Bacteriological assessment of tap water collected from different markets of Mymensingh , Gazipur and Sherpur districts of Bangladesh with special focus on the molecular detection and antimicrobial resistance of the isolated Escherichia coli

The objectives of this study were to assess the bacteriological quality of tap water samples obtained from different markets of different upazillas of Mymensingh, Sherpur & Gazipur district. For achieving the above mentioned objectives, methods of heterotrophic plate count (HPC) and total coliform count (TCC) were applied. Moreover, isolated E. coli from tap water samples were characterized by using biochemical test, molecular method and antimicrobial susceptibility tests. HPC was highest in market tap water collected from Kaligonj and TCC was highest in market tap water of collected from Mymensingh sadar. The geometric mean of HPC of Mymensingh, Gazipur and Sherpur districts water was 8.4x10 5 , 2.5 x10 6 and 6.8 x10 5 C.F.U/100 ml. All isolates of E. coli (n=20) were amplified by using 16S rRNA gene based PCR. In respect to antimicrobial susceptibility testing, most of the E. coli isolates were susceptible to norfloxacin, ampicilin, tetracycline, streptomycin and ciprofloxacin. Furthermore, a few E. coli isolates were intermediate resistant to gentamycin and ciprofloxacin. However, a few of the E. coli isolates were resistant to erythromycin and amoxycilin. Moreover, out of 20 E. coli isolates 3 (15%) isolates were detected as multidrug resistant. This study indicated the presence of multidrug resistant E. coli isolates in tap water in Mymensingh, Sherpur and Gazipur districts that warrants particular attention.


Introduction
According to the World Health Report (2002), every year more than 3.4 million people die as a result of water related diseases indicating these as the leading cause of disease and death around the world.In the diseaseprone, humid, tropical region of Bangladesh, outbreaks of diarrheal diseases, often on an epidemic scale, are not unusual and the possible role of water-borne pathogens in these outbreaks has been emphasized.Among waterborne diseases of bacterial origin typhoid fever, bacillary dysentery and diarrhea are common in Bangladesh (Islam et al., 2010).Despite the availability and promotion of the use of safe water sources, waterrelated diseases remain an important cause of mortality and morbidity in Bangladesh and it is suggested that intake of contaminated water acts an important mode of pathogen transmission (Kabir et al., 2015).Even if disinfection is practiced in water supply systems, but failure of the disinfection system due to poor management could result in serious health hazards and post contamination.It is estimated that the 1% of drinking water is getting polluted with various organic and inorganic matters.The organic matters which are responsible for the contamination of water are fecal wastes of poultry and livestock farms, pesticides, herbicides, and many industrial wastes, minerals and biological agents such as bacteria, virus, fungus, algae etc. Enterobacteriaceae in the water of river Manzanares at Cumana (Venezuela) and a high degree of enteric species of organisms was present in water (Mieres and Bastardo, 1975).Chemical contaminants of the most of the water sources are man-made.In urban area the major contaminants that contribute to chemical contamination of the water sources include industrial effluents, sludge from sewage treatment plants, raw untreated sewage from urban populations and industry, suspended solids, biodegradable organics, pathogens, priority pollutants, refractory organics, heavy metals and dissolved inorganics (Chatterjee et al., (2005).In rural area water pollution is also caused by silts and other suspended solids such as soil, wash off plowed fields, agriculture run-offs, and eroded river banks when rains, sewage and wastes of houses (Kabir et al., 2015).In China, about 90% of surface waters and over 60% of drinking water sources in urban areas have been polluted by different extents of organic substances, ammonia nitrogen, phenols, pesticides and pathogenic microorganisms (Wang et al., 2000).In South Asian countries, rivers such as in the Kathmandu valley, the Yamuna River at Delhi, and peripheral rivers (mainly Buriganga River) of Dhaka was more severely polluted by urban activities destined to unplanned urbanization and industrialization, inadequate sewerage, and lack of effective pollution control measures (Karn et al., 2001).The World Health Organization has estimated that up to 80% of all sickness and disease in the world is caused by inadequate sanitation, polluted water or unavailability of water.It was estimated that nearly 1.5 billion people lack safe drinking water and that at least 5 million deaths per year can be attributed to water-borne disease.Health effects associated with water supplies in developing countries are evaluated to be based on four bacterial indicators of tropical drinking-water quality (faecal coliforms, Escherichia coli, Enterococci and faecal Streptococci) and their relationship to the prevalence of diarrhoeal disease in Cebu, Philippines (Moe et al., 1991).The contaminated water or inadequate supply of safe drinking water causes various gastrointestinal diseases like diarrhoea, dysentery and water-borne diseases like cholera, typhoid.It is now evident that most of the enteric diseases of human and animals are transmitted through contaminated food and water (Johnson et al., 2003).So to get rid from suspended biological agents and to ensure the supply of pure drinking water, water must need prior treatment or purified before consumption.From this view point of public health, it is highly imperative that potable water supply system should be safe.Water may be polluted at its sources by excreta or sewage, which is almost certain to have pathogenic microorganisms.Potable water system can become polluted with coli form and pathogenic bacteria due to lack of hygiene and sanitation.As a result, microbiological examination of water should routinely be carried out to monitor and control the quality and safety of drinking water.Although the concept of safe water is under consideration in Bangladesh, unfortunately science-based little information is available.Therefore, the present study was conducted to determine the bacteriological quality, molecular detection and antibiotic sensitivity of isolated E. coli of tap water collected from different market of different upazilla of Mymensingh, Gazipur, Sherpur districts.

Collection and transportation of samples
A total of 20 tap water samples were collected in sterile glass bottles from different upazilas of Mymensingh, Gazipur and Sherpur district of Bangladesh and transported to the laboratory in ice box containing ice freezer packs.From each sampling point, 250 ml samples were taken for analyses.The bacteriological tests were undertaken within 6 hours after collection to avoid the growth or death of microorganisms in the sample.With regard to the bacteriological analysis, water samples were collected, labeled and transported to the Microbiology laboratory of Bangladesh Agricultural University.

Heterotrophic plate count (HPC)
For the determination of heterotrophic plate count, 100 μl of serial tenfold dilution of tap water samples were transferred and spreaded on plate count agar media using micro pipette for each dilution.The diluted samples were spreaded as quickly as possible on the surface of plate count agar with a sterile glass spreader.One sterile glass spreader was used for each plate.The plates were then taken in an incubator at 37ºC for 24 hours.After incubation at 37ºC for 24 hours plates exhibiting 30-300 colonies were counted.The average number of colonies in a particular dilution was multiplied by the dilution factor to obtain the heterotrophic plate count (HPC).The heterotrophic plate count was calculated according to ISO (1995).The result of total bacterial count was expressed as the number of organism or colony forming units per milliliter (C.F.U/ml) of water samples.

Total coliform count
The most probable number (MPN) test for water examination for the presence of coliforms was performed according to the procedures described by Harley and Prescott (2002).An estimate of the number of coliforms (Most Probable Number) can also be done in the presumptive test.In this procedure, 15 lactose broth tubes were inoculated with the water samples.Five tubes received 10 ml of water, 5 tubes received 1ml of water, and 5 tubes received 0.1ml of water.A count of the number of tubes showing gas production was then made, and the figure was compared to a table developed by American Public Health Association.The number was the most probable number (MPN) of coliforms per 100 ml of the water sample.

Detection of fecal coliforms
The positive presumptive cultures were transferred to lactose broth, which is specific for fecal coliform bacteria.Any presumptive tube which showed gas production after 24 (+/ 2) hours incubation at 44.5°C (+/ 0.2°C) confirmed the presence of fecal coliform bacteria in that tube and was recorded as a positive confirmed tube.

Isolation and identification of E. coli
The bacteriological examination followed detail study of colony characteristics including the morphological and biochemical properties.In order to find out different types of microorganisms in samples well isolated individual of bacterial colonies were fished out in pure culture from the EMB and MacConkey agar subsequently identified according to the Bergan's manual of determinative bacteriology (1984).Gram's staining was performed to determine the size, shape and arrangement of bacteria.Gram's staining reaction was performed according to the methods described by Merchant and Packer (1976).The organism if E. coli revealed gram-negative, pink color, large rod shape appearance, arranged in single or paired.The isolated organisms with supporting growth characteristics on various media were subjected to different biochemical tests; sugar fermentation test for acid or acid and gas, indole production test, Motility tests, methyl-red and Voges-Proskauer (VP) test.In all cases standard methods as described by Cowan (1985) were followed for conducting these tests.

Molecular identification by polymerase chain reaction (PCR)
Bacterial DNA template was prepared by using boiling method (Englen and Kelley, 2000).All the samples were examined by two pairs of primers (Table 1) to detect 16S rRNA gene of E. coli.In case of E. coli, the PCR reactions were carried out using a thermocycler (ASTEC, Japan) with the following programme: initial denaturation with 1 cycle of 5 min at 95°C, 30 cycles each consisting of denaturation with 45 sec at 94°C, annealing with 45 sec at 52°C, extension with 1 min at 72 °C and a final extension step of 5 min at 72 °C.PCR products were separated on 2% agarose (Invitrogen, USA) gel, stained with ethidium bromide and photographed using a gel documentation system (BioRad).

Statistical analysis
The data on heterotrophic plate count (HPC) and Total coliform count (TCC) obtained from the bacteriological examination of tap water collected from different selected upazila of Bangladesh were analyzed in completely randomized design (CRD) using computer package subjected to Analysis of Variance using SPSS Software (Version 16, 2007).The differences between means were evaluated by Duncan's Multiple Range Test (Gomez and Gomez, 1984).

Discussion
The objective of this research work was to assess the bacteriological quality of tap water samples collected from different upazilla markets of Mymensingh, Gazipur and Sherpur district.Heterotrophic plate count (HPC) and total coliform count (TCC) are commonly used to assess the general microbiological quality of tap water.The geometric mean of HPC of Mymensingh, Gazipur and Sherpur districts water was 8.4x105, 2.5 x106 and 6.8 x105 C.F.U/100 ml.In this study, it was found that HPC was highest in market tap water collected from Kaligonj (Gazipur) and TCC was highest in market tap water of collected from Mymensinghsadar.
According to the world health report (2002), drinking water quality specifications world-wide recommend HPC limits from 100 to 500 cfu/ml in tap water.In this study, HPC was too high in case all types of tap water and high HPC measurements might be due to availability of favourable conditions for the bacterial growth in pipe system.The present study also revealed that tap water from different sources were contaminated with E. coli and other unidentified bacteria.A number of factors might be involved for such contamination.In Mymensingh the pipe system is very old and most of the pipes are poor in condition.There are leakage and breakage through which contaminants from outside the pipe might enter and get mixed with the supplied water.Due to lack of adequate water these pipes are often out of pressure.There is also an illegal practice of drawing water from pipes by suction.As a result, the pressure in the water main becomes less than the atmospheric pressure.Both of these phenomena might cause easier entrance of contaminants into pipelines.Moreover, due to improper layout of water supply lines and sewer lines there might be crossing between them.This might cause fecal contamination.Thus, it is very much possible that even if the water, while entering the pipes, satisfy the specification, it might no longer potable and palatable at the user's end.The findings of the present study correlate with the findings of Islam et al. (2010).A total of 20 isolates were identified as E. coli on the basis of cultural and biochemical characteristics and 16S rRNA gene based PCR from tap water samples used in this study.The colony characteristics of the isolated E. coli in different media resemble the colony characteristics of E. coli as stated by Hamner et al. (2007).The fermentation reaction by the isolates of E. coli in five basic sugars (dextrose, sucrose, fructose, maltose, and mannitol) was positive.Moreover, MR reaction and catalase tests were also positive for E. coli.The organism was able to ferment lactose, dextrose and mannitol, sucrose and maltose completely.The result of sugar fermentation tests agreed with the findings of Sandhuet al. (1996).These respective authors reported that although E. coli ferments all 5 basic sugars but it partially fermented sucrose and maltose.Variation of the results might be due to genetic factors and nature of inhabitant of the organisms.Malaney and Weiser (1962) isolated E. coli from pond water.Dragas and Tratnik (1975) stated that 21.5% of water was contained E. coli.Lin et al. (1974) and Mieres and Bastardo (1975) isolated E. coli from river water.Johnson et al. (2003) detected E. coli and Salmonella in surface water.Abdel-Magid (1997) concluded that if the total coliform count becomes too numerous in water it should warrant more attention.Kravitz et al. (1999) found coliforms in all unimproved and semi-improved water sources and they considered these types of water as non-potable.They however found that E. coli.was absent in majority of the improved water sources.The findings of the present study obviously demonstrated that protection of water sources is very important and the avoidance of contamination can promote hygienic quality of water supplies, where disinfection is not possible.Nogueria et al. (2003) and Shelton et al. (2006) found fecal pollution of water samples.The findings of the present study correlate with the findings of Nogueria et al. (2003) who found highest load of coliform organism in tap water samples.Analogously Opara (2005) found coliform organisms in two rural communities and the quality of rural water supplied was found to be bacteriologically unsatisfactory.Recent studies of Shayo et al. (2007) obtained high coliform count in a rural district and overall, water supplies in the village.Campos et al. (2002) analyzed the microbiological quality of water samples collected from selected houses and could not detect coliforms.On the other hand, Vollared et al. (2005) reported that one third of the households, were significantly associated with water contaminated with >100 fecal coliforms /100 ml.They did not however found any association with water source or any environment was encountered.Campos et al. (2002) analyzed the microbiological quality of water samples collected from selected houses and found that total coliform count was absent.E. coli is able to acquire resistance easily; therefore it is a good bioindicator model for surveillance studies of antimicrobial resistance.In antimicrobial susceptibility testing are in partial agreement with Islam et al. (2010) and Kabir et al. (2013).A total of 20 E. coli isolates, 3(15%) were multidrug resistant.These findings are in partial agreement with Nazir et al. (2005).Thus, resistant strains might be emerged by genetic recombination against one or more antimicrobial agent(s).

Conclusions
The result validates the fact that the unhygienic and poor condition of tap water in studied areas and sanitary point of view and it has evidenced clearly the undesirable level of bacterial contamination such as E. coli which may have acquired from different sources.Detection of pathogenic E. coli in tap water samples revealed the fact that the water used in households may endanger public health.