[Campylobacteriosis of man : livestock as reservoir for Campylobacter species]. / Die Campylobacteriose des Menschen : Nutztiere als Reservoir für Campylobacter-Arten.

Over the last few years, infections with Campylobacter have significantly increased in Europe and Germany and these bacteria have even surpassed Salmonella as the most prevalent bacteria, causing gastroenteritis. Especially contamination during the handling and consumption of meat products seems to be the most important risk factor which plays a prominent role for transmission to man. In addition, contact with pets and other animals, drinking raw or improperly pasteurized milk, and the tenacity of Campylobacter in different environments, especially water, have also to be considered for an adequate risk assessment. Besides gastroenteritis, arthralgia, and Guillain-Barré syndrome are important clinical complications of Campylobacter infections in man. At the same time, it is mostly unclear why the course of infection in man and in reservoir animals differs significantly, especially as only a few classical bacterial virulence factors have been identified so far. For these reasons, the development of efficient prevention strategies is of utmost importance in order to control campylobacteriosis.

Resveratrol Prevents Campylobacter jejuni-Induced Leaky gut by Restoring Occludin and Claudin-5 in the Paracellular Leak Pathway.

Campylobacter jejuni is a bacterial human pathogen causing gastroenteritis and sequelae like irritable bowel syndrome. Epidemiologists count the human campylobacteriosis by C. jejuni as the most common foodborne zoonosis and bacterial diarrheal disease worldwide. Based on bioinformatics predictions for potential protective compounds in campylobacteriosis, the question was raised whether the plant-based polyphenol resveratrol is sufficient to attenuate intestinal epithelial damage induced by C. jejuni. We investigated this by performing experimental infection studies in an epithelial cell culture and the secondary abiotic IL-10-/- mouse model. In C. jejuni-infected human colonic HT-29/B6 cell monolayers, transepithelial electrical resistance (TER) was decreased and the paracellular marker flux of fluorescein (332 Da) increased. Concomitantly, the tight junction (TJ) proteins occludin and claudin-5 were re-distributed off the tight junction domain. This was accompanied by an increased induction of epithelial apoptosis, both changes contributing to compromised barrier function and the opening of the leak pathway induced by C. jejuni. In parallel, the recovery experiments with the application of resveratrol revealed a functional improvement of the disturbed epithelial barrier in both models in vitro and in vivo. During treatment with resveratrol, TJ localization of occludin and claudin-5 was fully restored in the paracellular domain of HT-29/B6 cells. Moreover, resveratrol decreased the rate of epithelial apoptosis. These resveratrol-induced molecular and cellular effects would therefore be expected to improve epithelial barrier function, thereby minimizing the so-called leaky gut phenomenon. In conclusion, the induction of the leak pathway by C. jejuni and the restoration of barrier function by resveratrol demonstrates its effectiveness as a potential preventive or therapeutic method of mitigating the leaky gut associated with campylobacteriosis.

Structural and functional analysis of the riboflavin synthesis genes encoding GTP cyclohydrolase II (ribA), DHBP synthase (ribBA), riboflavin synthase (ribC), and riboflavin deaminase/reductase (ribD) from Helicobacter pylori strain P1.

The functions of the riboflavin synthesis gene homologues ribA, ribBA, ribC, and ribD from Helicobacter pylori strain P1 were confirmed by complementation of defined Escherichia coli mutant strains. The H. pylori ribBA gene, which is similar to bifunctional ribBA genes of Gram-positive bacteria, fully complemented the ribB mutation and partially restored growth in a ribC mutant. However, ribBA did not complement the ribA mutation in E. coli, thus explaining the presence of the additional separate copy of the ribA gene in the H. pylori chromosome. In E. coli exclusively ribA conferred hemolytic activity and gave rise to production of molecules with fluorescence characteristics similar to flavins, as observed earlier. The E. coli hemolysin ClyA was not involved in causing the hemolytic phenotype. No riboflavin synthesis genes on plasmids conferred iron uptake functions to a siderophore-deficient mutant of E. coli. Marker exchange mutagenesis of the genes in H. pylori was not successful indicating that riboflavin synthesis is essential for basic metabolic functions of the gastric pathogen.

Identification and molecular analysis of superoxide dismutase isoforms in Helicobacter pylori.

Three electromorphs of iron superoxide dismutase (FeSOD) were identified among 29 Helicobacter pylori isolates by native gel electrophoresis and activity staining. The electromorphs designated isoforms A, B, and C are characterized by slow, intermediate and fast electrophoretic migration, respectively, which was not observed under denaturing conditions. The isoforms were not associated with virulence determinants and with the outcome of disease. Sequence analysis of the sodB gene in strains producing different FeSOD isoforms and comparison of deduced protein sequences revealed that differences in the electric migration behavior are associated with exchange of charged amino acids, suggesting that faster migration is caused by a more negative total charge of the proteins. Electrophoretic migration of native FeSOD was not influenced by changes in the iron cofactor concentration, oxidative stress, and different media, indicating that FeSOD isoforms represent stable strain-specific markers.

Cloning and characterization of the fur gene from Helicobacter pylori.

The fur homologue of Helicobacter pylori was isolated by screening a plasmid-based, genomic DNA library using the Fur titration assay (FURTA). The analysis of the DNA sequence revealed significant homology with Fur proteins from various other bacterial species. The highest degree of homology was observed for the Fur protein from Campylobacter jejuni. The H. pylori fur gene on a plasmid could partially complement the fur mutation in Escherichia coli strain H1681. The repressor activity depended on addition of iron to the medium indicating that iron acts as a co-repressor for the H. pylori protein similar to Fur from other bacteria. Comparison of Fur from H. pylori strain NCTC11638 with the recently published genomic DNA sequence of another strain (26695) confirmed the identity of the fur homologue and revealed that the fur locus is highly conserved in both strains.

Hemin-dependent growth and hemin binding of Bartonella henselae.

Bartonella henselae causes cat-scratch disease and bacillary angiomatosis peliosis. The bacteria reside in erythrocytes of asymptomatic cats, which represent the natural reservoir for this pathogen. B. henselae is usually grown on blood-enriched media. Growth experiments on Brucella medium without blood demonstrated that heme compounds are essential for the growth of B. henselae and can completely substitute the addition of blood components. The heme precursor protoporphyrin IX alone, or in combination with FeCl(2) or FeCl(3), as well as transferrin or lactoferrin did not support growth, indicating that B. henselae cannot synthesize heme itself. Hemin supported growth even when free iron was chelated, indicating that hemin is also used as an iron source. Binding assays showed that hemin starvation increased the binding capacity of B. henselae for hemin, providing evidence that the bacteria carry a specific hemin uptake system, which might be regulated by hemin.

Identification of iron-regulated genes of Helicobacter pylori by a modified fur titration assay (FURTA-Hp).

The Escherichia coli-based Fur titration assay (FURTA), although a powerful tool for identification of genes regulated by the ferric uptake regulator (Fur), was unsuccessful for the gastric pathogen Helicobacter pylori. The FURTA was modified by construction of an E. coli indicator strain producing H. pylori Fur only. The promoter regions of the ferric citrate receptor homolog fecA2 and the riboflavin synthesis gene ribBA were both positive in the modified FURTA, but negative in the original FURTA. Transcription of fecA2 and ribBA was demonstrated to be iron-repressed in H. pylori. This type of modification should allow FURTA analysis for bacteria with Fur binding sequences poorly recognized by E. coli Fur.

The role of Helicobacter pylori virulence factors in interleukin production by monocytic cells.

Helicobacter pylori infection results in chronic gastritis, which is initiated by the release of cytokines like interleukin (IL)-12 and IL-8 from mononuclear cells, and IL-8 from gastric epithelial cells. The severity of gastritis is influenced both by host factors and by bacterial factors such as the Cag proteins and the vacuolating cytotoxin VacA. Amounts of IL-12 and IL-8 produced by monocytic THP-1 cells differed considerably between the eight H. pylori isolates tested, but in contrast to H. pylori-induced IL-8 production by gastric epithelial cells, did not correlate to the Cag and VacA types of the strains. Apparently, in addition to Cag and VacA, other bacterial factors determine the extent in which H. pylori induced IL production in monocytes.

Susceptibility in vitro of Helicobacter pylori to cetylpyridinium chloride.

The antimicrobial agent cetylpyridinium chloride (CPC) which is used in therapy of oro-pharyngeal infections and for antiseptic treatment of the oral cavity is active against different bacterial species. Determination of the minimal inhibitory concentration (MIC) using the agar dilution technique revealed that the gastric pathogen Helicobacter pylori in vitro is highly susceptible to CPC as indicated by an MIC of 10 microM (3.4 microg ml(-1)) which was significantly lower than the MIC of CPC against other bacterial species, which were analyzed in comparison to H. pylori. Bacteria of the genus Campylobacter, various Streptococcus spp., Staphylococcus aureus and Escherichia coli showed higher MICs ranging from 100 microM to 2 mM. In summary, this finding renders CPC-containing drugs candidates possibly useful for eradication or for the prevention of transmission of the gastric pathogen.

Visualization of capsule formation by Erwinia amylovora and assays to determine amylovoran synthesis.

Exopolysaccharide (EPS) synthesis by Erwinia amylovora depends on environmental and genetic predispositions. To measure the amount of the acidic EPS amylovoran synthesized by E. amylovora cell cultures, a turbidity assay using cetylpyridinium salt was developed. The EPS produced by bacteria grown on solid media was additionally characterized by its water content. The amylovoran capsules were visualized in situ by staining with fluorescein isothiocyanate (FITC)-labelled lectin from Abrus precatorius, which reacts with the galactose residue of the EPS side chain. The staining and the turbidity assays were applied to suspension cell cultures or to cells from colonies and did not require any purification steps. Lectin staining was superior to electron microscopic (EM) techniques for visualization of capsules. For EM, the capsule was stabilized with polycationic ferritin. In contrast to lectin staining, only a small fraction of the cells was found to be EPS-coated in the EM assay. An increase in capsulation and in amylovoran production was found in conjunction with mutations in a ribosomal protein conferring resistance to streptomycin. Furthermore, the presence of sorbitol in the growth environment resulted in high synthesis of amylovoran. Cells in the stationary growth phase continued to produce amylovoran. Apparently, the strong dependence of the fireblight pathogen on capsules requires the capacity for EPS synthesis in all growth stages in order to escape plant defence reactions.

Selective gelatinase inhibition reduces apoptosis and pro-inflammatory immune cell responses in Campylobacter jejuni-infected gnotobiotic IL-10 deficient mice.

Increased levels of the matrix metalloproteinases-2 and -9 (also referred to gelatinase-A and -B, respectively) can be detected in intestinal inflammation. We have recently shown that selective gelatinase blockage by the synthetic compound RO28-2653 ameliorates acute murine ileitis and colitis. We here investigated whether RO28-2653 exerts anti-inflammatory effects in acute Campylobacter jejuni-induced enterocolitis of gnotobiotic IL-10(-/-) mice generated following antibiotic treatment. Mice were perorally infected with C. jejuni (day 0) and either treated with RO28-2653 (75 mg/kg body weight/day) or placebo from day 1 until day 6 post infection (p.i.) by gavage. Irrespective of the treatment, infected mice displayed comparable pathogen loads within the gastrointestinal tract. Following RO28-2653 administration, however, infected mice exhibited less severe symptoms such as bloody diarrhea as compared to placebo controls. Furthermore, less distinct apoptosis but higher numbers of proliferating cells could be detected in the colon of RO28-2653-treated as compared to placebo-treated mice at day 7 p.i. Remarkably, gelatinase blockage resulted in lower numbers of T- and B-lymphocytes as well as macrophages and monocytes in the colonic mucosa of C. jejuni-infected gnotobiotic IL-10(-/-) mice. Taken together, synthetic gelatinase inhibition exerts anti-inflammatory effects in experimental campylobacteriosis.

The intestinal micro-environment imprints stromal cells to promote efficient Treg induction in gut-draining lymph nodes.

De novo induction of Foxp3⁺ regulatory T cells (Tregs) is particularly efficient in gut-draining mesenteric and celiac lymph nodes (mLN and celLN). Here we used LN transplantations to dissect the contribution of stromal cells and environmental factors to the high Treg-inducing capacity of these LN. After transplantation into the popliteal fossa, mLN and celLN retained their high Treg-inducing capacity, whereas transplantation of skin-draining LN into the gut mesenteries did not enable efficient Treg induction. However, de novo Treg induction was abolished in the absence of dendritic cells (DC), indicating that this process depends on synergistic contributions of stromal and DC. Stromal cells themselves were influenced by environmental signals as mLN grafts taken from germ-free donors and celLN grafts taken from vitamin A-deficient donors did not show any superior Treg-inducing capacity. Collectively, our observations reveal a hitherto unrecognized role of LN stromal cells for the de novo induction of Foxp3⁺ Tregs.

Can microbiota transplantation abrogate murine colonization resistance against Campylobacter jejuni?

Enterocolitis caused by Campylobacter jejuni represents an important socioeconomic burden worldwide. The host-specific intestinal microbiota is essential for maintaining colonization resistance (CR) against C. jejuni in conventional mice. Notably, CR is abrogated by shifts of the intestinal microbiota towards overgrowth with commensal E. coli during acute ileitis. Thus, we investigated whether oral transplantation (TX) of ileal microbiota derived from C. jejuni susceptible mice with acute ileitis overcomes CR of healthy conventional animals. Four days following ileitis microbiota TX or ileitis induction and right before C. jejuni infection, mice displayed comparable loads of main intestinal bacterial groups as shown by culture. Eight days following ileitis induction, but not ileal microbiota TX, however, C. jejuni could readily colonize the gastrointestinal tract of conventional mice and also translocate to extra-intestinal tissue sites such as mesenteric lymph nodes, spleen, liver, and blood within 4 days following oral infection. Of note, C. jejuni did not further deteriorate histopathology following ileitis induction. Lack of C. jejuni colonization in TX mice was accompanied by a decrease of commensal E. coli loads in the feces 4 days following C. jejuni infection. In summary, oral ileal microbiota TX from susceptible donors is not sufficient to abrogate murine CR against C. jejuni.

Pro-inflammatory potential of Escherichia coli strains K12 and Nissle 1917 in a murine model of acute ileitis.

Non-pathogenic Escherichia coli (Ec) strains K12 (EcK12) and Nissle 1917 (EcN) are used for gene technology and probiotic treatment of intestinal inflammation, respectively. We investigated intestinal colonization and potential pro-inflammatory properties of EcK12, EcN, and commensal E. coli (EcCo) strains in Toxoplasma (T.) gondii-induced acute ileitis. Whereas gnotobiotic animals generated by quintuple antibiotic treatment were protected from ileitis, mice replenished with conventional microbiota suffered from small intestinal necrosis 7 days post-T. gondii infection (p.i.). Irrespective of the Ec strain, recolonized mice revealed mild to moderate histopathological changes in their ileal mucosa. Upon stable recolonization with EcK12, EcN, or EcCo, development of inflammation was accompanied by pro-inflammatory responses at day 7 p.i., including increased ileal T lymphocyte and apoptotic cell numbers compared to T. gondii-infected gnotobiotic controls. Strikingly, either Ec strain was capable to translocate to extra-intestinal locations, such as MLN, spleen, and liver. Taken together, Ec strains used in gene technology and probiotic treatment are able to exert inflammatory responses in a murine model of small intestinal inflammation. In conclusion, the therapeutic use of Ec strains in patients with broad-spectrum antibiotic treatment and/or intestinal inflammation should be considered with caution.

Campylobacter jejuni infection of infant mice: acute enterocolitis is followed by asymptomatic intestinal and extra-intestinal immune responses.

Campylobacter (C.) jejuni is among the leading bacterial agents causing enterocolitis worldwide. Despite the high prevalence of C. jejuni infections and its significant medical and economical consequences, intestinal pathogenesis is poorly understood. This is mainly due to the lack of appropriate animal models. In the age of 3 months, adult mice display strong colonization resistance (CR) against C. jejuni. Previous studies underlined the substantial role of the murine intestinal microbiota in maintaining CR. Due to the fact that the host-specific gut flora establishes after weaning, we investigated CR against C. jejuni in 3-week-old mice and studied intestinal and extra-intestinal immunopathogenesis as well as age dependent differences of the murine colon microbiota. In infant animals infected orally immediately after weaning C. jejuni strain B2 could stably colonize the gastrointestinal tract for more than 100 days. Within six days following infection, infant mice developed acute enterocolitis as indicated by bloody diarrhea, colonic shortening, and increased apoptotic cell numbers in the colon mucosa. Similar to human campylobacteriosis clinical disease manifestations were self-limited and disappeared within two weeks. Interestingly, long-term C. jejuni infection was accompanied by distinct intestinal immune and inflammatory responses as indicated by increased numbers of T- and B-lymphocytes, regulatory T-cells, neutrophils, as well as apoptotic cells in the colon mucosa. Strikingly, C. jejuni infection also induced a pronounced influx of immune cells into extra-intestinal sites such as liver, lung, and kidney. Furthermore, C. jejuni susceptible weaned mice harbored a different microbiota as compared to resistant adult animals. These results support the essential role of the microflora composition in CR against C. jejuni and demonstrate that infant mouse models resemble C. jejuni mediated immunopathogenesis including the characteristic self-limited enterocolitis in human campylobacteriosis. Furthermore, potential clinical and immunological sequelae of chronic C. jejuni carriers in humans can be further elucidated by investigation of long-term infected infant mice. The observed extraintestinal disease manifestations might help to unravel the mechanisms causing complications such as reactive arthritis or Guillain-Barré syndrome.

Campylobacter jejuni induces extra-intestinal immune responses via Toll-like-receptor-4 signaling in conventional IL-10 deficient mice with chronic colitis.

Campylobacter jejuni is one of the predominant causes for foodborne bacterial infections worldwide. We investigated whether signaling of C. jejuni-lipoproteins and -lipooligosaccharide via Toll-like-receptor (TLR) -2 and -4, respectively, is inducing intestinal and extra-intestinal immune responses following infection of conventional IL-10(-/-) mice with chronic colitis. At day 3 following oral infection, IL-10(-/-) mice lacking TLR-2 or TLR-4 harbored comparable C. jejuni strain ATCC 43431 loads in their colon. Interestingly, infected TLR-4(-/-) IL-10(-/-) mice displayed less compromized epithelial barrier function as indicated by lower translocation rates of live gut commensals into mesenteric lymphnodes (MLNs), and exhibited less distinct B lymphocyte responses in their colonic mucosa as compared to naїve IL-10(-/-) controls. Furthermore, in extra-intestinal compartments such as MLNs and spleens, abundance of myeloid cells was less distinct whereas relative percentages of activated T helper cells and cytotoxic T cells were higher in spleens and dendritic cells more abundant in MLNs of infected IL-10(-/-) animals lacking TLR-4 as compared to IL-10(-/-) controls. Taken together, in conventionally colonized IL-10(-/-) mice, TLR-4, but not TLR-2, is involved in mediating extra-intestinal pro-inflammatory immune responses following C. jejuni infection. Thus, conventional IL-10(-/-) mice are well suited to further dissect mechanisms underlying Campylobacter infections in vivo.

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10 ameliorates acute DSS colitis.

In experimental models of and humans with intestinal inflammation, increased levels of the matrix-degrading gelatinases MMP-2 and -9 in inflamed tissues can be detected. The synthetic collagen analogue (Gly-Pro-Hyp)10, (GPO)10, has been identified as a relevant binding structure for proMMP-2/-9 and promotes enzymatic activity of proMMP-2. Since targeted MMP strategies might offer promising anti-inflammatory treatment options, we for the first time studied in vivo actions exerted by (GPO)10 applying an acute dextrane sulfate sodium (DSS) induced colitis model. Seven-day intraperitoneal (GPO)10 treatment ameliorated clinical symptoms and histopathological colonic changes as compared to placebo controls with severe colitis. (GPO)10-treated mice displayed a diminished influx of neutrophils, and T- and B-lymphocytes into their colonic mucosa whereas numbers of regulatory T-cells and regenerative cells were higher as compared to placebo controls. Furthermore, IL-6 secretion was down-regulated in ex vivo colonic biopsies derived from (GPO)10-treated mice whereas higher concentrations of the anti-inflammatory cytokine IL-10 in extra-intestinal compartments such as MLN and spleen could be detected. Strikingly, influx of inflammatory cells into lungs was abolished following (GPO)10 application. We therefore propose (GPO)10 as a promising effective and safe treatment option of intestinal and extra-intestinal inflammatory conditions in humans.

Mesenteric fat - control site for bacterial translocation in colitis?

In Crohn's disease bacteria could be detected in the adjacent mesenteric fat characterized by hypertrophy of unknown function. This study aimed to define effector responses of this compartment induced by bacterial translocation during intestinal inflammation. Dextran sulfate sodium-induced colitis served as a model of intestinal inflammation. Translocation of peptides and bacteria into mesenteric fat was evaluated. Innate functions of mesenteric fat and epithelium were characterized at whole tissue, cellular, and effector molecule levels. Orally applied peptides translocated in healthy wild-type (WT) mice. Bacterial translocation was not detected in healthy and acute but increased in chronic colitis. Mesenteric fat from colitic mice released elevated levels of cytokines and was infiltrated by immune cells. In MyD88(-/-) mice bacterial translocation occurred in health and increased in colitis. The exaggerated cytokine production in mesenteric fat accompanying colonic inflammation in WT mice was less distinct in MyD88(-/-) mice. In vitro studies revealed that fat not only increases cytokine production following contact with bacterial products, but also that preadipocytes are potent phagocytes. Colonic inflammation is accompanied by massive cytokine production and immune cell infiltration in adjacent adipose tissue. These effects can be considered as protective mechanisms of the mesenteric fat in the defense of bacterial translocation.

What you eat is what you get: Novel Campylobacter models in the quadrangle relationship between nutrition, obesity, microbiota and susceptibility to infection.

Enterocolitis caused by Campylobacter jejuni-infections represents an important socioeconomic burden worldwide. Recent results from novel murine infection models reveal that the intestinal microbiota is essential for maintaining colonization resistance against C. jejuni. We extended these studies to investigate the role of nutrition and obesity in susceptibility to C. jejuni-infection. Gnotobiotic (GB) mice generated by antibiotic treatment, which were fed with a human cafeteria diet (CAF), as well as obese (ob/ob) mice with a conventional microbiota harbored higher Escherichia coli loads in their colon as compared to respective controls. Following oral infection, C. jejuni 43431 ATCC readily colonized the intestines of CAF and ob/ob mice, whereas GB mice fed with a standard chow (MUD) eradicated the pathogen within days. Furthermore, live C. jejuni translocated into mesenteric lymph nodes of CAF, but not MUD mice. Strikingly, stably infected animals developed enterocolitis as indicated by increased numbers of immune and apoptotic cells in the colon in situ. We conclude that a specific human diet and obesity render mice susceptible to C. jejuni infection. The corresponding murine models are excellently suited for the study of C. jejuni pathogenesis and will help to get further insights into interplays between C. jejuni, microbiota, diet, obesity and immunity.

The distinct roles of MMP-2 and MMP-9 in acute DSS colitis.

Expression of gelatinases A and B, also referred to matrixmetalloproteinases (MMP)-2 and -9, respectively, is increased in inflamed tissues of experimental intestinal inflammation and humans with inflammatory bowel disease (IBDs). Given that we recently reported that treatment with the selective gelatinase inhibitor RO28-2653 ameliorates acute dextrane sulfate sodium (DSS) colitis, we asked whether gelatinase A or B expression is pivotal in mediating large intestinal inflammation. Results from our study reveal that symptoms of acute DSS colitis as well as histopathological colonic changes were ameliorated in MMP-2-, but not MMP-9-deficient mice, and were paralleled by a diminished influx of immune cells. In MMP-2-deficient mice, we observed lower expression of pro-inflammatory cytokines including interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-6 in colonic biopsies and less overgrowth of the colonic lumen by potentially pro-inflammatory enterobacteria from the commensal gut microbiota. We conclude that rather MMP-2 than MMP-9 is causative for the establishment of DSS colitis in mice. The discrepancy of these data to prior reports might be due to substantial differences in the intestinal microbiota composition of the mice bred at different animal facilities impacting susceptibility to inflammatory stimuli. Consequently, a detailed survey of the gut microbiota should be implemented in immunological/inflammatory studies in the future in order to allow comparison of data from different facilities.