Evaluation of Two New Chromogenic Media, CHROMagar MRSA and S. aureus ID, for Identifying Staphylococcus aureus and Screening Methicillin-Re
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微生物临床杂志 2005年第8期
Department of Clinical Bacteriology, Karolinska University Hospital, Huddinge, Karolinska Institute, Stockholm, Sweden
ABSTRACT
Thirty-nine methicillin-resistant Staphylococcus aureus (MRSA) isolates with diverse genetic backgrounds and two reference strains were correctly identified as S. aureus on CHROMagar MRSA and S. aureus ID media. Growth inhibition on CHROMagar MRSA was noted. A combination of cefoxitin disk and S. aureus ID was found suitable for rapid MRSA screening.
TEXT
Methicillin-resistant Staphylococcus aureus (MRSA) has, during the last 3 decades, evolved as one of the most important causes of hospital infections worldwide. In Sweden, the prevalence of MRSA is still low in comparison with the prevalences in other European countries (14). Since 2000, however, an increasing number of patients carrying MRSA have been identified in Stockholm. To cope with the clinical requirement for rapid MRSA diagnosis, a screening method for MRSA, based on selective broth and real-time PCR, was developed (5). To further improve our diagnostic procedure, two new chromogenic media were investigated in the present study. The aim was to evaluate their accuracy for S. aureus identification and the sensitivity for MRSA isolation. Furthermore, a combination of a cefoxitin disk and S. aureus ID (SAID), one of the new chromogenic media, was developed and evaluated for rapid MRSA screening.
Culture media. S. aureus ID (bioMerieux, Marcy-l'Etoile, France) and CHROMagar MRSA (CHROM agar Microbiology, Paris, France) (CMRSA) were evaluated. SAID was supplied from the manufacturer as prepoured culture plates. CMRSA was produced by adding a proprietary supplement containing a cephamycin antibiotic to dehydrated CHROMagar Staph aureus (CHROM agar Microbiology, Paris, France). Columbia blood agar base (Acumedia, Baltimore, Md.) supplemented with 5% horse blood (blood agar) and mannitol-salt agar (Mast Diagnostics, Bootle, United Kingdom) supplemented with cefoxitin (4.0 μg/ml) (MSA) were used as reference media. For the cefoxitin disk diffusion test, Isosensitest agar (Oxoid Ltd., Basingstoke, United Kingdom) and SAID were used. Enrichment of clinical samples was conducted as previously described (5).
Quantitative growth of MRSA and accuracy of reactions of S. aureus on selective media. Forty-one strains of MRSA were tested, including two culture collection strains, CCUG 31966 and CCUG 46147, and 39 clinical isolates. Each clinical isolate presented a unique pulsed-field gel electrophoresis banding pattern, and from the results of other typing methods, multilocus sequence typing (4) in which 15 types were identified and SCC-mec typing (9) in which all of the SCC-mec types described by Oliveira et al. in 2002 (9) were found, it became evident that MRSA isolates of different genetic lineages were represented. The isolates were stored at –70°C. They were cultured once on blood agar, suspended in phosphate-buffered saline, and diluted to approximately 103 CFU/ml. One hundred microliters of the diluted bacterial solution was spread on blood agar, SAID, and CHROMagar MRSA. The plates were incubated at 37°C and examined after 1 and 2 days of incubation.
All the strains were correctly identified as S. aureus on SAID and CMRSA, i.e., the colonies were green on SAID and mauve on CMRSA. On blood agar and SAID, the numbers of CFU were unchanged between the 1-day and 2-day incubations, but on CMRSA, a rise of approximately 2 to 10 CFU was observed after 2 days of incubation in four cases. For the clinical isolates, the ranges, means, standard deviations, and 95% confidence intervals of the means on the different media after 1 and 2 days of incubation are shown in Table 1. No significant difference was detected on SAID compared with blood agar (P = 0.135; t test of the difference between the means). For CMRSA, however, the number of CFU was, on average, only 34% of the number observed on blood agar, a highly significant difference (P < 0.0001). The percentages of CFU on SAID and CMRSA compared to CFU on blood agar (mean of duplicate tests) were 95% and 48%, respectively, for strain CCUG 31966 (a homogeneous, highly resistant strain) and 75% and 9%, respectively, for CCUG 46147 (a heterogeneous, weakly resistant strain).
MRSA screening of clinical samples. Before the combination assay of cefoxitin disk and SAID medium was applied to clinical samples, the 41 strains of MRSA and 20 strains of methicillin-sensitive S. aureus (clinical isolates) were tested to compare the zones of inhibition on SAID with those on Isosensitest agar. The test was performed as described by the Swedish Reference Group for Antibiotics by use of a 10-μg cefoxitin disk (10, 13). The result is shown in Fig. 1. As seen, the inhibition zones of the two media are comparable and a zone diameter of <22 mm can be used for MRSA screening on both.
A total of 425 consecutive samples for MRSA screening were tested. After the samples were enriched in a selective broth overnight, a PCR assay directed for the nuc gene was conducted to identify samples containing S. aureus. Only nuc-positive broths were subcultured on agar as previously described (5). In the present study, subculture on blood agar and MSA was performed in parallel with subculture on SAID with a cefoxitin disk. The agar plates were inoculated with a plastic loop containing 10 μl broth or, in rare cases in which the broth was not turbid, with a cotton swab. After a dense inoculation of half of the plate, another loop was used for spreading to the other half. A 10-μg cefoxitin disk was placed on the densely inoculated area. The plates were incubated at 37°C and examined after 1 and 2 days. Using blood agar and MSA, the presumptive identification of S. aureus was based on colony morphology. When SAID was used, all green colonies were regarded as presumptive S. aureus. If there was a semiconfluent or confluent growth of green colonies around the cefoxitin disk, the inhibition zone was measured. From each plate, up to 10 CFU were tested for DNase activity and gram staining. DNase-positive, gram-positive cocci were further tested for methicillin resistance with a disk diffusion method using a 10-μg cefoxitin disk on Isosensitest agar as recommended by the Swedish Reference group for Antibiotics (10, 13). Isolates with an inhibition zone of <22 mm were confirmed to be MRSA by a multiplex PCR (1).
The broth PCR for the nuc gene was positive for 76 of the 425 samples (17%). S. aureus was finally identified in 46 of them, and the sensitivity for S. aureus detection was the same on SAID alone as on blood agar together with MSA. Two MRSA isolates were identified.
Green colonies on SAID were found on 55 of the 76 plates. There seemed to be just one strain/plate with a green color in 52 cases and two different strains in 3 cases, giving a total of 58 isolates with green color. The majority, 46, were isolates of S. aureus. Six isolates were identified as Enterococcus spp., three as Micrococcus spp., two as Pseudomonas spp., and one as Stenotrophomonas maltophilia. Thus, in this study, when the culture on SAID was preceded by enrichment in a selective broth for MRSA, the positive predictive value of SAID for identification of S. aureus was 46/58 (79%).
Among the 46 cases in which S. aureus was found, it was possible to read a zone of inhibition at 22 mm in 19 cases but impossible to read any zone of inhibition in 25 cases due to sparse growth. In the two cases in which MRSA isolates were found, the colonies were growing up to the edge of the cefoxitin disk (6 mm).
Chromogenic media for the identification of S. aureus and MRSA. CHROMagar Staph aureus has been evaluated in several studies and a high sensitivity for S. aureus has been reported (2, 6-8, 12). In two studies, 4.0 μg/ml of oxacillin was added to the agar in an attempt to make the medium selective for MRSA (7, 8). In both studies, however, growth inhibition in several MRSA strains was observed. Recently, CMRSA, which is CHROMagar Staph aureus with the addition of a cephamycin antibiotic, was developed. When 216 strains from a Dutch collection of MRSA isolates were cultured on CMRSA, all were correctly identified as S. aureus (3). SAID is another commercially available chromogenic agar medium for the detection of S. aureus. A sensitivity of 98.7% has been reported (12). In a study by Perry et al., CMRSA (M-Tech Diagnostics, Warrington, United Kingdom), SAID supplemented with cefoxitin (4.0 μg/ml), and two other media containing selective antibiotics were evaluated for MRSA (11). With each single medium, >10% of the MRSA could not be detected. The failure may be explained by shortcomings of the selective media in cases where the inoculum amount is low. In accordance to these findings, growth inhibition of MRSA on the selective medium was also observed in our study. Therefore, the application of a cefoxitin disk on a chromogenic medium without antibiotic is an attractive alternative to the selective medium, since the growth inhibition of MRSA on the whole agar surface is avoided.
REFERENCES
Brakstad, O. G., J. A. Maeland, and Y. Tveten. 1993. Multiplex polymerase chain reaction for detection of genes for Staphylococcus aureus thermonuclease and methicillin resistance and correlation with oxacillin resistance. APMIS 101:681-688.
Carricajo, A., A. Treny, N. Fonsale, M. Bes, M. E. Reverdy, Y. Gille, G. Aubert, and A. M. Freydiere. 2001. Performance of the chromogenic medium CHROMagar Staph Aureus and the Staphychrom coagulase test in the detection and identification of Staphylococcus aureus in clinical specimens. J. Clin. Microbiol. 39:2581-2583.
Diederen, B., I. van Duijn, A. van Belkum, P. Willemse, P. van Keulen, and J. Kluytmans. 2005. Performance of CHROMagar MRSA medium for detection of methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 43:2581-2583.
Enright, M. C., N. P. Day, C. E. Davies, S. J. Peacock, and B. G. Spratt. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38:1008-1015.
Fang, H., and G. Hedin. 2003. Rapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay. J. Clin. Microbiol. 41:2894-2899.
Gaillot, O., M. Wetsch, N. Fortineau, and P. Berche. 2000. Evaluation of CHROMagar Staph aureus, a new chromogenic medium, for isolation and presumptive identification of Staphylococcus aureus from human clinical specimens. J. Clin. Microbiol. 38:1587-1591.
Kluytmans, J., A. Van Griethuysen, P. Willemse, and P. Van Keulen. 2002. Performance of CHROMagar selective medium and oxacillin resistance screening agar base for identifying Staphylococcus aureus and detecting methicillin resistance. J. Clin. Microbiol. 40:2480-2482.
Merlino, J., M. Leroi, R. Bradbury, D. Veal, and C. Harbour. 2000. New chromogenic identification and detection of Staphylococcus aureus and methicillin-resistant S. aureus. J. Clin. Microbiol. 38:2378-2380.
Oliveira, D. C., and H. de Lencastre. 2002. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 46:2155-2161.
Olsson-Liljequist, B., P. Larsson, M. Walder, and H. Mirner. 1997. Antimicrobial susceptibility testing in Sweden. III. Methodology for susceptibility testing. Scand. J. Infect. Dis. Suppl. 105:13-23. [Online.] http://www.srga.org.
Perry, J. D., A. Davies, L. A. Butterworth, A. L. Hopley, A. Nicholson, and F. K. Gould. 2004. Development and evaluation of a chromogenic agar medium for methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 42:4519-4523.
Perry, J. D., C. Rennison, L. A. Butterworth, A. L. Hopley, and F. K. Gould. 2003. Evaluation of S. aureus ID, a new chromogenic agar medium for detection of Staphylococcus aureus. J. Clin. Microbiol. 41:5695-5698.
Skov, R., R. Smyth, A. R. Larsen, N. Frimodt-Moller, and G. Kahlmeter. 2005. Evaluation of cefoxitin 5 and 10 μg discs for the detection of methicillin resistance in staphylococci. J. Antimicrob. Chemother. 55:157-161.
Tiemersma, E. W., S. L. Bonzwaer, O. Lyytikainen, J. E. Degener, P. Schrijnemakers, N. Bruinsma, J. Monen, W. Witte, and H. Grundman. 2004. Methicillin-resistant Staphylococcus aureus in Europe, 1999-2002. Emerg. Infect. Dis. 10:1627-1634.(Gran Hedin and Hong Fang)
ABSTRACT
Thirty-nine methicillin-resistant Staphylococcus aureus (MRSA) isolates with diverse genetic backgrounds and two reference strains were correctly identified as S. aureus on CHROMagar MRSA and S. aureus ID media. Growth inhibition on CHROMagar MRSA was noted. A combination of cefoxitin disk and S. aureus ID was found suitable for rapid MRSA screening.
TEXT
Methicillin-resistant Staphylococcus aureus (MRSA) has, during the last 3 decades, evolved as one of the most important causes of hospital infections worldwide. In Sweden, the prevalence of MRSA is still low in comparison with the prevalences in other European countries (14). Since 2000, however, an increasing number of patients carrying MRSA have been identified in Stockholm. To cope with the clinical requirement for rapid MRSA diagnosis, a screening method for MRSA, based on selective broth and real-time PCR, was developed (5). To further improve our diagnostic procedure, two new chromogenic media were investigated in the present study. The aim was to evaluate their accuracy for S. aureus identification and the sensitivity for MRSA isolation. Furthermore, a combination of a cefoxitin disk and S. aureus ID (SAID), one of the new chromogenic media, was developed and evaluated for rapid MRSA screening.
Culture media. S. aureus ID (bioMerieux, Marcy-l'Etoile, France) and CHROMagar MRSA (CHROM agar Microbiology, Paris, France) (CMRSA) were evaluated. SAID was supplied from the manufacturer as prepoured culture plates. CMRSA was produced by adding a proprietary supplement containing a cephamycin antibiotic to dehydrated CHROMagar Staph aureus (CHROM agar Microbiology, Paris, France). Columbia blood agar base (Acumedia, Baltimore, Md.) supplemented with 5% horse blood (blood agar) and mannitol-salt agar (Mast Diagnostics, Bootle, United Kingdom) supplemented with cefoxitin (4.0 μg/ml) (MSA) were used as reference media. For the cefoxitin disk diffusion test, Isosensitest agar (Oxoid Ltd., Basingstoke, United Kingdom) and SAID were used. Enrichment of clinical samples was conducted as previously described (5).
Quantitative growth of MRSA and accuracy of reactions of S. aureus on selective media. Forty-one strains of MRSA were tested, including two culture collection strains, CCUG 31966 and CCUG 46147, and 39 clinical isolates. Each clinical isolate presented a unique pulsed-field gel electrophoresis banding pattern, and from the results of other typing methods, multilocus sequence typing (4) in which 15 types were identified and SCC-mec typing (9) in which all of the SCC-mec types described by Oliveira et al. in 2002 (9) were found, it became evident that MRSA isolates of different genetic lineages were represented. The isolates were stored at –70°C. They were cultured once on blood agar, suspended in phosphate-buffered saline, and diluted to approximately 103 CFU/ml. One hundred microliters of the diluted bacterial solution was spread on blood agar, SAID, and CHROMagar MRSA. The plates were incubated at 37°C and examined after 1 and 2 days of incubation.
All the strains were correctly identified as S. aureus on SAID and CMRSA, i.e., the colonies were green on SAID and mauve on CMRSA. On blood agar and SAID, the numbers of CFU were unchanged between the 1-day and 2-day incubations, but on CMRSA, a rise of approximately 2 to 10 CFU was observed after 2 days of incubation in four cases. For the clinical isolates, the ranges, means, standard deviations, and 95% confidence intervals of the means on the different media after 1 and 2 days of incubation are shown in Table 1. No significant difference was detected on SAID compared with blood agar (P = 0.135; t test of the difference between the means). For CMRSA, however, the number of CFU was, on average, only 34% of the number observed on blood agar, a highly significant difference (P < 0.0001). The percentages of CFU on SAID and CMRSA compared to CFU on blood agar (mean of duplicate tests) were 95% and 48%, respectively, for strain CCUG 31966 (a homogeneous, highly resistant strain) and 75% and 9%, respectively, for CCUG 46147 (a heterogeneous, weakly resistant strain).
MRSA screening of clinical samples. Before the combination assay of cefoxitin disk and SAID medium was applied to clinical samples, the 41 strains of MRSA and 20 strains of methicillin-sensitive S. aureus (clinical isolates) were tested to compare the zones of inhibition on SAID with those on Isosensitest agar. The test was performed as described by the Swedish Reference Group for Antibiotics by use of a 10-μg cefoxitin disk (10, 13). The result is shown in Fig. 1. As seen, the inhibition zones of the two media are comparable and a zone diameter of <22 mm can be used for MRSA screening on both.
A total of 425 consecutive samples for MRSA screening were tested. After the samples were enriched in a selective broth overnight, a PCR assay directed for the nuc gene was conducted to identify samples containing S. aureus. Only nuc-positive broths were subcultured on agar as previously described (5). In the present study, subculture on blood agar and MSA was performed in parallel with subculture on SAID with a cefoxitin disk. The agar plates were inoculated with a plastic loop containing 10 μl broth or, in rare cases in which the broth was not turbid, with a cotton swab. After a dense inoculation of half of the plate, another loop was used for spreading to the other half. A 10-μg cefoxitin disk was placed on the densely inoculated area. The plates were incubated at 37°C and examined after 1 and 2 days. Using blood agar and MSA, the presumptive identification of S. aureus was based on colony morphology. When SAID was used, all green colonies were regarded as presumptive S. aureus. If there was a semiconfluent or confluent growth of green colonies around the cefoxitin disk, the inhibition zone was measured. From each plate, up to 10 CFU were tested for DNase activity and gram staining. DNase-positive, gram-positive cocci were further tested for methicillin resistance with a disk diffusion method using a 10-μg cefoxitin disk on Isosensitest agar as recommended by the Swedish Reference group for Antibiotics (10, 13). Isolates with an inhibition zone of <22 mm were confirmed to be MRSA by a multiplex PCR (1).
The broth PCR for the nuc gene was positive for 76 of the 425 samples (17%). S. aureus was finally identified in 46 of them, and the sensitivity for S. aureus detection was the same on SAID alone as on blood agar together with MSA. Two MRSA isolates were identified.
Green colonies on SAID were found on 55 of the 76 plates. There seemed to be just one strain/plate with a green color in 52 cases and two different strains in 3 cases, giving a total of 58 isolates with green color. The majority, 46, were isolates of S. aureus. Six isolates were identified as Enterococcus spp., three as Micrococcus spp., two as Pseudomonas spp., and one as Stenotrophomonas maltophilia. Thus, in this study, when the culture on SAID was preceded by enrichment in a selective broth for MRSA, the positive predictive value of SAID for identification of S. aureus was 46/58 (79%).
Among the 46 cases in which S. aureus was found, it was possible to read a zone of inhibition at 22 mm in 19 cases but impossible to read any zone of inhibition in 25 cases due to sparse growth. In the two cases in which MRSA isolates were found, the colonies were growing up to the edge of the cefoxitin disk (6 mm).
Chromogenic media for the identification of S. aureus and MRSA. CHROMagar Staph aureus has been evaluated in several studies and a high sensitivity for S. aureus has been reported (2, 6-8, 12). In two studies, 4.0 μg/ml of oxacillin was added to the agar in an attempt to make the medium selective for MRSA (7, 8). In both studies, however, growth inhibition in several MRSA strains was observed. Recently, CMRSA, which is CHROMagar Staph aureus with the addition of a cephamycin antibiotic, was developed. When 216 strains from a Dutch collection of MRSA isolates were cultured on CMRSA, all were correctly identified as S. aureus (3). SAID is another commercially available chromogenic agar medium for the detection of S. aureus. A sensitivity of 98.7% has been reported (12). In a study by Perry et al., CMRSA (M-Tech Diagnostics, Warrington, United Kingdom), SAID supplemented with cefoxitin (4.0 μg/ml), and two other media containing selective antibiotics were evaluated for MRSA (11). With each single medium, >10% of the MRSA could not be detected. The failure may be explained by shortcomings of the selective media in cases where the inoculum amount is low. In accordance to these findings, growth inhibition of MRSA on the selective medium was also observed in our study. Therefore, the application of a cefoxitin disk on a chromogenic medium without antibiotic is an attractive alternative to the selective medium, since the growth inhibition of MRSA on the whole agar surface is avoided.
REFERENCES
Brakstad, O. G., J. A. Maeland, and Y. Tveten. 1993. Multiplex polymerase chain reaction for detection of genes for Staphylococcus aureus thermonuclease and methicillin resistance and correlation with oxacillin resistance. APMIS 101:681-688.
Carricajo, A., A. Treny, N. Fonsale, M. Bes, M. E. Reverdy, Y. Gille, G. Aubert, and A. M. Freydiere. 2001. Performance of the chromogenic medium CHROMagar Staph Aureus and the Staphychrom coagulase test in the detection and identification of Staphylococcus aureus in clinical specimens. J. Clin. Microbiol. 39:2581-2583.
Diederen, B., I. van Duijn, A. van Belkum, P. Willemse, P. van Keulen, and J. Kluytmans. 2005. Performance of CHROMagar MRSA medium for detection of methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 43:2581-2583.
Enright, M. C., N. P. Day, C. E. Davies, S. J. Peacock, and B. G. Spratt. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38:1008-1015.
Fang, H., and G. Hedin. 2003. Rapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay. J. Clin. Microbiol. 41:2894-2899.
Gaillot, O., M. Wetsch, N. Fortineau, and P. Berche. 2000. Evaluation of CHROMagar Staph aureus, a new chromogenic medium, for isolation and presumptive identification of Staphylococcus aureus from human clinical specimens. J. Clin. Microbiol. 38:1587-1591.
Kluytmans, J., A. Van Griethuysen, P. Willemse, and P. Van Keulen. 2002. Performance of CHROMagar selective medium and oxacillin resistance screening agar base for identifying Staphylococcus aureus and detecting methicillin resistance. J. Clin. Microbiol. 40:2480-2482.
Merlino, J., M. Leroi, R. Bradbury, D. Veal, and C. Harbour. 2000. New chromogenic identification and detection of Staphylococcus aureus and methicillin-resistant S. aureus. J. Clin. Microbiol. 38:2378-2380.
Oliveira, D. C., and H. de Lencastre. 2002. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 46:2155-2161.
Olsson-Liljequist, B., P. Larsson, M. Walder, and H. Mirner. 1997. Antimicrobial susceptibility testing in Sweden. III. Methodology for susceptibility testing. Scand. J. Infect. Dis. Suppl. 105:13-23. [Online.] http://www.srga.org.
Perry, J. D., A. Davies, L. A. Butterworth, A. L. Hopley, A. Nicholson, and F. K. Gould. 2004. Development and evaluation of a chromogenic agar medium for methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 42:4519-4523.
Perry, J. D., C. Rennison, L. A. Butterworth, A. L. Hopley, and F. K. Gould. 2003. Evaluation of S. aureus ID, a new chromogenic agar medium for detection of Staphylococcus aureus. J. Clin. Microbiol. 41:5695-5698.
Skov, R., R. Smyth, A. R. Larsen, N. Frimodt-Moller, and G. Kahlmeter. 2005. Evaluation of cefoxitin 5 and 10 μg discs for the detection of methicillin resistance in staphylococci. J. Antimicrob. Chemother. 55:157-161.
Tiemersma, E. W., S. L. Bonzwaer, O. Lyytikainen, J. E. Degener, P. Schrijnemakers, N. Bruinsma, J. Monen, W. Witte, and H. Grundman. 2004. Methicillin-resistant Staphylococcus aureus in Europe, 1999-2002. Emerg. Infect. Dis. 10:1627-1634.(Gran Hedin and Hong Fang)