Abstract
Resistance to lincomycin and clindamycin in the clinical isolateEnterococcus faecium HM1025 is due to a ribosomal methylase encoded by an ermAM-like gene and the plasmid-mediated inactivation of these antibiotics. We have cloned and determined the nucleotide sequence of the gene responsible for the inactivation of lincosamides, linB. This gene encodes a 267-amino-acid lincosamide nucleotidyltransferase. The enzyme catalyzes 3-(5′-adenylation) (the adenylation of the hydroxyl group in position 3 of the molecules) of lincomycin and clindamycin. Expression oflinB was observed in both Escherichia coli andStaphylococcus aureus. The deduced amino acid sequence of the enzyme did not display any significant homology with staphylococcal nucleotidyltransferases encoded by linA andlinA′ genes. Sequences homologous to linB were found in 14 other clinical isolates of E. faecium, indicating the spread of the resistance trait in this species.
Lincosamide antibiotics include lincomycin, naturally produced by several actinomycetes, and clindamycin, a semisynthetic derivative obtained by the chlorination of lincomycin. These antibiotics are active against many gram-positive cocci and anaerobes; they inhibit protein synthesis by blocking the peptidyltransferase activity of the 50S subunit of the bacterial ribosome (11). Resistance to lincosamides is usually due to alteration of the ribosome following the N6dimethylation of a specific adenine in the 23S rRNA, which confers cross-resistance to macrolide, lincosamide, and streptogramin B type antibiotics, i.e., the MLSB phenotype (22, 32). In contrast to this broad-spectrum resistance, resistance specific to lincosamides, gained by bacterial modification of those antibiotics, has been reported. Phosphorylation (1) and nucleotidylation (2, 26) of the hydroxyl group in position 3 of lincosamide molecules (24, 26) have been detected in several species ofStreptomyces. Inactivation of lincosamides was also observed in strains of staphylococci, streptococci, enterococci, and lactobacilli of animal origin (10, 12, 13) and in staphylococci isolated from humans (5, 20, 21). Clinical isolates of Staphylococcus haemolyticus BM4610 andStaphylococcus aureus BM4611 are highly resistant to lincomycin (MIC = 64 μg/ml) and are apparently susceptible to clindamycin (MIC = 0.12 μg/ml). In these strains, lincosamideO-nucleotidyltransferases encoded by two closely related genes named linA (lincosamide inactivation nucleotidylation) and linA′, respectively, were characterized (4, 5). These genes encode two 161-amino-acid isoenzymes that differ by 14 amino acids. These enzymes inactivate lincomycin and clindamycin by converting them to lincomycin 3-(5′-adenylate) and clindamycin 4-(5′-adenylate) by using ATP, GTP, CTP, or UTP as a nucleotidyl donor and MgCl2 as a cofactor (5). The distribution of linA and linA′ genes was studied by using DNA-DNA hybridization, and related sequences were found in strains belonging to various species of staphylococci (20).
In this paper, we report the nucleotide sequence of a newlinB gene that confers resistance to lincosamides on a clinical strain of Enterococcus faecium, HM1025, by inactivating the compounds, and we further report on our study of the biochemical mechanism of the resistance.