Isolation of Vibrio cholerae from clinical and drinking water samples during Cholera Outbreak in Khairpur Sindh Pakistan.

Background: Cholera is a diarrheal disease that is caused by the bacterium Vibrio cholera (V. cholerae) that transmits through contaminated water, food, oral-faecal route and spread through poor sanitation system. This study aims to investigate the reports of clinical and drinking water samples during 2014 to 2016 cholera outbreak in Pakistan. Methodology: This study was conducted in District Khairpur including 660 samples (360 from clinical and 300 from drinking water). All samples were enriched in Alkaline peptone water for 6 hours and then streaked on Thiosulfate-citrate-bile salts-sucrose (TCBS) agar, incubated at 35ºC for 24 hours. Moreover, microbiological, biochemical, serological techniques were used for the identification of V. cholerae and further identification was performed using PCR. Results: Out of 360 clinical samples, 76(21.11%) were positive for V. cholerae. The species-specific outer membrane protein W precursor (ompW) gene was amplified and shown at the correct size (588 bp) through agarose gel electrophoresis. The serotyping revealed that the isolates belonged to serogroup Inaba, O1. Cholerae O1 strains shown typical El Tor phenotype similar to V. cholerae El Tor strain N16961 (PBR VP+) used as the reference strain in this study. All age groups were affected where the highest onset was seen among those aged 19 years and above. The culture of drinking water (n=300), observed negative on TCBS agar. Conclusion: As far as our knowledge, this is the first time when the presence of V. cholerae El Tor peak has been reported for causing cholera outbreaks in Khairpur Sindh, Pakistan. However, these findings can be used for further investigations and recognizing control measures.


Introduction
Cholera is a very serious form of human-caused diarrhea by Gram-negative, motility, bacterial filamentous, and coma-sized called V. cholerae 1 . More than 200 cholera serogroups, only serge O1 and O139 groups are primarily responsible for cholera. Cholera has infected South Asia for at least 1,000 years, besides it has caused seven epidemics throughout the world since 1817 2,3 .
Serogroup O1 contains classical and El Tor biotypes (based on special chemical tests and phase sensitivity) and possess three different serotypes, Inaba Ogawa and Hikojima 4 . The classical biotype caused six cholera outbreaks from 1817 to 1923 4,5 . Later, the El Tor biotype replaced the classical biotype that created the seventh cholera epidemic in 1961, and since then, V. cholerae O1 El Tor is the world's leading cholera organism 6,7 . A few years later, serogroup O139 was replaced by O1 serogroup, and now it does not appear 8,9 . In recent years, cholera has become even more common in developing lands where sanitation is poor and safe drinking water is scarcely available. For example, the most recent outbreak was reported in Yemen on April 27, 2017, and the disease resulted in a total of 332,658 suspected cases of cholera and 1,759 deaths as of 2017 10 . Cholera may be caused by infected people and water reservoirs such as water and rivers 11 . Cholera infection is most common in drinking water when V. cholerae is found naturally or in the feces of an infected person. Transfers from person to person, even to health care workers during an epidemic, are documented 12,13 . V. cholerae pathogenicity is caused by a genetic code of proteins that are directly or indirectly involved in the bacteria virulence 14 .
At the time of infection, V. cholerae also releases cholera toxin, a protein that causes diarrhea. In 2010, there was an increase in cholera cases due to the worst floods that threatened public health throughout Pakistan, with 164 confirmed cases in the laboratory, where cholera cases had previously been hardly reported 15 . Cholera remains a major health problem in Pakistan, with many cases every year, especially during the rainy season. Because of poor living conditions and sanitation practices, Pakistan is facing health risks, including gastroenteritis [16][17][18] .
This study aims to investigate the reports of clinical and drinking water samples during 2014 to 2016 cholera outbreak in Pakistan. Our findings may also provide important insights into the outcome of V. cholerae O1 El Tor isolates [17][18] .
DNA extraction was carried out by boiling method as described by Sepp and colleagues 24 . Well, isolated colonies from fresh culture were suspended in Tris-EDTA (TE buffer) and boiled for 10 minutes. After that, the tubes were placed on ice for 2 minutes, centrifuged at 13000 rpm for 10 minutes at 4ºC, and the supernatant was transferred to clean sterile 1.5 ml tubes and stored at -20ºC.

Polymerase chain reaction (PCR)
Hot-start PCR was performed for amplification of ompW gene from indigenous V. cholerae strains and ATCC 14035 V. cholerae strain used as a positive control. The parameters were 1 cycle predenaturation at 95ºC for 5 minutes, 35 cycles denaturation at 95ºC for 1 minutes, annealing at 61ºC for 30 seconds, extension at 72ºC for 1 minutes and final extension at 72ºC for 10 minutes. A Hybrid thermal cycler was used for all reactions. The amplicons were stored at -20ºC 15 .

Agarose gel electrophoresis
Agarose Gel Electrophoresis was performed as described in Shah et al 15 . The gel was observed by using the ABI Gel doc system and photographed. The amplified DNA products were visualized using 1% agarose gel electrophoresis under a UV transilluminator (BioRad USA).

Calculation of isolation rate of V. cholerae
The isolation rate was calculated using the following equation 24 .

Sero-grouping and bio-typing of indigenous V. cholerae
Sero-groping of the indigenous strains with polyvalent antisera showed that the V. cholerae belong to serogroup Inaba, O1. The biotype classification of the V. cholerae O1 strains revealed a typical El Tor phenotype similar to that of El Tor strain N16961 (PBR VP+). In our study, the isolated strains were Polymyxin B resistant, therefore, confirmed as El Tor biotype.

Genotypic characterization of indigenous V. cholera-DNA Extraction (n=76)
Genomic DNA from isolates was extracted as described in materials and methods. Agarose gel electrophoresis revealed the presence of genomic DNA in all the indigenous samples (not shown). All the samples showed a band approximately of similar size.

Amplification of ompW Gene from indigenous V. cholerae
The agarose gel electrophoresis revealed the successful amplification of own gene in all the clinical isolates at the correct position of 588 bp comparable to the ompW gene of V. cholerae ATCC14035 used as a positive control in this study ( Figure 2).

Discussion
The present study aimed to examine the phenotypic and genotypic characteristics of native Although cholera often affects outside of gender or age, however, these factors may play a role in the incidence of cholera, which can affect the diagnosis. This, therefore, puts certain social groups at high risk of contracting and spreading the disease in a particular setting. As the men in the study area were more exposed to foreign activities they were the bread earning members in their families; as a result, the incidence rate was higher in this study group. It is important to note here that no drinking water samples were found suitable for V. cholerae even in the high incident months, i.e. May to August (determined in this study) of cholera accompanied by reports of high outbreaks of cholera during the pre-rainy season of the year, with the secondhighest number in the post-rainy season 27 . The adverse effects of the culture of water samples in our study may be due to the fact that V. cholerae may live in contaminated water for a long time in an inactive state and infect only in the human stomach 28,29 . This poses a threat to human health due to its no recovery from water as non-culturable status in water, or it may be simply a reaction to unfavorable conditions and can be counterproductive to heterotrophic invaders and bacteriophages by adopting this unique survival strategy of an unculturable state 30 .

Conclusion
In the present study, of 360 clinical cell samples, 76 clinical isolates were identified as V. cholerae serogroup O1 Inaba, biotype El Tor. Genotypic mutations have shown the presence of virulence gene ompW in isolates. Additional studies are required to further investigate the mechanism of infection and factors promoting the overall prevalence of V. Cholerae, as it seems potentially invasive in the population of Khairpur.

Conflicts of Interest
The authors have declared that no competing interests exist.