Journal of Microbiological Methods 112 (2015) 46–48
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Evaluation of an immunochromatographic assay for detection of PBP2a on non-Staphylococcus aureus clinical isolates Benoit Mantion a, Laurent Cavalié a, Marie-Françoise Prère a,b,c,⁎ a b c
Laboratoire de Bactériologie-Hygiène, Chu-Purpan, Toulouse, France Faculté de médecine, Université Paul Sabatier, Toulouse, France CNRS UMR5100, Université Paul Sabatier, Toulouse, France
a r t i c l e
i n f o
Article history: Received 19 February 2015 Received in revised form 26 February 2015 Accepted 27 February 2015 Available online 11 March 2015
a b s t r a c t We evaluated the immunochromatographic assay PBP2a Culture Colony Test (Alere™) on 50 samples of methicillin resistant and sensitive non-Staphylococcus aureus isolates belonging to ten species. Because it is rapid and reliable, this test should be advantageous as routine test in place of mecA/C PCR. © 2015 Elsevier B.V. All rights reserved.
Keywords: Non-Staphylococcus aureus Methicillin PBP2a mecA/C Immunochromatographic assay
Non-Staphylococcus aureus (nSa) isolates from human infections are more and more frequent especially in hospital infections and present a larger antibiotic multi-resistance than S. aureus (Sa). They are responsible of a wide range of infections depending on species. That implicates, for clinical laboratories, to possess a rapid and robust method of methicillin resistance detection to help clinicians in starting up the treatment. The conventional disk diffusion method is simple. However, especially for detecting methicillin resistance, the results of these culture tests may be influenced by variations in the incubation time, pH and salt concentrations of media (Velasco et al., 2005). Furthermore this conventional method requires at least 24 h to provide definitive susceptibility results, so the appropriate antibiotic treatment may be delayed. The most frequent mechanism for methicillin resistance in Staphylococcus results from acquisition of the mecA gene that is harbored by a mobile genetic element, the chromosomal SCCmec cassette. The mecA gene has been first reported in Staphylococcus fleurettii, a commensal bacterium of animals (Tsubakishita et al., 2010). SSCmec has then been found in a large variety of S. species. More recently, a variant of the mecA gene, known as mecC (previously mecALGA251), was identified in isolates of S. aureus from both animals and humans (García-Álvarez et al., 2011). The mecA/C gene codes for the exogenous penicillinbinding protein PBP2a which cannot bind methicillin and allows ⁎ Corresponding author at: Laboratoire de Bactériologie-Hygiène, Chu-Purpan, 31059 Toulouse cedex9, France. E-mail address: [email protected] (M.-F. Prère).
http://dx.doi.org/10.1016/j.mimet.2015.02.014 0167-7012/© 2015 Elsevier B.V. All rights reserved.
Staphylococcus to grow in the presence of betalactams (Pinho et al., 2001). So, a test based on PBP2a detection offers pertinent and early information. The immuno-chromatographic (ICA) assay Alere™ PBP2a Culture Colony Test previously named Clearview®PBP2a Exact Test Evaluation (Inverness Medical Innovations, Scarborough, MA) has been evaluated on various collections of S. aureus isolates with excellent results in terms of specificity and sensibility. Whatever the level of methicillin resistance, the results of the PBP2a test were in complete agreement with the results of the mecA gene PCR test (Nonhoff et al., 2012). The aim of the present study was to evaluate the performance of the commercially available ICA PBP2a Culture Colony Test (Alere®) on colonies of nSa. The 50 Staphylococcus isolates for testing included the most frequent species of non-S. aureus found in sepsis in our hospital: Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus capitis, Staphylococcus hominis, Staphylococcus warneri, Staphylococcus cohnii, Staphylococcus lugdunensis, Staphylococcus intermedius, Staphylococcus saprophyticus, and Staphylococcus auricularis. Half (25/50) were methicillin resistant by phenotypic detection. Species identification was performed first using Maldi-Tof mass spectrometry (microflex™ LT, BrukerDaltonics Inc.). When species determination was negative or imprecise, identification was carried out by sequencing a fragment of 16S ribosomal RNA rrs gene (Prère and Fayet, 2011). The phenotypic test for methicillin resistance was performed using the agar disk diffusion method as recommended by guidelines (Société Française de Microbiogie, 2014) with 5 μg oxacillin and 30 μg cefoxitin disks (OXOID®) on Mueller Hinton agar (MHE bioMérieux®). Genotypic
B. Mantion et al. / Journal of Microbiological Methods 112 (2015) 46–48
detection of methicillin resistance was performed using a PCR method for detection of the both mecA and mecC genes (MRSA HainLife science®) (Prère et al., 2006). The PBP2a test is an immunochromatographic membrane assay based on a nitrocellulose strip containing a monoclonal PBP2a antibody and a control antibody fixed on two distinct lines. The test was performed according to the manufacturer's instructions. For each strain, colonies were tested from both Columbia Agar with 5% sheep blood (COLS+ OXOID®) and Mueller Hinton agar (MHE bioMérieux®). Results were read visually at 5 min. We investigated in this study ten species of nSa. For each species, we tested methicillin resistant and methicillin susceptible strains. The repartition of strains among the different species and according to resistance or sensitivity reflected the observed clinical frequency in our hospital. For example, S. haemolyticus and S. epidermidis strains were more frequently resistant and conversely, S. lugdunensis strains were more often sensitive (Mateo et al., 2005). We obtained the same results for the ICA PBP2a test from colonies growing on the two media used. Whatever the species of nSa, the results of PBP2a tests were identical to those of mecA/C PCR detection (Table 1) and to the phenotypic categorization in 100% of the cases. Detection of mec genes and that of the PBP2a protein are regarded as gold standards for the diagnosis of methicillin resistance. This is indeed the predominant mechanism responsible for methicillin resistance in Staphylococcus. But in S. aureus species other types of resistance to beta-lactams have been described, resulting in strains known as borderline oxacillin-resistant S. aureus (BORSA) or intrinsically resistant S. aureus. Resistance in most of BORSA isolates is attributed to the hyperproduction of a beta-lactamase (Gál et al., 2001). Intrinsic beta-lactam resistance was found correlated to increased level of production of PBP4 due to mutation in the promoter of the pbp4 gene (Henze and Berger-Bächi, 1995). Another poorly known mechanism of resistance is a small multidrug resistance pump. Some isolates of S. aureus and S. epidermidis possess the qacC gene. QacC, the qacC gene product, functions as a proton gradientdependent pump and confers resistance to some antiseptics, to ethidium bromide and to a number of beta-lactam antibiotics (Fuentes et al., 2005). Recently Ba and coworkers (Ba et al., 2014) identified methicillin resistant Sa (MRSA) isolates that lacked mecA or mecC genes but were still phenotypically resistant to penicillinase-resistant beta-lactam antibiotics. A number of amino acid substitutions present in the endogenous PBPs 1, 2 and 3 were found in these resistant isolates. In summary, it is clear that there are multiple distinct mechanisms for beta-lactam resistance in Staphylococcus; so mec-negative isolates with resistance to multiple beta-lactams may be misclassified as methicillin susceptible based on negative detection of mecA/C or PBP2a. That needs to be taken
Table 1 Results of PCR mecA/C and test PBP2a on the two groups of strains in function of the phenotypic determination of methicillin resistance. Species
S. S. S. S. S. S. S. S. S. S.
epidermidis haemolyticus hominis capitis warneri cohnii lugdunensis intermedius saprophyticus auricularis
Methicillin resistant strains
Methicillin sensitive strains
Nb tested
mecA/C
PBP2a
Nb tested
mecA/C
PBP2a
8 4 2 2 3 2 1 1 1 1
+ + + + + + + + + +
+ + + + + + + + + +
2 2 3 3 2 3 6 2 1 1
− − − − − − − − − −
− − − − − − − − − −
Nb tested: number of strains tested in each species. mecA/C: PCR determination. PBP2a: Alere™ test.
47
into consideration by diagnostic laboratories because it could potentially lead to treatment failure. The clinical performance of the Alere™ PBP2a Culture Colony Test was established in a multi-center study in 2009 with expected results on strains of MRSA and methicillin susceptible Sa (manufacturer's information). In a more recent study the authors reported a 100% positive predictive value of methicillin resistance in Sa strains (Nonhoff et al., 2012). However, there was no published report of testing on nonS. aureus isolates. In view of the increasing number of human infections due to nSa, especially in immuno-compromised patients, obtaining presumptive information rapidly about the methicillin resistance status is an important issue. The results of our present study indicated that the Alere™ test is highly sensitive for the identification of methicillin resistance due to PBP2a in various species of Staphylococcus found in human infections. It is rapid, reliable, easily performed and simple to interpret; this makes it very useful especially in cases of bacteremia. Moreover, this test can help to detect methicillin resistant nSa in cultures of clinical samples from skin, soft tissue and osteoarticular infections. Indeed, different species with the same aspect or the same species with different antibiotypes can coexist in such specimens. Therefore we propose that the test should be accepted as routine test in place of the mecA/C PCR test. Conflict of interest All authors declare that they have no conflict of interest. Acknowledgments We would like to express our gratitude to Olivier Fayet for the critical reading of the manuscript. This work was presented in part at the 34th Reunion Interdisciplinaire de Chimiotherapie anti-infectieuse, Paris, France, 2014. References Ba, X., Harrison, E.M., Edwards, G.F., Holden, M.T., Larsen, A.R., Petersen, A., Skov, R.L., Peacock, S.J., Parkhill, J., Paterson, G.K., Holmes, M.A., 2014. Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene. J. Antimicrob. Chemother. 69, 594–597. Fuentes, D.E., Navarro, C.A., Tantaleán, J.C., Araya, M.A., Saavedra, C.P., Pérez, J.M., Calderón, I.L., Youderian, P.A., Mora, G.C., Vásquez, C.C., 2005. The product of the qacC gene of Staphylococcus epidermidis CH mediates resistance to betalactam antibiotics in gram-positive and gram-negative bacteria. Res. Microbiol. 156, 472–477. Gál, Z., Kovács, P., Hernádi, F., Barabás, G., Kiss, L., Iglói, A., Szabó, I., 2001. Investigation of oxacillin-hydrolyzing beta-lactamase in borderline methicillin-resistant clinical isolates of Staphylococcus aureus. Chemotherapy 47, 233–238. García-Álvarez, L., Holden, M.T., Lindsay, H., Webb, C.R., Brown, D.F., Curran, M.D., Walpole, E., Brooks, K., Pickard, D.J., Teale, C., Parkhill, J., Bentley, S.D., Edwards, G.F., Girvan, E.K., Kearns, A.M., Pichon, B., Hill, R.L., Larsen, A.R., Skov, R.L., Peacock, S.J., Maskell, D.J., Holmes, M.A., 2011. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect. Dis. 11, 595–603. Henze, U.U., Berger-Bächi, B., 1995. Staphylococcus aureus penicillin-binding protein 4 and intrinsic beta-lactam resistance. Antimicrob. Agents Chemother. 39, 2415–2422. Mateo, M., Maestre, J.R., Aguilar, L., Cafini, F., Puente, P., Sánchez, P., Alou, L., Giménez, M.J., Prieto, J., 2005. Genotypic versus phenotypic characterization, with respect to susceptibility and identification, of 17 clinical isolates of Staphylococcus lugdunensis. J. Antimicrob. Chemother. 56, 287–291. Nonhoff, C., Roisin, S., Hallin, M., Denis, O., 2012. Evaluation of Clearview Exact PBP2a, a new immunochromatographic assay, for detection of low-level methicillin-resistant Staphylococcus aureus (LL-MRSA). J. Clin. Microbiol. 50, 3359–3360. Pinho, M.G., de Lencastre, H., Tomasz, A., 2001. An acquired and a native penicillinbinding protein cooperate in building the cell wall of drug-resistant staphylococci. Proc. Natl. Acad. Sci. U. S. A. 98, 10886–10891. Prère, M.F., Fayet, O.A., 2011. A specific polymerase chain reaction test for the identification of Streptococcus pneumoniae. Diagn. Microbiol. Infect. Dis. 70, 45–53. Prère, M.F., Baron, O., Cohen, Bacrie S., Fayet, O., 2006. Genotype MRSA, a new genetic test for the rapid identification of staphylococci and detection of mecA gene. Pathol. Biol. (Paris) 54, 502–505.
48
B. Mantion et al. / Journal of Microbiological Methods 112 (2015) 46–48
Société Française de Microbiologie, 2014. Comite de l'Antibiogramme — Recommandations. www.sfm-microbiologie.org (Edition de Mai 2014). Tsubakishita, S., Kuwahara-Arai, K., Sasaki, T., Hiramatsu, K., 2010. Origin and molecular evolution of the determinant of methicillin resistance in staphylococci. Antimicrob. Agents Chemother. 54, 4352–4359.
Velasco, D., del Mar Tomas, M., Cartelle, M., Beceiro, A., Perez, A., Molina, F., Moure, R., Villanueva, R., Bou, G., 2005. Evaluation of different methods for detecting methicillin (oxacillin) resistance in Staphylococcus aureus. J. Antimicrob. Chemother. 55, 379–382.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Evaluation of an immunochromatographic assay for detection of PBP2a on non-Staphylococcus aureus clinical isolates Benoit Mantion a, Laurent Cavalié a, Marie-Françoise Prère a,b,c,⁎ a b c
Laboratoire de Bactériologie-Hygiène, Chu-Purpan, Toulouse, France Faculté de médecine, Université Paul Sabatier, Toulouse, France CNRS UMR5100, Université Paul Sabatier, Toulouse, France
a r t i c l e
i n f o
Article history: Received 19 February 2015 Received in revised form 26 February 2015 Accepted 27 February 2015 Available online 11 March 2015
a b s t r a c t We evaluated the immunochromatographic assay PBP2a Culture Colony Test (Alere™) on 50 samples of methicillin resistant and sensitive non-Staphylococcus aureus isolates belonging to ten species. Because it is rapid and reliable, this test should be advantageous as routine test in place of mecA/C PCR. © 2015 Elsevier B.V. All rights reserved.
Keywords: Non-Staphylococcus aureus Methicillin PBP2a mecA/C Immunochromatographic assay
Non-Staphylococcus aureus (nSa) isolates from human infections are more and more frequent especially in hospital infections and present a larger antibiotic multi-resistance than S. aureus (Sa). They are responsible of a wide range of infections depending on species. That implicates, for clinical laboratories, to possess a rapid and robust method of methicillin resistance detection to help clinicians in starting up the treatment. The conventional disk diffusion method is simple. However, especially for detecting methicillin resistance, the results of these culture tests may be influenced by variations in the incubation time, pH and salt concentrations of media (Velasco et al., 2005). Furthermore this conventional method requires at least 24 h to provide definitive susceptibility results, so the appropriate antibiotic treatment may be delayed. The most frequent mechanism for methicillin resistance in Staphylococcus results from acquisition of the mecA gene that is harbored by a mobile genetic element, the chromosomal SCCmec cassette. The mecA gene has been first reported in Staphylococcus fleurettii, a commensal bacterium of animals (Tsubakishita et al., 2010). SSCmec has then been found in a large variety of S. species. More recently, a variant of the mecA gene, known as mecC (previously mecALGA251), was identified in isolates of S. aureus from both animals and humans (García-Álvarez et al., 2011). The mecA/C gene codes for the exogenous penicillinbinding protein PBP2a which cannot bind methicillin and allows ⁎ Corresponding author at: Laboratoire de Bactériologie-Hygiène, Chu-Purpan, 31059 Toulouse cedex9, France. E-mail address: [email protected] (M.-F. Prère).
http://dx.doi.org/10.1016/j.mimet.2015.02.014 0167-7012/© 2015 Elsevier B.V. All rights reserved.
Staphylococcus to grow in the presence of betalactams (Pinho et al., 2001). So, a test based on PBP2a detection offers pertinent and early information. The immuno-chromatographic (ICA) assay Alere™ PBP2a Culture Colony Test previously named Clearview®PBP2a Exact Test Evaluation (Inverness Medical Innovations, Scarborough, MA) has been evaluated on various collections of S. aureus isolates with excellent results in terms of specificity and sensibility. Whatever the level of methicillin resistance, the results of the PBP2a test were in complete agreement with the results of the mecA gene PCR test (Nonhoff et al., 2012). The aim of the present study was to evaluate the performance of the commercially available ICA PBP2a Culture Colony Test (Alere®) on colonies of nSa. The 50 Staphylococcus isolates for testing included the most frequent species of non-S. aureus found in sepsis in our hospital: Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus capitis, Staphylococcus hominis, Staphylococcus warneri, Staphylococcus cohnii, Staphylococcus lugdunensis, Staphylococcus intermedius, Staphylococcus saprophyticus, and Staphylococcus auricularis. Half (25/50) were methicillin resistant by phenotypic detection. Species identification was performed first using Maldi-Tof mass spectrometry (microflex™ LT, BrukerDaltonics Inc.). When species determination was negative or imprecise, identification was carried out by sequencing a fragment of 16S ribosomal RNA rrs gene (Prère and Fayet, 2011). The phenotypic test for methicillin resistance was performed using the agar disk diffusion method as recommended by guidelines (Société Française de Microbiogie, 2014) with 5 μg oxacillin and 30 μg cefoxitin disks (OXOID®) on Mueller Hinton agar (MHE bioMérieux®). Genotypic
B. Mantion et al. / Journal of Microbiological Methods 112 (2015) 46–48
detection of methicillin resistance was performed using a PCR method for detection of the both mecA and mecC genes (MRSA HainLife science®) (Prère et al., 2006). The PBP2a test is an immunochromatographic membrane assay based on a nitrocellulose strip containing a monoclonal PBP2a antibody and a control antibody fixed on two distinct lines. The test was performed according to the manufacturer's instructions. For each strain, colonies were tested from both Columbia Agar with 5% sheep blood (COLS+ OXOID®) and Mueller Hinton agar (MHE bioMérieux®). Results were read visually at 5 min. We investigated in this study ten species of nSa. For each species, we tested methicillin resistant and methicillin susceptible strains. The repartition of strains among the different species and according to resistance or sensitivity reflected the observed clinical frequency in our hospital. For example, S. haemolyticus and S. epidermidis strains were more frequently resistant and conversely, S. lugdunensis strains were more often sensitive (Mateo et al., 2005). We obtained the same results for the ICA PBP2a test from colonies growing on the two media used. Whatever the species of nSa, the results of PBP2a tests were identical to those of mecA/C PCR detection (Table 1) and to the phenotypic categorization in 100% of the cases. Detection of mec genes and that of the PBP2a protein are regarded as gold standards for the diagnosis of methicillin resistance. This is indeed the predominant mechanism responsible for methicillin resistance in Staphylococcus. But in S. aureus species other types of resistance to beta-lactams have been described, resulting in strains known as borderline oxacillin-resistant S. aureus (BORSA) or intrinsically resistant S. aureus. Resistance in most of BORSA isolates is attributed to the hyperproduction of a beta-lactamase (Gál et al., 2001). Intrinsic beta-lactam resistance was found correlated to increased level of production of PBP4 due to mutation in the promoter of the pbp4 gene (Henze and Berger-Bächi, 1995). Another poorly known mechanism of resistance is a small multidrug resistance pump. Some isolates of S. aureus and S. epidermidis possess the qacC gene. QacC, the qacC gene product, functions as a proton gradientdependent pump and confers resistance to some antiseptics, to ethidium bromide and to a number of beta-lactam antibiotics (Fuentes et al., 2005). Recently Ba and coworkers (Ba et al., 2014) identified methicillin resistant Sa (MRSA) isolates that lacked mecA or mecC genes but were still phenotypically resistant to penicillinase-resistant beta-lactam antibiotics. A number of amino acid substitutions present in the endogenous PBPs 1, 2 and 3 were found in these resistant isolates. In summary, it is clear that there are multiple distinct mechanisms for beta-lactam resistance in Staphylococcus; so mec-negative isolates with resistance to multiple beta-lactams may be misclassified as methicillin susceptible based on negative detection of mecA/C or PBP2a. That needs to be taken
Table 1 Results of PCR mecA/C and test PBP2a on the two groups of strains in function of the phenotypic determination of methicillin resistance. Species
S. S. S. S. S. S. S. S. S. S.
epidermidis haemolyticus hominis capitis warneri cohnii lugdunensis intermedius saprophyticus auricularis
Methicillin resistant strains
Methicillin sensitive strains
Nb tested
mecA/C
PBP2a
Nb tested
mecA/C
PBP2a
8 4 2 2 3 2 1 1 1 1
+ + + + + + + + + +
+ + + + + + + + + +
2 2 3 3 2 3 6 2 1 1
− − − − − − − − − −
− − − − − − − − − −
Nb tested: number of strains tested in each species. mecA/C: PCR determination. PBP2a: Alere™ test.
47
into consideration by diagnostic laboratories because it could potentially lead to treatment failure. The clinical performance of the Alere™ PBP2a Culture Colony Test was established in a multi-center study in 2009 with expected results on strains of MRSA and methicillin susceptible Sa (manufacturer's information). In a more recent study the authors reported a 100% positive predictive value of methicillin resistance in Sa strains (Nonhoff et al., 2012). However, there was no published report of testing on nonS. aureus isolates. In view of the increasing number of human infections due to nSa, especially in immuno-compromised patients, obtaining presumptive information rapidly about the methicillin resistance status is an important issue. The results of our present study indicated that the Alere™ test is highly sensitive for the identification of methicillin resistance due to PBP2a in various species of Staphylococcus found in human infections. It is rapid, reliable, easily performed and simple to interpret; this makes it very useful especially in cases of bacteremia. Moreover, this test can help to detect methicillin resistant nSa in cultures of clinical samples from skin, soft tissue and osteoarticular infections. Indeed, different species with the same aspect or the same species with different antibiotypes can coexist in such specimens. Therefore we propose that the test should be accepted as routine test in place of the mecA/C PCR test. Conflict of interest All authors declare that they have no conflict of interest. Acknowledgments We would like to express our gratitude to Olivier Fayet for the critical reading of the manuscript. This work was presented in part at the 34th Reunion Interdisciplinaire de Chimiotherapie anti-infectieuse, Paris, France, 2014. References Ba, X., Harrison, E.M., Edwards, G.F., Holden, M.T., Larsen, A.R., Petersen, A., Skov, R.L., Peacock, S.J., Parkhill, J., Paterson, G.K., Holmes, M.A., 2014. Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene. J. Antimicrob. Chemother. 69, 594–597. Fuentes, D.E., Navarro, C.A., Tantaleán, J.C., Araya, M.A., Saavedra, C.P., Pérez, J.M., Calderón, I.L., Youderian, P.A., Mora, G.C., Vásquez, C.C., 2005. The product of the qacC gene of Staphylococcus epidermidis CH mediates resistance to betalactam antibiotics in gram-positive and gram-negative bacteria. Res. Microbiol. 156, 472–477. Gál, Z., Kovács, P., Hernádi, F., Barabás, G., Kiss, L., Iglói, A., Szabó, I., 2001. Investigation of oxacillin-hydrolyzing beta-lactamase in borderline methicillin-resistant clinical isolates of Staphylococcus aureus. Chemotherapy 47, 233–238. García-Álvarez, L., Holden, M.T., Lindsay, H., Webb, C.R., Brown, D.F., Curran, M.D., Walpole, E., Brooks, K., Pickard, D.J., Teale, C., Parkhill, J., Bentley, S.D., Edwards, G.F., Girvan, E.K., Kearns, A.M., Pichon, B., Hill, R.L., Larsen, A.R., Skov, R.L., Peacock, S.J., Maskell, D.J., Holmes, M.A., 2011. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect. Dis. 11, 595–603. Henze, U.U., Berger-Bächi, B., 1995. Staphylococcus aureus penicillin-binding protein 4 and intrinsic beta-lactam resistance. Antimicrob. Agents Chemother. 39, 2415–2422. Mateo, M., Maestre, J.R., Aguilar, L., Cafini, F., Puente, P., Sánchez, P., Alou, L., Giménez, M.J., Prieto, J., 2005. Genotypic versus phenotypic characterization, with respect to susceptibility and identification, of 17 clinical isolates of Staphylococcus lugdunensis. J. Antimicrob. Chemother. 56, 287–291. Nonhoff, C., Roisin, S., Hallin, M., Denis, O., 2012. Evaluation of Clearview Exact PBP2a, a new immunochromatographic assay, for detection of low-level methicillin-resistant Staphylococcus aureus (LL-MRSA). J. Clin. Microbiol. 50, 3359–3360. Pinho, M.G., de Lencastre, H., Tomasz, A., 2001. An acquired and a native penicillinbinding protein cooperate in building the cell wall of drug-resistant staphylococci. Proc. Natl. Acad. Sci. U. S. A. 98, 10886–10891. Prère, M.F., Fayet, O.A., 2011. A specific polymerase chain reaction test for the identification of Streptococcus pneumoniae. Diagn. Microbiol. Infect. Dis. 70, 45–53. Prère, M.F., Baron, O., Cohen, Bacrie S., Fayet, O., 2006. Genotype MRSA, a new genetic test for the rapid identification of staphylococci and detection of mecA gene. Pathol. Biol. (Paris) 54, 502–505.
48
B. Mantion et al. / Journal of Microbiological Methods 112 (2015) 46–48
Société Française de Microbiologie, 2014. Comite de l'Antibiogramme — Recommandations. www.sfm-microbiologie.org (Edition de Mai 2014). Tsubakishita, S., Kuwahara-Arai, K., Sasaki, T., Hiramatsu, K., 2010. Origin and molecular evolution of the determinant of methicillin resistance in staphylococci. Antimicrob. Agents Chemother. 54, 4352–4359.
Velasco, D., del Mar Tomas, M., Cartelle, M., Beceiro, A., Perez, A., Molina, F., Moure, R., Villanueva, R., Bou, G., 2005. Evaluation of different methods for detecting methicillin (oxacillin) resistance in Staphylococcus aureus. J. Antimicrob. Chemother. 55, 379–382.
