JAC Advance Access originally published online on June 16, 2006
Journal of Antimicrobial Chemotherapy 2006 58(2):305-309; doi:10.1093/jac/dkl248
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Occurrence of integrons and antimicrobial resistance genes among Salmonella enterica from Brazil
1 Oswaldo Cruz Institute Avenida Brasil 4365, 21045-900 Rio de Janeiro, Brazil 2 WHO Collaborating Centre for Antimicrobial Resistance in Foodborne Pathogens, Danish Institute for Food and Veterinary Research Bülowsvej 27, DK-1790 Copenhagen V, Denmark
*Corresponding author. Tel: +45-72-34-60-00; Fax: +45-72-34-60-01; E-mail: faa{at}dfvf.dk
Received 27 February 2006; returned 26 March 2006; revised 23 May 2006; accepted 24 May 2006
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Abstract |
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Objectives: To determine the occurrence of antimicrobial resistance genes and role of integrons among 135 antimicrobial-resistant Salmonella enterica from Brazil.
Methods: The presence of antimicrobial resistance genes, class 1 and 2 integrons and gene cassettes was analysed by PCR and sequencing. The genetic location of class 1 integrons was determined in 25 isolates by hybridization and plasmid transfer experiments.
Results: Fifty-five of the isolates were positive for class 1 integrons. Integron-positive isolates represented 17 different serovars and were mainly from human (n = 28) and animal (n = 13) sources. The gene cassette arrangements could be determined in 51 of the positive isolates, which harboured one [dfrA22, aadA1 or orf3 (putative trimethoprim resistance)], two [aadA1-dfrA1, aac(6')-Ib-orf1 (unknown function) or aacA4-aadA1], three [dfrA15b-cmlA4-aadA2, orf2 (unknown function)-dfrA5-orfD] or four [orf4-aacA4-blaOXA-30 (interrupted by an IS1 element)-aadA1] cassettes in their variable region. Only one isolate harboured a class 2 integron with the gene cassette array dfrA1-sat-aadA1. Several integron unrelated resistance genes were also detected in the isolates. Sulphonamide resistance was primarily mediated by sul2 and sul3, tetracycline resistance by tet(B) and tet(A), chloramphenicol resistance by catA1, streptomycin resistance by strA and ampicillin resistance by blaTEM. blaCTX and blaCMY-2 were found in cephalosporin-resistant isolates. Mating and hybridization experiments demonstrated that a high-molecular-weight plasmid mediated the gene transfer of integrons and additional resistance determinants.
Conclusions: The present study revealed that integron-mediated resistance genes contributed to the multiresistance phenotype observed in the isolates, but most resistance genes were located outside the integron structure, as independent genes. However, they might be located on the same conjugative plasmid.
Keywords: gene cassettes , Salmonella , resistance determinants
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Introduction |
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Multiresistant Salmonella isolates of different serovars are increasingly common worldwide.1 Integrons are genetic elements comprising a site-specific recombination system capable of integrating and expressing resistance genes contained in cassette-like structures.2 The most frequently reported are class 1 and class 2 integrons, which have been shown to contribute to the spread of antimicrobial resistance genes.3
The present study was conducted to investigate the occurrence of antimicrobial resistance genes and integrons (class 1 and 2) as well as horizontal transfer of antimicrobial resistance among Salmonella isolated from different sources in Brazil.
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Materials and methods |
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A total of 135 antimicrobial-resistant Salmonella enterica isolates were collected during 1999–2003 from different Brazilian regions. The isolates were from humans (48), animal feed (41), food-producing animals (23), foodstuff (11) and other sources (12). Antimicrobial susceptibility was re-tested at the Danish Institute for Food and Veterinary Research by determining MICs using a commercially dehydrated panel Sensititre (Trek Diagnostic Systems, UK).4 The number of isolates, serovar and antimicrobial susceptibility are shown in Table 1.
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PCR amplifications for class 1 and 2 integrons were performed as described previously.5 Characterization of gene content and their order into class 1 integrons was performed in three steps: (i) representative amplicons of different sizes were sequenced; (ii) primers were designed; and (iii) PCR was performed to determine the gene cassette content in all other isolates with corresponding size amplicons. Sequences were analysed using VECTOR NTI Suite 8 (InforMax, Inc.) and compared with the sequences in GenBank (http://www.ncbi.nlm.nih.gov/BLAST).
Detection of antimicrobial resistance genes was performed for sulfamethoxazole-resistant isolates (sul2 and sul3); ampicillin-resistant isolates (blaTEM, blaOXA, blaCTX and blaCMY); chloramphenicol/florfenicol-resistant isolates (catA1, cmlA and floR); tetracycline-resistant isolates [tet(A), tet(B), tet(C), tet(D) and tet(G)], streptomycin-resistant isolates (aadA, strA and strB), gentamicin-resistant isolates [ant(2'')-Ia, aac(3)-IIa, aac(3)-IIIa and aac(3)-IVa] and neomycin-resistant isolates [aph(3)-Ia, aph(3)-IIa and aph(3)-IIIa]. Primers were those previously reported4–12 and the following: aac6-Ib 5'-ATCGGCTGATTGAAGCCTCAA-3'; orf1 5'-TTCCCGATTTCATCGAAGA-3'; dfrA22 5'-CATTTATCTGGTCGCTGC-3' and 5'-ACGACGTGCATAGACGGA-3'; dfrA1 5'-GGCAGATTTGGTAACTATAA-3'; orfD 5'-CGCATTCTGCGGTC-3'; aadA1-74 5'-GAGTCGATACTTCGGCGA-3'; dfrA15b 5'-CAGATGAGCACTTCCAAGA-3'; aadA2 5'-ATGACGCTTAGCACCTC-3'.
Mating experiments were performed for 17 class-1-integron-positive and streptomycin-resistant isolates as donors into Escherichia coli 1005 (RIFR NALR). Transfer of class 1 integron and additional resistance genes was confirmed by PCR. Plasmid DNA was extracted using an alkaline lysis method in 25 integron-positive isolates and 10 transconjugants.
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Results and discussion |
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A total of 55 isolates harboured a class 1 integron. We found class 1 integrons in serovars where they have not previously been reported (Anatum, Kottbus, Saintpaul and Senftenberg), confirming the widespread occurrence of these genetic structures.
A class 2 integron (with dfrA1, sat1 and aadA1) was identified in one isolate of Salmonella Heidelberg, which also contained a class 1 integron. To our knowledge, class 2 integrons in Salmonella have been identified so far only in Salmonella Typhimurium13 and Salmonella Paratyphi.14
Sequencing of class 1 integrons revealed 17 different gene cassettes in 10 different combinations (Figure 1). aadA1 was the most frequently observed gene cassette, but 20 class 1 and 2 integron-negative isolates also carried aadA1. Thus, class 1 integrons are not alone responsible for the spread of streptomycin/spectinomycin resistance.
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Six different open reading frames (orf, orf1, orf2, dfrA25, orf4 and orfD), with unknown function, were found as gene cassettes. aac(6')-Ib was followed by orf1 in nine isolates. orf2 was followed by dfrA5 and orfD. orfD is one among the six ORFs that have previously been described associated with class 1 integrons.3,15 orf3 is based on 85% amino acid homology to dfrA5, probably a new trimethoprim resistance gene. orf4 was followed by the aacA4 gene cassette, a blaOXA-30 interrupted by an IS1 element and an aadA1 gene cassette in one isolate. blaOXA-30 was found inserted in a class 1 integron of Salmonella Agona. The association between blaOXA-30 and class 1 integrons has previously been described in Salmonella Typhimurium.16
Table 2 shows the distribution of antimicrobial resistance genes among the Salmonella isolates.
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All 84 ampicillin-resistant isolates contained either blaTEM (80 isolates) or blaCMY (four isolates). blaCTX was found in association with the blaTEM gene in 15 isolates. Fifteen representative positive amplicons generated with each one of the bla primers were sequenced (five blaTEM, seven blaCTX and three blaCMY). Comparison with GenBank revealed identities to blaTEM-1 (4 isolates), blaTEM-104 (1 isolate), blaCTX-M-8 (1 isolate), blaCTX-M-9 (6 isolates) and blaCMY-2 (3 isolates), respectively. Resistance to ampicillin in Salmonella is usually mediated by TEM ß-lactamases as also found in this study. blaCMY-2 encoding cephalosporin resistance was found in Salmonella Heidelberg ceftriaxone-resistant isolates. This gene has been identified in many Gram-negative enteric pathogens, including Salmonella.1 Six isolates contained blaCTX-M-9 and one blaCTX-M-8. We always found blaCTX concomitantly with blaTEM and as an integron-independent gene.
We found five different trimethoprim resistance genes (dfrA1, dfrA5, dfrA12, dfrA15b and dfrA22) as gene cassettes in class 1 integrons. This is to our knowledge the first description of dfrA22 as a gene cassette in Salmonella. The most prevalent trimethoprim gene among Gram-negative bacteria seems to be dfrA1, which occurs as a cassette of both class 1 and class 2 integrons.17 However, in our study nearly 41% of the trimethoprim-resistant isolates were integron-negative. In three of these isolates there was a functional inactivation on the strA coding region due to the insertion of a dfrA14 gene.
Sulfamethoxazole resistance was found in 121 isolates. sul1 was detected as part of a class 1 integron in 55 isolates. sul2 alone was found in 61 isolates. Both sul1 and sul2 were present in 22 isolates. sul3 alone was detected in 6 isolates. Class 1 integrons were also detected in two sulfamethoxazole-susceptible isolates. sul1 and sul2 were almost equally common. In Salmonella Weltevreden sul2 was found to be the most common cause of sulphonamide resistance.4 sul3 was recently detected among Salmonella strains isolated from livestock and food in Germany.18
Tetracycline resistance genes were detected among all the 119 tetracycline-resistant isolates. The distribution was as follows: 58 tet(B), 25 tet(A), 23 tet(D) and 10 tet(C). tet genes were also found in combinations of tet(A) + tet(B) (one Salmonella 3,10:h:–), tet(A) + tet(C) (one Salmonella Worthington) and tet(B) + tet(D) (one Salmonella Rissen). None of the isolates was positive for tet(G). Previous studies identified tet genes of classes B and C to occur most frequently in Salmonella.19
The chloramphenicol resistance genes catA1 and cmlA1 were found in 45 and 7 isolates, respectively, of a total of 59 chloramphenicol-resistant isolates. Two isolates harboured cmlA4 as an integron gene cassette as well. Seven isolates were negative for both genes. The floR gene was found in five of six florfenicol-resistant isolates. In five isolates more than one gene was responsible for chloramphenicol resistance.
A total of 116 isolates were resistant to at least one of the six aminoglycosides tested. aadA was found in 44 streptomycin/spectinomycin-resistant isolates, while strA was found either in association with aadA (18 isolates) or alone in 38 streptomycin-resistant isolates. strB was found to occur only in association with strA in three isolates. Both genes can confer streptomycin (but not spectinomycin) resistance and are widely distributed in Salmonella and other Gram-negative species.20 Interestingly, the therapeutic use of streptomycin has declined in both human and veterinary medicine, but the persistence might be caused by co-selection.
Gentamicin resistance was mediated by aac(3)-IIa in 17 isolates, or by aac(3)-IVa (5 isolates) or ant(2'')-Ia (2 isolates) when the resistance profile included also apramycin or kanamycin, respectively. In three gentamicin-resistant isolates no genes encoding gentamicin resistance could be detected. Five of six apramycin-resistant isolates contained aac(3)-IVa, which also encodes gentamicin resistance. Neomycin resistance was encoded by aph(3')-Ia (24 of 26 isolates).
Mating experiments showed that nine Salmonella isolates were able to transfer the class 1 and 2 integrons, and also additional resistance genes. Transconjugants acquired resistance also to streptomycin/spectinomycin, gentamicin, neomycin, apramycin, ampicillin, cephalosporins, chloramphenicol and tetracycline. Transconjugant resistance profiles proved, by PCR, the co-transfer of aadA1, blaTEM, blaCTX, aac(3)-IIa, aphA-1, catA1, tet(A), tet(B) and tet(C) genes. Except aadA1, none of the additional genes was part of the integron; however, they were probably located on the same plasmid. We found that class 1 integrons were located on a high-molecular-weight transferable plasmid (
150 kb) in 11 of 25 isolates.
In conclusion, integron-mediated resistance genes contributed to the multiresistance phenotype observed in the isolates, but most resistance genes were located outside the integron structure. However, they might be located on the same conjugative plasmid. Despite the low occurrence of resistant Salmonella in Brazil, it would be prudent to identify reasons and possible sources for the emergence of these resistant bacteria. A better understanding of the molecular mechanisms by which antimicrobial resistance emerges and spreads should enable us to design intervention strategies to reduce its progression.
Genbank submissions
The DNA sequences of the gene cassette arrangements containing the four new ORFs (orf1, orf2, orf3 and orf4) were submitted to GenBank, with accessions numbers DQ267940, DQ278190, DQ278188 and DQ278189, respectively.
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None to declare.
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Acknowledgements |
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We are grateful to Berith Kummerfelt for technical assistance and to Kirsten Vestergaard for technical assistance in DNA sequencing. This work was supported by the WHO Global Salmonella Survey Program, CAPES Foundation and CNPq (Brazilian National Council for Scientific and Technological Development).
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