Advances in cholera research: from molecular biology to public health initiatives

The aquatic bacterium Vibrio cholerae is the etiological agent of the diarrheal disease cholera, which has plagued the world for centuries. This pathogen has been the subject of studies in a vast array of fields, from molecular biology to animal models for virulence activity to epidemiological disease transmission modeling. V. cholerae genetics and the activity of virulence genes determine the pathogenic potential of different strains, as well as provide a model for genomic evolution in the natural environment. While animal models for V. cholerae infection have been used for decades, recent advances in this area provide a well-rounded picture of nearly all aspects of V. cholerae interaction with both mammalian and non-mammalian hosts, encompassing colonization dynamics, pathogenesis, immunological responses, and transmission to naïve populations. Microbiome studies have become increasingly common as access and affordability of sequencing has improved, and these studies have revealed key factors in V. cholerae communication and competition with members of the gut microbiota. Despite a wealth of knowledge surrounding V. cholerae, the pathogen remains endemic in numerous countries and causes sporadic outbreaks elsewhere. Public health initiatives aim to prevent cholera outbreaks and provide prompt, effective relief in cases where prevention is not feasible. In this review, we describe recent advancements in cholera research in these areas to provide a more complete illustration of V. cholerae evolution as a microbe and significant global health threat, as well as how researchers are working to improve understanding and minimize impact of this pathogen on vulnerable populations

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

Diversity and Distribution of Archaea in the Mangrove Sediment of Sundarbans

Mangroves are among the most diverse and productive coastal ecosystems in the tropical and subtropical regions. Environmental conditions particular to this biome make mangroves hotspots for microbial diversity, and the resident microbial communities play essential roles in maintenance of the ecosystem. Recently, there has been increasing interest to understand the composition and contribution of microorganisms in mangroves. In the present study, we have analyzed the diversity and distribution of archaea in the tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencing. The extraction of DNA from sediment samples and the direct application of 16S rRNA gene amplicon sequencing resulted in approximately 142 Mb of data from three distinct mangrove areas (Godkhali, Bonnie camp, and Dhulibhashani). The taxonomic analysis revealed the dominance of phyla Euryarchaeota and Thaumarchaeota (Marine Group I) within our dataset. The distribution of different archaeal taxa and respective statistical analysis (SIMPER, NMDS) revealed a clear community shift along the sampling stations. The sampling stations (Godkhali and Bonnie camp) with history of higher hydrocarbon/oil pollution showed different archaeal community pattern (dominated by haloarchaea) compared to station (Dhulibhashani) with nearly pristine environment (dominated by methanogens). It is indicated that sediment archaeal community patterns were influenced by environmental conditions

Immunomodulatory role of outer membrane vesicles of Shigella in mouse model

In our previous studies, we discussed the protective efficacy of the two types of vaccine formulation namely SOMVs (single-serotype outer membrane vesicles) and MOMVs (multi-serotype outer membrane vesicles). Here, we compared the immunogenic roles of these two types of formulations and also studied general immunomodulation by Shigella OMVs in adult BALB/c mice. The production of various pro-inflammatory (TNF-α, IL-1β, IL-6, IL-12, IL-18, IFN-γ) and anti-inflammatory (IL-4 and IL-10) cytokine profile were assessed by in vivo, ex vivo and in vitro studies. MOMVs treated mice showed significantly enhanced cytokine production compared to SOMVs treated mice. MOMVs treatment has also upregulated iNOS mRNA synthesis in macrophages. Overall the OMVs of Shigella were found to show a mixed Th1/Th2 response and maintain the balance between pro-inflammation and anti-inflammation in mice. This will be crucial in the development of the next generation OMVs based vaccine against shigellosis

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

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

List of Primers used in construction of <i>chiA2</i> deleted <i>V. cholerae</i> strain and cloning <i>chiA2</i> in pBAD-TOPO TA expression vector.

<p>List of Primers used in construction of <i>chiA2</i> deleted <i>V. cholerae</i> strain and cloning <i>chiA2</i> in pBAD-TOPO TA expression vector.</p

Utilization of mucin as nutrient for growth by <i>V. cholerae</i>.

<p><i>V. cholerae</i> wild type, Δ<i>chiA2</i> mutant and the complemented strain were separately grown in LB upto log phase. The log phase cultures of the wild type <i>V. cholerae</i>, Δ<i>chiA2</i> mutant and the complemented strain were centrifuged and washed in PBS. The minimal media supplemented with 2% (w/v) porcine mucin and fresh LB broth were inoculated separately with equal number of washed <i>V. cholerae</i> wild type and Δ<i>chiA2</i> mutant and complemented strain. The number of bacteria survived with increasing incubation time was detected by plate count method for all of the strains. The viable counts of bacteria were graphically represented. <b>A</b>. Viable count when mucin used as nutrient. <b>B</b>. Viable count when grown in LB broth. Each of the experiment was repeated thrice (n = 3) and the data expressed as means ± SEM.</p

HPLC analysis of end products of ChiA2-treated mucin.

<p>The mucin-ChiA2 Reactions were carried as mentioned in the materials and methods section. The end products were analyzed using a 4.6 nm×250, 5 µm Zorbax carbohydrate analysis column (Agilent Technologies, Waldbrown, Germany) connected to a Shimadzu Prominence 20A; HPLC system. <b>A</b>. This figure shows the HPLC chromatogram of the end products of ChiA2 treated mucin. The chromatogram shows four distinct peaks and an unresolved region. Peak 1 with retention time 5.4 minutes indicates presence of GlcNAc. Peak 2 indicates presence of (GlcNAc)2 which is divided into two separate peaks: peak 2A and peak 2B. Peak 2A with retention time 6.0 minutes probably is for the β enantiomer and peak 2B with retention time 6.57 minutes is for α enantiomer of (GlcNAc)₂. Peak 3 with retention time 7.6 minutes indicates the presence of (GlcNAc)₃. <b>B</b>. This is the HPLC chromatogram of standards of GlcNAc, (GlcNAc)₂, (GlcNAc)₃ and (GlcNAc)₆. The chromatogram shows 4 distinct peaks. Peak 1 with retention time 5.5 minutes is for GlcNAc. Peak 2 with retention time 6.6 minutes is for α-(GlcNAc)₂. Peak 3 with retention time 7.5 minutes is for (GlcNAc)₃ and peak 4 with retention time 11.1 minutes is for (GlcNAc)₆.</p