The polymerase chain reaction (PCR) was used to study the prevalence of the macrolide resistance genes ermA, ermB, ermC, msrA/msrB, ereA and ereB, in 851 clinical isolates of Staphylococcus aureus and 75 clinical isolates of Enterococcus faecium that were erythromycin resistant. The isolates were from 24 European university hospitals. In S. aureus, the ermA gene was more common in methicillin-resistant S. aureus (MRSA) isolates (88%) than in methicillin-susceptible S. aureus (MSSA) isolates (38%), and occurred mainly in strains with constitutive MLS(B) expression. In contrast, ermC was more common in MSSA (47%) than in MRSA (5%), occurring mainly in strains with inducible expression. The ereB gene was only found in MRSA isolates expressing a constitutive MLS(B) phenotype (1%). The ereA gene was not detected. Macrolide resistance by efflux due to the msrA/msrB gene was only detected in MSSA isolates (13%). In contrast to S. aureus, erythromycin resistance in E. faecium was almost exclusively due to the presence of the ermB gene (93%).

A relationship between resistance to methicillin and resistance to fluoroquinolones, rifampin, and mupirocin has been described for Staphylococcus aureus. Differences in resistance rates may be explainable by a higher spontaneous mutation rate (MR) or a faster development of resistance (DIFF) in methicillin-resistant S. aureus (MRSA). No differences in MR, DIFF, and mutations in grlA and gyrA were detected between methicillinsusceptible S. aureus and MRSA. The higher resistance rates in MRSA are not the result of hypermutability of target genes or a faster emergence of different mutations and may be the consequence of clonal spread of multiresistant MRSA.

Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus isolates were exposed to subinhibitory MICs of ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin during a 10-day period. Subculturing led to resistance development, regardless of the initial potencies of the quinolones. None of the quinolones was associated with a significantly slower rate of resistance development.Fluoroquinolone resistance in gram-positive cocci is related to mutations in the DNA gyrase and topoisomerase IV genes (8-12, 16, 23) and the active efflux of agents (1, 3, 13, 21-25, 31, 44). Because fluoroquinolones differ in both their target affinity (8,16,(33)(34)(35)(36)39) and their activation of efflux pumps (7,13,21,22,24,25,31,42), one can speculate that the phenotypic expression of quinolone resistance will also differ. Studies have shown that fluoroquinolone resistance can be selected for in pneumococci and staphylococci (5,6,37).In order to analyze the ability of newer fluoroquinolones to cause resistance development in Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus, we repeatedly exposed six clinical strains of each species to ciprofloxacin, sparfloxacin, gatifloxacin, moxifloxacin, clinafloxacin, and gemifloxacin.Approximately 5 ϫ 10 7 CFU of each of the 18 strains was added to tubes containing 9.9 ml of appropriate broth containing antibiotic concentrations ranging from 3 doubling dilutions above to 3 doubling dilutions below the MIC of each of the six agents. The tubes were then incubated for 24 h at 37°C. Aliquots from the test tubes containing the highest drug concentration that permitted visible growth were used following a 1:100 dilution to inoculate a second set of serial drug dilutions. After overnight incubation, the bacteria were transferred again. Finally, after 10 serial transfers, the bacteria for which the MICs were the highest were collected, stored, and also subcultured on quinolone-free agar for 10 days to assess the stability of resistance.MICs were determined by the microdilution methodology according to NCCLS guidelines (29, 30). Ciprofloxacin MIC determinations were conducted in the presence and absence of reserpine (20 g/ml; tests were repeated three times) for all of the original isolates (n ϭ 18) as well as for all of the selected mutants (n ϭ 108) (7).S. pneumoniae and S. aureus isolates were analyzed before and after transfers for mutations in the quinolone-resistance determining regions (QRDRs) of parC or grlA and gyrA, respectively (19,40,41).The MIC results from subculturing as well as the mutations in the QRDRs of S. pneumoniae and S. aureus are summarized in the Tables 1 to 3. Subculturing with newer quinolones led to resistance development in all three species. This is in line with previous reports with regard to cephalosporins, macrolides, and older quinolones in pneumococci (5, 6, 37).Resistance was stable in all cases; i.e., the MICs for the 108 selected mutants remained within 1 doubling dilution after 10 tr...

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Of the 419 Moraxella catarrhalis isolates collected during the 1997-1999 European SENTRY surveillance study, 385 (92%) were ␤-lactamase positive. Twenty-two (5.7%) produced BRO-2 ␤-lactamase. Twenty-one new mutations were found in the putative promoter region of the bro genes. Nineteen percent of all isolates tested were complement sensitive. Resistance to ␤-lactams is not linked to the phylogenetic lineages associated with susceptibility to complement.Moraxella catarrhalis was long considered a harmless commensal of the respiratory tract and uniformly susceptible to penicillins. In recent years, however, its pathogenic potential has come to light (9,20,27). The production of a new ␤-lactamase enzyme, designated BRO (from Branhamella and Moraxella), by this bacterium was an event that occurred virtually simultaneously around the world (9,13,19,20). Two distinct BRO ␤-lactamase enzymes, namely, BRO-1 and BRO-2, have since been found in strains of M. catarrhalis. These enzymes are identical in substrate and inhibition profile but differ by a single amino acid (2-5). The increased level of resistance conferred by BRO-1 compared to BRO-2 appears to be related to the relative amount of each enzyme produced by the strains (5). This difference in production may be the result of differences in the promoter strength of the two genes. Bootsma et al. (5) reported a 21-bp deletion in the promoter region of the ␤-lactamase gene from a BRO-2-producing strain. In addition, Richter et al. (22) suggested that additional mutations or deletions or insertions in the promoter sequence might explain the variations seen in antibiotic susceptibilities within the populations of BRO-1-and BRO-2-producing M. catarrhalis isolates.The first goal of this study, therefore, was to characterize the BRO ␤-lactamases and their putative promoter regions in the European M. catarrhalis isolates collected between 1997 and 1999 during the European SENTRY surveillance study. The relative percentages of the two enzyme types were also determined.Besides protecting the bacteria against ␤-lactam antibiotics, ␤-lactamase can also have indirect pathogenic effects. For example, it can block the antibiotic treatment of concomitant infections with more dangerous pathogens (e.g., pneumococci), as experimentally confirmed by . This, together with the discovery of M. catarrhalis as a pathogen in its own right, has aroused scientific interest in other possible virulence factors. One of these virulence factors is the ability of M. catarrhalis to resist the action of complement (9,12,17,20,(24)(25)(26). Based on molecular typing data, i.e., pulsed-field gel electrophoresis and PCR-restriction fragment length polymorphism analysis, it has been suggested that complement-resistant M. catarrhalis isolates form a genetically distinct lineage with a different pathogenic potential within the species (3,26,28). The second aim of this study, therefore, was to determine the percentage of complement-resistant M. catarrhalis isolates in the recent SENTRY surveillance study and t...