Single-day and multi-day exposure to orogastric gavages does not affect intestinal barrier function in mice.

Animals involved in common laboratory procedures experience minor levels of stress. The direct effect of limited amounts of stress on gastrointestinal function has not been reported yet. Therefore, this study aimed to assess the effect of single-day and multi-day orogastric gavages on gut physiology in mice. To this end, 12-wk-old female C57Bl6/J mice were randomized to receive treatment with sterile water (200 µL) delivered by orogastric gavages twice daily for a total of 1 or 10 day(s). Control animals did not receive any treatment. Subsequently, gastrointestinal function was assessed by measuring fecal pellet production. Furthermore, ex vivo intestinal barrier and secretory function of the distal colon, proximal colon, and terminal ileum were quantified in Ussing chambers. In mice, single-day gavages did neither influence corticosterone levels nor gastrointestinal function. In mice exposed to multi-day gavages, corticosterone levels were slightly but significantly increased compared with controls after 10 days of treatment. Gastrointestinal motor function was altered, as evidenced by increased fecal pellet counts and a small increase in fecal water content. However, exposure to repeated gavages did not lead to detectable alterations in gastrointestinal barrier function as quantified by the paracellular flux of the probe 4 kDa FITC-dextran as well as transepithelial resistance measurements. Thus, the administration of drugs via single-day or multi-day orogastric gavages leads to no or minor stress in mice, respectively. In both cases, it does not hamper the study of the intestinal barrier function and therefore remains a valuable administration route in preclinical pharmacological research.NEW & NOTEWORTHY Exposure of mice to serial orogastric gavages over the course of 10 days leads to a small but significant increase in plasma corticosterone levels, indicating the presence of a limited amount of stress that is absent after a single-day treatment. This minor stress after multi-day gavages results in increased fecal pellet production and fecal water content in exposed compared with nontreated mice but does not affect the intestinal barrier function in the distal colon, proximal colon, or terminal ileum.

Helicobacter bizzozeronii infection in a girl with severe gastric disorders in México: case report.

BACKGROUND: Gastric non-Helicobacter pylori helicobacters (NHPH) naturally colonize the stomach of animals. In humans, infection with these bacteria is associated with chronic active gastritis, peptic ulceration and MALT-lymphoma. H. bizzozeronii belongs to these NHPH and its prevalence in children is unknown. CASE PRESENTATION: This case report describes for the first time a NHPH infection in a 20-month-old girl with severe gastric disorders in Mexico. The patient suffered from melena, epigastric pain, and bloating. Gastroscopy showed presence of a Hiatus Hill grade I, a hemorrhagic gastropathy in the fundus and gastric body, and a Forrest class III ulcer in the fundus. Histopathologic examination revealed a chronic active gastritis with presence of long, spiral-shaped bacilli in the glandular lumen. Biopsies from antrum, body and incisure were negative for presence of H. pylori by culture and PCR, while all biopsies were positive for presence of H. bizzozeronii by PCR. Most likely, infection occurred through intense contact with the family dog. The patient received a triple therapy consisting of a proton pump inhibitor, clarithromycin, and amoxicillin for 14 days, completed with sucralfate for 6 weeks, resulting in the disappearance of her complaints. CONCLUSION: The eradication could not be confirmed, although it was suggested by clear improvement of symptoms. This case report further emphasizes the zoonotic importance of NHPH. It can be advised to routinely check for presence of both H. pylori and NHPH in human patients with gastric complains.

Comparative genomics of Flavobacterium columnare unveils novel insights in virulence and antimicrobial resistance mechanisms.

This study reports the comparative analyses of four Flavobacterium columnare isolates that have different virulence and antimicrobial resistance patterns. The main research goal was to reveal new insights into possible virulence genes by comparing the genomes of bacterial isolates that could induce tissue damage and mortality versus the genome of a non-virulent isolate. The results indicated that only the genomes of the virulent isolates possessed unique genes encoding amongst others a methyl-accepting chemotaxis protein possibly involved in the initial colonization of tissue, and several VgrG proteins engaged in interbacterial competition. Furthermore, comparisons of genes unique for the genomes of the highly virulent (HV) carp and trout isolates versus the, respectively, low and non-virulent carp and trout isolates were performed. An important part of the identified unique virulence genes of the HV-trout isolate was located in one particular gene region identified as a genomic island. This region contained araC and nodT genes, both linked to pathogenic and multidrug-resistance, and a luxR-gene, functional in bacterial cell-to-cell communication. Furthermore, the genome of the HV-trout isolate possessed unique sugar-transferases possibly important in bacterial adhesion. The second research goal was to obtain insights into the genetic basis of acquired antimicrobial resistance. Several point-mutations were discovered in gyrase-genes of an isolate showing phenotypic resistance towards first and second-generation quinolones, which were absent in isolates susceptible to quinolones. Tetracycline-resistance gene tetA was found in an isolate displaying acquired phenotypic resistance towards oxytetracycline. Although not localized on a prophage, several flanking genes were indicative of the gene's mobile character.

Biopsy Sampling in Upper Gastrointestinal Endoscopy: A Survey from 10 Tertiary Referral Centres Across Europe.

BACKGROUND: Guidelines give robust recommendations on which biopsies should be taken when there is endoscopic suggestion of gastric inflammation. Adherence to these guidelines often seems arbitrary. This study aimed to give an overview on current practice in tertiary referral centres across Europe. METHODS: Data were collected at 10 tertiary referral centres. Demographic data, the indication for each procedure, endoscopic findings, and the number and sampling site of biopsies were recorded. Findings were compared between centres, and factors influencing the decision to take biopsies were explored. RESULTS: Biopsies were taken in 56.6% of 9,425 procedures, with significant variation between centres (p < 0.001). Gastric biopsies were taken in 43.8% of all procedures. Sampling location varied with the procedure indication (p < 0.001) without consistent pattern across the centres. Fewer biopsies were taken in centres which routinely applied the updated Sydney classification for gastritis assessment (46.0%), compared to centres where this was done only upon request (75.3%, p < 0.001). This was the same for centres stratifying patients according to the OLGA system (51.8 vs. 73.0%, p < 0.001). More biopsies were taken in centres following the MAPS guidelines on stomach surveillance (68.1 vs. 37.1%, p < 0.001). Biopsy sampling was more likely in younger patients in 8 centres (p < 0.05), but this was not true for the whole cohort (p = 0.537). The percentage of procedures with biopsies correlated directly with additional costs charged in case of biopsies (r = 0.709, p = 0.022). CONCLUSION: Adherence to guideline recommendations for biopsy sampling at gastroscopy was inconsistent across the participating centres. Our data suggest that centre-specific policies are applied instead.

Gastric Helicobacter suis Infection Partially Protects against Neurotoxicity in A 6-OHDA Parkinson's Disease Mouse Model.

The exact etiology of Parkinson's disease (PD) remains largely unknown, but more and more research suggests the involvement of the gut microbiota. Interestingly, idiopathic PD patients were shown to have at least a 10 times higher prevalence of Helicobacter suis (H. suis) DNA in gastric biopsies compared to control patients. H. suis is a zoonotic Helicobacter species that naturally colonizes the stomach of pigs and non-human primates but can be transmitted to humans. Here, we investigated the influence of a gastric H. suis infection on PD disease progression through a 6-hydroxydopamine (6-OHDA) mouse model. Therefore, mice with either a short- or long-term H. suis infection were stereotactically injected with 6-OHDA in the left striatum and sampled one week later. Remarkably, a reduced loss of dopaminergic neurons was seen in the H. suis/6-OHDA groups compared to the control/6-OHDA groups. Correspondingly, motor function of the H. suis-infected 6-OHDA mice was superior to that in the non-infected 6-OHDA mice. Interestingly, we also observed higher expression levels of antioxidant genes in brain tissue from H. suis-infected 6-OHDA mice, as a potential explanation for the reduced 6-OHDA-induced cell loss. Our data support an unexpected neuroprotective effect of gastric H. suis on PD pathology, mediated through changes in oxidative stress.

Distinct transcriptome signatures of Helicobacter suis and Helicobacter heilmannii strains upon adherence to human gastric epithelial cells.

The porcine Helicobacter suis and canine-feline H. heilmannii are gastric Helicobacter species with zoonotic potential. However, little is known about the pathogenesis of human infections with these Helicobacter species. To gain more insight into the interactions of both zoonotic Helicobacter species with human gastric epithelial cells, we investigated bacterial genes that are differentially expressed in a H. suis and H. heilmannii strain after adhesion to the human gastric epithelial cell line MKN7. In vitro Helicobacter-MKN7 binding assays were performed to obtain bacterial RNA for sequencing analysis. H. suis and H. heilmannii bacteria attached to the gastric epithelial cells (i.e. cases) as well as unbound bacteria (i.e. controls) were isolated, after which prokaryotic RNA was purified and sequenced. Differentially expressed genes were identified using the DESeq2 package and SARTools pipeline in R. A list of 134 (83 up-regulated and 51 down-regulated) and 143 (60 up-regulated and 83 down-regulated) differentially expressed genes (padj ≤ 0.01; fold change ≥ 2) were identified for the adherent H. suis and H. heilmannii strains, respectively. According to BLASTp analyses, only 2 genes were commonly up-regulated and 4 genes commonly down-regulated in both pathogens. Differentially expressed genes of the H. suis and H. heilmannii strains belonged to multiple functional classes, indicating that adhesion of both strains to human gastric epithelial cells evokes pleiotropic adaptive responses. Our results suggest that distinct pathways are involved in human gastric colonization of H. suis and H. heilmannii. Further research is needed to elucidate the clinical significance of these findings.

Review: Other Helicobacter species.

This review covers the most important, accessible, and relevant literature published between April 2019 and April 2020 in the field of non-Helicobacter pylori Helicobacter species (NHPH). The initial part of the review covers new insights regarding the presence of gastric and enterohepatic NHPH in humans and animals, while the subsequent section focuses on the progress in our understanding of animal models, the pathogenicity and omics of these species. Over the last year, the clinical relevance of gastric NHPH infections in humans was highlighted. With regard to NHPH in animals, the ancestral source of Helicobacter suis was further established showing that Cynomolgus macaques are the common ancestor of the pig-associated H. suis population, and 3 novel Helicobacter species isolated from the gastric mucosa of red foxes were described. "Helicobacter burdigaliensis" sp nov. and "Helicobacter labetoulli" sp nov. were proposed as novel enterohepatic Helicobacter species associated with human digestive diseases. An analysis of Helicobacter cinaedi recurrent infections in humans proposed long-term antibiotic therapies. Several studies using rodent models further elucidated the mechanisms underlying the development of NHPH-related disease, as well as intestinal immunity in inflammatory bowel disease models. Omics approaches supported Helicobacteraceae taxonomy and unraveled the transcriptomic signatures of H. suis and Helicobacter heilmannii upon adherence to the human gastric epithelium. With regard to virulence, data showed that the nuclear remodeling promoted by cytolethal distending toxin of Helicobacters involves the MAFB oncoprotein and is associated with nucleoplasmic reticulum formation in surviving cells.

Review: Other Helicobacter species.

This article is a review of the most important, accessible, and relevant literature published between April 2018 and April 2019 in the field of Helicobacter species other than Helicobacter pylori. The initial part of the review covers new insights regarding the presence of gastric and enterohepatic non-H. pylori Helicobacter species (NHPH) in humans and animals, while the subsequent section focuses on the progress in our understanding of the pathogenicity and evolution of these species. Over the last year, relatively few cases of gastric NHPH infections in humans were published, with most NHPH infections being attributed to enterohepatic Helicobacters. A novel species, designated "Helicobacter caesarodunensis," was isolated from the blood of a febrile patient and numerous cases of human Helicobacter cinaedi infections underlined this species as a true emerging pathogen. With regard to NHPH in animals, canine/feline gastric NHPH cause little or no harm in their natural host; however they can become opportunistic when translocated to the hepatobiliary tract. The role of enterohepatic Helicobacter species in colorectal tumors in pets has also been highlighted. Several studies in rodent models have further elucidated the mechanisms underlying the development of NHPH-related disease, and the extra-gastric effects of a Helicobacter suis infection on brain homeostasis was also studied. Comparative genomics facilitated a breakthrough in the evolutionary history of Helicobacter in general and NHPH in particular. Investigation of the genome of Helicobacter apodemus revealed particular traits with regard to its virulence factors. A range of compounds including mulberries, dietary fiber, ginseng, and avian eggs which target the gut microbiota have also been shown to affect Helicobacter growth, with a potential therapeutic utilization and increase in survival.

The effect of a commercial competitive exclusion product on the selection of enrofloxacin resistance in commensal E. coli in broilers.

The effect of a competitive exclusion product (Aviguard®) on the selection of fluoroquinolone resistance in poultry was assessed in vivo in the absence or presence of fluoroquinolone treatment. Two experiments using a controlled seeder-sentinel animal model (2 seeders: 4 sentinels per group) with one-day-old chicks were used. For both experiments, as soon as the chicks were hatched, the birds of two groups were administered Aviguard® and two groups were left untreated. Three days later, all groups were inoculated with an enrofloxacin-susceptible commensal E. coli strain. Five days after hatching, two birds per group were inoculated with either a bacteriologically fit or a bacteriologically non-fit enrofloxacin-resistant commensal E. coli strain. In experiment 2, all groups were orally treated for three consecutive days (days 8-10) with enrofloxacin. Throughout the experiments, faecal excretion of all inoculated E. coli strains was determined on days 2, 5, 8, 11, 18 and 23 by selective plating (via spiral plater). Linear mixed models were used to assess the effect of Aviguard® on the selection of fluoroquinolone resistance. The use of Aviguard® (P < 0.01) reduced the excretion of enrofloxacin-resistant E. coli when no enrofloxacin treatment was administered. However, this beneficial effect disappeared (P = 0.37) when the birds were treated with enrofloxacin. Similarly, bacterial fitness of the enrofloxacin-resistant E. coli strain used for inoculation had an effect (P < 0.01) on the selection of enrofloxacin resistance when no treatment was administered, whereas this effect was no longer present when enrofloxacin was administered (P = 0.70). Thus, enrofloxacin treatment cancelled the beneficial effects from administrating Aviguard® in one-day-old broiler chicks and resulted in an enrofloxacin-resistant flora. RESEARCH HIGHLIGHTS The effect of Aviguard® on the selection of enrofloxacin resistance was assessed in vivo. Without enrofloxacin, Aviguard® reduced the selection of enrofloxacin resistance. When enrofloxacin was administered, it cancelled the beneficial effect of Aviguard®.

Studying the effect of administration route and treatment dose on the selection of enrofloxacin resistance in commensal Escherichia coli in broilers.

Objectives: Factors potentially contributing to fluoroquinolone resistance selection in commensal Escherichia coli strains in poultry were studied through a series of in vivo experiments. The effect of the initial prevalence of enrofloxacin resistance in the E. coli gut microbiota, effect of the bacterial fitness of the enrofloxacin-resistant strain and effect of treatment with enrofloxacin (effect of dose and effect of route of administration) were assessed. Methods: Four in vivo studies with broiler chickens were performed. Right after hatching, the chicks were inoculated with either a bacteriologically fit or a bacteriologically non-fit fluoroquinolone-resistant strain as either a minority or the majority of the total E. coli population. Six days later, the chicks were treated for three consecutive days either orally or parenterally and using three different doses (under-, correct- and over-dose) of enrofloxacin. The faecal shedding of E. coli strains was quantified by plating on agar plates either supplemented or not supplemented with enrofloxacin. Linear mixed models were used to assess the effect of the aforementioned variables on the selection of enrofloxacin resistance. Results: The factors that significantly contributed were treatment ( P < 0.001), bacterial fitness of the resistant donor strain ( P < 0.001), administration route ( P = 0.052) and interactions between bacterial fitness and administration route ( P < 0.001). Conclusions: In the currently used models, fluoroquinolone resistance selection was influenced by treatment, bacterial fitness of the inoculation strain and administration route. The use of oral treatment seems to select more for fluoroquinolone resistance, particularly in the model where a non-fit strain was used for inoculation.

A novel isolation protocol and probe-based RT-PCR for diagnosis of gastric infections with the zoonotic pathogen Helicobacter suis.

BACKGROUND: Helicobacter suis is a very fastidious microorganism associated with gastritis, gastric ulcers, and mucosa-associated lymphoid tissue lymphoma in humans. In vitro isolation of this agent from human patients has so far been unsuccessful. MATERIALS AND METHODS: A probe-based real-time PCR (RT-PCR) for the rapid detection of H. suis in gastric biopsies was developed. Secondly, a mouse-passage-based protocol was optimized for isolation of low numbers of viable H. suis bacteria. Mice were inoculated with different numbers of viable H. suis (102 -108 ) and kept for 4 weeks to allow multiplication of this pathogen. RESULTS: The probe-based real-time PCR (RT-PCR) exhibited a high degree of diagnostic specificity and analytical sensitivity, high linear correlations (r2 between 0.995 and 0.999), and high amplification efficiencies (>90%) for H. suis. No cross-reactivity was detected with human, porcine, non-human primate, and murine DNA nor with DNA from other bacteria including Helicobacter spp. and Campylobacter spp. H. suis was successfully re-isolated from the stomach of mice inoculated with at least 104 viable H. suis, using a biphasic medium (pH 5), consisting of Brucella agar with Brucella broth on top, both supplemented with vitox supplement, Campylobacter-selective supplement, amphotericin (5 µg/mL), HCl (0.05%), fetal bovine serum (20%), and linezolid (5 µg/mL). Linezolid was necessary to inhibit proliferation of contaminants, including lactobacilli. CONCLUSION: The methods described above can be implemented for detection or isolation of H. suis from human gastric biopsies.

Comparative virulence of in vitro-cultured primate- and pig-associated Helicobacter suis strains in a BALB/c mouse and a Mongolian gerbil model.

BACKGROUND: Helicobacter suis (H. suis) is the most prevalent gastric non-H. pylori Helicobacter species in humans. This bacterium mainly colonizes the stomach of pigs, but it has also been detected in the stomach of nonhuman primates. The aim of this study was to obtain better insights into potential differences between pig- and primate-associated H. suis strains in virulence and pathogenesis. MATERIALS AND METHODS: In vitro-isolated H. suis strains obtained from pigs, cynomolgus monkeys (Macaca fascicularis), and rhesus monkeys (Macaca mulatta) were used for intragastric inoculation of BALB/c mice and Mongolian gerbils. Nine weeks and six months later, samples of the stomach of inoculated and control animals were taken for PCR analysis and histopathological examination. RESULTS: The cynomolgus monkey-associated H. suis strain only colonized the stomach of mice, but not of Mongolian gerbils. All other H. suis strains colonized the stomach in both rodent models. In all colonized animals, severe gastric inflammation was induced. Gastric lymphoid follicles and destruction of the antral epithelium were observed in infected gerbils, but not in mice. Infection with both pig- and primate-associated H. suis strains evoked a similar marked Th17 response in mice and gerbils, accompanied by increased CXCL-13 expression levels. CONCLUSIONS: Apart from the cynomolgus monkey-associated strain which was unable of colonizing the stomach of Mongolian gerbils, no substantial differences in virulence were found in rodent models between in vitro-cultured pig-associated, cynomolgus monkey-associated and rhesus monkey-associated H. suis strains. The experimental host determines the outcome of the immune response against H. suis infection, rather than the original host.

Species-specific immunity to Helicobacter suis.

BACKGROUND: Helicobacter (H.) suis is mainly associated with pigs, but is also the most prevalent gastric non-H. pylori Helicobacter species found in humans. Both H. pylori and H. suis may cause persistent infection of the stomach. Several immune evasion mechanisms have been proposed for H. pylori, which focus to a great extent on its major virulence factors, which are absent in H. suis. The aim of this study was to gain more knowledge on immune evasion by H. suis. MATERIALS AND METHODS: Cytokine expression kinetics were monitored in the stomach of BALB/c mice experimentally infected with H. suis. The cytokine expression profile in the stomach of naturally H. suis-infected pigs was also determined. Subsequently, the effect of H. suis on murine and porcine dendritic cell (DC) maturation and their ability to elicit T-cell effector responses was analyzed. RESULTS: Despite a Th17/Th2 response in the murine stomach, the inflammatory cell influx was unable to clear H. suis infection. H. suis-stimulated murine bone marrow-derived dendritic cells induced IL-17 secretion by CD4+ cells in vitro. Natural H. suis infection in pigs evoked increased expression levels of IL-17 mRNA in the antrum and IL-10 mRNA in the fundus. In contrast to mice, H. suis-stimulated porcine monocyte-derived dendritic cells were unable to express MHCII molecules on their cell surface. These semimature DCs induced proliferation of T-cells, which showed an increased expression of TGF-ß and FoxP3 mRNA levels. CONCLUSIONS: Helicobacter suis might evade host immune responses by skewing toward a Treg-biased response.

Divergence between the Highly Virulent Zoonotic Pathogen Helicobacter heilmannii and Its Closest Relative, the Low-Virulence "Helicobacter ailurogastricus" sp. nov.

Helicobacter heilmannii naturally colonizes the stomachs of dogs and cats and has been associated with gastric disorders in humans. Nine feline Helicobacter strains, classified as H. heilmannii based on ureAB and 16S rRNA gene sequences, were divided into a highly virulent and a low-virulence group. The genomes of these strains were sequenced to investigate their phylogenetic relationships, to define their gene content and diversity, and to determine if the differences in pathogenicity were associated with the presence or absence of potential virulence genes. The capacities of these helicobacters to bind to the gastric mucosa were investigated as well. Our analyses revealed that the low-virulence strains do not belong to the species H. heilmannii but to a novel, closely related species for which we propose the name Helicobacter ailurogastricus. Several homologs of H. pylori virulence factors, such as IceA1, HrgA, and jhp0562-like glycosyltransferase, are present in H. heilmannii but absent in H. ailurogastricus. Both species contain a VacA-like autotransporter, for which the passenger domain is remarkably larger in H. ailurogastricus than in H. heilmannii. In addition, H. ailurogastricus shows clear differences in binding to the gastric mucosa compared to H. heilmannii. These findings highlight the low-virulence character of this novel Helicobacter species.

Helicobacter suis affects the health and function of porcine gastric parietal cells.

The stomach of pigs at slaughter age is often colonized by Helicobacter (H.) suis, which is also the most prevalent gastric non-H. pylori Helicobacter (NHPH) species in humans. It is associated with chronic gastritis, gastric ulceration and other gastric pathological changes in both hosts. Parietal cells are highly specialized, terminally differentiated epithelial cells responsible for gastric acid secretion and regulation. Dysfunction of these cells is closely associated with gastric pathology and disease. Here we describe a method for isolation and culture of viable and responsive parietal cells from slaughterhouse pigs. In addition, we investigated the interactions between H. suis and gastric parietal cells both in H. suis-infected six-month-old slaughter pigs, as well as in our in vitro parietal cell model. A close interaction of H. suis and parietal cells was observed in the fundic region of stomachs from H. suis positive pigs. The bacterium was shown to be able to directly interfere with cultured porcine parietal cells, causing a significant impairment of cell viability. Transcriptional levels of Atp4a, essential for gastric acid secretion, showed a trend towards an up-regulation in H. suis positive pigs compared to H. suis-negative pigs. In addition, sonic hedgehog, an important factor involved in gastric epithelial differentiation, gastric mucosal repair, and stomach homeostasis, was also significantly up-regulated in H. suis positive pigs. In conclusion, this study describes a successful approach for the isolation and culture of porcine gastric parietal cells. The results indicate that H. suis affects the viability and function of this cell type.

Other Helicobacters and gastric microbiota.

This article aimed to review the literature from 2015 dealing with gastric and enterohepatic non-Helicobacter pylori Helicobacter species (NHPH). A summary of the gastric microbiota interactions with H. pylori is also presented. An extensive number of studies were published during the last year and have led to a better understanding of the pathogenesis of infections with NHPH. These infections are increasingly reported in human patients, including infections with H. cinaedi, mainly characterized by severe bacteremia. Whole-genome sequencing appears to be the most reliable technique for identification of NHPH at species level. Presence of NHPH in laboratory animals may influence the outcome of experiments, making screening and eradication desirable. Vaccination based on UreB proteins or bacterial lysate with CCR4 antagonists as well as oral glutathione supplementation may be promising strategies to dampen the pathogenic effects associated with gastric NHPH infections. Several virulent factors such as outer membrane proteins, phospholipase C-gamma 2, Bak protein, and nickel-binding proteins are associated with colonization of the gastric mucosae and development of gastritis. The development of high-throughput sequencing has led to new insights in the gastric microbiota composition and its interaction with H. pylori. Alterations in the gastric microbiota caused by the pH-increasing effect of a H. pylori infection may increase the risk for gastric cancer.

Residues of chlortetracycline, doxycycline and sulfadiazine-trimethoprim in intestinal content and feces of pigs due to cross-contamination of feed.

BACKGROUND: Cross-contamination of feed with low concentrations of antimicrobials can occur at production, transport and/or farm level. Concerns are rising about possible effects of this contaminated feed on resistance selection in the intestinal microbiota. Therefore, an experiment with pigs was set up, in which intestinal and fecal concentrations of chlortetracycline (CTC), doxycycline (DOX) and sulfadiazine-trimethoprim (SDZ-TRIM) were determined after administration of feed containing a 3 % carry-over level of these antimicrobials. RESULTS: The poor oral bioavailability of tetracyclines resulted in rather high concentrations in cecal and colonic content and feces at steady-state conditions. A mean concentration of 10 mg/kg CTC and 4 mg/kg DOX in the feces was reached, which is higher than concentrations that were shown to cause resistance selection. On the other hand, lower mean levels of SDZ (0.7 mg/kg) and TRIM (< limit of detection of 0.016 mg/kg) were found in the feces, corresponding with the high oral bioavailability of SDZ and TRIM in pigs. CONCLUSIONS: The relation between the oral bioavailability and intestinal concentrations of the tested antimicrobials, may be of help in assessing the risks of cross-contaminated feed. However, future research is needed to confirm our results and to evaluate the effects of these detected concentrations on resistance selection in the intestinal microbiota of pigs.

Role of γ-glutamyltranspeptidase in the pathogenesis of Helicobacter suis and Helicobacter pylori infections.

Helicobacter (H.) suis can colonize the stomach of pigs as well as humans, causing chronic gastritis and other gastric pathological changes including gastric ulceration and mucosa-associated lymphoid tissue (MALT) lymphoma. Recently, a virulence factor of H. suis, γ-glutamyl transpeptidase (GGT), has been demonstrated to play an important role in the induction of human gastric epithelial cell death and modulation of lymphocyte proliferation depending on glutamine and glutathione catabolism. In the present study, the relevance of GGT in the pathogenesis of H. suis infection was studied in mouse and Mongolian gerbil models. In addition, the relative importance of H. suis GGT was compared with that of the H. pylori GGT. A significant and different contribution of the GGT of H. suis and H. pylori was seen in terms of bacterial colonization, inflammation and the evoked immune response. In contrast to H. pyloriΔggt strains, H. suisΔggt strains were capable of colonizing the stomach at levels comparable to WT strains, although they induced significantly less overall gastric inflammation in mice. This was characterized by lower numbers of T and B cells, and a lower level of epithelial cell proliferation. In general, compared to WT strain infection, ggt mutant strains of H. suis triggered lower levels of Th1 and Th17 signature cytokine expression. A pronounced upregulation of B-lymphocyte chemoattractant CXCL13 was observed, both in animals infected with WT and ggt mutant strains of H. suis. Interestingly, H. suis GGT was shown to affect the glutamine metabolism of gastric epithelium through downregulation of the glutamine transporter ASCT2.

Purification of Helicobacter suis Strains From Biphasic Cultures by Single Colony Isolation: Influence on Strain Characteristics.

BACKGROUND: Helicobacter (H.) suis causes gastritis and decreased weight gain in pigs. It is also the most prevalent non-Helicobacter pylori Helicobacter species in humans with gastric disease. H. suis is extremely fastidious, and so far, biphasic culture conditions were essential for isolation and culture, making it impossible to obtain single colonies. Hence, cultures obtained from an individual animal may contain multiple H. suis strains, which is undesirable for experiments aiming for instance at investigating H. suis strain differences. MATERIALS AND METHODS: Pure cultures of H. suis were established by growing bacteria as colonies on 1% brucella agar plates, followed by purification and enrichment by biphasic subculture. Characteristics of these single colony-derived strains were compared with those of their parent strains using multilocus sequence typing (MLST) and by studying bacterium-host interactions using a gastric epithelial cell line and Mongolian gerbil model. RESULTS: The purification/enrichment procedure required a nonstop culture of several weeks. For 4 of 17 H. suis strains, MLST revealed differences between parental and single colony-derived strains. For three of four single colony-derived strains tested, the cell death-inducing capacity was higher than for the parental strain. One single colony-derived strain lost its capacity to colonize Mongolian gerbils. For the four other strains tested, colonization capacity and histopathologic changes were similar to what has been described when using strains with only a history of limited biphasic culture. CONCLUSIONS: A method was developed to obtain single colony-derived H. suis strains, but this procedure may affect the bacterial genotype and phenotype.