Genotypic Evolution in a Quinolone-Resistant <i>Neisseria gonorrhoeae</i> Isolate from a Patient with Clinical Failure of Levofloxacin Treatment

Abstract

Recently, a reduction in the antimicrobial susceptibility of clinical isolates of Neisseria gonorrhoeae to newer fluoroquinolones including levofloxacin in vitro has been recognized in Japan. We examined the quinolone resistance mechanisms in N. gonorrhoeae isolates from a patient with clinical failure of levofloxacin treatment. Man with gonococcal urethritis was treated with oral 100 mg levofloxacin 3 times daily for 7 days. However, clinical failure of the treatment was observed. The minimum inhibitory concentration of levofloxacin for the posttreatment isolate (4.0 μg/ml) was 4-fold higher than that for the pretreatment isolate (1.0 μg/ml). To analyze quinolone resistance mechanisms in the set of isolates, we performed DNA sequencing of the quinolone reisistance-determining regions within the gyrA and parC genes. Moreover, we assayed the intracellular levofloxacin and norfloxacin accumulation level in these isolates. The pretreatment isolate contained three substitutions compared to susceptible wild-type isolate, including serine to phenylalanine at position 91 and aspartic acid to asparagine at position 95 in the GyrA protein, and serine to proline at position 88 in the ParC protein. The posttreatment isolate had four substitutions, including the same three substitutions and an additional glutamic acid to glutamine substitution at position 91 in ParC. There was no significant difference in the level of accumulation of levofloxacin and norfloxacin between the pretreatment and posttreatment isolates. Our results indicate that levofloxacin selects a mutant having an additional alteration within the gene cording for the ParC protein during treatment, which may have enhanced quinolone resistance in the organism.

Molecular characterization of Neisseria gonorrhoeae identifies transmission and resistance of one ciprofloxacin‐resistant strain

“…All phenotypically determined ciprofloxacin-resistant isolates comprised SNPs that are highly correlated with ciprofloxacin resistance, i.e. in the Ser91 and the Asp95 codons of the wild-type gyrA gene sequence (14)(15)(16)(17)(18)(19)21,22). This method basedon Pyrosequencing technology is rapid, high-throughput, sensitive, accurate, and costeffective for identification of ciprofloxacin resistance in N. gonorrhoeae.…”

“…However, treatment failures with fluoroquinolones have been shown to be due to mutations primarily in the gyrA gene, and especially substitutions in a short quinolone resistance determining region (QRDR) of this gene (14,19,20). Consequently, it may be sufficient to analyze a short QRDR of the gyrA gene for identification of N. gonorrhoeae isolates with decreased susceptibility and resistance to ciprofloxacin (14)(15)(16)(17)(18)(19)21,22).In 2003, a presumed epidemiological core group comprising domestic cases of mainly young (<25 years of age) heterosexuals with urogenital gonorrhoea was identified in one Swedish county. All preserved N. gonorrhoeae isolates from these cases were assigned to serovar IB-10 and showed a high level of resistance to ciprofloxacin.…”

“…Since the mid-1990s, the prevalence of isolates resistant to ciprofloxacin, previously the drug of first choice for treatment of uncomplicated gonorrhoea, has been increasing in Sweden and in many other countries (SWEDRES 2004 http://www.strama.se (2)(3)(4)(5)13)). In N. gonorrhoeae, the ciprofloxacin resistance is mainly due to single nucleotide polymorphisms (SNPs) in the gyrA gene and the parC gene, which code for the target enzymes DNA gyrase and topoisomerase IV, respectively (14)(15)(16)(17)(18)(19). The level of resistance appears to be correlated with the location and number of mutations in these genes.…”

Use of Applied Biosystems 7900HT Sequence Detection System and Taqman Assay for Detection of Quinolone-Resistant Neisseria gonorrhoeae

“…With widespread use of nucleic acid amplification tests (NAATs), antimicrobial resistance detection will require molecular methods, as has been described for other organisms (1,7,14,(16)(17)(18)25). QRNG detection is a good model for this approach since the resistance mechanisms are based on stepwise accumulation of point mutations correlating with increased MICs (20,22,23).…”

Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing

“…The level of resistance appears to be correlated with the location and number of mutations in these genes. Treatment failures have been shown to be associated with quinolone resistance due to mutations primarily in the gyrA gene [5][6][7]. Moreover, resistance to ciprofloxacin in many isolates of N. gonorrhoeae is emerging, and genotyping these mutations is of great importance for resistance surveillance.…”