DSpace at VNU: Detection of the sul1, sul2, and sul3 genes in sulfonamide-resistant bacteria from wastewater and shrimp ponds of north Vietnam

S CIE N CE OF T H E TOT AL E N V I RO N ME N T ( 00 ) 7–3 84 a v a i l a b l e a t w w w s c i e n c e d i r e c t c o m w w w e l s e v i e r c o m / l o c a t e / s c i t o t e n v Detection of the sul1, sul2, and sul3 genes in sulfonamide-resistant bacteria from wastewater and shrimp ponds of north Vietnam Phan Thi Phuong Hoa a,b , Lisa Nonaka a , Pham Hung Viet c , Satoru Suzuki a,⁎ a Center for Marine Environmental Studies (CMES), Ehime University, Matsuyama, 790-8577, Japan United Graduate School of Agricultural Science, Ehime University, Japan c Research Center for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Hanoi, Vietnam b AR TIC LE I N FO ABS TR ACT Article history: To assess the presence and distribution of the sul genes (sul1, sul2, and sul3) and plasmids in Received March 2008 human-mediated environments of north Vietnam, we examined a total of 127 sulfonamide- Received in revised form June 2008 resistant (SR) bacterial isolates from four shrimp ponds (HNAQs), a city canal (HNCs) and Accepted June 2008 three fish ponds that received wastewater directly from swine farms (HNPs) Results from Available online August 2008 the SR isolates revealed that sul genes were most frequently detected in the HNPs (92.0%), Keywords: the most prevalent gene in all three environments (57.0, 33.0 and 60.0% in HNPs, HNAQs, and followed by HNCs (72.0%), and the HNAQs (43.0%) Among the sul genes detected, sul1 was Sulfonamide HNCs, respectively) followed by sul2 (51.0, 19.0, and 20.0%, respectively) and sul3 (14.0, 6.0, Resistance and 8.0%, respectively) All combinations of paired different sul genes were detected, with sul the combination between sul1 and sul2 being the most frequent in all three environments Vietnam (20.0, 8.0, and 8.0% in HNPs, HNAQs, and HNCs, respectively) The combination of three sul genes was detected at low frequencies (2–3%) in the HNPs and HNAQs, and was absent in the HNCs The sul genes were more frequently located on the chromosome than on plasmids The identification of SR isolates positive for the sul genes and plasmids showed that Acinetobacter was the most dominant Our study revealed that the sul genes were common in SR bacteria from the aquatic environments we examined from northern Vietnam Wastewater from swine farms might be “hot spots” of the sul genes and plasmids and may be reservoirs for the exchange of the sul genes among bacteria © 2008 Elsevier B.V All rights reserved Introduction Sulfonamides, synthetic antibiotics, have been widely used to treat bacterial and protozoan infections in humans, domestic animals, and aquaculture since their introduction to clinical practice in 1935 (Perreten and Boerlin, 2003; Le and Munekage, 2004; Blahna et al., 2006) Sulfonamides work by inhibiting folate biosynthesis by competing with the natural substrate pamino-benzoic acid for binding to dihydropteroate synthase (DHPS), an enzyme in the folic acid synthesis pathway Through this process, sulfonamides inhibit the formation of dihydrofolic acid (Perreten and Boerlin, 2003) Bacterial resistance to sulfonamides, however, can occur through mutations in the chromosomal DHPS gene (folP) or through acquisition of an alternative DHPS gene (sul), whose product has a low affinity for sulfonamides (Perreten and Boerlin, 2003) Of the two pathways, the sul genes are the most prevalent mechanism of sulfonamide resistance (Enne et al., 2002; Perreten and Boerlin, 2003) Sulfonamide resistance (SR) is globally prevalent within human and animal pathogens ⁎ Corresponding author Tel./fax: +81 89 927 8552 E-mail address: ssuzuki@agr.ehime-u.ac.jp (S Suzuki) 0048-9697/$ – see front matter © 2008 Elsevier B.V All rights reserved doi:10.1016/j.scitotenv.2008.06.023 378 SC IE N CE OF T H E TOT AL E N V I RO N ME N T ( 00 ) 7–3 84 (Kerrn et al., 2002; Perreten and Boerlin, 2003; Antunes et al., 2005), however, the presence of the sul genes is not equally distributed among bacterial populations (Kerrn et al., 2002; Antunes et al., 2005; Hammerum et al., 2006) Monitoring programs for antimicrobial resistance, including SR in agents of zoonoses and in indicator bacteria of normal intestinal and pathogenic flora of animals (e.g., Escherichia coli, Enterococus spp) have been established in Europe, North America, and Latin America (Boerlin et al., 2005) However, the persistence and spread of an antibiotic resistance gene may occur not only to animal intestinal and pathogenic microbiota but also to environmental bacteria (Aminov and Mackie, 2007) Antibiotic resistance genes can be widely spread by horizontal gene transfer within the environment Recently, some studies revealed the wide distribution of tetracycline resistance in coastal areas of Japan and Korea (Nonaka et al., 2000; Kim et al., 2003, 2004); however, limited information exists on the presence of the sul genes in environmental bacteria of Asia with only one known study on the sul2 among resistant Acinetobacter sp from Thailand (Agersø and Petersen, 2007) In Vietnam, the unregulated use of antimicrobials in plant protection, animal husbandry, aquaculture facilities, and human and veterinary medicine may be a significant source of antibiotic resistance (Le and Munekage, 2004; Le et al., 2005; Cabello, 2006) Antibiotic resistance may be further enhanced through polyculture practices that have been commonly used in Southeast Asia and Vietnam In Vietnam, a recycling farm system, typically consisting of vegetable, aquaculture and cage (VAC), has been in practice since the 1980s, in which animal (usually pigs, chickens, and ducks) manure is directly transported to fish ponds, and to vegetable and rice fields The VAC system has been considered to be a very economical method of farming (Hop, 2003); however the heavy use of antibiotics in farm animals and subsequent waste discharge of antibiotic residues and antibiotic-resistant bacteria has been shown to increase bacterial antibiotic resistance in the surrounding environment (Petersen and Dalsgaard, 2003; Heuer and Smalla, 2007) The VAC system and other habitats such as city canals, receive many anthropogenic sources, and may therefore be a mixture of bacteria and drug resistance genes In contrast, the common shrimp ponds used in Vietnam which receive more natural water have very specific sources of drug contamination In this study, we tested the hypothesis that these different aquatic habitats varied in their sul gene profile, and that habitats with higher inputs (i.e VAC and city canal) are expressed as a mixture of the sul genes To accomplish this, we assessed the distribution of the sul genes and plasmids in SR bacterial isolates from water samples collected from different aquatic environments (fish ponds of VAC swine farms, shrimp ponds and a city canal) of north Vietnam Materials and methods 2.1 Sample collection and isolation of sulfonamide-resistant (SR) bacteria Water samples from nine sites were taken, including three fish ponds in three different VAC swine farms (HNPs), four shrimp ponds (HNAQs), and a city canal (2 sites) (HNCs) in January 2007 The three site types (HNP, HNAQ, and HNC) were chosen for their importance and common use throughout Vietnam and other countries of Asia The HNP sites (1–3) were from medium-sized swine farms with 50–70 pigs, where pig waste was directly discharged into a fish pond The HNAQ sites (1–4), which are approximately 200 km away from HNPs, consisted of intensive shrimp culture The HNC sites (2–3) received untreated hospital and municipal wastewater Field collection of water samples was taken using a metal bucket after washing three times with water from the site The sample water passed through a 150 µm-mesh plankton net, then stored in sterile bottle (500 ml) The sample was maintained on ice and analyzed in the lab within 3–4 h after sampling The isolation of SR bacteria, those growing on agar plates containing 60 μg/ml sulfamethoxazole in this study, was performed by using the plate spread method (Nonaka et al., 2000) In brief, 1.0 ml of each water sample was suspended in ml of phosphate-buffered saline (PBS) and serial 10-fold dilutions (100, 10− 1, 10− 2, 10− 3, and 10− 4) were prepared In this study, nutrient broth (Difco, Detroit, MD, USA) for fresh water samples collected from the HNPs and HNCs and marine broth (Difco, Detroit, MD, USA), for brackish water samples collected from the HNAQs, plus 1.5% agar plates (called NA-nutrient agar, MA-marine agar) were used These media were supplemented with 60 μg/ml sulfamethoxazole (SIGMA-ALDRICH, St Louis MO USA) to select sulfamethoxazole-resistant (SMXR) bacteria After 5days of incubation at 30 °C, 10 to 15 SR isolates were randomly picked from the double plates We obtained a total of 127SR bacterial isolates, including 49 isolates in HNPs, 53 isolates in HNAQs, and 25 isolates in HNCs and used them for subsequent analyses 2.2 DNA extraction SR isolates were cultured in 10 ml nutrient broth (for HNPs and HNCs isolates), or marine broth (for HNAQs isolates) supplemented with 60 μg/ml of sulfamethoxazole, and incubated at 30 °C overnight DNA extraction from a 1.5ml volume of cultured SR isolates was performed by using the protocol described previously by Kim et al (2004) Table – Primers for PCR, amplicon size and positive control Gene sul1 sul2 sul3 Primer name Primer sequence Amplicon size (bp) Positive control Reference Sul1-F Sul1-R Sul2-F Sul2-R pVP440sul3F pVP440sul3R 5′-CGGCGTGGGCTACCTGAACG-3′ 5′-GCCGATCGCGTGAAGTTCCG-3' 5′-GCGCTCAAGGCAGATGGCATT-3′ 5′-GCGTTTGATACCGGCACCCGT-3′ 5′-TCAAAGCAAAATGATATGAGC-3′ 5′-TTTCAAGGCATCTGATAAAGAC-3′ 433 433 293 293 787 787 R388 R388 RSF1010 RSF1010 pUVP4401 pUVP4401 Kerrn et al (2002) Kerrn et al (2002) Kerrn et al (2002) Kerrn et al (2002) Heuer and Smalla (2007) Heuer and Smalla (2007) S CIE N CE OF T H E TOT AL E N V I RO N ME N T ( 00 ) 7–3 84 379 Table – Distribution of the sul genes in the three environments investigated: three swine farms (HNPs), four shrimp ponds (HNAQs), and two-canal sites (HNCs) sul gene combination HNPs (n = 49) HNAQs (n = 53) HNCs (n = 25) No of No of No of isolates (%) isolates (%) isolates (%) Single gene sul1 sul2 sul3 Twosul1 + sul2 genes sul1 + sul3 sul2 + sul3 Three- sul1 + sul2 + genes sul3 None Total sul1 sul2 sul3 Total of SR isolates positive for sul genes 2.3 15 13 10 1 (31.0) (27.0) (6.0) (20.0) (4.0) (2.0) (2.0) 12 4 0 (23.0) (8.0) (2.0) (8.0) (0.0) (0.0) (3.0) 12 2 1 (48.0) (8.0) (0.0) (8.0) (4.0) (4.0) (0.0) 28 25 45 (8.0) (57.0) (51.0) (14.0) (92) 30 18 10 23 (57.0) (34.0) (19.0) (6.0) (43) 15 18 (28.0) (60.0) (20.0) (8.0) (72.0) Plasmid extraction The Kieser plasmid isolation protocol (KPIP), using a modification by Sobecky et al (1997) was used for screening the incidence of plasmids from all environmental isolates in this study A supercoiled DNA ladder (Invitrogen, USA) was used as a marker E coli K-12 containing plasmids R388 (33926bp) and RSF1010 (8684bp) were used as plasmid size standards and positive control strains, respectively The determination of plasmid size was carried out following the method by Beeson et al (2002) 2.4 PCR amplification The presence of the sul1, sul2 and sul3 was examined by using PCR with gene-specific primers (Table 1) Amplification of the sul1 and sul2 genes was performed with the primers and conditions described previously (Kerrn et al., 2002) Primers for the sul3 were as described by Heuer and Smalla (2007) Amplification condition of the sul3 was as follows: heating at 94 °C for 5min, 30cycles at 94 °C for 60s, 55 °C for 60s and 72 °C for 60s, followed with one cycle at 72 °C for PCR was performed by GeneAmp PCR System 9700 Gel electrophoresis Fig – Plasmid-size distribution in SR bacterial isolates was performed on 1.2% agarose gel Plasmids R388, RSF1010, and pUVP4401 that contained the sul1, sul2, and sul3, respectively, were used as positive controls for these genes When a PCR product showed a single clear band having the same migration profile as the corresponding gene control, the isolate was counted as positive for that gene 2.5 Hybridizations To examine whether the sul genes were coded on plasmids and to confirm PCR products positive for the sul1, sul2, and sul3, Southern blotting was performed following the protocols of Sambrook and Russell (2001) Hybridization probes were PCR products of the sul1, sul2 and sul3, which were labeled by DIG-Chem-Link (Roche Diagnostics, Mannheim, Germany) 2.6 Identification of SR isolates All SR isolates possessing plasmids and the sul genes (43 isolates) were identified by 16S rRNA gene (primers F984GC, R1378) as described by Heuer et al (1997) The PCR products were purified and sequenced by using a Big Dye terminator version 3.1 cycle reaction kit (Applied Biosystems, Foster City, CA, USA) on a 3100 ABI Prism DNA sequencer (Applied Biosystems, Foster City, CA, USA) For identification Fig – Incidence of single and multiple plasmids in SR bacterial isolates with plasmids 380 SC IE N CE OF T H E TOT AL E N V I RO N ME N T ( 00 ) 7–3 84 Table – Rate of the sul genes on plasmids Gene Plasmid carrying number/ sul-positive number (%) The sul genes located on plasmids (%) 28/61 a (46.0) 22/40 a (55.0) 10/12 a (83.0) b/28 (32.0) 10 b/22 (45.5) b/10 (10.0) sul1 sul2 sul3 a b sul1, 2, or positive number of the total 127 SR isolates Number of SR isolates owning the sul genes located on plasmids of bacteria, DNA sequences were analyzed by the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information web site (NCBI, http://www.ncbi nlm.nih.gov/) significant (p b 0.05) Genes sul2 and sul3 were most frequently detected in the HNPs (51.0 and 14.0%, respectively), whereas sul1 was the most frequent in the HNCs (60.0%); all three genes showed lowest detections in the HNAQs (34.0, 19.0, and 6.0% for sul1, sul2, and sul3, respectively) The combinations of two different sul genes were also detected, with the combination of sul1 and sul2 being the most frequent in all three environments (20.0, 8.0, and 8.0% in HNPs, HNAQs, and HNCs, respectively) The combination of three sul genes was detected, but at low frequencies (2–3%) in the HNPs and HNAQs, and was absent in the HNCs In total, the sul genes were most frequently detected in SR isolates from the HNPs (92.0%), followed by HNCs (72.0%), and the HNAQs (43.0%) 3.2 2.7 Plasmid profile of SR isolates Statistical analyses A Fisher's exact test was used to test for differences in the sul gene distribution in 127 SR isolates A p value of b 0.05 was considered as statistically significant Results 3.1 Distribution of the sul genes The number and percentage of isolates carrying the sul genes are summarized in Table The sul1, sul2, and sul3 genes were detected from all the three environments Overall, sul1 was the most prevalent gene in all the three environments (57.0, 34.0 and 60.0% in HNPs, HNAQs and HNCs, respectively) followed by sul2 (51.0, 19.0, and 20.0%, respectively), and sul3 (14.0, 6.0, and 8.0%, respectively) The order of sul1 N sul2 N sul3 was statistically The distribution of plasmids is shown in Fig The percentages of SR isolates bearing one plasmid were highest in the HNAQs (47.0%), followed by the HNCs (40.0%) and the HNPs (29.0%) The SR isolates bearing two plasmids were detected almost in identical frequencies in the three environments investigated The percentages of SR isolates bearing 3–5 plasmids were relatively high, with the highest in HNPs (46.0%) and somewhat lower in HNCs and HNAQs (30.0 and 29.0%, respectively) The isolates bearing ≥ plasmids were most frequently detected in the HNCs (10.0%), followed by HNPs (6.0%) and HNAQs (5.0%) We also observed a broad range of plasmid sizes from to N 16 kbp (Fig 2) The percentage of plasmids with a size N 16 kbp was highest from the HNPs (52.0%) and HNAQs (44.0%), whereas isolates from HNCs showed varying size ranges The percentages of occurrence of 1–5 kbp, 6–10kbp, N 16 kbp were more or less similar (20–30%) Fig – Detection of the sul genes by Southern hybridization (a, c, and e), agarose gel profile of DNA extracted from the sul gene positive strains; (b, d, and f), Southern hybridization with the sul probes; number at the top indicates sample ID of SR isolates possessing plasmid; PC, positive control for each genes; M, super-coiled DNA ladder (a and c) or lambda HindIII (e); arrow, chromosome position 381 S CIE N CE OF T H E TOT AL E N V I RO N ME N T ( 00 ) 7–3 84 Results on the frequency of plasmid occurrence and the sul gene location in each sampling site showed that plasmid positive-rates were 63% in HNPs, 40% in HNCs and HNAQs The detection rate of the sul genes on plasmid was 38.7% in HNPs, 19% in HNAQs and 30% in HNCs Among sul1-positive isolates, as shown in Table 3, 46% isolates possessed plasmids, while in the cases of sul2 and sul3-positive isolates, isolates carrying plasmids were 55% and 83%, respectively 3.3 Location of sul1, sul2 and sul3 Southern hybridization experiments were performed to determine the location of the sul genes on plasmids (Fig 3), which could confirm PCR results on the sul genes Results are summarized in Table 3, showing that 9/28 isolates had sul1 located on plasmids, of which eight isolates had sul1 on N 16 kbp-sized plasmids (Fig 3b); 10/22 isolates of sul2 were Table – List of the sul-positive isolates carrying plasmids Sampling site HNPs ∑ HNAQs ∑ HNCs ∑ a sul1 Closest species Acinetobacter sp PD 12 (AY673994) Acinetobacter sp.u21 (EU375649) Aeromonas punctata (EU082831) Aeromonas sp MCCB 113 (EU394215) Arthrobacter sp PD8 (EF412972) Brachybacterium sp SSCS6 (AB21098) Cellulosimicrobium sp 3-H (EU307933) Escherichia coli strain LD09 (EU130557) Acinetobacter junii (AY269228) Acinetobacter sp.u21 (EU375649) Acinetobacter baumannii ATCC 17978 (EU052267) Bacillus sp T27 (1) (EU111708) Bacillus sp.Tianshan222–3 (EU305637) Bacillus sp B105 (EU384288) Pseudoalteromonas byunsanesis (EF492006) Pseudoalteromonas sp L-fcr-129 (EU420061) Acinetobacter sp PD 12 (AY673994) Aeromonas sp MCCB 113 (EU394215) sul2 Identity No (%) 98–100 99–100 99 97–98 99 98 99 96–99 96 99 98 98 99 97 Closest species 4a(2)b Acinetobacter sp PD 12 (AY673994) Acinetobacter sp.u21 (EU375649) 1(1) Acinetobacter sp (U37348) 2(1) Acinetobacter sp Zf-78-II (AM292068) 1(1) Aeromonas sp MCCB 113 (EU394215) Arthrobacter sp PD8 (EF412972) 1(1) Enterobacter sp ZJUPD1 (EU430750) 2(1) Pseudomonas sp Y12A (EU306338) Vitreosciella stercoraria (DQ217656) Wautersiella falsesenii subsp Genomovar (AM084342) 14(7) 1(1) Acinetobacter johnsonii (EU16914) Acinetobacter sp.u21 (EU375649) Acinetobacter sp BHSN (EU293155) Acinetobacter junii (AY269228) Acinetobacter baumannii ATCC 17978 (EU052267) Bacillus sp T27 (EU111708) 96 97 Pseudoalteromonas byunsanesis (EF492006) Identity (%) 98–99 100 98 95 97 99 98 99 99 99 No Acinetobacter sp Hop10 1(1) (AF015300) 97 1(1) Acinetobacter sp PD 12 (AY673994) 98 99 99 99 1(1) 1 Aeromonas enteropelogenes (EF465529) 1(1) Arthrobacter sp PD8 (EF412972) 1(1) Shigella sp MAC17169 (DQ874994) 100 12(7) 1(1) 99 100 95 1 98 98 96 99 8(1) 1(1) 98 2(1) 98 1(1) 4(1) Identity No (%) 99 10(1) Acinetobacter sp.u21 (EU375649) Aeromonas sp MCCB 113 (EU394215) Closest species 2(1) Acinetobacter sp.u21 2(2) (EU375649) 99 Indicates a total number of SR isolates Indicates the number of SR isolates owning the sul genes on plasmid b sul3 2(2) 10(1) 0(0) 0(0) 382 SC IE N CE OF T H E TOT AL E N V I RO N ME N T ( 00 ) 7–3 84 located on various sized plasmids (Fig 3d) One isolate (the strain named 21 in Fig 3f) had sul3 on N 16 kbp-sized plasmids, and one isolate from HNPs (the strain named in Fig 3b and d) showed sul1 and sul2 located on the same plasmid 3.4 Identification of SR isolates positive for both the sul genes and plasmids The results are summarized in Table All SR isolates positive for both the sul genes and plasmids were identified and belonged to thirteen genera (Acinetobacter, Aeromonas, Arthrobacter, Bacillus, Brachybacterium, Cellulosimicrobium, Enterobacter, Escherichia, Pseudoalteromonas, Pseudomonas, Sigella, Vitreosciella and Wautersiella) Among bacteria identified, eleven genera were found in the HNPs, three in HNAQs, and two in HNCs Acinetobacter was a common genus in all sampling sites HNPs contained human enteric bacteria, E coli, whereas HNAQs contained Pseudoalteromonas which was predominantly found in water Discussion The distribution and spread of drug resistance genes in aquatic environments of Vietnam are of interest and are important from the viewpoint of gene transfer and public health The integrated aquaculture–agriculture (VAC) system widely used across Vietnam is a form of polyculture that incorporates the recycling of animal manure for the culture of fish and vegetables This system, however, may allow mixing of aquatic and fecal bacteria and transport such bacteria to humans We revealed here the distribution profile of the sul genes in the aquatic environments of northern Vietnam The percentage of plasmid-bearing isolates from environmental water samples of Vietnam measured here (40.0–63.0%) were relatively high compared to those from other environments and in other countries, which ranged from 24.0–52.0% (Sobecky, 2002) In all of the three environments examined, 60.0–70.0% of the plasmid-bearing isolates carried multiple plasmids (≥ plasmids) (Fig 1), whereas previous studies showed that N 50.0% of the plasmid-bearing isolates carried a single plasmid (Sobecky et al., 1997) Furthermore, the percentage of SR isolates carrying from 3–5 plasmids (29.0– 46.0%) was extremely high compared to previous studies (b 10%) (Beeson et al., 2002; Sobecky et al., 1997) In terms of plasmid size, we also showed a relatively high percentage of plasmids with the size N 16 kbp (Fig 2) This is similar to the observations on the distribution of plasmid size detected in marine bacteria (Sobecky et al., 1997) It is probable that SR bacteria found in northern Vietnam are a potential pool of plasmids The frequency distribution of the sul genes in the three environments investigated generally followed sul1 N sul2 N sul3 (p b 0.05) This is in agreement with our observation on the distribution of the sul genes in SR bacteria isolated from the five VAC farms of north Vietnam (Unpublished data) The sul3 gene was always found at low frequencies in all the three environments, whereas the sul1 and sul2 were high In the previous studies of the sul genes from clinical populations of specific bacterial species, the three sul genes did not occur at similar frequencies, and in most of the experiments the pattern of gene frequency distribution was sul2 N sul1 N sul3, [e.g., Enterobacteriaceae (Frank et al., 2007), E coli (Blahna et al., 2006), Haemophilus influenzae, (Enne et al., 2002)] Our samples from Vietnam were different from these previous reports with respect to the frequency of occurrence In spite of previous reports showing the sul2 gene as more prevalent (e.g., Enne et al., 2002; Blahna et al., 2006; Frank et al., 2007), the sul1 was the most prevalent gene in this study The abundance of the sul genes in environmental samples from Vietnam is comparable to those observed from clinical origins SR bacteria may carry either a single or more sul genes Two or three sul gene combinations were found in a single bacterial cell in this study In previous studies, a considerable proportion of SR isolates carrying ≥2 sul genes were observed (Antunes et al., 2005; Hammerum et al., 2006; Frank et al., 2007) In this study, the combination of the sul1 and sul2 was detected in all sampling sites, with frequencies as high as those in isolates from a clinical origin in other countries (Kerrn et al., 2002; Guerra et al., 2004; Bean et al., 2005; Frank et al., 2007) The isolates having three genes (i.e sul1, sul2 and sul3) were detected in VAC swine farm and shrimp pond samples The co-presence of the sul1, sul2 and sul3 in a single cell has rarely been reported elsewhere (Antunes et al., 2005; Hammerum et al., 2006) The high frequency of various combinations of the sul genes in environmental samples observed here appears to be a specific characteristic of Vietnam, which may be a result of the material recycled in the integrated aquaculture–agriculture system This farming system might be a reservoir of drug resistance genes, and various sul genes are suggested to flow in the VAC-pond from different sources, such as from humans, animals and natural environment From the HNPs, the sul genes were detected in most of the SR isolates (92%), agreeing with previous studies from other countries which also showed very high frequencies of the sul genes in isolates from swine farms in other countries (Perreten and Boerlin, 2003; Boerlin et al., 2005; Hammerum et al., 2006) Among the three environments investigated, HNPs might be the “hot spots” of plasmids and the sul genes Further research is necessary to determine if this is unique to the type of environment examined The mechanism of SR is likely not caused by only the sul genes, because the sul-negative isolates were found in this study In particular, from the HNAQs, more than half of the SR isolates did not possess the sul genes, which may be due to the heavy use of prophylactic antibiotics in the aquaculture of shrimp (Le and Munekage, 2004; Cabello, 2006) Drug-resistant bacteria in the environment should occur by various mechanisms such as efflux pumps, modification of the antibiotic target (e.g., mutations in the chromosomal DHPS gene), and modification of the antibiotics, some of which are induced by selective pressure For the past several years many different kinds of anti-microbial drugs have been applied to aquaculture in Vietnam In shrimp culture within Vietnam, the residue of various drugs such as oxytetracycline, chloramphenicol, furazolidone, afatoxin, and sulfamethoxazole have been found to be high (Le and Munekage, 2004; website of the ministry of fishery, Vietnam: www.fistenet.gov.vn) and thus may lead to the diversity of SR mechanisms in shrimp culture environments S CIE N CE OF T H E TOT AL E N V I RO N ME N T ( 00 ) 7–3 84 In all sampling sites, the percentage of SR isolates that were positive to the sul genes and carry plasmids was very high; however the percentages of isolates carrying the sul genes located on plasmids was relatively low (Table 3) This suggests that most sul genes are located on the chromosome Of the three sul genes, sul2 was detected on plasmids with the highest frequency (45.5%); this is consistent with other studies that showed sul2 is often located on plasmids with a wide hostrange (Scholz et al., 1989; Smalla et al., 2000) The identification of SR isolates positive for the sul genes and plasmids in this study showed that Acinetobacter was dominant in all investigated habitats (Table 4), and thus, this genus could be the main reservoir of the sul genes and plasmids A total of genera (13) identified in this study are higher than that of genera (7) identified in the previous report in Vietnamese aquaculture (Le et al., 2005) Among the bacteria identified in this study, Acinetobacter, Aeromonas, Arthrobacter, Cellulosimicrobium, Escherichia, Shigella, Vitreosciella, and Wautersiella carried the sul genes on plasmids Bacillus and Pseudoalteromonas were found in HNAQs, whereas Aeromonas was frequently observed in HNCs and HNPs The diversity of the sul-positive bacteria with plasmids was high in HNPs (11 genera), but not in HNQAs (3 genera) and HNCs (2 genera) This suggests that HNPs might be a potential pool of the sul genes and plasmids with a corresponding diversity of enteric, pathogenic, and environmental bacteria Although detection rates of the sul genes on plasmids in this study were not high, it does not suggest a limited potential of horizontal gene transfer in the environment The sul genes may be transferred by means of integrons and transposons or by using phage and mobile plasmids as vehicles for their dissemination (Smalla and Sobecky, 2002) Previous studies have shown that sul1, sul2 and sul3 were usually detected in association with integrons and conjugative plasmids The sul1 was typically associated with class integrons (Rådström and Swedberg, 1988; Aminov and Mackie, 2007; Heuer and Smalla, 2007), the sul2 was often found on a broad host-range of plasmids (Smalla et al., 2000), and the sul3 was recently detected on a conjugative plasmid and was shown to be associated with class integrons (Perreten and Boerlin, 2003; Antunes et al., 2005; Hammerum et al., 2006; Antunes et al., 2007) This suggests that all three sul genes have a potential of being horizontally transferred and widely spread in the environment This study revealed that the sul genes were common in aquatic environments of northern Vietnam, especially in integrated aquaculture–agriculture areas used for rearing fish This suggested that reuse of untreated wastewater from the integrated aquaculture–agriculture system as well as from a city canal (untreated hospital and municipal wastewater) might have high potential for microbial contamination with the abundance of drug resistant genes that are potentially detrimental to human and ecosystem health Acknowledgements We thank K Smalla, Federal Biological Research Centre for Agriculture and Forestry (BBA), Braunschweig, Germany, for supplying positive controls We thank also P A Sobecky and T H Hazen, School of Biology, Georgia Institute of Technology, 383 USA, for advising the modification of Kieser plasmid isolation protocol T Miller and A Subramanian, Ehime University, Japan are appreciated for their critical reading of this paper We also thank the anonymous reviewers for their critical comments to improve the quality of the manuscript This work was partly supported by 21-COE and Global-COE programs from the MEXT, and the Grant-in-Aid from JSPS 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