Clonal Spread of Pediatric Isolates of Ciprofloxacin-Resistant, emm Type 6 Streptococcus pyogenes

Abstract

Twenty-four community isolates of Streptococcus pyogenes resistant to ciprofloxacin and susceptible to levofloxacin, gatifloxacin, and moxifloxacin were studied. Sequence determination of the quinolone resistance-determining regions in the gyrA and parC genes revealed a T/G mutation in parC leading to a Ser79Ala substitution in ParC. All isolates were of the emm type 6, and 18 and 2 of them were indistinguishable or closely related, respectively, on the basis of pulsed-field gel electrophoresis.

Streptococcus pyogenes is an important human pathogen that causes both mild infections, such as pharyngitis and impetigo, and severe diseases, such as toxic shock syndrome and necrotizing fascitis. Infection by this bacterial species can also give rise to sequelae such as rheumatic fever and acute glomerulonephritis (7). Penicillin is the antibiotic of choice for the treatment of S. pyogenes pharyngitis (4). Erythromycin represents an alternative in patients allergic to β-lactams. Fluoroquinolone resistance in S. pyogenes has been attributed to mutations in the genes for the type IV topoisomerase and/or the DNA gyrase. The mutations occur most frequently in codons S79 for ParC and S81 for GyrA (2, 13, 16). It has been proposed that active efflux was also involved in fluoroquinolone resistance (5, 10). M genotyping and pulsed-field gel electrophoresis (PFGE) are currently used for the study of clonality of S. pyogenes. The goals of this study were to determine the mechanism of ciprofloxacin resistance of 24 clinical isolates of S. pyogenes and to determine their genetic relatedness.

The strains were isolated from May 2000 to February 2001 from outpatients at the Hospital de Basurto, Bilbao, Spain. They were compared to 16 ciprofloxacin-susceptible strains isolated during the same time period. All the isolates were recovered from the throat of pediatric patients, and a single specimen per patient was included in the study. Strains were identified as S. pyogenes by colony morphology, beta-hemolysis on blood agar, and commercial latex agglutination (Slidex Strepto A; bioMérieux). Antimicrobial susceptibility to ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin was determined by microdilution in Mueller-Hinton broth supplemented with 3% lysed horse blood according to the NCCLS guidelines (11). Ciprofloxacin susceptibility testing was also carried out in the presence of the efflux inhibitor reserpine (10 μg/ml). An arbitrary breakpoint for resistance to ciprofloxacin of >2 μg/ml was used for quinolone resistance (12). Isolates were analyzed for mutations in the quinolone resistance-determining regions of parC and gyrA. A 614-bp PCR fragment internal to gyrA and a 520-bp fragment amplified from parC obtained with specific primers (16) were sequenced directly on both strands with an automated sequencer (CEQ 2000 DNA Analysis System; Beckman Coulter). Clonal relatedness of the isolates was studied by PFGE after digestion of total DNA with SmaI (6) with a CHEF-DR II apparatus (Bio-Rad) and emm typing. PFGE patterns were compared according to the criteria of Tenover et al. (14). The M type of S. pyogenes was determined by amplification and sequencing of the 5′ region of the emm gene according to the Centers for Disease Control and Prevention recommendations (http://www.cdc.gov/ncidod/biotech/strep/protocols.htm) with the modifications described by Hasenbein et al. (9). The PCR products were purified with the QIAquick PCR purification kit (QIAGEN) and sequenced with primer emmseq2 by Sistemas Genómicos SA (Valencia, Spain). The degree of identity required for emm gene designation was ≥95% for a portion of 200 bp within the 5′ region of the gene using a BLAST search (http://www.cdc.gov/ncidod/biotech/strep/strepblast.htm).

We have analyzed 24 pediatric S. pyogenes isolates whose ciprofloxacin MICs were >2 μg/ml that were susceptible to the other fluoroquinolones tested, including levofloxacin, gatifloxacin, and moxifloxacin. The ciprofloxacin MICs were not lower in the presence of reserpine. Sequence analysis of the parC gene in all resistant isolates showed that ciprofloxacin resistance was due to a base pair change (TCC/GCC) at position 366 that resulted in amino acid substitution Ser79Ala. No mutations in the quinolone resistance-determining region of gyrA were detected.

To investigate a putative clonal spread of ciprofloxacin-resistant S. pyogenes, we analyzed the sequence of the emm gene of the 24 strains resistant to ciprofloxacin and of 16 ciprofloxacin-susceptible isolates collected simultaneously and at the same site. The M type 6 was detected in all the resistant S. pyogenes isolates. It is one of the predominant M types (5%) among S. pyogenes isolates (1) and is known for its propensity to cause meningitis (15). Four PFGE patterns were observed among the resistant strains: 18 isolates were indistinguishable, 2 isolates were closely related to the previous set of isolates, and 1 strain was unrelated to the outbreak; three strains were nontypeable by SmaI analysis with a pattern characterized by the presence of undigested DNA. Both emm typing and PFGE demonstrated that there are few clones of ciprofloxacin-resistant S. pyogenes among the pediatric patients attending the Hospital de Basurto. The 16 isolates susceptible to fluoroquinolones had no mutations in parC, and in gyrA they had unrelated PFGE patterns that were distinct from those of the resistant strains and belonged to six emm types: emm22, emm75, emm4, emm9, emm12, and emm28.

There are no NCCLS breakpoints for streptococci and ciprofloxacin. However, ciprofloxacin resistance is becoming a matter of concern, with reports of prevalence rates that might represent a warning signal (3, 12, and G. A. Storch and R. C. Orscheln, Abstr. 41st Annual Meeting IDSA, abstr. 213, 2003). Storch and Orscheln described a high rate (10.7%) of fluroquinolone resistance in S. pyogenes isolates from children. Perez-Trallero et al. have reported that ciprofloxacin resistance is more prevalent among erythromycin-resistant isolates (12). We did not observe this association of resistance characters, since only three strains were resistant to both antimicrobials (data not shown). This could reflect local epidemiological difference, since geographical variation in macrolide resistance has been observed in Spain (12).

To the best of our knowledge, this is the first description of the clonal spread of ciprofloxacin-resistant S. pyogenes among a pediatric population. Albertí et al. (S. Albertí, G. Cortés, C. García-Rey, C. Rubio, F. Baquero, et al., Abstr. 14th Eur. Congress Clin. Microbiol. Infect. Dis., abstr. O201, 2004) have observed that ciprofloxacin resistance in S. pyogenes is mainly caused by an emm type 6 clone in Spain. They found that 67% of the resistant isolates were of M type 6 and that 50% of the M 6 type strains had indistinguishable PFGE patterns. We found that all 24 ciprofloxacin-resistant strains were of M type 6 and that 75% had an identical restriction pattern. These differences could result from temporal and geographical differences in the sampling or might be related to the pediatric origin of the samples.

The use of fluoroquinolones can select resistant mutants in nontarget bacterial species (8) and are not indicated to treat infections in children. Thus, emergence of clinical isolates resistant to ciprofloxacin, in particular among pediatric patients, is a cause of concern and emphasizes the need for judicious use of antibacterial agents. Our results indicating geographical spread of a ciprofloxacin-resistant clone of S. pyogenes stress the necessity for surveillance studies to prevent dissemination of these microorganisms.