Munirul Alam

Viable but nonculturable Vibrio cholerae O1 in biofilms in the aquatic environment and their role in cholera transmission

Vibrio cholerae persists in aquatic environments predominantly in a nonculturable state. In this study coccoid, nonculturable V. cholerae O1 in biofilms maintained for 495 days in Mathbaria, Bangladesh, pond water became culturable upon animal passage. Culturability, biofilm formation, and the wbe, ctxA, and rstR2 genes were monitored by culture, direct fluorescent antibody (DFA), and multiplex PCR. DFA counts were not possible after formation of biofilm. Furthermore, wbe, but not ctxA, were amplifiable, even after incubation for 54 and 68 days at room temperature (≈25°C) and 4°C, respectively, when no growth was detectable. Slower biofilm formation and extended culturability were observed for cultures incubated at 4°C, compared with ≈25°C, suggesting biofilm production to be temperature dependent and linked to loss of culturability. Small colonies appearing after incubation in microcosms for 54 and 68 days at 25°C and 4°C, respectively, were wbe positive and ctxA and rstR2 negative, indicating loss of bacteriophage CTXΦ. The coccoid V. cholerae O1 observed as free cells in microcosms incubated for 495 days could not be cultured, but biofilms in the same microcosms yielded culturable cells. It is concluded that biofilms can act as a reservoir for V. cholerae O1 between epidemics because of its long-term viability in biofilms. In contrast to biofilms produced in Mathbaria pond water, V. cholerae O1 in biofilms present in cholera stools and incubated under identical conditions as the Mathbaria pond water biofilms could not be cultured after 2 months, indicating that those V. cholerae cells freshly discharged into the environment are significantly less robust than cells adapted to environmental conditions.

Genomic diversity of 2010 Haitian cholera outbreak strains

The millions of deaths from cholera during the past 200 y, coupled with the morbidity and mortality of cholera in Haiti since October 2010, are grim reminders that Vibrio cholerae, the etiologic agent of cholera, remains a scourge. We report the isolation of both V. cholerae O1 and non-O1/O139 early in the Haiti cholera epidemic from samples collected from victims in 18 towns across eight Arrondissements of Haiti. The results showed two distinct populations of V. cholerae coexisted in Haiti early in the epidemic. As non-O1/O139 V. cholerae was the sole pathogen isolated from 21% of the clinical specimens, its role in this epidemic, either alone or in concert with V. cholerae O1, cannot be dismissed. A genomic approach was used to examine similarities and differences among the Haitian V. cholerae O1 and V. cholerae non-O1/O139 strains. A total of 47 V. cholerae O1 and 29 V. cholerae non-O1/O139 isolates from patients and the environment were sequenced. Comparative genome analyses of the 76 genomes and eight reference strains of V. cholerae isolated in concurrent epidemics outside Haiti and 27 V. cholerae genomes available in the public database demonstrated substantial diversity of V. cholerae and ongoing flux within its genome.

Toxigenic Vibrio cholerae in the Aquatic Environment of Mathbaria, Bangladesh

ABSTRACT Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.

El Tor cholera with severe disease: a new threat to Asia and beyond.

During epidemics of cholera in two rural sites (Bakerganj and Mathbaria), a much higher proportion of patients came for treatment with severe dehydration than was seen in previous years. V. cholerae O1 isolated from these patients was found to be El Tor in its phenotype, but its cholera toxin (CT) was determined to be that of classical biotype. Whether the observed higher proportion of severe dehydration produced by the El Tor biotype was due to a shift from El Tor to classical CT or due to other factors is not clear. However, if cholera due to strains with increased severity spread to other areas where treatment facilities are limited, there are likely to be many more cholera deaths.

Occurrence and Characteristics of Class 1 and 2 Integrons in Pseudomonas aeruginosa Isolates from Patients in Southern China

ABSTRACT Class 1 and 2 integrons were detected in 45.8% (54/118) and 19.5% (23/118) of our tested Pseudomonas aeruginosa isolates, respectively. Three strains were positive for both the integrons. This is the first report of class 2 integrons in P. aeruginosa and also of isolates carrying class 1 and 2 integrons simultaneously.

Development and validation of a mismatch amplification mutation PCR assay to monitor the dissemination of an emerging variant of Vibrio cholerae O1 biotype El Tor

A mismatch amplification mutation PCR assay was developed and validated for rapid detection of the biotype specific cholera toxin B subunit of V. cholerae O1. This assay will enable easy monitoring of the spread of a new emerging variant of the El Tor biotype of V. cholerae O1.

Seasonal Cholera Caused by Vibrio cholerae Serogroups O1 and O139 in the Coastal Aquatic Environment of Bangladesh

ABSTRACT Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.

Gut Microbiota of Healthy and Malnourished Children in Bangladesh

Poor health and malnutrition in preschool children are longstanding problems in Bangladesh. Gut microbiota plays a tremendous role in nutrient absorption and determining the state of health. In this study, metagenomic tool was employed to assess the gut microbiota composition of healthy and malnourished children. DNA was extracted from fecal samples of seven healthy and seven malnourished children (n = 14; age 2–3 years) were analyzed for the variable region of 16S rRNA genes by universal primer PCR followed by high-throughput 454 parallel sequencing to identify the bacterial phyla and genera. Our results reveal that the healthy children had a significantly higher number of operational taxonomic unit in their gut than that of the malnourished children (healthy vs. malnourished: 546 vs. 310). In malnourished children, bacterial population of the phyla Proteobacteria and Bacteroidetes accounted for 46 and 18%, respectively. Conversely, in healthy children, Proteobacteria and Bacteroidetes accounted for 5% and 44, respectively (p < 0.001). In malnourished children, the phylum Proteobacteria included pathogenic genera, namely Klebsiella and Escherichia, which were 174-fold and 9-fold higher, respectively, than their healthy counterpart. The predominance of potentially pathogenic Proteobacteria and minimal level of Bacteroidetes as commensal microbiota might be associated to the ill health of malnourished children in Bangladesh.

Metagenomic profile of gut microbiota in children during cholera and recovery

BackgroundThe 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.MethodBacterial 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.Results454 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.ConclusionCholera 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.

Biofilms in water, its role and impact in human disease transmission

Understanding the mechanism of biofilm formation is the first step in determining its function and, thereby, its impact and role in the environment. Extensive studies accomplished during the past few years have elucidated the genetics and biochemistry of biofilm formation. Cell-to-cell communication, that is, quorum sensing, is a key factor in the initiation of biofilm. Occurrence of viable but nonculturable bacteria, including Vibrio cholerae in biofilms has been reported and most likely such cells were overlooked previously because appropriate methods of detection were not employed. For this reason discovery and investigation of this important bacterial ecological niche in the environment were impeded.