Prevalence of Arcobacter and other pathogenic bacteria in river water in Nepal

Published: 10 July 2019This study aims to determine the diversity of pathogenic bacteria in the Bagmati River, Nepal, during a one-year period. A total of 18 river water samples were collected from three sites (n = 6 per site) along the river. Bacterial DNA, which were extracted from the water samples, were analyzed for bacterial 16S rRNA genes by next-generation sequencing for 13 of 18 samples, and by quantitative PCR targeting Arcobacter for all 18 samples. The 16S rRNA sequencing identified an average of 97,412 ± 35,909 sequences/sample, which were then categorized into 28 phyla, 61 classes, and 709 bacterial genera. Eighteen (16%) genera of 111 potential pathogenic bacteria were detected with abundance ratios of >1%; Arcobacter, Acinetobacter, and Prevotella were the dominant genera. The Arcobacter abundance ratios were 28.6% (n = 1), 31.3 ± 15.8% (n = 6), and 31.8 ± 17.2% (n = 6) at the upstream, midstream, and downstream sites, respectively. Arcobacter was detected in 14 (78%) of 18 samples tested, with concentrations ranging from 6.7 to 10.7 log10 copies/100 mL, based on quantitative PCR. Our results demonstrate the poor bacterial quality of the Bagmati River water, suggesting a need for implementing more measures to reduce fecal contamination in the river water.Rajani Ghaju Shrestha, Sarmila Tandukar, Dinesh Bhandari, Samendra P. Sherchan, Yasuhiro Tanaka, Jeevan B. Sherchand and Eiji Haramot

Detection of Cryptosporidium, Giardia, fecal indicator bacteria, and total bacteria in commercial jar water in Kathmandu Valley, Nepal

Introduction: Jar water is a convenient and common source of drinking water in the Kathmandu Valley. However, studies including detailed microbial analyses of this source of potable water are lacking. In this study, jar water samples were examined for the occurrence of Cryptosporidium, Giardia, fecal indicator bacteria, and total bacteria. Methods: Thirty different brands of jars were collected in September 2014. Escherichia coli and total coliforms w ere determined using a Colilert reagent. Ten of the 30 brands w ere selected to test for Cryptosporidium, Giardia, and total bacteria. Bacterial DNA extraction from water samples w as performed using the Cica Geneus DNA Extraction Kit, follow ed by quantitative polymerase chain reaction (qPCR) targeting the 16S rRNA gene of bacterial DNA. Protozoan detection was accomplished by concentrating the samples using the electronegative membrane vortex method, followed by immunomagnetic separation and fluorescent staining. Results: E. coli w as detected in 10% of the samples, with a maximum concentration of 2 most probable number (MPN)/100 mL, whereas total coliforms were detected in 97% of the samples, with a maximum and mean concentration of 7.3 × 10 2 and 3.8 × 10 1 MPN/100 mL, respectively. Total coliforms concentrations in 40% of the samples ranged from 10 2 to 10 3 MPN/100 mL. Cryptosporidium and Giardia w ere not detected in any of the tested samples. Concentrations of total bacteria in the samples ranged from 10 4 to 10 6 cells/100 mL. Conclusions: Ninety-seven percent of the jar water brands were unsuitable for drinking without proper treatment based on the guideline values of the National Drinking Water Quality Standards (NDW Q S) of Nepal. There is no guideline value for total bacteria in NDW Q S however, high concentrations can be indicative of poor control on regrowth of bacteria and recontamination or inefficient water treatment methods.Malla B, Ghaju Shrestha R, Bhandari D, Tandukar S, Shrestha S, Yoshinaga H, Inoue D, Sei K, Nishida K, Tanaka Y, Sherchand JB, Haramoto

Co-infection by waterborne enteric viruses in children with gastroenteritis in Nepal

Enteric viruses are highly contagious and a major cause of waterborne gastroenteritis in children younger than five years of age in developing world. This study examined the prevalence of enteric virus infection in children with gastroenteritis to identify risk factors for co-infections. In total, 107 stool samples were collected from patients with acute gastroenteritis along with samples of their household drinking water and other possible contamination sources, such as food and hand. The presence of major gastroenteritis-causing enteric virus species (group A rotaviruses, enteroviruses, adenoviruses, and noroviruses of genogroup I) in stool and water samples was examined using quantitative polymerase chain reaction. Among the 107 stool samples tested, 103 (96%) samples contained at least one of the four tested enteric viruses, and the combination of group A rotaviruses and enteroviruses was the most common co-infection (52%, n = 54/103). At least one viral agent was detected in 16 (16%) of 103 drinking water samples. Identical enteric viruses were detected in both the stool and water samples taken from the same patients in 13% of cases (n = 13/103). Group A rotaviruses were most frequently found in children suffering from acute diarrhea. No socio-demographic and clinical factors were associated with the risk of co-infection compared with mono-infection. These less commonly diagnosed viral etiological agents in hospitals are highly prevalent in patients with acute gastroenteritis.Sarmila Tandukar, Jeevan B. Sherchand, Surendra Karki, Bikash Malla, Rajani Ghaju Shrestha, Dinesh Bhandari, Ocean Thakali and Eiji Haramot

Validation of host-specific Bacteroidales quantitative PCR assays and their application to microbial source tracking of drinking water sources in the Kathmandu Valley, Nepal

Aims: To validate host-specific Bacteroidales assays to identify faecal-sourcecontamination of drinking water sources in the Kathmandu Valley, Nepal.Methods and Results: A total of 54 composite faecal-source samples werecollected from human sewage, ruminants, pigs, dogs, chickens and ducks,which were analysed by quantitative polymerase chain reaction using human-specific (BacHum, HF183 SYBR, gyrB and HF183 TaqMan), ruminant-specific(BacCow and BacR), pig-specific (Pig2Bac and PF163) and dog-specific assays(BacCan SYBR). The BacHum, BacR and Pig2Bac assays were judged the bestperforming human-specific, ruminant-specific and pig-specific assaysrespectively. The BacCan SYBR assay highly cross-reacted with other species,resulting in poor performance. Furthermore, these validated assays wereapplied to microbial source tracking (MST) of 74 drinking water samples. Outof these, 20, 12 and 4% samples were judged contaminated by human,ruminant and pig faeces respectively. Detection ratios of human and ruminantfaecal markers were relatively higher in built-up and agricultural areasrespectively.Conclusion: BacHum, BacR and Pig2Bac assays were found suitable for MSTand both, human and animal faecal contaminations of drinking water sourceswere common in the valley.Significance and Impact of the study: MST could be an effective tool forpreparing the faecal pollution strategies as these are site specific.B. Malla, R. Ghaju Shrestha, S. Tandukar, D. Bhandari, D. Inoue, K.Sei, Y. Tanaka, J.B. Sherchand and E. Haramot