Fresh Water Habitat Pollution by Treated Sewage ...

Available online at www.sciencedirect.com

APCBEE Procedia 5 (2013) 363 – 367

ICESD 2013: January 19-20, Dubai, UAE

Fresh Water Habitat Pollution by Treated Sewage Effluent in Relation to Multiple-Antibiotic-Resistant Bacteria I. Y. Mahmouda , S. N. Al-Bahryb, S. K. Al-Musharafic a

Department of Biological Science and Chemistry, University of Nizwa, Nizwa, Oman Department of Biology, College of Science, Sultan Qaboos University, Muscat, Oman c Sur College of Applied Sciences, Sur, Oman

b

Abstract Oman is located in an arid zone with low rainfall and water demand is a major concern. Water desalination and wastewater recycling are alternative water sources. Since antibiotics become highly diluted in aquatic environment, their detection becomes difficult. Antibiotic resistant bacteria were used as bio-indicators of treated sewage effluent (TSE) contamination from industrial and residential sources. Access TSE was dumped directly into a valley which transformed into a small pond. TSE dumping point, pond water soil and snail samples were collected for analysis. Snail samples were the highest total count of heterotrophic bacteria compared to the soil and water samples. A total of 90 isolates belong to 8 genera were found. Most of the isolates were from snails and pond water. Resistance of the isolates was tested to 12 antibiotics. More than 50% were resistant to at least one antibiotic with maximum to 11 antibiotics. Escherichia spp and Enterobacter spp isolates from soil, snail and pond water samples had similar antibiograms. Most isolates were resistant to ampicillin followed by tetracycline. This study is important to evaluate the degree of pollution originated from TSE using antibiotic resistant bacteria as bioindicator of pollution.

©2013 2013The Published ElsevierbyB.V. Selection © Authors.by Published Elsevier B.V. and/or peer review under responsibility of Asia-Pacific Chemical, Environmental Engineering Society Selection andBiological peer review&under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society Keywords: Pollution; sewage effluent; antibiotic resistance; bioindicators

1. Introduction

Corresponding author. Tel.: +968-25446234; fax: +968-25443400 E-mail address: [email protected].

2212-6708 © 2013 The Authors. Published by Elsevier B.V. Selection and peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society doi:10.1016/j.apcbee.2013.05.062

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I. Y. Mahmoud et al. / APCBEE Procedia 5 (2013) 363 – 367

During the last four decades Oman has experienced a rapid development in all aspects of life including social, industrial and agricultural sectors which resulted for more demand for water consumption. In order to solve this problem and at the same time meeting the essential needs of the country, desalination projects and recycling of sewage water were implemented. Multiple resistant bacteria (MARBs) to commonly used antibiotics have been reported in Oman [1, 2, 3, 4]. MRBs from Treated sewage effluent (TSE) were also found to contaminate terrestrial and marine environments. Although pharmaceutical compounds, such as antibiotics, may exist in sewage, it becomes highly diluted and undetectable in TSE [1]. Consequently, resistant strains will be released to the environment through human activities. For example, the discharge of sewage effluent into the environment is considered to be one of the routes to transfer MARBs to soil and aquatic organisms. Isolation of MARBs from soil and aquatic organisms indicates pollution which may be a threat to the public health and environment. Therefore MARBs are used as bioindicators of contaminated effluents [2-4]. The aim of this study is to examine the man-made pond from TSE for the presence of MARBs. 2. Experimental procedures This study was conducted in Darseit, Oman near a major sewage water treatment plant (STP). Excess TSE was discharged forming pond water. Five TSE samples were taken from the damping point, while twenty samples, of each category: pond water, surrounding soil, and water were taken from different 4 sites within each category. A total of 65 samples (250 ml water samples and 250 gm for snails and surrounding soil) were collected according to APHA, AWWA and WEF procedure [5]. The samples were collected and immediately stored in a cool box. Total count of bacteria was made using membrane filtration technique. Opportunistic and microbial pathogens were isolated and identified biochemically. All bacterial isolates were tested for their resistance to 12 antibiotics: Amikacin (Ak), Ampicillin (Amp), Carbenicillin (Cn), Chloamphenicol (C), Gentamycin (Gm), Kanamycin (K), Minocylin (Min), Neomycin (N), Sulphamethoxazole (Smx), Streptomycin (S), Tetracyclin (Te), Tobramycin (Tob). An E. coli strain (ATCC 25922) and Pseudomonas aeruginosa strain (ATCC 27853) were used as controls to measure the inhibition zone diameter of the isolates according to the disk diffusion method [6]. 3. Results Total counts of heterotrophic bacteria from four sources revealed that snail samples have the highest count followed by pond water, soil and TSE. There was a significant difference in total count of heterotrophic bacteria in all samples (p < 0.05) (Fig 1). A total of 90 isolates belong to 8 genera were found. These include Escherichia, Salmonella, Proteus, Enterobacter, Pantoea and Citrobacter spp. Pseudomonas, and Aeromonas spp. Salmonella spp. Escherichia spp and Enterobacter spp had the highest frequencies of isolation. The lowest frequency of isolation were found in Psudomonas spp (Fig 2). Citrobacter spp, Pantoea spp and Pseudomonas spp, were absent from the TSE disposal point, while Pantoea spp were absent in soil and pond water samples. Enterobacter spp and Escherichia coli had the highest frequency of isolation in soil samples (Fig 3). Escherichia spp, Enterobacter spp, Citrobacter and Salmonella showed maximum resistance to antibiotics. Aeromonas was the lowest (Fig 4). Resistance to each antibiotic also varied among the isolates. Escherichia spp and Enterobacter spp isolated from soil, snail and pond water, had the same frequency of resistance to antibiotics. Resistant frequency of Aeromonas spp was 100% for Amp, S and Tob. While Pseudomonas spp was 100% resistant to Amp, Cn, C, Te. Citrobacter spp. Salmonella spp had the highest frequency to Amp (100%), Te (62.5%) and Min (60%). Among all antibiotics the highest frequency of resistance was Amp (83.3 %) followed by Cn (66.7 %). None of the isolates were resistant to Smx (Fig 5).


Fig. 1. Total heterotrophic bacterial count from the four sources.

Fig. 2. Percentage frequency of isolation.

Fig. 3. Percentage of bacterial genera from the four sources.

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Fig. 4. Number of antibiotic resistance relative to each isolate.

Fig. 5. Resistant percentage frequency to 12 antibiotics.

4. Discussion In this study, some of TSE samples exceeded the recommended wastewater standards [7]. The TSE disposal point was highly contaminated with coliform. Similar findings were reported by Bruni et al [8]. The presence of indicator-microorganisms in water is evidence that the water is polluted with faecal material from humans and other warm-blooded animals. In this study pathogenic and opportunistic, Salmonella, Enterobacter spp and Escherichia spp demonstrated the highest frequency of isolation in the samples with snails being the highest. The isolates were also resistant to antibiotics. Snails have proven to be very useful in detecting the changes in habitat and water quality because these organisms are filter feeders accumulating pollutants [9]. Almost all of the isolates in this study were resistant to Amp and Cn. Other investigators showed similar resistance to Amp from sewage isolates [1, 3, 4; 10]. Although similar bacteria which were isolated from environmental samples in Oman were resistant to Smx, none of the isolates were resistant to this antibiotic [1,


2, 3, 4]. Existence of MARBs in all samples including snails is a clear indication that environmental pollution is originated from contaminated effluents. Similar results have been reported by others using MARBs as bioindicators [1, 2, 3, 4]. Undoubtedly, the improper and unnecessary use of antimicrobial drugs in humans and animals has promoted the development of resistant strains that are now affecting the environment. It is clear that the opportunistic and pathogenic microbes which are resistant to antibiotics, and originated in the humans have already contaminated the environment and infected wild life causing serious public health concerns. References [1] Al-Bahry SN, Al-Zadjali MA, Mahmoud IY, Elshafie AE. Biomonitoring marine habitats in reference to antibiotic resistant bacteria and ampicillin resistance determinants from oviductal fluid of the nesting green sea turtle, Chelonia mydas. Chemosphere 2012;87:1308 1315. [2] Al-Bahry SN, Mahmoud IY, Al-Belushi KI, Elshafie AE, Al-Harthy A, Bakheit CK. Coastal sewage discharge and its impact on fish with reference to antibiotic resistant enteric bacteria and enteric pathogens as bio-indicators of pollution. Chemosphere, 2009;77: 1534-1539. [3] Al-Bahry SN, Mahmoud IY, Al-Zadjali M, Elshafie A, Al-Harthy A, Al-Alawi W. Antibiotic resistant bacteria as bio-indicator of polluted effluent in the green turtles, Chelonia mydas in Oman. Mar Env Res 2010;71:139-144. [4] Al-Bahry SN, Mahmoud IY, Al-Musharafi SK. Antibiotic resistant bacteria used as bioindicators of environmental pollution produced by tertiary treated sewage effluent. 11th International Conference on Modelling, Monitoring and Management of Water Pollution. New Forest, UK. www.witpress.com, ISSN 1743-3541 (on-line). WIT Transactions on Ecology and The Environment, Vol. 164, © 2012 WIT Press. doi:10.2495/WP120271. UK. Page 313-321. 2012. [5] APHA, AWWA, WEF . (Clescerl. L . S . , A . E Greenberg , and A. D Eaton (eds)). Standard Methods for the examination of water and wastewater. 20th ed. United book press. Washington. USA; 1998. [6] NCCLS (National Committe for Clinical Laboratory Standard). Performance Standards for Antimicrobial Disk Susceptibility Tests. Sixth ed. Approved Standard M2-A6. National Committee for Clinical Laboratory Standards, Wayne, PA. 1997. [7] MRMWR (Ministry of Regional Municipalities and Water Recourses). Regulation for wastewater reuse and discharge and Ministry of Regional Municipalities and Water Recourses; 1998. [8] Bruni V, Maugeri, TL, and Monticelli L. Faecal pollution indicators in the Terra Nova Bay (Rosa Sea, Antaractica). Marine Pollut. Bull 1997;34: 908-912. [9] Smith R, Jeffree R, John J, Clayton, P. Review of methods for water quality assessment of temporary stream and lake systems. Australian Centre for Mining Environmental Research. Chemosphere 2004;55:227-255. [10] Lee HK, Kang DR. Species identification of the genus Aeromonas isolated from freshwater fish culture-ponds and seawater in Korea and their antibiotic resistance patterns. J Korean Soc Microbiol 2000;34: 393 400.

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