Sharon A. Crawford

Practical Disk Diffusion Method for Detection of Inducible Clindamycin Resistance in Staphylococcus aureus and Coagulase-Negative Staphylococci

ABSTRACT Resistance to macrolides in staphylococci may be due to active efflux (encoded by msrA) or ribosomal target modification (macrolide-lincosamide-streptogramin B [MLSB] resistance; usually encoded by ermA or ermC). MLSB resistance is either constitutive or inducible following exposure to a macrolide. Induction tests utilize closely approximated erythromycin and clindamycin disks; the flattening of the clindamycin zone adjacent to the erythromycin disk indicates inducible MLSB resistance. The present study reassessed the reliability of placing erythromycin and clindamycin disks in adjacent positions (26 to 28 mm apart) in a standard disk dispenser, compared to distances of 15 or 20 mm. A group of 130 clinical isolates of Staphylococcus aureus and 100 isolates of erythromycin-resistant coagulase-negative staphylococci (CNS) were examined by disk approximation; all CNS isolates and a subset of S. aureus isolates were examined by PCR for ermA, ermC, and msrA. Of 114 erythromycin-resistant S. aureus isolates, 39 demonstrated constitutive resistance to clindamycin, while 33 showed inducible resistance by disk approximation at all three distances. Only one isolate failed to clearly demonstrate induction at 26 mm. Of 82 erythromycin-resistant CNS isolates that contained ermA or ermC, 57 demonstrated constitutive clindamycin resistance, and 25 demonstrated inducible resistance, at 20 and 26 mm. None of the 42 S. aureus isolates or 18 CNS isolates containing only msrA and none of the erythromycin-susceptible isolates yielded positive disk approximation tests. Simple placement of erythromycin and clindamycin disks at a distance achieved with a standard disk dispenser allowed detection of 97% of S. aureus strains and 100% of CNS strains with inducible MLSB resistance in this study.

Detection of Inducible Clindamycin Resistance of Staphylococci in Conjunction with Performance of Automated Broth Susceptibility Testing

ABSTRACT This study has shown that inducible clindamycin resistance in staphylococci can be detected by disk testing on sheep blood agar inoculum purity plates used with the bioMerieux VITEK 2. Tests of 150 erythromycin-resistant isolates correlated with standard D-zone tests on Mueller-Hinton agar and with PCR for erm(A), erm(C), and msr(A).

Susceptibility of Neisseria meningitidis to 16 Antimicrobial Agents and Characterization of Resistance Mechanisms Affecting Some Agents

ABSTRACT Neisseria meningitidis represents a pathogen of great public health importance in both developed and developing countries. Resistance to some antimicrobial agents used either for therapy of invasive infections or for prophylaxis of case contacts has long been recognized, although specific guidelines for susceptibility testing have not been fully developed. We have examined the susceptibilities of a collection of 442 meningococcal clinical isolates from 15 countries to 16 antimicrobial agents. These included isolates recovered between 1917 and 2004, with representatives of all major serogroups. All isolates were tested by the Clinical and Laboratory Standards Institute (formerly NCCLS) broth microdilution method using Mueller-Hinton lysed horse blood broth, while a subset of 102 isolates was tested by agar dilution using Mueller-Hinton sheep blood agar. Most isolates provided adequate growth for MIC determinations by both broth and agar methods. Growth in broth was enhanced by CO2 incubation and was required for two strains (1.7%). MICs of the study drugs compared favorably between the broth and agar methods (79 to 100% essential agreement), and MICs also generally agreed closely (92 to 100% essential agreement, excluding azithromycin) between broth tests incubated in the two different atmospheres. Elevated penicillin and ampicillin MICs (≥0.12 μg/ml and ≥0.25 μg/ml, respectively) occurred in 14.3% and 8.6% of strains and were associated with polymorphisms of the penA gene encoding a modified penicillin-binding protein 2. None of the 442 isolates produced beta-lactamase. Elevated tetracycline and doxycycline (but not minocycline) MICs were associated with efflux-mediated resistance encoded by tet(B) in 13 strains. Resistance to sulfisoxazole in 21.7% of strains and to trimethoprim-sulfamethoxazole in 21.0% resulted from polymorphisms of folP encoding a modified dihydropteroate synthetase. Seven strains were resistant to rifampin due to mutations in the rpoB gene, and two strains were resistant to chloramphenicol due to production of chloramphenicol acetyltransferase mediated by catP. Two strains had reduced quinolone susceptibility due to mutations of gyrA. The determination of the susceptibilities of a large group of meningococcal strains (including strains with characterized resistance mechanisms) to 16 antimicrobial agents has served as the essential first step in defining susceptibility testing breakpoints specific for this organism.

In Vitro Activity of Daptomycin against Vancomycin-Resistant Enterococci of Various Van Types and Comparison of Susceptibility Testing Methods

ABSTRACT The increasing prevalence of vancomycin-resistant enterococcal (VRE) infections and the limited number of antimicrobial agents for their treatment emphasize a need for new, more effective agents. In this study, the in vitro activity of daptomycin was determined against a collection of 156 VRE from seven different institutions. Van types were characterized by PCR, and pulsed-field gel electrophoresis was performed to exclude isolates with >85% relatedness by dendrogram. Included were 126 Enterococcus faecium (109 vanA, 17 vanB) isolates, 5 Enterococcus faecalis (3 vanA, 2 vanB) isolates, 2 Enterococcus avium (vanA) isolates, 1 Enterococcus durans (vanA) isolate, 10 Enterococcus gallinarum (vanC1) isolates, and 12 Enterococcus casseliflavus (vanC2) isolates. MICs of daptomycin and five additional agents were determined by the NCCLS broth microdilution method with Mueller-Hinton (MH) broth containing supplemental calcium. MICs were also determined using two investigational E-test strip formulations, and disk diffusion testing was performed by the standard NCCLS method. The MIC of daptomycin at which 50% of the isolates tested were inhibited for this isolate collection was 4 μg/ml, and the MIC at which 90% of the isolates tested were inhibited was 8 μg/ml. Two isolates of vanA E. faecium were resistant to linezolid, and one isolate was resistant to quinupristin-dalfopristin. MICs of daptomycin determined by the E test with and without added calcium varied by 8- to 16-fold, and disk diffusion zones varied by 3 to 6 mm according to the calcium content of the commercial MH agar lots used in the study. This study has shown daptomycin to have good activity against a diverse collection of contemporary VRE isolates. However, improved standardization of the calcium content of MH agar will be important for reliable testing of daptomycin by clinical laboratories using either the E test or disk diffusion methods.

Abiotrophia bacteremia in a patient with neutropenic fever and antimicrobial susceptibility testing of Abiotrophia isolates.

We report a case of bacteremia due to Abiotrophia species in a patient with neutropenic fever and cancer who was receiving levofloxacin prophylaxis, followed by empirical therapy with cefepime; the organism was resistant to both antibiotics. We provide susceptibility data on 20 additional bloodstream isolates of Abiotrophia species.

In vitro activities of moxalactam and cefotaxime against aerobic gram-negative bacilli.

The in vitro activities of two new beta-lactam antibiotics, moxalactam disodium (LY 127935) and cefotaxime (HR-756), were compared with cefoxitin, cefamandole, cefuroxime, cephalothin, and, in some instances, carbenicillin, gentamicin, and amikacin against aerobic gram-negative bacilli. Test isolates included normally cephalosporin-resistant members of the Enterobacteriaceae and Pseudomonas spp. and a variety of nonfermentative or oxidase-positive bacteria. Both moxalactam and cefotaxime demonstrated impressive in vitro activities against both groups of microorganisms. The two new drugs were clearly more active than any of the other beta-lactam antibiotics against species of Escherichia, Citrobacter, Enterobacter, Klebsiella, Proteus, Providencia, Pseudomonas, and Serratia. An additive or synergistic effect could also be demonstrated with the majority of Pseudomonas and Serratia isolates when either moxalactam or defotaxime was combined with amikacin.

Comparison of moxalactam (LY127935) and cefotaxime against anaerobic bacteria.

The in vitro activities of moxalactam (LY127935 [6059S]) and cefotaxime were compared with those of cefoxitin, cefamandole, cefuroxime, carbenicillin, and penicillin by agar dilution susceptibility testing of a variety of anaerobic bacteria. Moxalactam proved to be the most active agent tested against Bacteroides fragilis and other species of the B. fragilis group. Moxalactam and cefotaxime showed activity similar to the other drugs against the remaining species of Bacteroides, Fusobacterium, Actinomyces, Propionibacterium, and Veillonella. Penicillin was the most effective drug tested against most species of Clostridium, the anaerobic gram-positive cocci, and Eubacterium lentum.

Assessment of Two Commercial Susceptibility Test Methods for Determination of Daptomycin MICs

ABSTRACT Daptomycin is a lipopeptide antibiotic with activity against several important gram-positive bacterial pathogens, including drug-resistant staphylococci and enterococci. Because the mechanism of action of daptomycin is calcium-dependent depolarization of the cell membrane, susceptibility testing requires medium supplemented with a physiological level of calcium. This study assessed two Food and Drug Administration-cleared commercial test devices for determination of daptomycin MICs, Etest and JustOne. A collection of 220 selected isolates, including Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus faecalis, E. faecium, E. avium, E. durans, E. casseliflavus, and E. gallinarum, were tested by both methods. Included in the collection were 22 S. aureus and 14 Enterococcus sp. isolates that were recovered from patients and were nonsusceptible on the basis of the daptomycin MICs. As the reference method for comparison, all isolates were tested by the Clinical and Laboratory Standards Institute broth microdilution method incorporating cation-adjusted Mueller-Hinton broth with 50 μg/ml calcium. Daptomycin MICs agreed, within 1 twofold dilution, for 97% of the isolates by Etest and for 100% by JustOne. However, daptomycin MICs determined by Etest were 1 dilution lower than the reference MICs for 65% of the Enterococcus sp. isolates tested. This resulted in 28.5% very major (VM) errors (4/14) with enterococci (all E. faecium) but none (0/22) with staphylococci. Use of JustOne yielded MICs that were 1 dilution lower than the reference MICs for 69% of the staphylococci and 25% of the enterococci. This resulted in 13.6% VM errors (3/22) with staphylococci and 14.3% VM errors (2/14) with enterococci. The manufacturer-recommended JustOne inoculum preparation resulted in mean colony counts of only 5 × 104 to 1 × 105 CFU/ml in the wells of the strip. Increasing the inoculum to 3 × 105 to 4 × 105 CFU/ml eliminated two of five VM errors upon retesting. No major interpretive errors occurred with either device. In summary, daptomycin MICs generated by the Etest or JustOne method generally agreed within 1 dilution of the reference daptomycin MICs. However, both devices produced slightly lower MICs that resulted in some VM errors.

Mutations in folP Associated with Elevated Sulfonamide MICs for Neisseria meningitidis Clinical Isolates from Five Continents

ABSTRACT Sulfonamide resistance in meningococci is associated with mutations in the chromosomal gene folP, which encodes dihydropteroate synthase. Several mutations associated with resistance have been previously described, including amino acid substitutions at codons 31 and 194, a glycine-serine insertion at codons 195 and 196, and, recently, an additional mutation at nucleotide 682 (C682A). In this study, sulfisoxazole MICs were determined for 424 geographically diverse clinical isolates of Neisseria meningitidis, including all major subtypes. A subset of 134 isolates with MICs ranging from 0.5 to >64 μg/ml were assayed for the C682A mutation by real-time PCR, and 25 isolates were selected for folP gene sequencing. All isolates for which the sulfisoxazole MIC was ≥8 possessed the C682A mutation by real-time PCR or folP sequencing, and 34 of 35 isolates with a MIC of ≤2 lacked this mutation. Of 16 sequenced isolates for which the sulfisoxazole MIC was ≥4, 15 possessed previously described mutations, including 10 at codon 31, 1 at codon 194, and 4 with the 2-amino-acid insertion codons 195 and 196; all 16 possessed the C682A mutation. The C682A mutation predicted elevated sulfonamides MICs for a large number of geographically diverse clinical isolates of meningococci. Detection of this mutation by real-time PCR or other methods may allow more wide-scale detection of meningococcal isolates with for which the sulfonamide MICs are elevated without resorting to multiple assays or folP gene sequencing, providing a simple, high-throughput screening method for use in public health and epidemiologic settings.

Ceftazidime levels in human breast milk.

Ceftazidime in the breast milk of 11 puerperal women were determined by bioassay. Each patient received 2 g of ceftazidime intravenously every 8 h for 5 days. The mean (+/- standard deviation) ceftazidime concentrations in the milk were: 3.8 +/- 2.0 (before the next dose), 5.2 +/- 3.0 (1 h after), and 4.5 +/- 1.7 micrograms/ml (3 h after). Ceftazidime was excreted in breast milk at relatively constant levels between days 2 and 4 of therapy.