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.

Comparative measurements of plasma binding capacity and concentration of human sex hormone binding globulin.

Comparisons are made of the plasma binding capacity and concentration of sex hormone binding globulin. Concentration was measured by electroimmunodiffusion standardised in terms of mass of the protein and binding capacity by two methods measuring the binding of 5 alpha-dihydrotestosterone. Isolation of steroid bound by SHBG was by either ammonium sulphate precipitation or cellulose filter discs. Both binding methods correlate highly with electroimmunodiffusion indicating they respond similarly to changes in the plasma concentration of the protein. However, they do not equally reflect the actual concentration. Estimates of the molecular mass of the protein of 188000 and 100000 from the precipitation and disc methods respectively, suggest the former measures less of the protein present than does the latter. A parallel reduction in binding capacity and concentration is seen in obese post-menopausal females. This previously unreported finding suggests that the reduced plasma binding capacity of sex hormone binding globulin in obesity is not due to altered or impaired steroid binding.

A rapid procedure for the isolation of human sex steroid binding protein.

A three stage procedure is described which provides a rapid, simple, and reliable means to isolate sex steroid binding protein from human serum. Purification is achieved by the sequential use of affinity chromatography, isoelectric focusing in granulated gels and ion exchange chromatography. Between 3--4 mg of protein can be recovered from one litre of pregnancy serum without four days. A molecular radius of 2.98 nm and an apparent molecular mass of 91 000 were obtained for the purified native protein by polyacrylamide gel electrophoresis. A value of 50 000 was obtained for the molecular mass of the reduced protein by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The purified protein was used to prepare a monospecific rabbit antiserum and a murine monoclonal antibody. Partial characterisation of the latter has been achieved.