1 Belgian Reference Centre for Group A Streptococcus, and 2 Department of Epidemiology and Community Medicine, University of Antwerp, Campus Drie Eiken, Antwerpen, Belgium
Received 20 September 2004; returned 7 December 2004; accepted 8 December 2004
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Abstract |
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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 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 (
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.
Keywords: S. pyogenes , antibiotic resistance , molecular mechanisms
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Introduction |
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When introduced into clinical use, fluoroquinolones were largely targeted against Gram-negative bacteria, with limited activity against Gram-positive pathogens.6 However, newer fluoroquinolones possess increased activity against Gram-positive bacteria and have superior pharmacokinetic/pharmacodynamic properties over older fluoroquinolones, such as ciprofloxacin, norfloxacin and ofloxacin.7 Fluoroquinolone resistance in S. pyogenes is rare but has been reported in other streptococci, including Streptococcus pneumoniae, mediated either by efflux pump hyperexpression that mainly affects the older fluoroquinolones or by mutations in parC/parE and gyrA/gyrB encoding topoisomerase IV and DNA gyrase, respectively.8,9 First-step mutations usually occur in the topoisomerase preferentially targeted by the selector fluoroquinolone; for instance, in Gram-positive bacteria, selection by older fluoroquinolones results in first-step mutations in parC. The newer fluoroquinolones, however, require a second-step or additional mutations at the same or additional drug target sites to generate full clinical resistance.9,10
Four case reports of single fluoroquinolone-resistant S. pyogenes carrying mutations in parC and/or gyrA have been described,1114 and in another recent study, S. pyogenes with reduced susceptibility to fluoroquinolones were identified in Spain, although the molecular mechanisms were not studied.15 We studied the prevalence, clonality, and the molecular mechanisms mediating fluoroquinolone resistance in a large series of clinical S. pyogenes isolated from pharyngitis patients in Belgium during 1 January 199931 December 2002.
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Materials and methods |
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During 19992002, the Belgian Reference Centre for Group A Streptococcus received 2955 putative S. pyogenes isolates. These were recovered from tonsillopharyngitis patients from private laboratories covering 10 Belgian provinces, along with a standardized record of patients' age, residential addresses and dates of isolation. At the reference centre, 2793 isolates were confirmed to be S. pyogenes by standard methods.
Susceptibility testing
Initially, all isolates were subjected to ciprofloxacin MIC testing, and those exhibiting ciprofloxacin MIC 2 mg/L (defined as fluoroquinolone non-susceptible) were tested further with a panel of fluoroquinolones by an agar dilution method. Breakpoints used (except for norfloxacin) were those defined by the NCCLS for S. pyogenes or S. pneumoniae,16
and were as follows (susceptible/resistant, in mg/L): norfloxacin (
4/
16) (Sigma Chemical Co., St Louis, MO, USA), ofloxacin (
2/
8) (Sigma), levofloxacin (
2/
8) (Aventis, Romainville, France) and moxifloxacin (
1/
4) (Bayer, Leverkusen, Germany). All fluoroquinolone non-susceptible isolates were also screened for changes in any fluoroquinolone MICs in the presence of 25 mg/L of reserpine (Sigma), an efflux pump-blocker. All MIC determinations, including the reserpine assay, were performed in duplicate. In addition, all 2793 isolates were screened for penicillin, macrolide and tetracycline resistance by disc diffusion and MIC testing using NCCLS breakpoints.16
Genotyping of isolates
Clonality among the fluoroquinolone non-susceptible isolates and a random selection of fluoroquinolone-susceptible isolates (n=627, MIC < 2 mg/L) was investigated by pulsed-field gel electrophoresis (PFGE) using SmaI and by emm typing by the reverse line blotting method, as described previously.17,18 From the fluoroquinolone non-susceptible S. pyogenes, 55 isolates that included representatives of all PFGE types were analysed for mutations in the quinolone resistance determining regions (QRDRs) of parC, gyrA, and 54 isolates in parE, gyrB, by direct double-strand sequencing (3730 DNA Analyser, Applied Biosystems, Foster City, CA, USA) with the BigDye Terminator Version 3.1 Kit (Applied Biosystems) using published primers.11,19 In addition, the QRDRs of parC of 59 fluoroquinolone-susceptible isolates (MIC 0.125 mg/L, n=9; MIC 0.25 mg/L, n=32; MIC 0.5 mg/L, n=16; MIC 1 mg/L, n=2) were also sequenced. Nucleotide sequence alignment was carried out using SeqMan (DNASTAR Inc., Madison, WI, USA).
Tests for statistical significance
Pearson's 2-test was used to attribute statistical significance to the differences in the yearly prevalence of fluoroquinolone non-susceptible S. pyogenes in children (
16 years of age) versus adults, and the prevalence change over years in these population groups. Multiple logistic regression analyses were used to determine the effect of calendar time on the association between prevalence of fluoroquinolone non-susceptibility and Belgian provinces. P values >
0.05 (two-sided) were deemed significant.
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Results |
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Of the 2793 S. pyogenes isolates recovered from tonsillopharyngitis patients during 19992002, the age of the patient was known in 2651 cases. Children and adults constituted 56% and 39% of the patients sampled. The paediatric population studied (1999, 60%; 2000, 51%; 2001, 58%; and 2002, 55%) was further subdivided into the following age groups: <1 year (n=11), 14 years (n=483), 59 years (n=737) and 1016 years (n=341).
Identification of fluoroquinolone non-susceptible S. pyogenes in paediatric patients
One hundred and fifty-two (5.4%) S. pyogenes were identified as fluoroquinolone non-susceptible (ciprofloxacin MIC 2 mg/L). The yearly distribution of fluoroquinolone non-susceptible S. pyogenes according to patient age is shown in Table 1. The prevalence of non-susceptible isolates increased significantly in the year 2000 among children (P=0.01) and decreased in 2002 among both children (P < 0.001) and adults (P < 0.001). Despite the decreased prevalence in 2002, the proportion of fluoroquinolone non-susceptible S. pyogenes contributed by children increased linearly from 39% in year 1999 to 58% in 2002. Distribution of the non-susceptible isolates among children according to age-group was as follows: <1 year (n=1), 14 years (n=25), 59 years (n=34) and 1016 years (n=24).
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The 152 fluoroquinolone non-susceptible S. pyogenes were classified into nine emm types, of which emm6 and emm75 constituted >90% of the isolates (Table 2). The nine emm serotypes were further segregated into 15 PFGE types based on the criteria of Tenover et al.20 Five fluoroquinolone non-susceptible PFGE types were identified as clones on the criteria of being isolated from more than one patient. Of these, the two major clones, 5 and 15 belonging to the emm6 and emm75 serotypes, constituted >85% of the fluoroquinolone non-susceptible isolates and clone 5 alone constituted 74% of all fluoroquinolone non-susceptible S. pyogenes (Table 2). Clone 5 constituted 98% of all isolates of the emm6 serotype, and clone 15, 91% of all isolates of the emm75 serotype. Of the fluoroquinolone-susceptible isolates studied, emm6 and emm75 serotypes were identified in only three (emm6) and seven (emm75) isolates (Table 3).
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In vitro activities of the older and newer fluoroquinolones and of other antibiotic classes against the 152 fluoroquinolone non-susceptible S. pyogenes are presented in Table 4. Thirty-eight (25%) and 102 (67%) of the fluoroquinolone non-susceptible isolates showed reduced/borderline susceptibility to levofloxacin (MIC 4 mg/L) and to moxifloxacin (MIC 1 mg/L).
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Only five (3%) fluoroquinolone non-susceptible S. pyogenes showed resistance to macrolides, of which one isolate each of emm6, emm28 and emm76 also showed resistance to tetracycline and were considered to be multidrug-resistant.
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Discussion |
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We studied the molecular mechanisms underlying fluoroquinolone non-susceptibility in our isolates. All fluoroquinolone non-susceptible S. pyogenes analysed carried parC mutations, occasionally accompanied by parE mutations. Detection of mutations in parC was consistent with the current widespread use of older fluoroquinolones that preferentially target ParC. In accordance with previous observations in S. pneumoniae,21 the majority of our isolates (95%) showed a ParC amino acid substitution at S79 (to A, F or Y), confirming its role as the key residue mediating fluoroquinolone resistance. Substitutions of S79, D91, A121 and S140 have been reported in the previous case reports of fluoroquinolone non-susceptible S. pyogenes, although three high-level fluoroquinolone-resistant isolates also showed a substitution in GyrA.1114 These data, as well as studies on S. pneumoniae, suggest that first-step (or low-level fluoroquinolone-resistance-conferring) mutations serve as precursors for the development of high-level resistance by mutations at additional drug target sites under selection pressure of fluoroquinolones.9,22 Together with our data of S. pyogenes clones non-susceptible to older fluoroquinolones and having only borderline susceptibility or intermediate resistance to newer fluoroquinolones, a more judicious use of the newer fluoroquinolones is clearly warranted.
It is interesting to note that the S79 substitutions were also detected in two susceptible isolates. One explanation for fluoroquinolone-susceptibility in the presence of the same mutations that have been earlier linked to resistance, is additional mutations/polymorphisms in other regions of parCthat we have not analysedthat together allow fluoroquinolone binding. Identification of isolates belonging to the same clone and carrying the same parC/parE mutation(s) but having varied ciprofloxacin MICs also suggests existence of other additional mechanisms. For example, there could be additional mutations in the topoisomerase genes outside the regions analysed, or the presence of a fluoroquinolone efflux pump that is either inherently resistant to reserpine-induced inhibition or has mutations in the gene that have rendered it reserpine-resistant.19,23
We have shown, for the first time, multi-clonal spread of fluoroquinolone non-susceptible S. pyogenes, with emm6 and emm75 as the predominant serotypes. There are several explanations for the dissemination of these clones in Belgian provinces. These might have emerged under selective pressure of the older fluoroquinolones like ciprofloxacin that have limited activity on S. pyogenes.24 However, although carriage rates in adults are quite low, S. pyogenes constitutes normal flora in up to 35% of children. Fluoroquinolones are contraindicated in children based on reports of fluoroquinolone-induced cartilage damage in juvenile animal models and musculo-skeletal adverse effects in paediatric patients.25 However, data from the USA suggest widespread off-label use of ciprofloxacin for select life-threatening conditions, as well as topical use of ciprofloxacin and ofloxacin in the treatment of acute otitis media with otorrhoea through tympanostomy tubes in paediatric patients.25,26 In Belgium, we have no information about off-label use of systemic fluoroquinolones, but prescriptions for topical fluoroquinolones (ear or eye drops) that are approved for treatment of otitis externa and otitis media with otorrhoea, are estimated to exceed 15 000 per year. The high carriage rate of S. pyogenes and pneumococci in children and our data clearly indicate a real risk if fluoroquinolones become available for regular use in paediatric patients, particularly for respiratory tract infections.
On the other hand, the geographical drift and the temporal shift of the fluoroquinolone non-susceptible emm6 and emm75 serotypes noted in our study might merely represent the natural fluctuations of predominant S. pyogenes serotypes observed in the community.27,28 In a recent study, a shift of the predominant emm serotypes was shown to occur very rapidly and almost completely in closed-community pharyngitis patients.28 The M-protein seromarker is an important surface virulence factor and is epidemiologically associated with particular clinical syndromes. However, despite being well described, the mechanisms of the fluctuations within the predominant S. pyogenes serotypes are poorly understood. The parC mutations in the emm6 and emm75 subclones we detected here do not seem to confer a significant fitness cost to the organism, nor do they seem to be responsible for the success of the clones, which most likely results from some other genetic variation that segregates with our fluoroquinolone non-susceptible emm6 and emm75 subclones. Such non-virulence related markers could thus be a useful adjunct in the selection and the understanding of mechanisms underlying the success of S. pyogenes clones that would likely impact drug targeting to this organism.
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Acknowledgements |
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