Inflammatory diarrhea due to enteroaggregative Escherichia coli: evidence from clinical and mice model studies

Background　 This study was conducted to determine the role of enteroaggregative Escherichia coli (EAEC) in inflammatory diarrhea among hospitalized patients in Kolkata. The inflammatory pathogenesis of EAEC was established in mice model and histopathological studies. Presence of fecal leucocytes (FLCs) can be suspected for EAEC infection solely or as a mixed with other enteric pathogens.　 Methods　 Active surveillance was conducted for 2 years on 2 random days per week with every 5th patient admitted to the Infectious Diseases Hospital (IDH). Diarrheal samples were processed by conventional culture, microscopy, ELISA and molecular methods. Two EAEC isolated as sole pathogens were examined in mice after induced intestinal infection. The intestinal tissue samples were processed to analyze the histological changes.　 Results　 Of the 2519 samples screened, fecal leucocytes, erythrocytes and occult blood were detected in 1629 samples. Most of the patients had acute watery diarrhea (75%) and vomiting (78%). Vibrio cholerae O1 was the main pathogen in patients of 5–10 years age group (33%). Shigellosis was more in children from 2–5 years of age (19%), whereas children <2 years appeared to be susceptible for infection caused by EAEC (16%). When tested for the pathogenicity, the EAEC strains colonized well and caused inflammatory infection in the gut mucosa of BALB/C mice.　 Conclusion　 This hospital-based surveillance revealed prevalence of large number of inflammatory diarrhea. EAEC was the suspected pathogen and <2 years children appeared to be the most susceptible age group. BALB/C mice may be a suitable animal model to study the EAEC-mediated pathogenesis

Emerging trends in the etiology of enteric pathogens as evidenced from an active surveillance of hospitalized diarrhoeal patients in Kolkata, India

Background: This study was conducted to determine the etiology of diarrhoea in a hospital setting in Kolkata. Active surveillance was conducted for 2 years on two random days per week by enrolling every fifth diarrhoeal patient admitted to the Infectious Diseases and Beliaghata General Hospital in Kolkata. Results: Most of the patients (76.1%) had acute watery diarrhoea in association with vomiting (77.7%) and some dehydration (92%). Vibrio cholerae O1, Rotavirus and Giardia lamblia were the important causes of diarrhoea. Among Shigella spp, S. flexneri 2a and 3a serotypes were most predominantly isolated. Enteric viruses, EPEC and EAEC were common in children <5 year age group. Atypical EPEC was comparatively higher than the typical EPEC. Multidrug resistance was common among V. cholerae O1 and Shigella spp including tetracycline and ciprofloxacin. Polymicrobial infections were common in all age groups and 27.9% of the diarrhoea patients had no potential pathogen. Conclusions: Increase in V. cholerae O1 infection among <2 years age group, resistance of V. cholerae O1 to tetracycline, rise of untypable S. flexnerii, higher proportion of atypical EPEC and G. lamblia and polymicrobial etiology are some of the emerging trends observed in this diarrhoeal disease surveillance

Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

BACKGROUND: Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL). METHODOLOGY/PRINCIPAL FINDINGS: We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/-0.3 n = 3), CHA6.8 (FA ratio 1.08+/-0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/-0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/-0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/-0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/-0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure. CONCLUSION: Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model

Haitian Variant Vibrio cholerae O1 Strains Manifest Higher Virulence in Animal Models

Vibrio cholerae causes fatal diarrheal disease cholera in humans due to consumption of contaminated water and food. To instigate the disease, the bacterium must evade the host intestinal innate immune system; penetrate the mucus layer of the small intestine, adhere and multiply on the surface of microvilli and produce toxin(s) through the action of virulence associated genes. V. cholerae O1 that has caused a major cholera outbreak in Haiti contained several unique genetic signatures. These novel traits are used to differentiate them from the canonical El Tor strains. Several studies reported the spread of these Haitian variant strains in different parts of the world including Asia and Africa, but there is a paucity of information on the clinical consequence of these genetic changes. To understand the impact of these changes, we undertook a study involving mice and rabbit models to evaluate the pathogenesis. The colonization ability of Haitian variant strain in comparison to canonical El Tor strain was found to be significantly more in both suckling mice and rabbit model. Adult mice also displayed the same results. Besides that, infection patterns of Haitian variant strains showed a completely different picture. Increased mucosal damaging, colonization, and inflammatory changes were observed through hematoxylin-eosin staining and transmission electron microscopy. Fluid accumulation ability was also significantly higher in rabbit model. Our study indicated that these virulence features of the Haitian variant strain may have some association with the severe clinical outcome of the cholera patients in different parts of the world

Curcumin Alleviates Matrix Metalloproteinase-3 and -9 Activities during Eradication of Helicobacter pylori Infection in Cultured Cells and Mice

Current therapy-regimens against Helicobacter pylori (Hp) infections have considerable failure rates and adverse side effects that urge the quest for an effective alternative therapy. We have shown that curcumin is capable of eradicating Hp-infection in mice. Here we examine the mechanism by which curcumin protects Hp infection in cultured cells and mice. Since, MMP-3 and -9 are inflammatory molecules associated to the pathogenesis of Hp-infection, we investigated the role of curcumin on inflammatory MMPs as well as proinflammatory molecules. Curcumin dose dependently suppressed MMP-3 and -9 expression in Hp infected human gastric epithelial (AGS) cells. Consistently, Hp-eradication by curcumin-therapy involved significant downregulation of MMP-3 and -9 activities and expression in both cytotoxic associated gene (cag)+ve and cag-ve Hp-infected mouse gastric tissues. Moreover, we demonstrate that the conventional triple therapy (TT) alleviated MMP-3 and -9 activities less efficiently than curcumin and curcumin's action on MMPs was linked to decreased pro-inflammatory molecules and activator protein-1 activation in Hp-infected gastric tissues. Although both curcumin and TT were associated with MMP-3 and -9 downregulation during Hp-eradication, but unlike TT, curcumin enhanced peroxisome proliferator-activated receptor-γ and inhibitor of kappa B-α. These data indicate that curcumin-mediated healing of Hp-infection involves regulation of MMP-3 and -9 activities

Role of ChiA2 in <i>V. cholerae in vitro</i> and <i>in vivo</i> proliferation.

<p><b>A</b>. Comparative study of the survival of wild type and Δ<i>chiA2</i> mutant in human intestinal HT29 cell line. The graph represents a dose dependent survival of wild type <i>V. cholerae</i> and Δ<i>chiA2</i> mutant and also a complemented strain. <b>B</b>. Comparative study of dose dependent mice intestinal survival of wild type <i>V. cholerae</i>, Δ<i>chiA2</i> mutantand the complemented strain. For the experiment, 5 days old suckling mice were fed with different dilutions of the mentioned strains and sacrificed after 16 hours. The viability of the strains was measured by cell count method. Bars: (□) PBS; (▪) wild type <i>V. cholerae</i>; ( ) Δ<i>chiA2</i> mutant () complemented strain. Each of the experiment was repeated thrice (n = 3) and the data expressed as means ± SEM.</p

The <i>Vibrio cholerae</i> Extracellular Chitinase ChiA2 Is Important for Survival and Pathogenesis in the Host Intestine

<div><p>In aquatic environments, <i>Vibrio cholerae</i> colonizes mainly on the chitinous surface of copepods and utilizes chitin as the sole carbon and nitrogen source. Of the two extracellular chitinases essential for chitin utilization, the expression of <i>chiA2</i> is maximally up-regulated in host intestine. Recent studies indicate that several bacterial chitinases may be involved in host pathogenesis. However, the role of <i>V. cholerae</i> chitinases in host infection is not yet known. In this study, we provide evidence to show that ChiA2 is important for <i>V. cholerae</i> survival in intestine as well as in pathogenesis. We demonstrate that ChiA2 de-glycosylates mucin and releases reducing sugars like GlcNAc and its oligomers. Deglycosylation of mucin corroborated with reduced uptake of alcian blue stain by ChiA2 treated mucin. Next, we show that <i>V. cholerae</i> could utilize mucin as a nutrient source. In comparison to the wild type strain, Δ<i>chiA2</i> mutant was 60-fold less efficient in growth in mucin supplemented minimal media and was also ∼6-fold less competent to survive when grown in the presence of mucin-secreting human intestinal HT29 epithelial cells. Similar results were also obtained when the strains were infected in mice intestine. Infection with the Δ<i>chiA2</i> mutant caused ∼50-fold less fluid accumulation in infant mice as well as in rabbit ileal loop compared to the wild type strain. To see if the difference in survival of the Δ<i>chiA2</i> mutant and wild type <i>V. cholerae</i> was due to reduced adhesion of the mutant, we monitored binding of the strains on HT29 cells. The initial binding of the wild type and mutant strain was similar. Collectively these data suggest that ChiA2 secreted by <i>V. cholerae</i> in the intestine hydrolyzed intestinal mucin to release GlcNAc, and the released sugar is successfully utilized by <i>V. cholerae</i> for growth and survival in the host intestine.</p></div