Emergence of high-level fluoroquinolone resistance in emm6 Streptococcus pyogenes and in vitro resistance selection with ciprofloxacin, levofloxacin and moxifloxacin.

OBJECTIVES: To investigate the prevalence of fluoroquinolone resistance in Streptococcus pyogenes and its in vitro selection by ciprofloxacin and the respiratory fluoroquinolones, levofloxacin and moxifloxacin. METHODS: S. pyogenes (n = 5851) recovered from pharyngitis and invasive infections during 2003-06 in Belgium were screened for fluoroquinolone non-susceptibility (ciprofloxacin MIC > or =2 mg/L) and further studied for mutations in the topoisomerase genes, reserpine-sensitive efflux, clonality by PFGE and emm typing. Fourteen well-characterized fluoroquinolone-non-susceptible or -susceptible isolates were exposed stepwise to increasing levels of ciprofloxacin, levofloxacin and moxifloxacin. Selected mutants with increased MICs were analysed for resistance mechanisms. Mutation frequencies at 2x and 4x MIC of moxifloxacin and levofloxacin were estimated for a clinical emm6 parent strain carrying mutations in both parC and gyrA. RESULTS: Prevalence of fluoroquinolone-non-susceptible S. pyogenes (n = 437; 7.47%) increased significantly from 2.08% and 5.08% to 13.11% during 2003-05 and decreased to 8.93% in 2006 (chi(2) test; P < or = 0.001). emm6 constituted 80.09% of the total fluoroquinolone-non-susceptible isolates. Of the 71 S. pyogenes sequenced, 70 harboured first-step parC or gyrA mutations correlating with ciprofloxacin MICs 2-8 mg/L. Reserpine-sensitive efflux was not observed. One emm6parC mutant (Ser79Ala) also showed a second-step mutation in gyrA (Ser81Tyr), with MICs of ciprofloxacin, levofloxacin and moxifloxacin of 32, 8 and 1 mg/L, respectively. Mean mutation frequencies under moxifloxacin selection were 500- to 30 000-fold higher for this strain than those for an emm6 control strain. Selection of the emm6 double mutant with moxifloxacin generated a mutant with a moxifloxacin MIC of 64 mg/L and a levofloxacin MIC of 128 mg/L, and an additional Asp83Tyr substitution in ParC. CONCLUSIONS: We report an emergence of levofloxacin and high-level ciprofloxacin resistance associated with a second-step gyrA mutation in a clinical emm6 S. pyogenes. The observed high mutation frequency and in vitro selection of high-level resistance to the respiratory fluoroquinolones in the emm6 double mutant is of concern.

Genotypic diversity, antimicrobial resistance, and virulence factors of human isolates and probiotic cultures constituting two intraspecific groups of Enterococcus faecium isolates.

The intraspecific relationships among a collection of Enterococcus faecium isolates comprising probiotic cultures and human clinical isolates were investigated through the combined use of two high-resolution DNA-fingerprinting techniques. In addition, the incidences of antimicrobial resistance and virulence traits were investigated. A total of 128 E. faecium isolates from human clinical or nonclinical sources or used as probiotic cultures were subjected to fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting and pulsed-field gel electrophoresis (PFGE) analysis of SmaI macrorestriction patterns. Susceptibilities to 16 antimicrobial agents were tested using broth microdilution, and the presence of the corresponding resistance genes was investigated using PCR. Multiplex PCR was used to detect the presence of the enterococcal virulence genes asa1, gelE, cylA, esp, and hyl. The results of the study showed that two intraspecific genomic groups (I and II) were obtained in FAFLP analysis. PFGE analysis demonstrated high variability within these two groups but also indicated that some probiotic cultures were indistinguishable and that a number of clinical isolates may be reisolations of commercial probiotic cultures. Compared to group II, which contained the majority of the probiotic isolates and fewer human clinical isolates, higher phenotypic and genotypic resistance frequencies were observed in group I. Two probiotic isolates were phenotypically resistant to erythromycin, one of which contained an erm(B) gene that was not transferable to enterococcal recipients. None of the probiotic E. faecium isolates demonstrated the presence of the tested virulence genes. The previously reported observation that E. faecium consists of two intraspecific genomic groups was further substantiated by FAFLP fingerprinting of 128 isolates. In combination with antimicrobial resistance and virulence testing, this grouping might represent an additional criterion in assessing the safety of new potential probiotic E. faecium isolates.

Clonal spread of fluoroquinolone non-susceptible Streptococcus pyogenes.

BACKGROUND: Fluoroquinolones are an important group of antibiotics widely used in adults, and, despite the absence of official approval, these drugs are also used in children. So far, resistance to fluoroquinolones in Streptococcus pyogenes is very rare. METHODS: During a national surveillance programme in Belgium from 1999 to 2002, 2793 non-duplicate S. pyogenes recovered from tonsillopharyngitis patients were screened for fluoroquinolone resistance. Mutations in topoisomerase genes and the presence of any efflux pump activity were investigated to elucidate the fluoroquinolone resistance mechanisms. Clonality was assessed by pulsed-field gel electrophoresis (PFGE) and emm typing. RESULTS: Non-susceptibility to fluoroquinolones, defined as ciprofloxacin MIC > or = 2 mg/L, was identified in 152 (5.4%) of 2793 S. pyogenes. Fifty-five (36%) fluoroquinolone non-susceptible isolates were investigated for known resistance mechanisms; all showed mutations in parC, and 29 (19%) isolates also in parE; antibiotic efflux was not noted. Two major PFGE types comprised 88% of fluoroquinolone non-susceptible S. pyogenes and belonged to serotypes emm6 and emm75. Overall, emm6 and emm75 constituted >90% of all fluoroquinolone non-susceptible isolates and showed a significant temporal and geographical shift within Belgian provinces. Although fluoroquinolone-susceptible S. pyogenes also showed fluctuations in the predominant S. pyogenes serotypes, emm6 or emm75 were under-represented in this population. Approx. 55% of the fluoroquinolone non-susceptible isolates were recovered from children ( < or =16 years). CONCLUSIONS: We show here, for the first time, a multi-clonal spread of fluoroquinolone non-susceptible S. pyogenes exhibiting a known resistance mechanism. Non-susceptibility to fluoroquinolones in paediatric isolates is of concern.

Macrolide- and telithromycin-resistant Streptococcus pyogenes, Belgium, 1999-2003.

We found a 13% macrolide resistance in 3,866 Streptococcus pyogenes isolated from tonsillopharyngitis patients; 59% macrolide-resistant isolates were distributed in 5 clones, suggesting the importance of both resistance gene transfer and clonal dissemination in the spread of these organisms. We also report one of the largest collections of telithromycin-resistant isolates.

Bacitracin-resistant clone of Streptococcus pyogenes isolated from pharyngitis patients in Belgium.

We report 16 bacitracin-resistant Streptococcus pyogenes isolates recovered from pharyngitis patients in Belgium, 14 of which belonged to a particular emm type (emm28). All 16 isolates were constitutively resistant to macrolides and carried erm(B). The emergence of a bacitracin-resistant S. pyogenes clone raises questions about the continued reliability of bacitracin susceptibility testing for S. pyogenes identification.

Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in enterococci and survey for virulence determinants among European hospital isolates of Enterococcus faecium.

A multiplex PCR for the simultaneous detection of five virulence genes (asa1, gelE, cylA, esp, and hyl) in enterococci was developed. The presence of these genes was investigated in 153 clinical and 118 fecal Enterococcus faecium isolates from inpatients at an increased risk of developing infections (such as patients in intensive care units and hematology wards) from 13 hospitals in eight European countries. Of the 271 E. faecium isolates, 135 were vancomycin resistant E. faecium (VREF) isolates and 136 were vancomycin susceptible E. faecium (VSEF) isolates. Susceptibilities to ampicillin, gentamicin, streptomycin, vancomycin, teicoplanin, ramoplanin, quinupristin-dalfopristin, and linezolid were tested by the microdilution method. Overall, the prevalence of esp was significantly higher (P = 0.03) in clinical VREF isolates (92%) than in fecal VREF isolates (73%). In Italy, the prevalence of esp was significantly higher (P = 0.02) in VREF isolates (91%) than in VSEF isolates (68%), whereas in the United Kingdom, hyl was significantly more prevalent (P = 0.01) in VREF isolates (71%) than in VSEF isolates (29%). No significant differences were found for the other countries. Pulsed-field gel electrophoresis was used to check the clonality among the strains tested and showed the spread of two center-specific (esp-positive) VREF clones in Italy and one center-specific (hyl-positive) clone in the United Kingdom. These clones were resistant to ampicillin, gentamicin, and streptomycin. The multiplex PCR reported in this study is a convenient and rapid method for the simultaneous detection of the virulence genes asa1, gelE, cylA, esp, and hyl in enterococci. Molecular analysis showed the intrahospital spread of esp-positive VREF clones (in Italy) and hyl-positive VREF clones (in the United Kingdom); the role of hyl remains to be elucidated.

Oropharyngeal carriage of macrolide-resistant viridans group streptococci: a prevalence study among healthy adults in Belgium.

OBJECTIVES: Viridans group streptococci (VGS) are gaining significance as reservoirs of resistance determinants for respiratory tract pathogens. Our aim was to investigate healthy adults for oropharyngeal carriage of VGS that are resistant to macrolides, as well as to other common antibiotics. METHODS: Macrolide-resistant VGS were isolated from throat samples of 154 healthy Belgian adults, and phenotyped and genotyped for erm(A), erm(B) and mef(A). In vitro susceptibilities to 10 antimicrobials and the presence of tetracycline resistance genes were also determined. RESULTS: Carriage was detected in 71% of the population screened, from whom 157 unique, macrolide-resistant VGS were isolated. A constitutive (cMLS) phenotype was present in 105 isolates, of which 102 isolates carried either erm(B) or erm(B) + mef(A). The remaining three isolates did not present with any of the genes studied. All 45 isolates showing the M phenotype carried mef(A). The least abundant inducible (iMLS) isolates (n = 7) carried either erm(B) or erm(B) + mef(A). The most abundant macrolide-resistant VGS species was Streptococcus mitis (51%). Co-resistance to tetracycline was identified in 114 isolates, of which tet(M) was present in 105, tet(O) in two and both tet(M) and tet(O) in one, while the remaining six isolates did not present with either gene. tet(M) was also present in four tetracycline susceptible and two intermediately resistant isolates. Fluoroquinolone resistance (ciprofloxacin MIC > or = 4 mg/L) was detected in 16 isolates. Resistance to telithromycin, penicillin and chloramphenicol was appreciably low. CONCLUSIONS: This study highlights a high oropharyngeal carriage of macrolide-resistant VGS and its co-resistance to tetracycline and fluoroquinolones among healthy Belgian adults.