The international spread of methicillin-resistant Staphylococcus aureus.

A genetic analysis of representative methicillin-resistant Staphylococcus aureus (MRSA) being isolated in London and Dublin hospitals has demonstrated that the epidemic strains from two London hospitals are different from the Dublin strains, but indistinguishable from the epidemic strains of eastern Australia. The possibility that some strains of MRSA are more likely than others to spread within hospitals is discussed.

Comparison of phage-mediated and conjugative transfer of staphylococcal plasmids in vitro and in vivo.

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.

Conjugative, staphylococcal plasmids carrying hitch-hiking transposons similar to Tn554: intra- and interspecies dissemination of erythromycin resistance.

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.

Transfer of plasmid-borne aminoglycoside-resistance determinants in staphylococci.

Aminoglycoside-resistance determinants in staphylococci are borne on conjugative and non-conjugative plasmids. The conjugative plasmids were found in methicillin-resistant strains of Staphylococcus aureus isolated recently in Darwin and Sydney, Australia and in Houston, Texas, USA. These plasmids and the class-2 conjugative plasmid reported by Archer and Johnston (1983) had similar patterns of EcoR1 restriction-endonuclease fragments, encoded resistance to gentamicin, kanamycin and neomycin, transferred to a non-lysogenic recipient in conditions that promoted close cell-to-cell contact and mobilised a small, non-conjugative plasmid. A further plasmid, pWG14, encoding resistance to kanamycin, neomycin, streptomycin, erythromycin and lincomycin, also displayed conjugative properties but did not mobilise the small, non-conjugative plasmid. The transfer frequency of all conjugative plasmids was stimulated by the addition of polyethylene glycol, particularly at concentrations above 20%, to mixtures of donor and recipient broth cultures. Polyethylene glycol appeared to promote close cell-to-cell contact between donor and recipient cells. A representative of the most common aminoglycoside-resistance plasmids in Australian isolates of methicillin-resistant S. aureus was non-conjugative and transferred by a bacteriophage-mediated system to a lysogenic recipient. With the exception of plasmid pWG14, the conjugative plasmids were also transferred by a bacteriophage-mediated system. Furthermore, cultural conditions that favoured conjugative transfer of plasmids inhibited bacteriophage-mediated transfer and vice versa. The efficacy of the two transfer systems for analysing the plasmids of gentamicin-resistant, methicillin-resistant isolates of S. aureus has been compared.

A conjugative plasmid encoding production of a diffusible pigment and resistance to aminoglycosides and macrolides in Staphylococcus aureus.

A methicillin-resistant strain of Staphylococcus aureus, WG512, was isolated in 1968 and has been shown to harbour a plasmid, pWG14, which encodes the production of a diffusible pigment and resistance to kanamycin, neomycin, streptomycin, lincomycin, erythromycin and spectinomycin. Plasmid pWG14 has only been detected as a slowly migrating band of DNA during agarose-gel electrophoresis, and this was thought to be the open-circular form of the plasmid with an estimated molecular weight of 30-34 Mdal. The transfer properties of this plasmid were compared with a non-conjugative plasmid encoding tetracycline resistance and the class 2, conjugative plasmid described by Archer and Johnston (1983). Plasmid pWG14 shared many of the conjugative properties of the latter plasmid, including the ability to transfer at high frequency on the surface of a filter membrane and to transfer between non-lysogenic strains in the absence of Ca++ and in the presence of citrate of DNase I. However, unlike the class 2 plasmid, pWG14 was able to transfer in broth culture at low frequency and did not mobilise the non-conjugative plasmid. Furthermore, the class 2 plasmid and pWG14 were shown to belong to different incompatibility groups. A derivative of WG512, which had lost the ability to produce the diffusible pigment along with plasmid pWG14 but had retained the resistance determinants of plasmid pWG14 in the chromosome, was used to demonstrate that the conjugative mechanism of pWG14 was a property of the plasmid and not the resistance determinants.

Evolution of Australian isolates of methicillin-resistant Staphylococcus aureus: a problem of plasmid incompatibility?

Methicillin-resistant Staphylococcus aureus (MRSA), currently causing problems in Australian hospitals, have chromosomal penicillinase and carry a new family of incompatibility group I (IncI) plasmids that encode resistance to nucleic acid-binding compounds (NAB). These plasmids may carry additional determinants for penicillinase production and resistance to gentamicin and trimethoprim. By comparison, earlier MRSA isolates from Australia were NAB-sensitive and the penicillinase determinants were carried on IncI plasmids. The possibility that these newer MRSA isolates have the same 'clonal' origin as other MRSA isolates has been investigated. Forcible maintenance of IncI penicillinase plasmids and NAC-resistance plasmids in the same cells resulted in various recombination events. Similar recombination events to those generated in the laboratory have been found in MRSA isolates.

Distribution of plasmid-borne resistance to nucleic acid binding compounds in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) which are epidemic in Australian hospitals have been found to be genetically different from strains previously isolated in Australia and in Europe. A characteristic feature of the recent Australian isolates is a plasmid which encodes resistance to nucleic acid binding compounds (NAB) such as propamidine isethionate. Of 753 MRSA strains isolated from around the world in 1980-1983, c. 60% were NAB resistant. The determinant for this resistance could be transferred from 90% of strains to a recipient strain in mixed culture. A plasmid analysis revealed that, in general, the largest plasmid of MRSA isolates coded for NAB resistance and may carry other determinants for penicillinase production and resistance to gentamicin, trimethoprim, neomycin, tetracycline, cadmium and mercury. Some plasmids exhibited unusual behaviour with the appearance of deletion mutants after transfer and, in one case, a high-frequency alteration in the expression of gentamicin resistance from high-level to low-level resistance, correlated to a deletion of c. 0.5 megadalton of plasmid DNA. These results demonstrate that these NAB-resistance plasmids are not unique to Australian MRSA strains but are widely distributed throughout the world.

Genetics and epidemiology of methicillin-resistant Staphylococcus aureus isolated in a Western Australian hospital.

Strains of methicillin-resistant Staphylococcus aureus (MRSA) isolated in the Royal Perth Hospital (RPH) in Western Australia have been analysed genetically and three main types were characterized: (i) strains similar to those isolated in Europe before 1973. These strains caused small outbreaks in the RPH during the period 1966-1974, but have not been isolated in recent years, except from one patient with reactivation of osteomyelitis after 16 years; (ii) strains of the type prevalent in eastern and northern Australia, one of which caused a difficult-to-control outbreak in the RPH in 1982. Strains of this type have previously been isolated only from patients who had been in hospitals in eastern and northern Australia, but recently were isolated also from other patients--which indicates that this type of MRSA is now present in the Western Australian community; and (iii) strains, which are genetically different from either of the above two types, were isolated from patients who had been in hospitals in Southeast Asia, but have not yet caused an outbreak in the RPH.

Genetic analysis of methicillin-resistant Staphylococcus aureus from a Western Australian hospital.

The isolates from an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) at Royal Perth Hospital (RPH) during a 3 month period in 1982 have been compared genetically with MRSAs isolated at the same hospital during 1969-1973. The 1982 isolates are genetically similar to isolates from eastern Australia and appear to have been introduced by a patient transferred from a hospital in another Australian state. Methicillin-resistant Staph. aureus isolated during 1969-1973 were genetically distinguishable from the 1982 isolates but were similar to strains reported from elsewhere in the world during these years. This indicates that the MRSA strains currently prevalent in Australia are either a new type or are related to previous MRSAs but have undergone considerable genetic change. These results give further support to suggestions that the strains of MRSA currently being isolated in Australian hospitals have special properties which have facilitated their spread.

"Australian" methicillin-resistant Staphylococcus aureus in a London hospital?

The Royal Free Hospital in London has experienced the spread of a phage 85 methicillin-resistant Staphylococcus aureus (MRSA). An analysis of a sample has demonstrated that the plasmids carried by these strains, and the type and location of their drug-resistance determinants, are similar to those of MRSA strains which have been causing problems in some Australian hospitals.

Transposition of gentamicin resistance to staphylococcal plasmids encoding resistance to cationic agents.

Plasmid pWG115 isolated from a methicillin-resistant Staphylococcus aureus encodes resistance to cationic surface-active agents and trimethoprim. It has a molecular weight of ca 14.6 megadaltons and can be transferred to other strains of staphylococci in mixed-culture transfer with propamidine isethionate as a selective agent. Gentamicin resistance in Australian methicillin-resistant Staph. aureus isolates can be either chromosomal or plasmid-borne. The most common gentamicin resistance plasmid is 18.0 megadaltons and also encodes resistance to trimethoprim and cationic surface-active agents. This suggested that pWG115 was related to gentamicin resistance plasmids and that it may provide a target for the postulated gentamicin resistance transposon. This paper demonstrates that the chromosomal gentamicin resistance determinant from WG523 can transpose into pWG115 to generate an 18.0 megadalton plasmid, phenotypically indistinguishable from the naturally occurring gentamicin resistance plasmids such as pWG53. EcoR1 restriction enzyme analysis demonstrated that gentamicin resistance can transpose into at least two sites on pWG115. One of these sites generates EcoR1 restriction fragments identical to pWG53. The 5.2 kilobase pair (3.4 megadalton) element involved confers low-level resistance to gentamicin, cross resistance to tobramycin and kanamycin, and has been designated Tn3851.

Analysis of plasmids mediating gentamicin resistance in methicillin-resistant Staphylococcus aureus.

Gentamicin-resistance plasmids in methicillin-resistant Staphylococcus aureus isolated from four Australian hospitals have been studied. All the plasmids conferred resistance to gentamicin, tobramycin, kanamycin and all but one conferred resistance to quarternary ammonium compounds. Plasmids which only carried these resistance determinants were ca 15.3 megadaltons and were only found in isolates from one hospital. The most common plasmid was ca 18.0 megadaltons and in addition encoded trimethoprim resistance. Two plasmids, one of ca 19.6 megadaltons and one of ca 28.5 megadaltons were found to carry the penicillinase determinant. However only the larger of these encoded heavy metal ion resistance and was sensitive to quaternary ammonium compounds. EcoR1 analysis indicated that all but the ca 28.5 megadalton plasmid were closely related. The EcoR1 analysis of the ca 28.5 megadalton plasmid indicated that it could have resulted from recombination between a gentamicin resistance determinant and a penicillinase plasmid.

Genetics of drug resistance in methicillin-resistant Staphylococcus aureus from Australian hospitals.

The drug-resistance determinants in methicillin-resistant Staphylococcus aureus (MRSA) from three different hospitals in Eastern Australia have been examined. With one exception, all the isolates had chromosomal determinants for penicillinase and resistance to cadmium (Cd), mercury (Hg), phenyl mercuric acetate, methicillin, tetracycline, erythromycin, lincomycin and low level streptomycin. The strain which was the exception differed in that it did not have chromosomal resistance to Cd and lincomycin. In addition, the strains often contained plasmids which belonged to one of three categories: a small cryptic plasmid of either c. 1.4 Mdal, c. 1.7 Mdal or c. 1.9 Mdal; a chloramphenicol resistance plasmid of c. 2.8 Mdal; and a gentamicin resistance plasmid within the range of c. 15.3 to c. 28.5 Mdal. The predominant gentamicin-resistant plasmid in isolates from two hospitals had a molecular weight of c. 18 Mdal, whereas the isolates from the third hospital had a plasmid of molecular weight c. 15.3 Mdal. The only other gentamicin resistance plasmids detected were associated with penicillinase determinants. In one isolate, this corresponded to a plasmid of c. 19.6 Mdal and in the other to a plasmid of c. 28.5 Mdal. These results indicate that MRSAs which are prevalent in Eastern Australian hospitals are substantially different in the location of their drug resistance determinants to earlier strains reported in the literature.

Gentamicin resistance in methicillin-resistant Staphylococcus aureus.

Gentamicin resistance has been studied in methicillin-resistant Staphylococcus aureus (MRSA) strains, from Royal Melbourne Hospital (RMH) and Sydney. Gentamicin resistance was transferred in mixed cultures to a plasmid free strain, and the determinants were examined. The Sydney strain had high level resistance to gentamicin, tobramycin, kanamycin and neomycin which was carried on a c.34 megadalton plasmid. The gentamicin resistant RMH isolates all had a determinant which conferred low level resistance to gentamicin, tobramycin and kanamycin and appeared to be chromosomal in one isolate, on a plasmid of c.28.5 megadaltons in another and on a plasmid of c.18 megadaltons in the other isolates. It is suggested that a gentamicin resistance transposon is being transferred in the MRSA at RMH.