Conjugative transfer of staphylococcal antibiotic resistance markers in the absence of detectable plasmid DNA

Abstract

Eleven Staphylococcus aureus clinical isolates were tested for transfer of resistance markers by transduction and filter mating. The resistance markers of six of the strains could be transferred only by transduction; however, the five remaining strains transferred their resistance both by transduction and filter mating. The resistance markers that were cotransferred in filter matings (transfer of resistance to penicillin and streptogramin A was accompanied, in each case, by the transfer of one or more markers, i.e., resistance to aminoglycosides, cadmium, or tetracycline, depending on the donor) were not cotransduced. The ylococcal Diseases, in press). Evidence for the chromosomal location of these genes includes their low frequency of transduction (<1 x 10-9 transductants per PFU) and the absence of extrachromosomal DNA in the transductants. Further evidence was obtained from hybridization experiments with probes consisting of aminoglycoside resistance genes. These probes did not hybridize with the plasmids isolated from the wild-type strains; hybridization with cellular DNA from both the wild-type strains and the plasmid-free transductants supported the hypothesis of the chromosomal location of these genes (13a).Although the transfer by conjugation of the antibiotic resistance genes in the absence of detectable plasmids occurs in various bacterial genera (4,7,18,20,21,23,24,34,36,44,49,50,56), reports of such conjugative transfer in staphylococci have so far been limited to plasmid-borne determinants (2,3,15,19,33,37,53,54).We report in this study the transfer by conjugation of apparently chromosome-borne resistance markers from wild-type S. aureus strains into staphylococcal recipients belonging to different species. MATERIALS AND METHODSBacterial strains. The bacterial strains used in this study are listed in bacterial growth, mating experiments, and the preparation of all of the selective media except those containing trimethoprim. Nutrient broth no. 2 (Oxoid Ltd., London, England) and nutrient broth agar (Oxoid) were used for bacteriophage propagation and transduction experiments. Mueller-Hinton agar (Institut Pasteur Production, Paris, France) was used for disk susceptibility tests and preparation of selective media containing trimethoprim.Mating procedures on agar media. A 0.3-ml portion of a mixture containing 1 x 107 CFU of donors and 10-fold more recipients, both obtained from late-logarithmic-phase broth cultures, was spread either on nitrocellulose filters (type HAEP; pore size, 0.45 ,um; Millipore Corp., Bedford, Mass.), as described by Horodniceanu et al. (20), or directly on agar plates. After 18 h of incubation at 37°C, the cells on each filter were suspended in 1 ml of BHI, and the suspension was spread on BHI agar or Mueller-Hinton agar containing the appropriate selective drug. Controls, consisting of donor or recipient cells alone, were treated similarly. For two crosses, the donor and recipient cultures were separated during incubation (18 h at 37°C) by a nitrocellulose filter: a filter w...

Integrative and Sequence Characteristics of a Novel Genetic Element, ICE 6013 , in Staphylococcus aureus

“…An additional line of investigation is needed to understand mechanisms that promote conjugation in staphylococci. Previous work on the conjugative transfer of antibiotic resistance genes between S. aureus strains that lacked detectable plasmids, and where transformation and transduction had been inhibited, hinted at the presence of conjugative elements in the chromosome (28). ICE6013 represents a demonstrably integrative genetic element for staphylococci that has hallmark components of a conjugative system.…”

Transmissible mupirocin resistance inStaphylococcus aureus

“…Centrifugation in density gradients also failed to reveal CCC plasmid DNA though endonuclease cleavage of this DNA seems to reveal extra-chromosomal DNA associated with the transfer of high level mupirocin resistance. Conjugative plasmids bearing genes for gentamicin resistance are well established in S. aureus and the conjugative transfer of aminoglycoside resistance in the absence of plasmid DNA has been recorded (El Sohl et al 1986). Conjugative transposons which might also explain the phenomenon have not yet been reported in S. aureus (Murphy, 1988) though well established in streptococci (Clewell, 1986).…”

ATP Hydrolysis and Pristinamycin IIA Inhibition of the Staphylococcus aureus Vga(A), a Dual ABC Protein Involved in Streptogramin A Resistance

“…Screening for plasmid DNA mutagenesis was achieved by using E. coli XL1-Blue supercompetent cells (Stratagene). E. coli strain AG100A, susceptible to SgA by disruption of the AcrAB pump (26), and Staphylococcus epidermidis strain BM3302 (27) were used as recipients for testing the functionality of the genes cloned into pIP1840, as described previously (14). Overexpression and purification of the recombinant proteins were carried out using BL21(DE3) pDIA17 (28) transformed with vga(A) constructs cloned into the pIVEX2.3 vector (Roche Applied Science).…”