Identification and susceptibility testing of presumed staphylococci in blood cultures require >24 h by routine phenotypic methods. Delayed appropriate antibiotic therapy results in increased mortality in critically ill patients, while unnecessary vancomycin use may result in suboptimal therapy and unnecessary selection for resistant organisms (1, 6, 10).
We tested the BD GeneOhm StaphS/R assay, a rapid nucleic acid test to differentiate coagulase-negative Staphylococcus (CoNS) isolates from methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA, respectively) isolates, with samples drawn immediately from 145 consecutive positive blood culture bottles (Bactec 9240; BD Systems) which contained gram-positive cocci in clusters visible by microscopy. There were no discordant results between the BD GeneOhm StaphS/R assay, culture, coagulase testing (as previously described), and susceptibility testing (by the CLSI disc diffusion method) in 46 MSSA isolates and 87 CoNS isolates (2, 7). However, only 50% (12/24) of the MRSA isolates identified by culture were correctly identified as MRSA by the BD GeneOhm assay. Genotyping of these by using published typing methods (5) showed that 11 of the 12 isolates corresponded to the predominant Australian nosocomial clone (AUS2/3 clone; ST 239-MRSA-III) and that one corresponded to a community-acquired clone prevalent in eastern Australia (South West Pacific clone; ST 30-MRSA-IV) (9).
We modified a published assay targeting mecA and nuc, including an internal control comprising a unique probe-binding region (based on Drosophila melanogaster; GenBank accession no. AY119135; nt 144 to 175) flanked by binding sites for our nuc primers (11). Using 5 μl of extracted DNA (BioRobot EZ1 QIAamp tissue protocol), this S. aureus methicillin resistance assay (SAM2-PCR) correctly identified 98.6% (216/219) of the S. aureus isolates (including 64/65 culture-proven MRSA isolates) within 2 hours of a positive Gram stain. Incorrect labeling caused two errors, and failed extraction caused another. No true assay failures occurred, with 100% concordance on repeat testing.
SAM2-PCR correctly identified 100% (439/439) of all non-Staphylococcus aureus isolates as being nuc negative (Table 1). In eight methicillin-susceptible CoNS (MSSE) isolates, mecA was detected, while mecA was not detected in 9/274 methicillin-resistant CoNS (MRSE) isolates. These SAM2-PCR results were confirmed as being correct by an independent PCR for all eight MSSE isolates and five of nine MRSE isolates (8). Three of the four remaining discordant specimens had no DNA present (extraction failures), with one assay failure occurring (0.2% [1/658]). Determination of methicillin resistance in CoNS isolates using the SAM2-PCR was therefore more accurate than that using direct susceptibility testing, which was incorrect in 3.2% (13/412).
TABLE 1.
Results of SAM2-PCR compared with those of culture and susceptibility identification
| Isolate identification by culture and susceptibility testing | No. of isolates identified by PCR with indicated resultb
|
|||
|---|---|---|---|---|
| nuc+ mec+ | nuc+ mec− | nuc− mec+ | nuc− mec− | |
| MSSA (n = 154) | 1 | 152 | 0 | 1 |
| MRSA (n = 65) | 64 | 0 | 0 | 1 |
| MSSE (n = 138) | 0 | 0 | 8 | 130 |
| MRSE (n = 274) | 0 | 0 | 265 | 9 |
| Othera (n = 27) | 0 | 0 | 0 | 27 |
Nonstaphylococcal (Micrococcus spp., Streptococcus spp., Enterococcus spp.).
+, positive; −, negative.
The BD GeneOhm StaphS/R assay targets the right extremity (i.e., junctional target) of the SCCmec region. False negatives have been documented secondary to variations in this region, new variants of the SCCmec (type IV and V) and nontypeable strains (3, 4). This approach will always be vulnerable to epidemiological variations. A nuc/mec assay with an internal control to detect inhibition (not observed in our study) seems robust and reliable, outperforming the culture methods in our hands. It may, therefore, be prudent for simple duplex assays to remain the “gold standard” for rapid detection of S. aureus and MRSA from blood culture bottles.
Acknowledgments
We acknowledge Lin Cai for assistance with genotyping of the MRSA isolates and Andrew Ginn for help with the cloning of the internal control.
Footnotes
Published ahead of print on 22 October 2008.
REFERENCES
- 1.Bhorade, S. M., J. Christenson, A. S. Pohlman, P. M. Arnow, and J. B. Hall. 1999. The incidence of and clinical variables associated with vancomycin-resistant enterococcal colonization in mechanically ventilated patients. Chest 1151085-1091. [DOI] [PubMed] [Google Scholar]
- 2.Clinical and Laboratory Standards Institute. 2007. Standards performance standards for antimicrobial susceptibility testing; 17th informational supplement. M100-S17. Clinical and Laboratory Standards Institute, Wayne, PA.
- 3.Francois, P., M. Bento, G. Renzi, S. Harbarth, D. Pittet, and J. Schrenzel. 2007. Evaluation of three molecular assays for rapid identification of methicillin-resistant Staphylococcus aureus. J. Clin. Microbiol. 452011-2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Heusser, R., M. Ender, B. Berger-Bachi, and N. McCallum. 2007. Mosaic staphylococcal cassette chromosome mec containing two recombinase loci and a new mec complex, B2. Antimicrob. Agents Chemother. 51390-393. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Kondo, Y., T. Ito, X. X. Ma, S. Watanabe, B. N. Kreiswirth, J. Etienne, and K. Hiramatsu. 2007. Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr, and major differences in junkyard regions. Antimicrob. Agents Chemother. 51264-274. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Kumar, A., D. Roberts, K. E. Wood, B. Light, J. E. Parrillo, S. Sharma, R. Suppes, D. Feinstein, S. Zanotti, L. Taiberg, D. Gurka, and M. Cheang. 2006. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit. Care Med. 341589-1596. [DOI] [PubMed] [Google Scholar]
- 7.Lee, C. C., W. J. Lin, H. I. Shih, C. J. Wu, P. L. Chen, H. C. Lee, N. Y. Lee, C. M. Chang, L. R. Wang, and W. C. Ko. 2007. Clinical significance of potential contaminants in blood cultures among patients in a medical center. J. Microbiol. Immunol. Infect. 40438-444. [PubMed] [Google Scholar]
- 8.Murakami, K., W. Minamide, K. Wada, E. Nakamura, H. Teraoka, and S. Watanabe. 1991. Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J. Clin. Microbiol. 292240-2244. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Nimmo, G., J. Pearson, P. Collignon, K. Christiansen, G. Coombs, J. Bell, M.-L. McLaws, et al. 2005. Prevalence of MRSA among Staphylococcus aureus isolated from hospital inpatients: report from the Australian Group for Antimicrobial Resistance (AGAR). http://www.health.gov.au/internet/wcms/publishing.nsf/content/cda-cdi3103d.htm. [PubMed]
- 10.Stryjewski, M. E., L. A. Szczech, D. K. Benjamin, Jr., J. K. Inrig, Z. A. Kanafani, J. J. Engemann, V. H. Chu, M. J. Joyce, L. B. Reller, G. R. Corey, and V. G. Fowler, Jr. 2007. Use of vancomycin or first-generation cephalosporins for the treatment of hemodialysis-dependent patients with methicillin-susceptible Staphylococcus aureus bacteremia. Clin. Infect. Dis. 44190-196. [DOI] [PubMed] [Google Scholar]
- 11.Thomas, L. C., H. F. Gidding, A. N. Ginn, T. Olma, and J. Iredell. 2007. Development of a real-time Staphylococcus aureus and MRSA (SAM−) PCR for routine blood culture. J. Microbiol. Methods 68296-302. [DOI] [PubMed] [Google Scholar]