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Antimicrobial Agents and Chemotherapy, April 2006, p. 1594-1598, Vol. 50, No. 4
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.4.1594-1598.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

New Real-Time PCR Assay Using Locked Nucleic Acid Probes To Assess Prevalence of ParC Mutations in Fluoroquinolone-Susceptible Streptococcus pneumoniae Isolates from France

Jean-Winoc Decousser,1,2* Imen Methlouthi,2 Patrick Pina,3 Anne Collignon,4 Pierre Allouch,2 on behalf of the ColBVH Study Group

Laboratoire de Biologie, Centre Hospitalier de Dourdan, Dourdan, France,1 Service d'Hygiène Hospitalière, Centre Hospitalier de Versailles André Mignot, Versailles, France,2 Service de Médecine B, Hôpital de Plaisir Grignon, Plaisir Grignon, France,3 Laboratoire de Microbiologie, Centre Hospitalier Universitaire Jean Verdier, Bondy, France4

Received 3 August 2005/ Returned for modification 4 September 2005/ Accepted 5 January 2006


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ABSTRACT
 
A real-time PCR assay with locked nucleic acid probes was developed to screen mutations at codons 79 and 83 of the Streptococcus pneumoniae parC gene. Only silent mutations were detected among 236 French invasive fluoroquinolone-susceptible strains. This test could be useful for some high-risk patients or in national surveys.


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TEXT
 
The worldwide spread of multidrug-resistant clones has led to the increasing use of fluoroquinolones (FQs) in the therapy of Streptococcus pneumoniae infections (21). In some countries the increase of FQ resistance (FQR) in that species and some clinical failures have been reported (5, 9, 13, 16, 18). The mechanisms of FQR mostly correspond to stepwise mutations in the quinolone resistance-determining regions (QRDR) of ParC and GyrA, two subunits of the FQ targets (DNA gyrase and the topoisomerase IV) (29). The low-level resistance first-step parC mutants (FSPC) were implicated in the selection following levofloxacin therapy of the highly resistant parC-gyrA double mutants (7, 16). FSPC mutants are classified as susceptible to FQs according to the standard breakpoints (19, 26, 27, 28). Sequencing of QRDR parC gene was considered the gold standard. We described a real-time PCR assay with TaqMan probes including locked nucleic acid (LNA) bases to detect mutations in codon 79 or 83 of parC, the two codons being mostly implicated in FQR in S. pneumoniae (5, 8, 9, 16). The LNA base is a bicyclic RNA analogue that increases the stability of the DNA/LNA mixmer (14). When probes include LNA bases, the melting temperature of the duplex DNA target/probe and then the specificity of the test increase. This assay was tested for an epidemiological survey that included a panel of controls and S. pneumoniae invasive strains, using sequencing as control method.

Control strains including wild-type and ParC mutant strains were tested repeatedly (Table 1) . A sample of 236 clinical FQ-susceptible strains was randomly selected from the annual survey program performed between 2000 and 2003 in 105 general hospitals as part of the Collège de Bactériologie-Virologie Hygiène des Hôpitaux (ColBVH) Study Group and screened for mutations. The MICs were determined by an agar dilution method and interpreted according to Clinical and Laboratory Standards Institute guidelines as previously described (11, 12, 22, 23). Two specific primers and TaqMan probes were designed to detect wild-type alleles at positions 79 and 83 of the parC QRDR of S. pneumoniae (nucleotide positions 3852 to 3854 and 3864 to 3866, respectively) (Table 2). DNA crude extracts were prepared from a loopful of overnight plate culture suspended in 200 µl of distilled water, using a rapid DNA extraction kit (QIAamp DNA MiniKit; QIAGEN, Courtaboeuf, France) according to the manufacturer's instructions. The PCR experiments were performed on a Smart Cycler (Cepheid, Sunnyvale, Calif.); 5 µl of DNA extract was transferred into a 20-µl PCR mixture containing 0.25 µM concentrations of the primers parC3 and parC4, 0.25 µM concentrations of each probe, and 12.5 µl of the Smart Kit (Eurogentec, Seraing, Belgium), including deoxynucleoside triphosphates, DNA polymerase (Hot Start Goldstar DNA polymerase), and MgCl2. The thermal cycling protocol consisted of 10 min at 95°C, followed by 45 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C. A mutation was suspected if at least one probe failed to hybridize. The parC QRDR was then sequenced as previously described (11, 24). Sequence analysis was carried out by using BLAST software (www.ncbi.nlm.nih.gov) and the parC gene of S. pneumoniae from GenBank accession no. Z67739 as a reference.


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  		TABLE 1. Bacterial control strains used in this study


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  		TABLE 2. Amplification primers and TaqMan probes used in this study

The control strains showed reliable hybridization results (data not shown). Hybridization results were in agreement with the parC QRDR sequences (Table 1 and Fig. 1). Strains Col594 and UA1680 presented an additional mutation inside the site recognized by the parC4 primer or the 79FAM-1 probe. All clinical strains were susceptible to levofloxacin, and 6.3% (15 of 236) of them showed only one (13 strains) or no (2 strains) fluorescence signal (Tables 3 and 4). The PCR results were confirmed by the sequencing: a silent mutation in one target site (11 strains) and mutation inside the sequences recognized by the probes (codon 77, 81, or 86; 5 strains) or the primers (codon 91 or 95; 2 strains showing no increase in the MICs of FQs).


Figure 1
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  		FIG. 1. Examples showing the real-time detection of a wild-type strain (ATCC 49619) containing no mutation in codons 79 (A) and 83 (B) and the lack of a signal with the 79FAM-1 probe when a Ser79Phe mutant (UA1066) was tested (A). For every sample, both probes (79FAM-1 and 83ROX-1) were tested in the same assay.


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  		TABLE 3. Results of PCR assays with TaqMan probes and analysis of sequencing data for clinical strains


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  		TABLE 4. Fluoroquinolone MICs of 236 S. pneumoniae strains isolated from blood cultures of adults

Using the current interpretative standards, 29 to 97% of strains with parC mutations were susceptible to levofloxacin (19, 27). Some authors have proposed to decrease its breakpoint (28). The French Society of Microbiology has recommended to test norfloxacin (breakpoint of 16 µg/ml) (15). Unfortunately, this compound is a good substrate for efflux pumps, representing other FQR mechanisms not clearly implicated in clinical failures (16, 25). Molecular methods have been developed to detect FSPC (restriction fragment length polymorphism, PCR-oligonucleotide ligation, DNA microarray, denaturing high-pressure liquid chromatography, etc.), but they were time-consuming and costly (1, 4, 6, 8). Our assay presents several advantages: the use of the real-time PCR tools available in numerous laboratories; the practicability and rapidity of this technology; the targeting of the wild-type sequence, allowing detection of the new mutation; and the robustness of the test in terms of sensibility according to these first results. The lack of an internal control could be offset by the low frequency of the double parC mutant (Ser79, Asp83): the total lack of fluorescence signal should be interpreted with caution. Nevertheless, this screening test could not replace sequencing, with silent mutations leading to an overestimation of significant mutations. The DNA extraction step was still time-consuming and needs automation. Its use could be reserved for high-risk fluoroquinolone resistance settings or for epidemiological surveys (30). First-step GyrA mutants are not detected by this assay; these mutants seem to be infrequent and resistant to levofloxacin (MIC ≥ 8 µg/ml) (27). We also provided for the first time epidemiological data regarding the prevalence of FSPC mutants in France. No significant mutation at the targeted codons was identified. In terms of MICs, the impact of Arg95Cys or Asn91Asp was weak, according to previously published data (3, 8). This low level of FSPC mutant contrasts with the prevalence of 4.7% found in the United States by Davies et al. (8). The difference was more dramatic in the group of strains showing the higher levofloxacin MIC (2 µg/ml), which represented 2.6% of the American sample and 12.2% in the present study: 71% (10 of 14) versus 0% (0 of 29). It could be explained by (i) the difference in the levofloxacin MIC distribution and the low frequency of high-level levofloxacin-resistant strains (0.4% versus 1.8% in the United States), (ii) the methodology (systematic screening versus sampling) and origin of the strains (bloodstream versus noninvasive strains), and (iii) the low level of levofloxacin consumption in France compared to the United States, which was significantly associated with the increasing rate of FQR in this country (2, 5, 8, 9, 12, 17, 20, 27, 30). However, these results should be confirmed with a larger number of strains.

In addition to antibiotic susceptibility testing, this real-time PCR assay could be useful as a screening test for detecting candidates for QRDR sequencing among S. pneumoniae strains.


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ACKNOWLEDGMENTS
 
We thank P. Courvalin from the Unité des Agents Antibactériens, Institut Pasteur de Paris, Paris, France, for providing control strains and the microbiologists throughout France who participated to the ColBHV Study Group: J. Akli (Blois), C. Alba-Sauviat (Chaumont), G. Aubert (Saint Etienne), A. Amirault (Vierzon), J. Assens (St. Afrique), J. P. Aubry (Quimperle), P. Aucher (Saint Jean D'Angely), C. Auvray (Charleville Mezieres), A. Bailly (Albi), A. Barrans (Sete), D. Barraud (Gonesse), C. Benoit (Fontainebleau), E. Bichier (Saumur), H. Biessy (La Rochelle), M. Bietrix (Martigues), P. Bineau (Saint Dizier), V. Blanc (Antibes), S. Bland (Annecy), A. Boisivon (St. Germain en Laye), Y. Boucaud-Maitre (Lyon), C. Bouguigny-Saison (Soissons), P. Brisou (Toulon Naval), S. Brovedani (Rambouillet), M. Caillaux (Tourcoing), B. Cancet (Villeneuve sur Lot), J. Carre-Cavelier (Bayeux), G. L. Cartolano (St. Germain En Laye), J. Cartron (Dreux), G. Chambreuil (La Roche sur Yon), P. Chantelat (Vesoul), A. Chapelle (Aubenas), C. Chaplain (Saint Denis), H. Chardon (Aix En Provence), B. Chaurang (Neuilly Sur Seine), A. Clarac (Foix), P. Clergeau (Sallanches), E. Collot (Bar Le Duc), P. Courrier (Metz Armees), M. F. Danjoux (Tarbes), J. P. Darchis (Compiegne), H. De Montclos (Bourg En Bresse), A. Decoste (Lomme Les Lille), C. Delamare (Thionville), J. M. Delarbre (Mulhouse), P. Deligne (Remiremont), F. Delubac (Annonay), M. C. Demachy (Meaux), H. Demontclos (Bourg en Bresse), J. Deregnaucourt (Paris), M. A. Desailly-Chanson (La Roche Sur Yon), J. Didion (Metz), F. Doucet-Populaire (Versailles), A. Dublanchet (Villeneuve St. Georges), B. Dubourdieu (Rodez), S. Dubourdieu (Gisors), A. Dupond (Laon), C. Durand (Provins), C. Eloy (Troyes), P. Emerique (Remiremont), F. Evreux (Le Havre), D. Fevre (Vienne), J. Flipo (Wissembourg), N. Fonsale (St. Etienne), A. Fremaux (Creteil), C. Fuhrmann (Lyon), S. Gabriel (Monaco), M. Galanti (Coulommiers), G. Gallou (Falaise), F. Gandhilhon-Crepet (Monbrison), I. Ganivala (Montauban), E. Gardien (Morlaix), A. Garnotel (Marseille-Armees), M. Gavignet (Bourges), F. Geffroy (Quimper), C. Grasmick (Cahors), B. Gravagna (Lyon), G. Grise (Elbeuf), C. Guier (St. Valler), P. Guiet (Nemours), A. Heidt (Hagueneau), M. Helfre (Firminy), J. Heurte (Beauvais), E. Heusse (Bayeux), M. C. Jaffar Bandjee (Saint Denis Reunion), D. Jan (Laval), E. Jaouen (Sable Sur Sarthe), G. Khatib (Bagnols Sur Ceze), J. P. Lafargue (Dax), R. Lamarca (Narbonne), V. Larroque (Carcassonne), E. Laurens (Cholet), A. Le Coustumier (Cahors), F. Le Turdu (Argenteuil), J. Y. Leberre (Saint Nazaire), E. Lecaillon Thibon (Perpignan), H. Lefrand-Crepin (Avignon), P. Lemaitre (Creil), C. Lemble (Selestat), M. Leneveu (Meulan), A. Lepilleur (St. Dizier), A. Mandjee (Romans), A. Mangeol (Montfermeil), M. F. Marchal (Annemasse), M. Marcolin (Arras), A. Marmonier (Le Mans), T. Masseron (Lyon), R. Meley (Saint Etienne), O. Menouni (Montceau les Mines), M. Menouar (Rang Du Flier), A. Michel (Marseille), M. Mora (Frejus), B. Moreau (Cayenne), A. Morel (Le Havre), O. Morvan (Saint Brieuc), D. Neri Schiavini (Cannes), G. Otterbein (Bry Sur Marne), X. Palette (Plaisir), B. Pangon (Versailles), J. Paul (Boulogne sur Mer), C. Payen (Brignoles), M. Perrin (Thionville), D. Pierrejean (Auch), P. Pouedras (Vannes), D. Pressac (Tulle), G. Rast (Poissy), D. Reisz (Montceau les Mines), F. Richardin (Mantes La Jolie), Y. Rio (Metz), P. Roos (Thionville), P. Roussellier (Salon De Provence), A. Rousset (Beaune), M. Rouviere (Mende), O. Sabot (Belley), A. Saly (St. Denis de la Réunion), S. Samaille (Saint Omer), R. Sanchez (Perigueux), A. Scanvic (Argenteuil), Y. Scat (Paris), A. Secher (Chartres), H. Sep-Hieng (Avranches), D. P. Simeon (Langres), V. Simha (Hyeres), C. Sire-Bidault (Chalon sur Saone), A. Smati (Aubenas), A. Sommabere (Brive), P. Stoessel (Neufchateau), P. Stolidi (Aubagne), F. Templier (Armentieres), J. P. Thellier (Chateau Thierry), A. Thore (Beaune), J. Tous (Chambery), A. Trevoux (Mulhousse), A. Vachee (Roubaix), E. Vallee (Eaubonne), J. Vaucel (St. Brieuc), A. Verhaeghe (Dunkerque), M. Villemain (Aurillac), M. Viot (Nice), I. Vray (Voiron), and J. F. Ygout (Lorient).


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FOOTNOTES
 
* Corresponding author. Mailing address: Laboratoire de Biologie, Centre Hospitalier de Dourdan, 2 Rue du Potelet, 91415 Dourdan, France. Phone: (33) 1 60 81 58 92. Fax: (33) 1 60 81 58 97. E-mail: jean-winoc.decousser{at}wanadoo.fr. Back


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Antimicrobial Agents and Chemotherapy, April 2006, p. 1594-1598, Vol. 50, No. 4
0066-4804/06/$08.00+0     doi:10.1128/AAC.50.4.1594-1598.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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