwide, vastly harboured by raw meat as their common food reservoir. Both microbes are prevalent in meat processing environments in the form of biofilms that contribute to cross-contamination and foodborne infection. This study applied raw meat juice (chicken juice and pork juice) as a minimally processed food model to study its effects on bacterial biofilm formation. Meat juice was collected during the freeze-thaw process of raw meat and sterilized by filtration. In 96-well polystyrene plates and glass chambers, supplementation of over 25% meat juice (v/v) in laboratory media led to an increase in biofilm formation of Campylobacter and Salmonella. During the initial attachment stage of biofilm development, more bacterial cells were present on surfaces treated with meat juice residues compared to control surfaces. Meat juice particulates on abiotic surfaces facilitated biofilm formation of Campylobacter and Salmonella under both static and flow conditions, with the latter being assessed using a microfluidic platform. Further, the deficiency in biofilm formation of selected Campylobacter and Salmonella mutant strains was restored in the presence of meat juice particulates. These results suggested that meat juice residues on the abiotic surfaces might act as a surface conditioner to support initial attachment and biofilm formation of Campylobacter and Salmonella. This study sheds light on a possible survival mechanism of Campylobacter and Salmonella in meat processing environments, and indicates that thorough cleaning of meat residues during meat production and handling is critical to reduce the bacterial load of Campylobacter and Salmonella.

Survey of small intestinal and systemic immune responses following murine Arcobacter butzleri infection

BackgroundArcobacter (A.) butzleri has been described as causative agent for sporadic cases of human gastroenteritis with abdominal pain and acute or prolonged watery diarrhea. In vitro studies revealed distinct adhesive, invasive and cytotoxic properties of A. butzleri. Information about the underlying immunopathological mechanisms of infection in vivo, however, are scarce. The aim of this study was to investigate the immunopathological properties of two different A.butzleri strains in a well-established murine infection model.ResultsGnotobiotic IL-10−/− mice, in which the intestinal microbiota was depleted by broad-spectrum antibiotic treatment, were perorally infected with two different A. butzleri strains isolated from a diseased patient (CCUG 30485) or fresh chicken meat (C1), respectively. Eventhough bacteria of either strain could stably colonize the intestinal tract at day 6 and day 16 postinfection (p.i.), mice did not exert infection induced symptoms such as diarrhea or wasting. In small intestines of infected mice, however, increased numbers of apoptotic cells could be detected at day 16, but not day 6 following infection with either strain. A strain-dependent influx of distinct immune cell populations such as T and B cells as well as of regulatory T cells could be observed upon A. butzleri infection which was accompanied by increased small intestinal concentrations of pro-inflammatory cytokines such as TNF, IFN-γ, MCP-1 and IL-6. Remarkably, inflammatory responses following A. butzleri infection were not restricted to the intestinal tract, given that the CCUG 30485 strain induced systemic immune responses as indicated by increased IFN-γ concentrations in spleens at day 6, but not day 16 following infection.ConclusionUpon peroral infection A. butzleri stably colonized the intestinal tract of gnotobiotic IL-10−/− mice. The dynamics of distinct local and systemic inflammatory responses could be observed in a strain-dependent fashion pointing towards an immunopathogenic potential of A. butzleri in vivo. These results indicate that gnotobiotic IL-10−/− mice are well suited to further investigate the molecular mechanisms underlying arcobacteriosis in vivo.