JAC Advance Access published online on June 11, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn233
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Original research |
Analysis and distribution of class 1 and class 2 integrons and associated gene cassettes among Escherichia coli isolates from swine, horses, cats and dogs collected in the BfT-GermVet monitoring study
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute, Höltystr. 10, 31535 Neustadt-Mariensee, Germany
* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: stefan.schwarz{at}fli.bund.de
Received 24 April 2008; returned 16 May 2008; revised 19 May 2008; accepted 19 May 2008
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Abstract |
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Objectives: In the BfT-GermVet monitoring study, 417 Escherichia coli isolates collected during 2004–06 in Germany from various disease conditions of pigs (n = 87), horses (n = 102) or cats/dogs (n = 228) were investigated for their susceptibility to 24 antimicrobial agents. This study dealt with the identification of integron-associated resistance genes among these isolates.
Methods: Class 1 and class 2 integrons were detected by PCR. The variable parts of the integrons were cloned and sequenced. Transformation and conjugation experiments were conducted to confirm a plasmid location of the integrons.
Results: Class 1 and/or class 2 integrons, alone or in different combinations, were detected in 79 of the 417 E. coli isolates. Four trimethoprim resistance genes (dfrA1/12/14/17), five streptomycin/spectinomycin resistance genes (aadA1/2/4/5/6), two streptothricin resistance genes (estX, sat2), one gentamicin/tobramycin/kanamycin resistance gene (aadB) and one chloramphenicol resistance gene (catB3) were detected. Seven different cassette arrangements were identified within class 1 integrons: aadA1 (21 isolates), dfrA1 + aadA1 (18 isolates), dfrA17 + aadA5 (9 isolates), dfrA12 + orfF + aadA2 (8 isolates), aadB + aadA1 (1 isolate), dfrA14 + recombined aadA6 (1 isolate) and dfrA1 + catB3 + aadA4 (1 isolate). Three different cassette arrangements in class 2 integrons, dfrA1 + sat2 + aadA1 (24 isolates), estX + sat2 + aadA1 (6 isolates) and estX + sat2 + 
aadA1 (1 isolate), were identified. The plasmid location of class 1 and/or class 2 integrons was confirmed in 37 isolates.
Conclusions: Class 1 and/or class 2 integrons carrying resistance gene cassettes were detected in 18.9% of the isolates tested. This molecular analysis complements the phenotypic susceptibility testing conducted in the BfT-GermVet monitoring study and helps to explain the persistence of resistance genes even without direct selective pressure.
Key Words: antimicrobial multiresistance , food animals , pets , companion animals , conjugation , plasmids
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsReferences |
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During the years 2004–06, the BfT-GermVet monitoring study was conducted in Germany. The study represented a complementary study to the National Resistance Monitoring Programme GERM-Vet of the Federal Office for Consumer Protection and Food Safety (BVL) and as such included only bacteria from disease conditions of animals not monitored in the GERM-Vet programme. In the BfT-GermVet study, 1626 bacterial isolates from 31 different disease conditions of cattle, swine, horses and cats/dogs were analysed.1 Among them, a total of 417 Escherichia coli isolates was collected from swine (urinary/genital tract infections including mastitis-metritis-agalactia syndrome), horses (genital tract infections) and dogs/cats (infections of the respiratory tract, urinary/genital tract or gastrointestinal tract) and investigated for their MICs to 24 antimicrobial agents or combinations of antimicrobial agents.2
Recently published BfT-GermVet follow-up studies have dealt with the molecular analysis of selected isolates for the resistance genes present and/or the molecular relationships of the respective bacteria.3,4 In the present study, we investigated the E. coli isolates collected in the BfT-GermVet study for the presence of class 1 and class 2 integrons and associated resistance gene cassettes.
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Materials and methods |
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Of the 417 E. coli isolates included in this study, 87 were from infections of the urinary/genital tract (including metritis-mastitis-agalactia syndrome) of swine, 102 from genital tract infections of horses and the remaining 228 from different disease conditions of cats/dogs, including infections of the respiratory tract (n = 28), the urinary/genital tract (n = 100) or the gastrointestinal tract (n = 100).2
All 417 isolates were analysed for class 1 and class 2 integrase genes as well as the sulphonamide resistance gene sul1, which represents part of the 3'-conserved segment (CS) of class 1 integrons, by previously reported PCR assays.5 PCR primers derived from the respective 5' and 3' CS sequences of the integrons served for the detection of the gene cassettes located within these class 1 and class 2 integrons.5 Same-sized amplicons were compared by restriction analysis with two to three restriction endonucleases. At least one representative of each type of amplicon was cloned and sequenced. Cloning experiments were performed with the vector pCR 2.1 and competent E. coli TOP10 cells (Invitrogen, Groningen, The Netherlands) or with the vector pPCR-Script Amp SK(+) and competent E. coli XL-10 Gold kan cells (Stratagene, La Jolla, CA, USA). The complete sequences of the amplicons were determined by primer walking, starting with the M13 universal and reverse primers. Sequence comparisons were carried out using the BLAST® programs blastn and blastp (http://www.ncbi.nlm.nih.gov/BLAST/; 21 April 2008, date last accessed) and with the ORF finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html; 21 April 2008, date last accessed). The nucleotide sequences of the amplicons have been deposited in the European Molecular Biology Laboratory database under accession numbers AM932671 [GenBank] –AM932678 [GenBank] , AM939644 [GenBank] and AM990340.
Transformation and conjugation experiments were conducted to confirm a plasmid location of the integrons. Conjugation was performed by filter mating with the rifampicin-resistant E. coli HK225 as recipient strain and a donor:recipient ratio of 1:5.5 Transconjugants were selected on LB agar plates containing rifampicin (100 mg/L) and trimethoprim (10 mg/L) or rifampicin (100 mg/L) and spectinomycin (100 mg/L). Transformation or electrotransformation into E. coli HB101 cells was performed, as described previously.5 The transformants were selected on LB agar supplemented with 10 mg/L trimethoprim and/or 100 mg/L spectinomycin. Transconjugants and transformants were tested for their antimicrobial susceptibilities by agar disc diffusion according to the CLSI (formerly NCCLS) document M31-A2.6
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Results and discussion |
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Prevalence of integrons and associated gene cassettes
A total of 90 class 1 and/or class 2 integrons were detected in 79 (18.9%) of the 417 E. coli isolates. Among them, single class 1 integrons were detected in 47 isolates and single class 2 integrons in 21 isolates. One isolate harboured two different class 1 integrons, whereas 10 isolates harboured a class 1 and a class 2 integron. Seven different cassette arrangements within class 1 integrons and another three arrangements within class 2 integrons were identified. Among the gene cassettes detected, those carrying genes for combined streptomycin/spectinomycin resistance (aadA genes) and trimethoprim resistance (dfrA genes) were most prevalent (Figure 1 and Table 1).
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Analysis of class 1 integrons and their gene cassettes
The first type of class 1 integron harboured a single gene cassette carrying the gene aadA1 coding for a 257 amino acid aminoglycoside adenyltransferase. Compared with all other database entries, the deduced amino acid sequence shows two substitutions in comparison with the next closely related, same-sized AadA1 proteins: Cys-30 is a Tyr and Ala-178 is an Asp in the AadA1 protein sequences of E. coli (YP001096431, AAC33912 [GenBank] and ABD59948 [GenBank] ) or Salmonella Bredeney (CAP69661 [GenBank] ).
The second type of integron harboured a dfrA1 and an aadA1 cassette. Although the 157 amino acid DfrA1 protein sequence was indistinguishable from those of several database entries, the 259 amino acid AadA1 protein differed by a single amino acid substitution from the next best same-sized database matches: the His-211 is an Arg in the AadA1 sequences of E. coli (CAI57692 [GenBank] and ABG46351 [GenBank] ) or Salmonella spp. (BAF63518 [GenBank] ).
The third type harboured unique dfrA17 and aadA5 cassettes. The 157 amino acid DfrA17 showed a single amino acid substitution at position 12, whereas the 262 amino acid AadA5 protein exhibited two substitutions at positions 167 and 235: the Ser-12 in the DfrA17 protein was Asn, and the Leu-167 and Gly-236 in the AadA5 protein were Phe and Asp, respectively, in the corresponding DfrA17 or AadA5 proteins of a large number of bacteria, including mostly Enterobacteriaceae, but also Pseudomonas aeruginosa or Acinetobacter spp.
The fourth type of class 1 integron harboured a dfrA12, an orfF and an aadA2 gene cassette. Neither the deduced amino acid sequences of the DfrA12 and AadA2 proteins nor the nucleotide sequence of the orfF differed from already known database entries. Database searches revealed that such integrons have been identified in various Gram-negative bacteria such as Salmonella, E. coli or Citrobacter freundii, but also in Staphylococcus warneri.
The fifth type carried aadB and aadA1 gene cassettes and an insertion element. Although the 177 amino acid AadB protein matched numerous database entries, the 259 amino acid AadA1 protein differed in its deduced amino acid sequence from all other known AadA1 proteins by a single substitution: Pro-162 was Leu in the next closely related AadA1 protein sequences of E. coli (YP001096431, AAC33912 [GenBank] and ABD59948 [GenBank] ) or Salmonella Bredeney (CAP69661 [GenBank] ). An insertion sequence-like element of 1375 bp was found to be integrated into the 2L integrase-binding domain of the 59-base element of the aadA1 gene cassette. This element exhibited a single reading frame for a putative transposase of 347 amino acids and was bounded by imperfect inverted repeats of 17 and 19 bp. No directly repeated sequences, which are characteristic for many insertion sequences, were detected at the integration site. A closely related element, whose transposase was identical in 341 (98.3%) of the 347 amino acids to the one found in this study, has previously been detected in P. aeruginosa from Spanish hospitals.7 In P. aeruginosa, this element was found to be integrated in a non-coding region downstream of a blaVIM-2 gene cassette.
The sixth type of class 1 integron harboured a typical dfrA14 gene cassette coding for a 157 amino acid DfrA14 dihydrofolate reductase and an unusual gene cassette previously described in P. aeruginosa as an aadA6/aadA10 fusion product.8 In this latter cassette, the 59-base element is missing, and the aadA6 reading frame for a 277 amino acid protein extends 22 bp into the 3' CS of the integron. However, a closer look at the aadA6 reading frame9 revealed that a recombination between the terminal part of the aadA6 gene and the attI site of an empty integron10 is an alternative explanation for the development of this structure. We identified a 27 bp region that showed 77.8% identity to the aadA6 reading frame and 74.1% identity to the attI site and might have served for the recombination (Figure 2). The only so-far known aadA10 gene cassette11 also lacks a 59-base element, shows an extension of the aadA10 reading frame into the 3' CS sequence and hence may also be the result of a recombination between a formerly intact aadA10 cassette and an empty integron (Figure 2).
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The seventh type of class 1 integron consisted of an already known dfrA1 cassette, but also novel types of catB3 and aadA4 cassettes. The 210 amino acid CatB3 protein showed two substitutions: Gly-122 and Val-190 were Glu and Met, respectively, in the most closely related CatB3 proteins of Enterobacteriaceae, Aeromonas hydrophila, Bordetella bronchiseptica, P. aeruginosa or Acinetobacter baumannii. The 262 amino acid AadA4 protein exhibited a single amino acid exchange: Ala-121 was Thr in the next closely related AadA4 proteins found in Enterobacteriaceae, P. aeruginosa or Acinetobacter spp.
Analysis of class 2 integrons and their gene cassettes
Among the class 2 integrons, only three cassette arrangements were detected. The first comprised already known dfrA1 and sat2 gene cassettes, but also a novel aadA1 cassette coding for a 258 amino acid AadA1 protein. In comparison with the regular 259 amino acid AadA1 proteins, this novel variant lacked Glu at position 231. An integron-borne aadA1 gene, which also lacks the codon for Glu-231, has recently been described in Salmonella Bredeney from slaughter pigs in Brazil.12 Moreover, a 1 bp deletion followed by a 1 bp insertion 11 bp further downstream led to the substitution of four consecutive codons. The sequence 27-Val-His-Leu-Tyr-30 present in all other AadA1 proteins was modified to 27-Arg-Thr-Phe-Asp-30 in the present case.
The second type of class 2 integron harboured a novel variant of an estX cassette and the aforementioned sat2 and aadA1 cassettes. The 280 amino acid EstX protein described in the databases as streptothricin acetyltransferase differs by a single amino acid substitution at position 241 from the so-far known EstX proteins: Glu in the present case versus Asp in all other EstX sequences.
The third type of class 2 integron also harboured estX, sat2 and aadA1 gene cassettes, but differed slightly upon restriction analysis from the aforementioned second type. We observed that one of the PvuI, BamHI and BclI fragments was ~200 bp smaller than that seen in the type 2 amplicon. The sequence analysis revealed the presence of a 198 bp deletion immediately upstream of the translational stop codon of the aadA1 gene, which then codes for a 192 amino acid protein. Susceptibility testing of the clone carrying this amplicon confirmed that this aadA1 gene does not confer streptomycin/spectinomycin resistance. Further analysis of the estX gene cassette revealed one amino acid exchange at position 41: Gly in the present case versus Pro in the other known EstX sequences. The sat2 gene cassette corresponded exactly to those seen in the other two types of class 2 integrons described in this study.
Plasmid location and distribution among different animal hosts
Although only 9 isolates were plasmid-free, class 1 or class 2 integrons were transferred by conjugation or transformation in 28 and 9 isolates, respectively. Among the integron-carrying plasmids, 29 proved to be conjugative and exhibited sizes of 
90 kb. The 37 plasmids harbouring class 1 or class 2 integrons usually also mediated other resistance properties such as resistances to tetracycline (n = 21), ampicillin (n = 20), chloramphenicol (n = 5), chloramphenicol+florfenicol (n = 3), neomycin (n = 6), trimethoprim (n = 4) or gentamicin (n = 3). All nine plasmids that harboured a class 2 integron also conferred sulphonamide resistance. The observation that the same integrons have been detected in E. coli isolates from different animal sources (Table 1) points towards the transferability of these integrons. In this regard, the location of the integrons on mostly conjugative plasmids might facilitate their exchange. The co-location of other resistance traits on the same plasmid strongly supports the possibility of co-selection and persistence of resistance genes, even in the absence of a direct selective pressure. This aspect is further supported by the presence of the streptothricin resistance genes sat2 and estX in a considerable number of E. coli isolates. Streptothricin antibiotics have not been used as veterinary therapeutics, but have been used as growth promoters since the mid-1980s in the former German Democratic Republic.13
Comparisons with the results obtained in recent studies conducted in the Netherlands,14 Switzerland,15 Portugal16 or Korea17 revealed that E. coli isolates from animal or environmental sources also carried a wide range of gene cassette arrays in class 1 and class 2 integrons. Similar to the results obtained in the present study, class 1 integrons carrying either an aadA1 cassette alone or dfrA1-aadA1 cassettes were seen most frequently in the E. coli isolates from Europe,14–16 whereas the cassette arrays dfrA17-aadA5 and dfrA12-orfF-aadA2 were most prevalent among the Korean E. coli isolates.17 Regardless of the origin of the E. coli isolates, the cassette array dfrA1-sat2 (sat/sat1)-aadA1 was detected most frequently in class 2 integrons.14–17 As far as the transferability of the integrons has been tested, 70% of the class 1 integrons14,17 and 66% of the class 2 integrons14 were transferable by conjugation. In the present study, only 24 of the 59 class 1 integrons (40.7%) and 5 of the 31 class 2 integrons (16.1%) were transferable by conjugation. Nevertheless, conjugative transfer seems to be an efficient way for the horizontal dissemination of such integrons across species and genus borders.
In summary, the data obtained in this study represent a molecular complement to the previously published MIC data of a representative set of E. coli isolates from defined disease conditions of animals.2 Sequencing of the resistance genes revealed the presence of three novel aadA1 variants and one functionally inactive aadA1 gene in addition to novel aadA4, aadA5, dfrA17, catB3 and estX variants.
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Funding |
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This study was financially supported by the Bundesverband für Tiergesundheit (BfT) e.V.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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We thank Friederike Kops, Kerstin Meyer and Regina Ronge for excellent technical assistance.
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References |
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