ABSTRACT
We report the cloning and sequencing of vanA genes present in the high-level vancomycin- and teicoplanin-resistant clinical isolates Oerskovia turbata 892 and Arcanobacterium (Corynebacterium) haemolyticum 872. The presence of vanA was detected by Southern blotting and PCR and confirmed by DNA sequencing. vanA-like sequences were encoded on plasmids of 15 and 20 kb respectively. The A. haemolyticum 872 DNA sequence was identical to the published vanA sequence of vancomycin-resistant Enterococcus faecium BM4147, but the O. turbata 892 sequence showed three coding changes. Induction experiments indicated that vancomycin resistance in A. haemolyticum 872 and O. turbata 892 was constitutive. SDS-PAGE analysis of membrane proteins showed the presence of a c. 39 kD protein in both clinical isolates whose expression was unaltered in the presence of vancomycin, while a similar protein in E. faecium BM4147 was inducible. Since A. haemolyticum and O. turbata are naturally susceptible to vancomycin, the high-level constitutive resistance seen in these isolates appears to be mediated by vanA. This is the first report confirming the presence of vanA in genera other than Enterococcus.
Subject(s)
Actinomycetaceae/genetics , Actinomycetales/genetics , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Carbon-Oxygen Ligases , Genes, Bacterial/genetics , Ligases/genetics , Vancomycin/pharmacology , Actinomycetaceae/drug effects , Actinomycetales/drug effects , Actinomycetales Infections/microbiology , Base Sequence , Blotting, Southern , Cloning, Molecular , DNA Primers , Drug Resistance, Microbial/genetics , Feces/microbiology , Humans , Microbial Sensitivity Tests , Molecular Sequence Data , Plasmids , Polymerase Chain ReactionABSTRACT
One hundred and forty-two coagulase-negative staphylococci (CNS) isolated from dialysate effluent or skin of patients receiving continuous ambulatory peritoneal dialysis (CAPD) were typed by extended antibiogram (16 antibiotics) and biotype (26 reactions). These isolates were then typed by supplementary methods to determine the most suitable typing method for an epidemiological study of antibiotic resistance. These included phage typing, reverse phage typing, plasmid typing, whole-cell protein typing by SDS-PAGE with analysis by densitometry, and immunoblotting. The percentage of isolates typed successfully by the supplementary methods were: phage typing 20%, reverse phage typing 0%, plasmid typing 66%, SDS-PAGE 100%, immunoblotting 100%. The discrimination of each method was: phage typing 20%, plasmid typing 37%, SDS-PAGE 69%, immunoblotting 57%. Reproducibility was 88% for phage typing and 97% for plasmid typing. The reproducibility of the whole-cell protein typing was 83% if the same extracts were used but only 43% when separate protein extracts were analysed on separate occasions. However, strain relatedness was highly reproducible. The determination of an antibiogram-biotype profile was not a sufficiently accurate typing method for an epidemiological study of antibiotic resistance. Whole-cell protein typing by SDS-PAGE or immunoblotting was technically demanding but was the most effective of the supplementary methods for detecting erroneous discrimination and false matching produced by antibiogram-biotype combinations.
Subject(s)
Bacterial Typing Techniques , Staphylococcus/classification , Bacterial Proteins/analysis , Bacteriophage Typing , Blotting, Western , Coagulase , Densitometry , Electrophoresis, Polyacrylamide Gel , Evaluation Studies as Topic , Humans , Peritoneal Dialysis, Continuous Ambulatory/adverse effects , Peritonitis/microbiology , Plasmids/genetics , Staphylococcus/enzymologyABSTRACT
A total of 50 strains of Staphylococcus aureus, including 41 methicillin-resistant S. aureus (MRSA) strains, were characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins. The protein patterns contained 40-50 discrete bands and were highly reproducible. Partial patterns were used as the basis of a computer-assisted numerical analysis. The MRSA strains clustered into four phenons at the 83% similarity level; and further division of phenon 1, at the 86% similarity level, resulted in a total of six clusters. All of the MRSA isolates from an MRSA epidemic in the United Kingdom were found to cluster in phenon 1 together with 9 of the 12 MRSA isolates from eastern Australia and 3 other MRSA isolates from the United Kingdom. The remaining three eastern Australian isolates clustered separately in phenon 2. Phenon 3 appeared to be exclusive to strains that were both susceptible and resistant to methicillin and that reacted with group V phages, and phenon 4 comprised 11 isolates, all of which were other MRSA isolates from the United Kingdom. We conclude that computer-assisted numerical analysis by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins provides additional criteria for the study of the epidemiology and the evolution of MRSA.
