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NADPH-dependent superoxide production by intact human neutrophils is inhibited by DPI (diphenyleneiodonium), when stimulated by either FMLP (N-formylmethionyl-leucyl-phenylalanine) or PMA (phorbol 12-myristate 13-acetate). Addition of 10 microM-DPI abolished the reduction of both the FAD and the cytochrome b components of the NADPH oxidase. DPI inhibition of the oxidase was associated with defective aerobic killing of staphylococci by human neutrophils. Anaerobic killing, phagocytosis, chemotaxis and motility were relatively unaffected by 10 microM-DPI. Degranulation of the azurophil and specific granules, induced by the soluble stimuli FMLP or PMA, and by particulate stimuli was decreased by the presence of DPI. The above effects of DPI on human neutrophils are similar to those found in chronic granulomatous disease.

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...

Fluorescein-labelled Staphylococcus aureus were used to follow changes in phagolysosome (PL) pH of bovine and human neutrophils following phagocytosis. Under aerobic conditions there was an alkalinisation of the PL followed by a slow decline. Under anaerobic conditions no alkalinisation of the PL was seen, and pharmacological inhibition of the NADPH oxidase with diphenyleneiodonium (DPI) resulted in a rapid acidification of the PL following phagocytosis. The inclusion of amiloride, an inhibitor of Na+/H+ antiporter activity, produced a more rapid alkalinisation phase following phagocytosis under aerobic conditions and reduced, but did not abolish, the acidification phases seen under anaerobic conditions or following treatment of neutrophils with DPI. The results suggest that PL pH is influenced by NADPH oxidase activity and to a lesser extent by a Na+/H+ antiporter. The antibacterial effectiveness of neutrophil granule proteins may be affected under conditions that influence the functioning of these two systems.

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...

were white to cream and light pink colored, respectively, while strain A2-1c T displayed a 37 bright pink color. All three strains were aerobic chemoheterotrophic mesophiles with a broad 38 temperature range of growth and a moderately acidic pH optimum. Sugars and complex 39 proteinaceous substrates were the preferred carbon and energy source. A few polysaccharides 40 were degraded. The major quinone in all three strains was MK-8. As minor compound MK-7 41 occurred in strain A2-1c T . Major fatty acids were iso-C 15:0 and iso-C 17:1 7c. In addition, iso-42 C 17:0 occurred in significant amounts. The DNA G+C content of strains 277 T , 307, and A2-1c T 43 was 59.6, 59.9, and 58.5 mol%, respectively. Based on these characteristics, the three isolates

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The penetration of three antibiotics, penicillin, chloramphenicol and erythromycin into bovine neutrophils, either alone or containing previously ingested Staphylococcus aureus, was determined, and their intracellular activity against these bacteria was measured. Uptake of radiolabelled antibiotics was assessed by rapidly separating neutrophils from extracellular antibiotic by centrifugation through silicone oil. Intracellular activity was estimated by comparing the numbers of bacteria surviving intracellularly in neutrophils exposed to antibiotic for 3 h at ten times the MBC, with those surviving intracellularly in untreated neutrophils. Penicillin was slightly concentrated within the neutrophils, reaching a maximum intracellular concentration 1.75 times that of the extracellular concentration; this is the C/E ratio. Chloramphenicol entered to a greater extent with a maximum C/E ratio of 7.08. Erythromycin became highly concentrated within the neutrophils with a C/E ratio of 11.46 after 90 min incubation. The presence of ingested staphylococci significantly reduced the uptake of chloramphenicol, but had no significant effect on the penetration of the other antibiotics. Intracellular activity studies indicated that, at ten times MBC, only penicillin had any significant activity against intracellular staphylococci, reducing survival by 28%. This work demonstrates that penetration of certain antibiotics can be altered by the presence of ingested staphylococci and that high intracellular levels of antibiotics do not necessarily ensure good intracellular activity against pathogenic micro-organisms.