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Antimicrobial Agents and Chemotherapy, February 2005, p. 836-839, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.836-839.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Dissemination of Sulfonamide Resistance Genes (sul1, sul2, and sul3) in Portuguese Salmonella enterica Strains and Relation with Integrons

Patrícia Antunes,1,2 Jorge Machado,3 João Carlos Sousa,2 and Luísa Peixe2*

Faculdade de Ciências da Nutrição e Alimentação,1 Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Porto,2 Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisbon, Portugal3

Received 5 July 2004/ Returned for modification 16 September 2004/ Accepted 17 October 2004


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ABSTRACT
 
In 200 sulfonamide-resistant Portuguese Salmonella isolates, 152 sul1, 74 sul2, and 14 sul3 genes were detected. Class 1 integrons were always associated with sul genes, including sul3 alone in some isolates. The sul3 gene has been identified in isolates from different sources and serotypes, which also carried a class 1 integron with aadA and dfrA gene cassettes.


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TEXT
 
Salmonella enterica is a zoonotic pathogen transmitted through the food chain to humans, with contaminated foods of animal origin being important sources of infection. In particular, S. enterica serotype Typhimurium definitive phage type 104 (DT104), with resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracyclines (R-type ACSSuT), has emerged as a global health problem. The use of antimicrobial agents in food animals has been a major factor in the emergence of Salmonella with decreased susceptibility to antibiotics (19). A basic role in the spread of antimicrobial resistance in Salmonella has been attributed to class 1 (6, 17) and class 2 (4, 13) integrons. Sulfonamide resistance in gram-negative bacilli generally arises from the acquisition of either of the two genes sul1 and sul2, encoding forms of dihydropteroate synthase that are not inhibited by the drug (2). The sul1 gene is normally found linked to other resistance genes in class 1 integrons, while sul2 is usually located on small nonconjugative plasmids (18) or large transmissible multiresistance plasmids (2). Recently, a new plasmid-borne sulfonamide resistance gene called sul3 has been discovered (15). The objective of this study was to evaluate the incidence of sulfonamide resistance genes and class 1 and class 2 integrons among sulfamethoxazole-resistant nontyphoidal Salmonella isolates from different sources in Portugal.

For this study all the sulfonamide-resistant isolates (200) from a total of 1,183 Portuguese Salmonella isolates apparently epidemiologically unrelated collected during 2002 and 2003 were selected. The strain collection was obtained from the National Center of Salmonella (Lisbon, Portugal) and from clinical and food microbiology laboratories dispersed in our country. The MIC of sulfamethoxazole was determined by the agar dilution method, according to the NCCLS (12), with Mueller-Hinton agar 2 (bioMérieux, Marcy-l'Etoile, France). The breakpoint used was the one defined by the NCCLS (12) for the family Enterobacteriaceae (≥512 µg/ml). These 200 isolates were recovered from human clinical sources (120), food products (73), the environment (five), and unknown sources (two) and belong to several serogroups, principally B, C, and D. By a PCR assay performed according to the work of Pritchett et al. (16), all the Salmonella isolates contained the invA gene and 66 were identified as belonging to the DT104 phage type. Clonality among the isolates was assessed by pulsed-field gel electrophoresis (PFGE) following XbaI digestion of genomic DNA according to the standard 1-day protocol set by the Centers for Disease Control and Prevention (CDC) (1). Isolates with electrophoretic patterns that differed by three bands at most were assigned to the same clone.

All the sulfonamide-resistant isolates were screened by PCRs, which were performed with primers specific for sul1 (9), sul2 (9), and sul3 (15) genes. In 200 sulfonamide-resistant isolates, 152 (76%) sul1, 74 (37%) sul2, and 14 (7%) sul3 genes were detected. In 34 isolates, more than one gene coding for sulfonamide resistance was present: sul1 and sul2 in 24; sul1 and sul3 in four; and sul1, sul2, and sul3 in six (Table 1). The sequencing data from one of the PCR products obtained with primers for the sul3 gene showed identity with the newly described sul3 gene (15). The 14 sul3-positive Salmonella isolates were from human clinical samples (six), from foods of animal origin (six), and from environmental sources (two), obtained from geographically dispersed regions in Portugal and belonging to three Salmonella serotypes. The PFGE profiles showed one clone which includes the four S. enterica serotype Rissen isolates, all from swine end products, and three clones were identified among the nine serotype Typhimurium isolates, with one of them carrying sul1, sul2, and sul3 genes isolated from swine end products, humans, and the environment (Table 2; Fig. 1).


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  		TABLE 1. Distribution of sulfonamide resistance genes in Salmonella isolates and relation to class 1 and class 2 integrons


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  		TABLE 2. Characterization of Salmonella isolates with the sul3 gene



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  		FIG. 1. PFGE patterns of sul3-carrying Salmonella isolates. Lanes: 1, 15, and 17, Salmonella serotype Braenderup H9812 (CDC); 2, SFCL-5 (type A); 3, BF033 (type A); 4, SFS-2 (subtype A2); 5, BF128 (subtype A2); 6, IH459/02 (type B); 7, AF30 (subtype B3); 8, IE352/03 (subtype B3); 9, IE357/03 (subtype B3); 10, IH752/03 (subtype B3'); 11, IH779/03 (subtype B3"); 12, IH184/03 (type C); 13, BH212 (type C); 14, AF29 (type D); 16, IH842/03 (type E).

The presence of class 1 integrons was tested by PCR, with the primers 5'CS-3'CS (10) and the specific primer int1 (9) in all the sulfamethoxazole-resistant isolates. To characterize the conserved segment 3'CS, the presence of sul1 and qacE{Delta}1 genes was determined in all the isolates by PCR, with specific primers (17). To determine the content of the variable regions of the integrons, sequencing and PCR with the 5'CS primer in combination with reverse primers for several genes were performed. Class 2 integrons were detected by PCR with primers specific for int2 (11). PCR analysis revealed that, of 200 sulfonamide-resistant isolates, 149 (75%) contained class 1 integrons and 5 (3%) contained class 1 and class 2 integrons (Table 1). Five of the sulfamethoxazole-resistant strains carried class 1 integrons, which lacked the qacE{Delta}1 and sul1 genes at the 3'CS.

Among the 154 isolates carrying class 1 integrons, 149 presented the sul1 gene, found alone (116 isolates) or simultaneously with sul2 (23 isolates) or sul3 (four isolates) and with sul2 and sul3 (six isolates) (Table 1). In the five strains with class 1 integrons, which lacked the qacE{Delta}1 and sul1 genes, four carried a sul3 gene and the other one carried a sul2 gene. The sul1 gene is frequently located on class 1 integrons, which could be confirmed by the fact that, of the 152 sul1-positive isolates, 149 (98%) harbored class 1 integrons. Interestingly, the sul3 gene occurs in Salmonella isolates carrying class 1 integrons, with identical cassette genes (dfrA12 and aadA2) in all but one isolate (dfrA1 and aadA1), which confer resistance to the same antimicrobial agents (Table 2).

Conjugation assays on an agar plate were carried out with the recipient strain Escherichia coli K802N. Transconjugants were selected on Mueller-Hinton agar 2 (bioMérieux) containing sulfamethoxazole (256 µg/ml) plus nalidixic acid (64 µg/ml). Sulfonamide resistance was transferred from 6 out of the 14 sul3-positive isolates; transconjugants were confirmed by PCR specific for sul3. Resistance to other antimicrobial agents and class 1 integrons was also cotransferred in those isolates (Table 2).

Class 1 integrons and sulfonamide resistance genes are disseminated among Salmonella in contrast with class 2 integrons. A significant proportion (77%) of isolates resistant to sulfonamides carried class 1 integrons; in nearly all cases (98%) the sul1 gene was a consistent marker for the presence of this class of integrons. The presence in all isolates with class 1 integrons of at least one of the sul genes provides, when bacteria are submitted to selective pressure by sulfonamides, a useful tool for the maintenance and further extension of resistance to other antimicrobial agents.

In our Salmonella isolates the sul1 gene was the most frequent mechanism of resistance to sulfonamides. In contrast, recently the spread of sul2 seems to have increased in other European countries (2, 9), as the gene was reported to be more widespread among clinical isolates of E. coli than the sul1 gene.

A new sulfonamide resistance gene, named sul3, has been detected in E. coli isolates from pigs in Switzerland (15) and also among German E. coli isolates from various animals and foods (7) and Salmonella isolates (8). The sul3 gene was also identified in two different human clinical isolates of E. coli in Sweden (5). In our study, the newly described sul3 gene has now been identified in 14 Salmonella isolates from three serotypes collected from human and nonhuman sources in Portugal, being mainly observed in isolates from swine food products. The consumption of sulfonamides for veterinary use is generally widespread in Portugal, particularly for swine production. So, the appearance of a newly described gene and the simultaneous presence of several sul genes may reflect its high usage in food-producing animals, as verified in our country by Pena et al. (14). The association of sul3 genes with conjugative plasmids in the food-borne isolates of the serotype Rissen clone and serotype Typhimurium and in one human Salmonella isolate of another serotype could facilitate the further spread of this gene to other bacteria.

Interestingly, the sul3 gene occurs in Salmonella carrying class 1 integrons with aadA and dfrA gene cassettes, which allows isolates to survive exposure to sulfamethoxazole and trimethoprim, a combination frequently used in therapeutics. It is of note that serotype Typhimurium was the main serotype carrying the sul3 gene and the only serotype associated with the three sul genes. The persistence of several sulfonamide resistance genes may be the result of the successive pressure exerted by sulfonamides and other antimicrobial agents that are also commonly used and may be mitigated by the fact that not all sulfonamide-resistant determinants exert a fitness cost, as described for the sul2-encoding plasmid (3).


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ACKNOWLEDGMENTS
 
We are very grateful to V. Perreten (Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland) for the control strain with plasmid pVP440, Centro Nacional de Salmonella (Lisbon, Portugal) for serotyping of the strains, and CDC for the PFGE protocols and the control strain S. enterica serotype Braenderup H9812.

This work was partially supported by Fundação Calouste Gulbenkian, Portugal (project no. 49975).


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha, n°164, 4050-047 Porto, Portugal. Phone: 351 22 2078972. Fax: 351 22 2003977. E-mail: lpeixe{at}ff.up.pt. Back


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Antimicrobial Agents and Chemotherapy, February 2005, p. 836-839, Vol. 49, No. 2
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.2.836-839.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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