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In order to maintain water quality standards of drinking water in rural communities, it is necessary to have a reliable technique for monitoring pathogens in such water. Few studies denoted the existence of no correlations between faecal bacterial pathogens and faecal indicator bacteria such as Escherichia coli and their bacteriophages. Hence in the present work, a novel technique using Salmonella specific phages was designed to detect Salmonella contamination in tropical rivers. Three different methods were used in which the first one was a standard conventional technique and the other two were novel methods viz., (i) Isolation of the bacterium on semi selective media (ii) Isolation of Salmonella specific bacteriophage (Direct and Enrichment method) and (iii) Enrichment of known Salmonella specific phage in the water sample. The study area was "Chittar", a small tropical river system in extreme Southern India. Water samples were collected from five different sites during 2 different time periods (20th December, 2012 and 20th January, 2013) with an interval of 1 month. In the Salmonella isolation method using semi selective media, E. coli was predominately present along with Salmonella sp. in the samples S23, S24 and S25 which showed that the water was highly contaminated by anthropogenic activities. In the second method by using isolation of Salmonella specific bacteriophage (Direct and Enrichment method), the direct method has not produced any significant results. The results of the other two phage based methods coincided with that of the standard method and they successfully mapped the Salmonella population in the river water. But they were unable to quantify presence of Salmonella sp.
Key words : Salmonella sp., E.coli, bacteriophage, River water, pollution,
In India, Salmonella enterica serotype typhi remains the predominant species causing enteric fever (Jesudason et al., 1996, Pillai and Prakash, 1993). It can be seen that all enteric fever/typhoid and some of the water-based infections depend on fecal access to domestic water sources. Salmonella typhi causes a protracted bacteremic illness referred to as typhoid fever(de Jong et al., 2012). Since humans are the only reservoirs for S. typhi (Madanagopalan et al., 1975), infection most often acquires through ingestion of food or water contaminated with feces and urine of infected persons and chronic carriers increasing across all endemic areas (Weill, 2010). A multidrug resistant (MDR) variable of Salmonella typhi emerged as the cause of epidemic typhoid fever in some Asian countries including India during the late 1980s (Jesudason et al., 1996) and a multi-drug resistant variety of typhoid fever in India. John (1996) stated that the rivers are the major drinking water source for the rural India as a large population of Indians lives along the river banks. The consumption of polluted surface water in developing nations is a major cause of diarrheal disease related mortality. The prevalence of multi-antimicrobial resistant E. coli in the river Ganga water poses increased risk of infections in the human population (Ram et al., 2007). The river Ganges in Varanasi during the period December 1985 to November 1987 showed the presence of enteropathogenic bacteria indicating that the water of the river Ganges is potentially hazardous and unfit for drinking. In order to maintain water quality standards in rural communities, it is necessary to have a simple, rapid, cheap and reliable technique for monitoring the microbiological quality of such drinking water. Microbial indicators of water quality are used to monitor the presence and level of fecal pollution or contamination of the water and to assess its health risk. Bacteriophage is a generic term for a large group of agents with the ability to lyse growing bacterial cultures and they represent the largest of all virus groups and found in many diverse natural habitats in enormous numbers (Ackermann, 1987). Bacteriophages whose hosts are strains of E. coli are coliphages. They have been found to be ubiquitous inhabitants of the intestinal tract of man and animals and are encountered wherever fecal contamination occurs (D'Herelle, 1929). Coliphages have been suggested to be better indicators of enteroviruses. The bacteriophages are used as indicators of bacterial and viral contamination of water (Griffin et al., 2000, Miller et al., 1998, Meloni et al., 2003). Few studies denoted the appearance of no correlations between fecal bacterial pathogens such as Salmonella and Shigella species and fecal indicator bacteria such as Escherichia coli and their bacteriophages in fresh and marine waters. The present study tries to bridge the gap in tracing Salmonella contamination in the river system by using Salmonella specific phages. The study area "Chittar" is a small river system in extreme South India. It is a major tributary of the river Thamirabarani. The river takes its origin in the eastern slopes of the Western Ghats in the Courtallam hills at an altitude of 1750m above mean sea level. From its origin, the river climbs down for about six Kms, turns North and flows for about 16 Kms before turning towards the east. Its total length is about 80 Km. It joins the river Thamirabarani near Sivalapperi village. The river Chittar makes many patches of Tenkasi regions in South India, fertile.
Materials and Methods
The sampling sites for the current research work was decided based on the anthropogenic activities. A total of five sampling sites was determined and water samples were collected. The first site was selected at Shenbagadevi falls (08° 55N, 77° 17E) as the river comes down the hills of Western Ghats without witnessing anthropogenic activities. After reaching the second site The Courtallam main falls (08° 34 N, 77° 28 E) where the river starts to witness pollution due to heavy exposure to human activities being an important tourist spot. The third sample point was picked as the river leaves the Courtallam town and starts to experience lesser inflows of pollution, The Courtallam bridge (08° 13 N, 77° 39 E). After running 6 km approximately, the river enters the densely polluted Tenkasi town; the entry point into the town by the river marks the fourth sampling site "Tenkasi Yanaipalam" (08° 58 N, 77° 21 E). The fifth and the final spot was selected at Tenkasi Vaikapalam (08° 43N, 77° 46 E) as in this region the river leaves Tenkasi town after witnessing heavy pollution. Samples were drawn from all the five sites in a single day in the month of December 20, 2012 & January 20, 2013. The five samples were named as S11, S12, S13, S14, S15 for the samples collected on December 20, 2012 and S21, S22, S23, S24, S25 for the samples collected on January 20, 2013 for our convenience. For each sampling site 25ml of water samples was collected in a sterile plastic container and was kept at 4 °C for few hours until transferred to the laboratory for processing.
Isolation of Salmonella typhi
From the collected samples, the presence of Salmonella typhi was investigated by conventional colony isolation technique where the sample was plated onto a semi selective medium which inhibits the growth of other unwanted and unrelated bacteria. Following the methods of Zajc-Satler and Gragas (1977), each sample was serially diluted up to 10-6 dilution and was plated on the XLD media by performing spread plate technique. The plates were incubated at 37°C for 24 hours, the colony morphology and the number of colonies were recorded and used as the standard method for comparison.
Salmonella typhi (MTCC 3917) was obtained from Microbial Type Cell Culture Centre (MTCC), Chandhigarh and used as the standard host for the isolation of Salmonella phages.
Direct Isolation Method
Phage lysate was prepared using the methods of Grabow and Coubrough (1986) with modifications. The samples were allowed to stand still to settle larger suspended particles. The supernatant was centrifuged at 7000 rpm for 10 min to remove residual bacterial cells and debris. The remnants were filtered through 0.22µm syringe filter and used as their respective cell lysate for plaque assay.
Isolation of the phages by enrichment method
A sample of 10 ml volume and 5 ml of Logarithmic phase cells of Salmonella serovar typhi MTCC No 3917, a potential recipient was mixed with 50 ml double strength Trypticase Soy broth (Himedia, Mumbai) and incubated at 37°C on a rotary shaker to enrich the phages specific to the host. After 24 hours of incubation, 4ml of chloroform was added to the mixture and incubated at 4°C for 4hours. Later 10 ml of the supernatant was centrifuged at 8000 g for 15 min and filtered through 0.22µm PVDF which has the expected phage, free of live bacterial cells (Cell lysate) and was stored at 4°C. The cell lysate was stored in a refrigerator at 4°C for further use as per the methods of Bao et al., (2011) with modifications.
Enrichment of STP A phage in the water sample
From the collected river water samples, 10 ml was filtered through masculine cloth and mixed with 100ml of XLD broth for specific enrichment of enteric bacterium. After enrichment of the enteric bacterium, 1ml of Salmonella specific phage STP A (Unpublished data) previously isolated and characterized in the laboratory was added to a concentration of 108PFU/ml, incubated for 3 hours. Cell lysate was prepared and assayed for the phage titre using the plaque assay technique.
The plaque assay was carried out by double agar layer diffusion method following Mc Laughlin et al. (2006). The cell lysate (0.1ml) and exponentially grown host cells (0.2 ml) were suspended in 3 ml aliquots of molten soft agar (0.7% agar) held at 45°C. Mixed and uniformly dispensed over the hard agar (1.5% agar), incubated at 37 ± 2°C for 12 h. A positive control S. typhi specific bacteriophage was used. Negative control was maintained by using sterile phage free water as lysate.
The water samples collected from the different regions of Chittar river flowing through the Tenkasi region were analyzed for its microbial water quality using bacteriophages. The samples were collected during 2 different time periods with an interval of 1 month and were analyzed for the presence of Salmonella sp. by three different methods viz. (i) Isolation of the bacterium in semi selective media (ii) Isolation of Salmonella specific bacteriophage (Direct and Enrichment method) and (iii) Enrichment of Salmonella specific phage in the water sample. Isolation of Salmonella isolation on semi selective media was used as the standard method for quantification of Salmonella sp. in the river water. The samples were collected on two different days December 20, 2012 & January 20, 2013 which showed high variation in the microbial load (Fig - 1). For the isolation of the bacterium in semi selective media, the colonies that appeared in the XLD agar indicated the following colony morphology - round, mucoid, yellow coloured which corresponds that the colony may be suspected for E. coli in the samples S14 and S15 with an average of 20.6±14.5x103 and 53±26.1x103 CFU/ml respectively. The samples S23, S24 and S25 showed the development of mucoid yellow coloured colonies with an average of 29.6±16.8x103, 23±14x105 and 17±8.1x106 CFU/ml respectively. Along with it, red colored colonies with an average of 12±6.5X102, 12.7±14.3X102 and 20.3±18.1X104 CFU/ml in the respective samples S23, S24 and S25 represented Salmonella sp.
Figure - 1. Plaque assays for phage lystae prepared by isolation on semi selective media technique against the bacterial host Salmonella typhi. A-Coutrallam Main falls(S12), B- Coutrallam Bridge(S13), C-Yanaipalam (S14), D-Vaikapalam falls(S15), E-Coutrallam Main falls(S22), F-Coutrallam Bridge(S23), G-Yanaipalam (S24), H-Vaikapalam falls(S25).
The cell lysate prepared by the direct isolation method without enrichment produced no visible plaques in the plaque assay for the 9 water samples except for the S25 with an average of 13.1±17.3x102 PFU/ml. The cell lysate prepared by the enrichment of phages using Salmonella typhi (MTCC 3917) produced visible plaques in the samples S23, S24 and S25 with an average titre of 7.3±5.5x106, 16±7.2x104 and 10.3±7x109 PFU/ml respectively and no visible plaques were visualized in the samples S11, S12, S13, S14, S15, S21 and S22. The cell lysate produced by the enrichment of STP A phage in the enriched enteric bacterium present in the water produced visible plaques in all the samples. The samples S23, S24 and S25 revealed an increase in the average titre of the phages to 10.6±12.6x108, 27±12.1x109 and 8.6±7.3x1012 PFU/ml respectively. The samples S11, S12, S13, S14, S15, S21 and S22 produced a titre of 21.6±19x103, 15.6±9.7x103, 38.3±28x103, 35.6±17.8x103, 8.6±8.6x103, 10.6±10.1x103and 18.3±10x103 PFU/ml respectively on an average. The control sample prepared by diluting the crude known Salmonella phage of 1ml in 100ml XLD broth in sterile condition yielded a phage titre of 11.6±9.0x103 PFU/ml. For a positive control, a phage lysate which contained phages specific to Salmonella typhi was used. This phage lysate was serially diluted up to 10-6 dilutions and was plated by a double agar method which yielded 64 numbers of plaques in the dilution of 10-6. For a negative control, the sterile distilled water was used as a phage lysate. This yielded no visible phages in the negative control plate. Statistical analysis was done using the Graphpad prism 6.01 (Trail) statistical and graphical software. The correlation between the Salmonella isolation method using semi selective media with the other methods were done using the Pearson correlation method. The phage enriched isolation and the STP A enrichment method in the river water correlated with the Salmonella isolation method using semi selective media which was used as the standard for the estimation of Salmonella sp. The Pearson r value for phage enriched isolation in the river water and the STP A enrichment method was 0.9924 & 0.9925 respectively. The P value was statistically significant (alpha = 0.01) < 0.0001 & < 0.0001 against the standard method. The phage direct isolation method does not correlate to the standard method which has the Pearson r value of 0.7373 and the p value was 0.0150 representing statistically nonsignificant against the standard method (Table-1) (Figure-2). The correlation between the presence of E.coli and Salmonella sp. was statistically nonsignificant with the r value - 0.8172 and the P value - 0.0912.
Fever is one of the most common illnesses in all age groups in India where poor sanitation facilities prevails (Nandagopal et al., 2010). Probably one of the most important reservoirs of Salmonella-infections germs is the water of rivers and lakes (Mersch-Sundermann and Wundt, 1987). Tobias and Heinemeyer (1994) proved that the presence of Salmonella did not correlate to an increasing presence of fecal indicator bacteria. Sugumar et al. (2008) predicted that the relationship between fecal indicators and Salmonella was not significant (p > 0.05). These reports were substantiated by other workers Krometis et al. (2010) & Sudhanandh et al. (2012). Present work on statistical analysis showed minimal correlation existence between E.coli and Salmonella population, but none of the samples have produced results enumerating only Salmonella sp. leaving E. coli which in the other sense can be treated that the coliphage/E.coli enumeration is enough to trace fecal Salmonella contamination. Similarly as there are samples which have the traces of E.coli without Salmonella population, Salmonella cannot be used as an indicator for fecal contamination. The present identification of the pathogens present in the river water using coliphage as the indicator organism seems to be unsuitable for tracing the presence of Salmonella sp. This study has introduced a simple method using Salmonella phages to trace Salmonella sp. contamination in river water. Three different methods compared viz., (i) Isolation of Salmonella bacterium in semi selective media (ii) Isolation of Salmonella specific bacteriophage (Direct and Enrichment method) and (iii) Enrichment of STP A phage in the water sample to identify Salmonella contamination in river water. In the bacterium isolation method, only the sample S14 and S15 collected on December 20, 2012 have produced few E.coli colonies which indicates that the water encounters fecal contamination once it reaches the Tenkasi city. Whereas the E.coli contamination was not seen in the other samples collected on December 20, 2012. All the five samples were free of Salmonella sp. For the samples collected on January 20, 2013, E.coli presence was found along with Salmonella sp. in the samples S23, S24 and S25 which showed that the water was highly contaminated by anthropogenic activities. The results were highly contradictory on the two different days. Presence of lesser fecal contamination on 20 December, 2012 when compared to the 20, January 2013 may be due the runoff of the bacterial contamination during the heavy inflow of water from the hills into the river on December 20, 2012. Both the days the water samples S11, S12, S21 and S22 were free of fecal contamination because the river flowing through the hills did not witness any anthropogenic activities. Our results coincides with the early works of Murugesan et al. (2007) who found the significantly poor pollution with the biological quality in the river Chittar. The total and fecal coliforms exceeded the permissible limits indicating a poor status of the river. In the second method by using Isolation of Salmonella specific bacteriophage (Direct and Enrichment method), the direct method has not produced any significant results which was unable to trace smaller no of phages in a given sample. Hence this method was not advisable and appropriate for tracing or isolating phages from the river samples. Another method of enrichment produced valid results where existed a 100% correlation between the standard method and this method. The samples S11, S12, S13, S14, S15, S21 and S22 did not produced any visible plaques which inturn indicate that the samples were avoided of Salmonella sp. and the samples S23, S24 and S25 showed the presence of Salmonella in the water sample. This result correlates with the results of the standard method. In the third method of enrichment of STP A phage Salmonella contamination in river water persisted. All the samples produced visible plaques on intentional addition of STP A. The increase in the titre of the added STP A indicated the presence of Salmonella sp. within it. The increase in the titre of the phage was due to the presence of Salmonella sp. in the sample which acted as the host for the added STP A phage. The enrichment of the phage is due to the specific enrichment of the available Salmonella bacterium present in the sample. The samples S23, S24 and S25 produced an increased phage titre. This interprets that the sample has Salmonella sp. within it. The samples S11, S12, S13, S14, S15, S21 and S22 produced a titre of 21x103, 5x103, 9x103, 51x103, 18x103, 7x103 and 29x103. This titre is same as the control without the enrichment of the phage with the absence of Salmonella phage. This suggests that the samples were free of Salmonella contamination. Among the three methods, all the methods have produced similar results except the method with direct isolation of the phage. Although this method was not suited for this river it can perform well in waters densely polluted with microbes and this also has an advantage of having the ability the show the actual titre of the phages present in the water naturally which in turn predicts the quantity of the bacterium. The other two phage based methods can be used to trace the presence of Salmonella sp. in the water,but they were not able to quantify the amount of Salmonella sp. present because both the methods uses enrichment protocols in their methodology which can alter or even manipulate the actual titre of the bacterium/phages present in the sample.