Hideaki Muramatsu

Detection of mutations in quinolone resistance-determining regions in levofloxacin- and methicillin-resistant Staphylococcus aureus: effects of the mutations on fluoroquinolone MICs

The minimum inhibitory concentrations (MICs) of 18 antibiotics were determined for 66 clinical isolates of staphylococci. Genotypes, mutations in the quinolone resistance-determining regions (QRDRs), and effect of efflux were determined in the 18 levofloxacin-resistant isolates, for which the MICs of levofloxacin were high (> or =8 microg/ml). The increased levofloxacin resistance mainly resulted from some combinations of mutations in the QRDRs, although NorA-mediated efflux may play a minor role in resistance. A combination of mutations in GrlA (Ser80Phe), GrlB (Pro451Ser), and GyrA (Ser84Leu) was found in 4 methicillin-resistant Staphylococcus aureus (MRSA) isolates that were unrelated genotypically. The mutations in grlA QRDR varied in the isolates classified as being in an identical pulsed-field gel electrophoresis (PFGE) group, although the grlB, gyrA, and gyrB QRDRs were the same. These results suggest that the patterns of amino acid mutations in the QRDRs can provide distinct epidemiologic information from PFGE genotypes in fluoroquinolone-resistant MRSA. A combination of at least three mutations in GrlA, GrlB, and/or GyrA is required to increase the MICs of fluoroquinolones, although all of the levofloxacin-resistant MRSA retained the MICs of sitafloxacin in the range of 1 to 2 microg/ml.

Characterization of Fluoroquinolone and Carbapenem Susceptibilities in Clinical Isolates of Levofloxacin-Resistant Pseudomonas aeruginosa

Background:Pseudomonas aeruginosa can rapidly acquire resistance to antibiotics, including fluoroquinolones and carbapenems. Methods: We characterized fluoroquinolone, carbapenem and other β-lactam susceptibilities and analyzed fluoroquinolone and carbapenem resistance in 16 clinical isolates of levofloxacin-resistant P. aeruginosa. Results: All levofloxacin-resistant isolates showed high MICs (≧32 µg/ml) for fluoroquinolones including norfloxacin, levofloxacin, sparfloxacin, gatifloxacin and pazufloxacin, whereas the MICs for sitafloxacin were between 2 and 16 µg/ml. These isolates had both a Thr83Ile mutation in GyrA and a Ser87Leu mutation in ParC. An additional mutation, Glu469Asp in GyrB, was detected in 3 isolates. Three of 16 isolates found during antibiotic therapy showed resistance to carbapenems (MIC, 16–32 µg/ml) because of a reduced production of OprD. Fluoroquinolones, β-lactams and sulfamethoxazole-trimethoprim were used for 3 months before the isolation of levofloxacin-resistant P.aeruginosa. Conclusions: Emergence of resistant isolates to both fluoroquinolones and carbapenems during antibiotic therapy is a serious clinical problem. Our results suggest that susceptibilities to fluoroquinolones as well as carbapenems should be monitored during a prolonged course of antibiotic therapy against P. aeruginosa infection.

Characterization of Pseudomonas aeruginosa Isolates from Patients with Urinary Tract Infections During Antibiotic Therapy

We characterized susceptibilities and genotypes in a series of Pseudomonas aeruginosa isolates from five cases of urinary tract infections (UTIs) to evaluate clonal shifts of carbapenem resistance. In one case, a series of isolates showed different susceptibility patterns for carbapenems but an identical genotype. In another case, genotypes varied among 4 P. aeruginosa isolates from recurrent UTIs over 9 months. Although the patient had been treated with no antibiotic immediately before isolation, the susceptibility patterns for carbapenems and ceftazidime varied. Further analysis in these two cases of outer membrane protein profiles showed that loss of OprD production resulted in reduced susceptibilities to carbapenems in all of the carbapenem-resistant isolates. Loss of OprD production was likely due to oprD gene inactivation in both of cases, since the carbapenem-resistant isolates showed no cross resistance to levofloxacin and chloramphenicol compared with the carbapenem-susceptible isolates. There was another case in which all isolates showed similar susceptibility patterns for carbapenems and ceftazidime, and an identical genotype during the intermittent use of antibiotics over 5 months. In two cases, a single course of antibiotic therapy resulted in eradication of P. aeruginosa. Our results suggest that clonal shifts of carbapenem resistance in P. aeruginosa may result from loss of OprD during antibiotic treatment. Therefore, it is important for clinicians to monitor susceptibilities to antibiotics, especially carbapenems, in P. aeruginosa isolated during therapy.

Release of exotoxin A, peptidoglycan and endotoxin after exposure of clinical Pseudomonas aeruginosa isolates to carbapenems in vitro.

Background: Production of several cell-associated components and extracellular enzymes can play important roles in the pathogenesis of Pseudomonas aeruginosa infections. Methods: We characterized the time course of morphological changes, production of exotoxin A (ETA) and release of peptidoglycan (PG) and endotoxin (ET) in clinical P. aeruginosa isolates after exposure to carbapenems including imipenem, panipenem, meropenem and biapenem at 0.5, 2, 8 and 32 µg/ml. Results: The amount of ETA in the supernatant of bacterial cultures exposed to carbapenems correlated with the number of viable cells, independently of morphological changes. Formation of ovoid cells and rapid cell lysis induced by carbapenems above the minimal inhibitory concentrations (MICs) decreased ETA production and ET release, while filamentation and prolonged cell lysis increased ETA production and/or ET release. Neither the number of viable cells nor bacterial morphology was related to the amount of PG released throughout the 6-hour observation period. Conclusion: Exposure to concentrations of carbapenems above the MIC resulted in rapid cell lysis of P. aeruginosa and decreased ETA levels and ET release, while filamentation and prolonged cell lysis induced by exposure to sub-MICs of carbapenems were associated with increased ETA production and/or greater ET release.

Effect of basic amino acids on susceptibility to carbapenems in clinical Pseudomonas aeruginosa isolates

We evaluated effects of medium composition, including basic amino acid content and pH, on susceptibility to carbapenems such as imipenem, panipenem and meropenem, in clinical isolates of Pseudomonas aeruginosa. Susceptibility to carbapenems was reduced by basic amino acids in the medium, while susceptibilities to ceftazidime and aztreonam were not. Among carbapenems, susceptibility to panipenem was most sharply reduced by addition of basic amino acids to 1:16 Mueller-Hinton agar (MHA). In 174 of 175 clinical isolates, MICs for carbapenems were affected to different degrees by medium composition. One isolate, in which MICs for carbapenems did not differ between MHA and 1:16 MHA, showed reduced production of porin (OprD). Our results suggest that susceptibility to individual carbapenems, especially panipenem, is difficult to evaluate based on MICs for other carbapenems determined on MHA. For a better prediction of antibiotic efficacy, it may be important to evaluate the susceptibility for each carbapenem individually.

Detection of Macrolide Resistance in Streptococcus pneumoniae

We determined the susceptibilities of recent clinical isolates of Streptococcus pneumoniae to 19 antibiotics. The frequency of erythromycin nonsusceptibility was high, i.e. 8/13 (61.5%), 10/14 (71.4%) and 11/11 isolates (100%) from 13 penicillin-susceptible, 14 penicillin-intermediate and 11 penicillin-resistant S. pneumoniae, respectively. Macrolide resistance was detected by polymerase chain reaction (PCR) and disk diffusion methods. Of these erythromycin-nonsusceptible pneumococcal isolates, 13/29 (44.8%) isolates contained genomic copies of mefA and showed non-‘D’-shaped zones of inhibition observed around rokitamycin and/or clindamycin disks. Sixteen out of 29 isolates (55.2%) contained copies of ermB and showed typical ‘D’-shaped zones of inhibition, except one isolate. Although the macrolide resistance determinants, mefA and ermB, could be identified by PCR and disk diffusion methods, PCR methods were more reliable in elucidating these determinants. The susceptibility pattern to 14-, 15- and 16-membered macrolides and clindamycin differed between the mefA+ and ermB+ isolates. All isolates were susceptible to levofloxacin, sparfloxacin and vancomycin. The MICs of sitafloxacin were lowest among the fluoroquinolones examined for 38 isolates.

Fluoroquinolone Resistance in Clinical Isolates of Klebsiella oxytoca

Background: Prevalence of fluoroquinolone-resistant Klebsiella oxytoca has been reported worldwide. Methods: We recovered ten clinical K. oxytoca isolates from patients with acute cystitis, asymptomatic bacteriuria or acute bacillary diarrhea in Japan. Out of ten isolates, one fluoroquinolone-susceptible isolate was included as a control. Fluoroquinolone resistance was characterized genetically by PCR and DNA sequencing methods. Outer membrane protein (OMP) profiles were determined by SDS-PAGE. Results: In nine clinical isolates of levofloxacin-resistant K. oxytoca, nucleotide sequences in the quinolone-resistance-determining regions showed amino acid mutations such as Thr83Ile and Asp87Gly in GyrA and Ser80Ile in ParC. Combined effects of reduced 36-kDa OMP production and amino acid mutations in GyrA and ParC were shown by two K. oxytoca isolates exhibiting higher minimum inhibitory concentrations for fluoroquinolones than other fluoroquinolone-resistant isolates. Conclusions: In clinical K. oxytoca isolates, the various mechanisms of fluoroquinolone resistance may include reduced 36-kDa OMP production as well as GyrA and ParC mutations.