1 Faculdade de Ciências da Nutrição e Alimentação, Universidade do Porto, Porto; 2 Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, no. 164, 4050-047 Porto; 3 Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal
Received 3 February 2004; returned 5 April 2004; revised 14 May 2004; accepted 19 May 2004
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Abstract |
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Methods: The isolates were examined for susceptibility to antimicrobial agents. The relationships between resistance genes, class 1 integrons, plasmids and isolates were screened by molecular methods such as polymerase chain reaction and restriction fragment length analysis.
Results: A blaOXA-30 gene, located in a class 1 integron, was detected in all isolates. This integron was present on a conjugative plasmid in all but one isolate. By pulsed-field gel electrophoresis, it was determined that all strains share the same chromosomal type.
Conclusions: This study demonstrates the spread of an OXA-30-producing S. typhimurium in Portugal, suggesting dissemination of a resistant clone through the food chain.
Keywords: salmonellosis , food-borne diseases , class 1 integrons , antimicrobial resistance , ß-lactams
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Introduction |
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During an antibiotic resistance survey of Salmonella isolates from human and food samples in Portugal in 2002/2003, we detected nine isolates with reduced susceptibility to co-amoxiclav and synergy of clavulanic acid with cefotaxime and cefepime. In this study, we have characterized the molecular basis of ß-lactam resistance in these isolates, and have determined the relationship between the isolates, and the resistance genes, class 1 integrons and plasmids carried by them.
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Materials and methods |
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Seven human S. typhimurium isolates from unrelated clinical cases (one outbreak and six sporadic cases) were obtained from stools in four geographically distant hospital units in Portugal between September 2002 and May 2003. Five isolates were recovered from children with typical gastroenteritis, without any history of antimicrobial therapy. Salmonella 172/03 was isolated from a 3-year-old child with gastroenteritis following co-amoxiclav treatment for recurrent otitis. The remaining isolate, Salmonella BIO104H, was isolated from a 1.5-month-old infant, fed with mother's breast milk, that presented inflammation of the mammary gland on admission to the hospital. Although the patient's clinical condition improved, after flucloxacillin administration, the clinical course was complicated, on day 5, by severe gastroenteritis (bloody and mucous diarrhoea). During the same period, two S. typhimurium isolates were recovered from pork end products: one from a pork sausage collected in the north of Portugal and one from a food mainly consisting of fried bacon and pork grease, collected in the southern countryside (Table 1).
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Antimicrobial susceptibility testing and investigation of ß-lactamase expression
The MICs of 10 antimicrobial agents (streptomycin, kanamycin, gentamicin, ampicillin, nalidixic acid, ciprofloxacin, chloramphenicol, tetracycline, sulfamethoxazole and trimethoprim) were determined by the agar dilution method.9 Susceptibility to ß-lactam agents was assessed both by the disc diffusion10 and Etest (AB Biodisk, Solna, Sweden) methods. A double-disc synergy test for the detection of ESBL production was carried out using a co-amoxiclav disc placed next to ceftazidime, cefotaxime, cefuroxime, aztreonam, ceftriaxone and cefepime discs on MuellerHinton agar 2 (bioMérieux).
The pIs of the ß-lactamases expressed by the isolates were determined by isoelectric focusing in a PhastSystem (Pharmacia AB, Uppsala, Sweden) with ampholine gels of pH 39 (Phast gels, Amersham Biosciences, Uppsala, Sweden). ß-Lactamase bands were visualized with nitrocefin (100 µM) (Oxoid, Basingstoke, UK). Detection of ß-lactamase genes by PCR was carried out using primers designed for the detection of genes encoding OXA group III11 and TEM-type12 enzymes.
Conjugation and plasmid analysis
Conjugative transfer of plasmids from S. typhimurium isolates into Escherichia coli K802N (hsdR, hsdM, gal, met, supE, gyrA) or E. coli K802N (RifR) was attempted using agar plates. Transconjugants were selected on MuellerHinton agar 2 (bioMérieux) containing ampicillin (64 mg/L) plus nalidixic acid (64 mg/L) (or 100 mg/L rifampicin if the donor was nalidixic acid-resistant) and sulfamethoxazole (256 mg/L) plus nalidixic acid (64 mg/L) or rifampicin (100 mg/L). Plasmids harboured by S. typhimurium isolates and E. coli transconjugants were extracted using a rapid methodology (QIAGEN Plasmid Midi Kit; Qiagen, Hilden, Germany). The sizes and relatedness of plasmids harboured by the transconjugants were estimated by single restriction analysis using EcoRI, HindIII and BamHI.
Detection and characterization of class 1 integrons
The presence of class 1 integrons in the isolates was confirmed by PCR using the primers 5'CS-3'CS.13 To determine the content of the variable regions of the integrons, the 5'CS primer was used in combination with a reverse primer for the streptomycin resistance gene [ant(3')-Ia]4 and a reverse primer for OXA group III.11 Restriction fragment length polymorphism (RFLP) analysis using TaqI was also carried out on all class 1 integron PCR amplicons. To confirm the association of integrons with conjugative plasmids, PCRs using the 5'CS and reverse OXA group III primers were also carried out using plasmids extracted from transconjugants as the template. The presence of sul1 and qacE1 genes was confirmed by PCR, using specific primers.4
PCR amplicons obtained with 5'CS and 3'CS primers from four isolates (two from humans and two from food products) were purified and sequenced by the dideoxy-chain termination method with an ALF Express automatic DNA sequencer (Amersham Pharmacia Biotech, Uppsala, Sweden), using the primers 5'CS and 3'CS, and the internal primer (5'-GGATTAACAGAAGCATGGCT-3').
Pulsed-field gel electrophoresis
Clonality amongst the isolates was assessed by PFGE following XbaI digestion of genomic DNA according to the standard 1-day protocol of the CDC. Genomic DNA from Salmonella braenderup H9812 obtained from the CDC was also restricted with XbaI and used as a size standard. Isolates with electrophoretic patterns that differed by less than three bands, at most, were assigned to the same type.
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Results |
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PCR using various primer combinations to enable amplification of class 1 integron variable regions showed that in each isolate, a gene encoding a ß-lactamase of the OXA group III, which includes OXA-1, OXA-4, OXA-30 and OXA-31,11 and the ant(3')-Ia gene were carried in a class 1 integron of about 2000 bp. All of the integrons yielded the same TaqI RFLP profile (data not shown) and sul1 and qacE1 genes in the conserved segment 3'CS. The sequence of each amplicon revealed two gene cassettes, with blaOXA-30 being 5' proximal to aadA1 (GenBank accession no. AY534545).
The integron carrying blaOXA-30 was associated with a conjugative plasmid (>70 kb) in eight of the S. typhimurium isolates (Figure 1) which were able to transfer blaOXA-30 to E. coli K802N. The ninth isolate, 236/03, which expresses two ß-lactamases, was only able to transfer the pI 5.4 ß-lactamase gene. PCR analysis revealed that this gene is from the blaTEM group. Six of the conjugative plasmids carrying blaOXA-30 confer the ACSSulT resistance profile on the E. coli recipient, and restriction digests for these six plasmids were identical. In two isolates, BIO67H and 172/03, amoxicillin, streptomycin and sulfamethoxazole resistance, as well as tetracycline resistance in 172/03, was encoded on the conjugative plasmids, which were different from each other, and from the plasmid carrying the ACSSulT resistance profile according to RFLP analysis (Table 2 and Figure 1).
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Discussion |
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There have only been a few reports of OXA-30 ß-lactamase-producing strains of Shigella flexneri,15 E. coli1618 and S. typhimurium2 and all are from human sources. The association of blaOXA-30 with class 1 integrons has recently been described in S. typhimurium19 and E. coli18 from human patients. The data presented here confirm that class 1 integrons carrying blaOXA-30 can be found in S. typhimurium isolates from products having animal origins. All the evidence presented suggests that the dissemination of OXA-30-producing S. typhimurium isolates throughout Portugal could be explained by the clonal spread of a particular strain. As the majority of Salmonella infections result in asymptomatic or self-limited diarrhoeal illness, it is probable that the real number of cases associated with this clone has been underestimated. Based on all available information, it is most likely that the OXA-30-producing S. typhimurium strain has been transmitted from food animals to humans. The fact that the two pork products were produced in geographically distinct regions minimizes the likelihood of independent contamination of these products by humans during processing.
It is tempting to speculate that acquisition of blaOXA-30 may relate to therapeutic use of ß-lactams in pig production. However, persistence of multidrug-resistant strains of Salmonella in farm animals may be further encouraged by the use of other antimicrobials, a common practice in the veterinary field. Our results indicate the potential human health hazard of multiresistant S. typhimurium isolated from food and corroborate the increasing levels of antibiotic resistance in this microorganism. In addition, blaOXA-30 was shown to be transferable through conjugation. Under such circumstances, further spread of blaOXA-30 is likely to occur. Also of special concern is the high level of resistance to extended-spectrum cephalosporins due to the high level of expression of OXA-30, as described by Oliver et al.16 The establishment and dissemination of a S. typhimurium clone that is different from the widespread multidrug-resistant clone of S. typhimurium DT104, with decreased susceptibility to therapeutically important broad-spectrum ß-lactams, is a cause for concern.
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Acknowledgements |
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Footnotes |
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References |
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