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International Journal of Antimicrobial Agents 30 (2007) 183–185

Short communication

Characterisation of levofloxacin-resistant clinical isolatesof Streptococcus pyogenes in Bilbao, Spain

Rodrigo Alonso a,∗, Estibaliz Mateo a, Guillermo Ezpeleta b, Ramon Cisterna a,b

a Departamento de Inmunologıa, Microbiologıa y Parasitologıa, Facultad de Farmacia, Universidaddel Paıs Vasco, Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spainb Servicio de Microbiologıa, Hospital de Basurto, 48013 Bilbao, Spain

Received 30 November 2006; accepted 5 April 2007

bstract

The resistance mechanisms and clonal relationship were determined for two Streptococcus pyogenes isolates with high-level resistance

o levofloxacin as well as to other fluoroquinolones. DNA amplification and sequencing revealed a serine-81→phenylalanine substitutionn GyrA and a double substitution in ParC of serine-79→phenylalanine and aspartic acid-91→asparagine. Pulsed-field gel electrophoresisnalysis and emm typing determined that both isolates were emm type 28 and were genetically indistinguishable.

2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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eywords: Fluoroquinolone; Resistance; parC; gyrA; Streptococcus pyogen

. Introduction

Streptococcus pyogenes is the aetiological agent of a wideange of infections, including streptococcal sore throat, skinnd soft-tissue infections and the post-infectious syndromesf glomerulonephritis and acute rheumatic fever. Penicillins the drug of choice for the treatment of these infectionsecause S. pyogenes remains susceptible to this antibioticespite its intensive use.

The increasing use of fluoroquinolones due to theirood activities against some bacterial pathogens has ledo the emergence of S. pyogenes isolates with reducedusceptibilities to these antibiotics [1–3]. Increased resis-ance of S. pyogenes to ciprofloxacin has been reported inpain and Belgium at high rates [4,5]. The mechanism ofuoroquinolone resistance in isolates with decreased suscep-

ibility has been associated with mutations in the quinolone

esistance-determining region (QRDR) of parC alone. High-evel resistance has been associated with mutations in theRDRs of both parC and gyrA [2,3,6,7] and appears to be

∗ Corresponding author. Tel.: +34 945 01 39 04; fax: +34 945 01 30 14.E-mail address: rodrigo.alonso@ehu.es (R. Alonso).

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924-8579/$ – see front matter © 2007 Elsevier B.V. and the International Societyoi:10.1016/j.ijantimicag.2007.04.001

nalogous to that described in Streptococcus pneumoniae andther streptococci [8,9]. This report describes the character-sation of two highly fluoroquinolone-resistant strains of S.yogenes from Bilbao, Spain.

. Materials and methods

Strains were isolated from the wound (skin ulcer) ofwo outpatients who had previously received courses of lev-floxacin. Strain ESGA-1242 was isolated in March 2004rom a woman with a history of osteoporosis. She hadeen treated for a skin ulcer due to coagulase-negativetaphylococci 1 month prior to isolation of S. pyogenes, aondition that was treated with levofloxacin and rifampicindosage unknown) at that time. Strain ESGA-1473 wassolated in April 2004 from a man with a history ofypertension and alcoholic hepatitis. He was not takingntibiotics at the time the culture was obtained but had

een treated with levofloxacin for pulmonary infection0 days prior to isolation of the fluoroquinolone-resistant. pyogenes strain. In addition, he had suffered multipleecurrent pulmonary infections requiring several courses of

of Chemotherapy. All rights reserved.

184 R. Alonso et al. / International Journal of Antimicrobial Agents 30 (2007) 183–185

Table 1Characteristics of Streptococcus pyogenes strains showing high-level resistance to fluoroquinolones

Country Source MICs (�g/mL) Amino acid alteration in QRDR emm Reference

CIP LEV GAT MOX ParC GyrA

Germany Wound ≥32 ≥32 4 4 S79F, D91N S140P S81F 89 [15]USA Blood 32 16 4 2 S79F, A121V S81Y 12 [2]USA Blood ≥32 ≥32 N.D. N.D. S79Y S81F, M99V N.D. [3]Spain N.D. ≥32 ≥32 N.D. 1.5 S79F, A121V S81Y 12.26 [16]S

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pain Wound 32 32 8 4

IC, minimum inhibitory concentration; CIP, ciprofloxacin; LEV, levofletermining region; N.D., no data.

ntibiotics. The strains were identified as S. pyogenes byolony morphology, �-haemolysis on blood agar and com-ercial latex agglutination (Slidex Strepto A; bioMerieux,arnes-la-Coquette, France). Antimicrobial susceptibility to

iprofloxacin, levofloxacin, sparfloxacin, gatifloxacin, mox-floxacin and gemifloxacin was determined by microdilutionn Mueller–Hinton broth supplemented with 3% lysed horselood and the results were interpreted according to Clinicalnd Laboratory Standards Institute guidelines [10]. Strepto-occus pneumoniae ATCC 49619 was used as the controltrain.

Chromosomal DNA was used as the template for poly-erase chain reaction (PCR) amplification of a 520 bp

ragment of parC and a 614 bp fragment of gyrA containinghe QRDRs of both genes with pairs of primers previ-usly described [3]. PCR products were purified with theIAquick PCR purification kit (Qiagen, Hilden, Germany)

nd sequenced by Sistemas Genomicos SA (Valencia, Spain).he GenBank accession numbers for the wild-type sequencessed for comparison purposes were AF220946 for parC andF220945 for gyrA.The genetic relatedness of both isolates was investigated

y pulsed-field gel electrophoresis (PFGE) followingigestion of bacterial DNA by SmaI using a CHEF-DRI apparatus (Bio-Rad Laboratories, Hercules, CA) [11].omparison of banding patterns was performed by visual

nspection, and interpretation of relatedness was based onhe criteria of Tenover et al. [12]. The M type of S. pyogenesas determined by amplification and sequencing of the′ region of the emm gene according to the recommen-ations of the Centers for Disease Control and Preven-ion (http://www.cdc.gov/ncidod/biotech/strep/protocolmm-type.htm) with the modifications described by Hasen-ein et al. [13]. The PCR products were purified andequenced with primer emmseq2. The degree of identityequired for emm gene designation was ≥95% for a portion of00 bp within the 5′ region of the gene using a BLAST searchhttp://www.cdc.gov/ncidod/biotech/strep/strepblast.htm).

. Results

The two isolates were resistant to all tested fluoroquino-ones, as follows: ciprofloxacin, 32 �g/mL; levofloxacin,

rflan

S79F, D91N S81F 28 This report

; GAT, gatifloxacin; MOX, moxifloxacin; QRDR, quinolone resistance-

2 �g/mL; sparfloxacin, 32 �g/mL; gatifloxacin, 8 �g/mL;oxifloxacin, 4 �g/mL; and gemifloxacin, 1 �g/mL.DNA amplification and sequencing revealed mutations

n the QRDR of both the parC and the gyrA gene. Thereere two substitutions in parC: Ser79Phe and Asp91Asn.Ser81Phe substitution was found in gyrA gene. Muta-

ional changes in both parC and gyrA have been describedy several authors in S. pyogenes isolates showing high-evel resistance to fluoroquinolones (Table 1). However, thesp91Asn substitution by itself has not been shown to con-

er fluoroquinolone resistance [14]. In addition, numerousilent mutations were present within and surrounding thearC QRDR of the isolates, and these substitutions were theame as those described by Orscheln et al. [14] in emm type 28solates. In addition, a silent mutation was found at positionly-103 (GGG → GGC) of gyrA. Although only anecdo-

al cases of high-level fluoroquinolone-resistant S. pyogenesave been published [2,3,15,16], our results are consistentith previous reports demonstrating that high-level fluoro-uinolone resistance results from alterations in the QRDR ofoth parC and gyrA genes. In our isolates, mutations observedn parC were identical to those previously identified, such aseplacement of Ser-79 by Phe and of Asp-91 by Asn [15].he mutation observed in gyrA (Ser81Phe) was identical to

hat reported by Reinert et al. [15].Both isolates were typed by PFGE and DNA sequencing

f the M-protein gene (emm). Isolates were found to be emmype 28, and PFGE analysis revealed that both isolates wereenetically indistinguishable. emm type 28 is frequently asso-iated with invasive infections and has even been reported ashe second most common invasive type [17]. Although bothatients attended the same centre, no epidemiological rela-ionship was found between the two patients from whom theighly resistant isolates were obtained.

This report of two S. pyogenes isolates with high-leveluoroquinolone resistance is a reminder that the neweruoroquinolones should be used appropriately. Fluoro-uinolone resistance in S. pyogenes is currently rare ands represented by sporadic isolation over a geographicallyiverse area. The isolation frequency of fluoroquinolone-

esistant �-haemolytic streptococci may increase as theuoroquinolone selective antimicrobial pressure continues,nd continuous surveillance of the level of resistance isecessary.

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cknowledgments

This work was supported by the Health Department ofhe Basque Government (grant 200311052). R.A. was theecipient of a postdoctoral fellowship from the Basque Gov-rnment.

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