JAC Advance Access originally published online on November 9, 2006
Journal of Antimicrobial Chemotherapy 2007 59(1):23-27; doi:10.1093/jac/dkl419
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The tetracycline resistance determinant Tet 39 and the sulphonamide resistance gene sulII are common among resistant Acinetobacter spp. isolated from integrated fish farms in Thailand
1 Danish Institute for Food and Veterinary Research Bülowsvej 27, DK-1790 Copenhagen V, Denmark 2 Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark
*Corresponding author. Tel: +45-72-34-6273; Fax: +45-72-34-6001; E-mail: ya{at}dfvf.dk
Received 29 May 2006; returned 19 July 2006; revised 16 September 2006; accepted 18 September 2006
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Abstract |
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Objectives: To determine the genetic basis for tetracycline and sulphonamide resistance and the prevalence of class I and II integrons in oxytetracycline-resistant Acinetobacter spp. from integrated fish farms in Thailand.
Methods: A total of 222 isolates were screened for tetracycline resistance genes [tet(A), tet(B), tet(H), tet(M) and tet(39)] and class II integrons by PCR. One hundred and thirty-four of these isolates were also sulphonamide resistant and these isolates were screened for sulphonamide resistance genes (sulII and sulIII) as well as class I integrons. Plasmid extraction and Southern blots with sulII and tet(39) probes were performed on selected isolates.
Results: The recently identified tetracycline resistance gene tet(39) was demonstrated in 75% (166/222) of oxytetracycline-resistant Acinetobacter spp. from integrated fish farms in Thailand. Isolates that were also sulfamethoxazole-resistant contained sulII (96%; 129/134) and/or sulI (14%; 19/134) (as part of class I integrons). sulII and tet(39) were located on plasmids differing in size in the isolates tested.
Conclusions: The study shows tet(39) and sulII to be common resistance genes among clonally distinct Acinetobacter spp. from integrated fish farms and these bacteria may constitute reservoirs of resistance genes that may increase owing to a selective pressure caused by the use of antimicrobials in the overlaying animal production.
Keywords: aquatic environments , integrons , tet(39) , tetA(39)
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionGenBank submissionTransparency declarationsReferences |
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Integrated fish farming is practiced throughout Southeast Asia. The farming systems are relatively confined units with little water exchange. The fish feed on manure and photosynthetic organisms whose growth is supported by nutrient release from the overlaying animal production.1 The livestock integrated with the fish production is often reared intensively and antimicrobials are used for growth promoting, and prophylactic and therapeutic treatment. Antimicrobial residues and antimicrobial-resistant bacteria are therefore entering the aquatic environment, and selection of antimicrobial-resistant bacteria may occur throughout the system.2 Thus, a significant increase over time of resistance to six different antimicrobials was observed in Acinetobacter spp. in a study on the impact of integrated fish farming on antimicrobial resistance in a pond environment.2 Especially, resistance to tetracycline and sulfamethoxazole increased and after 2 months the resistance levels of both compounds reached 100%.2 Integrated fish farming constitutes a production system where bacteria of animal and environmental origin are living in close contact. The role of environmental bacteria as a reservoir for antimicrobial resistance genes may therefore be important in the spread of resistance genes from such a system.
Acinetobacter spp. are Gram-negative coccibacilli, non-motile, non-fermentative and easily isolated from the aquatic environment.3 Acinetobacter spp. have an increasing importance as opportunistic pathogens in clinical settings.4 Resistance to antimicrobials such as tetracycline, sulphonamides, trimethoprim, erythromycin and ciprofloxacin has been reported among Acinetobacter spp. from aquatic environments.2,5–7
Four classes of genes encoding tetracycline resistance by specific active efflux have been described in Acinetobacter spp., tet(A), tet(B), tet(H)5,6 and, most recently, tet(39) that were identified among oxytetracycline-resistant Acinetobacter spp. isolates from aquatic environments in Denmark and, in one case, a clinical isolate from The Netherlands.8 Other tetracycline resistance genes occasionally reported in clinical Acinetobacter baumannii isolates include tet(M), a widespread gene encoding tetracycline resistance by ribosomal protection, and adeB, which confers multidrug resistance by an unspecific efflux pump mechanism.9,10 sulI has been demonstrated as part of class I integrons among Acinetobacter spp. of clinical and environmental origin.7,11,12
The aim of this study was to determine the genetic basis for tetracycline and sulphonamide resistance and the prevalence of class I and II integrons in oxytetracycline-resistant Acinetobacter spp. from integrated fish farms in Thailand. Determination of the prevalence of resistance genes and genetic elements such as integrons is important when studying spread and horizontal gene transfer of resistance within and between bacterial species of different ecological niches.
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Materials and methods |
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Integrated fish farms and sampling procedure
Isolates were obtained and tested for antimicrobial resistance in a previous study.2 In short, Acinetobacter spp. were isolated from water-sediment and chicken manure samples on Baumann agar plates13 and identification was verified by colony hybridization with a genus-specific 16S rRNA-targeted alkaline phosphatase-labelled oligonucleotide probe.14 Determination of antimicrobial resistance was performed by a disc diffusion method according to standardized guidelines.15 Samples were collected three to four times during a 3 month period from four integrated fish farms and two times from four control farms. The farms were located in the same province in Thailand but were geographically apart. Only isolates resistant to oxytetracycline were included in this study.
Screening for resistance genes and integrons by PCR
A total of 222 isolates were screened by PCR for the presence of the tetracycline resistance genes tet(39), tet(A) and tet(B) and for class II integrons as previously described.8,16,17 Isolates where none of the targeted tetracycline resistance genes [tet(39), tet(A) and tet(B)] was detected were in addition screened for the tetracycline resistance genes tet(H) and tet(M).18 The sulfamethoxazole-resistant isolates were screened for the sulphonamide resistance genes sulII and sulIII as previously described.19,20 sulI was detected as part of class I integrons as previously described.21 In short, three PCR sets were used, primers: qacE
1-F/qacE1-B targeting qacE1; Sul-1-B/qacE1-F targeting the sulI and qacE1 region; and Att-1-F/3'CS-B targeting the variable region of class I integrons.21
The performance of each PCR assay was secured by using the reference strain for the respective tet gene as a positive control. For a negative control a sample containing PCR mixture with no DNA was used. The respective genes were verified by sequencing the PCR products of randomly selected isolates [tet(39) and sulII, four isolates of each; tet(A), one isolate] and selected sequences were submitted to GenBank. All primers and references are listed in Table 1. The following positive control strains were used for PCR: tet(39), Acinetobacter spp. (LUH5605);8 Escherichia coli NCTC50078/RP4 with tet(A) and E. coli HB101/pRT1 with tet(B) obtained from Dr M. C. Roberts; tet(H) cloned on a blunt-ended TOPO vector in E. coli HB101 and Staphylococcus intermedius with tet(M) obtained from Dr S. Schwarz, sulI (class I integron) Salmonella Typhimurium DT104; Salmonella enterica strains containing class II integron, sulII and sulIII, respectively obtained from Dr R. Helmuth.
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Plasmid isolation and Southern blot
Six isolates with both tet(39) and sulII originating from four integrated fish farms and two control farms were screened for plasmids by use of a commercial kit, Qiagen Plasmid Midi Kit (100) (Qiagen, Germany), following the manufacturer's guidelines. The plasmid profiles were analysed by gel electrophoresis on a 0.8% TBE gel at 45 V for 18.5 h. Southern blots on plasmids were performed. The blots were hybridized with two digoxigenin-labelled DNA probes for the presence of tet(39) using the PCR product (701 bp) amplified by the primers tet(39)-1 and tet(39)-2 and for sulII (285 bp) using the PCR product amplified by sulII-1 and sulII-2, respectively (Table 1).
RAPD–PCR typing
Random amplified polymorphic DNA (RAPD)–PCR was performed on all tet(39)-harbouring isolates originating from one integrated fish farm (B2) (n = 19) and on 16 tet(39)-positive isolates from control farm C4. The Ready-To-GoTM RAPD Analysis Beads and the RAPD Analysis primer 2 (5'-d[GTTTCGCTCC]-3') from Amersham Bioscience (Little Chalfont, UK) were used. The cycle profile was 1 cycle at 95°C for 5 min, followed by 45 cycles at 95°C for 1 min, 36°C for 1 min and 72°C for 2 min.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionGenBank submissionTransparency declarationsReferences |
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A total of 222 oxytetracycline-resistant Acinetobacter spp. were included in the study. The isolates originated from 43 of 46 samples obtained from four integrated fish–chicken farms (two with layer hens and two with broilers) and three control fish farms with no history of antimicrobial consumption.2 Of these isolates 134 were in addition resistant to sulfamethoxazole (Table 2).
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In Table 3 the number of different resistance patterns for all isolates included in this study is given. Among the 222 isolates 25 different resistance patterns were recorded. The isolates from the integrated farms demonstrated between 9 and 14 different patterns and the isolates from control farms between 2 and 7 patterns, which shows that the collection comprised independent isolates.
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tet(39) was detected in 75% (166/222) of the isolates, tet(A) in 8.6% (19/222) and no isolates contained tet(B). Although some of the isolates obtained at the same sampling time (Figure 1: lanes 8–12; lanes 23, 24 and 31; lanes 27 and 29) demonstrated a similar profile, the RAPD analysis of all tet(39)-harbouring isolates from integrated farm B2 collected at three sampling times during 3 months and 16 tet(39)-harbouring isolates from control farm C4 collected at one sampling time demonstrated a diverse clonal origin (Figure 1). tet(A) was found alone (n = 17) or in combination with tet(39) (n = 2) (Table 2). None of the isolates screened for tet(H) and tet(M) (n = 39) contained any of the tet genes. The genetic basis of sulphonamide resistance could be detected in 96% of the isolates where 129 out of 134 sulfamethoxazole-resistant isolates had sulII; 19 isolates had in addition class I integrons and thereby the sulphonamide resistance gene sulI. sulIII was not detected among any of the sulfamethoxazole-resistant isolates. None of the isolates contained class II integrons. Eighteen of the 19 isolates with class I integrons were isolated from the broiler-fish farms (both water-sediment and chicken manure samples), and one isolate was from chicken manure of a layer-fish farm. None of the water-sediment samples of the control fish farms and the layer-fish farm contained class I integrons. tet(39) and sulII were present in most isolates and could therefore be the genetic basis of tetracycline and sulphonamide resistance, respectively. tet(39) was located on plasmids in all six isolates tested (
15–50 kb); in five of these isolates sulII was located on plasmids differing in size from the plasmids carrying tet(39) (>50 kb). No hybridization to plasmids was found for the remaining isolate indicating that sulII was either chromosomal or located on a plasmid not purified by this method. The tet(39) gene has recently been identified and so far has only been demonstrated in Acinetobacter spp. (aquatic sources and in one case a clinical specimen).8 This is, to our knowledge, the first study on the distribution of tet(39). tet(39) was found widely distributed among both isolates from the water-sediment samples and manure, and since this gene has been found associated with horizontally transferable plasmids8 these plasmids may spread tet(39) among Acinetobacter spp. and maybe to other species as well. The number of recorded resistance patterns from each sample farm was at least nine (Table 3). This suggests to us that the tet(39) gene is widely distributed among unrelated Acinetobacter spp. of aquatic origin in Thailand. This was further supported by the RAPD–PCR analysis of all tet(39)-harbouring isolates from integrated fish farm B2 and 16 tet(39)-harbouring isolates from control farm C4 (Figure 1). The RAPD–PCR analysis demonstrated diversity both between isolates from the same farms and between isolates from different farms.
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hindIII. Lanes 2–5: isolates from farm B2, sampling time 1. Lanes 6–14: isolates from farm B2, sampling time 2. Lanes 15–20: isolates from farm B2, sampling time 3. Lanes 22–37, isolates from farm C4, sampling time 1. Lane 38: positive control DNA E. coli.tet(A) was most frequently (13/42, 31%) demonstrated among isolates from layer manure and only in two cases did an isolate originate from a control farm. None of the targeted tetracycline resistance genes was detected in 18% of the isolates. These isolates may contain unknown tetracycline resistance determinants or multidrug mechanisms like adeB.9 The sulphonamide resistance gene sulII was found to be widely distributed in isolates from both fish ponds and manure. The gene was recently demonstrated in an A. baumannii isolate from a hospital in South Africa,23 and is frequently found among Enterobacteriaceae.24,25 The more frequent prevalence of class I integrons and tet(A) in Acinetobacter spp. of animal origin may indicate that the chicken flocks spread class I integrons and tet(A) to the aquatic water ponds.
The tet(39) and sulII gene occurred at the same frequency in the resistant isolates in all farms, including control fish farms with no consumption of antimicrobials before or during the sampling period. The integrated fish–chicken farm B1 was a newly started facility where the occurrence of tetracycline- and sulfamethoxazole-resistant Acinetobacter spp. increased rapidly from <5% to 100% within the first 2 months of production.2 The prevalence of tet(39) among the oxytetracycline-resistant Acinetobacter spp. was almost constant during the sampling period: day 18 (days numbered after fish production started), 67% (2/3); day 31, 80% (4/5); day 45, 80% (8/10); and day 80, 69% (18/26). The oxytetracycline-resistant isolates that were also sulfamethoxazole-resistant and contained the sulII gene increased during the period: day 18, 67% (2/3); day 31, 60% (3/5); day 45, 90% (9/10); and day 80, 96% (25/26). This may indicate that these genes can be maintained in Acinetobacter spp. without a selective pressure and may explain the rapid increase in sulfamethoxazole- and oxytetracycline-resistant Acinetobacter spp. in the new production facility.2
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GenBank submission |
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The following partial sequences of tetA(39) (accession numbers DQ195074 [GenBank] , DQ195075 [GenBank] and DQ195076 [GenBank] ) and sulII (accession number DQ195077 [GenBank] ) described in this study have been submitted to GenBank.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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This study was funded by grants from the Danish Research Council for Agricultural and Veterinary Research (no. 23-02-0169 and no. 23-02-0153). We would like to thank Inge M. Hansen, Anette Nielsen, Maria Louise Johannsen and Diana Anette Allen for excellent technical assistance.
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References |
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