Genomics and Population Dynamics of Vibrio cholerae Causing Pandemic Cholera

Abstract

Cholera remains a major public health threat in many developing countries of Asia, Africa and the Americas. The causative agent of cholera, the Gram-negative bacterium Vibrio cholerae, is distributed ubiquitously in estuarine and marine water all over the world. Among all enteric pathogens, only cholera bacterium has the potential to cause pandemics. Seasonal cholera outbreaks in Bangladesh and the Ganges delta region of the Bay of Bengal are influenced by several biotic and abiotic factors. Over the past few decades, numerous epidemiological studies have been conducted to elucidate the ecology and epidemiology of V. cholerae, with strong emphasis on its survival, transmission, and pathogenesis. However, the global burden of cholera is increasing steadily in many parts of the world and massive cholera outbreaks in non-endemic countries have been reported recently. This study, therefore, employed a holistic approach to understand the emergence and evolution of pathogenic V. cholerae and microbial community dynamics in natural reservoir. Conventional microbiological and molecular methods were employed along with genome sequencing of V. cholerae isolates and whole genome shotgun sequencing of water samples to achieve the goal. Phenotypic and genetic characterization of 97 V. cholerae O1 isolates from Mathbaria, Bangladesh, revealed the presence of cholera toxin gene and other virulence factors, and showed six antibiotic resistance patterns with reduced susceptibility to ciprofloxacin and azithromycin. Multilocus variable number tandem repeat analysis (MLVA) of 222 environmental and clinical isolates identified genotypic variability in V. cholerae O1 isolated in two distinct areas of Bangladesh, i.e., Mathbaria and Chhatak. Multiple genetic lineages were detected among toxigenic V. cholerae O1 strains from Mathbaria; however, the outbreak in Chhatak was attributed to clonal expansion, commonly known as ‘founder flush’. Thus, results of this study support ‘person-to-person’ or accelerated mode of transmission of cholera during an outbreak. Whole genome sequencing and analysis of 165 V. cholerae isolates from Bangladesh identified multiple clones of toxigenic V. cholerae O1 carrying genotype ctxB1, ctxB3, and ctxB7. Comparative genomics of V. cholerae O1 and non-O1/O139 strains revealed a substantial number of GIs and MGEs, suggesting genomic rearrangement plays an important role in the dynamic of pathogenic V. cholerae. Comparative genomics also detected greater genetic diversity in environmental V. cholerae O1 strains compared to clinical strains. Extensive genetic analysis of 91 V. cholerae O1 isolates from Mexico, isolated between 1991 and 2008, identified a truncated pro-phage in the upstream region of CTX, TCP variant, and predominance of V. cholerae with ctxB3 during 2004 and 2008. Given the fact that very few clones may have been introduced to Mexico from Asia and Africa, core genome phylogeny also identified genetically different clones that might have evolved over time in the aquatic environment of Mexico. Presence of West African-South American genomic island (WASA)-1 in V. cholerae O1 isolated in Mexico and lack of SXT/R391 ICE is remarkable compared to atypical El Tor strains isolated in Asia and Africa. Whole genome shotgun sequencing and bioinformatics analysis of 20 pond water samples from Mathbaria, Bangladesh, identified 549 different species of bacteria, 50 viruses, 11 fungi, and 9 protista, commonly found in both fresh and coastal waters. Five Vibrio species were detected, of which V. cholerae and V. mimicus were predominant during both the epidemic and non-epidemic seasons of cholera. Interestingly, CTXΦ and other Vibrio phages were consistently detected throughout the study period, 2013 -2014. Like several previous studies, results of this study also showed environmental factors, i.e., temperature and salinity have direct influence in the microbial community dynamics. Another interesting observation was detection and identification of Vibrio phages during both epidemic and inter-epidemic periods. Toxigenic and non-toxigenic V. cholerae, other Vibrio species, symbiotic microbes, and predatory phages were found to exhibit a complex synchronization of the microbial community with environmental factors influencing the abundance of V. cholerae related to seasonal epidemics.