Penicillin-binding properties and characteristics of penicillin-binding proteins (PBPs) were investigated in several clinical isolates of Streptococcus pneumoniae differing in their susceptibilities to penicillin (minimal inhibitory concentration [MIC], 0.03 to 0.5 jig/ml) and compared with the penicillin-susceptible strain R36A (MIC, 0.07 ,ug/ml). Several changes accompanied the development of resistance: the relative affinity to penicillin of whole cells, isolated membranes, and two major PBPs after in vivo or in vitro labeling decreased (with increasing resistance). Furthermore, one additional PBP (2') appeared in four of five relatively resistant strains with an MIC of 0.25 ,ug/ml and higher. PBP 3 maintained the same high affinity toward penicillin in all strains under all labeling conditions.
During 1984 we conducted a population-based survey of culture-confirmed invasive disease due to Streptococcus pneumoniae among persons who lived in the Oklahoma City, Oklahoma, metropolitan area (population, 846,000) through the 20 clinical laboratories in the area. There were 139 residents identified with invasive pneumococcal disease (11 with meningitis and 128 with other bacteremic infections), for an infection rate of 16.4 per 100,000 population (meningitis, 1.3 cases per 100,000; other bacteremias, 15.1 cases per 100,000). Cases peaked in January-May and December (75% of cases). Rates were highest among infants less than 12 months old (97 cases per 100,000) and persons greater than or equal to 80 years old (87 cases per 100,000). Seventeen (12.2%) of the pneumococcal isolates were relatively penicillin resistant. These isolates were most prevalent among elderly persons greater than or equal to 70 years old (six [17.6%] of 34) and young children 0-4 years old (7 [15.9%] of 44) compared with persons 5-69 years old (four [6.6%] of 61).
The inhibitory and bactericidal activities of carbenicillin, ticarcillin, moxalactam, cefoperazone, azlocillin, piperacillin, ceftazidime, and three aminoglycosides, alone and in various combinations, were determined against 60 isolates of Pseudomonas aeruginosa from the sputum of patients with cystic fibrosis. Ceftazidime was the most active ,B-lactam, with minimum inhibitory and bactericidal concentrations for 90o of isolates of 4 ,ug/ml. Moxalactam was the least active of the new -lactams, with activity equivalent to that of carbenicillin; each had a minimum inhibitory concentration for 90% of isolates of 64 ,ug/ml and a minimum bactericidal concentration for 90% of isolates of 128 p.g/ml. Of the recently introduced 3-lactam derivatives, azlocillin, cefoperazone, ceftazidime, moxalactam, and piperacillin have a higher order of activity than their predecessors against not only the Enterobacteriaceae but also against Pseudomonas aeruginosa (2,10,13,15,18). Thus, they might be useful in the treatment ofP. aeruginosa pulmonary infections which are currently difficult to manage in patients with cystic fibrosis (CF). This possibility led to the current study of the activities of these new 3-lactams, alone and in combination with each other or gentamicin, tobramycin, and amikacin, against 60 isolates of P. aeruginosa from the sputum of patients with CF. MATERIALS AND METHODSA total of 60 isolates of P. aeruginosa, including mucoid and nonmucoid strains, was identified by standard microbiological methods. The organisms were then stored on tryptic soy agar slants and were checked for purity and subcultured monthly.Moxalactam (Eli Lilly & Co.), piperacillin (Lederle Laboratories), carbenicillin (Roerig-Pfizer Co.), azlocillin (Delbay Research Corp.), ticarcillin (Beecham Laboratories), cefoperazone (Pfizer Inc.), ceftazidime (Glaxo Research Group), gentamicin (Schering Corp.), tobramycin (Eli Lilly), and amikacin (Bristol Laboratories) were furnished as dry powders. Stock solutions were prepared from these powders and were used immediately or stored at -70°C for no longer than 2 weeks.Minimum inhibitory concentrations (MICs) were determined by microbroth dilution, and combination studies were done by microbroth checkerboard methods. Mueller-Hinton broth was used for all determinations. Mueller-Hinton broth was adjusted to optimal calcium and magnesium concentrations and a pH of 7.2 to 7.4 (6). The preparation and inoculation of microbroth plates were accomplished with the MIC-2000 system (Dynatech Corp.). Plates were used immediately or stored at -70°C for no longer than 2 weeks. Microbroth plates received an inoculum consisting of a 10' dilution of a log-phase culture adjusted to the density of a 0.
Thirty-three clinical isolates of Streptococcus pneumoniae were tested for susceptibility to penicillin and ampicillin by a standard agar dilution method. Results were compared to those obtained using a micro-broth dilution technique in which Mueller-Hinton broth was supplemented with 5% difibrinated whole sheep blood. Among the 33 strains, 2 were resistant (minimal inhibitory concentration, 8 ,ug/ml), 10 were relatively resistant (minimal inhibitory concentration, 0.12 to 0.5 ,ug/ml), and 20 were susceptible (minimal inhibitory concentration, <0.06 /Lg/ml) to penicillin by both methods. Only one strain showed a twodilutional-step difference by micro-broth and agar dilution testing resulting in categorization as relatively resistant by the former method but susceptible by the latter. A 1-,ug oxacillin disk correctly identified 11 of the 12 resistant strains. The micro-broth dilution technique is a reliable, simple method for penicillin or ampicillin susceptibility testing of pneumococci and economically feasible to perform manually or with a semiautomated system. Penicillin-resistant isolates of Streptococcus penumoniae have been noted with increasing frequency (1, 11) and have been clinically significant in patients with meningitis (7, 10). Because of this, disk diffusion susceptibility testing with a 1-,ug oxacillin disk is recommended as a screening test on all S. pneumoniae isolates from cerebrospinal fluid. Although quantitative susceptibility testing would be desirable for strains apparently resistant by this method, the recommended technique is agar dilution, which is cumbersome and expensive when a small number of isolates require testing (15). Our objective was to evaluate a micro-broth dilution technique for the antimicrobial susceptibility testing of S. pneumoniae. We report this method and compare the results with those obtained with the agar dilution technique and oxacillin disk diffusion test.MATERIALS AND METHODS Organisms. Thirty-three strains of S. pneumoniae were studied. Thirty-one strains were isolated from patients in the Oklahoma area. The remaining two strains were resistant South African isolates. Identifications were based on colony morphology, Gram stain, bile solubility, and optochin susceptibility (2). All strains were initially screened for susceptibility to penicillin by using a 1-,ug oxacillin disk, and thosefound to be resistant were tested for fi-lactamase production by the chromogenic cephalosporin method (9).All were f4-lactamase negative. Laboratory reference strains of Escherichia coli and Staphylococcus aureus were used as controls throughout the experiments. Disk diffusion testing. Oxacillin disks (1 jig;Pfizer, Inc.) were tested against all strains using the methodology described by the National Committee for Clinical Laboratory Standards (8). Interpretation of zone sizes with oxacillin was as follows: c12 mm, resistant; 13 to 19 mm, indeterminate; -20 mm, susceptible (14).Agar dilution testing. The methodology employed was essentially that described by an international co...
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