A study of prevalence of pathogenic bacteria, particularly, fecal coliforms and their antibiotic resistance pattern in environmental water samples of a tertiary-care hospital, Ahmedabad.
The antibiotic-resistant bacteria (ARB) are creating a major public health issue, globally, owing to their presence and spread, especially in aquatic ecosystems that are known for the ARB and antibiotic resistance genes (ARGs). Although several studies have detected ARB in drinking water systems, most of the earlier studies had focused on cultivable bacteria and/or indicator organisms. There is a scarce knowledge regarding the effects of ARGs in the drinking water bodies, and, recently, they were found to be the emerging contaminants. The emergence of antimicrobial-resistant strains of pathogenic bacteria has become a great threat to the public health.  However, in our region, the study of antibiotic resistance of bacteria from environment such as soil, water, or from fish is scanty.
E. coli is a part of the normal flora of human and animal fecal matters, which may contaminate the soil and water.  E. coli in water sources is accountable for disease outbreaks [3-5] and mortality worldwide in recent years. Infection with these bacteria may be transmitted through an accidental ingestion or deliberate consumption and direct contact with E. coli-contaminated water. Although most E. coli strains are harmless, some are pathogenic and can cause diseases  such as diarrhea, urinary tract infections, respiratory problems, and even life-threatening bloodstream illnesses  among others, as new pathogenic strains of these bacteria have been emerging. The increased resistance shown by E. coli till date  has become a major issue in the treatment of infections by causing difficulties in the mode of treatment through severe infections, which in turn affects the efficiency of the presently followed treatments.  Water bodies act as a source of these drug-resistant E. coli strains,  because they contain the ARB and ARGs, which spread the resistance to opportunistic pathogens,  such as E. coli bacteria. This study aims to know the prevalence of pathogenic bacteria in environmental water in our hospital and their antibiotic resistance pattern.
Materials and Methods
This prospective study that was done to determine the prevalence of antibiotic resistance in environmental bacteria from water samples of different areas of General Hospital, Sola, Ahmedabad, India, in the Department of Microbiology, GMERS Medical College, Sola, Ahmedabad, from December 2013 to March 2014. Total 50 water samples were analyzed for the determination of the prevalence of antibiotic-resistant coliforms. All samples were collected from different sites [Table 1].
Samples from RO system, tapwater, and leakage pipe line were treated as drinking water. Before the collection of water sample from the tap, first of all, it was sterilized by spirit lamp and then collected in a dry, clean, autoclaved, leak-proof, and sterile glass container. Each sample was labeled with the name of the area, and the date of collection. All the samples were stored in refrigerator at a temperature of 4[degrees]C for further use. Water samples were collected for the analysis of fecal contamination and to detect the most probable number (MPN) of fecal coliform by multiple-tube fermentation technique. Water sample was added in MacConkey broth [with indicator bromocresol purple (BCP)]; double strength as 50 mL of sample was inoculated into a bottle containing 50 mL of double strength broth; 10 mL of sample was inoculated into each of the five bottles containing 10 mL of double strength broth, and 1 mL of sample was inoculated into each of the five tubes containing 5 mL of single strength (SS) broth. The tubes and bottles were incubated at 44[degrees]C for 24 to 48 h in incubator. At the end of the 24-h incubation period, each tube and bottle were examined for the presence of turbidity, color change to yellow, and gas. If present, gas can be seen in the Durham tube. Negative tubes and bottles were reincubated for further 24-h period. At the end of this period, the tubes and bottles were checked again for turbidity, color change, and gas production. The MPN was found from the test result by referring to MPN table. [12-15]
Inoculation Procedure for Presumptive Fecal Coliform Count from Drainage (Sewage) Water Sample
Before inoculation of the water sample into SS MacConkey's broth, a serial dilution of the sample was done using phosphate buffer saline. Serial dilution up to [10.sup.-5], [10.sup.-6], and [10.sup.-7] were prepared.
1. One milliliter of undiluted sample was inoculated into the tube containing 5 mL of SS broth.
2. One milliliter of diluted sample (each [10.sup.-5], [10.sup.-6], and [10.sup.-7]) was inoculated into each of the five tubes containing 5 mL of SS broth.
Determination of MPN for Drainage (Sewage) Water
After the incubation, the number of tubes in which lactose fermentation with acid and gas production occurred was counted. The MPN was found from the test result by referring to the MPN table for the MPN of fecal coliforms, bacteria isolated from drainage (sewage) water (also called McRady's table) MPN procedure for irrigation water, EPA, 2002, as mentioned below.
* When calculating the coliform concentration for drainage water, the MPN index (3 x 5 tubes) from the table was first found. The calculation of the coliform concentration uses the following formula: (MPN index number/lowest dilution) x 100.
* For example, if we had used [10.sup.-2], [10.sup.-3], and [10.sup.-4] dilutions and our MPN index was 2 (2/[10.sup.-2]) x 100 = 2.0 x [10.sup.4]. Antibiotic sensitivity test (AST) was done by Kirby-Bauer
Disc diffusion method on Mueller-Hinton agar for 16-24 h incubation. All E. coli that were isolated were subjected for AST, which was performed by disc diffusion test, according to NCCLS guideline.  Gram-negative panel consisted of ampicillin + sulbactum (20/10), cotrimoxazole (30 [micro]g), chloramphenicol (30 [micro]g), ciprofloxacin (5 [micro]g), tetracycline (30 [micro]g), ofloxacin (5 [micro]g), amikacin (30 [micro]g), gentamicin (10 [micro]g), cefoxitin (30 [micro]g), cefotaxime (30 [micro]g), and ceftazidime (30 [micro]g).
In this study, of the 50 samples collected from civil hospital, 22 samples (44%) showed contamination with fecal coliform. Of the 22 positive samples, 12 samples showed contamination with E. coli and 10 samples with Klebsiella spp.
Of the 22 positive samples, 10 were from drinking water, 8 from tap water, 2 from drainage water, and 2 from leakage water. So, in our study, we observed more positivity rate in drainage water [Figure 1].
In our study, a total of 12 E. coli were observed in different sources; their numbers shown in different sources were as follows: in drinking water (five), tap water (three), leakage water (two), and drainage water (two). In this study, thermotolerant E. coli were observed in the highest number when compared with Klebsiella [Table 2].
All the bacteria isolated from drinking water (100%) were sensitive to cotrimoxazole (BA), ciprofloxacin (RC), ofloxacin (ZN), chloramphenicol (CH), gentamicin (GN), amiacin (AK), and nitrofurantoin (NI). E. coli from drainage water (100%) were resistant to cotrimoxazole (BA), cefotaxime (CF), piperacillin (PC), nitrofurantoin (NI), and gentamicin (GM) [Table 3].
All the Isolated bacteria (100%) were sensitive to amikacin, whereas Klebsiella spp. was 100% sensitive to cotrimoxazole (BA), cefotaxime (CF), ciprofloxacin (RC), ofloxacin (ZN), gentamicin (GM), and amikacin (AK). Klebsiella species, on the other hand, showed high resistance frequency to ampicillin (60%); only 10% of the Klebsiella strain was resistant to chloramphenicol (CH), tetracycline (TE) [Table 4]. The highest sensitivity frequency of E. coli was observed with amikacin (100%), ofloxacin (91.66%), and ciprofloxacin (91.66%). E. coli were 50% sensitive and showed resistance to ceftizoxime (CI).
The widespread emergence of antibiotic resistance, particularly, multidrug resistance, among bacterial pathogens has become one of the most serious challenges in clinical therapy. Environment containing antibiotic residues exert selection pressure and contribute to the appearance of resistant bacteria. In light of the potential health risks, many studies have focused on antibiotic-resistant bacteria from various ecosystems. 
In this study, 12 thermotolerant E. coli and 10 Klebsiella spp. isolates that were isolated from different water sources (drinking water, tap water, leakage water, and drainage water) were further analyzed for the prevalence of antibiotic-resistant members. The result of the tests revealed that all of the isolates of E. coli (100%) were sensitive to amikacin, whereas isolates of Klebsiella (100%) were sensitive to cotrimoxazole (BA), cefotaxime (CF), ciprofloxacin (RC), ofloxacin (ZN), gentamicin (GM), and amikacin(AK).
Overall resistance was most frequently observed to ampicillin (16.6%), chloramphenicol (16.6%), tetracycline (33.3%), cotrimoxazole (16.6%), piperacillin (41.66%), and ciprofloxacin (8.34%). Most of these antibiotics have been widely used for therapeutic purposes against bacterial infections in humans and animals and as growth promoters in agriculture and aquaculture. [17-19]
The results are also in accordance with other reports. A similar study made in the water supplies of rural Venda communities, South Africa, also showed that more than 95% and 75% of E. coli isolates were sensitive to ciprofloxacin and cotrimoxazole, respectively. Similarly, TNAS  and Biyela and Bezuidenhout  isolated E. coli from river water and demonstrated that none of the isolates were resistant to ciprofloxacin.
The results of the study also showed that nearly 40% (10 out of 25) of the drinking water sources were contaminated. The majority of the isolates showed remarkable resistance for cefotaxime (CF) and piperacillin (PC) than resistance in any other source. Antibiotic usage and public awareness creation activities are required to improve the observed efficacy of these two antibiotics in the study area.
Multidrug-resistant bacteria were also observed from water samples collected at General Hospital. Of the 12 E. coli isolates tested for antibiotic resistance, eight of them (66.66%) showed multiple resistances for two to eight antibiotics.
This study is compared with other studies as shown in the Table 5
The presence of a large number of coliforms of environmental source in the absence of bacterial pathogens is of no consequence, because these organisms are usually considered as harmless. However, this is not necessarily true if the bacteria in question possess transferable drug resistance. Once these organisms enter the gastrointestinal tract of humans, they may colonize the human gut themselves and transfer their resistance to already colonized bacteria to the sensitive pathogens with which their host may become infected. 
Among 50 (100%) water samples of hospitalized area tested, 22 (44%) were positive for the presence of fecal coliform. On the basis of the results of our study, it has been recommended that of the 22 samples, 12 (24%) were positive for thermotolerant E. coli contamination, whereas 10 (20%) were positive for Klebsiella spp. contamination. In our study, the numbers of bacteria from the sources were drinking water (10), tap water (eight), leakage water (two), and drainage (sewage) water (two). Of the 10 drinking water samples, five were positive for thermotolerant E. coll and five were positive for Klebsiella. Among the total isolates, 13 showed multiple resistance to two to eight antibiotics.
This study revealed that amikacin, ofloxacin, and ciprofloxacin are the best antibiotics to treat E. coli infection. Antibiotic susceptibility studies revealed that water from hospital contain antibiotic-resistant E. coli strain, which may serve as a reservoir for ARGs in water environment.
This study will provide us a baseline reference for ARB spread in environment and community and help us in devising preventive tools for the emergence of resistant bacteria in hospitals, which will help the hospital administration in implementing guidelines for reducing the emergence of ARBs and the morbidity and mortality arising out of them.
Awareness creation on water quality and sanitation, together with the construction of protected water sources, should be encouraged to reduce the risk of water-borne diseases in the area and to monitor the widespread antibiotic resistance among the environmental bacterial isolates.
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Source of Support: Nil, Conflict of Interest: None declared.
Nidhi K Sood, Parul C Patel, Sachin M Patel, Asha H Mandalia
Department of Microbiology, GMERS Medical College, Sola, Ahmedabad, Gujarat, India.
Correspondence to: Parul C Patel, E-mail:email@example.com
Received April 10, 2015. Accepted June 7, 2015
Table 1: Water collection sites Number of Water collection samples No. sites collected 1 Reverse osmosis 25 (RO) plants 2 Tap water from 17 washroom 3 Leakage pipeline 5 4 Drainage (sewage) 3 pipeline Table 2: Comparison of the number of E. coli and Klebsiella in the different sources of water Drinking Tap Leakage Drainage water water water water Escherichia coli 5 3 2 2 Klebsiella spp. 5 5 0 0 Table 3: Sensitivity (%) of E. coli and Klebsiella spp. against 13 selected antibiotics in different sources Escherichia coli Drinking water Tap water Drug S (%) R (%) S (%) R (%) AS 80 20 66.6 33.3 BA 100 0 100 0 CF 40 60 100 0 PC 40 60 66.6 33.3 CH 100 0 66.6 33.3 RC 100 0 100 0 CI 80 20 100 0 TE 40 60 66.6 33.3 ZN 100 0 100 0 GM 100 0 100 0 AK 100 0 100 0 NI 100 0 100 0 Escherichia coli Leakage water Drainage water Drug S (%) R (%) S (%) R (%) AS 50 50 50 50 BA 100 0 0 100 CF 100 0 0 100 PC 100 0 0 100 CH 50 50 100 0 RC 100 0 100 0 CI 50 50 50 50 TE 100 0 50 50 ZN 100 0 50 50 GM 100 0 0 100 AK 100 0 100 0 NI 100 0 0 100 Klebsiella spp. Drinking water Tap water Drug S (%) R (%) S (%) R (%) AS 20 80 40 60 BA 100 0 100 0 CF 100 0 100 0 PC 80 20 80 20 CH 100 0 60 40 RC 100 0 100 0 CI 80 20 80 20 TE 100 0 40 60 ZN 100 0 100 0 GM 100 0 100 0 AK 100 0 100 0 NI 100 0 100 0 Table 4: Comparison of resistance in Escherichia coli in this study with another study Antibiotics Escherichia coli Escherichia coli: Ref.  S (%) R (%) S (%) R (%) Cotrimoxazole (BA) 83.33 16.67 88 4 Ciprofloxacin (RC) 91.66 8.34 96 4 Tetracycline (TE) 66.66 33.34 42 42 Table 5: Comparison of the multidrug-resistant bacteria in this study with other studies Antibiotics Resistance (%) This study Two to eight antibiotics 66.66 Debmandal et al.  Three or more antibiotics 57 Bahiru et al.  Three or more antibiotics 96.30 Figure 1: Bacterial isolates from different sources. Isolate number Total sample Bacterial isolates Drinking water 25 10 Tap water 17 8 Leakage water 5 2 Drainage water 5 2 Note: Table made from bar graph.
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|Title Annotation:||Research Article; Ahmedabad, India|
|Author:||Sood, Nidhi K.; Patel, Parul C.; Patel, Sachin M.; Mandalia, Asha H.|
|Publication:||International Journal of Medical Science and Public Health|
|Date:||Dec 1, 2015|
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