Approximately 75% of coagulase-negative staphylococci are resistant to methicillin, but it is suspected that even more resistance exists that is not detected by standard susceptibility assays. To determine the most accurate assay for measuring resistance, we compared the detection of mecA by PCR with detection by National Committee for Clinical Laboratory Standards methods using oxacillin as the class drug. Strains from 11 species of coagulase-negative staphylococci were selected such that 84% were susceptible by the broth microdilution method. Of 45 mecA-positive strains, 1 strain was unable to express the mecA gene product after induction and was not included in further analyses. For microdilution with 2% NaCl, the disk test without salt, and agar screen containing 4% NaCl plus 6 g of oxacillin per ml, the sensitivities in detecting the 44 mecA-positive strains were 50, 84, and 70%, respectively, at 24 h and 77, 82, and 100%, respectively, at 48 h. The specificities of microdilution, disk, and agar screen in detecting the 97 strains lacking mecA were 100, 89, and 100%, respectively, at 24 h. Only the disk test proved to be less specific at 48 h (81%). Furthermore, for 10 of the mecA-positive strains plus an additional 8 strains subsequently added to the analyses, the MICs were 2 g/ml at 24 h by the broth microdilution method; all 18 strains were positive for mecA by PCR. Thus, an oxacillin MIC of >2 g/ml indicated resistance and is probably a more appropriate breakpoint than the current National Committee for Clinical Laboratory Standards breakpoint of 4 g/ml for coagulase-negative staphylococci. Strains for which MICs are <2 g/ml may be methicillin resistant and should be verified as susceptible by oxacillin agar screening with incubation for 48 h.
Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries.
During 1994 and 1995, 157 isolates of Streptococcus pyogenes from patients with invasive disease were consecutively collected in the San Francisco Bay area to determine the frequency of antimicrobial resistance. Susceptibility testing was performed according to the guidelines of the National Committee for Clinical Laboratory Standards by the disk method and by broth microdilution. For comparison of susceptibility patterns, an additional 149 strains were randomly collected from patients with pharyngitis. For San Francisco County, 32% of the isolates from invasive-disease-related specimens but only 9% of the isolates from throat cultures from the same period were resistant to erythromycin (P = 0.0007). Alameda County and Contra Costa County had rates of resistance of ≤10% from isolates from all cultures. When the data were analyzed by hospital, the San Francisco County Hospital had a statistically higher rate of erythromycin resistance (39%) among the strains from serious infections compared to those from other counties (P = <0.0003). For tetracycline, high rates of resistance were observed in San Francisco County for both isolates from patients with invasive disease (34%) and pharyngitis (21%) in the same period. Using pulsed-field gel electrophoresis, two clones, one at the San Francisco County Hospital and a second in the entire area, were identified. The latter clone exhibited resistance to bacitracin. Of 146 strains that were tested by microdilution, all were susceptible to penicillin. Clindamycin resistance was not seen among the erythromycin-susceptible strains, but two of the 39 erythromycin-resistant strains were also resistant to clindamycin. An additional 34 strains showed resistance to clindamycin when exposed to an erythromycin disk in the double-disk diffusion test, suggesting that the mechanism of erythromycin resistance is due to an erm gene. This study demonstrates a high rate of resistance to macrolides and tetracycline among S. pyogenes isolates in San Francisco County and shows that macrolide resistance is more common in strains from patients with invasive disease than in strains from those with pharyngitis.
MYCO/F Lytic medium (MFL), a liquid medium developed for use with the BACTEC 9240 blood culture system, was compared to the Isolator system (IS) for the recovery of fungi and to the BACTEC 13A medium for the recovery of mycobacteria. Recovery of bacteria was compared to routine BACTEC Plus Aerobic/F (AF) blood cultures. Microbial growth was detected in 203 (17%) of 1,166 blood cultures. Fifty-seven specimens were positive for fungi: 35 were positive with both IS and MFL; six were positive with IS only (three Candida albicans, one Histoplasma capsulatum, one Candida glabrata, and one Fusarium species isolate); three were positive with AF only (two C. albicans and one Candida parapsilosis isolate); and 13 were positive with MFL only (five C. glabrata, three C. albicans, two Candida krusei, two Candida tropicalis, and one C. parapsilosis isolate; P > 0.05 versus IS). Eighteen of 19 blood cultures positive for H. capsulatum grew in both IS and MFL, although the time to detection for MFL was greater. The mean time to detection for all fungi was 8.15 days for IS and 12.07 days for MFL. Seven hundred forty specimens were also cultured for mycobacteria with MFL and 13A. Forty-four grew mycobacteria; 38 were positive with both 13A and MFL; and 16 were positive with MFL only. Mycobacterium avium was recovered from 41 specimens; 36 were positive for both systems and 5 were positive for MFL alone. MFL was also compared to the AF bottle for the same 740 specimens. MFL and AF both detected 34 of the 40 clinically significant bacteria, while IS detected only 15 of 40. In summary, MFL is an excellent medium for the recovery of fungi, mycobacteria, and bacteria; however, the time to detection of H. capsulatum is increased.Blood cultures have become one of the most critically important and frequently performed tests in the clinical microbiology laboratory (1,5,9). Various culture systems may be used to recover microorganisms from these specimens, but for optimal recovery, a fully automated system and media that are directly inoculated with the specimens are most desirable (1, 11). Several systems are available for the detection of fungemia, which perform relatively well for the recovery of yeasts (3, 7-9, 12, 15). The Isolator system (IS) (Wampole Laboratories, Cranbury, N.J.) has been the method of choice for the recovery of Histoplasma capsulatum, although other systems do show promise (11; D. Fuller, J. Daily, T. Davis, G. Denys, C. Hazelrigg, and M. York, Abstr. 97th Gen. Meet. Am. Soc. Microbiol., abstr. C-231, p. 160, 1997). Several automated systems reliably detect mycobacteria from blood, but often specimens must first be processed by lysis-centrifugation, as with the IS, and the pellet must be inoculated into the medium. Many automated systems exist for the detection of bacteria in blood, and some have well-documented recovery from sterile body fluids as well (1, 9). Often patients with a higher probability for fungemia will also be at risk for mycobacteremia. Therefore, a fully automated blood culture system with a med...
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