Twenty five strains of Enterobacteriaceae (five each of Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii and Providencia stuartii) were exposed to aztreonam and ceftazidime at 1/2 and 1 x MIC in liquid medium for 22 h, and also to 3, 5 and 10 x MIC and 16 mg/l of each agent in agar. Any putative mutant with an increase in the MIC of greater than or equal to 4 fold was examined for beta-lactamase expression and outer membrane protein profile. Mutants were selected on agar at approximately 10(7); however in liquid medium not all strains yielded mutants. Mutants lacking an outer membrane protein (OMP) with a molecular weight of 40,000 (+/- 5000) were selected with both agents, as were mutants expressing constitutive Richmond and Sykes Class 1 beta-lactamase. For the Ent. cloacae mutants increased beta-lactamase gave rise to MICs above the breakpoint of both agents, whereas with the other species ceftazidime susceptibility was more affected. Strains that were OMP- rarely had MICs above the breakpoint, unless there was also increased beta-lactamase expression, as in species such as M. morganii. Hence the major mechanism of resistance in these strains would appear to be beta-lactamase mediated rather than due to altered expression of outer membrane proteins.

Twenty-five strains of Enterobacteriaceae (five each of Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii, and Providencia stuartii) and five strains of Pseudomonas aeruginosa were exposed to various concentrations of cefpirome or ceftazidime in agar. Mutants with a greater than four-fold increase in the MIC were examined for changes in beta-lactamase expression and outer membrane protein (OMP) profile. Both agents selected mutants with decreased susceptibility to the selecting antibiotic and other beta-lactams at a frequency of 10(-7)-10(-8). The MICs of all beta-lactams were higher for the resistant mutants of E. cloacae and P. aeruginosa than for the other species. Both agents selected mutants expressing derepressed class I beta-lactamase, but this was more common with ceftazidime. Only a few mutants of P. aeruginosa and E. cloacae had an MIC of cefpirome that was above the recommended breakpoint concentration. Some mutant strains of Enterobacteriaceae lacked an OMP of molecular size similar to OmpF, but the MIC of cefpirome was below the breakpoint concentration for all these strains.

The susceptibility of 47 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) to cefpirome, ceftazidime and methicillin was determined with Isosensitest media, with/without 5% NaCl and incubation at 30 degrees, 37 degrees and 44 degrees C for 24 and 48 h. At 24 h the MIC50 of cefpirome was 8 mg/l compared to 64 mg/l ceftazidime; at 48 h this increased to 32 mg/l cefpirome. The addition of 10 mg/l clavulanic acid or sulbactam lowered the MIC of cefpirome (at 48 h) by greater than four-fold in 23% and 11% of the strains, respectively. Cefpirome had primary affinity for penicillin-binding protein (PBP) 1 and 2 in five MRSA and one methicillin-susceptible Staphylococcus aureus. PBP 2a was present in all MRSA and was not saturated by 64 mg/l cefpirome. Clavulanic acid at a concentration of 10 mg/l bound to PBP 2 by greater than 50% in all strains, and when combined with cefpirome, the density of PBP 2a was also reduced but not completely abolished. The data from this study suggests that the mechanism of synergy of a beta-lactamase inhibitor plus a cephalosporin for MRSA may be due to an additive effect against PBPs and not just inhibition of a beta-lactamase. No cefpirome-resistant mutants could be selected from a methicillin-susceptible Staphylococcus aureus, but mutants were selected from an MRSA (expressing homogeneous methicillin resistance) for which MICs of cefpirome were 8 to 32 mg/l.

The susceptibility of 47 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) to FCE 22101, imipenem and methicillin was determined with Iso-Sensitest media, with or without NaCl and with incubation at 30 degrees and 37 degrees C and for 24 and 48 h. All strains had a MIC 8 mg/l of methicillin under at least one of the culture conditions. The MIC90 of FCE 22101 was 1 mg/l, and that of imipenem 16 mg/l. The affinity of FCE 22101 for the penicillin-binding proteins (PBPs) of five clinical isolates of MRSA and S. aureus 13136 p-m+ was examined in envelope preparations and whole cell assay under the growth conditions listed above. PBP 2' was detected in all MRSA, and the clinical isolates had an I50 of 4 mg/l FCE 22101. These in-vitro data suggest that FCE 22101 may be active against MRSA in clinical use.

Twenty-five strains of Enterobacteriaceae (five each of Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Morganella morganii, and Providencia stuartii) were exposed to FCE 22101 in agar containing 3, 5, or 10 x the MIC. Any putative mutant with a greater than or equal to four-fold increase in the MIC was examined for beta-lactamase expression and outer membrane protein (OMP) profile. Mutant colonies were selected at a frequency of 10(-7)-10(-11) with decreased susceptibility to FCE 22101 and other beta-lactams, but after one subculture on antibiotic-free agar the mutants from 13 of the 25 strains reverted to wild-type. Only 19 stable mutants were selected from the other 12 wild-type strains, of which 15 lacked an OMP of similar molecular size to OmpF, and/or a low size OMP of approximately 18 kDa. None of the mutants had a significant alteration in expression of Richmond & Sykes class I beta-lactamase. In a separate section of the study in which 50 strains of Pseudomonas aeruginosa were examined, it was found that FCE 22101, at a concentration of 4 mg/L, induced beta-lactamase expression such that, after 24 h exposure, 24 of the 50 strains had a greater than or equal to four-fold rise in the MIC of several anti-pseudomonal beta-lactams.