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Antimicrobial Agents and Chemotherapy, February 2009, p. 776-778, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01128-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of a Novel Trimethoprim Resistance Gene, dfrK, in a Methicillin-Resistant Staphylococcus aureus ST398 Strain and Its Physical Linkage to the Tetracycline Resistance Gene tet(L)

Kristina Kadlec and Stefan Schwarz^*

Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute (FLI), Höltystr. 10, 31535 Neustadt-Mariensee, Germany

Received 22 August 2008/ Returned for modification 10 October 2008/ Accepted 6 November 2008

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
A novel trimethoprim resistance gene, designated dfrK, was detected in close proximity to the tetracycline resistance gene tet(L) on the ca. 40-kb plasmid pKKS2187 in a porcine methicillin (meticillin)-resistant Staphylococcus aureus isolate of sequence type 398. The dfrK gene encodes a 163-amino-acid dihydrofolate reductase that differs from all so-far-known dihydrofolate reductases.

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) represent a major problem in human medicine by causing both healthcare-associated and community-associated infections (5). A large number of MRSA clones has been identified (4, 11), one of which, consisting of strains of the sequence type 398 (ST398), has gained particular attention during recent years because of its association with pigs and its ability to colonize pig farmers and other people in close contact with pigs (18-21, 23). Although MRSA ST398 strains rarely cause infections in pigs, few reports described their involvement in specific disease conditions of pigs (15, 20). Recently, five multiresistant porcine MRSA ST398 strains from infections of the skin or the urinary/genital tract, including metritis-mastitis-agalactia syndrome, have been identified in the BfT-GermVet study (15, 16). PCR screening for antimicrobial resistance genes identified the β-lactam resistance gene mecA as well as the tetracycline resistance genes tet(K), tet(L), and/or tet(M) in various combinations in these isolates (16). In addition, three strains were resistant to gentamicin and kanamycin via the gene aacA-aphD [aac(6')-Ie-aph(2')-Ia], and one isolate was resistant to macrolides and lincosamides via the gene erm(A) (16). However, none of the known staphylococcal trimethoprim resistance genes, dfrS1, dfrD, and dfrG, could be detected by PCR in the three trimethoprim-resistant isolates.

One of these MRSA ST398 isolates, namely isolate 2187, was chosen for the identification of the presumably new trimethoprim resistance gene. This strain was resistant only to penicillins (penicillin G, ampicillin, and oxacillin [MICs of 8 µg/ml]), tetracycline (MIC of 64 µg/ml), and trimethoprim (MIC > 256 µg/ml). Plasmid profiling and protoplast transformation assays were conducted as previously described (8). Staphylococcus aureus RN4220 served as the recipient strain, and transformants were selected on regeneration medium containing 10 µg/ml trimethoprim. An analysis of the transformants revealed that a ca. 40-kb plasmid, designated pKKS2187, was associated with trimethoprim resistance. The susceptibility testing and PCR analysis of the transformants carrying this plasmid confirmed that plasmid pKKS2187 also mediated tetracycline resistance via a tet(L) gene. To identify the pKKS2187-associated trimethoprim resistance gene, plasmid pKKS2187 was digested with BglII, and the resulting fragments were cloned into the BamHI site of pBluescript II SK+. Escherichia coli JM109 was transformed with these recombinant plasmids, and clones were selected by blue-white screening with subsequent cultivation on Luria-Bertani agar (Oxoid, Wesel, Germany) supplemented with 10 µg/ml trimethoprim. Clones growing on these selective plates also exhibited tetracycline resistance and carried a common ca. 7-kb BglII fragment.

The sequence analysis of this 7,045-bp BglII fragment showed that its terminal parts consisted of IS257 sequences located in the same orientation (Fig. 1). Between these IS257 elements, a structure was detected that closely resembled the small tet(L)-carrying plasmid pBC16 with an additional segment that contained a novel trimethoprim resistance gene (Fig. 1). The repU gene was interrupted by the integration of the IS257 elements, and a typical 8-bp direct repeat, 5'-TGCTGAAA-3', was detected at the integration sites. The functional deletion of the repU gene ensured that the replication properties of the entire plasmid were specified solely by the original large plasmid without interference from the integrated small plasmid. This finding closely resembled the situation of small tet(K)-carrying plasmids being integrated via IS257 into larger staphylococcal plasmids (22).

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FIG. 1. Comparison of the tet(L)-dfrK segment of plasmid pKKS2187 (accession no. FM207105) with plasmid pBC16 from B. cereus (accession no. AAA84922). The arrows indicate the extents and directions of the transcription of the genes repU (plasmid replication), tet(L) (tetracycline resistance), dfrK (trimethoprim resistance), and pre/mob (plasmid recombination/mobilization). It should be noted that the repU gene of pBC16 is intact and has been displayed only in a disrupted form for better comparison to the pKKS2187 sequence. The IS257 elements are shown as black boxes, with the white arrow indicating the transposase gene tnp. The 8-bp direct repeats at the IS257 integration sites are shown in boxes. The regions of >99% homology between pBC16 and pKKS2187 are marked by gray shading. The sequences at the junctions of pBC16-homologous and -nonhomologous parts in pKKS2187 are shown for comparison to the corresponding pBC16 sequence between the two maps. The BglII cleavage site in the IS257 element is indicated as Bgl. A size scale in kilobase pairs is given below each map.

Downstream of the 3' end of the repU gene in pKKS2187, a reading frame for a 458-amino-acid (aa) Tet(L) protein was found. This reading frame was preceded by a translational attenuator for inducible tet(L) gene expression that consisted of a small reading frame for a 20-aa peptide and two pairs of inverted repeated sequences. The tet(L) gene in pKKS2187 was indistinguishable from that of the tetracycline resistance plasmids pBC16 from Bacillus cereus (13), pTB19 from Bacillus stearothermophilus (12), and pLS1 from Streptococcus agalactiae (9). Another 282 bp downstream of the tet(L) gene, a reading frame for a 163-aa dihydrofolate reductase, designated DfrK, was detected. This gene mediated trimethoprim resistance, as confirmed by an at least 2,048-fold increase in the trimethoprim MIC to 512 µg/ml for the recombinant E. coli JM109 clone carrying the 7-kb BglII fragment compared to that of the original E. coli JM109 strain. An analysis of the dfrK nucleotide sequence revealed that this gene showed 86.2% identity to the gene dfrG from Staphylococcus aureus (17) and 81.2% identity to the dfrD genes found in Staphylococcus haemolyticus (3) and Listeria monocytogenes (1). On the amino acid level, identities of 87.9 and 77.2% were noted between DfrK and DfrG and between DfrK and DfrD, respectively. Distinctly lower levels of 49.3% nucleotide sequence identity between dfrK and the Tn4003-associated dfrS1 gene (14) and 38.7% amino acid identity between DfrK and DfrS1 were seen. The phylogenetic tree shown in Fig. 2 confirmed that DfrD, DfrG, and DfrK form a separate branch that is only distantly related to DfrS1 and DfrC. DfrC is a trimethoprim-susceptible dihydrofolate reductase that is considered a potential precursor of the trimethoprim-resistant DfrS1 protein (2). The DfrB proteins identified in MRSA strains (6, 7) also represent trimethoprim-susceptible dihydrofolate reductases.

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FIG. 2. Phylogenetic tree of the staphylococcal DfrS1 (14), DfrD (1, 3), DfrG (17), and DfrK (this study) proteins, which are involved in trimethoprim resistance, and the DfrB (6, 7) and DfrC (2) proteins, which represent trimethoprim-sensitive dihydrofolate reductases. Branch lengths are scaled according to amino acid exchanges observed in a multisequence alignment. The numbers at the major branch points refer to the percentage of times that a particular node was found in 10,000 bootstrap replications.

Further downstream of dfrK, a reading frame for a 420-aa Pre/Mob protein indistinguishable from those of plasmids pBC16 (accession no. AAA84922) and pUB110 (10) was detected (Fig. 1). The entire integrated segment between the IS257 elements closely resembled the 4,630-bp plasmid pBC16. Two segments of 2,488 and 2,172 bp were detected that differed from the corresponding pBC16 sequences by 1 and 4 bp, respectively (Fig. 1). Between these two pBC16-homologous segments, a stretch of 1,589 bp, including the dfrK gene, was detected. The dfrK-flanking regions did not show homology to sequences deposited in the databases and did not contain additional reading frames for proteins of known function. Exactly at the junctions between pBC16-homologous and -nonhomologous sequences, direct duplications of 21 bp, which showed a single mismatch, were detected (Fig. 1). Since this 21-bp sequence also was present in pBC16, it might have served for the integration of the dfrK region into a pBC16-like plasmid. The physical linkage between dfrK and tet(L) allows the maintenance of a plasmid like pKKS2187 under selective pressure by either tetracyclines or trimethoprim, both of which are widely used in veterinary medicine.

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
The sequence of the 7,045-bp segment of plasmid pKKS2187 has been deposited in the EMBL database under accession number FM207105.

 
We thank Kerstin Meyer for excellent technical assistance.

 
* Corresponding author. Mailing address: Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute (FLI), Höltystr. 10, 31535 Neustadt-Mariensee, Germany. Phone: 49-5034-871-241. Fax: 49-5034-871-246. E-mail: stefan.schwarz{at}fli.bund.de

Published ahead of print on 17 November 2008.

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 

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Antimicrobial Agents and Chemotherapy, February 2009, p. 776-778, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.01128-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

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• Kadlec, K., Schwarz, S. (2009). Novel ABC Transporter Gene, vga(C), Located on a Multiresistance Plasmid from a Porcine Methicillin-Resistant Staphylococcus aureus ST398 Strain. Antimicrob. Agents Chemother. 53: 3589-3591 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kadlec, K. Articles by Schwarz, S. Search for Related Content PubMed PubMed Citation Articles by Kadlec, K. Articles by Schwarz, S.