Bacterial resistance to antimicrobial agents generally involves drug inactivation, target site modification, impermeability, or efflux mechanisms. Macrolide antibiotic resistance in Staphylococcus aureus and coagulase-negative staphylococci (CNS) may be due to an active efflux mechanism encoded by msrA (conferring resistance to macrolides and type B streptogramins only) (16, 17) or may be due to ribosomal target modification, affecting macrolides, lincosamides, and type B streptogramins (MLS B resistance). erm genes encode enzymes that confer inducible or constitutive resistance to MLS agents via methylation of the 23S rRNA, reducing binding by MLS agents to the ribosome (15). Resistance is induced by the binding of a macrolide to upstream translational attenuator sequences, leading to changes in mRNA secondary structure, exposure of the ribosomal binding site, and translation of the erm methylase. Alterations in these 5Ј upstream sequences, including deletions, duplications, and other mutations, lead to constitutive expression of the methylase gene and constitutive MLS B resistance (1,15,24). Strains with inducible MLS B resistance (MLS B i) strains demonstrate in vitro resistance to 14-and 15-member macrolides (e.g., erythromycin), while appearing susceptible to 16-member macrolides, lincosamides, and type B streptogramins; strains with constitutive MLS B resistance (MLS B c strains) show in vitro resistance to all of these agents (15).MLS antibiotics are commonly used in treatment of staphylococcal infections. Clindamycin is a frequent choice for some staphylococcal infections, particularly skin and soft-tissue infections, and as an alternative in the penicillin-allergic patient. Inducible MLS B resistance is not recognized by using standard susceptibility test methods, including standard broth-based or agar dilution susceptibility tests. Failure to identify inducible MLS B resistance may lead to clinical failure of clindamycin therapy (3). Conversely, labeling all erythromycin-resistant staphylococci as clindamycin resistant prevents the use of clindamycin in infections caused by truly clindamycin-susceptible staphylococcal isolates.Low levels of erythromycin are the most effective inducer of inducible MLS B resistance (23). To detect MLS B i strains, there are special disk approximation tests that incorporate erythromycin induction of clindamycin resistance (23). These tests involve the placement of an erythromycin disk in close proximity to a disk containing clindamycin or lincomycin. As the erythromycin diffuses through the agar, resistance to the lincosamide is induced, resulting in a flattening or blunting of the lincosamide zone of inhibition adjacent to the erythromycin disk, giving a D shape to the zone (D-zone effect). Jenssen and colleagues suggested that closer spacing than in a standard disk dispenser was necessary to discern inducible resistance, with optimal spacing of 10 to 15 mm (6); 20-mm spacing of disks and a higher concentration of erythromycin (30 g) have also been suggested (18).

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 erythromycinresistant isolates correlated with standard D-zone tests on Mueller-Hinton agar and with PCR for erm(A

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 CO 2 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 g/ml and >0.25 g/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.

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.

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 g/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.