Effective evaluations of antimicrobial susceptibility tests (ASTs) require robust study design. The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing has recognized that many published studies reporting the performance of commercial ASTs (cASTs) suffer from major design and/or analysis flaws, rendering the results difficult or impossible to interpret. This minireview outlines the current consensus of the Methods Development and Standardization Working Group of the CLSI Subcommittee on Antimicrobial Susceptibility Testing regarding best practices for systematic evaluation of the performance of an AST, including the analysis and presentation of essential data intended for publication.

Ciprofloxacin-resistant mutants of Streptococcus pneumoniae 7785 were generated by stepwise selection at increasing drug concentrations. Sequence analysis of PCR products from the strains was used to examine the quinolone resistance-determining regions of the GyrA and GyrB proteins of DNA gyrase and the analogous regions of the ParC and ParE subunits of DNA topoisomerase IV. First-step mutants exhibiting low-level resistance had no detectable changes in their topoisomerase quinolone resistance-determining regions, suggesting altered permeation or another novel resistance mechanism. Nine of 10 second-step mutants exhibited an alteration in ParC at Ser-79 to Tyr or Phe or at Ala-84 to Thr. Third- and fourth-step mutants displaying high-level ciprofloxacin resistance were found to have, in addition to the ParC alteration, a change in GyrA at residues equivalent to Escherichia coli GyrA resistance hot spots Ser-83 and Asp-87 or in GyrB at Asp-435 to Asn, equivalent to E. coli Asp-426, part of a highly conserved EGDSA motif in GyrB. No ParE changes were observed. Complementary analysis of two S. pneumoniae clinical isolates displaying low-level resistance to ciprofloxacin revealed a ParC change at Ser-79 to Phe or Arg-95 to Cys but no changes in GyrA, GyrB, or ParE. A highly resistant isolate, in addition to a ParC mutation, had a GyrA alteration at the residue equivalent to E. coli Asp-87. Thus, in both laboratory strains and clinical isolates, ParC mutations preceded those in GyrA, suggesting that topoisomerase IV is a primary topoisomerase target and gyrase is a secondary target for ciprofloxacin in S. pneumoniae.

We investigated the roles of DNA gyrase and topoisomerase IV in determining the susceptibility of Streptococcus pneumoniae to gemifloxacin, a novel fluoroquinolone which is under development as an antipneumococcal drug. Gemifloxacin displayed potent activity against S. pneumoniae 7785 (MIC, 0.06 g/ml) compared with ciprofloxacin (MIC, 1 to 2 g/ml). Complementary genetic and biochemical approaches revealed the following. (i) The gemifloxacin MICs for isogenic 7785 mutants bearing either parC or gyrA quinolone resistance mutations were marginally higher than wild type at 0.12 to 0.25 g/ml, whereas the presence of both mutations increased the MIC to 0.5 to 1 g/ml. These data suggest that both gyrase and topoisomerase IV contribute significantly as gemifloxacin targets in vivo. (ii) Gemifloxacin selected first-step gyrA mutants of S. pneumoniae 7785 (gemifloxacin MICs, 0.25 g/ml) encoding Ser-81 to Phe or Tyr, or Glu-85 to Lys mutations. These mutants were cross resistant to sparfloxacin (which targets gyrase) but not to ciprofloxacin (which targets topoisomerase IV). Second-step mutants (gemifloxacin MICs, 1 g/ml) exhibited an alteration in parC resulting in changes of ParC hot spot Ser-79 to Phe or Tyr. Thus, gyrase appears to be the preferential in vivo target. (iii) Gemifloxacin was at least 10-to 20-fold more effective than ciprofloxacin in stabilizing a cleavable complex (the cytotoxic lesion) with either S. pneumoniae gyrase or topoisomerase IV enzyme in vitro. These data suggest that gemifloxacin is an enhanced affinity fluoroquinolone that acts against gyrase and topoisomerase IV in S. pneumoniae, with gyrase the preferred in vivo target. The marked potency of gemifloxacin against wild type and quinolone-resistant mutants may accrue from greater stabilization of cleavable complexes with the target enzymes.Gemifloxacin (SB-265805) is a new fluoroquinolone which displays impressive activity against Streptococcus pneumoniae (5, 24), the principal cause of community-acquired pneumonia and a major player in meningitis, otitis, sinusitis, and exacerbations of chronic bronchitis (3). The drug is effective in vitro, not only against penicillin-susceptible isolates of S. pneumoniae but also against penicillin-resistant strains, which are now commonly encountered in the clinic (5, 9, 24). A tentative breakpoint for S. pneumoniae of 0.5 g/ml has been proposed (35). Recent work has shown that gemifloxacin also retains activity against multidrug-resistant S. pneumoniae, including strains resistant to ciprofloxacin (16), a fluoroquinolone widely used in treating gram-negative infections but which has borderline activity against pneumococci (31). Although the incidence of quinolone-resistant pneumococci is presently low (7), such strains could become important with increased quinolone usage. The mechanism underlying the susceptibility of ciprofloxacin-resistant strains to gemifloxacin is not known.Fluoroquinolones act by inhibiting DNA gyrase and topoisomerase IV, two enzymes that operate by a double-strand DNA break mech...

b Ceftaroline, the active metabolite of the prodrug ceftaroline-fosamil, is an advanced-generation cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA). This investigation provides in vitro susceptibility data for ceftaroline against 1,971 S. aureus isolates collected in 2012 from seven countries (26 centers) in the Asia-Pacific region as part of the Assessing Worldwide Antimicrobial Resistance and Evaluation (AWARE) program. Broth microdilution as recommended by the CLSI was used to determine susceptibility. In all, 62% of the isolates studied were MRSA, and the ceftaroline MIC 90 for all S. aureus isolates was 2 g/ml (interpretive criteria: susceptible, <1 g/ml). The overall ceftaroline susceptibility rate for S. aureus was 86.9%, with 100% of methicillin-sensitive S. aureus isolates and 78.8% of MRSA isolates susceptible to this agent. The highest percentages of ceftaroline-nonsusceptible MRSA isolates came from China (47.6%), all of which showed intermediate susceptibility, and Thailand (37.1%), where over half (52.8%) of isolates were resistant to ceftaroline (MIC, 4 g/ml). Thirty-eight ceftaroline-nonsusceptible isolates (MIC values of 2 to 4 g/ml) were selected for molecular characterization. Among the isolates analyzed, sequence type 5 (ST-5) was the most common sequence type encountered; however, all isolates analyzed from Thailand were ST-228. Penicillin-binding protein 2a (PBP2a) substitution patterns varied by country, but all isolates from Thailand had the Glu 239 Lys substitution, and 12 of these also carried an additional Glu 447 Lys substitution. Ceftaroline-fosamil is a useful addition to the antimicrobial agents that can be used to treat S. aureus infections. However, with the capability of this species to develop resistance to new agents, it is important to recognize and monitor regional differences in trends as they emerge.

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Fluoroquinolones are being increasingly used for acute lower respiratory tract infection where Streptococcus pneumoniae is the most important bacterial pathogen. S. pneumoniae becomes resistant to quinolone antibiotics by mutations in a small section of the parC and gyrA genes. In this study, we investigated the mutation rates and spectrum of resistance when ciprofloxacin and gemifloxacin were the selective agents. When ciprofloxacin was the selective agent, parC mutants arose at a rate of 1.1 x 10(-9) mutations per cell division. There were two double mutants: parC + gyrA and parC + gyrB, and these mutations arose in as few as five generations. When gemifloxacin was the selective agent, all but one of the colonies growing on the x2 MIC plate had no mutations in gyrA or parC. The only mutation identified was in gyrA, and it appeared at a rate of 1.6 x 10(-11). When the gemifloxacin MIC of strains with mutations in parC was determined, there was no change from the susceptible parent. These data indicate that S. pneumoniae becomes resistant to gemifloxacin through mutation in gyrA rather than parC. Because gyrA mutations arise at a lower rate than parC mutations, it is likely that resistance to gemifloxacin will emerge more slowly than is seen with those quinolones that become resistant through an initial mutation in parC. The rate at which second-step mutants emerged was 1.3 x 10(-8) for parC Serine 79 Tyrosine and 7.2 x 10(-9) for gyrA Serine 81 Phenylalanine, 12 and 450 times higher, respectively, than for first-step rates, suggesting that mutation in either gene readies the genome for further mutation.

This study suggests that MRSA populations with a WT PBP2a and those with nPBD variations overlap significantly and that PBP2a sequence-independent factors contribute to ceftaroline susceptibility. Whereas characterization of isolates with a ceftaroline MIC of 2 mg/L enriched for isolates with nPBD variations, it was not a discrete population. In contrast, the rare isolates containing a substitution in the transpeptidase-binding pocket were readily differentiated.

Detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that has been used to detect mutations in human DNA. We report on the first study to use this technique as a tool to detect mutations in genes encoding antibiotic resistance in bacteria. Three methicillin-sensitive and three methicillin-resistant clinical Staphylococcus aureus isolates, susceptible to ciprofloxacin (MIC Leu, Ser-112-->Pro, Glu-88-->Lys in GyrA, Glu-84-->Val, Ser-80-->Phe in GrlA, Pro-456-->Ser in GyrB and Glu-422-->Asp, Pro-451-->Ser, Asp-432-->Gly in GrlB. Mutations could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run) and reliable technique for characterizing antibiotic resistance in bacteria.