Simultaneous species identification and detection of methicillin resistance in staphylococci using triplex real-time PCR assay

Diagnostic Microbiology and Infectious Disease 56 (2006) 13 – 18 www.elsevier.com/locate/diagmicrobio

Simultaneous species identification and detection of methicillin resistance in staphylococci using triplex real-time PCR assay Negar Shafiei Sabet, Geetha Subramaniam, Parasakthi Navaratnam, Shamala Devi Sekaran4 Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia Received 6 January 2006; accepted 28 February 2006

Abstract For rapid identification of methicillin-resistant Staphylococcus aureus, molecular methods are generally targeting mecA and speciesspecific genes. Sa442 DNA fragment is a popular species-specific target. However, recently, there have been few reports on S. aureus isolates that are negative for Sa442 fragment; therefore, use of single gene or DNA-fragment–specific polymerase chain reaction (PCR) for identification of microbial isolate may result in misidentification. This study includes CoA gene in parallel with Sa442 marker for identification of S. aureus. This further improves the specificity of the assay by checking for 2 determinants simultaneously for the identification of S. aureus and can prevent misidentification of S. aureus isolates lacking Sa442 DNA fragment. In this study, the newly developed triplex real-time PCR assay was compared with a quadruplex conventional gel-based PCR assay using the same primer sets in both assays. The dual-labeled TaqMan probes (ProOligo, France) for these primers were specifically designed and used in a real-time PCR assay. The clinical isolates (n = 152) were subjected to both PCR assays. The results obtained from both assays proved that the primer and probe sets were 100% sensitive and 100% specific for identification of S. aureus and detection of methicillin resistance. This triplex real-time PCR assay represents a rapid and powerful method for S. aureus identification and detection of methicillin resistance. D 2006 Elsevier Inc. All rights reserved.

1. Introduction Staphylococcus aureus is well known as a major pathogen, causing a variety of nosocomial and communityacquired infections (Karchmer, 2000; Hiramatsu et al., 2002). Coagulase production is the principle criterion used in the clinical microbiology laboratory for the identification of S. aureus. The coagulase status of an isolate is not always easily established in a timely fashion, increasing delay in definitive identification of S. aureus (Schmitz et al., 1997). Because methicillin resistance mediated by PBP2a (PBP2V) is often heterogeneously expressed in staphylococci, polymerase chain reaction (PCR) detection of mecA gene is more accurate than the standard susceptibility methods, especially for coagulase negative staphylococci (CoNS) and S. aureus isolates with low-level resistance due to other mechanisms (Carroll et al., 1996). Molecular methods for rapid identification of methicillin-resistant S. aureus

4 Corresponding author. Tel.: +60-3-79675759; fax: +60-3-79676672. E-mail address: [email protected] (S.D. Sekaran). 0732-8893/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2006.02.013

(MRSA) are generally based on detection of mecA gene and S. aureus species-specific gene (Unal et al., 1992). There are several genes that have been used to identify S. aureus (e.g., nuc, gyrA, femA, and coa) (Emori and Gaynes, 1993; Kearns et al., 1999; Martineau et al., 1998; Phonimdaeng et al., 1990; Zambardi et al., 1994). Sa442 DNA fragment, originally described by Martineau et al. (1998), is a popular DNA target for identification of S. aureus by PCR (Grisold et al., 2002; Reischl et al., 2000; Shrestha et al., 2002; Tan et al., 2001). Several PCRbased assays targeting this DNA fragment alone or in combination with a mecA for identification of MRSA have been described (Martineau et al., 1998; Reischl et al., 2000). However, recently, there are few reports on S. aureus isolates that are negative for the Sa442 fragment (Klaassen et al., 2003; Su¨tterlin et al., 2003); these results proved that the use of single gene in the PCR assay for species identification may result in misidentification (Klaassen et al., 2003). Because S. aureus resembles CoNS on visual examination of agar plates and the coagulase status of an isolate is not always easily established in a timely fashion, the inclusion of Coa gene that can be used as a marker for

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Table 1 Characteristics of the primer used in this study Primer name

Gene

Sequence

5V position

Length (bp)

Sa-1 forward Sa-2 reverse Sa-3 forward Sa-4 reverse Sa442-F forward Sa442-R reverse X-F forward Y-R reverse

mecA mecA Coa Coa Sa442 Sa442 16Sr RNA 16Sr RNA

CGG TAA CAT TGA TCG CAA CGT TCA (sense strand) CTT TGG AAC GAT GCC TAA TCT CAT (antisense strand) GTA GAT TGG GCA ATT ACA TTT TGG AGG (sense strand) CGC ATC TGC TTT GTT ATC CCA TGT A (antisense strand) TCG GTA CAC GAT ATT CTT CAC (sense strand) ACT CTC GTA TGA CCA GCT TC (antisense strand) GGA ATT CAA A[T/G, 1:1] G AAT TGA CGG GGG C (sense strand) CGG GAT CCC AGG CCC GGG AAC GTA TTC AC (antisense strand)

315 528 82 160 13 192 911 1371

24 21 27 25 21 20 25 27

identification of S. aureus along with Sa442 fragment is favorable. This has the advantage of improving specificity of the assay by checking for 2 determinants simultaneously for the identification of S. aureus and preventing misidentification of isolates lacking Sa442 fragment. Therefore, the Coa gene was included into the assays for the correct and reliable identification of the S. aureus. In the first part of the study, quadruplex PCR assay that targets 4 genes—mecA (methicillin-resistant determinant), Coa (pathognomonic for S. aureus), Sa442 fragment (species-specific marker), and 16S rRNA (internal control)—was developed. In the second part of the study, a triplex real-time PCR assay was developed using 3 sets of primers that have been evaluated in the first part of the study. The TaqMan probes (ProOligo, France) were designed specifically for each set of primers. TaqMan probes were labeled with different fluorophores, which can be detected at different wavelength. This will further increase the specificity of the PCR assay. Use of differently colored fluorescent labels allows simultaneous detection of different products within a reaction. The objective of this study is to develop multiplex PCR assays that will enable simultaneous species confirmation and detection of methicillin resistance while determining the coagulase status of staphylococci isolates in a single rapid test. 2. Materials and methods 2.1. Bacterial strains The strains used in this study includes 9 ATCC strains and 152 staphylococcal clinical isolates. ATCC strains used as reference strains were ATCC 14990 Staphylococcus epidermidis, ATCC 15305 Staphylococcus saprophyticus, ATCC 35663 Staphylococcus xylosus, ATCC 27840 Staphylococcus capitis subsp. capitis, ATCC 43809 Staphylococcus lugdunensis, ATCC 29970 Staphylococcus

haemolyticus, ATCC 27844 Staphylococcus hominis subsp. hominis, and ATCC 43300 S. aureus (MRSA). Clinical isolates were obtained from the University of Malaya Medical Center (Kuala Lumpur, Malaysia). Among the 152 clinical isolates investigated, there were 48 MRSAs, 45 methicillin-sensitive S. aureus (MSSA), 48 methicillinresistant coagulase negative strains (MRCoNs), and 11 methicillin-sensitive coagulase negative strains (MSCoNs). 2.2. Staphylococci identification and antimicrobial susceptibility testing The staphylococci isolates used in this study were identified as S. aureus or CoNS by colony morphology, Gram stain characteristics, catalase reaction, coagulase production, and the result of API Staph. System (bioMe´rieux, France). Resistance to methicillin was detected using the method recommended by the National Committee for Clinical Laboratory Standards (Wayne, PA). Disk diffusion was performed on Mueller–Hinton agar using Kirby–Bauer method (Oxoid, Basingstoke, UK), with 1 Ag of oxacillin discs (Oxoid). MICs of methicillin were determined by agar dilution method on Mueller–Hinton supplemented with NaCl (Promega, Madison, WI). 2.3. DNA isolation The original protocol for DNA extraction was adapted from that described by Unal et al. (1992). Briefly, an aliquot of an overnight culture (approximately 108 cells) was suspended in 50 AL of lysostaphin (100 Ag/mL in sterile deionized water, Sigma, St. Louis, MO). After incubation at 37 8C for 10 min, 50 AL of proteinase K (100 Ag/mL in sterile deionized water, Roche Molecular Bio-chemicals, Mannheim; Germany) and 150 AL of 100 mmol/L Tris buffer (Promega), pH 7.5, were added. The suspension was incubated at 37 8C for a further 10 min, boiled for 5 min, and centrifuged at 1300 rpm for 2 min; the supernatant was directly transferred to the PCR mixture.

Table 2 Characteristics of the probe used in the real-time PCR assay Probe name

Gene

Sequence

5V position

GenBank accession no.

mecA CoA Sau3A

mecA Coa Sa442

TTC CAG GAA TGC AGA AAG ACC AAA GCA (sense strand) CGC TAG GCG CAT TAG CAG TTG CAT C (sense strand) TAC TGA AAT CTC ATT ACG TTG CAT CGG AAA CA (sense strand)

398 133 95

X52593 X16457 AF033191

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15 cycles of 94 8C for 30 s, 68 8C for 30 s, and 72 8C for 30 s. Thereafter, further 20 cycles were completed as above but using an annealing temperature of 60 8C with final extension at 72 8C for 2 min. The PCR were performed in an eppendorf thermocycler unit. Ten microliters of PCR-amplified reaction mixture was resolved by electrophoresis on a 2% agarose gel (Promega). The sizes of amplification product were estimated by comparison with 100-bp molecular size standard ladder (New England BioLabS, UK). Fig. 1. An ethidium bromide-stained gel demonstrating the typical banding patterns observed in quadruplex PCR assay. Lane 1—ATCC MRSA (positive control). Lane 2—MRSA. Lane 3—MSSA. Lane 4—MRCoNs. Lane 5—MSCoNs. Lane 6—S. epidermidis. Lane 7—S. saprophyticus. Lane 8—S. lugdunensis. Lane 9—S. xylosus. Lane 10—Staphylococcus homonis. Lane 11—S. haemolyticus. Lane 12—S. capitis (capitis). Lane 13—S. capitis (ureolyticus). Lane 14—E. coli. Lane 15—Klebsiella sp. Lane 16—100 bp DNA marker. Lane 17—negative control.

2.4.2. DNA sequencing Polymerase chain reaction products were purified using the PCR Purification Kit (Qiagen, Germany), and PCR DNA sequencing was carried out using an automated DNA sequencer (an ABI Prism 377 DNA sequencer; PerkinElmer ABI, Wellesley, MA).

2.4. Quadruplex PCR assay Primer pairs used in this study are SA-1 and SA-2 (Kearns et al., 1999) for detection of 214-base pair (bp) fragment within the mecA gene, SA-3 and SA-4 (Kearns et al., 1999) for a 117-bp fragment from Coa gene, and Sa442-F and Sa442-R (Tan et al., 2001) for a 179-bp fragment within the S. aureus-specific genomic marker (Thean et al., 2001), and X-F and Y-R (Geha et al., 1994) for a 479-bp fragment within 16S rRNA gene. The 16S rRNA gene common to all bacteria was included as internal control. Amplification of this portion of 16Sr RNA gene is used as control for DNA extraction as well as failure of PCR to amplify target sequences. To have the quality assurance for each PCR batch that was performed, the negative control without template DNA and the positive control with defined targets were included to check for false-positive and falsenegative results, respectively. Primers used in this study are listed in Table 1. 2.4.1. Quadruplex PCR amplification conditions The PCR reagent mixture consisted of 4 mmol/L MgCl2, 200 Amol/L dNTP (MBI Fermentas, Vilnius, Lithuania), 15 pmol of each primer, and 1 U Taq DNA polymerase (MBI). One-microliter template was directly transferred to PCR reaction mixture. Amplification conditions consisted of an initial denaturation step, 1 min at 94 8C, followed by

Table 3 Multiplex PCR and MIC results obtained with 152 clinical isolates of Staphylococci Identity MRSA MSSA MRCoNs MSCoNs

No. of isolate with mecA

Coa

Sa 442

No. of isolates examined

Oxacillin resistance MIC range (Ag/mL)

48 0 48 0

48 45 0 0

48 45 0 0

48 45 48 11

128 – 256 0.25 – 1 8 – 256 0.06 – 0.125

Fig. 2. The representative results obtained in triplex real-time PCR assay. Five different colors represent 5 different samples that were tested for Sa442, mecA, and Coa genes simultaneously. The samples of this panel were positive control (MRSA ATCC 43300 strain, dark green amplicon curve), 3 MRSA clinical samples (light green, red, and yellow amplicon curve), and negative control (blue line). Pos. = positive amplification; Neg. = negative amplification.

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Fig. 3. Example showing the Texas Red fluorescence detection of MRSA strain; serial dilutions of MRSA strain corresponding to amplicon curves that show sensitivity of real-time PCR assay.

2.5. Probe chemistry and design of the triplex real-time PCR assay In the fluorescent TaqMan assay, the probe is labeled at the 5V end with a fluorescent reporter molecule such as the fluorescein and at the 3V end with another fluorescent molecule such as the quencher for the reporter; when the 2 fluorophores are fixed at opposite ends of 20–30 nucleotide probe and the reporter fluorophore is excited by an outside light source, the normal fluorescence of the reporter is absorbed by the nearby quencher and no reporter fluorescence is detected. During real-time PCR, the fluorogenic probe and PCR amplimers first hybridize to their DNA targets, the probe will be cleaved by the 5V exonuclease activity of Taq DNA polymerase, and the signal from the degraded fluorogenic probe can be continuously monitored throughout the course of the gene amplification (Oliver et al., 2001). TaqMan probes were designed specifically for each primer sets. TaqMan probes (ProOligo) that have been used in this study were labeled as follows: mecA probe with FAM (fluorescein) at 5V end and BHQ-1 (black-whole quencher 1) at 3V end, Coa probe with Texas Red and BHQ-2, and Sau3A probe with TET and BHQ-1 (Table 2).

Laboratories, Hercules, CA) detection system that combines rapid thermal cycling and probe-specific detection of the amplified product. 2.7. Determination of specificity and sensitivity of both PCR assays The sensitivity of the assays was determined using serial dilution of the genomic DNA. The extracted DNA was serially diluted in sterile deionized water. The concentration of the DNA was adjusted in the PCR reaction to the final concentration starting from 2  105 genomic molecules in the first tube, 2  104 in the second one, 2  103 in the third, 2  102 in the fourth, 2  101 in the fifth, and 2  100 in the final tube in the dilution series. The specificity of the assay was determined using the collection of the Gram-positive and Gram-negative strains available in our laboratory. The Gram-positive strains include 8 Staphylococci ATCC strains and Streptococcus spp. Among the Gram-negative strains that have been tested for specificity were Pseudomonas spp, Acinetobacter spp, Escherichia coli, and Klebsiella spp.

2.6. The triplex real-time PCR conditions

3. Results

Real-time PCR amplification was performed in a 50-AL reaction volume containing 7.5 mmol/L MgCl2, 200 Amol/L dNTP, 0.2 Amol/L of SA-1 primer, 0.4 Amol/L SA-2, 0.25 Amol/L SA-3, 0.25 Amol/L SA-4, 0.5 Amol/L Sa442-F, 0.6 Amol/L Sa442-R, 0.2 Amol/L of each hybridization probe, and 2 AL of template DNA in the single tube. The methodology in this real-time PCR assay uses a 2-step PCR amplification protocol, whereby the annealing step comprises the hybridization and extension steps. The thermal cycling protocol was as follows: 3 min at 95 8C for initial denaturation, 30 cycles of 2 steps consisting of 30 s at 95 8C for denaturation, and 45 s at 55 8C for annealing. Real-time PCR was performed using iCycler iQ (BioRad

All 152 clinical isolates were correctly identified using the conventional quadruplex gel-based PCR assay and real-time triplex PCR assay. An ethidium bromide-stained gel demonstrating the typical banding patterns observed with MRSA, MSSA, MRCoNs, and MSCoNs is shown in Fig. 1. The results obtained by quadruplex PCR assay have been summarized in Table 3. The specific PCR product for the 16S rRNA primer set (internal control) was found in all of the isolates tested by conventional quadruplex gel-based PCR assay. Representative results obtained by triplex real-time PCR assay for the clinical isolates that were tested simultaneously for Sa442 (A), mecA (B), and Coa (C) genes are shown in Fig. 2.

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Detection of methicillin resistance in S. aureus isolates using mecA-specific set by conventional quadruplex gelbased PCR assay and real-time triplex PCR assay shows 100% sensitivity and specificity as compared with conventional susceptibility test. Detection of S. aureus-specific set for all S. aureus species showed 100% sensitivity and specificity using both assays. This finding is in accordance with those of Martineau et al. (1998) who reported 100% sensitivity and specificity for Sa442 fragment. The Coa gene was detected only in S. aureus isolates (n = 93) with specificity of 100% and was not detected in any of CoNS (n = 59). Real-time triplex PCR assay could detect as low as 20 fg of genomic DNA (Fig. 3). The quadruplex PCR assay could detect up to 2000 pg of genomic DNA only.

4. Discussion The emergence of methicillin resistance in S. aureus is of great concern because MRSA are often multidrug resistant (Chambers, 2001). Infections with MRSA are known to be associated with considerable morbidity and mortality (Cosgrove et al., 2003). Coagulase is produced by all strains of S. aureus. Its production is the principle criterion used in clinical microbiology laboratory for identification of S. aureus in human infections, and it is an important virulence factor. In routine diagnostic laboratory, a Gram stain is performed followed by the tube coagulase test. The tube coagulase test is checked after 4 h of incubation, and if the test is negative, a further 20 h of incubation is required; hence, the result of the test is only available after 24 h, thus, delaying definitive identification of S. aureus. Hence, in routine diagnostic laboratories, species identification is primarily based on biochemical characteristics of cultured organisms, which are time-consuming procedures. Molecular-based methods are becoming increasingly useful in clinical microbiology laboratories to increase accuracy of identification and to obtain reliable results more rapidly (Kearns et al., 1999). Species-specific 442-bp chromosomal fragment has been recently the specific choice for identification of S. aureus in multiplex PCR (Martineau et al., 1998) and real-time PCR assays (Reischl et al., 2000, Shrestha et al., 2002, Tan et al., 2001). There are few cases that state the absence of Sa442 fragment from S. aureus isolates in PCR assays. These isolates were coagulase positive and presented a pattern of biochemical reactions typical for S. aureus (Su¨tterlin et al., 2003). Hence, PCR assay for identification of S. aureus may result in misidentification when it is solely based on the Sa442 fragment. The primary mechanism of resistance to methicillin in S. aureus is the production of novel penicillin binding protein termed as PBP2a or PBP2V that has low affinity for h-lactam antibiotics (Hiramatsu et al., 2001). Expression of methicillin resistance in clinical microbiology laboratory setting is subjected to environmental conditions (i.e.,

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temperature, pH, salt concentration) (Geha et al., 1994). Conditional expression of PBP2a may cause ambiguities in susceptibility tests, thus, emphasizing the need to develop a rapid and sensitive method for the detection of methicillin resistance in staphylococci, which is not dependent on growth conditions. mecA PCRs are most robust, and the reliable test for detecting oxacillin-resistant staphylococci and the PCR assays are used as bgold standardQ for detection of methicillin resistance (Martineau et al., 2000). Numerous molecular methods have been developed for the detection of MRSA; the most recent assays includes multiplex gel-based PCR assay targeting mecA gene encoding PBP2V and species-specific genes. These methods along with immunologic methods for detection of PBP2 are used as standard confirmatory tests for MRSA. The gel-based conventional PCR methods are laborious in terms of post-PCR work and have problems of carryover contamination. These disadvantages have been overcome by the advent of real-time PCR defined as the ability to monitor the amplified product during amplification. Real-time PCR developed previously have been focusing on mecA gene, Sa-442 singly (Shrestha et al., 2002; Tan et al., 2001), or in combination (Reischl et al., 2000). Real-time PCR developed in this study includes Coa gene in addition to Sa442 for the identification of S. aureus, thereby preventing misidentification of S. aureus isolates that are not carrying Sa442 fragment. A few investigators reported false-negative results with usage of Sa442 fragment as species-specific target (Klaassen et al., 2003; Su¨tterlin et al., 2003). Using Sa442 primer and probe set in this study, no false-negative results were observed. Our findings are in accordance with those of Martineau et al. (1998) who reported 100% sensitivity and specificity for Sa442 fragment. However, the possibility of the presence of S. aureus isolates lacking this fragment exists; therefore, the usage of Sa442 fragment as a sole target for the identification of S. aureus is no longer recommended. In our study, the Coa primer and probe sets were 100% sensitive and specific. Using this primer–probe set, the additional information about the coagulase status of an isolate will be obtained, therefore, will be helpful on identifying CoNS isolates carrying mecA gene. The developed real-time triplex PCR assay is able to differentiate the isolates into 4 categories: MRSA, MSSA, MRCoNs, and MSCoNs. The triplex real-time PCR assay using 3 dual-labeled TaqMan probes (mecA, Coa, species specific) could detect 3 genes (mecA, Coa, and Sa442) at a time. TaqMan probes that have been chosen in this study were labeled with different fluorophore, which can be detected at different wavelength. This will further increase the specificity of the PCR assay. The triplex real-time PCR assay with the detection of 20 fg of genomic DNA was found to be far more sensitive as compared with the conventional quadruplex PCR assay. This triplex real-time PCR assay could be adapted for direct detection from positive blood cultures or from normally sterile clinical specimens (e.g., blood), thereby allowing diagnosis to be made much faster than conventional

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methods of identification. However, this assay would be of limited value to the specimens containing mixtures of S. aureus and CoNS. The time frame from DNA extraction until the results are obtained is 3 h, as compared with the conventional gel-based quadruplex PCR assay that may take 6 –8 h. The real-time PCR has the additional advantage of producing the results immediately after the PCR reaction is completed without the need for opening the PCR tube, therefore, minimizing the risk of carryover contamination. This triplex real-time PCR assay may represent a feasible and practical tool for use in routine diagnostic laboratories as a rapid, sensitive, and improved assay for accurate identification of S. aureus isolates with respect to specificity. Application of this rapid and sensitive technique in a routine diagnostic microbiology laboratory can contribute to reduction of empirical therapy with broad-spectrum antibiotics. This reduction consequently will decrease the antibiotic consumption and emergence of new resistance profile in microorganisms.

Acknowledgment This study was funded by IRPA grant 0-02-03-0112 PR 0047/19-10 and partly by Vote F F0100/2004A. References Carroll KC, Leonard RB, Newcomb-Gayman PL, Hillyard DR (1996) Rapid detection of staphylococcal mecA gene from Bactec blood culture bottles by the polymerase chain reaction. Am J Clin Pathol 106:600 – 605. Chambers HF (2001) The changing epidemiology of Staphylococcus aureus? Emerg Infect Dis 7:178 – 182. Cosgrove SE, Sakoulas G, Perencevich EN, Schwaber MJ, Karchamer AW, Carmeli Y (2003) Comparison of mortality associated with methicillinresistant and methicillin susceptible Staphylococcus aureus bacteremia: a meta-analysis. Clin Infect Dis 36:53 – 59. Emori TG, Gaynes RP (1993) An overview of nosocomial infections including the role of the microbiology laboratory. Clin Microbiol Rev 6:428 – 442. Geha DJ, Uhl JR, Gustaferro CA, Persing DH (1994) Multiplex PCR for identification of methicillin-resistant Staphylococci in the clinical laboratory. J Clin Microbiol 32:1768 – 1772. Grisold AJ, Leitner E, Mqhlbauer G, Marth E, Kessler H (2002) Detection of methicillin-resistant Staphylococcus aureus and simultaneous confirmation by automated nucleic acid extraction and real-time PCR. J Clin Microbiol 40:2392 – 2397.

Hiramatsu K, Cui L, Kuroda M, Ito T (2001) The emergence and evolution of methicillin-resistant Staphylococcus aureus. Trends Microbiol 9: 486 – 493. Hiramatsu K, Okuma K, Ma XX, Yamamoto M, Hori S, Kapi M (2002) New trends in Staphylococcus aureus infections: glycopeptide resistance in hospital and methicillin resistance in the community. Curr Opin Infect Dis 15:407 – 413. Karchmer AW (2000) Nosocomial blood stream infections: organisms, risk factors and implications. Clin Infect Dis 31(Suppl. 4):S139 – S143. Kearns AM, Seiders PR, Wheeler J, Freeman R, Steward M (1999) Rapid detection of methicillin-resistant Staphylococci by multiplex PCR. J Hosp Infect 43:33 – 37. Klaassen CHW, de Valk HA, Horrevorts AM (2003) Clinical Staphylococcus aureus isolate negative for the Sa442 fragment. J Clin Microbiol 41:4493. Martineau F, Picard FJ, Roy PH, Ouellette M, Bergeron MG (1998) Species-specific and ubiquitous-DNA–based assays for rapid identification of Staphylococcus aureus. J Clin Microbiol 36:618 – 623. Martineau F, Picard FJ, Lansac N, Menard C, Roy HP, Ouellette M, Bergeron MG (2000) Correlation between the resistance genotype determined by multiplex PCR assays and the antibiotic susceptibility patterns of Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob Agents Chemother 44:231 – 238. Oliver G, Philip JRD, Philip PB, Fatime I, Martin A, David N (2001) Quantitative detection of S. pneumoniae in nasopharyngeal secretions by real time PCR. J Clin Microbiol 39:3129 – 3134. Phonimdaeng P, O’Reilly M, Nowlan P, Brmley AJ, Foster TJ (1990) The coagulase of Staphylococcus aureus 8325-4. Sequence analysis and virulence of site-specific coagulase-deficient mutants. Mol Microbiol 4:393 – 404. Reischl U, Linde H-J, Metz M, Leppmeier B, Lehn N (2000) Rapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using real-time PCR. J Clin Microbiol 38:2429 – 2433. Schmitz FJ, Mackenzie CR, Hofmann B, verhoef J, Finken eigen M, Hewz HP, Kohrer K (1997) Specific information concerning, taxonomy, pathogenicity and methicillin resistance of staphylococci obtained by multiplex PCR. J Med Microbiol 46:773 – 778. Shrestha NK, Tuohy MJ, Hall GS, Isada CM, Procop GW (2002) Rapid identification of Staphylococcus aureus and the mecA gene from BacT/ALERT blood culture bottles by using the Light Cycler system. J Clin Microbiol 40:2659 – 2661. Sqtterlin K, Englert R, Schmidt-Wieland T, Schmitt J, Reischl U, Lehn N (2003) Sporadic cases of Staphylococcus aureus organisms negative for a species-specific 442-base pair chromosomal fragment. J Clin Microbiol 41:3449. Tan TY, Corden S, Barnes R, Cookson B (2001) Rapid identification of methicillin-resistant Staphylococcus aureus from positive blood cultures by real-time fluorescence PCR. J Clin Microbiol 39:4529 – 4531. Unal S, Hoskins J, Flokowitsch JE, Wu CYE, Preston DA, Skatrud PL (1992) Detection of methicillin-resistant staphylococci by using the polymerase chain reaction. J Clin Microbiol 30:1685 – 1691.