Failure of Coagulase-negative Staphylococci to Transfer Antibiotic Resistance to Staphylococcus aureus 1030 in Mixed Cultures

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One hundred and fifty-three isolates of coagulase-negative staphylococci obtained from human skin failed to transfer resistance to either cadmium ions (46 isolates), trimethoprim (37 isolates), erythromycin (25 isolates) or tetracycline (45 isolates) to Staphylococcus aureus strain 1030 or to each of 10 of its lysogenic derivatives in mixed cultures. Thirty-three trimethoprim-resistant coagulase-negative staphylococcal isolates obtained from the human intestine also failed to transfer this resistance to the recipients in mixed cultures.

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... In contrast, phage-mediated conjugation was inhibited on human skin but transferred resistance plasmids in moist conditions in the presence of free Ca"^+ (Townsend et al., 1986). This mechanism would appear to be limited to intraspecies transfer based on the species specificity of staphylococcal phages and previous failure to detect interspecies transfer under conditions that favoured phage-mediated conjugation (Fawcett, Lord and Lacey, 1981). ...
... Other studies had failed to detect interspecies transfer of resistance among staphylococci (Fawcett et aL, 1981). This led to suggestions that similar plasmids in different staphylococcal species arose by convergent evolution or by being descendants of a highly conserved '^primordal" plasmid (Lacey, 1984). ...
... This led to suggestions that similar plasmids in different staphylococcal species arose by convergent evolution or by being descendants of a highly conserved '^primordal" plasmid (Lacey, 1984). However, the failure to detect interspecies transfer between staphylococci was most probably the result of using transfer conditions more suited to phage-mediated conjugation (Fawcett et aL, 1981) as well as the presence of restriction barriers. Plasmids pWG4 and pWG25 serve as an example. ...
Two staphylococcal plasmids, pWG4 and pWG25, encode production of a diffusible pigment and resistance to erythromycin and spectinomycin. The former was found occurring naturally in a clinical isolate of Staphylococcus aureus and the latter in S. epidermidis. Both plasmids are conjugative, capable of high-frequency, interspecies transfer, only isolated in the open-circular form and identical in molecular weight and pattern of restriction-endonuclease fragments. The only difference between the plasmids is in the expression of resistance, pWG4 encoding inducible and pWG25 constitutive erythromycin resistance. The resistance determinants of both plasmids behave as hitch-hiking transposons in cultural conditions that favour phage-mediated or phage-independent conjugation, always inserting a copy of themselves into the recipient's chromosome, except in S. epidermidis in which the chromosomal insertion site may be absent. The resistance determinants have been cloned and located on a 4 X 7 kbp EcoR1/HindIII restriction fragment which has a restriction map similar to that of the right arm of Tn554 (Murphy and Lofdahl, 1984). The hitch-hiking transposon of plasmid pWG25 has been designated Tn3853.
... Surveys of coagulasenegative staphylococci and animal isolates of S. aureus found little or no evidence for transferable resistance to human isolates of S. aureus, raising doubts about these organisms as a source of an ti bio tic resistance. However, the experiments were performed under conditions that allowed transfer by a phage-mediated mechanism only (Lacey, 1980b;Fawcett et al., 1981). ...
A strain of Staphylococcus aureus was constructed with which to compare transfer of resistance plasmids by the mechanisms of phage-mediated conjugation and conjugation. Transfer by each mechanism could be distinguished by the patterns of resistances transferred. Conjugation was favoured on dry absorbent surfaces, e.g., human skin, tissue and surgical gauze, whereas phage-mediated conjugation was favoured in fluids, e.g., milk and urine. The degree of hydration of the mating cells is postulated as one factor determining whether plasmids are transferred by phage-mediated conjugation or conjugation. Preliminary evidence indicates that topical creams and ointments affect the conjugative transfer of plasmids.
The plasmid DNA profiles were compared to phenotypically-similar, antibiotic-resistant strains of Staphylococcus aureus and Staphylococcus epidermidis associated with nosocomial infections in a Melbourne hospital. Whereas resistance to gentamicin, tobramycin and kanamycin was encoded by one of 3 plasmids [pSK1,18 megadalton (Md); pSK4, 22 Md; pSK9,17 Md] in S. aureus no similar plasmids were detected in S. epidermidis. Mediated exclusively by the chromosome in S. aureus tetracycline resistance was encoded either by the chromosome or by a 2.8 Md plasmid in strains of S. epidermidis. The inability to detect common resistance plasmids in strains of S. aureus and S. epidermidis recovered from this outbreak is in contrast to recent observations with staphylococci from other geographic areas; nevertheless, on the basis of restriction endonuclease analyses of 3 Md chloramphenicol resistance plasmids, it is suggested that a common gene pool does exist within isolates of S. aureus and S. epidermidis from Melbourne hospitals.
The ability of Staphylococcus epidermidis to transfer antimicrobial resistance to Staphylococcus aureus was tested by mixed culture on filter membranes. Two of six clinical isolates examined were able to transfer resistance to S. aureus strains 879R4RF, RN450RF, and UM1385RF. Subsequent S.aureus transconjugants resulting from matings with S. epidermidis donors were able to serve as donors to other S. aureus strains at similar frequencies. Cell-free and mitomycin C-induced filtrates of donors and transconjugants showed no plaque-forming ability. Addition of DNase I, citrate, EDTA, calcium chloride, and human sera to mating mixes and agar showed no effect on transfer. Nonviable donor cells were unable to transfer resistance and transfer did not occur at 4 degrees C. Cell-to-cell contact was required since transfer did not occur in broth or when filters of donor and recipient, respectively, were placed back-to-back so cells were not in direct contact. Analysis of DNA from S. epidermidis isolate UM899, its subsequent S. aureus transconjugants, and cured derivatives demonstrated that all resistance markers which transferred resided on plasmids. Mating experiments suggested a central role for the gentamicin plasmid pAM899-1 in the transfer process. It is concluded that our results are consistent with a conjugative transfer of resistance from S. epidermidis to S. aureus analogous to plasmid transfer demonstrated in streptococcal species for plasmids such as pAM beta 1. This represents a novel mechanism for gene exchange among staphylococci.
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We have demonstrated previously that application of topical erythromycin, an antibiotic commonly used for the treatment of acne, results in an increased density of cutaneous erythromycin-resistant (Emr) coagulase-negative staphylococci; however, it is unknown if this increase results in an overall higher density of total cutaneous staphylococci or if upon cessation of erythromycin use, Emr coagulase-negative staphylococci remain at an increased density compared with the pretreatment density. To investigate this, 2% erythromycin or vehicle was applied to each subject's forehead (n = 225) twice a day by laboratory personnel for a period of 6 weeks. Samples were obtained for culture from the forehead, anterior nares, and back of the subjects at baseline and at weeks 6, 9, and 12 of the study. Cultures were performed on differential media. Plates into which erythromycin was incorporated (8 micrograms/ml) were used to identify Emr coagulase-negative staphylococci. The species of all Emr coagulase-negative staphylococci were determined, and an antibiogram for 16 antibiotics was obtained. The baseline prevalence of Emr coagulase-negative staphylococci on the forehead and nose was about 80% at the two study sites, whereas that on the back was 50%. The baseline density of Emr coagulase-negative staphylococci on the forehead, nose, and back was approximately 20% of the total flora. Following 6 weeks of erythromycin treatment, the prevalence of Emr coagulase-negative staphylococci on the forehead and nose was nearly 100% and the densities were 73 and 62%, respectively; the prevalence and density for the back were 78 and 42%, respectively. The most prevalent erythromycin resistance gene expressed by the Emr coagulase-negative staphylococci was ermC. There was no increase in the numbers of Staphylococcus aureus, gram-negative rods, or yeasts, nor was there increased resistance to any other antibiotic except clindamycin. The density of total aerobic organisms also remained static. There were no changes in the prevalence or density of Emr coagulase-negative staphylococci in the vehicle group. A statistically significant decrease in the prevalence and density of Emr coagulase-negative staphylococci in the erythromycin group was observed within 3 weeks posttreatment and by 6 weeks posttreatment, the prevalence and density returned to baseline values. These data demonstrate that the increased prevalence and density of Emr coagulase-negative staphylococci as a result of topical 2% erythromycin use are transient on both population and individual levels.
Transfer of pen- and chl-plasmids does not only occur between strains of Staph, aureus but also betwen strains of Staph, aureus and Staph, epidermidis. In Staph, aureus, the frequency of transfer of a tet-plasmid is less frequent than the transfer of pen- and chl-plasmids. Lysogenization of the donor strain by a sero-group-B-phage has no influence on the frequency of transfer. The transfer is not inhibited by gamma-globulin. gamma-globulin inhibits unspecifically phage adsorption and also the transduction by phage 80. The transfer is also possible when an encapsulated recipient strain is used. Phages are inhibited by the capsular material (SCHEER and KOFT, 1975); a transduction to an encapsulated strain is impossible. These results speak against a phage-mediated transfer in mixed cultures. A dilution of the incubation mixture leads to an unproportional decrease of the frequency of transfer. A prerequisite to the transfer is probably a close cell-to-cell contact.
Transduction of resistance from a multiply antibiotic-resistant strain of Staphylococcus epidermidis sub-group II was studied using the typing phage 108. The effect of increasing doses of ultraviolet radiation on the transducing phage was used to indicate the chromosomal or plasmid nature of the genes. Tetracycline and chloramphenicol resistance behaved as plasmid genes and streptomycin resistance as a chromosomal marker. It was also possible to transduce penicillin resistance (Pc) due to penicillinase production (bla+) using a low level of benzylpenicillin (0.03 microgram ml-1) for recovery. Approximately 10(-5) transductant colonies per phage input were obtained and ultraviolet kinetics indicated that Pc was plasmid carried. Pc transductants fell into two categories. In one group PC was stable as in the donor strain and transductants had the same phage sensitivity as the recipient. In the other, Pc was unstable at 37 degrees C and the instability was enhanced by growth at approximately 43.5 degrees C; these transductants also gained genes for restriction and modification of certain phages. Transductants that subsequently lost bla+ also lost the restriction and modification characters.
Four strains of Staphylococcus epidermidis from clinical sources were capable of serving as donors for the transduction of either penicillinase production, ethidium bromide resistance, or tetracycline resistance. Three typing phages served as transducing phages and, depending upon the combination of transducing phage, donor strain, and recipient strain, the rates of transduction ranged between 10(-5) and 10(-9). In one strain, cotransduction of penicillinase production and ethidium bromide resistance was observed. Although ultraviolet irradiation kinetics indicated that both the tetracycline resistance and the penicillin resistance determinants were located on plasmids, only resistance to tetracycline could be eliminated by growth in the presence of curing agents or at elevated temperature. However, evidence was obtained by agarose gel electrophoretic studies that both the tetracycline resistance and the penicillin resistance determinants are located on separate plasmids in this organism.
Phages were isolated from Staphylococcus epidermidis after exposure of cultures to mitomycin C. Using certain of these phages, genes controlling resistance to novobiocin, streptomycin, and erythromycin and genes involved in the synthesis of adenine and riboflavin were transferred from donor to recipient strains of S. epidermidis. Interspecific transduction of the Novr marker was accomplished between S. epidermidis and S. aureus. Attempts to transfer penicillin resistance were not successful.