Comparative Evaluation of Thermotolerant Escherichia coli

Comparative Evaluation of Thermotolerant Escherichia coli

Bangladesh J Microbiol, Volume 34, Number 1, June 2017, pp 07-14 Original Article Comparative Evaluation of Thermotolerant Escherichia coli, Enteroco...

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Bangladesh J Microbiol, Volume 34, Number 1, June 2017, pp 07-14

Original Article Comparative Evaluation of Thermotolerant Escherichia coli, Enterococci and Total Coliform as Indicators of Water Quality Jannatun Nayma1, Fatema Moni Chowdhury1, Sunjukta Ahsan2 and Marufa Zerin Akhter2* 1Department

of Microbiology and Biotechnology, Jagannath University, Dhaka, 2Department of Microbiology, University of Dhaka

The present study was aimed to evaluate thermotolerant Escherichia coli and enterococci as alternative water quality indicator bacteria to assess the microbiological quality of surface water and supplied tap water of Dhaka city. Membrane filtration count of total coliform, E. coli and enterococci were obtained from various surface water bodies and supplied tap water. To find out the correlation of these indicators with the presence of water borne pathogens, counts of Salmonella and Vibrio were assessed concomitantly. The identifications of E. coli, Salmonella and Vibrio were performed by biochemical tests; whereas, for enterococci, identification by PCR method was applied. According to USEPA set rule for single sample analysis, the maximum concentration of d”235 E. coli or d”62 enterococci per 100 ml is considered a safe recreational or surface water standard for fresh water bodies. Following this, the present study found that out of the 22 surface water samples studied from 12 different surface water bodies in and around Dhaka city, 13 crossed the limit for E. coli and 10 crossed the limit for enterococci. On the other hand, all of the surface water bodies showed an exceedingly high number of total coliform bacteria regardless of the presence or absence of pathogens. Apart from only one sample, no correlation was observed between the presence of total coliform and the presence of the two pathogenic bacteria studied. On the other hand, E. coli and enterococci showed a better correlation in number with the presence of the pathogens; E. coli showing a better correlation than enterococci. Supplied tap water samples were examined from 8 different locations of Dhaka city. The international set rule is 0 enterococci or E. coli or total coliform or fecal coliform per 100 ml of potable water. All tap water samples showed considerable numbers of both total coliform and E. coli that always exceeded the limit allowed. However, out of the 8 samples, only 3 carried enterococci. Salmonella and Vibrio were obtained from most of the samples. In tap water the occurrence of E. coli showed more correlation with the presence of pathogens than with enterococci. This study suggested that for microbiological quality assessment of surface and tap waters of Dhaka city, total coliform has lost its credibility. Hence, E. coli, enterococci or other alternate water quality indicators suitable for subtropical region should be included in water quality testing. Key words: Alternate water quality indicator bacteria, Thermotolerant Escherichia coli, Enterococcus faecalis, Total coliform

Introduction Polluted water is a major environmental issue worldwide. In Bangladesh, about 80% of the disease outbreaks are waterborne. According to the British advocacy group WaterAID, Waterrelated diseases are responsible for 24 per cent of all deaths1. Every year, gastroenteritis and diarrhoeal diseases kill 110,000 children below the age of five. Over 4,100 children die from diarrhoea every year in Bangladesh and water-related diseases cause nearly a quarter of all deaths. Major waterborne diseases are cholera, bacterial and protozoal diarrhoea, hepatitis A and E, typhoid fever etc. Causes of this overwhelming incidence of waterborne diseases in Bangladesh can be attributed to the rudimentary water purification and sewage discharge systems, ignorance of the common people about proper waste and sewage disposal, traditional habit of using open field for excreta disposal and defecation. The matter of concern is, some underground

water of Bangladesh that are collected through tube well has been reported to have coliform bacteria. Faecal indicators are recommended in both temperate and tropical regions as microbiological water quality monitoring tools. However, various studies particularly in the tropics have expressed doubts in their reliability. This has been largely attributed to different conditions of the tropics like temperature, solar radiation, higher nutrient levels and the presence of a more diverse microbial community in the tropics. Various researches carried out in tropical countries question the appropriateness of using total coliform, fecal coliform and even E. coli (37°C) as indicator bacteria. An ever increasing number of studies have revealed that fecal bacteria, E. coli (37°C) and some pathogens such as Salmonella, Shigella and Campylobacter, have become naturalized to secondary habitats like soil, sands, sediments, water, phytoplanktons and zooplanktons2-9. The ubiquitous appearance

*Corresponding author: Dr. Marufa Zerin Akhter, Department of Microbiology, University of Dhaka, Tel: 008801817630976, Fax: 0088-02-9667222, e-mail: [email protected]

Nayma et. al

of total and fecal coliforms in water samples of Bangladesh has necessitated the evaluation of other indicator bacteria.

4°C to 6°C. Microbiological tests were carried out as promptly as possible after collection to avoid unpredictable changes.

In Bangladesh, total coliform and in a very few instances, fecal coliform bacteria are used as the only water quality indicators. Total coliforms are widespread in nature. All members of this group can occur in human feces, but some can also be present in natural habitats. Even fecal coliforms contain a genus, Klebsiella, with species that are not necessarily fecal in origin. High concentrations of organic materials can support bacterial populations, a portion of which are capable of responding positively to the total coliform and fecal coliform tests. Under the circumstances, the use of these traditional indicators is no longer justified in tropical waters. The present study aimed to assess the feasibility of thermotolerant Escherichia coli, Enterococcus faecalis and E. faecium as alternative indicator bacteria in Bangladesh. Surface water bodies in and around Dhaka city and supplied piped water were assessed to correlate the appropriateness of using different indicator bacteria.

Membrane Filtration Method Enterococci on Selective Media Membrane filtration was carried out on EF medium, a selective and differential medium for Enterococcus faecium and E. faecalis. After incubation at 37°C for 48 hours E. faecalis forms pink or red brown colonies 0.5 – 2 mm in diameter, while E. faecium forms yellow colonies. Maroon colonies were carefully selected to obtain pure culture of Enterococcus faecalis. Stock culture Pure isolates were transferred into 1 ml nutrient broth in 1.5 ml eppendorf tube and grown overnight. Then glycerol was added to be stored at -70°C. Microscopic examination for the identification of the isolates The size, shape, arrangement, presence of endospore, staining properties, etc. of the vegetative cells of the selected strains were determined through microscopic examination.

Materials and Methods Sampling sites

Biochemical studies for identification of the isolates

Surface water samples were collected from the following stagnant water bodies of lakes and ponds: Shahidullah Hall pond, University of Dhaka; Jagannath Hall pond, University of Dhaka; Bangla Academy pond; Mirpur pond; Sohrawardi Uddan pond; Dhanmondi lake; Gulshan lake; Mirpur lake and Dhaka Cantonment lake. Samples were also collected from the following rivers in and around Dhaka: the Buriganga river; the Shitolokkha river; the Turag river and the Brahmaputra river. Tap water samples were collected from the following areas of Dhaka city: Microbiology lab tap water, University of Dhaka; Shanir Akhra tap water; Sahidullah Hall canteen tap water, University of Dhaka; Jagannath Hall tap water, University of Dhaka; Agrani Bank, Dhaka University branch tap water; Mirpur area tap water; Cantonment area tap water; Uttara area tap water.

Biochemical tests were performed according to the methods described in Manual of Methods for General Bacteriology by American Society of Microbiology11. Colony Polymerase chain Reaction (Colony PCR) of the isolated enterococci One well isolated colony from fresh culture was taken in a PCR tube and mixed with 20 µl distilled water. This tube was incubated in the PCR thermocycler set at 100°C for 10 minutes for disintegration of the cells and allowing genomic DNA to be released from the cells. Cell debris was pelleted by centrifugation at 10,000 rpm for 5 minutes. The tube was cooled down to normal temperature and the supernatant was used as the sample DNA. PCR cocktails for 50µl reaction mixtures for each contained 2× reaction mixture containing 25µl premix taq, 1.2 µl forward primer, 1.2 µl reverse primer, 1µl sample DNA, 21.6 µl H2O. PCR amplification was performed with 30 or 40 temperature cycles under standard conditions. At the end of the cycling steps a 10min extension at 72°C was performed, and then samples were maintained at 4°C. Five µl of the PCR products was analyzed on 1% agarose gels. The PCR primers used were specific for enterococci and is described in table 1.

Sampling Procedure and Sample Transportation The water samples were collected from relatively fresh flow and from a depth of 4.0 cm - 6.0 cm by pre-sterilized glass bottle or PET bottle. Standard procedures were followed for sampling10. Three representative samples were collected from each source aseptically. All samples were labeled at the spot and transported to the laboratory at the earliest convenience. Transportation to the laboratory was done in ice box in a temperature ranging from

Table 1. Primers used in the PCR method to amplify enterococci specific product Primer

Sequence (5´to 3´)





Tm (°C)

Amplicon size (bp)



112 bp

Ke et al., 199912

Comparative Evaluation of Thermotolerant Escherichia coli, Enterococci

Result Membrane filtration count Table 2. Relative number (c.f.u per 100 ml) of total coliform, thermotolerant E. coli, Enterococcus faecalis, Vibrio and Salmonella in six pond water samples around Dhaka University Total coliform Shahidullah Hall pond sample 1 Shahidullah Hall pond sample 2 Jagannath Hall pond sample 1 Jagannath Hall pond sample 2 Bangla Academy pond Sohrawardi Uddan pond


Bacterial count (cfu/100ml) Thermotolerant Enterococcus E. coli faecalis 250.0 400 8.0 122 400.0 80 0.0 80 600.0 50 500 10



480 200 60 50 140 70

0 66 0 0 10 50

Table 3. Relative number (c.f.u per 100 ml) of Total coliform, thermotolerant E. coli, Enterococcus faecalis, Vibrio and Salmonella in five water samples of Dhanmondi Lake, Dhaka Bacterial count (cfu/100ml) Total coliform Dhanmondi lake sample-1 Dhanmondi lake sample-2 Dhanmondi lake sample-3 Dhanmondi lake sample-4 Dhanmondi lake sample-5


Thermotolerant E. coli 0 4 40 30 50

Enterococcus faecalis 166 144 42 26 40



82 40 12 8 10

44 80 26 20 0

Table 4. Relative number of Total coliform, thermotolerant E. coli, Enterococcus faecalis, Vibrio and Salmonella in five lake water samples of Dhaka City Total coliform Mirpur Lake -1 Mirpur Lake -2 Cantonment lake -1 Cantonment lake -2 Gulshan lake


Bacterial count (cfu/100ml) Thermotolerant Enterococcus Vibrio E. coli faecalis TNTC TNTC 400 TNTC 44 TNTC 500 46 TNTC 500 TNTC TNTC TNTC 0 46

Salmonella 120 180 0 0 0

Table 5. Relative number of Total coliform, thermotolerant E coli, Enterococcus faecalis, Vibrio and Salmonella of four river water in and around Dhaka City Bacterial count (cfu/100ml) Total coliform Turag sample-1 Turag sample-2 Brahmaputra sample-1 Brahmaputra sample-2 Shitolokkha Buriganga


Thermotolerant E. coli TNTC TNTC 44 38 TNTC TNTC 9

Enterococcus faecalis 12 40 22 24 400 TNTC



29 32 25 32 20 50

21 20 20 21 10 10

Nayma et. al

Figure 1. Relative number (c.f.u per 100 ml) of Total coliform, thermotolerant E coli, Enterococcus faecalis, Vibrio and Salmonella of eight tap water sampls of Dhaka City. SHC Shahidullah Hall Canteen; JHC Jagannath Hall canteen; AB Agrani Bank DU; SA Shanir Akhra; CT Cantonment; MR Mirpur; UT Uttara; ML Microbiology Lab PCR results for identification of enterococci

PCR, the highly specific maroon to deep red colonies of 0.5-2 mm dia on EF agar plate (Figure 3) and also the cell morphology and arrangements in Gram staining (Figure 4).

PCR amplification products of 112 bp of enterococci specific gene were obtained which is shown in Figure 2. The confirmation of Enterococcus faecalis was done by combined results of these

Figure 2. PCR amplification product of enterococci. A 112 bp PCR amplification product was obtained which is specific for enterococci. The molecular weight marker seen on the left lane is 100 bp DNA marker (NEB, UK)

Figure 3. Maroon colonies of Enterococcus faecalis on EF agar

Figure 4. Cell morphology and arrangement of Enterococcus faecalis under microscope 10

Comparative Evaluation of Thermotolerant Escherichia coli, Enterococci

Discussion In the tropics, the classical indicators of water pollution are suspected to originate from non-faecal sources (such as soil) and to proliferate in tropical aquatic habitats under favourable situations and thus can be detectable at levels which may not reflect the original extent of faecal contamination2-9.

conditions. Preliminary evidence suggests that E. coli and enterococci can be detected at tropical locales such as Puerto Rico, Hawaii, and Guam in waters where there is no apparent source of contamination from warm-blooded animals21. EPA and others are evaluating whether the current indicator bacteria grow and persist in natural tropical environments.

The enterococcus group includes two strains of the fecal streptococci that are human specific, namely, Streptococcus faecalis and Streptococcus faecium. Studies on marine and fresh water bathing beaches indicated that swimming-associated gastroenteritis was related directly to the quality of the bathing water and that enterococci were the most efficient bacterial indicator of water quality13,14. Streptococcus faecalis has the advantage over E. coli in that it survives better in the aquatic environment.

Historically, fecal coliforms and E. coli have been used as indicators of choice when monitoring recreational water quality22. Recent studies have shown that high densities of E. coli and enterococci recovered from recreational waters have a stronger correlation with swimming-associated gastrointestinal disease than do densities of fecal coliform bacteria23. Although enterococci have been traditionally used to monitor marine bathing water24, both of these indicators have been referenced as being equally acceptable for monitoring freshwater22,25. Therefore, studies of both marine water and freshwater have been undertaken to support the idea that enterococci may be the more relevant indicator of water quality22.

Many members of the total coliform group and some so-called faecal coliforms (e.g. species of Klebsiella and Enterobacter) are not specific to faeces, and even E. coli has been shown to grow in some natural aquatic environments8,15-19. Hence, the primary targets representing faecal contamination in temperate waters are now considered to be E. coli and enterococci.

The abundance of enterococci in human and animal feces, the ease with which they are cultured, and their correlation with human health outcomes in fresh and marine waters have led to their widespread use as tools for assessing recreational water quality worldwide13,26-29. However, many reports also state that E. coli should be the indicator of choice for fresh water quality30.

The complexity of the tropical environments in relation to the expectable indicator performances has not been taken into consideration in our country so far. We have continued accepting and relying on guidelines established for temperate regions. Therefore, in many instances, the bacterial indicators that are currently in use do not represent the actual number of fecal bacteria in the water samples giving false positive results. There is thus scope and need to reassess, evaluate and explore existing novel methods and parameters for fecal contamination monitoring in the tropical waters. Hence, the present study was undertaken to assess microbiological qualities of several surface water bodies and supplied tap water of Dhaka city and to make a comparative assessment of the credibility of the traditional water quality indicators like total coliform with thermotolerant E. coli and enterococci. The presence of enterococci is a valuable bacterial indicator for determining the extent of fecal contamination of recreational surface waters20.

Previous studies have shown that E. coli (37°C) can become naturalized to the microbial community in tropical, subtropical, and temperate soil and sand31,32,33. This likely limits the use of this bacterium as an indicator of water quality. Moreover, these culture-based methods cannot differentiate among sources of fecal bacteria34. It has been proved that the conventional indicators of fecal origin i.e. coliform bacteria (total and fecal coliforms), used to evaluate microbiological quality of waters provide erroneous information35. They do not adequately reflect the occurrence of pathogens in disinfected wastewater effluent due to their relatively high susceptibility to chemical disinfection and failure to correlate with protozoan parasites and enteric viruses36. As well as, coliforms are generally considered unreliable indicators of faecal contamination because many are capable of growth in the environment. Thus, the public health is not protected by using these common indicators such as total coliform and fecal coliform.

Indicators vary in their ability to reliably predict potential risks to human health. Some indicators have been shown to have a greater statistical relationship to disease than others. Also, current indicators are based on fecal contamination and might not accuratelty assess the potential for disease from other pathogens that can cause skin, upper respiratory tract, eye, ear, nose and throat disease21. More research on the use of other bacteria and viruses as indicators is being conducted at the federal, state, and local levels in the USA. Despite variability in the ability of indicators to reliably predict potential risks to human health, EPA studies indicate that enterococci and E. coli are the most effective available primary indicators for predicting the presence of gastrointestinal illness-causing pathogens, and for marine waters, enterococci is most appropriate.

The present study was an effort to verify the authenticity of using total coliforms as surface water quality indicators in a tropical country like Bangladesh and also to verify the feasibility of E. coli and Enterococcus faecalis as an alternative water quality indicator. It was observed that in all the surface water samples tested, the total coliform exceeded the countable limit beyond the allowed limit, even in the water bodies where faecal contamination was not that obvious. Moreover, total coliform numbers did not show any correlation with the numbers of the two waterborne pathogens tested, namely Vibrio and Salmonella

One area of current scientific debate is whether indicator bacteria react differently under various climatic and environmental 11

Nayma et. al

spp. This justifies the statements of many previous findings that stated these indicators are becoming acclimatized to and proliferate in the tropical water bodies. Thermotolerant E. coli (45°C) and Enterococcus faecalis, on the other hand, showed a correlation with the presence of these two pathogens. Their number was always within a countable limit, and always exceeded the number of pathogens suggesting their validity as a more acceptable surface water quality indicator in our country. Different countries and different organizations have set different allowable limits of different indicators. The present data were analyzed taking the limit of USEPA37 for single count measurement for fresh recreational water, which considers 62 cfu / 100ml to be the highest permissible limit for enterococci, and 235 cfu / 100 ml for E. coli. At present the USEPA does not consider total coliform as a recreational water quality indicator any more.

faecalis. Cross contamination of the water supply lines with sewage or drain water is likely to occur. However, the total and thermotolerant E. coli were always present in these tap water samples. In this case, however, thermotolerant E. coli correlated more with the presence of the pathogenic bacteria. It might seem unrealistic why coliforms are present in supplied treated piped water. Unfortunately, this is the reality in Bangladesh. Many previous and current research papers show similar statistics of the presence of total coliform, faecal coliform and even Salmonella, Shigella and Vibrio cholera in supply water of Dhaka38,39,40. In such a context, the supplied piped water of Dhaka city cannot be certified to be safe for drinking without prior treatment. Although, except for the slum dwellers, people of Dhaka city do not directly drink supplied piped water without treatment, it is the duty of concerned authority to provide absolutely safe drinking water through the pipeline. At least the bacterial load and indicator load must be lowered down to an acceptable limit. The number of total coliform, thermotolerant E. coli, Enterococcus faecalis, Salmonella and Vibrio that the present study found out indicated a very high level of harmful disease causing bacteria in the supplied drinking water which is not at all acceptable under any circumstances. Mahbub et al.40 reported that among the 45 piped water samples they studied, 57.78% samples exceeded the BDS standard and WHO guideline for coliform bacteria and 51.11% for E. coli bacteria. Total Coliform and E. coli count in water samples ranged from <1.8 to >1600(MPN) / 100 ml. These values for Total coliform and E .coli are unacceptable for drinking water41. They also found that most of the pump water of WASA which use deep tube well is free of bacterial load and the highest amount was found in house tap water. They concluded that the source of contamination mainly is the distribution system of water of Dhaka city. Therefore, quality of pipelines, integrity of pipes and junctions between pipes, proper and adequate chlorination must be checked and maintained at a regular basis. Otherwise, this poor quality of piped water cannot be upgraded. There is a conception among the city dwellers that the piped water is not meant for drinking purpose, which is a wrong concept. Unfortunately, their conception about the health risk for drinking piped water is correct. Our piped water must be made safe for drinking. This is possible as it does not require a much investment. It is necessary to monitoring piped water at the treatment point and at different locations of the city regularly and taking adequate corrective measures wherever any contamination source is observed.

In all the samples (except S2) studied, total coliform always exceeded the permissible limit showing matt growth on the membrane filter, even in diluted samples. It showed no correlation with the presence of Vibrio and Salmonalla. On the other hand, E. coli and Enterococcus faecalis showed a much better correlation with the presence of these pathogens in all the samples studied. Among the stagnant water bodies studied, Dhanmondi lake was apparently the cleanest. This observation correlated with the findings showing much less number of both E. coli and Enterococcus faecalis as well as of the pathogens (Table 3). Other lakes and ponds showed quite a high number of these indicators and in some crossing the recommended limit. Mirpur and Gulshan lakes showed a very high number of Vibrio as well as E. coli and Enterococcus faecalis (Table 4). Amongst the river water studied, Brahmaputra and Shitalakhya were the samples outside Dhaka. Brahmaputra water sample was collected from Mymensingh and the river is apparently the cleanest of all. This organoleptic observation was reflected in the results as well showing the least number of E. coli and Enterococcus faecalis as well as the pathogens (Table 5). The Shitalakhya and the Buriganga rivers showed a very high number of Vibrio as well as the indicators E. coli and Enterococcus faecalis (Table 5). Both the water samples of the Turag river showed a very high number of E. coli. However, enterococci did not cross the limit in these two samples (Table 5) The tap water samples crossed the limit of E. coli and total coliform in most of the samples (Figure 1). Salmonella and Vibrio were present as well. The tap water samples from various sites of Dhaka city were tested along with the surface waters, to observe the numbers of these three indicators so that the authenticity of these indicators can be compared and justified. According to established guidelines of WHO, USEPA enterococci and E. coli should not be present in any 100 mL sample of raw drinking water. Our pipeline waters are treated by chlorination, still they possessed both of the indicators and sometimes pathogens as well. However, only a very few of the tap water samples showed the presence of Enterococcus

Conclusion It is seen from the results of this study that, both thermotolerant E. coli and enterococci showed a correlation with the presence of Vibrio and Salmonella in various fresh surface water bodies. However, thermotolerant E. coli showed better correlation. Total coliform number exceeded the upper limit set for recreational water bodies and did not correlate with the pathogen numbers. All the tap water samples crossed the limit (which is 0 per 100 ml) of E. 12

Comparative Evaluation of Thermotolerant Escherichia coli, Enterococci

coli and total colifom. However, only 3 samples (JHC, SA, ML) crossed the limit for enterococci (Figure 1). Most of the samples had Salmonella and Vibrio. Hence, the supplied piped water must be made drinkable for the sake of public health. The level of indicators as well as pathogens isolated from piped water from different areas of Dhaka city is not at all acceptable. The appropriate concerned authorities of Bangladesh must include alternative water quality indicators (enterococci, thermotolerant Escherichia coli) as well as some suggested pathogenic bacteria in their routine survey and assessment of water quality testing, as it is quite apparent from this and some other previous studies that the validation of still using total coliform as water quality indicator has become questionable. Acknowledgement: The authors are grateful to the University Grants Commission of Bangladesh for financial support. References


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