Indication of transposition of a mobile DNA element containing the vat(D) and erm(B) genes in Enterococcus faecium.
ABSTRACT: The vat(D) and erm(B) genes encoding streptogramin resistance in Enterococcus faecium transferred together, and a direct physical link between erm(B) and vat(D) was detected. Both the vat(D) and erm(B) probes hybridized to fragments of different sizes in the donor and transconjugants, which indicated a transposition event.
Project description:Sixteen isolates of Enterococcus faecalis were recovered from retail poultry samples (seven chickens and nine turkeys) purchased from grocery stores in the greater Washington, D.C., area. PCR for known streptogramin resistance genes identified vat(E) in five E. faecalis isolates (three isolates from chickens and two isolates from turkeys). The vat(E) gene was transmissible on a ca. 70-kb plasmid, along with resistance to erythromycin, tetracycline, and streptomycin, by conjugation to E. faecalis and Enterococcus faecium recipient strains. DNA sequencing showed little variation between E. faecalis vat(E) genes from the chicken samples; however, one E. faecalis vat(E) gene from a turkey sample possessed 5 nucleotide changes that resulted in four amino acid substitutions. None of these substitutions in the vat(E) allele have previously been described. This is the first report of vat(E) in E. faecalis and its transferability to E. faecium, which indicates that E. faecalis can act as a reservoir for the dissemination of vat(E)-mediated streptogramin resistance to E. faecium.
Project description:In a survey of retail meat samples, twelve quinupristin-dalfopristin-resistant (MICs, > or =4 mg/liter) Enterococcus faecium isolates that carried a vat(E) gene were recovered. DNA sequence comparison revealed five new variations in the vat(E) allele among 12 isolates, which were designated vat(E-4) through vat(E-8); two isolates had vat(E-1). There was no correlation between the number of base changes and the quinupristin-dalfopristin MIC.
Project description:Fifty-nine erm(B)-positive Enterococcus faecium strains isolated from pigs, broilers, and humans were typed using multilocus sequence typing (MLST), and the coding sequence of the erm(B) gene was determined. Identical erm(B) gene sequences were detected in genetically unrelated isolates. Furthermore, genetically indistinguishable strains were found to contain different erm(B) alleles. This may suggest that horizontal exchange of the erm(B) gene between animal and human E. faecium strains or the existence of a common reservoir of erm(B) genes might be more important than direct transmission of resistant strains.
Project description:A gene which mediates inducible resistance to macrolides, lincosamides, and streptogramin B antibiotics, designated erm(33), was detected on the Staphylococcus sciuri plasmid pSCFS1. Analysis of the erm(33) reading frame suggested that this gene was the product of a recombination between an erm(C) gene and an erm(A) gene. Such a recombination event is a novel observation for erm genes.
Project description:In this study we describe a multiplex PCR assay for the detection of nine clinically relevant antibiotic resistance genes of Staphylococcus aureus. Conditions were optimized to amplify fragments of mecA (encoding methicillin resistance), aacA-aphD (aminoglycoside resistance), tetK, tetM (tetracycline resistance), erm(A), erm(C) (macrolide-lincosamide-streptogramin B resistance), vat(A), vat(B), and vat(C) (streptogramin A resistance) simultaneously in one PCR amplification. An additional primer pair for the amplification of a fragment of the staphylococcal 16S rDNA was included as a positive control. The multiplex PCR assay was evaluated on 30 different S. aureus isolates, and the PCR results correlated with the phenotypic antibiotic resistance data obtained by the broth microdilution assay. The multiplex PCR assay offers a rapid, simple, and accurate identification of antibiotic resistance profiles and could be used in clinical diagnosis as well as for the surveillance of the spread of antibiotic resistance determinants in epidemiological studies.
Project description:Whole-genome sequencing of Staphylococcus xylosus strain JW2311 from bovine mastitis milk identified the novel 49.3-kb macrolide-lincosamide-streptogramin B (MLSB) resistance plasmid pJW2311. It contained the macrolide resistance gene mph(C), the macrolide-streptogramin B resistance gene msr(A), and the new MLSB resistance gene erm(48) and could be transformed into Staphylococcus aureus by electroporation. Functionality of erm(48) was demonstrated by cloning and expression in S. aureus.
Project description:The search for a specific rRNA methylase motif led to the identification of the new macrolide, lincosamide, and streptogramin B resistance gene erm(43) in Staphylococcus lentus. An inducible resistance phenotype was demonstrated by cloning and expressing erm(43) and its regulatory region in Staphylococcus aureus. The erm(43) gene was detected in two different DNA fragments, of 6,230 bp and 1,559 bp, that were each integrated at the same location in the chromosome in several S. lentus isolates of human, dog, and chicken origin.
Project description:High-level, acquired macrolide resistance in mycobacteria is conferred by mutation within the 23S rRNA gene. However, several mycobacteria are naturally resistant to macrolides, including the Mycobacterium smegmatis group and Mycobacterium tuberculosis complex. Thus, the aim of this study was to characterize this resistance. Intrinsic macrolide resistance in M. smegmatis was inducible and showed cross-resistance to lincosamides but not to streptogramin B (i.e., ML resistance). A similar phenotype was found with Mycobacterium microti and macrolide-resistant Mycobacterium fortuitum. A search of the DNA sequence data for M. smegmatis strain mc(2)155 identified a novel erm gene, erm(38), and expression analysis showed that erm(38) RNA levels increased >10-fold after a 2-h incubation with macrolide. Inducible ML resistance was not expressed by an erm(38) knockout mutant, and complementation of this mutant with intact erm(38) in trans resulted in high-level ML resistance (e.g., clarithromycin MIC of >512 micro g/ml). Thus, the results indicate that erm(38) confers the intrinsic ML resistance of M. smegmatis. Southern blot analysis with an erm(38)-specific probe indicated that a similar gene may be present in macrolide-resistant M. fortuitum. This finding, with the presence of the erm(37) gene (Rv1988) in the M. tuberculosis complex, suggests that such genes are widespread in mycobacteria with intrinsic macrolide resistance.
Project description:We characterized two new streptogramin A resistance genes from quinupristin-dalfopristin-resistant Enterococcus faecium JS79, which was selected from 79 E. faecium isolates lacking known genes encoding streptogramin A acetyltransferase. A 5,650-bp fragment of HindIII-digested plasmid DNA from E. faecium JS79 was cloned and sequenced. The fragment contained two open reading frames carrying resistance genes related to streptogramin A, namely, genes for an acetyltransferase and an ATP efflux pump. The first open reading frame comprised 648 bp encoding 216 amino acids with a predicted left-handed parallel ?-helix domain structure; this new gene was designated vatH. [corrected] The second open reading frame consisted of 1,575 bp encoding 525 amino acids with two predicted ATPase binding cassette transporters comprised of Walker A, Walker B, and LSSG motifs; this gene was designated vgaD. vgaD is located 65 bp upstream from vatH, [corrected] was detected together with vatH [corrected] in 12 of 179 quinupristin-dalfopristin-resistant E. faecium isolates, and was located on the same plasmid. Also, the 5.6-kb HindIII-digested fragment which was observed in JS79 was detected in nine vgaD- and vatH-containing [corrected] E. faecium isolates by Southern hybridization. Therefore, it was expected that these two genes were strongly correlated with each other and that they may be composed of a transposon. Importantly, vgaD is the first identified ABC transporter conferring resistance to streptogramin A in E. faecium. Pulsed-field gel electrophoresis patterns and sequence types of vgaD- and vatH-containing [corrected] E. faecium isolates differed for isolates from humans and nonhumans.
Project description:The ErmB macrolide-lincosamide-streptogramin B (MLS) resistance determinant from Clostridium difficile 630 contains two copies of an erm(B) gene, separated by a 1.34-kb direct repeat also found in an Erm(B) determinant from Clostridium perfringens. In addition, both erm(B) genes are flanked by variants of the direct repeat sequence. This genetic arrangement is novel for an ErmB MLS resistance determinant.