University of Strathclyde, Glasgow1
Antibiotic Resistant Bacteria (ARB) and their genes (ARGs) are considered as well known contaminants in waste water and emerging contaminants in drinking water treatment plants  and water distribution system . Due to the presence of pathogenic bacteria and their genes, availability of safe drinking water has become one of the key concerns of the world.
Drinking water pollution is not only prevalent in developing low income countries but also occurs in developed nations. Multiple drug resistance bacteria are found in water sample having plasmid mediate resistance . In India and Pakistan, New Delhi metallo-beta-lactamase 1 (NDM-1) genes are reported in bacteria in chlorinated treated drinking water . The same genes are also found in UK including England, Scotland, Ireland , and in USA in Michigan and Ohio . Both prokaryotes and eukaryotes [3, 16, 23] are found in water. Some studies reported the presence of ARGs in treated drinking water as well [20, 25].
During treatment process, it is not possible to fully eradicate the ARGs . Some resistance bacteria are found dominant in potable drinking water as compare to source water. It is also because of the fact that these bacteria live in pipelines and shed in water . Plumbing system in buildings contains more bacteria as compare to source water and even in the presence of suitable amount of residual disinfectants, drinking water contains diverse type of bacteria . Presence of biofilm has a role in the development of ARGs  as exchange of genetic material occurs through horizontal gene transfer in these biofilms . Older biofilms are less susceptible to disinfection due to the physiological changes in population .
Main objective of this study is to find the antibiotic resistance bacteria in municipal water supply system in Glasgow at the consumer end. Our hypothesis is that resistant bacteria are present even after the application of disinfectant in treatment plant and presence of residual chlorine in distribution system. We have tried to find out the presence of transferable integrons (Int-1) and disinfectant resistant genes (qac) in the bacteria isolated from water distribution system of Glasgow. The confirmation of presence of these bacteria and their genes is an indication that water is not totally safe and there are chances of epidemics.
Materials and Methods:
Tap water samples were collected from kitchens in Glasgow and process by filtration . 148 bacterial isolates were tested for Tetracycline, Sulfamethoxazole, Amoxicillin and Ciprofloxacin by Agar Dilution Method. Each antibiotic was tested with Replica Plating method and MIC values were determined . Disinfectant susceptibility test was performed by Kirby-Bauer disc diffusion method  and suspension test. Free chlorine solutions with 5 concentrations were prepared. Bacterial was added at a concentration of 1 x 105 cfu/mL. At 0, 15 and 60 minute contact time, samples were plated on agar plate and cfu/mL was calculated. Free chlorine was determined by DPD method . Monochloramine suspension test was performed similarly using PBS of pH 8.0 [5,9,10] and concentration was determined by Indophenol method . DNA of bacterial isolates was extracted by freeze thawed cycles. PCR reaction was performed by primers for 16S rRNA , integrase gene  and qac genes . PCR products length was verified on 2% agarose gel.
Results and Discussion:
Among 148 isolates, 128 bacteria (86.5%) show single or multiple resistant while 20 bacteria (13.5%) have no resistant against any of the antibiotic tested. 96 bacteria (64.9%) have Amoxicillin (A) resistance and they grow at > 8 Âµg/mL of antibiotic. Amoxicillin resistance is the most common resistance among the bacteria isolated from tap water of Glasgow. Seven (4.7%) bacteria have quadruplet resistant (TSCA) against all four antibiotics indicating that they are multiple antibiotic resistance bacteria (MDR). 11 (7.4%) have triple resistant and 56 (37.8%) show resistant against any two antibiotics simultaneously. Sulfamethoxazole and amoxicillin resistant combination is the most common multidrug resistance among bacteria in tap water as 45 (30.4%) isolates show this resistant combination. The result confirms our hypothesis that ARBs are present in distribution system. Presence of int-1 genes shows that the higher number of resistant organisms is due to the transferability of genes among bacteria.
Chlorine is the most preferred disinfectant in water treatment plants but it has a role in the enrichment of ARBs. Due to this situation, currently available disinfectants are unable to completely eradicate the pathogens or bacteria from water as found in our study that not only bacteria are present in tap water but they have transferable antibiotic resistant genes. It may be because of the reason that during disinfection process, ARBs are killed but their genes retain their activity and transfer to other bacteria.
Presence of antibiotic resistance bacteria and transferable genes indicates that the genes are dispersing in the distribution system environment and due to this reason they are present in tap water. This study highlights the malfunction of residual disinfectant or there is an intrusion in distribution system. There is need to use some strategies to make water safe at consumer end. 1. Allion, A., Lassiaz, S., Peguet, L., Boillot, P., Jacques, S., Peultier, J. and Bonnet, M.-C. (2011) A long term study on biofilm development in drinking water distribution system: comparison of stainless steel grades with commonly used materials. Revue de Mettalurgie, 108, 259-268.
2. APHA, AWWA, WEF, (1998) Standard methods for the examination of water and wastewater. American Public Health Association, Washington, DC.
3. Armstrong, J.L., Shigeno, D.S., Calmoris, J.J. and Seilder, R.J. (1981) Antibiotic-Resistant Bacteria in Drinking Water. Appl. Environ. Microbiol. 42, 277-283.
4. Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Lozupone, C.A., Turnbaugh, P.J., Fiefer, N. and Knight, R. (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. The Proceedings of the National Academy of Sciences of the United States of America, 108 Suppl 1, 4516-22.
5. Chiao, T.-H., Clancy, T.M., Pinto, A., Xi, C. and Raskin, L. (2014) Differential resistance fo drinking water bacteial populations to monochloriamine disinfection. Environ. Sci. Tech. 48, 4038-4047.
6. CLSI 2012. Performance standards for antimicrobial disk susceptibility tests; Approved standardÂ—11th ed. CLSI document M02-A11. 32:1. Clinical Laboratory Standards Institute, Wayne, PA.
7. Diehl, D.L. and Lapara, T.M. (2010) Effect of Temperature on the Fate of genes encoding tetracycline resistance adn the integrase of class 1 integrons within anaerobic and aerobic digesters treating municipal waster water solids. Environ. Sci. Tech. 44, 9128-9133.
8. Dodd, M.C. (2012) Potential impacts of disinfection processes on elimination and deactivation of antibiotic resistance genes during water and wastewater treatment. J. Environ. Monitor. 14, 1754-1771.
9. Driedger, A.M., Rennecker, J.L. and Martinas, B.J. (2001) Inactivation of Cryptosporidium parvum oocysts with ozone and monochloramine at low temperature. Water Res. 35, 41-48.
10. Howard, K. and Inglis, T.J.J. (2005) Disinfection of Burkholderia pseudomallei in potable water. Water Res. 39, 1085-1092.
11. Jechalke, S., Schreiter, S., Wolters, B., Dealtry, S., Heuer, H. and Smalla, K. (2013) Widespread dissemination of class 1 integron components in soils and related ecosystems as revealed by cultivation-independent analysis. Front. Microbiol. 4, 420.
12. Kumarasamy, K.K., Toleman, M.A., Walsh, T.R., BagariaA, J., Butt, F.A., Balakrishnan, R., Chaudhary, U., Doumith, M., Giske, C.G., Irfan, S., Krishnan, P., Kumar, A.V., Maharjan, S., Mushtaq, S., Noorie, T., Paterson, D.L., Pearson, A., Perry, C., Pike, R., Rao, B., Ray, U., Sarma, J.B., Sharma, M., Sheridan, E., Thiruarayan, M. A., Turton, J., Upadhyay, S., Warner, M., Welfare, W., Livermor, D.M. and Woodford, N. (2010) Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis. 10, 597-602.
13. Lechevallier, M.W., Cawthon, C.D. and LEE, R.G. (1988) Factors promoting survival of bacteria in chlorinated water supplies. Appl. Environ. Microbiol. 53, 649-654.
14. Lederberg, J. and Lederberg, E.M. 1952. Replica plating and indirect selection of bacterial mutants. J. Bacteriol. 63, 399-406.
15. Lee, W., Westerhoff, P., Yang, X. and Shang, C. (2007) Comparison of colorimetric and membrane introduction mass spectrometry techniques for chloramine analysis. Water Res. 41, 3097-102.
16. Pinto, A.J., XI, C. and Raskin, L. (2012) Bacterial community structure in the drinking water microbiome is governed by filtration processes. Environ. Sci. Tech. 46, 8851-8859.
17. Pruden, A., Pei, R., Storteboom, H. and Carlson, K.H. (2006) Antibiotic resistance genes as emerging contaminants: Studies in Northern Colorado. Environ. Sci. Tech. 40, 7445-7450.
18. Rose, L.J., Rice, E.W., Hodges, L., Peterson, A. and Arduino, M. J. (2007) Monochloramine inactivation of bacterial select agents. Appl. Environ. Microbiol. 73, 3437-3449.
19. Schwartz, T., Kohnen, W., Jansen, B. and Obst, U. (2003) Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiol. Ecol. 43, 325-335.
20. Shi, P., Jia, S., Zhang, X. X., Zhang, T., Cheng, S. and Li, A. (2013) Metagenomic insights into chlorination effects on microbial antibiotic resistance in drinking water. Water Res. 47, 111-20.
21. Talukdar, P.K., Rahman, M., Rahman, M., Nabi, A., Islam, Z., Hoque, M.M., Endtz, H.P. and Islam, M.A. (2013) Antimicrobial resistance, virulence factors and genetic diversity of Escherichia coli isolates from household water supply in Dhaka, Bangladesh. PLoS One, 8, e61090.
22. Walsh, T.R., Weeks, J., Livermore, D.M. and Toleman, M.A. (2011) Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. The Lancet Infect. Dis. 11, 355-362.
23. Wang, H., Masters, S., Hong, Y., Stallings, J., Falkinham, J. O. lll, Edward, M.A., and Pruden, A. (2012) Effect of disinfectant, water age, and pipe material on occurence and persistence of Legionella, mycobacteria, Pseusomonas aeruginosa, and two amoebas. Environ. Sci. Tech. 46, 11566-11574.
24. Wang, H., Edwards, M.A., Falkinham, J.O., 3rd and Pruden, A. (2013) Probiotic approach to pathogen control in premise plumbing systems? A review. Enviro. Sci. Tech. 47, 10117-28.
25. Xi, C., Zhang, Y., Marrs, C.F., Ye, W., Simon, C., Foxman, B., and Niriagu, J. (2009) Prevalence of antibiotic resistance in drinking water treatment and distribution systems. Appl. Environ. Microbiol. 75(17), 5714-5718.