JAC Advance Access originally published online on April 17, 2007
Journal of Antimicrobial Chemotherapy 2007 59(6):1109-1113; doi:10.1093/jac/dkm098
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Comparative evaluation of VITEK 2® for antimicrobial susceptibility testing of group B Streptococcus
1 Groupe Hospitalier Cochin-Saint Vincent de Paul, Service de Bactériologie, Assistance Publique-Hôpitaux de Paris, 27 rue du Faubourg Saint Jacques, 75679 Paris, France 2 French National Reference Centre for Streptococci, Groupe Hospitalier Cochin-Saint Vincent de Paul, Assistance Publique-Hôpitaux de Paris, 27 rue du Faubourg Saint Jacques, 75679 Paris, France 3 Institut Cochin Department of Infectious Diseases-INSERM U567-UMR CNRS 810, Faculté de Médecine Descartes, Université Paris Descartes, 24 rue du Faubourg Saint Jacques, 75014 Paris, France 4 Unité de Biologie des Bactéries Pathogènes à Gram-positif, URA CNRS 2172, Institut Pasteur, 75724 Paris, France
* Correspondence address. Service de Bactériologie, Centre National de Référence des Streptocoques, Institut Cochin, INSERM567, Faculté de Médecine Descartes, 27 rue du Faubourg Saint Jacques, 75014 Paris, France. Tel: +33-1-58-41-15-60; Fax: +33-1-58-41-15-48; E-mail: claire.poyart{at}cch.aphp.fr
Received 28 January 2007; returned 1 March 2007; revised 10 March 2007; accepted 12 March 2007
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Objectives: Intrapartum antibiotic prophylaxis is recommended to prevent neonatal group B streptococcal (GBS) disease in colonized women, and penicillin or aminopenicillin constitute the first-line antibiotics. Most isolates are resistant to tetracycline, and resistance to macrolide–lincosamide–streptogramin (MLS) antibiotics is increasing. Therefore, laboratory testing for MLS resistance in GBS is now recommended for penicillin-allergic patients. The aim of this study was to compare the antimicrobial susceptibility of GBS as determined by the VITEK 2 system (bioMérieux, Marcy l'Étoile, France), agar diffusion methods and PCR-genotypic detection of resistance genes.
Methods: One hundred and ten unrelated selected GBS clinical isolates were studied. The antibiotics tested (VITEK 2 and agar diffusion method) were benzylpenicillin, ampicillin, erythromycin, clindamycin, co-trimoxazole, tetracycline, kanamycin, streptomycin and vancomycin. A standardized double-disc (DD) diffusion test was performed for MLS antibiotics. Genotypic characterization of tetracycline, MLS and aminoglycoside resistance genes was performed by PCR.
Results: All strains were susceptible to benzylpenicillin, ampicillin and vancomycin [category agreement (CA) between VITEK 2 and the diffusion method was 100%]. Ninety-five (86%) strains were resistant to tetracycline (CA was 98.9%). Eighty-one strains (73.6%) harboured an MLS resistance phenotype; 50 (61.8%) an MLSB-constitutive phenotype, 25 (30.8%) an MLSB-inducible phenotype and 6 (7.4%) an M phenotype. The agreement between data of VITEK 2 and the DD diffusion test for the detection of MLSB-constitutive, MLSB-inducible and M phenotype isolates was 76%, 36% and 100%, respectively. Almost all discrepancies were due to failure to detect erythromycin resistance by VITEK 2.
Conclusions: VITEK 2 allows accurate determination of GBS susceptibility for the majority of antibiotics, but has to be improved for erythromycin. Thus, the DD diffusion test remains the most simple and reliable method for macrolide resistance detection among this streptococcal species.
Keywords: Streptococcus agalactiae , antibiotics , macrolides–lincosamides–streptogramins , PCR
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Group B Streptococcus (GBS; Streptococcus agalactiae) is a normal constituent of the intestinal flora and is present in the vaginal flora of 20% to 40% of healthy women.1 However, it is also a leading cause of invasive infections (pneumonia, septicaemia and meningitis) in neonates and a serious cause of mortality or morbidity in adults with underlying diseases.1 As antibiotic administration to women during labour drastically reduces the incidence of early onset disease in neonates, maternal intrapartum prophylaxis for pregnant women colonized with GBS has been recommended for several years.2 Penicillin or aminopenicillin constitute the first-line antibiotics; however, in the case of allergy to these antibiotics, women will receive a macrolide or clindamycin.3 Erythromycin resistance rates of GBS have been increasing worldwide. Studies in France reported rates of 15% in 1999 and 18% in 2000,4–6 and higher rates of up to 40% have been published in other countries.7–9 Concerns over the increasing incidence of macrolide resistance in GBS have recently prompted the Centers for Disease Control and Prevention to recommend routine erythromycin and clindamycin susceptibility testing in their guidelines for the prevention of perinatal GBS disease.3 Many laboratories worldwide have adopted the VITEK 2 automated system (bioMérieux, Marcy l'Étoile, France) for rapid identification and susceptibility testing of most clinical isolates including GBS. Identification is made on the basis of biochemical reactions using colorimetric reading and MIC determinations by analysis of MIC patterns with the Advanced Expert System (AES) software. The aim of this study was to compare the antimicrobial susceptibility of GBS as determined by the VITEK 2 system, agar diffusion methods and PCR-genotypic detection of resistance genes, with a particular focus on macrolide-resistant strains.
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Bacterial strains
ß-Haemolytic colonies and suspected non-haemolytic colonies were identified as GBS by using a commercial latex agglutination test (Oxoid S.A., Dardilly, France). A total of 110 human epidemiologically unrelated GBS clinical isolates were included in this study. These strains were isolated between 1998 and 2006 from two university general hospitals in Paris. Strains were stored at –80°C in 10% glycerol Todd Hewitt medium, subcultured twice on Columbia agar containing 5% horse blood (bioMérieux) and grown overnight at 37°C under a 5% CO2 atmosphere prior to testing. Quality control strains consisted of S. agalactiae NEM316,11 S. agalactiae BM110,12 Enterococcus faecalis JH2-213 and Staphylococcus aureus ATCC 29213. The positive control strains for PCR were E. faecalis CCH424 [tet(M); erm(B); aphA-3],13 S. agalactiae CCH425 [tet(O)],5 Listeria monocytogenes CCH426 [tet(S); tet(L) and aad6],14 Streptococcus pyogenes CCH427 [tet(T)],15 S. aureus CCH428 [tet(K)],16 S. agalactiae CCH429 [erm(A)],5 S. aureus CCH430 [erm(C)]17 and S. agalactiae CCH431 [mef(A)].5
Colonies from pure cultures were resuspended in 0.45% NaCl solution to obtain a suspension with a turbidity equivalent to that of a 0.5 McFarland standard (Densicheck; bioMérieux) and the same inoculum was used for all susceptibility testing techniques. Antibiograms (agar diffusion method) were performed on 5% sheep blood Mueller–Hinton (MH). After incubation for 18–24 h at 37°C in 5% CO2, zone diameters were measured, and categorization as susceptible, intermediate or resistant was in accordance with the Comité de l'Antibiogramme de la Société Française de Mibrobiologie (CASFM) recommendations.18
VITEK 2 susceptibility testing was performed according to the manufacturer's instructions with the same bacterial suspension using the AST-P532 card. The results obtained after a maximum of 15 h of incubation were analysed and interpreted by AES 4.02 software.
Standardized double-disc (DD) agar diffusion tests with erythromycin and clindamycin discs (Bio-Rad, Marnes-la-Coquette, France) placed at 15 mm (edge to edge) were performed on 5% sheep blood MH agar incubated for 18–24 h at 37°C in 5% CO2.19 Macrolide–lincosamide–streptogramin (MLS) resistance phenotypes were interpreted as follows: (i) the M phenotype was erythromycin-resistant and clindamycin-susceptible with no inducible resistance visible in the overlap zone; (ii) the MLSB-inducible (MLSB-i) phenotype was erythromycin-resistant and clindamycin-susceptible with a blunted D-shaped zone of inhibition; and (iii) the MLSB-constitutive (MLSB-c) phenotype was resistant to both erythromycin and clindamycin. MICs of erythromycin and clindamycin were determined by the agar dilution method on 5% sheep blood MH. Categorization was determined according to the CASFM breakpoints as follows: erythromycin, >4 mg/L; and clindamycin, >2 mg/L.18 These breakpoints are different from those determined by the CLSI (formerly NCCLS), as strains are resistant to erythromycin and clindamycin with MICs >1 mg/L.20
When discordant results occurred, the VITEK 2 and the reference tests were repeated in duplicate to check these discrepancies. The disc diffusion method was considered as the reference method for susceptibility testing. For MLS, however, the DD diffusion test was taken as the reference method. Moreover, genotypic characterization of the resistance determinants was performed, providing precise identification of the resistance genes present in the strains studied.
Analysis of susceptibility testing
Category agreement (CA): both methods defined the category of microbial susceptibility as susceptible (S), intermediate (I) or resistant (R), according to the breakpoints recommended by the CASFM.18 Interpretative category errors were assessed with each antibiotic: (i) a very major error (VME) was defined when the results were obtained as resistant with the reference method, but susceptible with VITEK 2; (ii) a major error (ME) was defined when the results were obtained as susceptible with the reference method, but resistant with VITEK 2; and (iii) a minor error (mE) was defined when the results were obtained as intermediate with the reference method, but susceptible or resistant with VITEK 2 (and vice versa).
Total DNAs were extracted using the Instagene matrix (Bio-Rad) and PCR amplifications with specific primers enabling detection of tetracycline [tet(M), tet(O), tet(S), tet(T), tet(L) and tet(K)], MLS [erm(B), erm(A), erm(C) and mef(A)] and kanamycin (aphA-3) resistance genes were performed as described previously.5 A PCR with the primer pair dltSF (5'-AGGAATACCAGGCGATGAA CCGAT-3') and dltSR (5'-TGCTCTAATTCTCCCCTTATGGC-3') targeting the GBS-specific dltS gene was included as an internal control.21
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All 110 GBS strains were susceptible to benzylpenicillin, ampicillin, vancomycin and teicoplanin, and for these antibiotics, there was 100% agreement between the susceptibility results of the VITEK 2 system and those of the diffusion method. VITEK 2 MICs of benzylpenicillin, ampicillin, vancomycin and teicoplanin were
0.12, 0.25, 1 and 0.5 mg/L, respectively. Twenty-five strains (22.7%) exhibited high-level resistance to kanamycin (MIC90 > 1024 mg/L) because of the presence of the aphA-3 gene. High-level resistance to kanamycin was always associated with tetracycline resistance. Strains highly resistant to gentamicin were not seen. No correlation analysis between VITEK 2 and the reference method was performed for aminoglycosides as this class of antibiotics is not included in the AST-P532 card for GBS susceptibility testing.
According to the diffusion technique, 95 (86.5%) strains were resistant to tetracycline and minocycline. Two main mechanisms of resistance to tetracycline have been reported in streptococci and enterococci: efflux by proton antiporters [Tet(L) and Tet(K)] and ribosome protection [Tet(M), Tet(O), Tet(S) and Tet(T)].22 The tet(M) resistance gene was detected in 84 tetracycline/minocycline-resistant strains (88%), tet(O) was detected alone in 10 strains (10.5%) and in association with tet(M) in 5 strains and tet(T) was detected in association with tet(M) in 1 strain. The determinants tet(L), tet(K) and tet(S) were not detected. For tetracycline, a CA of 98.9% (94/95) was found between VITEK 2 and the reference method. One strain harbouring the tet(M) and tet(O) resistance determinants was found intermediate by VITEK 2 (MIC = 2 mg/L) and susceptible by the disc agar diffusion method resulting in an mE.
Eighty-one isolates (73.6%) were resistant to erythromycin by the disc agar diffusion method and different resistance phenotypes were observed: 50 (61.7%) displayed the MLSB-c phenotype, 25 (30.8%) the MLSB-i phenotype and 6 (7.4%) the M phenotype (Table 1). In streptococci, two major mechanisms accounting for resistance to MLS antibiotics are recognized.23 Cross-resistance to all MLSB antibiotics is due to methylation of the 23S rRNA by a methyltransferase encoded by an erm (erythromycin resistance methylase) gene. Resistance to 14- and 15-membered macrolides is mediated by a proton-dependent active drug efflux system encoded by the mef (macrolide efflux) genes. We searched for the presence of sequences related to erm(B), erm(A), erm(C) and mef(A) by PCR. The MLSB resistance phenotype was due to the presence of the genes erm(B) or erm(A), which were distributed differently in strains expressing constitutive [47/50 (94%) and 3/50 (6%), respectively] or inducible [1/25 (4%) and 24/25 (96%), respectively] resistance (Table 1). All strains exhibiting an M phenotype harboured the mef(A) gene (Table 1). The determinant erm(C) was not detected. These results confirm that the genes erm(B) and, to a lesser extent, erm(A) are widely distributed among GBS strains. When susceptibility results of VITEK 2 were compared with those of the reference method and the DD test results, there was 100% CA for the 29 MLS-susceptible isolates. VITEK 2 was able to detect all six M phenotype strains. For these strains, VITEK 2 erythromycin MICs ranged from 2 to 4 mg/L and they were assessed resistant by AES, whereas clindamycin MICs were 0.25 mg/L and they were assessed susceptible. The VITEK 2 system was able to correctly detect MLSB-c isolates as being resistant to erythromycin for 38/50 strains (76%) and to clindamycin for 36/50 (72%). However, it was unable to detect erythromycin and clindamycin resistance (VITEK 2 MICs 0.5 compared with 
128 mg/L by the reference method) in 12 (24%) and 14 (28%) strains, respectively, resulting in VMEs. All these strains harboured the erm(B) resistance determinant. For the 25 MLSB-i strains, the VITEK 2 system had an accuracy of 36% with the DD test. In most cases (16/25), inducible erythromycin resistance was not detected by the VITEK 2 whatever the resistance determinant present in the strain (Table 1). VITEK 2 erythromycin MICs ranged from 0.25 to 8 mg/L when compared with 2–8 mg/L for the agar dilution method. As expected, all strains were susceptible to clindamycin with both methods. In contrast, inducibility of resistance to erythromycin was detected in all cases by the DD test, even when MICs as low as 2 mg/L were observed. The VITEK 2 system incorporates 0.25, 0.5 and 2 mg/L erythromycin and 0.5, 1 and 2 mg/L clindamycin wells into its antimicrobial susceptibility card and calculates the actual MIC by analysing the growth characteristics at those concentrations. A likely explanation for the incapacity of VITEK 2 to detect inducible resistance to erythromycin could be due to the fact that, in the presence of erythromycin at a concentration as low as 0.25 mg/L, growth of bacteria harbouring an inducible erm gene is delayed,24 therefore impairing erythromycin resistance detection in 15 h as it is programmed in the VITEK 2 system. These results are in agreement with a previous work carried out on genotypically uncharacterized strains, suggesting that VITEK 2 was not reliable for the detection of macrolide resistance.19 Similar problems likely exist for erythromycin and clindamycin susceptibility testing with VITEK 2 in other streptococci including Streptococcus pneumoniae (E. Varon and L. Gutmann, personnal communication).
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In vitro studies have shown that clindamycin resistance due to constitutive synthesis of the methylase can be obtained from inducible group A and group G streptococci strains harbouring erm(A) genes by a one-step clindamycin selection used at concentrations 4–40-fold the MIC. The constitutive phenotype is due to mutations that alter the structure of the attenuator controlling the expression of the erm gene.24,25 Such easy selection of mutants in the presence of antibiotics is clinically relevant for clindamycin therapy of infections caused by inducible erythromycin-resistant strains with heavy inoculum. Consistently, clindamycin treatment failure in staphylococcal infections because of the emergence of constitutively resistant mutants has been reported.26 The fact that MLSB-c GBS (resistant to both erythromycin and clindamycin) containing erm(A) exist4,5,8,9,27,28 strongly suggests that these mutants have already been selected in vivo and that they have spread over several years. Thus, detection of MLSB-i resistance is of particular importance. This can be done by the DD test; the presence of a D-shaped zone between erythromycin and clindamycin indicating the MLSB-i phenotype. However, in some cases, this antagonism is barely visible and PCR detection of erm resistance genes should be performed. Recommendations from the scientific authorities such as CLSI or EUCAST for the routine testing and interpretation of clindamycin or MLSB-i phenotype have been done or are in process. Strains that may appear clindamycin-susceptible should be interpreted as resistant to dissuade physicians from treating streptococcal infections with lincosamides.23
Traditionally, macrolides and specifically erythromycin have been considered to be the treatment and prophylaxis against GBS infection in patients allergic to ß-lactams. Owing to the increasing prevalence of MLS resistance in GBS, these antibiotics should no longer be used without susceptibility testing. This strengthens the importance of accurate MLS resistance detection among GBS and this work provides evidence that the DD diffusion test remains the most simple and reliable method for this detection. Although VITEK 2 allows accurate determination of GBS susceptibility for the majority of antibiotics, it has to be improved for macrolide resistance determination in this streptococcal species.
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None to declare.
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Part of this work was presented at the Forty-seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, USA, 2006. We thank Gilles Zambardi from bioMérieux for helpful discussions and Valérie Colas for providing the AST-P532 cards for the VITEK 2 automated system. This work was supported by research funds from INSERM, CNRS, University Paris 5 and Institut Pasteur (GPH No. 9).
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