Investigation of inducible clindamycin and telithromycin resistance in isolates of beta-hemolytic streptococci.

We evaluated the accuracy of an erythromycin-clindamycin double-disk test (D-zone test) and an erythromycin-telithromycin D-zone test for detection of inducible resistance in isolates of beta-hemolytic streptococci with erythromycin resistance. The results of these tests were compared to results of a broth microdilution (BMD) induction test using combinations of erythromycin and either clindamycin or telithromycin. Of 29 erythromycin-resistant, clindamycin-susceptible isolates, 16 were positive by the erythromycin-clindamycin D-zone test; all of these demonstrated inducible clindamycin resistance by BMD. Twelve isolates were D-zone test-negative, did not demonstrate inducible resistance by BMD, and were positive for a mef determinant. Of 39 erythromycin-resistant, telithromycin-susceptible isolates, 13 were erythromycin-telithromycin D-zone test-positive, 19 questionably positive (unclear blunting of the zone), and 7 were D-zone test-negative. The erythromycin-telithromycin D-zone test result did not correlate with inducible resistance by BMD or the presence of an erm or mef gene. These results demonstrate that the erythromycin-clindamycin D-zone and BMD induction tests accurately detect inducible clindamycin resistance, but the erythromycin-telithromycin D-zone test is not reliable for detecting inducible telithromycin resistance.

Testing for induction of clindamycin resistance in erythromycin-resistant isolates of Staphylococcus aureus.

Disk diffusion and broth microdilution (BMD) were used to perform clindamycin (CLI) induction testing on 128 selected nonduplicate isolates of Staphylococcus aureus. Disk diffusion testing involved placing CLI and erythromycin (ERY) disks approximately 12 mm apart (measured edge to edge) on a Mueller-Hinton agar plate that had been inoculated with an S. aureus isolate; the plate was then incubated for 16 to 18 h. Two distinct induction phenotypes (labeled D and D(+)) and four noninduction phenotypes (designated as negative [Neg], hazy D zone [HD], resistant [R], and susceptible [S]) were observed in disk diffusion results. A clear, D-shaped zone of inhibition around the CLI disk was designated as the D phenotype and was observed for 21 isolates while a D-shaped zone containing inner colonies growing up to the CLI disk was designated as D(+) (17 isolates). In addition, 10 isolates were CLI susceptible and ERY resistant but were not inducible and showed no blunting of the CLI zone (Neg phenotype). Isolates that were CLI and ERY resistant (constitutive macrolide-lincosamide-streptogramin B resistance) demonstrated either a double zone of inhibition with an inner ring of reduced growth up to the edge of the disks (HD phenotype; 33 isolates) or solid growth around the CLI and ERY disks (R phenotype; 16 isolates). Finally, 31 isolates were susceptible by disk testing to both CLI and ERY (S phenotype). PCR results showed that isolates with a D phenotype harbored ermA, isolates with a D(+) phenotype contained either ermC (16 isolates) or ermA and ermC (one isolate), and all 10 isolates with a Neg phenotype contained msrA. All isolates with an HD or R phenotype harbored at least one erm gene. Isolates showing the D(+) phenotype by disk diffusion were also detected by BMD using a variety of CLI and ERY concentrations; however, isolates with the D phenotype were more difficult to detect by BMD and will likely require optimization of ERY and CLI concentrations in multilaboratory studies to ensure adequate sensitivity. Thus, at present, disk diffusion is the preferred method for testing S. aureus isolates for inducible CLI resistance.

Identification of some charcoal-black-pigmented CDC fermentative coryneform group 4 isolates as Rothia dentocariosa and some as Corynebacterium aurimucosum: proposal of Rothia dentocariosa emend. Georg and Brown 1967, Corynebacterium aurimucosum emend. Yassin et al. 2002, and Corynebacterium nigricans Shukla et al. 2003 pro synon. Corynebacterium aurimucosum.

Sixty-three clinical isolates of charcoal-black-pigmented, gram-positive coryneform rods were received for identification by the Centers for Disease Control and Prevention (CDC) and were provisionally designated CDC fermentative coryneform group 4 (FCG4). Forty-five of these were characterized by morphological, physiologic, antimicrobial susceptibility, cellular fatty acids, 16S rRNA gene sequencing, and DNA-DNA hybridization analyses. Nitrate reduction, cellular fatty acid analysis, 16S rRNA gene sequencing, and DNA-DNA hybridization studies segregated these strains into two groups: FCG4a (8 strains) and FCG4b (37 strains). The FCG4a strains, only one of which was from a female genitourinary source, produced cellular fatty acid and biochemical profiles similar to those observed with reference strains of Rothia dentocariosa and Rothia mucilaginosa, while the FCG4b strains were similar to Corynebacterium species. DNA-DNA hybridization analysis demonstrated species-level relatedness among six FCG4a tested strains and showed that they were a charcoal-black-pigmented variant of R. dentocariosa. Sixteen isolates of the FCG4b group, mainly from female genitourinary tract specimens, as well as the type strains of two recently named species, Corynebacterium aurimucosum and Corynebacterium nigricans, were shown by DNA-DNA hybridization analysis and the sequencing of the 16S rRNA gene to be related at the species level and unrelated to the type strain of R. dentocariosa; therefore, the Corynebacterium-like strains were classified as a charcoal-black-pigmented variant of C. aurimucosum, because this name has nomenclatural priority over C. nigricans. These findings indicate that FCG4 represents a heterogeneous group that contains pigmented variants of both R. dentocariosa and C. aurimucosum; hence, the descriptions of both R. dentocariosa and C. aurimucosum have been amended to include charcoal-black-pigmented variants, and C. nigricans is a pro synonym of C. aurimucosum.

Utility of NCCLS guidelines for identifying extended-spectrum beta-lactamases in non-Escherichia coli and Non-Klebsiella spp. of Enterobacteriaceae.

NCCLS screening and confirmation methods for detecting extended-spectrum beta-lactamases (ESBLs) apply only to Escherichia coli and Klebsiella spp., yet ESBLs have been found in other members of the family Enterobacteriaceae. We evaluated the effectiveness of NCCLS methods for detecting ESBLs in 690 gram-negative isolates of Enterobacteriaceae that excluded E. coli, Klebsiella pneumoniae, and Klebsiella oxytoca. Isolates were collected between January 1996 and June 1999 from 53 U.S. hospitals participating in Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology). The antimicrobial susceptibility patterns of the isolates were determined by using the NCCLS broth microdilution method (BMD), and those isolates for which the MIC of ceftazidime, cefotaxime, ceftriaxone, or aztreonam was >or=2 microg/ml or the MIC of cefpodoxime was >or=8 microg/ml (positive ESBL screen test) were further tested for a clavulanic acid (CA) effect by BMD and the disk diffusion method (confirmation tests). Although 355 (51.4%) of the isolates were ESBL screen test positive, only 15 (2.2%) showed a CA effect. Since 3 of the 15 isolates were already highly resistant to the five NCCLS indicator drugs, ESBL detection would have an impact on the reporting of only 1.7% of the isolates in the study. Only 6 of the 15 isolates that showed a CA effect contained a bla(TEM), bla(SHV), bla(CTX-M), or bla(OXA) beta-lactamase gene as determined by PCR (with a corresponding isoelectric focusing pattern). Extension of the NCCLS guidelines for ESBL detection to Enterobacteriaceae other than E. coli and Klebsiella spp. does not appear to be warranted in the United States at present, since the test has poor specificity for this population and would result in changes in categorical interpretations for only 1.7% of Enterobacteriaceae tested.

Evaluation of the NCCLS extended-spectrum beta-lactamase confirmation methods for Escherichia coli with isolates collected during Project ICARE.

To determine whether confirmatory tests for extended-spectrum beta-lactamase (ESBL) production in Escherichia coli are necessary, we selected 131 E. coli isolates that met the National Committee for Clinical Laboratory Standards (NCCLS) screening criteria for potential ESBL production from the Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) strain collection. For all 131 isolates, the broth microdilution (BMD) MIC of at least one extended-spectrum cephalosporin was >/=2 micro g/ml. For 21 of 131 (16%) isolates, the ESBL confirmatory test was positive; i.e., the BMD MICs of ceftazidime or cefotaxime decreased by >/=3 doubling dilutions in the presence of clavulanic acid (CA) or the disk diffusion zone diameters increased by >/=5 mm around ceftazidime or cefotaxime disks in the presence of CA. All 21 isolates were shown by PCR to contain at least one of the genes bla(TEM), bla(SHV), and bla(OXA), and in isoelectric focusing (IEF) tests, all isolates demonstrated at least one beta-lactamase band consistent with a TEM, SHV, or OXA enzyme. Of the 21 isolates, 3 showed a CA effect for cefotaxime by BMD but not by disk diffusion testing. A total of 59 (45%) of the 131 isolates demonstrated decreased susceptibility to cefpodoxime alone (MIC = 2 to 4 micro g/ml), and none had a positive ESBL confirmatory test result. These were classified as false positives according to ESBL screen test results. For the remaining 51 (39%) isolates, the cefpodoxime MICs ranged from 16 to >128 micro g/ml and the MICs for the other extended-spectrum cephalosporins were highly variable. All 51 isolates gave negative ESBL confirmatory test results. Most showed IEF profiles consistent with production of both a TEM and an AmpC beta-lactamase, and representative isolates of several phenotypic groups showed changes in porin profiles; these 51 isolates were considered true negatives. In all, only 16% of 131 E. coli isolates identified as potential ESBL producers by the current NCCLS screening criteria were confirmed as ESBL producers. Thus, changing the interpretation of extended-spectrum cephalosporins and aztreonam results from the susceptible to the resistant category without confirming the presence of an ESBL phenotype would lead to a large percentage of false resistance results and is not recommended. However, by increasing the cefpodoxime MIC screening breakpoint to >/=8 micro g/ml, 45% of the false-positive results could be eliminated. NCCLS has incorporated this change in the cefpodoxime screening breakpoint in its recent documents.

In vitro activities of Daptomycin, Linezolid, and Quinupristin-Dalfopristin against a challenge panel of Staphylococci and Enterococci, including vancomycin-intermediate staphylococcus aureus and vancomycin-resistant Enterococcus faecium.

We assessed the in vitro activities of daptomycin, linezolid, and quinupristin-dalfopristin (QD) against a contemporary challenge panel of 88 staphylococcal and 90 enterococcal isolates. The staphylococci selected included vancomycin-intermediate Staphylococcus aureus (VISA), methicillin-resistant S. aureus, and coagulasenegative staphylococci. Enterococcal isolates included vancomycin-resistant Enterococcus faecium (VREF) containing either vanA, vanB1, or vanD. The MICs of daptomycin, linezolid, and QD were determined using commercial broth microdilution panels. All three VISA isolates were susceptible to daptomycin, linezolid, and QD. QD was the most active agent against staphylococcal isolates (MIC50 < or = 0.5 microg/ml and MIC90 = 1 microg/ml), including those with decreased susceptibility to vancomycin. QD was also the most active agent against VREF (MIC90 < or = 0.5 microg/ml). No differences were seen for susceptibility of vanA, vanB1, and vanD VREF strains for daptomycin, linezolid, or QD. Daptomycin was the most effective against E. faecalis. On the basis of manufacturer-suggested interpretive criteria, 92% of isolates were susceptible (MIC90 = 4 microg/ml). All isolates tested were susceptible to at least one antimicrobial agent for which interpretive criteria have been defined. Population analysis of three S. aureus isolates for which the daptomycin MICs were 8 microg/ml showed a pattern of homogeneous resistance.