In recent years there has been a renewed interest concerning the ways in which the gastrointestinal tract – its functional integrity and microbial residents – might influence human mood (e.g. depression) and behavioral disorders. Once a hotbed of scientific interest in the early 20th century, this area lay dormant for decades, in part due to its association with the controversial term ‘autointoxication’. Here we review contemporary findings related to intestinal permeability, small intestinal bacterial overgrowth, lipopolysaccharide endotoxin (LPS) exposure, D-lactic acid, propionic acid, and discuss their relevance to microbiota and mental health. In addition, we include the context of modern dietary habits as they relate to depression, anxiety and their potential interaction with intestinal microbiota.

Background

The aim of this study is to expand existing knowledge about the CRC-associated microbiome among Han Chinese, and to further discover the variation pattern of the human CRC microbiome across all population.

Findings

Using pyrosequencing-based molecular monitoring of bacterial 16S rRNA gene from eight tumor/normal tissue pairs of eight Chinese CRC patients, we analyzed and characterized the basic features of the CRC-associated microbiome. Firstly, we discovered an increasing diversity among tumor-associated bacterial communities. Secondly, in 50% of Chinese CRC patients, we found a significant increase of Roseburia (P = 0.017), and a concurrent decrease of both Microbacterium (P = 0.009) and Anoxybacillus (P = 0.009) in tumor tissue.

Conclusions

We discovered a novel CRC microbiome pattern in Chinese. Both the over-represented Roseburia bacteria at tumor sites and the over-represented Microbacterium and Anoxybacillus bacteria away from tumor sites were both closely related in Chinese CRC patients. Across several populations reported in this study and previously, we observed both common and distinctive patterns of human CRC microbiome’s association with a high-risk of CRC.

Background

The diverse bacterial communities colonizing the gut (gastrointestinal tract) of infants as commensal flora, which play an important role in nutrient absorption and determining the state of health, are known to alter due to diarrhea.

Method

Bacterial community dynamics in children suffering from cholera and during recovery period were examined in the present study by employing metagenomic tool, followed by DNA sequencing and analysis. For this, bacterial community DNA was extracted from fecal samples of nine clinically confirmed cholera children (age 2–3 years) at day 0 (acute cholera), day 2 (antibiotic therapy), day 7 and, and day 28, and the variable region of 16S rRNA genes were amplified by universal primer PCR.

Results

454 parallel sequencing of the amplified DNA followed by similarity search of the sequenced data against an rRNA database allowed us to identify V. cholerae, the cause of cholera, in all nine children at day 0, and as predominant species in six children, accounting for 35% of the total gut microbiota on an average in all the nine children. The relative abundance (mean ± sem %) of bacteria belonging to phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, was 55 ± 7, 18 ± 4, 13 ± 4, and 8 ± 4, respectively, at day 0, while these values were 12 ± 4, 43 ± 4, 33 ± 3, and 12 ± 2, respectively, at day 28. As antibiotic therapy began, V. cholerae count declined significantly (p< 0.001) and was found only in four children at day 2 and two children at day 7 with the relative abundance of 3.7% and 0.01%, respectively, which continued up to day 28 in the two children. Compared to acute cholera condition (day 0), the relative abundance of Escherichia coli, Enterococcus, and Veillonella increased at day 2 (antibiotic therapy) while Bifidobacterium, Bacteroides, and Ruminococcus decreased.

Conclusion

Cholera results expulsion of major commensal bacteria of phyla Bacteroidetes, Firmicutes, and Actinobacteria, and increase of harmful Proteobacteria to colonize the gut during acute and convalescence states. The observed microbiota disruption might explain the prevalent malnutrition in children of Bangladesh where diarrheal diseases are endemic.

Background

Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of Johne’s disease, an enteric granulomatous disease. Recently, MAP has been associated with different autoimmune diseases such as Crohn’s disease, type 1 diabetes (T1D) and multiple sclerosis. Transthyretin (TTR) is a plasma transport protein for thyroid hormone and forms a complex with retinol-binding protein. Reduced TTR plasma levels in MAP infected ovines have been reported.

TTR exerts also a functional role in the pancreas promoting insulin release and protecting β-cells from death.

Our objective was to identify a protein that could be used as a diagnostic marker of T1D for determining disease progression and monitoring at-risk patients. We postulate that serological TTR levels would be reduced in T1D MAP exposed patients. Our hypothesis is based on the observation of cases of T1D patients with decreased TTR levels beside the reduced TTR plasma levels in ovines with Johne’s disease.

We quantified the plasma protein levels of TTR in 50 people with T1D and 51 age-matched healthy controls (HCs) by means of enzyme-linked immunosorbent assays (ELISA).

Findings

Our pilot study showed that plasma TTR levels were not significantly lower/higher in T1D Sardinian cases compared to the HCs.

Conclusion

These preliminary data indicate that plasma TTR may not be a good candidate biomarker for T1D diagnosis and further studies to elucidate the possible link are needed.

Background

Cytokine production and histopathological changes occur in the lungs of mice after intranasal inoculation with Campylobacter jejuni, but the levels of cytokines in different organs to which C. jejuni disseminates have not been studied.

Findings

Adult BALB/c mice were intranasally inoculated with C. jejuni 81–176 (test) or phosphate-buffered saline (control) (n=16 per group). The levels of cytokines in the organs (spleen, liver, and small and large intestines) to which C. jejuni disseminated were measured by ELISA. Two cytokine patterns were observed. First, increased proinflammatory cytokines, TNF-α, IL-1, and IL-2, were followed by anti-inflammatory cytokines, IL-4 and IL-10 in the spleen and large intestine. Second, in the liver and small intestine, there was a predominant production of anti-inflammatory cytokines, IL-4 and IL-10, with some increase in IL-2 levels. In the spleen and intestines, the levels of pro- and anti-inflammatory cytokines were concurrently increased.

Conclusion

Dissemination of C. jejuni is associated with the production of different cytokine profiles in different tissues, with the proinflammatory response appearing in the spleen and large intestine at an earlier time point than in the liver and small intestine. The organs produce different cytokine profiles in response to C. jejuni dissemination. These preliminary findings should be confirmed with a study involving a larger group of animals.

Motility helps many pathogens swim through the highly viscous intestinal mucus. Given the differing outcomes of Campylobacter concisus infection, the motility of eight C. concisus strains isolated from patients with Crohn’s disease (n=3), acute (n=3) and chronic (n=1) gastroenteritis and a healthy control (n=1) were compared. Following growth on solid or liquid media the eight strains formed two groups; however, the type of growth medium did not affect motility. In contrast, following growth in viscous liquid medium seven of the eight strains demonstrated significantly decreased motility. In media of increasing viscosities the motility of C. concisus UNSWCD had two marked increases at viscosities of 20.0 and 74.7 centipoises. Determination of the ability of UNSWCD to swim through a viscous medium, adhere to and invade intestinal epithelial cells showed that while adherence levels significantly decreased with increasing viscosity, invasion levels did not significantly change. In contrast, adherence to and invasion of UNSWCD to mucus-producing intestinal cells increased upon accumulation of mucus, as did bacterial aggregation. Given this aggregation, we determined the ability of the eight C. concisus strains to form biofilms, and showed that all strains formed biofilms. In conclusion, the finding that C. concisus strains could be differentiated into two groups based on their motility may suggest that strains with high motility have an increased ability to swim through the intestinal mucus and reach the epithelial layer.

Salmonella Typhi is a human restricted pathogen with a significant number of individuals as asymptomatic carriers of the bacterium. Salmonella infection can be effectively controlled if a reliable method for identification of these carriers is developed. In this context, the availability of whole genomes of carrier strains through high- throughput sequencing and further downstream analysis by comparative genomics approaches is very promising. Herein we describe the genome sequence of a Salmonella Typhi isolate representing an asymptomatic carrier individual during a prolonged outbreak of typhoid fever in Kelantan, Malaysia. Putative genomic coordinates relevant in pathogenesis and persistence of this carrier strain are identified and discussed.

Background

Escherichia coli is believed to participate in the etiology of Crohn’s disease (CD) and possibly of ulcerative colitis (UC), due at least in part to the observed rise in the number of these bacteria in the gut microbiota of CD and UC patients. Nevertheless, it is not fully understood whether this quantitative variation occurs equally throughout the mucosal and luminal spaces of the gut. To assess this question, stools and mucosa biopsies from distinct intestinal sites were cultured aiming at determining their E. coli concentration. The cultures were additionally screened for the presence of some virulence genes of pathogenic E. coli.

Results

Analyses of clinical materials from 14 controls (38 biopsies and 14 stools samples), 11 CD (25 biopsies and 11 stools samples) and 7 UC patients (18 biopsies and 7 stools samples) indicated no significant variation in the number of E. coli present in stools, but a rise of at least one log10 CFU/mg in biopsies from the ileum of CD patients and the sigmoid and rectum of CD and UC patients. The cultures were screened for the presence of E. coli attaching and effacing (eae), invasion plasmid antigen H (ipaH), aggregative adherence transcriptional activator (aggR), Shiga cytotoxins (stx), and heat labile enterotoxin (elt) and the following serine proteases autotransporters of Enterobacteriaceae (SPATE) genes: plasmid encoded toxin (pet), secreted autotransporter toxin (sat), Shigella extracellular protein (sepA), protein involved in intestinal colonization (pic) and Shigella IgA-like protease homolog (sigA). Six of the 10 genes screened were detected in the total of samples investigated: aggR, eae, pet, sat, sepA and sigA. No difference in the prevalence of any of these markers was observed in cultures from different clinical materials or groups of patients.

Methods

Bacterial quantitation was carried out following cultures of diluted samples suspensions in MacConkey agar, Wilkins Chalgren agar for anaerobes, E. coli/coliform chromocult agar, and blood agar. Screening for E. coli virulence genes was performed by multiplex PCR of DNA purified from total MacConkey undiluted broth cultures.

Conclusion

In CD and UC patients only the mucosa associated population of E. coli is augmented and the proliferation is prominent in the ileum of CD and rectum and sigmoid of both UC and CD patients which are sites where the lesions usually are observed. The augmented E. coli population in these sites presented a low number of the virulence markers, possibly meaning that they are not relevant for the disease process.

The large numbers of human intestinal microorganisms have a highly co-evolved relationship with the immune system. Dysbacteriosis of intestinal microbiota induces alterations of immune responses, and is closely related to disease development. Peyer’s patches are immune sensors in intestine which exert essential functions during development of inflammatory disease. However, interactions between commensal bacteria and PPs have been poorly characterized. In this study, changes of lymphocyte subpopulations and production of cytokines in PPs of mice with intestinal dysbacteriosis were investigated. The ceftriaxone-induced dysbacteriosis caused a notable change in populations of T lymphocytes, their subpopulations in PPs and expressions of various cytokines. Our results suggest intestinal dysbacteriosis in mice reduces immune tolerance in PPs and orients immune response towards humoral immunity.

Helicobacter pylori infection is the major cause of gastric cancer, which remains an important health care challenge. Recent investigation in gastric stem cell or progenitor cell biology has uncovered valuable information in understanding the gastric gland renewal and maintenance of homeostasis, they also provide clues for further defining the mechanisms by which gastric cancer may originate and progress. Lgr5, Villin-promoter, TFF2-mRNA and Mist have recently been identified as gastric stem/progenitor cell markers; their identification enriched our understanding on the gastric stem cell pathobiology during chronic inflammation and metaplasia. In addition, advance in gastric cancer stem cell markers such as CD44, CD90, CD133, Musashi-1 reveal novel information on tumor cell behavior and disease progression implicated for therapeutics. However, two critical questions remain to be of considerable challenges for future exploration; one is how H. pylori or chronic inflammation affects gastric stem cell or their progenitors, which give rise to mucus-, acid-, pepsinogen-, and hormone-secreting cell lineages. Another one is how bacterial infection or inflammation induces oncogenic transformation and propagates into tumors. Focus on the interactions of H. pylori with gastric stem/progenitor cells and their microenvironment will be instrumental to decipher the initiation and origin of gastric cancer. Future studies in these areas will be critical to uncover molecular mechanisms of chronic inflammation-mediated oncogenic transformation and provide options for cancer prevention and intervention. We review recent progress and discuss future research directions in these important research fields.

The recently developed rapid immunochromatographic tests (ICT) have the potential to provide a quick and easy diagnosis of Campylobacter enteritis in comparison to culture. In a previous study we found them sensitive but lacking in specificity. The aim of the present study was to focus on the problem of specificity and determine the positive predictive value (PPV) of a positive result of the ImmunoCard Stat! Campy (Meridian Bioscience, Cincinnati, OH, USA). For this purpose, the stools positive by ICT were cultured according to 3 different protocols: Karmali agar, Preston enrichment broth subcultured on Karmali agar, and a filtration method on a blood agar without antibiotics, all incubated for 7 days at 37°C. Out of 609 stools from adults and children with community acquired enteritis, the reference methods detected 25 positive cases (4.1%) (culture: 19, specific PCR and ELISA both positive: 6) and the ICT: 31 including the 25 true positives. The PPV was 80.6%. We conclude that ICT is a good method to screen Campylobacter positive stools but because of its lack of specificity the positive stools must be tested by another method.

The influence of resident gut microbes on xenobiotic metabolism has been investigated at different levels throughout the past five decades. However, with the advance in sequencing and pyrotagging technologies, addressing the influence of microbes on xenobiotics had to evolve from assessing direct metabolic effects on toxins and botanicals by conventional culture-based techniques to elucidating the role of community composition on drugs metabolic profiles through DNA sequence-based phylogeny and metagenomics. Following the completion of the Human Genome Project, the rapid, substantial growth of the Human Microbiome Project (HMP) opens new horizons for studying how microbiome compositional and functional variations affect drug action, fate, and toxicity (pharmacomicrobiomics), notably in the human gut. The HMP continues to characterize the microbial communities associated with the human gut, determine whether there is a common gut microbiome profile shared among healthy humans, and investigate the effect of its alterations on health. Here, we offer a glimpse into the known effects of the gut microbiota on xenobiotic metabolism, with emphasis on cases where microbiome variations lead to different therapeutic outcomes. We discuss a few examples representing how the microbiome interacts with human metabolic enzymes in the liver and intestine. In addition, we attempt to envisage a roadmap for the future implications of the HMP on therapeutics and personalized medicine.

The role of the gut microbiome in human health and disease with a particular emphasis on therapeutic use of probiotics under specific medical conditions was mainly highlighted in 1st Annual conference of Probiotic Association of India (PAi) and International Symposium on “Probiotics for Human Health - New Innovations and Emerging Trends” held on 27th-28th August, 2012 at New Delhi, India. There is increasing recognition of the fact that dysbiosis or alteration of this gut microbiome may be implicated in gastro-intestinal disorders including diarrheal diseases, ulcerative colitis, inflammatory bowel diseases, life style diseases viz. Diabetes Mellitus-2 and obesity etc. This report summarizes the proceedings of the conference and the symposium comprehensively. Although, research on probiotics has been continuing for the past few decades, the subject has been currently the major focus of attention across the world due to recent advances and new developments in genomics, transcriptomics, proteomics, metabolomics and emergence of new generation of high through put sequencing technologies that have immensely helped in understanding the probiotic functionality and mode of action from nutritional and health perspectives. There is now sufficient evidence backed up with good quality scientific clinical data to suggest that probiotic interventions could indeed be effective in various types of diarrheal diseases, other chronic gastrointestinal inflammatory disorders like pouchitis, necrotizing entero-colitis, allergic responses and lactose intolerance etc. This report makes a modest attempt to give all the stake holders involved in development of probiotic based functional/health foods an overview of the current status of probiotics research at the Global and National level. The most crucial issues that emerged from the lead talks delivered by the eminent speakers from India and abroad were the major focus of discussions in different plenary and technical sessions. By discussing some of these issues from scientific perspectives, the conference could achieve its prime objective of disseminating the current knowledge on the prospects of probiotics as potential biotherapeutics in the management of human health and diseases.

Campylobacter jejuni and coli are collectively regarded as the most prevalent cause of bacterial foodborne illness worldwide. An emerging species, Campylobacter ureolyticus has recently been detected in patients with gastroenteritis, however, the source of this organism has, until now, remained unclear. Herein, we describe the molecular-based detection of this pathogen in bovine faeces (1/20) and unpasteurized milk (6/47) but not in poultry (chicken wings and caeca). This is, to the best of our knowledge, the first report of the presence of this potential gastrointestinal pathogen in an animal source, possibly suggesting a route for its transmission to humans.

Background

Recently, we found that the probiotic strain Bacillus coagulans GBI-30, 6086 (GanedenBC30) improved indices of Clostridium difficile (C. difficile)-induced colitis in mice (Fitzpatrick et al., Gut Pathogens, 2011). Our goal was to determine if BC30 could also prevent the recurrence of C. difficile-induced colitis in mice, following initial treatment with vancomycin. During study days 0 through 5, mice were treated with antibiotics. On day 6, the C. difficile strain VPI 10463 was given by oro-gastric gavage at ≈ 5x104 CFU to induce colitis. Mice were treated on study days 6 to 10 with vancomycin (50 mg/kg) (vanco) or vehicle (saline) by gavage. On days 10 to16, mice were dosed by gavage with saline vehicle or BC30 (2 x 109 CFU per day). Mice were monitored for mortality, weight loss and diarrhea. On study days 14, 16 and 17, stools and colons were collected for analyzing other parameters of colitis.

Results

The mean stool consistency score in Vehicle/C.difficile/Vanco mice increased from 0.4 (day 10) to a range of 1.1 to 1.4 (days 14 to 17), indicating the recurrence of colitis. On days 13 through 17, the stool consistency scores for the vancomycin/BC30 mice were significantly lower (p< 0.05) than for the vancomycin/vehicle cohort of animals. On day 17, 88.9% of mice treated with BC30 had normal stools, while this value was 0% with vehicle treatment (p value = 0.0004). Colonic myeloperoxidase (Units/2 cm colon) was significantly (p < 0.05) reduced from 4.3 ± 0.7 (Vehicle/C.difficile/Vanco) to 2.6 ± 0.2 (BC30/C. Difficle/Vanco). The colonic histology score and Keratinocyte derived-chemokine level in the colon were also lower in BC30 treated mice.

Summary

In BC30-treated mice, there was evidence of better stool consistency, as well as improved biochemical and histological indices of colitis, following initial treatment of animals with vancomycin.

Conclusion

BC30 limited the recurrence of CD-induced colitis following vancomycin withdrawal in mice.

Background

Following Helicobacter pylori eradication in idiopathic parkinsonism (IP), hypokinesia improved but flexor-rigidity increased. Small intestinal bacterial-overgrowth (SIBO) is a candidate driver of the rigidity: hydrogen-breath-test-positivity is common in IP and case histories suggest that Helicobacter keeps SIBO at bay.

Methods

In a surveillance study, we explore relationships of IP-facets to peripheral immune/inflammatory-activation, in light of presence/absence of Helicobacter infection (urea-breath- and/or stool-antigen-test: positivity confirmed by gastric-biopsy) and hydrogen-breath-test status for SIBO (positivity: >20 ppm increment, 2 consecutive 15-min readings, within 2h of 25G lactulose). We question whether any relationships found between facets and blood leukocyte subset counts stand in patients free from anti-parkinsonian drugs, and are robust enough to defy fluctuations in performance consequent on short t½ therapy.

Results

Of 51 IP-probands, 36 had current or past Helicobacter infection on entry, 25 having undergone successful eradication (median 3.4 years before). Thirty-four were hydrogen-breath-test-positive initially, 42 at sometime (343 tests) during surveillance (2.8 years). Hydrogen-breath-test-positivity was associated inversely with Helicobacter-positivity (OR 0.20 (95% CI 0.04, 0.99), p<0.05).

In 38 patients (untreated (17) or on stable long-t½ IP-medication), the higher the natural-killer count, the shorter stride, slower gait and greater flexor-rigidity (by mean 49 (14, 85) mm, 54 (3, 104) mm.s-1, 89 (2, 177) Nm.10-3, per 100 cells.μl-1 increment, p=0.007, 0.04 & 0.04 respectively, adjusted for patient characteristics). T-helper count was inversely associated with flexor-rigidity before (p=0.01) and after adjustment for natural-killer count (-36(-63, -10) Nm.10-3 per 100 cells.μl-1, p=0.007). Neutrophil count was inversely associated with tremor (visual analogue scale, p=0.01). Effect-sizes were independent of IP-medication, and not masked by including 13 patients receiving levodopa (except natural-killer count on flexor-rigidity). Cellular associations held after allowing for potentially confounding effect of hydrogen-breath-test or Helicobacter status. Moreover, additional reduction in stride and speed (68 (24, 112) mm & 103 (38, 168) mm.s-1, each p=0.002) was seen with Helicobacter-positivity. Hydrogen-breath-test-positivity, itself, was associated with higher natural-killer and T-helper counts, lower neutrophils (p=0.005, 0.02 & 0.008).

Conclusion

We propose a rigidity-associated subordinate pathway, flagged by a higher natural-killer count, tempered by a higher T-helper, against which Helicobacter protects by keeping SIBO at bay.

Background

The foodborne pathogen Bacillus cereus can cause diarrhoeal food poisoning by production of enterotoxins in the small intestine. The prerequisite for diarrhoeal disease is thus survival during gastrointestinal passage.

Methods

Vegetative cells of 3 different B. cereus strains were cultivated in a real composite food matrix, lasagne verde, and their survival during subsequent simulation of gastrointestinal passage was assessed using in vitro experiments simulating transit through the human upper gastrointestinal tract (from mouth to small intestine).

Results

No survival of vegetative cells was observed, despite the high inoculum levels of 7.0 to 8.0 log CFU/g and the presence of various potentially protective food components. Significant fractions (approx. 10% of the consumed inoculum) of B. cereus vegetative cells survived gastric passage, but they were subsequently inactivated by bile exposure in weakly acidic intestinal medium (pH 5.0). In contrast, the low numbers of spores present (up to 4.0 log spores/g) showed excellent survival and remained viable spores throughout the gastrointestinal passage simulation.

Conclusion

Vegetative cells are inactivated by gastric acid and bile during gastrointestinal passage, while spores are resistant and survive. Therefore, the physiological form (vegetative cells or spores) of the B. cereus consumed determines the subsequent gastrointestinal survival and thus the infective dose, which is expected to be much lower for spores than vegetative cells. No significant differences in gastrointestinal survival ability was found among the different strains. However, considerable strain variability was observed in sporulation tendency during growth in laboratory medium and food, which has important implications for the gastrointestinal survival potential of the different B. cereus strains.

Mycobacterium avium subsp. paratuberculosis (MAP) is a zoonotic pathogen, a very slow growing bacterium which is difficult to isolate and passage in conventional laboratory culture. Although its association with Johne’s disease or paratuberculosis of cattle is well established, it has been only putatively linked to Crohn’s disease in humans. Further, MAP has been recently suggested to be a trigger for other autoimmune diseases such as type-1 diabetes mellitus (T1DM). Recently, some studies have indicated that exposure to MAP is associated with elevated levels of antibodies against MAP lysate although the exact mechanism and significance of the same remains unclear. Further, the cytokine profiles relevant in MAP associated diseases of humans and their exact role in the pathophysiology are not clearly known. We performed in vitro cytokine analyses after exposing different cultured human cells to the whole cell lysate of MAP and found that MAP lysate induces secretion of cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α by human peripheral blood mononuclear cells (PBMCs). Also, it induces secretion of IL-8 by cultured human stomach adenocarcinoma cells (AGS) and PANC-1(human pancreatic carcinoma cell line) cells. We also found that MAP lysate induced cytotoxicity in PANC-1cells. Collectively, these results provide a much needed base-line data set of cytokines broadly signifying a MAP induced cellular response by human cells.

Background

Helicobacter pylori are successful colonizers of the human gastric mucosa. Colonization increases the risk of peptic ulcer disease and adenocarcinoma. However, potential benefits of H. pylori colonization include protection against early-onset asthma and against gastrointestinal infections. Campylobacter jejuni are a leading cause of bacterial diarrhea and complications include Guillain-Barré syndrome. Here, we describe the development of reliable serological assays to detect antibodies against those two bacteria in Rhesus macaques and investigated their distribution within a social group of monkeys.

Methods

Two cohorts of monkeys were analyzed. The first cohort consisted of 30 monkeys and was used to establish an enzyme-linked immunosorbent assay (ELISA) for H. pylori antibodies detection. To evaluate colonization of those macaques, stomach biopsies were collected and analyzed for the presence of H. pylori by histology and culture. C. jejuni ELISAs were established using human serum with known C. jejuni antibody status. Next, plasma samples of the 89 macaques (Cohort 2) were assayed for antibodies and then statistically analyzed.

Results

An H. pylori IgG ELISA, which was 100% specific and 93% sensitive, was established. In contrast, the IgA ELISA was only 82% specific and 61% sensitive. The CagA IgG assay was 100% sensitive and 61% of the macaques were positive. In cohort 2, 62% macaques were H. pylori sero-positive and 52% were CagA positive. The prevalence of H. pylori IgG and CagA IgG increased with monkey age as described for humans. Of the 89 macaques 52% showed IgG against C. jejuni but in contrast to H. pylori, the sero-prevalence was not associated with increasing age. However, there was a drop in the IgG (but not in IgA) mean values between infant and juvenile macaques, similar to trends described in humans.

Conclusions

Rhesus macaques have widespread exposure to H. pylori and C. jejuni, reflecting their social conditions and implying that Rhesus macaques might provide a model to study effects of these two important human mucosal bacteria on a population.

Background

“Quorum sensing” (QS) is the phenomenon which allows single bacterial cells to measure the concentration of bacterial signal molecules. Two principle different QS systems are known, the Autoinducer 1 system (AI-1) for the intraspecies communication using different Acyl-homoserine lactones (AHL) and AI-2 for the interspecies communication. Aim of this study was to investigate QS of Escherichia coli Nissle 1917 (Mutaflor).

Results

While E. coli Nissle is producing AI-2 in a density dependent manner, no AI-1 was produced. To study the effect of AI-2 in the DSS (dextran sulphate sodium) induced mouse model of acute colitis, we silenced the corresponding gene luxS by intron insertion. The mutant bacterium E. coli Nissle::luxS was equally effective in colonizing the colon and the mutation turned out to be 100% stable during the course of the experiment. Isolating RNA from the colon mucosa and performing semiquantitative RT PCR, we were able to show that the expression of the pro-inflammatory cytokine IFN-y was suppressed in mice being infected with the E. coli Nissle wild type. Mice infected with the E. coli Nissle::luxS mutant showed a suppressed expression of IL-10 compared to uninfected mice, while the expression of the pro-inflammatory cytokines IL-6 and TNF-α was higher in these mice. The expression of mBD-1 was suppressed in mice being infected with the mutant in comparison to the mice not infected or infected with the wild type. No differences were seen in the histological examination of the colon sections in the different groups of mice.

Conclusions

E. coli Nissle is producing AI-2 molecules, which are influencing the expression of cytokines in the mucosa of the colon in the DSS mice. However, if QS has a direct influence on the probiotic properties of E. coli Nissle remains to be elucidated.

Background

Klebsiella pneumoniae carbapenemases (KPCs) producing bacteria have emerged as a cause of multidrug-resistant nosocomial infections worldwide. KPCs are plasmid-encoded enzymes capable of hydrolysing a broad spectrum of beta-lactams, including carbapenems and monobactams, therefore worryingly limiting antimicrobial treatment options. Analysis of circulating bacterial strains and KPC alleles may help understanding the route of KPC dissemination and therefore help containing the infection.

Methods

KPC-producing Klebsiella pneumoniae dissemination in two 1580- and 300- bed hospitals in Padua, Italy, from initial outbreak in 2009 to late 2011 was analysed. Molecular and clinical epidemiology, including bacterial strains, KPC-encoding plasmid sequences and associated resistance genes, involved hospital wards and relocation of patients were described. Routine antimicrobial susceptibility testing and MIC of carbapenems on clinical isolates were performed. Detection of resistance genes was obtained by PCR and sequencing. MLST, PFGE and ERIC were used for molecular genotyping. Plasmid analysis was obtained by digestion with restriction enzymes and deep sequencing.

Results

KPC-positive clinical samples were isolated from nearly 200 patients. In the initial outbreak intensive care units were almost exclusively involved, while medical, surgical and long-term wards were successively massively concerned. Analysis of KPC alleles, plasmids and bacterial sequence types (STs) indicated that during the initial outbreak KPC-3 in ST258 and KPC-2 in ST147 were each confined in one of the two surveilled hospitals. While KPC-2 dissemination was effectively contained, KPC-3 in ST258 cross-spreading was observed. The simultaneous presence of two carbapenemases, VIM-1 and KPC-2, in the same isolate was also observed in three patients. Total sequencing of plasmid content of two KPC-3 strains showed novel association of resistance plasmids.

Conclusions

The acquired molecular epidemiology demonstrated that 1) both acquisitions from outward sources and patient relocation within the hospitals were responsible for the observed spreading; 2) KPC-3-encoding Klebsiella pneumoniae ST258 prevailed over other strains. In addition, the described massive transfer of KPC-mediated resistance to non-intensive care units may anticipate spreading of resistance to the non-hospitalized population. Therefore, genotypic analysis alongside phenotypic identification of carbapenemase producers, also at the carriage state, is advisable to prevent and contain further carbapenemase resistance dissemination.

Background

Inflammatory Bowel Disease (IBD), which includes both Crohn’s disease (CD) and ulcerative colitis (UC), is caused by a complex interplay involving genetic predisposition, environmental factors and an infectious agent. Mycobacterium avium subsp. paratuberculosis (MAP) is a promising pathogen candidate since it produces a chronic intestinal inflammatory disease in ruminants that resembles CD in humans. MAP is a ubiquitous microorganism, although its presence in the food chain, especially in milk from infected animals, is what made us think that there could be an association between lactase persistence (LP) and IBD. The LCT mutation has brought adaptation to dairy farming which in turn would have increased exposure of the population to infection by MAP. NOD2 gene mutations are highly associated to CD.

Methods

In our study, CD and UC patients and controls from the North of Spain were genotyped for the lactase gene (LCT) and for three NOD-2 variants, R702W, G908R and Cins1007fs. MAP PCR was carried out in order to assess MAP infection status and these results were correlated with LCT and NOD2 genotypes.

Results

As for LP, no association was found with IBD, although UC patients were less likely to present the T/T−13910 variant compared to controls, showing a higher C-allele frequency and a tendency to lactase non-persistence (LNP). NOD2 mutations were associated to CD being the per-allele risk higher for the Cins1007fs variant. MAP infection was more extended among the healthy controls (45.2%) compared to CD patients (21.38%) and UC patients (19.04%) and this was attributed to therapy. The Asturian CD cohort presented higher levels of MAP prevalence (38.6%) compared to the Basque CD cohort (15.5%), differences also attributed to therapy. No interaction was found between MAP infection and LCT or NOD2 status.

Conclusions

We conclude that LP is not significantly associated with IBD, but that MAP infection and NOD2 do show not mutually interacting associations with IBD.

Background

Campylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.

Results

In the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.

Conclusion

These results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

Background

Vibrio cholerae is the causative organism of waterborne disease, cholera. V. cholerae has caused many epidemics and pandemics of cholera for many years. In this study, V. cholerae recovered from edible ice were investigated for their genetic diversity using Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and Repetitive Extragenic Palindromic (REP) PCR. Isolation was done using selective medium and the presumptive isolates were confirmed through biochemical and serological assays.

Results

Seventy-five isolates of V. cholerae were recovered from ice samples collected from different locations of Jakarta. Specifically, 19 of them were identified as O1 serotype, 16 were Ogawa, 3 isolates were Inaba and the remaining isolates were non-O1. The fingerprinting profiles of V.cholerae isolated from ice samples were very diverse.

Conclusion

This result showed that the ERIC sequence is more informative and discriminative than REP sequence for analysis of V. cholerae diversity.

Our understanding of the role of the microbiota in our gut and other sites in our body is rapidly emerging and could lead to many new and innovative approaches for health care. The promise of the potential role of probiotics for the prevention and treatment of enteric and other infections as an effective solution needs to be realized. The meeting report summarizes the insights and learning from a recent symposium, "Health Impact of Probiotics - Vision and Opportunities" conducted in Mumbai by the Yakult India Microbiota and Probiotic Science Foundation and P.D. Hinduja National Hospital, Mumbai. The symposium reflected its objective of unraveling the potential role of probiotics for health benefits through presentations and discussions. Experts clearly highlighted the role of probiotics in improving various aspects of health and in immune modulation. The report also captures the debate and discussions on the challenges that are likely to be encountered for the use of probiotics in the country.