DISTRIBUTION OF ESCHERICHIA COLI RESISTANT FLUOROQUINOLONES, SULFAMETHOXAZOLE AND TETRACYCLINE IN THE CHAO PHRAYA RIVER AND ITS TRIBUTARIES Honda R1, Watanabe T2, Masago Y3, Chulasak R4, Tanong K5, Tushara CGG1, Sawaittayothin V4, Chiemchaisri C5, Furumai H6. 1Environmental Science Center, The University of Tokyo, Japan 2Department of Food, Life and Environmental Sciences, Faculty of Agriculture, Yamagata University, Japan 3Department of Civil and Environmental Engineering, Tohoku University, Japan 4Environment Research and Training Center, Department of Environmental Quality Promotion, Thailand 5Department of Environmental Engineering, Faculty of Engineering, Kasetsart University, Thailand 6Research Center for Water Environment Technology, School of Engineering, The University of Tokyo, Japan Aims Wide distribution of pharmaceutical and personal care products (PPCPs) is being reported in urban water systems in the world. Increase in consumption of PPCPs expectedly affects water environment in Thailand as well. One of the subsequent concerns is occurrence of antibiotic-resistant bacteria in water environment. However, there was limited number of studies on antibiotic-resistant bacteria linked with antibiotic concentrations in water environment. Moreover, possibility in acquisition of antibiotic resistance in water environment is still veiled. In this study, we surveyed spatial distribution of E. coli resistant to fluoroquinolones (levofloxacin, norfloxacin and ofloxacin), sulfamethoxazole, tetracycline, and concentrations of the antibiotics in the Chao Phraya River and its tributaries, in order to clarify the origin of antibiotic resistances and their spreads in water environment. Methods Water samples were taken at 48 sites from the Chao Phraya River and its four tributaries of the Ping River, the Wang River, the Yom River and the Nan River in Thailand. E. coli strains in selected 32 samples were isolated by Chromocult Coliform Agar. The isolates were cultivated in ST media overnight and subsequently tested on sensitivity to 5 antibiotics: sulfamethoxazole, tetracycline, levofloxacin, norfloxacin and ofloxacin by Kirby-Bauer (KB) disk diffusion test. Sulfamethoxazole, tetracycline and two fluoroquinolone species: ciprofloxacin and norfloxacin in the 48 river water samples were extracted by SPE cartridges and eluted with methanol before analysis by LC-MS/MS. Results and Conclusions Spatial distribution of antibiotic-resistant E. coli isolates At 20 of 32 sites in the Chao Phraya River and tributaries, more than half of E. coli isolates were resistant to at least one antibiotics tested (Figure 1). No significant trend was found on ratios of resistant isolates from upstream to downstream. However, populated area possibly contributed to increase in ratios of E.coli resistant to multiple antibiotics. Ratios of isolates with resistance to more than 2 antibiotics were higher in the Chao Phraya River and at one site close to Chiang Mai city than other places. Especially, ratios of isolates resistant to fluoroquinolones were high at sampling sites close to populated area. Of total 396 E. coli isolates, 56% were resistant to at least one antibiotic. Only 38% of the isolates were sensitive to all tested antibiotics. Multiple resistances were found in 43% of the isolates, and 12% of the 396 isolates had resistance to all 5 antibiotics tested. Ratios of isolates with resistance to
sulfamethoxazole and tetracycline were higher than those to levofloxacin, norfloxacin and ofloxacin (Table 1). No significant correlation was found between concentrations of an antibiotic and ratios of isolates resistant to the antibiotic. Ciprolfoxacin and sulfamethoxazole were distributed extensively in the Chao Phraya River and tributaries. Norfloxacin was detected only at two sites in the Ping River. Tetracycline was not detected at any sites. Resistances to fluoroquinones Resistance to the three fluoroquinolone antibiotics: levofloxacin, norfloxacin and ofloxacin had significant correlation with over 0.87 of the correlation coefficients among each. This indicates a large portion of resistant E. coli to these antibiotics had cross-resistance to fluoroquinolone group. Resistant isolates to norfloxacin were extensively found at the sites except in the Nan River, while norfloxacin was detected only at two sites in the Ping River. According to National Antimicrobial Resistance Surveillance Center in Thailand, 51% of E. coli isolates in hospitals were resistant to norfloxacin. Therefore, one of the possible sources of the resistant isolates to norfloxacin in our survey was hospitals. Resistance to levofloxacin and ofloxacin was probably acquired as cross-resistance to norfloxacin. Another possible reason of the occurrence is cross-resistance to ciprofloxacin, another specie of fluoroquinolone. Ciprofloxacin was found at high concentrations in water samples from the upstream part of the Chao Phraya River and its tributaries. If the isolates have resistance to ciprofloxacin, they presumably have cross-resistance to norfloxacin, levofloxacin and ofloxacin. Resistances to sulfamethoxazole and tetracycline Resistant isolates to sulfamethoxazole and tetracycline were widely distributed in the target rivers. Meanwhile, tetracycline was not detected at any site while sulfamethoxazole was detected at many sites in the Chao Phraya River. However, resistance to sulfamethoxazole and tetracycline had moderate correlation with 0.72 of the correlation coefficient. This is not probably due to cross- resistance since the resistant mechanism of these two antibiotics differs. However, there was statistically significant dependence on resistance to them. This implies the presence of conditions to promote acquiring multiple resistances to the two antibiotics, such as transfer of a plasmid or transposon with resistant genes to both tetracycline and the sulfa drug. The river sediments might function as a reservoir of such genes. Tetracycline resistance genes, tet(M), tet(S) and tet(W), were reported to be widely distributed in the Mekong River (Kobayashi et al. 2007; Suzuki et al. 2008). Wide distribution of resistant isolates to the two antibiotics suggests the spread of the multiple- resistance gene in the rivers. References Kobayashi, T., Suehiro, F., Cach Tuyen, B., and Suzuki, S. (2007) Distribution and diversity of
tetracycline resistance genes encoding ribosomal protection proteins in Mekong river sediments in Vietnam. FEMS microbiology ecology, 59(3), 729–737.
Suzuki, S., Kobayashi, T., Suehiro, F., Tuyen, B. C., and Tana, T. S. (2008) High Occurrence Rate of
Tetracycline (TC)-Resistant Bacteria and TC Resistance Genes Relates to Microbial Diversity in Sediment of Mekong River Main Waterway. Microbes and Environments, 23(2), 149-152.
Figure 1 Ratios of antibiotic-resistant E. coli isolates in the Chao Phraya River and tributaries. Table 1 Antibiotic resistance of E. coli isolates from the Chao Phraya River and tributaries (n=396).
* LVX=levofloxacin, NFX=norfloxacin, OFX=ofloxacin, ST=sulfamethoxazole, TC=tetracycline.
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