Dissemination amongst humans and food products of animal origin of a Salmonella typhimurium clone expressing an integron-borne OXA-30 ß-lactamase

Patrícia Antunes1,2, Jorge Machado3, João Carlos Sousa2 and Luísa Peixe2,*

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: Characterization of the molecular basis for ß-lactam resistance and evaluation of the clonal relatedness among nine isolates of multidrug-resistant Salmonella typhimurium recovered from seven clinical human samples and two pork end products.

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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
ß-Lactam antibiotics are widely used in the treatment of salmonellosis in humans, particularly in children and neonates, as well as in food animals. It is accepted that the use of antimicrobials in food animals has been a major factor in the emergence and dissemination of Salmonella with decreased susceptibility to antibiotics, including ß-lactams.1 Resistance to broad-spectrum cephalosporins is widespread among strains of Salmonella with increasing reports of isolates that produce either an extended-spectrum ß-lactamase (ESBL), such as TEM- and SHV-type, CTX-M-type, or plasmid-mediated AmpC-type enzymes.2 Recently, an OXA-30 ß-lactamase produced by Salmonella typhimurium has been described.3 Resistance to multiple classes of antibacterial agents among Salmonella is increasingly associated with the presence of class 1 integrons carrying multiple resistance genes. blaPSE-1,4,5 blaCTX-M-26 and blaOXA-15,7 have previously been shown to be carried on integrons in this genus.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacterial strains

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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Table 1. Epidemiological information for the S. typhimurium isolates used in this study

 
The biochemical profile of Salmonella was confirmed using the API 32GN system (bioMérieux, Marcy l'Étoile, France). The serotype of each isolate was determined at the National Centre of Salmonella (Instituto Nacional de Saúde, Lisboa, Portugal). A PCR assay for identification of S. typhimurium DT104 and U302 phage types was conducted according to Pritchett et al.8

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 Mueller–Hinton 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 3–9 (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 Mueller–Hinton 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 qacE{Delta}1 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Resistance to amoxicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline (ACSSulT) was observed in eight of the S. typhimurium isolates, with the other (isolate 172/03) lacking chloramphenicol resistance. Additionally, resistance to nalidixic acid was observed in isolate 368/03, and resistance to trimethoprim plus gentamicin in isolate 236/03 (Table 2). Although the ACSSulT resistance profile is usually associated with S. typhimurium DT104,4,5 none of the isolates in this study was from the DT104 or U302 phage types according to the multiplex PCR test used as described by Pritchett et al.8


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Table 2. Antimicrobial susceptibility profiles of S. typhimurium isolates and transconjugants

 
The MICs of ß-lactams revealed resistance to aminopenicillins and carboxypenicillins, with activity of these agents being restored by clavulanic acid (Table 2). Although the isolates were susceptible to cephalosporins, a decreased activity of cefotaxime, cefepime and cefpirome was observed. A double-disc test revealed synergy between co-amoxiclav and cefepime, cefotaxime and cefuroxime. The clavulanic acid effect was more pronounced with cefepime in all of the isolates. Using isoelectric focusing, eight of the isolates were shown to express a single ß-lactamase with a pI value of 7.3. The ninth isolate, 236/03, expresses two detectable ß-lactamases, one with a pI of 7.3, and one with a pI of 5.4.

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 qacE{Delta}1 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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Figure 1. Restriction patterns of DNA plasmids isolated from E. coli transconjugants. Lanes: 1 and 26, {lambda} HindIII molecular marker; 2 to 9, plasmids digested with HindIII; 10 to 17, plasmids digested with EcoR1; 18 to 25, plasmids digested with BamH1. Isolate: 172/T, lanes 2, 10 and 18; 237/T, lanes 3, 11 and 19; 367/T, lanes 4, 12 and 20; 368/T, lanes 5, 13 and 21; C-11/T, lanes 6, 14 and 22; BIO104H/T, lanes 7, 15 and 23; LP-2/T, lanes 8, 16 and 24; BIO67H/T, lanes 9, 17 and 25.

 
PFGE showed that all of the OXA-30-producing S. typhimurium isolates are highly related. The PFGE profiles of human isolates differ from the food-borne isolates by less than three bands, suggesting clonality (Figure 2).



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Figure 2. PFGE patterns of four Salmonella typhimurium isolates. Lanes: 1, LP-2; 2, C-11; 3, BIO67H; 4, BIO104H; 5, Salmonella braenderup H9812 (CDC).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In Portugal, ~68 000 kg of antibiotics were used in animals in 1997,14 and mostly in pigs. Overall, the antimicrobials most frequently used in pigs are ß-lactams, tetracyclines and sulfamethazine. It is commonly accepted that the use of antimicrobials in food animals has been a major contributor in the emergence and dissemination of Salmonella with decreased susceptibility to antibiotics, including ß-lactams.1

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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We are very grateful to Rafael Cantón (Hospital Ramon et Cajal, Madrid, Spain) for critical reading of the manuscript, to Luís Marinho (Hospital S. Sebastião, Santa Maria da Feira, Portugal), Pedro Vale (Hospital de S. Tirso, Santo Tirso, Portugal), Ana Paula Castro (Hospital Distrital de Vila Real, Vila Real, Portugal) and Teresa Sardinha (Hospital Amadora-Sintra, Lisboa, Portugal) for providing the clinical data used in this study, to Centro Nacional de Salmonella (Lisboa, Portugal) for the serotyping of the strains and to CDC for the PFGE protocols and the control strain Salmonella braenderup H9812. This study was partially supported by Fundação Calouste Gulbenkian, Portugal (project no. 49975).


    Footnotes
 
* Corresponding author. Tel: +351-22-2078972; Fax: +351-22-2003977; Email: lpeixe{at}ff.up.pt


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 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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4 . Sandvang, D., Aarestrup, F. M. & Jensen, L. B. (1998). Characterisation of integrons and antibiotic resistance genes in Danish multiresistant Salmonella enterica Typhimurium DT104. FEMS Microbiology Letters 160, 37–41.[CrossRef][ISI][Medline]

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