High heterotrophic counts in potable water and antimicrobial resistance among indicator organisms in two peri-urban communities of Karachi, Pakistan
Shakoor et al. BMC Res Notes
High heterotrophic counts in potable water and antimicrobial resistance among indicator organisms in two peri-urban communities of Karachi, Pakistan
Sadia Shakoor 0 1
Imran Ahmed 0
Saima Mukhtiar 0
Israr Ahmed 0
Farzeen Hirani 1
Shazia Sultana 1
Rumina Hasan 0
0 Pathology & Laboratory Medicine, Aga Khan University , Karachi , Pakistan
1 Pediatrics & Child Health, Aga Khan University , Karachi , Pakistan
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 resistance 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.
Potable water; Household; Heterotrophic plate count; Peri-urban; Antimicrobial resistance
Safe drinking water is a basic human right [
sustainable development goal (SDG) 6 endorses efforts toward
provision of safe and affordable drinking water for all by
]. Lack of access to safe drinking water negatively
impacts human health and results in childhood illness,
infectious outbreaks, food security, and livelihood [
Deleterious health effects of water shortage are observed
in underserved communities, many of which are located
around populated megacities. Karachi is a coastal
megacity in southern Pakistan with a population of 16 million,
and an annual growth rate of 6% [
]. Karachi’s urban
geography is surrounded by a large peri-urban
agglomerate. A recent review identified Karachi as one of four
highly vulnerable megacities with regard to water supply
and urban water security [
Peri-urban communities are in transition between
urban and rural settings. Supply and quality of natural
resources such as water are often inadequate in
periurban communities [
] around expanding megacities
such as Karachi. Piped water may not always be available
and consumers often rely on groundwater sources, draw
water from distribution lines through suction pumps,
or purchase water from vendors [
]. Since only
municipal piped water may be chlorinated, the quality of water
reaching peri-urban areas suffers from high levels of
contamination. For similar reasons, measures to monitor
and maintain microbiological standards of water
overlook peri-urban areas. Therefore, little is known about
water sources, quantity, and quality of potable water in
these areas of 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
limited access to improved water sources [
pathogens in the study with high attributable fractions
for childhood diarrhea are water-borne, suggesting that
microbiological quality of potable water must be
examined. Monitoring of microbiological water quality is a
prerequisite for risk benefit analysis to define
practical safety limits for drinking water and human health
impact. In this non-hypothesis, descriptive study, we
have assessed potable water sources and
microbiological quality in the peri-urban communities of Bin Qasim
town of Karachi. The data provides insight into existing
water sources and contamination levels with the
ultimate purpose of identifying health risks and
highlighting opportunities for improvement of health outcomes
of peri-urban communities in Karachi.
Cattle Colony (CC) and Rehri Goth (RG) are peri-urban
localities around cattle-farming and fishing
communities, respectively, in the Bin Qasim town of Karachi
(Fig. 1a). The sites were included in the Global Enteric
Multicenter Study as surveillance sites for diarrhea in
children and have an established Health and
Demographic Surveillance System. The sites receive piped
water supply from the Kinjhar lake system (Fig. 1b).
Household water (500 mL) was collected in sterile plastic
containers with sodium thiosulfate from 50 households
each in CC and RG, from October 2015 to February 2016.
Water was collected directly (without prior run-off ) from
sources of consumption in households (Fig. 1c). Samples
were transported at room temperature within 4 h of
collection to the Aga Khan University clinical microbiology
laboratory for processing.
Microbiological methods and susceptibility testing
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
units (TUs). For heterotrophic plate counts (HPCs), 1 mL
of water was added to 19 mL of yeast extract agar (Oxoid)
and placed at 37 °C and results were read at 44 ± 4 h as
recommended by the Standing Committee of Analysts,
United Kingdom (SCA, UK) guidelines [
counts were read with a colony counter on the 100 mm
petri plate, the count limit for which was 5700 colony
forming units (CFU)/mL. For coliforms and
thermotolerant E. coli, 100 mL of water was filtered through a
47 mm 0.45 Millipore filter (Merck) and the membrane
placed onto Membrane lauryl sulfate agar (Conda
Laboratories). Two paired volumes were inoculated onto
MLSA and placed at 30 °C for 4 h followed by incubation
for 14 h at 37 °C (for presumptive coliform growth) and
44 °C (for presumptive E. coli growth). Presence of
coliforms and E. coli was recorded and counts were not
performed. For confirmation of coliforms and E. coli, oxidase
test and a combination of sulfide, and indole production
were used, respectively. For recovery of Salmonella and
Shigella, buffered peptone water was set up at 37 °C for
24 h, followed by Rappaport Vassiliadis broth (Oxoid)
enrichment and culture on xylose lysine deoxycholate
agar (Oxoid) to recover colored colonies (pink-red-yellow
with or without black centers).
Overall microbiological quality was considered
acceptable if no coliforms and/or E. coli were recovered from
100 mL of sample [
]. Since > 500 CFU/mL of HPC
inhibit recovery of coliforms and E. coli on lactose
containing media, HPC counts exceeding 500 CFU/mL were
also considered unacceptable [
], irrespective of the
recovery of coliforms and thermotolerant E. coli.
A subset of randomly selected coliforms and E. coli
were subjected to antimicrobial susceptibility
testing. Briefly, identification was confirmed with Activity
profile Index (API) 20 E (bioMereiux), and colonies of
selected isolates were suspended in sterile normal saline
to achieve a turbidity of 0.5 McFarland, and tested as per
manufacturer’s recommendations on the Gram negative
Vitek (bioMereiux) card. Antibiotics tested included
piperacillin, ceftazidime, ciprofloxacin, meropenem, and
trimethoprim-sulfamethoxazole. Extended Spectrum
Beta Lactamase (ESBL) production was determined by
the Vitek Advanced Expert System (AES) through
determination of susceptibility against third generation
cephalosporins and ceftazidime.
Data entry and analysis
All data was entered and frequencies and medians were
calculated using SPSS v19.0 (IBM). Median HPCs were
compared using Mann–Whitney U test.
Of 100 households included in the study, piped water
supply was available in 55% (n = 55; 38 in CC and 17
in RG), water was purchased from contractual tanker
hydrant suppliers in 36% (n = 36; 8 in CC and 28 in RG),
and groundwater was used in 9% (n = 9; 4 in CC and 5 in
RG). Households with piped water supply used pressure
boosting pumps to ensure continuous supply of water
(Fig. 1d). Neither boiling nor chlorination were used in
any of the households; 2 households in CC filtered piped
water. More than half (55%; n = 55, 40 in CC and 15 in
RG) used vessels for storage of drinking water; others
used these directly from the source.
Turbidity and microbiological acceptability
Turbidity was < 5 TUs in 67% of samples, 10 TU in 7% of
samples, and > 10 but < 20 TU in 26 samples, with visible
Microbiological quality was unacceptable in 96% of
samples. Heterotrophic Plate Counts (HPCs) and
recovery of coliforms and thermotolerant E. coli from
samples in CC and RG are shown in Table 1. Very high HPCs
inhibited growth of coliforms and E. coli in 4% of
samples, but these were considered unacceptable based on
their high HPCs. Salmonella spp. and Shigella spp. were
not recovered from any samples. Median HPCs did not
significantly differ between piped and purchased water
sources (p = 0.1).
Antimicrobial resistance was further evaluated in
randomly selected 113 indicator organisms recovered from
100 samples. Resistance rates against common
antibiotics are shown in Table 2. Data presented show a higher
resistance against cephalosporins, fluoroquinolones, and
cotrimoxazole in E. coli than in other coliforms.
Carbapenem resistance was not detected.
Results from our study show a very high recovery rate of
heterotrophic organisms from household potable water
in peri-urban Karachi, with a high fecal contamination
rate. Governance and infrastructural challenges in
periurban areas hinder sustainable water supply and
], which in turn lead to unsafe microbiological
quality of potable water. Our results reflect these
underlying challenges in peri-urban community dwellings of
Karachi. Routine monitoring of peri-urban water supply
systems is also not carried out, and resulting high levels
of microbial counts may lead to adverse human health
outcomes. These results therefore indicate that
periurban areas are in urgent need of attention with respect
to water supply, safety and sustainability.
Our results also show the multiplicity of methods
employed to obtain water. Piped water was not uniformly
accessible, and where available, had insufficient
hydraulic pressure and was interrupted, prompting residents to
employ pressure booster pumps (Fig. 1d). Negative
pressures in piped systems draw water from contaminated
groundwater and even leaky sewage lines [
Furthermore, use of water lifting and booster pumps further
increase contamination through ingress of air and surge
Previous assessments of water quality in urban Karachi
have established high levels of microbial contamination.
The 2014 Multiple Indicator Cluster Survey (MICS) [
demonstrated that 74.9% of households were using
contaminated water with coliform counts of > 1 CFU/mL.
In our study, rates are likely higher due to peri-urban
location. Moreover, our interpretation of
microbiological quality also included high HPCs as an indicator of
inhibited coliform growth [
]. Since the MICS
survey and the Pakistan National Quality Standards (NQS)
for Drinking Water [
] did not consider HPCs when
evaluating water quality, the proportion of unsafe water
quality may be underestimated. In our study, we
identified 4 samples with high HPCs and no coliform yield,
suggesting that such instances are likely to be
encountered with a finite frequency. We therefore recommend
that NQS should include HPCs and consider high HPC
counts (> 500 CFU/mL) to indicate water is unsafe for
High levels of HPCs have also been correlated with
seasonal changes, increasing in summer months and
during the wet season [
]. We collected water samples
in winter months with little or no rain in Karachi,
therefore our results are not likely to be high owing to seasonal
While point-of-use disinfection remains a viable option
safe potable use, technologies used may not be accessible,
affordable, reliable, or even safe [
]. Techniques such
as boiling are inherently unsafe due to the risk of burns
], especially in crowded households with large volume
needs. Chlorination of water by end users remains a
useful strategy, is affordable, but consistent use requires a
change in behavior and practices which need long-term
investments in community education [
Antimicrobial resistance in the environment is an
emerging concern. Higher rates of resistance among
thermotolerant E. coli than in other pathogens, which
may have multiple other sources than human and animal
guts (such as industrial wastewater, vegetable dead
matter, and soil) [
], reflect antibiotic resistance among the
human and animal populations as being the main
drivers for the observed resistance in water. This
suggestion requires further confirmation through culture and
molecular studies. Our results also show low prevalence
of extended spectrum beta lactamase mechanisms (as
indicated by third generation cephalosporin resistance)
among indicator coliforms. Since many bacteria may not
be recovered on culture, detection of antibiotic
resistance genes (ARGs) may be a better indicator of
prevalent transferrable resistant genes in water [
overall trends in quantity of antibiotic resistant bacteria
may correlate with ARGs [
], the potential role of
drinking water in transmission and acquisition of antibiotic
resistance in humans remains unknown.
Lack of adequate water supply and unsafe water has
important implications on citizenship and social
] of communities. We have highlighted the
problem of microbiologically unsafe potable water among
peri-urban households in Karachi, which can have
harmful physiological and psychological impact on the health
of peri-urban dwellers. Urgent operational preventive
measures to allow adequate resource allocation,
community engagement and educational measures at the
household level are necessary to avoid these health risks.
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. However, our
findings remain an important indicator of the need for
infrastructural as well as educational reforms to improve
water quality to prevent enteric infections.
Both poor water quality and presence of indicators of
human fecal contamination strongly suggest presence of
diarrheal pathogens which could not be recovered due
to use of only conventional methods. In addition,
overwhelming numbers of bacteria as indicated by very high
HPCs also interfere with pathogen detection and
coliform isolation [
API: activity profile index; ARG: antibiotic resistance gene; C: centigrade; CC:
Cattle Colony (Karachi peri-urban study area); CFU: colony forming units;
GEMS: Global Enteric Multicenter Study; E.coli: Escherichia coli; ESBL: extended
spectrum beta lactamase; HPC: heterotrophic plate count; IQR: interquartile
range; MICS: Multiple Indicator Cluster Survey; mL: milliliter; NQS: National
Quality Standards; RG: Rehri Goth (Karachi peri-urban study area); SCA:
Standing Committee of Analysts; SDG: sustainable development goals; spp: species;
TU: turbidity unit; UK: United Kingdom; WHO: World Health Organization.
SS1, SM, and IA1 analyzed and interpreted the data and compiled the
results. IA2 and SM performed laboratory work. FH and SS2 performed and
coordinated informed consents, sample collection, transport to the laboratory
and feedback to study households. SS1 wrote the manuscript. RH provided
guidance during all stages of the study. All authors read and approved the
We are grateful to Faisal Malik, Imran Ahmed Chaudhry, and Benazir Baloch for
providing mapping and artwork services.
The authors declare that have no competing interests.
Availability of data and materials
The datasets generated and/or analysed during the current study are available
from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
The study protocol was reviewed and approved by the Ethical Review
Committee for the Aga Khan University. Individual written informed consents were
obtained from household members of consenting age before water samples
The study was funded by the International Foundation for Science, Sweden.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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