Fluoroquinolone Resistance in Haemophilus influenzae Is Associated with Hypermutability

Abstract

Forty-three percent (12/28) of ciprofloxacin (CIP)-nonsusceptible respiratory isolates of Haemophilus influenzae were hypermutable, compared with 8.5% (3/35) in the CIP-susceptible control group (P ‫؍‬ 0.002). CIP-nonsusceptible mutants were obtained with hypermutable strains only; these mutants developed three resistance mechanisms in a step-by-step process: target modifications, loss of a porin protein, and increased efflux.Fluoroquinolone-resistant Haemophilus influenzae isolates have been described in several countries (4,5,8,17,19). In general, hypermutable isolates contribute to the emergence of antibiotic resistance (3, 9). Fluoroquinolone resistance in Staphylococcus aureus is related to hypermutability (23). We and other authors have reported a high prevalence of hypermutable H. influenzae isolates causing respiratory infections (22,24). We also noticed a high rate of fluoroquinolone resistance in these isolates (22) and reported a therapeutic failure in a community-acquired pneumonia case due to levofloxacinresistant H. influenzae (2).The main mechanism of fluoroquinolone resistance in H. influenzae is amino acid changes in the quinolone resistance-determining region (QRDR) of the topoisomerase II and I genes (5, 8, 9). Although not reported in H. influenzae, mutations resulting in overexpression of efflux pumps and permeability defects can also affect quinolone susceptibility (12).In this study, we investigated the hypothesis that ciprofloxacin (CIP) resistance in H. influenzae occurs mainly in hypermutable clinical isolates. To test this hypothesis, first we made an observational study, and after that, we subjected CIP-susceptible hypermutable isolates to selective pressure with CIP. We also characterized other mechanisms of resistance in highly fluoroquinolone-resistant isogenic mutants as overexpression of efflux pumps and permeability defects.The CIP reduced-susceptibility study group included 28 clinical isolates (3 from the United States [1] and 25 from Spain) chosen because they had amino acid changes in GyrA of the QRDR (21) and corresponding MICs for CIP of 0.12 to 32 g/ml. The CIP-susceptible control group contained 35 clinical strains without amino acid changes and corresponding MICs for CIP of Ͻ0.12 g/ml. All strains were noncapsulated and were isolated from respiratory specimens of adult patients with chronic respiratory infections (19,21).The study of mutation frequencies in response to rifampin was done in triplicate as described previously (22). Isolates were considered hypermutable if their mutation frequency was 10 Ϫ7 CFU/ml or higher (22). Forty-three percent of the strains (12/28) in the study group were hypermutable, compared to 8.5% (3/35) in the control group (odds ratio, 8; P, 0.0024). The average mutation frequencies in the two groups (4 ϫ 10 Ϫ6 CFU/ml for the study group and 7 ϫ 10 Ϫ8 CFU/ml for the control group) differed significantly (P, 0.017; Mann-Whitney U test). The mutation frequency distributions are shown in Fig. 1. A positive correlation was found between mutation...

Emergence of Fluoroquinolone-Resistant Haemophilus influenzae Strains among Elderly Patients but Not among Children

“…This suggested that a fluoroquinolone-resistant clone invaded in a city for a time required for the acquisition of genetic changes, such as the presence of the TEM-1 ␤-lactamase gene, mutations in PBP3, and resistance to tetracycline and chloramphenicol. Recently, Pérez-Vázquez et al showed that fluoroquinolone resistance in H. influenzae occurs mainly in hypermutable strains (15). The fluoroquinolone-resistant strains found in the present study could be hypermutable, leading to phenotypic changes within genotypes.…”

“…The recent increase in BLNAR strains has become a severe problem, leading to community-acquired infections (8,19). Fluoroquinolone-resistant H. influenzae isolates have occasionally been reported worldwide (1,4,13,15), but they have rarely been reported in Japan (9,10). Fluoroquinolones are effective against the causative agents of atypical pneumonia, such as Chlamydophila pneumoniae and Mycoplasma pneumoniae, as well as those of typical bacterial pneumonia, S. pneumoniae and H. influenzae.…”

“…For each strain, we obtained information on the patient's age and sex, the clinical source, and the name of the city where the strain was isolated, in accordance with the Act on the Protection of Personal Information in Japanese law. The isolates were obtained from the following clinical specimens (with the numbers of samples in parentheses): sputum (190), rhinorrhea (178), nasal cavity (19), pharyngeal fluid (33), otopyorrhea (15), vaginal secreta (6), conjunctival discharge (5), bronchial lavage fluid (5), blood (2), urine (2), lochia (1), and cerebrospinal fluid (1). Cities where strains were isolated were Sapporo (228 strains), Muroran (112 strains), Asahikawa (61 strains), Tomakomai (23 strains), Obihiro (17 strains), Hakodate (14 strains), and Iwamizawa (2 strains).…”

Antimicrobial Activities of Piperacillin-Tazobactam against Haemophilus influenzae Isolates, Including β-Lactamase-Negative Ampicillin-Resistant and β-Lactamase-Positive Amoxicillin- Clavulanate-Resistant Isolates, and Mutations in Their Quinolone Resistance-Determining Regions

“…Although CLSI has disk and/or MIC susceptibility testing breakpoints for multiple agents, for piperacillin-tazobactam it has only a MIC breakpoint (Յ1 g/ml) and a disclaimer comment that BLNAR H. influenzae should be considered resistant regardless of MIC results, despite the finding that piperacillin alone is very active against BLNAR H. influenzae (24). Furthermore, fluoroquinolone-resistant H. influenzae isolates have recently been occasionally reported worldwide (1,11,25,27), though they have rarely been reported in Japan (16,17). More recently, however, Yokota et al (32) reported that fluoroquinolone-resistant H. influenzae isolates have been emerging in the elderly in Japan, with some of the strains exhibiting levofloxacin MICs of 16 g/ml or higher.…”

Molecular Characterization of Fluoroquinolone Resistance in Nontypeable Haemophilus influenzae Clinical Isolates

“…Fluoroquinolones (FQs) are frequently used as antimicrobial therapy in respiratory tract infections in adults and have shown good activity against respiratory pathogens such as H. influenzae, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Moraxella catarrhalis (5,6). Since their first description in 1993, FQ-resistant H. influenzae isolates have been detected all over the world (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Although resistance in this bacterial pathogen remains low (8,17), treatment failure with ofloxacin or levofloxacin (LVX) has already been described (15,18).…”