Objective: Fecal contamination of potable water leads to unsafe water supply. Although many urban areas of large metropolitan cities receive safe water, peri-urban areas are often not monitored by public health authorities and water supply and quality remain unknown. We assessed microbiological quality and rates of antimicrobial resist- ance in viable indicator bacteria in two peri-urban communities of Karachi, Pakistan. Water samples were collected over 5 months (October 2015 to February 2016) from these peri-urban communities and samples were processed for microbiological quality as per Standing Committee of Analysts, United Kingdom and World Health Organization guidelines and criteria for drinking water. Results: Both communities received unimproved water. Potable water samples collected from 100 households showed that 96% of samples were unsafe for consumption. Extended spectrum beta lactamases production was found in 29.2% of fecal indicator organisms (coliforms). Use of unimproved water sources and unsafe potable water quality in peri-urban Karachi deserve immediate attention and upgrade. The study is instrumental in attracting the attention of authorities to the state of water resources in peri-urban communities in Karachi with a view to influence improvement of services and effects on human health. Keywords: Potable water, Household, Heterotrophic plate count, Peri-urban, Antimicrobial resistance Introduction and an annual growth rate of 6% . Karachi’s urban Safe drinking water is a basic human right  and sustain- geography is surrounded by a large peri-urban agglom- able development goal (SDG) 6 endorses efforts toward erate. A recent review identified Karachi as one of four provision of safe and affordable drinking water for all by highly vulnerable megacities with regard to water supply 2030 . Lack of access to safe drinking water negatively and urban water security . impacts human health and results in childhood illness, Peri-urban communities are in transition between infectious outbreaks, food security, and livelihood . urban and rural settings. Supply and quality of natural Deleterious health effects of water shortage are observed resources such as water are often inadequate in peri- in underserved communities, many of which are located urban communities  around expanding megacities around populated megacities. Karachi is a coastal megac- such as Karachi. Piped water may not always be available and consumers often rely on groundwater sources, draw ity in southern Pakistan with a population of 16 million, water from distribution lines through suction pumps, or purchase water from vendors . Since only munici *Correspondence: email@example.com pal piped water may be chlorinated, the quality of water Pathology & Laboratory Medicine, Aga Khan University, Karachi, Pakistan reaching peri-urban areas suffers from high levels of Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shakoor et al. BMC Res Notes (2018) 11:350 Page 2 of 6 contamination. For similar reasons, measures to monitor town of Karachi. The data provides insight into existing and maintain microbiological standards of water over- water sources and contamination levels with the ulti- look peri-urban areas. Therefore, little is known about mate purpose of identifying health risks and highlight- water sources, quantity, and quality of potable water in ing opportunities for improvement of health outcomes these areas of Karachi. of peri-urban communities in Karachi. Data from the Global Enteric Multicenter Study (GEMS) showed a high burden of childhood diarrhea in peri-urban Bin Qasim Town of Karachi, and extremely Main text limited access to improved water sources . Many Methods pathogens in the study with high attributable fractions Study sites for childhood diarrhea are water-borne, suggesting that Cattle Colony (CC) and Rehri Goth (RG) are peri-urban microbiological quality of potable water must be exam- localities around cattle-farming and fishing commu - ined. Monitoring of microbiological water quality is a nities, respectively, in the Bin Qasim town of Karachi prerequisite for risk benefit analysis to define practi - (Fig. 1a). The sites were included in the Global Enteric cal safety limits for drinking water and human health Multicenter Study as surveillance sites for diarrhea in impact. In this non-hypothesis, descriptive study, we children and have an established Health and Demo- have assessed potable water sources and microbiologi- graphic Surveillance System. The sites receive piped cal quality in the peri-urban communities of Bin Qasim water supply from the Kinjhar lake system (Fig. 1b). Fig. 1 Study areas in Karachi and water supply and consumption; a Map of Cattle Colony and Rehri Goth in the Bin Qasim Town of Karachi (Insets show location of Karachi within Pakistan and Pakistan in South Asia); b water distribution system in Karachi showing piped water supply to study areas from a natural lake east of Karachi; c water storage tanks used in some households; and d pressure booster pumps used by consumers to overcome low pressure and intermittent supply of piped water Shakoor et al. BMC Res Notes (2018) 11:350 Page 3 of 6 Sampling methods Vitek (bioMereiux) card. Antibiotics tested included Household water (500 mL) was collected in sterile plastic piperacillin, ceftazidime, ciprofloxacin, meropenem, and containers with sodium thiosulfate from 50 households trimethoprim-sulfamethoxazole. Extended Spectrum each in CC and RG, from October 2015 to February 2016. Beta Lactamase (ESBL) production was determined by Water was collected directly (without prior run-off ) from the Vitek Advanced Expert System (AES) through deter- sources of consumption in households (Fig. 1c). Samples mination of susceptibility against third generation cepha- were transported at room temperature within 4 h of col- losporins and ceftazidime. lection to the Aga Khan University clinical microbiology laboratory for processing. Data entry and analysis All data was entered and frequencies and medians were calculated using SPSS v19.0 (IBM). Median HPCs were Microbiological methods and susceptibility testing compared using Mann–Whitney U test. Water samples were processed within 24 h of receipt at the laboratory. Turbidity was measured by the turbidity tube method  and results were recorded in turbidity Results units (TUs). For heterotrophic plate counts (HPCs), 1 mL Household characteristics of water was added to 19 mL of yeast extract agar (Oxoid) Of 100 households included in the study, piped water and placed at 37 °C and results were read at 44 ± 4 h as supply was available in 55% (n = 55; 38 in CC and 17 recommended by the Standing Committee of Analysts, in RG), water was purchased from contractual tanker United Kingdom (SCA, UK) guidelines [10–12]. HPC hydrant suppliers in 36% (n = 36; 8 in CC and 28 in RG), counts were read with a colony counter on the 100 mm and groundwater was used in 9% (n = 9; 4 in CC and 5 in petri plate, the count limit for which was 5700 colony RG). Households with piped water supply used pressure forming units (CFU)/mL. For coliforms and thermo- boosting pumps to ensure continuous supply of water tolerant E. coli, 100 mL of water was filtered through a (Fig. 1d). Neither boiling nor chlorination were used in 47 mm 0.45 Millipore filter (Merck) and the membrane any of the households; 2 households in CC filtered piped placed onto Membrane lauryl sulfate agar (Conda Lab- water. More than half (55%; n = 55, 40 in CC and 15 in oratories). Two paired volumes were inoculated onto RG) used vessels for storage of drinking water; others MLSA and placed at 30 °C for 4 h followed by incubation used these directly from the source. for 14 h at 37 °C (for presumptive coliform growth) and 44 °C (for presumptive E. coli growth). Presence of coli- Turbidity and microbiological acceptability forms and E. coli was recorded and counts were not per- Turbidity was < 5 TUs in 67% of samples, 10 TU in 7% of formed. For confirmation of coliforms and E. coli, oxidase samples, and > 10 but < 20 TU in 26 samples, with visible test and a combination of sulfide, and indole production particulate matter. were used, respectively. For recovery of Salmonella and Microbiological quality was unacceptable in 96% of Shigella, buffered peptone water was set up at 37 °C for samples. Heterotrophic Plate Counts (HPCs) and recov- 24 h, followed by Rappaport Vassiliadis broth (Oxoid) ery of coliforms and thermotolerant E. coli from sam- enrichment and culture on xylose lysine deoxycholate ples in CC and RG are shown in Table 1. Very high HPCs agar (Oxoid) to recover colored colonies (pink-red-yellow inhibited growth of coliforms and E. coli in 4% of sam- with or without black centers). ples, but these were considered unacceptable based on Overall microbiological quality was considered accept- their high HPCs. Salmonella spp. and Shigella spp. were able if no coliforms and/or E. coli were recovered from not recovered from any samples. Median HPCs did not 100 mL of sample . Since > 500 CFU/mL of HPC significantly differ between piped and purchased water inhibit recovery of coliforms and E. coli on lactose con- sources (p = 0.1). taining media, HPC counts exceeding 500 CFU/mL were also considered unacceptable , irrespective of the recovery of coliforms and thermotolerant E. coli. Antimicrobial resistance A subset of randomly selected coliforms and E. coli Antimicrobial resistance was further evaluated in ran- were subjected to antimicrobial susceptibility test- domly selected 113 indicator organisms recovered from ing. Briefly, identification was confirmed with Activity 100 samples. Resistance rates against common antibiot- profile Index (API) 20 E (bioMereiux), and colonies of ics are shown in Table 2. Data presented show a higher selected isolates were suspended in sterile normal saline resistance against cephalosporins, fluoroquinolones, and to achieve a turbidity of 0.5 McFarland, and tested as per cotrimoxazole in E. coli than in other coliforms. Carbap- manufacturer’s recommendations on the Gram negative enem resistance was not detected. Shakoor et al. BMC Res Notes (2018) 11:350 Page 4 of 6 Table 1 Proportion of piped water supply source and microbiological acceptability of potable water samples from households in Cattle Colony (CC) and Rehri Goth (RG) peri-urban communities of Karachi, Pakistan CC RG Total % Piped source 76 (n = 38) 34 (n = 17) 55 (n = 55) Median HPC (IQR) 4004 CFU/mL (1647–5700) 5700 CFU/mL (2779–5700) 5700 (1781.5–5700) % Microbiologically unacceptable 92 (n = 46) 100 (n = 50) 96 (n = 96) % Samples with coliforms isolated 90 (n = 45) 94 (n = 47) 92 (n = 92) % Samples with thermotolerant E. coli isolated 26 (n = 13) 44 (n = 22) 35 (n = 35) 50 50 100 Table 2 Resistance to third generation cephalosporins, ciprofloxacin, and trimethoprim sulfamethoxazole among randomly selected organisms cultured from potable water in peri-urban households Organism Number % Resistant (n) Total Ceftazidime Ciprofloxacin Trimethoprim/ sulfamethoxazole E. coli 37 43.2 (16) 10.8 (4) 29.7 (11) 37 Enterobacter spp. 17 5.9 (1) 0 29.4 (5) 17 Klebsiella pneumoniae 56 26.8 (15) 0 8.9 (5) 56 Citrobacter spp. 3 33.3 (1) 0 0 3 Total 113 29.2 (33) 3.5 (4) 18.6 (21) 113 Discussion The 2014 Multiple Indicator Cluster Survey (MICS)  Results from our study show a very high recovery rate of demonstrated that 74.9% of households were using con- heterotrophic organisms from household potable water taminated water with coliform counts of > 1 CFU/mL. in peri-urban Karachi, with a high fecal contamination In our study, rates are likely higher due to peri-urban rate. Governance and infrastructural challenges in peri- location. Moreover, our interpretation of microbiologi- urban areas hinder sustainable water supply and manage- cal quality also included high HPCs as an indicator of ment , which in turn lead to unsafe microbiological inhibited coliform growth . Since the MICS sur- quality of potable water. Our results reflect these under - vey and the Pakistan National Quality Standards (NQS) lying challenges in peri-urban community dwellings of for Drinking Water  did not consider HPCs when Karachi. Routine monitoring of peri-urban water supply evaluating water quality, the proportion of unsafe water systems is also not carried out, and resulting high levels quality may be underestimated. In our study, we iden- of microbial counts may lead to adverse human health tified 4 samples with high HPCs and no coliform yield, outcomes. These results therefore indicate that peri- suggesting that such instances are likely to be encoun- urban areas are in urgent need of attention with respect tered with a finite frequency. We therefore recommend to water supply, safety and sustainability. that NQS should include HPCs and consider high HPC Our results also show the multiplicity of methods counts (> 500 CFU/mL) to indicate water is unsafe for employed to obtain water. Piped water was not uniformly consumption. accessible, and where available, had insufficient hydrau - High levels of HPCs have also been correlated with lic pressure and was interrupted, prompting residents to seasonal changes, increasing in summer months and employ pressure booster pumps (Fig. 1d). Negative pres- during the wet season . We collected water samples sures in piped systems draw water from contaminated in winter months with little or no rain in Karachi, there- groundwater and even leaky sewage lines . Further- fore our results are not likely to be high owing to seasonal more, use of water lifting and booster pumps further changes. increase contamination through ingress of air and surge While point-of-use disinfection remains a viable option effects. safe potable use, technologies used may not be accessible, Previous assessments of water quality in urban Karachi affordable, reliable, or even safe . Techniques such have established high levels of microbial contamination. as boiling are inherently unsafe due to the risk of burns Shakoor et al. BMC Res Notes (2018) 11:350 Page 5 of 6 , especially in crowded households with large volume needs. Chlorination of water by end users remains a use- Abbreviations ful strategy, is affordable, but consistent use requires a API: activity profile index; ARG : antibiotic resistance gene; C: centigrade; CC: change in behavior and practices which need long-term Cattle Colony (Karachi peri-urban study area); CFU: colony forming units; GEMS: Global Enteric Multicenter Study; E.coli: Escherichia coli; ESBL: extended investments in community education . spectrum beta lactamase; HPC: heterotrophic plate count; IQR: interquartile Antimicrobial resistance in the environment is an range; MICS: Multiple Indicator Cluster Survey; mL: milliliter; NQS: National emerging concern. Higher rates of resistance among Quality Standards; RG: Rehri Goth (Karachi peri-urban study area); SCA: Stand- ing Committee of Analysts; SDG: sustainable development goals; spp: species; thermotolerant E. coli than in other pathogens, which TU: turbidity unit; UK: United Kingdom; WHO: World Health Organization. may have multiple other sources than human and animal guts (such as industrial wastewater, vegetable dead mat- Authors’ contributions SS1, SM, and IA1 analyzed and interpreted the data and compiled the ter, and soil) , reflect antibiotic resistance among the results. IA2 and SM performed laboratory work. FH and SS2 performed and human and animal populations as being the main driv- coordinated informed consents, sample collection, transport to the laboratory ers for the observed resistance in water. This sugges - and feedback to study households. SS1 wrote the manuscript. RH provided guidance during all stages of the study. All authors read and approved the tion requires further confirmation through culture and final manuscript. molecular studies. Our results also show low prevalence of extended spectrum beta lactamase mechanisms (as Author details Pathology & Laboratory Medicine, Aga Khan University, Karachi, Pakistan. indicated by third generation cephalosporin resistance) Pediatrics & Child Health, Aga Khan University, Karachi, Pakistan. among indicator coliforms. Since many bacteria may not be recovered on culture, detection of antibiotic resist- Acknowledgements We are grateful to Faisal Malik, Imran Ahmed Chaudhry, and Benazir Baloch for ance genes (ARGs) may be a better indicator of preva- providing mapping and artwork services. lent transferrable resistant genes in water . Although overall trends in quantity of antibiotic resistant bacteria Competing interests The authors declare that have no competing interests. may correlate with ARGs , the potential role of drink- ing water in transmission and acquisition of antibiotic Availability of data and materials resistance in humans remains unknown. The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Conclusions Consent for publication Not applicable. Lack of adequate water supply and unsafe water has important implications on citizenship and social engage- Ethics approval and consent to participate ment  of communities. We have highlighted the prob- The study protocol was reviewed and approved by the Ethical Review Com- mittee for the Aga Khan University. Individual written informed consents were lem of microbiologically unsafe potable water among obtained from household members of consenting age before water samples peri-urban households in Karachi, which can have harm- were collected. ful physiological and psychological impact on the health Funding of peri-urban dwellers. Urgent operational preventive The study was funded by the International Foundation for Science, Sweden. measures to allow adequate resource allocation, commu- nity engagement and educational measures at the house- Publisher’s Note hold level are necessary to avoid these health risks. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Limitations Received: 1 March 2018 Accepted: 31 May 2018 Our study did not focus on variations in rainfall and seasonal effects, dissolved organic matter, and chemical composition of water, all of which impact water quality and may also impact risk to human health. 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