Ninety clinical Staphylococcus aureus isolates from separate patients were examined phenotypically and genotypically for susceptibility to methicillin/oxacillin. Thirty were methicillin/oxacillin susceptible and 60 were methicillin and oxacillin resistant (MRSA). The 60 MRSA isolates examined were subdivided into two groups according to their antibiotic profiles and comprised 30 non-multidrug-resistant (NMDR) isolates, resistant to less than two non-beta-lactam antibiotics, and 30 multidrug-resistant (MDR) isolates, resistant to three or more non-beta-lactam antibiotics. Phenotypic and genotypic analysis of methicillin/oxacillin showed that despite use of the guidelines published by the NCCLS for the testing of S. aureus susceptibility to methicillin/oxacillin, MIC values of some NMDR MRSA isolates fell below the NCCLS-recommended breakpoints. Etest strips failed to detect two NMDR MRSA isolates tested with oxacillin and four tested with methicillin. Lowering the NCCLS-recommended oxacillin screen agar concentration from 6 to 2 mg/L and temperature of incubation to 30 degrees C, improved the specificity and sensitivity of NMDR MRSA detection from 87% to 100%. On PFGE analysis these NMDR MRSA strains were genotypically different. Genotypic tests, such as multiplex PCR for the mecA/nuc genes and DNA hybridization for the mecA gene, or phenotypic monoclonal antibody-based tests to detect penicillin-binding protein 2a (PBP2a) offer advantages for problematic isolates in detecting or confirming low-level phenotypic heterogeneous mecA expression of oxacillin and methicillin resistance in NMDR MRSA.

Genetic investigations were carried out with 50 phenotypically selected strains of Pseudomonas aeruginosa from 18 patients attending an Australian cystic fibrosis (CF) center. The isolates were analyzed by restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE). Phylogenetic analysis of the macrorestriction patterns showed rates of genetic similarity ranging from 76 to 100%; 24 (48%) of the strains from 11 patients had greater than 90% similarity. A dominant strain emerged: 15 isolates from seven patients had identical PFGE patterns, and 4 other isolates were very closely related. The 50 isolates were grouped into 21 pulsotypes on the basis of visual delineation of a three-band difference. Ten of the 18 (56%) patients were infected with clonal or subclonal strains. Sequence analysis of PCR products derived from the mucA gene showed 20 mutations, with the number of mutations in individual isolates ranging from 1 to 4; 19 of these changes are reported here for the first time. Potentially functional changes were found in 22 (44%) isolates. Eight changes (five transversions and three single base deletions) led to premature stop codons, providing support for the presence of mucA mutations as one pathway to mucoidy. There was a trend toward an association between the dominant strain and lack of potentially functional mucA mutations (P ‫؍‬ 0.09 by the 2 test) but no relationship between genotype and phenotype. This is the first study of genetic variation in P. aeruginosa isolates from adult Australian CF patients. The findings highlight the need for further investigations on the transmissibility of P. aeruginosa in CF patients.Chronic lung infection with Pseudomonas aeruginosa leading to respiratory failure is the major cause of both morbidity and mortality in patients with cystic fibrosis (CF) (6). Genetic analyses by techniques such as restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE) have indicated that most CF patients are colonized for long periods with single clones or subclones of P. aeruginosa (4,7,9,17,21,23), and the appearance of new unrelated strains has often been associated with changes in antibiotic therapy (3). It has been believed that P. aeruginosa is generally environmentally acquired and that person-to-person spread occurs only rarely (3,5,9,11,16,20,21,23). However, recent reports of the transmission of virulent multidrug-resistant mucoid strains of P. aeruginosa in a large Australian pediatric CF clinic (1) and two adult CF clinics in the United Kingdom (10, 13) and the person-to-person spread of P. aeruginosa among holiday campers with CF in Germany (9) have raised concerns about the effectiveness of standard infection control practices for P. aeruginosa. Nonetheless, confirmation of substantial cross infection with epidemic strains would be needed to support a policy of segregation like the one already practiced for Burkholderia cepacia.Associated with the chronic colonization of P. aeruginosa in CF pat...

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In this study, the phenotypic and genotypic characteristics of 50 methicillin-resistant Staphylococcus aureus (MRSA) isolates (43 contemporary and 7 archaic strains from the mid-1960s) from four Sydney hospitals in the central Sydney area were compared. Phenotypic analysis based on antibiotic profiles and phage typing patterns categorized the MRSA isolates into three major groups: multidrug resistant (mMRSA), non-multidrug resistant (nmMRSA), and archaic. The nmMRSA isolates could be further subdivided into nmMRSA group 1, which was phage typeable and similar to the archaic group; nmMRSA group 2, which was non-phage typeable and only resistant to ciprofloxacin; and nmMRSA group 3, which was also nontypeable and generally resistant to other antibiotics. The characterization of all five phenotypic groups was then extended by genetic analysis. Restriction fragment length polymorphism (RFLP) analysis showed the 50 isolates could be sorted into 20 group-specific pulsotypes. mecI gene deletions and mutations at various percentages among the five MRSA groups were detected by sequencing. Several mec promoter mutations were also found. The overall findings indicated that nmMRSA strains may have independently acquired mec DNA and are more likely to be newly emergent strains than nmMRSA variants.

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