In Vitro Activity of Lascufloxacin against Streptococcus pneumoniae with Mutations in the Quinolone Resistance-Determining Regions

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Fluoroquinolones inhibit DNA synthesis by binding to DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) in Streptococcus pneumoniae (1). Fluoroquinolone resistance is usually due to a gradual accumulation of GyrA and ParC mutations in the quinolone resistance-determining regions (QRDRs) (1, 2). Previously, we reported that either a GyrA or ParC (first-step) mutation was detected in 20 of 41 (48.8%) susceptible strains with levofloxacin MICs of 1 or 2 μg/ml (3, 4). Several in vitro studies and a case report indicated that second-step mutants with both GyrA and ParC mutations could be selected by the exposure of first-step mutants to fluoroquinolones (5–7). Lascufloxacin was newly developed by Kyorin Pharmaceutical Co., Ltd. (Tokyo, Japan) as a respiratory fluoroquinolone. We evaluated the in vitro activity of lascufloxacin against S. pneumoniae, focusing on the selectivity of resistant strains after drug exposure in first-step mutants.

We used clinical isolates from patients in Japan between January 2006 and December 2008 for MIC measurements (3). The MICs were measured using the broth microdilution method with MIC plates customized by Eiken Chemical Co., Ltd. (Tokyo, Japan) according to the Clinical and Laboratory Standards Institute (CLSI) protocol. Susceptible strains with levofloxacin MICs of ≤2 μg/ml were chosen, and 33 clinical isolates were included in this study. The MIC₉₀ of lascufloxacin was 0.12 μg/ml (Table 1). Mutations in the QRDRs were detected by pyrosequencing. DNA was extracted using the boiling method reported previously, with minor modifications (8). PCR amplification for pyrosequencing was performed according to the following profile: 4 min at 94°C, 50 cycles consisting of 15 s at 94°C, 15 s at 55°C, and 20 s at 72°C, with a final extension step of 5 min at 72°C. Primers for gyrA reverse and parC forward had 5′ biotin (Bio) labels. PCR primers were as follows: gyrA forward, 5′-GAATGAATTGGGTGTGAC-3′; gyrA reverse, 5′-Bio-ATACGTGCCTCGGTATAA-3′; parC forward, 5′-Bio-GTTCAACGCCGTATTCTT-3′; and parC reverse, 5′-TGCCTCAGTATAACGCATAG-3′ (9). We evaluated the presence of mutations by pyrosequencing using PyroMark ID (Biotage, Uppsala, Sweden) according to the manufacturer's instructions. Primers for pyrosequencing were as follows: gyrA, 5′-GGTAAATACCACCCACACGG-3′; and parC, 5′-CTGTGACATACGAACCAT-3′ (3, 10). Of the 33 strains, 14 (42.4%) had a mutation in ParC, whereas no strains with only a GyrA mutation were found. The MICs of lascufloxacin and levofloxacin for first-step mutants were 0.06 to 0.12 μg/ml and 2 μg/ml, respectively.

To determine the frequency of the appearance of resistant strains after fluoroquinolone exposure, we used four clinical isolates (G21, G27, G39, and G11) selected from the 33 strains described above and four laboratory strains (NF9884, CF9842, SF9863, and GF9821) with first-step QRDR mutations (11). IID553 (wild type) was used as the parent strain of the first-step laboratory strains. We measured the MICs of levofloxacin, garenoxacin, moxifloxacin, and lascufloxacin using the agar dilution method according to the CLSI protocol. Lascufloxacin and garenoxacin were provided by Kyorin Pharmaceutical Co., Ltd., and levofloxacin and moxifloxacin were purchased from Sigma-Aldrich Japan (Tokyo, Japan) and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), respectively. Bacteria were incubated at 35°C for 3 days on Mueller-Hinton II agar (Becton Dickinson, Franklin Lakes, NJ) with 5% defibrinated sheep blood (Nippon Bio-Test Laboratories Inc., Tokyo, Japan) containing fluoroquinolones at 2×, 4×, 8×, and 16× MICs. The frequency of the appearance of resistant strains was calculated as the ratio of the number of colonies that appeared to that of bacteria inoculated (12). No differences were observed in the frequency of the appearance of resistant strains when the wild-type laboratory strain, IID553, was exposed to lascufloxacin, levofloxacin, and garenoxacin. Similar results were seen in comparisons between lascufloxacin and moxifloxacin. Conversely, the frequencies of resistance to lascufloxacin tended to be lower than those to levofloxacin and garenoxacin in both laboratory and clinical strains with first-step mutations (Table 2). Those to lascufloxacin were similar to those to moxifloxacin (Table 3). In addition, although the MICs of levofloxacin, garenoxacin, and moxifloxacin for strains selected after exposure of the clinical strains with only ParC mutations to the corresponding drugs were increased up to 16-, 32-, and 16-fold, respectively, the MICs of lascufloxacin were increased up to 4-fold compared with that of the parent strain (Tables 4 and 5). These results indicated that lascufloxacin was unlikely to result in the development of resistance in first-step mutants.

It was reported that gatifloxacin, clinafloxacin, and sitafloxacin, which inhibit both DNA gyrase and topoisomerase IV, had lower propensities to select resistant strains (12–14). The slight increases in the MICs of lascufloxacin in selected second-step mutants also suggested that lascufloxacin possessed dual target properties against both target enzymes in first-step mutants. On the other hand, if resistant strains were selected by the exposure of clinical strains with only ParC mutations to the corresponding drug, the increases in MICs of lascufloxacin were smaller than those of levofloxacin, garenoxacin, and moxifloxacin. These observations suggested that lascufloxacin has high potency against mutated DNA gyrase and topoisomerase IV. Altogether, the stable activity of lascufloxacin against first- and second-step mutants of S. pneumoniae was thought to be due to the dual target properties and the inhibition of the mutated enzymes. A recent study indicated that lascufloxacin showed strong activity against S. pneumoniae, including fluoroquinolone-resistant strains, and an enzymatic analysis indicated that lascufloxacin showed potent inhibitory activities against DNA gyrase and topoisomerase IV with mutations in Staphylococcus aureus as well as against those without mutations (15). This report was consistent with our proposal regarding the activity of lascufloxacin.

No additional mutations were observed in some of the strains selected by the exposure to fluoroquinolones (Tables 4 and 5). Although the gradual accumulation of GyrA and ParC mutations was the main cause of fluoroquinolone resistance, the increases in MICs in these strains were thought to be due to other mechanisms, such as GyrB and ParE mutations and an overexpression of efflux pumps, including PmrA and PatA/PatB ABC transporter (16, 17).

Lascufloxacin showed potent activity against first-step mutants. In addition, lascufloxacin was unlikely to select resistant strains after drug exposure of first-step mutants compared with levofloxacin and garenoxacin. The selectivity of resistant strains from first-step mutants was similar in the comparison between lascufloxacin and moxifloxacin. We cannot distinguish first-step mutants on the basis of drug susceptibility, because they may be susceptible according to the current CLSI breakpoint MIC (≤2 μg/ml) for levofloxacin. Lascufloxacin would contribute to preventing the emergence of resistance when treating pneumococcal infections in clinical settings. A clinical trial is currently in progress in Japan, and further clinical studies will clarify the efficacy of lascufloxacin against pneumococcal infection.

(This work was partly presented as a poster at ASM Microbe in 2016.)