The best of times,the worst of times

The development of resistance to antimicrobial agents by many bacterial pathogens has compromised traditional therapeutic regimens, making treatment of infections more difficult and frequently more expensive. Three factors have contributed to the development and spread of resistance: mutations in common genes that extend their spectrum of resistance, transfer of resistance genes among diverse microorganisms and increases in selective pressures in and outside of the hospital environment that enhance the development of resistant organisms. Some new resistance mechanisms are difficult to detect in the laboratory. Thus, resistant microorganisms may go unnoticed until they are widely disseminated in a hospital. The challenge for pharmacists, microbiologists and physicians is not only to contain the spread of existing resistant organisms, but also to prevent the emergence of new resistant pathogens by encouraging the rational and prudent use of antimicrobial agents.     

Detection of penicillin-resistant Streptococcus pneumoniae with commercially available broth microdilution panels.

We compared penicillin MICs obtained with three different commercially available broth microdilution panels (MicroScan, Sensititre, and Pasco) with MICs obtained with reference microdilution panels for 20 well-characterized pneumococci with decreased susceptibilities to penicillin (7 resistant and 13 intermediate). All panels were supplemented with 2 to 5% lysed horse blood (LHB) prepared in-house. Additional supplements included fastidious inoculum broth (FIB) for MicroScan panels and commercially prepared LHB (Difco) for Pasco panels. The percentages of penicillin-resistant strains (MIC 2 micrograms/ml) detected by the different methods follow: MicroScan-FIB, 0; MicroScan-LHB 0; Pasco in-house LHB, 71; and Sensititre-LHB, 100. The percentages of intermediate strains (MIC = 0.1 to 1.0 micrograms/ml) detected by the different methods follow: MicroScan-FIB, 31; MicroScan-LHB 23; Pasco in-house LHB, 46; and Sensititre-LHB, 85. Difco LHB supplement failed to support the growth of 86% of the strains in the Pasco panels. Of the commercially available panels evaluated, only Sensititre, supplemented with LHB prepared in-house could reliably detect penicillin-resistant pneumococci.     

Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum beta-lactamase detection methods

Extended-spectrum beta-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were > or =2 microg/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of > or =5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by > or =3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were > or =32 microg/ml, suggesting either the presence of an AmpC-type beta-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a beta-lactamase with a pI of > or =8.3 suggestive of an AmpC-type beta-lactamase; 6 of the 7 isolates were shown by PCR to contain both ampC-type and bla(OXA) genes. The IEF profiles of the remaining 16 isolates showed a variety of beta-lactamase bands, all of which had pIs of < or =7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type, bla(OXA), and bla(CTX-M) genes. In summary, 83.5% of the K. pneumoniae isolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained beta-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained beta-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.     

Development of interpretive criteria and quality control limits for macrolide and clindamycin susceptibility testing of Streptococcus pneumoniae.

A six-laboratory collaborative study was conducted to develop MIC and zone diameter quality control limits and interpretive criteria for antimicrobial susceptibility testing of Streptococcus pneumoniae with azithromycin, clarithromycin, dirithromycin, and clindamycin. The MICs of all of the agents plus erythromycin for 302 clinical isolates of pneumococci that had been selected with an emphasis on resistant strains were determined by use of the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution procedure. The zone diameters of the isolates were also determined for the same agents except erythromycin by the NCCLS disk diffusion test procedure. Repeated testing of S. pneumoniae ATCC 49619 with different sources and lots of media and disks allowed development of MIC and zone diameter quality control ranges for these agents. Interpretive criteria for the MIC of azithromycin were established and were as follows: susceptible, < or = 0.5 microgram/ml; intermediate, 1 microgram/ml; and resistant, > or = 2 micrograms/ml. The interpretive criteria advocated for the MICs of clarithromycin and clindamycin were as follows: susceptible, < or = 0.25 microgram/ml; intermediate, 0.5 microgram/ml; and resistant, > or = 1 microgram/ml. Comparison of MICs and disk diffusion zone diameters led to the development of interpretive criteria for the zone diameters for azithromycin, clarithromycin, and clindamycin that correlated well with these MIC breakpoints. Testing of this organism collection also led to the reestablishment of the erythromycin MIC breakpoints as being identical to those of clarithromycin, which resulted in equivalent cross-susceptibility and cross-resistance for the three macrolides that are currently marketed in the United States. Thus, the susceptibility of pneumococci to azithromycin and clarithromycin can be predicted accurately by testing only erythromycin in clinical laboratories. This recommendation, as well as the interpretive and quality control criteria that are described, have been accepted by NCCLS and are included in the latest NCCLS susceptibility testing guidelines.     

Comparison between testosterone oenanthate-induced azoospermia and oligozoospermia in a male contraceptive study. IV. Suppression of endogenous testicular and adrenal androgens

Administration of supraphysiological doses of testosterone to normal men causes inhibition of spermatogenesis, but while most become azoospermic, 30-55% maintain a low rate of spermatogenesis. We have investigated whether there are differences in endogenous androgen production, of testicular and adrenal origin, which may be related to the degree of suppression of spermatogenesis. Thirty-three healthy Caucasian men were given weekly i.m. injections of 200 mg testosterone oenanthate (TE), 18 became azoospermic, while 15 remained oligozoospermic. Urinary excretion of epitestosterone, a specific testicular product, was reduced to <10% of pretreatment values, with no differences between the groups. Similar results were obtained for other markers of testicular steroidogenesis. Urinary and plasma adrenal androgens were also reduced during TE treatment: a statistically significant decrease in both (P < 0.001 and P < 0.05 respectively) was seen in the azoospermic but not oligozoospermic responders. These results suggest that testicular steroidogenesis is decreased to <10% by the administration of supraphysiological doses of exogenous testosterone. Differences in the degree of ongoing steroidogenesis in the testis do not appear to account for incomplete suppression of spermatogenesis, thus differences in androgen metabolism may underlie this heterogeneous response. A small but significant reduction in secretion of adrenal androgens was also detectable, the relevance of which is unclear.      

Methods for improved detection of oxacillin resistance in coagulase-negative staphylococci: results of a multicenter study

A multilaboratory study was undertaken to determine the accuracy of the current National Committee for Clinical Laboratory Standards (NCCLS) oxacillin breakpoints for broth microdilution and disk diffusion testing of coagulase-negative staphylococci (CoNS) by using a PCR assay for mecA as the reference method. Fifty well-characterized strains of CoNS were tested for oxacillin susceptibility by the NCCLS broth microdilution and disk diffusion procedures in 11 laboratories. In addition, organisms were inoculated onto a pair of commercially prepared oxacillin agar screen plates containing 6 microg of oxacillin per ml and 4% NaCl. The results of this study and of several other published reports suggest that, in order to reliably detect the presence of resistance mediated by mecA, the oxacillin MIC breakpoint for defining resistance in CoNS should be lowered from >/=4 to >/=0.5 microg/ml and the breakpoint for susceptibility should be lowered from /=18 mm for susceptibility is suggested. Due to the poor sensitivity of the oxacillin agar screen plate for predicting resistance in this study, this test can no longer be recommended for use with CoNS. The proposed interpretive criteria for testing CoNS have been adopted by the NCCLS.     

Specimen volume versus yield in the BACTEC blood culture system.

During a 24-month period, 5,625 blood culture specimens were collected at the Seattle Veterans Administration Medical Center in 20-ml volumes and divided into separate 10-ml aliquots. The two aliquots were processed as duplicate sets (set 1, set 2) by the BACTEC system (Johnston Laboratories, Inc., Towson, Md.). Specimens (5 ml) from each set were inoculated into aerobic (6B) and anaerobic (7C/7D) vials. A total of 434 significantly positive blood cultures were found. In 342 of these positive cultures, yielding 379 isolates (112 members of the family Enterobacteriaceae, 104 staphylococci, 87 streptococci, 27 anaerobes, 20 yeasts, 14 pseudomonads, and 15 miscellaneous organisms), there was adequate specimen volume to fill all four vials. The utilization of set 1 would have resulted only in the failure to detect 65 of 379 (17.2%) significant isolates, 52 of 342 (15.2%) positive cultures, and 20 of 198 (10.1%) bacteremic episodes. There were no significant differences in the recovery of individual species in sets 1 and 2. Although the range of isolates recovered by the aerobic and anaerobic vials of each set differed, the percent yield of total isolates was similar, indicating total isolate yield was predominantly a function of specimen volume. The addition of set 2 most dramatically increased the recovery of Escherichia coli (30%), yeasts (33%), and anaerobes (42%).