Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/20359
Title: Phenotypic and Genotypic Charaterization of Indigenous Strains of Vibrio Cholerae Isolated from Khairpur, Sindh, Pakistan
Authors: Ali, Amjad
Keywords: Biological & Medical Sciences
Microbiology
Issue Date: 2021
Publisher: Shah Abdul Latif University, Khairpur.
Abstract: Cholera is a diarrheal disease caused by the bacterium Vibrio cholerae (V. cholerae), which is transmitted by contaminated water, food, and oral faeces, as well as poor hygiene. According to the World Health Organization, cholera is estimated to cause 3- 5 million cases and 100,000-120,000 deaths worldwide per year. Although the number of cholera outbreaks in Pakistan has been steadily increasing, a detailed understanding of the epidemiology is still lacking. This analysis aimed to classify V. cholerae isolates from the Khairpur District in Sindh, Pakistan, which is a large district (in terms of area) in the province's northern portion. This research aimed to see whether there were any responsible serogroups, biotypes, phenotypic and genotypic classification, whole-genome sequence analysis, and phylogenetic relationships between indigenous V. cholerae strains and modern strains isolated from Sindh's southern regions. 660 samples were obtained throughout the research (2014-2016). (360 from cholera patients and 300 drinking water samples). Normal microbiological, biochemical, and serological methods, as well as species specific PCR targeting the 585-bp virulence gene ompW, were used to identify the isolate. The optimum temperature for growth was 35oC and the optimum growth time was 24 hours. A gender bias was revealed among the clinical cases of male and female subjects. There was a male predominance over females (n=348, 96.6%) where the severely affected age group was 19 years to above. The month-wise index for positive V. cholerae samples shown that the maximum positive samples were attained from May to August. Out of 360 clinical samples, 76 (21.11%) were positive for Vibrio cholerae. The isolated V. cholerae belonged to the Inaba serogroup, O1. The biotype classification of 13 V. cholerae O1 strains showed normal El Tor phenotypes, which were close to the phenotype of V. cholerae El Tor strain N16961 (7th pandemic strain, PBr VP+ ), which was used as the reference strain in this study. Species-specific gene ompW was targeted for amplification by PCR and genetic identification of the isolates. The 585 bp ompW gene was present in all the 76 clinical strains of V. cholerae. We used MUMmer, NUCmer, and PROmer (MUMmer package) to determine the evolutionary relationship between the selected strains under the study. MUMmer was used for divergent regions between strains with the same topology, NUCmer stands for Nucleotides and is used to identify similar regions, and PROmer is used for the study of proteins. For multiple sequence alignment and phylogeny generation, we used Clustal Omega software. Multiple sequence alignment of V. cholerae genome unique sequences showed that reference sequence was not similar to the other regions of the test strains (AMVC from Khairpur, KTH from Jamshoro, and KCH from Karachi) mostly, in the same genomic coordinates. The phylogenetic tree showed that reference strain shared an ancestor relationship with KCH-17 where KTH and KCH strains were not clustered. The data generated through NUCmer displayed the information about conserved regions among all the test strains and their coordinates that belong to different regions. Multiple sequence alignment of V. cholerae conserved genome sequences showed that most of the test strains were similar to each other except KCH-10, AMVC-256, KCH 15, AMVC-58, and AMVC-64. The phylogenetic tree was generated that displayed that the reference sequence shared the same root with AMVC-256 and strain KCH-10 shared the same root with AMVC-64. It was clear that all three AMVCs were descended from the same ancestor along with KCH-10 and reference sequence. Most of the KCH strains were placed together in the tree except KCH-10. Multiple sequence alignment 14 of the regions translated into protein using the PROmer program gave a better understanding of evolution. The protein alignment showed that most of the test strains were similar except KCH-16 and AMVC-64. This divergence in the two strains was possibly due to evolution. It was also observed that two amino acids were deleted in the lower segment of the alignment in most of the sequences of the strains. The Phylogenetic tree showed that the reference sequence shared the same root with AMVC-256 where KCH-10 shared the same root with AMVC-64. It was clear that all three AMVCs strains isolated from Khairpur were descended from the same ancestor along with KCH-10 and reference sequence. Most of the KCH strains were placed together in the tree except KCH-10. Possibly it was separated from its group because of mutations and migration. Overall, the results of our study suggest the existence of genetically diverse V. cholerae O1 El Tor in Khairpur during the study period. It is important to mention here that none of the drinking water samples was found positive for V. cholerae even in the peak months for cholera i.e. May to August. This indicates the improved sanitation and water supply system in the study area and suggests the oro- fecal route and contamination as the mode of transmission of cholera in Khairpur. The negative culture results of water samples in our study could be attributed to the fact that V. cholerae can live for long periods in nutrient-depleted drinking water in a viable yet non-cultivable environment. Owing to its non detectability in its non-culturable form, this phenomenon appears to be a threat to human health. This study will provide awareness about personal hygiene and the threat of V. cholerae variants at District Khairpur. 15 This is the first time, to our knowledge, that V. cholerae El Tor has been linked to cholera outbreaks in Khairpur, Sindh, Pakistan. The pathogenic strains were thought to be V. cholerae O1 El Tor variant. The clinical strains shared a similar genetic heritage but were diverse, according to phylogenetic analysis. This research helps to better understand the epidemiology of V. cholerae in the study area, which can then be used to develop cholera prevention and management strategies in Pakistan.
Gov't Doc #: 25797
URI: http://prr.hec.gov.pk/jspui/handle/123456789/20359
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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