Cloning and characterization of a novel, plasmid-encoded trimethoprim-resistant dihydrofolate reductase from Staphylococcus haemolyticus MUR313

Abstract

In recent years resistance to the antibacterial agent trimethoprim (Tmp) has become more widespread, and several trimethoprim-resistant (Tmp r ) dihydrofolate reductases (DHFRs) have been described from gramnegative bacteria. In staphylococci, only one Tmp r DHFR has been described, the type S1 DHFR, which is encoded by the dfrA gene found on transposon Tn4003. In order to investigate the coincidence of high-level Tmp resistance and the presence of dfrA, we analyzed the DNAs from various Tmp r staphylococci for the presence of dfrA sequences by PCR with primers specific for the thyE-dfrA genes from Tn4003. We found that 30 of 33 isolates highly resistant to Tmp (MICs, >512 g/ml) contained dfrA sequences, whereas among the Tmp r (MICs, <256 g/ml) and Tmp s isolates only the Staphylococcus epidermidis isolates (both Tmp r and Tmp s ) seemed to contain the dfrA gene. Furthermore, we have cloned and characterized a novel, plasmid-encoded Tmp r DHFR from Staphylococcus haemolyticus MUR313. The dfrD gene of plasmid pABU17 is preceded by two putative Shine-Dalgarno sequences potentially allowing for the start of translation at two triplets separated by nine nucleotides. The predicted protein of 166 amino acids, designated S2DHFR, encoded by the longer open reading frame was overproduced in Escherichia coli, purified, and characterized. The molecular size of the recombinant S2DHFR was determined by ion spray mass spectrometry to be 19,821.2 ؎ 2 Da, which is in agreement with the theoretical value of 19,822 Da. In addition, the recombinant S2DHFR was shown to exhibit DHFR activity and to be highly resistant to Tmp.

dfrA20 , a Novel Trimethoprim Resistance Gene from Pasteurella multocida

“…2). Identities to the known DfrA proteins determined on the basis of a multisequence alignment varied between 20.0 and 37.7%, with the highest levels of identity to the Dfr proteins being found for Bacillus subtilis (37.7%) (7) and Staphylococcus haemolyticus (37.0%) (2). This observation confirmed that DfrA20 is only distantly related to other DfrA proteins.…”

Trimethoprim Resistance and Susceptibility Genes in Staphylococcus epidermidis

“…The MIC for TMP of the S. sciuri strain was much higher (1,600 .tg/ml) than those of resistant strains of other CNS species (100-400 ig/m1). The PCR-negative TMP', S. sciuri, may have a resistance determinant different from dfrA, like the dfrD gene possessed by a certain TMP' strain of S. haemolytics (8). When the primers for aacA-aphD were used in PCR, the results were in good correlation with those of the GM sensitivity test: all of the 65 GM' staphylococcal isolates (20 S. aureus, 37 S. epidermidis.…”

“…All of the 37 TMP' S. epidermidis isolates were also PCR positive, while all of the 43 TMP' isolates belonging to other species (27 S. aureus, 8 S. haemolyticus, 5 S. saprophyticus, 2 S. chonii, and 1 S. capitis) were negative. The sequence data reported by Dale et al (7)(8)(9) revealed that the TMP gene of S. aureus, dfrA, has only 5 nucleotides different from the TMP' gene of S. epidermidis, dfrC, and that the P5 and P6 primers for dfrA had only one base mismatch in the P6 primer to the dfrC sequence. Therefore, we assumed that the dfrC gene in TMP' S. epidermidis was amplified with the primers for dfrA by PCR.…”

Functional Cloning of Bacillus anthracis Dihydrofolate Reductase and Confirmation of Natural Resistance to Trimethoprim

“…This eventually affects RNA, DNA, and protein synthesis (11). Resistance to TMP is generally seen as the production of an extra DHFR found on plasmids or other genetic elements (5,9,13). In addition, TMP resistance can result from mutations in the chromosomal gene that result in a higher K i for the drug (1,8,13,17,21), as well as the production of an additional DHFR (7).…”