Nonmolecular Test for Detection of Low-Level Resistance to Fluoroquinolones in Streptococcus pneumoniae

Abstract

With respect to pneumococci, there is a need to detect first-step mutants with reduced fluoroquinolone (FQ) susceptibility from which second-step, resistant mutants are likely to be selected in the presence of antipneumococcal FQs. Here, we describe an interpretative disk diffusion test, of which three options are presented, that allows the distinction between first-and second-step mutants. Using five FQ disks (pefloxacin, norfloxacin, levofloxacin, ciprofloxacin, and sparfloxacin, option 1), all known mechanisms of altered FQ susceptibility found in first-step mutants (ParC, ParE, GyrA, or efflux) and in second-step mutants (ParC and GyrA or ParE and GyrA) can be accurately detected, making this option a useful epidemiological tool. Using three FQ disks (pefloxacin, norfloxacin, and levofloxacin, option 2), the most prevalent FQ-resistant mutants, but not the first-step GyrA mutants, can be detected. With only two FQ disks (norfloxacin and levofloxacin) in the third and simplest option, first-step mutants can be distinguished from second-step mutants, however, without differentiation of ParC, ParE, or efflux alterations.

Fatal Levofloxacin Failure in Treatment of a Bacteremic Patient Infected with Streptococcus pneumoniae with a Preexisting parC Mutation

“…By PCR and sequencing (12), the first isolate showed a mutation in the QRDR of parC (Ser79Phe), and the second one had an additional gyrA mutation (Ser81Phe). These mutations are most commonly found in fluoroquinolone-resistant S. pneumoniae strains (1,18). In addition, genetic relatedness was supported by several polymorphisms identified in the QRDRs of the two isolates: Met1Leu in parC and Ile460Val in parE.…”

“…Isolates with a single first-step parC mutation are frequently susceptible to fluoroquinolones because the MICs are at or below the Clinical and Laboratory Standards Institute (CLSI) breakpoints (4). However, a firststep parC mutation increases the likelihood of a subsequent gyrA mutation, high-level resistance, and therapeutic failure (1,18). At the same time, it has also been observed that once a parC mutation has taken place in an S. pneumoniae strain, the mutation prevention concentration (MPC) increases dramatically for all fluoroquinolones (17).…”

Real-Time PCR Detection of gyrA and parC Mutations in Streptococcus pneumoniae

“…The rapid detection of gyrA and parC mutations in a single run represents a faster and more reliable approach than the current phenotypic method (13), which is unable to detect S. pneumoniae strains that have a greater potential than wild-type strains to evolve toward resistance.…”

“…This evolution implies that microbiologists must be able to detect first-step, or "preresistant," mutants. The use of a nonmolecular test scheme for the detection of low-level resistance has been proposed, but this type of test lacks sensitivity and specificity (13). A powerful and highly specific technique for the detection of mutations within a DNA sequence is real-time PCR with fluorescent resonance energy transfer (FRET) hybridization probes.…”

“…They consisted of strain R6 and 3 R6 derivatives kindly provided by E. Varon (13), 10 mutants generated in vitro, reference strains CIP104485 and CP 1000, and 42 clinical isolates (37 from New Caledonia, 3 from Australia, and 2 from Tahiti). Table 1 summarizes the known mutations, the results of disk agar diffusion (Bio-Rad), and the MICs determined by agar dilution, as described previously (13). DNA was extracted with a QIAamp DNA mini kit (Qiagen).…”