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.

FIG. 1.

Distribution of 144 low-level and high-level fluoroquinolone-resistant strains of S. pneumoniae according to their genotypes, and MICs and inhibition zone diameters of six different fluoroquinolones (panels A to F). The strains are represented by colored disks, with the disk area corresponding to the number of strains and the color corresponding to a given FQ resistance mechanism. The rectangle above the abscissa represents the zone diameter range for 1,150 susceptible strains; the mean diameter is represented by the vertical bar inside the rectangle. CLSI breakpoints when available are represented by vertical lines. The vertical dotted line represents the cutoff value proposed for the detection of low-level-resistant mutants using pefloxacin (this work) and norfloxacin (recommended by the CA-SFM).

FIG. 2.

Disk diffusion test of fluoroquinolone susceptibility for five strains with defined resistance genotypes. The corresponding phenotypes are indicated in panels A to E. The position of the fluoroquinolone disks (5 μg) is indicated in panel F; the colored rectangles include the FQ disks to be tested in each of the three options (Table 3). PEF, pefloxacin; NOR, norfloxacin; LVX, levofloxacin; CIP, ciprofloxacin; SPX, sparfloxacin.