Science of the Total Environment 409 (2011) 2894–2901 Contents lists available at ScienceDirect Science of the Total Environment j o u r n a l h o m e p a g e : w w w e l s ev i e r c o m / l o c a t e / s c i t o t e n v Antibiotic contamination and occurrence of antibiotic-resistant bacteria in aquatic environments of northern Vietnam Phan Thi Phuong Hoa a, Satoshi Managaki b, Norihide Nakada b, Hideshige Takada b, Akiko Shimizu b, Duong Hong Anh c, Pham Hung Viet c, Satoru Suzuki a,⁎ a b c Center for Marine Environmental Studies (CMES), Ehime University, Matsuyama 790-8577, Japan Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Fuchu 183-8509, Japan Research Center for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Hanoi, Vietnam a r t i c l e i n f o Article history: Received September 2010 Received in revised form April 2011 Accepted 13 April 2011 Keywords: Sulfonamide Resistant bacteria Vietnam Acinetobacter Sul Animal farm a b s t r a c t The ubiquitous application and release of antibiotics to the environment can result in bacterial antibiotic resistance, which in turn can be a serious risk to humans and other animals Southeast Asian countries commonly apply an integrated recycling farm system called VAC (Vegetable, Aquaculture and Caged animal) In the VAC environment, antibiotics are released from animal and human origins, which would cause antibiotic-resistant bacteria (ARB) This study evaluated occurrence of ARB in the VAC environment in northern Vietnam, with quantitative analysis of antibiotic pollution We found that sulfonamides were commonly detected at all sites In dry season, while sulfamethazine was a major contaminant in pig farm pond (475–6662 ng/l) and less common in city canal and aquaculture sites, sulfamethoxazole was a major one in city canal (612–4330 ng/l) Erythromycin (154–2246 ng/l) and clarithromycin (2.8–778 ng/ml) were the common macrolides in city canal, but very low concentrations in pig farm pond and aquaculture sites High frequencies of sulfamethoxazole-resistant bacteria (2.14–94.44%) were found whereas the occurrence rates of erythromycin-resistant bacteria were lower (b 0.01–38.8%) A positive correlation was found between sulfamethoxazole concentration and occurrence of sulfamethoxazole-resistant bacteria in dry season The sulfamethoxazole-resistant isolates were found to belong to 25 genera Acinetobacter and Aeromonas were the major genera Twenty three of 25 genera contained sul genes This study showed specific contamination patterns in city and VAC environments and concluded that ARB occurred not only within contaminated sites but also those less contaminated Various species can obtain resistance in VAC environment, which would be reservoir of drug resistance genes Occurrence of ARB is suggested to relate with rainfall condition and horizontal gene transfer in diverse microbial community © 2011 Elsevier B.V All rights reserved Introduction It is known that antibiotics cause antibiotic-resistant bacteria (ARB) in hospital-inquired infection In recent years, antibiotics contamination is recognized as an emerging environmental pollution in aquatic environments, because of their potential adverse effects on the ecosystem and human health (Huang et al., 2001; Kümmerer, 2009) Majority of antibiotics used for human, plants and animals are excreted into the environment as intact or decomposed form via various pathways, including wastewater effluent discharge, runoff from land to which agricultural or human waste has been applied, and leaching (Zhang et al., 2009) Antibiotic residues in the environment impose selective pressure on bacterial populations, which results prevalence of resistant bacteria even at sub-inhibitory low concen- ⁎ Corresponding author Tel./fax: + 81 89 927 8552 E-mail address: ssuzuki@ehime-u.ac.jp (S Suzuki) 0048-9697/$ – see front matter © 2011 Elsevier B.V All rights reserved doi:10.1016/j.scitotenv.2011.04.030 trations Other pollutants are also known as selective agents (Stepanauskas et al., 2005, 2006) Additionally, the raw wastewater contaminated by antibiotics released into aquatic environments often carries human and animal pathogenic bacteria, in addition to commensal bacteria, and many of these organisms harbor antibiotic-resistance genes Therefore, water constitutes a way of dissemination of not only antibiotic-resistant bacteria, but also the resistance genes, which genetically change in natural bacterial ecosystems (Baquero et al., 2008; Rosenblatt-Farrell, 2009) The ARB has been found in various aquatic environments (Kümmerer, 2004; Kim and Aga, 2007; Schluter et al., 2007; Watkinson et al., 2007;Caplin et al., 2008; Vanneste et al., 2008) In particular, our previous studies showed that aquatic environment is potential reservoirs of ARB (Nonaka et al., 2000, 2007; Kim et al., 2003, 2004; Hoa et al., 2008) even in pristine conditions (Kobayashi et al., 2007; Rahman et al., 2008) On the other hand, a variety of antibiotics have been detected in the aquatic environments (Hirsch et al., 1999; Göbel et al., 2005; Zhang et al., 2009), from ng/l to μg/l levels, which are lower than P.T.P Hoa et al / Science of the Total Environment 409 (2011) 2894–2901 therapeutic levels (Göbel et al., 2005; Managaki et al., 2007, Zhang et al., 2009) In some cases, high concentration with mg/l order was found (Le and Munekage, 2004; Le et al., 2005) At the sub-therapeutic concentrations of antibiotics detected in the aquatic environments, the question is whether antibiotics could have an impact on bacterial populations (Kümmerer, 2009) Very few studies have investigated the relationship between antibiotic contamination and antibiotic resistance in aquatic environments relating to human and agricultural activities Furthermore the diversity of ARB within integrated recycling farm VAC (Vegetable, Aquaculture and Caged animal) systems is virtually unknown From these background, we hypothesized that Asian integrated agriculture environment should be polluted with various antibiotics derived from animal and human origins, which selects ARB in the environment To clarify this, we conducted monitoring of concentrations of residual antibiotics and ARB in the VAC environments Antibiotic residues were often detected in the human-impacted aquatic environments of Southeast Asian countries and China (Le and Munekage, 2004; Richardson et al., 2005; Managaki et al., 2007) In Vietnam, agriculture and aquaculture are the major economic activities, and excessive and unregulated use of antibiotics was commonly found in human medicine, management of livestock, and aquaculture (Le et al., 2005; Managaki et al., 2007; Duong et al., 2008) The Red River delta of northern Vietnam is an appropriate study site because of following conditions This area is one of the largest deltas in Southeast Asia, including provinces along with municipalities, the capital city of Hanoi, and the main seaport of Haiphong The delta is an agriculturally rich area, which is densely populated by 11,000,000 people (Berg et al., 2001) The heavy and unregulated use of antibiotics along with the discharge of untreated wastewater into the aquatic environments might cause significant contamination of both antibiotic residues and ARB On the basis of the information obtained during our pre-study onsite interview-based survey, we identified sulfonamides, trimethoprim, and macrolides as the target antibiotics In the present study, we first characterized the pattern of contamination by antibiotics and ABR in the rainy and dry seasons in the Red River delta area To better understand whether the antibiotic residues in the aquatic environment is an important source in selecting and creating an increasingly resistant bacteria in the environments, the statistical correlation between the concentration of antibiotic residues and ARB occurrence was analyzed A part of sulfonamide-resistant (SR) bacteria was isolated and classified in January when the relationship between sulfonamide and occurrence of SR bacteria was possibly observed The possession of sul genes was also monitored The variation and distribution of sul genes in manure were examined in Europe, and the molecular information for a method of detection is available (Heuer et al., 2009) Therefore sul genes were the priority to survey in this study Materials and methods 2.1 Sampling area and procedure Specific integrated aquaculture-agriculture system is major in Vietnam, at which freshwater fishponds directly receive excreta from intensive pig farms (termed “pig farm/fish ponds”) (Hoa et al., 2008) Sampling was performed at 10 sites in the Red River delta of northern Vietnam, including sites of a city canal in Hanoi (HNC-1–3), sites at pig farm/fish ponds in Hatay (HNP-1–3), and sites at coastal shrimp ponds of Haiphong (HNAQ-1–4) (Fig 1) The HNC sites were upper-, middle- and down-stream of the main canal of Hanoi City, and the HNP and HNAQ sites were representative farms of animal and shrimp culture The sampling sites were at least km away from each other These habitats were selected because they were representative of the aquatic environments exposed to antibiotics The city canal 2895 in Hanoi directly receives various types of untreated municipal wastewater, such as wastewater from households and hospital (Duong et al., 2008) According to the onsite interviews conducted during our field trips in both sampling periods, antimicrobials were rarely used for the freshwater fishponds in Hatay province, but very frequently used in the pig farms The categories of the drugs and their amounts varied widely among the individual pig farms We chose coastal shrimp ponds in Haiphong because this province is one of the centers of fish and shrimp cultivation, which is an industry that supplies seafood to domestic and foreign markets (Tran et al., 2006) The use of antibiotics in the Vietnamese shrimp industry has been reported by Le and Munekage (2004) and Le et al (2005) The investigated shrimp ponds were located near the estuary mouths, and the water level of the ponds was somewhat influenced by tidal movement; the water level in the ponds was adjusted by using pumps The collection of water samples was repeated twice, once in January (dry season) and then in July (rainy season) in 2007 A detailed description of the characteristics of water samples and the climate conditions of the sampling locations is provided in Tables S1 and The sampling procedure was identical to that described in our previous studies (Managaki et al., 2007; Hoa et al., 2008) Then, the samples were preserved on ice and analyzed in the laboratory within 3–6 h after sampling 2.2 Analysis of antibiotics The concentrations of sulfonamides, trimethoprim, and macrolides were determined by tandem mass spectrometry equipped with highperformance liquid chromatography (LC–MS/MS), which was performed after solid-phase extraction The detailed procedure has been described by Managaki et al (2007), and the outline of the procedure is as follows We extracted 50 ml (city canal samples), 20 ml (pig farm/fish pond samples), and 250 ml (shrimp pond samples) on 6-ml Oasis HLB sorbent cartridges (200 mg; Waters) (flow rate, b5 ml/min; pH, 4) After extraction, the cartridges were stored at − 30 °C, transported to the laboratory in Tokyo, and defrosted before elution of the antibiotics The cartridges were washed with ml of water– methanol (75:25) and dried in a nitrogen flow for 30 The analytes were eluted with × 1.5 ml of methanol–ethyl acetate (1:1) and × 1.5 ml of methanol containing 1% (v/v) ammonia A fixed amount 25 ng (500 pg/μl × 50 μl) of each antibiotic surrogate standard (sulfamethoxazole-d4, clarithromycin-d3, and roxithromycin-d9) was spiked to the sample extracts The extracts were evaporated to dryness using a rotary dryer and dissolved in ml of water–methanol (1:1), and the antibiotic contents were determined by separating the extracts by LC–MS/MS HPLC analyses were performed on a Hewlett-Packard G1310 The antibiotics were separated on a reverse-phase column (YMC Pro C18; μm, 150 mm × mm) that was operated at 30 °C at a flow rate of 0.15 ml/min The mobile-phase solvents were water-acidified with 1% (v/v) formic acid (eluent A) and methanol-acidified with 1% (v/v) formic acid (eluent B) to pH 2.5 by using a gradient program The antibiotics were detected using a triple-quadrupole mass spectrometer (TSQ Quantum 7000; Thermo Finnigan, Japan) equipped with electrospray ionization The analyses were performed in the positiveion mode The detection was performed in the selected-reactionmonitoring mode (SRM) using the most intense and specific fragment ions To compensate for matrix effects and experimental losses during sample treatment, we corrected the concentrations of the target antibiotics with the recovery values of the corresponding surrogates spiked in the same extracts The following compounds were used as recovery surrogate standards: sulfamethoxazole-d4 for all sulfonamides and trimethoprim; clarithromycin-d3 for clarithromycin, erythromycin-H2O, and azithromycin; and roxithromycin-d9 for roxithromycin The analytical precision was examined by performing 2896 P.T.P Hoa et al / Science of the Total Environment 409 (2011) 2894–2901 106E Red River Red River China 107E 21N Red River Delta Hanoi Vietnam HNC-3 HNC-2 HNC-1 Hanoi HNAQ-1 HNAQ-4 HNAQ-3 HNAQ-2 Laos HNP HNP-3 Haiphong HNP-2 HNP-1 20N Thailand Hue Hatay 25 50 km Fig Study area and sampling sites replicate analyses of 250 ml of water from the Tamagawa River The relative standard deviations of the target antibiotics ranged from 2% to 11% For the recovery studies, 25 ng of each antibiotic (500 pg/μl in 50 μl) and the recovery surrogate were spiked into 250 ml of water from the Tamagawa River Recoveries of the spiked standards ranged from 72% to 93% (SPY: 87% ± 6%, STZ: 91% ± 3%, SMR: 86% ± 3%, ; SMT: 84% ± 3%; SMZ: 86% ± 3%; SMX: 85% ± 6%; SDX: 86% ±2%; TRI: 72% ± 11%; AZI: 90% ±4%; ERY: 90% ±2%; CLA: 93% ± 6%; ROX: 93% ± 3%) Most of the target compounds showed recoveries of over 84%, with the exception of trimethoprim with 72% recovery If we consider such a trace amount (e.g., low ng/L) of the target compound from a complex environmental matrix, recovery of over 70% is normally acceptable for this type of monitoring study The limits of quantification were defined as 10 times the procedural blank value or 10 times the noise level of the baseline in the chromatograms if there were no peaks in the procedural blank analysis Limits of quantifications ranged from 0.1 ng/l to 1.2 ng/l Travel contamination and laboratory contamination were determined by analyzing the travel blanks and procedural blanks after extraction Calibration curves for each compound are shown in Fig S1 2.3 Enumeration of antibiotic-resistant bacteria (ARB) The colony forming unit (CFU) was measured by using the plate spreading method described in our previous study (Hoa et al., 2008) Briefly, m1 of each water sample was suspended in ml of phosphatebuffered saline (PBS), and serial 10-fold dilutions were prepared Nutrient broth (Difco, Detroit, MD, USA) with 1.5% agar was used for fresh water samples collected from HNPs and HNCs, and marine broth (Difco, Detroit, MD, USA) with 1.5% agar was used for the brackish water samples collected from HNAQs The total viable count was obtained from the antibiotic-free media, and the ARB was counted on media supplemented with 60 μg/ml of each antibiotic: sulfamethoxazole (SMX) and erythromycin (ERY) (both from Sigma-Aldrich, St Louis, MO, USA) These two compounds are appropriate representatives of sulfonamide and macrolide, which are commonly used drugs in Indochina and are well studied (Managaki et al., 2007) In this study, ‘resistance’ was determined as growth after days at 30 °C in the media containing antibiotic at concentrations of 60 μg/ml and additionally our previous study showed that 92%, 72% and 43% of SMX-resistant (SMXr, for 60 μg/ml) isolates from HNPs, HNCs and HNAQs, respectively contained the sul genes (Hoa et al., 2008) The criterion for indicating resistance has been designated to be 32–60 μg/ml (Hoa et al., 2008; Toleman et al., 2007), and this concentration is appropriate for comparison to other drugs (Nonaka et al., 2007) The bacterial count was determined by plating 0.1 ml of 10-fold dilution (10−3 and 10−4 dilution for most cases of total viable bacteria, and 10−1 and 10−2 dilution for the selection of ARB) CFUs were counted in double plates 2.4 Classification of SMXr isolates The bacterial isolates that were randomly picked from the duplicate plates in January samples (Hoa et al., 2008) were classified to the genus level by 16S rRNA gene sequencing Addition to 43 strains carrying sul genes reported in our previous study (Hoa et al., 2008), 78 SMXr strains were newly identified in this study The primers (F984, R1378) and polymerase chain reaction (PCR) conditions were the same as described by Heuer et al (1997) The 394-bp PCR product was purified and sequenced using the Big Dye terminator version 3.1 cycle reaction kit (Applied Biosystems, Foster City, CA, USA) on a 3100 ABI Prism DNA sequencer (Applied Biosystems) The DNA sequences obtained were analyzed by the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) website When the sequence of amplified PCR product showed similarity ≥ 95%, the isolate was recognized as the closest genus 2.5 Data analysis The correlation between the antibiotic concentration and incidence of ARB was calculated by Spearman correlation coefficients Continuous variables were compared by the t-test A p value b 0.05 was considered statistically significant Beside that linear correlation was used for calculation the correlation between SMX and trimethoprim concentrations detected P.T.P Hoa et al / Science of the Total Environment 409 (2011) 2894–2901 Results and discussion 3.1 Antibiotic contamination status We quantified the contamination of the antibiotic groups, sulfonamides, trimethoprim, and macrolides at 10 sites in different seasons (Table 1) Among sulfonamides, SMX and sulfamethazine were the major compounds detected Trimethoprim was also detected with same manner of sulfonamide Concentrations of SMX and trimethoprim were positively correlated (R2 = 0.758, p b 0.001), which is caused by combination use of sulfonamide with trimethoprim (Huovinen et al., 1986 Houvinen et al., 1995) Present study showed that SMX is used with trimethoprim in this area especially in human medicine, because of high concentration in city canal samples Positive correlation was not found between sulfamethazine and trimethoprim, suggesting that the combination of the two compounds in pig farming is not frequent (Table 1) Other four sulfonamides (sulfathiazole, sulfamerazine, sulfamethizol, and sulfadimethoxine) were not detected at any investigated sites, which have a similar tendency to our previous study conducted at the Mekong River delta, Vietnam and the Tamagawa River, Japan (Managaki et al., 2007) When the contamination was compared between rainy and dry seasons, clear difference was not observed (p = 0.96) despite that the precipitation in July (rainy season: 286 mm of rainfall/month) should dilute drugs much more than January (dry season: mm of rainfall/ month) This may be due to the intensive use of drugs in the rainy season, which could compensate for the large dilution effect by rain The concentrations of sulfonamides and trimethoprim residues in the city canal and the pig farm/fish ponds (16.1–4330.0 ng/l and 16.8– 6662.0 ng/l, respectively) were, on average, approximately 7-fold higher than those in the shrimp ponds (2.38–914 ng/l) (Table 1) The high concentrations of SMX, sufamethazine, and trimethoprim detected in the water samples of the city canal and the pig farm/fish ponds could be due to the unregulated consumption of these compounds by humans (Duong et al., 2008) and livestock (Managaki et al., 2007) The maximum detected concentration of sulfamethoxazole in the city canal (4330 ng/l) was higher than that in municipal wastewater in Switzerland (1900 ng/l) (Göbel et al., 2005), the Mekong River delta (360 ng/l) of southern Vietnam, and the Tamagawa River of Japan (132 ng/l) (Managaki et al., 2007), but lower than reported in the German study (9000 ng/l) conducted at 10 years ago (Hartig et al., 1999) In the present study, while the maximum concentration of SMX was detected in municipal raw wastewater, the maximum concentration of sulfamethazine was detected in the pig farm/fish ponds (Table 1) The contamination profile showed that sulfamethazine was a major contaminant in the pig farm/fish ponds, while SMX was a major sulfonamide in the city canal and the coastal shrimp ponds; these findings were repeatedly observed during the sampling trials, i.e., during both rainy and dry seasons (Table1) These findings suggested that sulfamethazine was intensively used in pig farms, and SMX was mainly used in human medicine A previous study also detected high concentrations of sulfamethazine (18,512–19,153 ng /l) in the Mekong River delta of Vietnam and suggested that this sulfamethazine had a livestock origin (Managaki et al., 2007) The studies by Le and Munekage (2004), Le et al (2005) and Managaki et al (2007) also suggested that SMX was primarily used for human medication and coastal aquaculture, but not for livestock production in Vietnam The data obtained in the present study showed that contamination by sulfonamides and trimethoprim did not occur frequently in the shrimp ponds Low concentrations of these drugs (0–914 ng/l) were detected in the shrimp ponds; however, the profile of the relative composition of sulfonamides suggested that the sulfonamides and trimethoprim detected in this study were derived from humans and livestock In terms of the number of compounds and residue concentrations, we found that the macrolide contamination in municipal raw 2897 wastewater was more severe than that of agricultural wastewater in both pig farm/fish ponds and the coastal shrimp ponds All the investigated macrolides were detected at relatively high concentrations in the city canal (azithromycin, 0–90.8 ng/l; ERY, 61.1–2246.0 ng/l; clarithromycin, 1.60–778.0 ng/l; and roxithromycin, 0–125 ng/l), while concentrations in the pig farm/fish ponds (ERY, 0–63.9 ng/l and clarithromycin, 0–0.40 ng/l) and the shrimp ponds (ERY, 0–0.28 ng/l) were very low (Table 1) Erythromycin and clarithromycin were abundant in the city canal and the pig farm/fish ponds, where the highest erythromycin concentration (2246 ng/l from city canal) was approximately 55-fold higher than that from the Mekong River delta (41 ng/l) (Managaki et al., 2007), and 3-fold higher than that from wastewater in Hong Kong (810 ng/l) (Gulkowska et al., 2008) The findings in this study suggested that human medication was the primary source of macrolide contamination in this area As a conclusion, the contamination data showed specific contamination patterns in the city canal, pig farm and aquaculture sites 3.2 Occurrence of ARB We enumerated SMX-resistant (SMXr) and ERY-resistant (ERYr) bacteria from the same samples that were evaluated for antibiotic concentrations Overall, the occurrence rates of SMXr bacteria were generally higher than those of ERYr bacteria at any site across the sampling times (Table 2), which was similar tendency to drug contaminations, that is, SMX was detected at higher concentration than ERY at all sites The occurrence rates of SMXr and ERYr bacteria in the city canal in January were higher than those in July, although other sites did not show differences between January and July (Table 2) This difference in city canal can be attributed to the high rainfall in July and the frequent wide-range floods during Hanoi's rainy season (Table S2) Antibiotic contamination was almost the same in both seasons as we mentioned above; however, there is a possibility that high rainfall in July disturbs microbial ecosystem in city canal, which could interfere the occurrence of ARB On the other hand, the water level was relatively lower and water exchange should not be frequent in January In intensive agricultural systems like the pig farm/fish ponds and the coastal shrimp ponds, the water level was less affected by rainfall because these ponds function as water storage and are not subject to frequent exchange This condition would facilitate adequate time to develop resistance by promoting cell-to-cell horizontal gene transfer The exposure time between bacteria and antibiotics in July might be as equal as that in January; and therefore their occurrence rates were nearly stable during the two seasons It was found in this study that occurrence of ARB is depending on rainfall condition in city canal, but the ARB are reserved through the year in pig farm and aquaculture sites without correlation to drug contamination The relationship of contamination and ARB will be discussed further below 3.3 Relationship between antibiotic concentration and occurrence of ARB Relationship between the use of antibiotic and occurrence of ARB is complicated A number of studies have shown a positive correlation between the use of antibiotics in humans and the development of antibiotic resistance in pathogenic bacteria (e.g β-lactam, aminoglycosides, fluoroquinolones, macrolides, reviewed in Cristino, 1999) In contrast, a number of studies have shown that heavy use of antibiotics did not necessarily accelerate the prevalence of resistance (Gaynes, 1997; Cristino, 1999; Kahlmeter et al., 2003; Le et al., 2005) In many cases, the association between these factors was not established because of various contributing factors, including cross-transmission, inter-hospital transfer of resistance, community contribution to resistance, or a complex relationship between resistance and the use of a variety of antibiotics (Gaynes, 1997; Cristino, 1999) On the other hand, some studies have shown that the consumption of 2898 Table Concentrations of sulfonamides, trimethoprim and macrolides detected from water samples Antibiotic concentrationa (ng/l) Sulfapyridine Sulfamethoxazole Sulfamethazine Sulfathiazol Sulfamerazine Sulfamethizol Sulfadimethoxine Trimethoprim Azithromycin Erythromycin Clarithromycin Roxithromycin A January HNC-1 HNC-2 HNC-3 HNP-1 HNP-2 HNP-3 HNAQ-1 HNAQ-2 HNAQ-3 HNAQ-4 LOQ n.d 21.50 57.50 n.d n.d n.d n.d n.d n.d n.d 7.00 612.00 2982.00 4330.00 422.00 625.00 328.00 13.40 n.d 2.38 n.d 0.02 16.10 47.90 66.20 6662.00 475.00 2501.00 n.d n.d n.d n.d 0.70 n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d 16.00 n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d 16.00 n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d 0.40 n.a n.a n.a n.a n.a n.a n.a n.a n.a n.a 0.08 23.00 222.00 1808.00 16.80 34.60 22.30 n.d n.d n.d n.d 0.10 n.d n.d 90.80 n.d n.d n.d n.d n.d n.d n.d 7.00 154.00 2246.00 2191.00 58.90 63.90 62.80 n.d n.d n.d n.d 1.00 2.80 778.00 674.00 0.20 0.12 0.40 n.d n.d n.d n.d 0.20 0.72 103.00 125.00 n.d n.d n.d n.d n.d n.d n.d 0.07 B July HNC-1 HNC-2 HNC-3 HNP-1 HNP-2 HNP-3 HNAQ-1 HNAQ-2 HNAQ-3 HNAQ-4 LOQ n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d 3.00 818.00 3847.00 3170.00 326.00 68.20 69.90 10.80 n.d 914.00 n.d 2.00 n.d 44.10 46.20 851.00 658.00 6.78 n.d n.d n.d n.d 0.30 n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d 7.00 n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d 5.00 n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d 8.00 n.a n.a n.a n.a n.a n.d n.a n.a n.a n.a 0.3 91.40 730.00 726.00 26.40 26.00 n.a n.d n.d 85.00 n.d 0.02 n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d 0.02 61.10 734.00 642.00 12.80 0.59 n.d 0.28 n.d n.d n.d 0.03 1.60 309.00 292.00 n.d 0.01 n.d n.d n.d n.d n.d 0.001 n.d 77.90 726.00 n.d n.d n.d n.d n.d n.d n.d 0.001 LOQ: limit of quantification; n.d., not detected; n.a., not available due to overlapped interfering peak a t-test (p N 0.05) showed no significant difference between antibiotic concentration detected at the campaigns, except for sulfamethazine concentration detected in HNPs P.T.P Hoa et al / Science of the Total Environment 409 (2011) 2894–2901 Site P.T.P Hoa et al / Science of the Total Environment 409 (2011) 2894–2901 2899 Table Antibiotic-resistant bacteria detected from water samples Site HNC-1 HNC-2 HNC-3 HNP-1 HNP-2 HNP-3 HNAQ-1 HNAQ-2 HNAQ-3 HNAQ-4 Total viable count Sulfamethoxazole-resistant bacteria Erythromycin-resistant bacteria CFU/ml CFU/ml (%) CFU/ml (%) Jan Jul Jan 1.00 × 106 4.80 × 106 1.80 × 106 1.80 × 105 3.40 × 104 1.60 × 105 1.70 × 104 1.90 × 104 5.14 × 104 2.90 × 104 1.50 × 106 2.05 × 106 2.44 × 106 7.40 × 105 No data 9.00 × 105 3.40 × 105 3.65 × 105 7.60 × 105 4.65 × 105 5.00 × 105 2.50 × 106 1.70 × 106 1.00 × 104 3.88 × 103 5.60 × 103 2.30 × 103 1.04 × 104 1.10 × 103 2.40 × 103 Jul (50.00) (52.08) (94.44) (5.55) (11.41) (3.50) (13.52) (54.70) (2.14) (8.27) Jan 9.14 × 104 1.31 × 105 8.92 × 104 7.10 × 104 9.62 × 103 2.83 × 104 6.73 × 104 5.16 × 104 6.59 × 104 7.97 × 104 (6.09) (6.39) (3.65) (9.59) (No data) (3.14) (19.79) (14.1) (8.7) (17.1) Jul No data 5.00 × 105 7.00 × 105 7.00 × 103 2.98 × 103 5.51 × 102 1.00 × 102 6.00 × 102 6.00 × 101 2.00 × 101 (No data) (10.41) (38.80) (3.88) (8.76) (0.34) (0.58) (0.31) (0.11) (0.06) 2.80 × 104 9.40 × 104 6.05 × 104 5.81 × 104 4.76 × 103 1.80 × 103 2.50 × 101 5.00 × 101 6.00 × 101 3.00 × 101 (1.86) (4.58) (2.47) (7.85) (No data) (0.20) (b0.01) (b0.01) (b0.01) (b0.01) No data is due to laboratory accident Each count is an average of duplicate counts t-test for the number of antibiotic-resistant bacteria (January N July) in HNCs is significant (p b 0.05), but not in HNAQs and HNPs (p N 0.05) that not only contamination of antibiotics but also other selective pressures act as inducing factors of ARB in aquatic environments, although further studies are needed to confirm these possibilities 3.4 Diversity of SMXr isolates and possession of sul genes SMXr bacteria (%) We examined the species composition of the 121 isolates sampled in January whose SMX resistance possibly correlated with SMX contamination These isolates were identified and classified into 25 different genera (Table 3) Among the identified SMXr bacteria, Acinetobacter was the most abundant (24%), followed by Aeromonas (19.8%), Bacillus (13.2%) and Pseudoalteromonas (10%); the other bacterial genera occupied small fraction (less than 10%) of the total isolates Acinetobacter was major in pig farm/fish pond, whereas Aeromonas was mainly detected in city canal Shrimp pond showed A (Jan) 100 90 80 70 60 50 40 30 20 10 Rs=0.803 10 100 1000 10000 1000 10000 SMX concentration (ng/L) SMXr bacteria (%) antibiotics in animal husbandry can also play a major role in the selection and dissemination of ARB in the environment For example, Angulo et al (2004) and Asai et al (2005) observed strong positive correlations between the usage of veterinary therapeutic antibiotics and antibiotic resistance in Escherichia coli isolates obtained from the feces of food-producing animals The findings by these studies raise great concerns regarding the long-term consequences of antibiotic use in agricultural ecosystems Furthermore, agricultural and aquacultural products are sometimes at risk from ARB through the food chain and from handlers (Levy and Marshall, 2004) The relationship between antibiotic contamination and ARB is not always observed Our statistical analysis revealed a significantly positive correlation between the occurrence rate of SMXr bacteria and the residual concentration of SMX (Rs = 0.803) in January, but this finding was not observed in July However, as shown in Fig 2, SMXr bacteria increased in January at only city canal cases Thus it is suggested that rainfall effect is appeared in city canal sites by the reason mentioned above There was no statistically significant correlation between ERY concentration and the occurrence rate of ERYr bacteria Erythromycin resistance is mediated by various mechanisms (Perichon and Courvalin, 2009), suggesting possibility of cross-resistance occurred by other chemicals The coastal shrimp ponds, brackish water environments, are habitats differed from the freshwater environments, the relationship between antibiotic contamination and ARB was not observed in this study We found that at several sites in the coastal shrimp ponds where antibiotic concentration was below or almost below the limit of detection (Table 1), high incidence of ARB was still observed both in rainy and dry seasons (Table 2); this was particularly the case with SMXr bacteria This result is in agreement with previous study in the coastal shrimp ponds of Vietnam by Le et al (2005); higher incidence of bacteria resistant to antibiotics was found in the ponds where antibiotic concentration was lower There are many possible reasons for this For example, first, higher incidence of ARB could be due to their persistence in the environments Previous studies (Enne et al., 2001 and Enne et al., 2002; Antunes et al., 2005; Bean et al., 2005) have shown strong evidence that SR bacteria can persist for a long time even after a great decrease in prescription of the antibiotic; this is due to the genetic linkage of the SR to other resistance determinants (Enne et al., 2001) Second, other factors could have impact on incidence of ARB Recent ecological studies have shown evidence for the co-selection of ARB with various other resistance determinants in aquatic environments (Stepanauskas et al., 2005 and Stepanauskas et al., 2006) Hence, ARB may be abundant even when the corresponding antibiotics are absent in the environment Third, horizontal gene transfer of SR genes might play an important role on their dissemination in the environment in the presence of coselection by other antibiotics (Bean et al., 2005) This study suggested 100 90 80 70 60 50 40 30 20 10 B (Jul) Rs=-0.610 10 100 SMX concentration (ng/L) Fig Relationship between sulfamethoxazole (SMX) concentration and the occurrence rate of sulfamethoxazole-resistant (SMXr) bacteria in January (A) and July (B) 2007 The value of RS was calculated by the test for Spearman rank correlation Symbol, rhombus enclosed with oval indicates city canal (HNCs), circle indicates pig farm/fish pond (NHPs) and triangle indicates shrimp pond (HNAQs) 2900 P.T.P Hoa et al / Science of the Total Environment 409 (2011) 2894–2901 Table Sulfamethoxazole-resistant strains from three environments and their sul gene possession Closest genus or strain name (total #) Isolate # in HNCs Isolate # in HNPs Isolate # in HNAQs (sul positive #) (sul positive #) (sul positive #) Acinetobacter (29) Aeromonas (24) Bacillus (16) Pseudoalteromonas (12) Halobacillus (8) Shewanella (4) Escherichia (4) Pseudomonas (3) Arthrobacter (2) Brachybacterium (2) Micobacterium (2) Rheinheimera (2) Agrococcus (1) Cellulosimicrobium (1) Citrobacter (1) Sandaracinobacter (1) Shigella (1) Staphylococcus (1) Tenacibaculum (1) Uruburuella (1) Vibrio (1) Vitreosciella (1) Wautersiella (1) Enterobacter (1) Marine bacterium Tw-9 (1) Total 25 genera (121 strains) (2) 15 (13) 0 0 (1) 0 0 (1) (0) (1) 0 0 0 0 17 (17) (7) (1) (1) (1) (1) (2) (2) (2) (2) (1) (1) 0 (1) (1) (1) (1) (1) (1) 10 (9) (1) 15 (9) 11 (4) (1) (0) 0 0 0 0 0 0 (1) (0) 0 (1) 22 (18 sul positive) 49 (44 sul positive) 50 (26 sul positive) variety of bacterial genera, suggesting the shrimp pond was highly diverse in microbial community In addition, referring to the distribution of the sulfonamide resistance genes (sul1, sul2 and sul3), which detected by PCR and Southern hybridization as reported in our previous study (Hoa et al., 2008), we detected a total of 23 genera conferring sul1, sul2 or sul3 genes; of these 23 genera, 13 genera, namely Pseudoalteromonas , Halobacillus, Arthrobacter, Brachybacterium, Microbacterium, Rheinheimera, Marine bacterium Tw-9, Agrococcus, Cellulosimicrobium, Sandaracinobacter, Tenacibaculum, Uruburuella, and Wautersiella were first reported in this study as sul-containing bacteria Interestingly, our results revealed a potential reservoir of sul-containing bacterial genera from various habitats; major genera were Acinetobacter (17/17 sul-positive) in the pig farm/ fish pond, Aeromonas (13/15 sul-positive) in the city canal and Bacillus (9/15 sul-positive) and Acinetobacter (9/10 sul-positive) in the coastal shrimp ponds The findings are in agreement with recent studies that Acinetobacter was a potential environmental reservoir for sulfonamide resistance genes in pig slurry, manured agricultural soils and fish ponds that contaminated by sulfonamides and other antibiotics (Petersen et al., 2002; Byrne-Beiley et al., 2009; Heuer et al., 2009) Among the sites, pig farm/fish ponds and shrimp ponds showed higher bacterial species diversity than from city canals (Table 3) A previous study by Suzuki et al., 2008) suggested that the occurrence rate of tetracycline-resistant bacteria positively correlated with bacterial species diversity, accounting for the increase in tetracycline-resistant bacteria in the environment with higher microbial diversity The SMXr bacteria in pig farm/fish ponds and shrimp ponds may also be a similar condition, and may be one of the reasons why the occurrence rate of SMXr bacteria in July and January were nearly equal Escherichia, Shigella, Staphylococcus and Enterobacter were found in HNPs (Table 3), and these possessed sul genes This suggests that pigs and /or humans released ARB which was earlier selected in animal and human bodies In the VAC environment, animals and humans are also candidates for the origins of ARB and resistance genes Bacillus was reported as the major bacteria possessing tet(M) in marine sediments in Japan (Rahman et al., 2008), which was found to be a potential reservoir sul genes in Vietnamese water in this study The three habitats investigated in this study showed a higher number of sul-processing bacteria; 18 sul-positive genera /18 total genera in the pig farm/fish pond, 5/6 in the city canal, and 7/9 in the shrimp ponds This finding suggests that the sul genes are widely distributed in various bacterial groups in the bacterial isolates, and more diverse than those genera reported in previous studies (Le et al., 2005; Rahman et al., 2008;Byrne-Beiley et al., 2009 Our study concluded that SMXr bacterial groups containing the sul genes in the environments were more diverse than known by previous studies Various species should be reservoirs of sul genes in VAC aquatic environments Conclusion This study provides new results on the contamination status by antibiotics in VAC environment in northern Vietnam Sulfonamide especially SMX and sulfamethazine were major drugs in city canal and pig farm/fish pond, respectively Sulfonamides were used intensively in rainy season Trimethoprim was used as combination drug with SMX Macrolides were detected in city canal, indicating human use origin Occurrence of ARB and diversity of ARB were also evaluated Result of city canal site showed higher occurrence of SMXr in dry season than rainy season; however, pig farm/fish pond and aquaculture sites showed constant rate of SMXr in rainy and dry seasons SMXr bacteria were diverse, which included first recorded genera as SMXr Acinetobacter and Aeromonas were the major SMXr in aquatic environment Many of SMXr possessed sul genes, suggesting reservoir of the sul genes This study first showed relationship of drug contamination and ARB diversity in rainy and dry seasons Acknowledgements This research was partly supported by the 21st Century and Global COE programs (MEXT) and Grant-in-Aids from JSPS (19405004 and 19310039) We thank Dr Nguyen Thi Mui, Hanoi University of Science, Vietnam, for providing us with the data on rainfall and temperature of Red River delta areas Dr 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populations (Kümmerer, 2009) Very few studies have investigated the relationship between antibiotic contamination and antibiotic resistance in aquatic environments relating to human and agricultural... without correlation to drug contamination The relationship of contamination and ARB will be discussed further below 3.3 Relationship between antibiotic concentration and occurrence of ARB Relationship