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. 2009 Mar 11;47(5):1443–1451. doi: 10.1128/JCM.01197-08

High Levels of mecA DNA Detected by a Quantitative Real-Time PCR Assay Are Associated with Mortality in Patients with Methicillin-Resistant Staphylococcus aureus Bacteremia ▿

Ya-Chi Ho 1,2, Shan-Chwen Chang 2, Su-Ru Lin 3, Wei-Kung Wang 2,3,*
PMCID: PMC2681853  PMID: 19279177

Abstract

Persistent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is known to be a poor prognostic factor. While several PCR assays for the detection of MRSA in various clinical samples were recently reported, the possibility that a quantitative PCR assay could be used to quantify and monitor MRSA bacteremia has not been explored. In this study, we established a quantitative real-time PCR assay for the mecA gene using known copy numbers of a plasmid containing mecA DNA as a standard and the previously described mecA-specific primers and probe (P. Francois et al., J. Clin. Microbiol. 41:254-260, 2003). We employed this assay to examine 250 sequential whole-blood samples from 20 adult patients, including 13 survivors and 7 nonsurvivors, with culture-proven MRSA bacteremia at the intensive care units of National Taiwan University Hospital between 1 July 2006 and 31 January 2007. The levels of mecA DNA in the nonsurvivors were significantly higher than those in the survivors during the three periods of bacteremia examined (days 0 to 2, 3 to 5, and 6 to 8) (P = 0.003 by two-tailed Mann-Whitney U test). Moreover, the nonsurvivors had higher mecA DNA levels than the survivors after 3 days and 7 days of anti-MRSA therapy (medians for nonsurvivors and survivors at 3 days, 5.86 and 4.30 log copies/ml, respectively; medians for nonsurvivors and survivors at 7 days, 5.21 and 4.36 log copies/ml, respectively; P = 0.02 and P = 0.04, respectively, by two-tailed Mann-Whitney U test). Together, these findings suggest that the level of mecA DNA in blood could potentially be used to monitor MRSA bacteremia and evaluate responses to therapy.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that is one of the most common causes of both community-acquired and nosocomial infections worldwide, especially in intensive care units (ICUs) (5, 8, 33, 49). In the past two decades, the proportion of MRSA infections has increased dramatically, and up to 60% of S. aureus isolates from ICUs were reported to be methicillin resistant (26). Compared with cases of bacteremia caused by methicillin-susceptible S. aureus (MSSA) strains, cases of bacteremia caused by MRSA strains have been shown to be associated with more persistent infections, more recurrent episodes, longer hospital stays, and higher rates of mortality (3, 6, 11, 13, 14, 27). Because of the high rates of mortality and the refractoriness of MRSA bacteremia to treatment, MRSA bacteremia has become a very challenging infectious disease (18, 21, 38, 39, 42).

It has recently been demonstrated that high bacterial loads in blood correlate with disease severity in patients with infections caused by Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis (4, 22, 35, 41), as well as in a murine model of Serratia marcescens infection (25). In the case of S. aureus-related infections, it was reported that positive blood cultures during follow-up and the persistence of fever were suggestive of a complicated course (6, 15, 37). However, the drastic decrease in the sensitivity of blood culture after the initiation of antimicrobial therapy, even when special culture media are used, has made the use of blood cultures to monitor responses to therapy and outcomes very difficult (17, 19, 31, 36). On the other hand, most of the real-time PCR assays used for the detection of MRSA have been qualitative in nature and have used specimens from blood culture bottles or nasal swabs rather than blood samples directly and therefore have provided little information regarding the MRSA load in blood (16, 20, 24, 43, 48).

In this study, we established a quantitative real-time PCR assay to quantify the mecA DNA load in blood by using a previously described mecA-specific primer pair and probe and known copy numbers of a plasmid containing the mecA gene as a standard (16). We then used this assay to investigate the mecA DNA load during the course of infection in 20 patients with culture-proven MRSA bacteremia. We applied this assay to monitor patients with MRSA bacteremia and demonstrated that high mecA DNA loads after 3 days and 7 days of therapy correlate with poor outcomes.

MATERIALS AND METHODS

Study participants and blood samples.

This was a prospective observational study. With the approval of the Institutional Review Board of the National Taiwan University Hospital (NTUH) and informed consent, adult patients who were admitted to the medical or surgical ICU of NTUH, a medical center with a 2,200-bed capacity, between 1 July 2006 and 31 January 2007 were enrolled as soon as a blood culture positive for MRSA that fulfilled the definition of the Clinical and Laboratory Standards Institute (CLSI) was reported (10). The identification of S. aureus was based on the colony morphology, Gram stain, a positive catalase reaction, and the result of the slide agglutination test (bio-Merieux) and/or from the results obtained with the Phoenix system (Becton Dickinson). This was followed by performance of a disk diffusion susceptibility test with oxacillin, vancomycin, teicoplanin, minocycline, erythromycin, clindamycin, gentamicin, trimethoprim-sulfamethoxazole, and fusidic acid. The course of MRSA bacteremia in each participant was carefully monitored according to clinical parameters and by the use of routine laboratory tests. Whole-blood samples in EDTA-containing tubes taken before and on the day of the report of MRSA bacteremia were retrospectively obtained from the central laboratory. After the report of MRSA bacteremia, blood samples taken were collected daily for the first week and two to three times a week thereafter.

Clinical data and definitions.

The following information was collected for each patient: age, gender, underlying conditions, port of entry, body temperature, white blood cell (WBC) count, C-reactive protein (CRP) level, reports of culture results, and antibiotic usage. The underlying illness was evaluated by use of the Charlson score (7), and clinical severity was evaluated by use of the acute physiology and chronic health evaluation II (APACHE II) and Pitt bacteremia scores (9).

Day 0 was the day on which the first blood sample positive for MRSA by culture was drawn, and the days prior to and after this time point were designated consecutively. Fever was defined as a tympanic temperature of ≥38°C. The anti-MRSA antibiotics available in NTUH included vancomycin, teicoplanin, and linezolid. Treatment delay was the lag between day 0 and the day when the anti-MRSA antibiotic treatment was initiated. The delay in catheter removal was the lag between day 0 and the day when a culprit central venous catheter, the vascular catheter tip of which yielded greater than 15 MRSA colonies by semiquantitative culture, was removed (30). Multiresistant MRSA is defined as resistance to three or more of the following: ciprofloxacin, erythromycin, clindamycin, gentamicin, trimethoprim-sulfamethoxazole, vancomycin, and linezolid (40). Patients whose cause of death fulfilled the definition of Staphylococcus bacteremia-related death (29), including (i) blood culture positive for MRSA at the time of death, (ii) death before the resolution of symptoms and signs, (iii) death within 14 days after the onset of MRSA bacteremia without another explanation, (iv) autopsy findings suggestive of MRSA-related death, and (v) MRSA bacteremia as a cause of death on the death certificate, were classified as the nonsurvivor group. Patients who survived the episode were classified as the survivor group.

Bacterium-spiked blood samples.

A previously described MRSA isolate (isolate N19) was grown from a single colony in Mueller-Hinton broth overnight and was serially diluted 10-fold in normal saline (45). One hundred microliters of each dilution was plated onto sheep blood agar plates, and the plates were incubated at 37°C overnight to determine the numbers of CFU. Increasing amounts of MRSA (1.74 × 103 to 1.74 × 108 CFU) were diluted with 1 ml normal saline or spiked with 1 ml whole blood from a healthy donor and subjected to DNA extraction. In addition, five aliquots of MRSA (2.36 × 106 CFU each) were each spiked with 1 ml whole blood from a healthy donor, stored at 4°C, and subjected to DNA extraction daily for 5 consecutive days.

DNA extraction.

One milliliter of MRSA-spiked whole blood or whole blood from patients was treated with 3 ml red blood cell lysis solution (Gentra Systems, Minneapolis, MN) at room temperature for 5 min, followed by centrifugation at 13,000 × g for 20 s to obtain the pellets, which were resuspended in TE (50 mM Tris [pH 8.0], 100 mM EDTA) containing 100 μg of lysostaphin (Sigma, St. Louis, MO) and subjected to DNA extraction with a QIAamp DNA minikit (Qiagen, Hilden, Germany) (16, 23). The final eluate was resuspended in 100 μl elution buffer and stored at −20°C until use. Whole-blood samples from healthy donors served as negative controls. DNA extraction and PCR were carried out in separate rooms, and precautions were taken to prevent contamination of the PCR mixture (28).

Primers, probes, and plasmid standards.

The sequences of the primers and probe for the mecA gene (primers F mecA and R mecA and probe P mecA) and for the femA gene of Staphylococcus epidermidis, designated femASe (primers F femA-SE and R femA-SE and probe P femA-SE), were described previously (16). The standard plasmid containing mecA DNA, mecA/pCRII-TOPO, was constructed by PCR amplification of a 909-bp fragment of the mecA gene with primers mecA121A (5′-TTATATAAGGAGGATATTGATG-3′) and mecA1029B (5′-GCCATCTTCATGTTGGAGC-3′) and template DNA extracted from isolate N19 and cloning of the DNA into pCRII-TOPO (Fig. 1A) (47). The femASe DNA standard plasmid, femA-SE/pCRII-TOPO, was constructed by PCR amplification of a 1,282-bp fragment of the femASe gene by using primers femASE367A (5′-TGGGAGTTATGAAGATGAAGT-3′) and femSEA1648B (5′-CCTTCTTAAAATCTATTTCTTTA-3′) and template DNA extracted from S. epidermidis (strain ATCC 1228) and cloning of the DNA into pCRII-TOPO. The copy number of the plasmid DNA was calculated on the basis of its concentration, determined with a spectrophotometer at 260 nm, and the molecular weight (47).

FIG. 1.

FIG. 1.

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Real-time PCR assay for quantification of mecA DNA derived from known numbers of CFU of MRSA in normal saline and stored whole-blood samples. (A) Schematic diagram of the construct, mecA/pCRII-TOPO, which contains a 909-bp region of the mecA gene (positions 121 to 1029 based on the sequence with GenBank accession number X552593). The relative positions of the previously described mecA-specific primers and probe are shown (16). The relationship between the input mecA DNA copies and threshold cycle number (CT value) of the assay is shown in the graph at the bottom of panel A. (B) Relationship between the numbers of CFU of MRSA diluted in normal saline (1.74 × 103 to 1.74 × 108 CFU/ml) and the mecA DNA copy number detected by the mecA real-time PCR assay. (C) Relationship between the mecA DNA copy number detected and the known amounts of MRSA (2.36 × 106 CFU) spiked in whole blood from a healthy control donor and stored at 4°C for 0 to 4 days. The results of one representative experiment of more than two that were performed are shown.

Quantitative real-time PCR.

An aliquot of purified DNA and known copy numbers of mecA DNA standards (5 to 5 × 108 copies) were subjected to real-time PCR. Briefly, a 25-μl reaction mixture containing 12.5 μl of 2× TaqMan universal PCR master mix, 2.5 μl of 10 pmol/μl forward primer (primer F mecA), 2.5 μl of 10 pmol/μl of reverse primer (primer R mecA), 2.5 μl of probe (probe P mecA), mecA DNA standards or purified DNA, and nuclease free-water (Promega) was prepared and subjected to real-time PCR with an ABI Prism 7900 sequence detector (Applied Biosystems, Foster City, CA). The amplification conditions were 50°C for 2 min and 95°C for 10 min, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min, as described previously (16). Another aliquot of purified DNA and known copy numbers of femASe DNA standards (5 to 5 × 108 copies) were subjected to real-time PCR by using the femASe-specific primers and probe. A positive result was defined by the cycle number required to reach the threshold, which was 10 times the standard deviation of the mean baseline emission calculated for cycles 3 to 10 (16, 47). Since 5 μl of the 100 μl DNA eluate, which was derived from 1 ml of whole-blood samples, was used in each reaction mixture, the mecA DNA copy number per reaction was multiplied by 20 to determine the number of copies of mecA DNA per ml whole blood. The sensitivity of the assay was 100 copies per ml whole blood. Samples positive for femASe, which consisted of 2 of the 253 samples tested, were considered contaminated and were discarded.

Statistical analysis.

A nonparametric test, the Mann-Whitney U test, in SPSS software (base 8.0; SPSS Inc., Chicago, IL) was used to compare continuous variables between two groups. Fisher's exact test was used to compare categorical variables between two groups.

RESULTS

Quantitative real-time PCR assay for mecA gene.

To establish a standard curve for quantification of the mecA gene, increasing copy numbers (5 to 5 × 108 copies per reaction) of a plasmid containing mecA DNA, mecA/pCRII-TOPO, were subjected to real-time PCR by using a previously described mecA-specific primer pair and probe (16) (Fig. 1A). To evaluate the mecA real-time PCR assay for its ability to quantify the mecA gene from an MRSA isolate, DNA extracted from known numbers of CFU of MRSA strain N19 (range, 1.74 × 103 to 1.74 × 108 CFU/ml) was subjected to the assay with the mecA DNA-containing plasmid as the standard. As shown in Fig. 1B, a linear relationship was observed between the number of CFU and the mecA DNA copy number, demonstrating the accuracy of this assay (correlation coefficient [r] = 0.9967).

To rule out the possibility of contamination by a common contaminant, S. epidermidis, a quantitative real-time PCR assay for the femASe gene was also established. A linear curve was observed as the amount of input plasmid containing femASe DNA increased from 5 to 5 × 108 copies per reaction mixture (data not shown). To examine whether the quantitative real-time PCR assays for mecA and femASe can distinguish reference strains of MRSA, MSSA, and S. epidermidis, DNA extracted from MRSA (strain N19), MSSA (ATCC 25923), and S. epidermidis (ATCC 12228) was subjected to these two assays. As expected, signals positive for mecA were detected with DNA extracted from the MRSA strain but not with DNA extracted from the MSSA strain or the S. epidermidis strain tested. In contrast, a positive femASe signal was detected only with DNA extracted from the S. epidermidis strain (data not shown).

Quantification of mecA DNA derived from MRSA in whole blood.

To investigate whether the mecA real-time PCR assay can quantify the mecA gene derived from MRSA in whole-blood samples, DNA was extracted from known numbers of CFU of MRSA strain N19 (1.74 × 103 to 1.74 × 108 CFU/ml) that had been spiked with whole blood from a healthy volunteer and subjected to the assay. A linear curve between the number of CFU and the mecA DNA copy number (r = 0.9995) was obtained (data not shown). The sensitivity of the assay was 100 copies per ml whole blood. To explore the possibility that mecA DNA extracted from MRSA in stored whole-blood samples can be quantified, 5 ml of whole blood from a healthy donor was spiked with 11.8 × 106 CFU of MRSA strain N19, divided into five aliquots, and stored in a refrigerator at 4°C. DNA extracted from one aliquot each (2.36 × 106 CFU) on days 0, 1, 2, 3, and 4 was subjected to the mecA real-time PCR assay. As shown in Fig. 1C, the levels of mecA DNA extracted from MRSA detected in whole blood that had been stored at 4°C for up to 4 days generally remained stable, suggesting the feasibility of quantifying mecA DNA derived from MRSA in stored whole-blood samples.

Quantification of mecA DNA in blood from patients with MRSA bacteremia.

We next employed this assay to quantify the mecA DNA levels in sequential whole-blood samples from four study participants, two nonsurvivors and two survivors, with culture-proven MRSA bacteremia. As shown in Fig. 2B and D, a trend toward a decrease in the mecA DNA level was found in the two patients who were successfully treated, whereas continuously high levels of mecA DNA were found in the two patients who died of MRSA bacteremia within 10 days of day 0 (i.e., within 10 days of the beginning of infection) (Fig. 2A and C). We then extended the assay to the other 16 study participants and studied a total of 250 sequential whole-blood samples from 20 patients, including 7 nonsurvivors and 13 survivors. The basic demographic and clinical features of the patients are summarized in Table 1.

FIG. 2.

FIG. 2.

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Quantification of mecA DNA in blood samples from four patients during the course of MRSA bacteremia. (A to D) Sequential whole-blood samples from four patients with blood culture-confirmed MRSA bacteremia were subjected to DNA extraction and the mecA real-time PCR assay. Patients ID32 (B) and ID10 (D) were successfully treated, and patients ID18 (A) and ID21 (C) died of MRSA bacteremia within 10 days. Closed rhomboids indicate mecA DNA detected (log copies/ml). (E and F) Levels of mecA DNA during the course of MRSA bacteremia in 7 nonsurvivors (E) and 13 survivors (F), presented as means ± standard errors. Day 0 was the day on which the first blood sample positive for MRSA by culture was drawn, and the days prior to and after that time point were designated consecutively. The levels of mecA DNA in blood were determined as described in Materials and Methods. The limit of detection of the assay was 100 copies mecA DNA per ml whole blood. B/C, blood culture. The bars beneath the panels indicate the times of anti-MRSA antibiotic therapy, and the asterisks at the ends of the bars indicate the day of death. *, **, and ***, P = 0.003; two-tailed Mann-Whitney U test.

TABLE 1.

Clinical characteristics of study patients with MRSA bacteremiaa

Characteristic Total (n = 20) Nonsurvivor group (n = 7) Survivor group (n = 13) P
Age (yr) 71 (34-89) 71 (34-89) 71 (50-89) 0.43
Sex (no. of M/no. of Fb) 15/5 5/2 10/3 1.00
Charlson score 4 (2-6) 3 (2-5) 4 (3-6) 0.50
No. (%) of patients with the following on ICU admission:
    Respiratory failure 18 (90) 7 (100) 11 (85) 0.52
    Septic shock 12 (60) 6 (86) 6 (46) 0.15
Score on ICU admission:
    APACHE II 18 (13-22) 20 (16-25) 17 (13-19) 0.28
    Pitt bacteremia 4 (2-5) 5 (2-7) 3 (2-4) 0.25
Score on day 0c:
    APACHE II 19 (16-23) 20 (19-23) 17 (15-20) 0.17
    Pitt bacteremia 4 (2-4) 4 (4-7) 3 (2-4) 0.03
No. of days of prior hospitalizationd 11 (10-29) 15 (5-33) 11 (9-27) 0.78
No. of days of prior ICU stayd 2 (0-12) 10 (2-13) 1 (0-6) 0.43
Site of MRSA infection (no. [%] of patients)
    Pneumonia 13 (65) 6 (86) 7 (54) 0.33
    Catheter 8 (40) 3 (43) 5 (38) 1.00
    Empyema 4 (20) 2 (29) 2 (15) 0.59
    Mediastinitis 4 (20) 3 (43) 1 (8) 0.10
    Endocarditis 2 (10) 1 (14) 1 (8) 1.00
    Urinary tract 2 (10) 1 (14) 1 (8) 1.00
    Osteomyelitis 1 (5) 0 (0) 1 (8) 1.00
Anti-MRSA antibiotic treatment (no. [%] of patients) 0.64
    Vancomycin 12 (60) 5 (71) 7 (54)
    Teicoplanin 8 (40) 2 (29) 6 (46)
Treatment delay (days)e 1 (0-2) 0 (0-1.5) 2 (0-2) 0.25
Delay in catheter removal (days)f 2.5 (0-8) 2.5 (0-8) 8 (3-8) 0.052
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a

Data are median (interquartile ranges) for the continuous variables and number of cases (percent) for the categorical variables; the two-tailed Mann-Whitney U test was used to calculate the former, and Fisher's exact test was used to calculate the latter.

b

F, female; M, male.

c

Day 0 was the day on which the first blood sample positive for MRSA by culture was drawn.

d

Days of hospitalization and ICU stay prior to day 0.

e

Treatment delay was the lag between day 0 and the day when anti-MRSA antibiotic treatment was initiated.

f

Delay in catheter removal was defined as the lag between day 0 and the day when a culprit central venous catheter was removed (30).

The patients, among whom there was a predominance of males and the median age was 71 years, were all critically ill with either septic shock (60%) or respiratory failure (90%). There was no significant difference between the two groups in the underlying illness according to the Charlson score or in the clinical severity according to the APACHE II and Pitt bacteremia scores on the day of ICU admission and day 0 (Table 1), except that the Pitt bacteremia score on day 0 was slightly higher for the nonsurvivor group than for the survivor group (P = 0.03, Mann-Whitney U test, two tailed). The patients developed MRSA bacteremia at a median of 11 days after hospitalization and 2 days after their stay in the ICU. There were no differences between the two groups in the numbers of days of hospitalization and the numbers of days of ICU stay prior to MRSA bacteremia or in the foci of MRSA infection (Table 1). Patients received anti-MRSA antibiotics on a median of day 1. Information on the antimicrobial therapy and susceptibility data for the isolates from the 20 patients are summarized in Table 2. There were no differences between the two groups in the type of antibiotics initially used to treat the MRSA infection, the delay of treatment, or the delay in catheter removal (Table 1). The nonsurvivors died on a median of day 2.5 after a median of 6 days of treatment. The survivors were monitored for a median of 105 days after a median of 15 days of treatment.

TABLE 2.

MRSA antimicrobial susceptibility profile and antibiotic therapy of study patients

Patient group and identifier MRSA antimicrobial susceptibilitya Antibiotic therapyb
Nonsurvivors
    ID18 S, VA, TE, MI, SXT; R, OX, EM, CC, GM VA on d0 to d4 (death)
    ID21 S, VA, TE, MI, SXT; R, OX, EM, CC, GM VA on d0 to d10 (death)
    ID10 S, VA, TE, MI, SXT; R, OX, EM, CC, GM VA on d2 to d3 (death)
    ID13 S, VA, TE, MI, SXT, GM; R, OX, EM, CC TE on d2 to d25 (death)
    ID28 S, VA, TE, MI, SXT; R, OX, EM, CC, GM VA on d1 to d6 (death)
    ID42 S, VA, TE; I, MI; R, SXT, OX, EM, CC, GM VA on d0 to d1 (death)
    ID43 S, VA, TE; I, MI; R, SXT, OX, EM, CC, GM TE on d0 to d16 (death)
Survivors
    ID11 S, VA, TE, MI, SXT, GM; R, OX, EM, CC TE on d1 to d8, LZ on d8 to d10
    ID32 S, VA, TE, MI, SXT, CC; R, OX, EM, GM VA on d1 to d12
    ID12 S, VA, TE, SXT, GM; R, MI, OX, EM, CC VA on d0 to d4, LZ on d5 to d14, TE on d14 to d17, LZ on d18 to d37
    ID14 S, VA, TE, MI; R, SXT, OX, EM, CC, GM VA on d0 to d29
    ID16 S, VA, TE, MI; R, SXT, OX, EM, CC, GM VA on d2 to d78, LZ on d78 to d245
    ID20 S, VA, TE, MI, SXT; R, OX, EM, CC, GM TE on d2 to d16
    ID25 S, VA, TE, MI, SXT; R, OX, EM, CC, GM TE on d2 to d21
    ID26 S, VA, TE; R, MI, SXT, OX, EM, CC, GM VA on d2 to d15, d22 to d30
    ID27 S, VA, TE, MI, SXT, GM; R, OX, EM, CC TE on d3 to d16
    ID30 S, VA, TE; R, MI, SXT, OX, EM, CC, GM VA on d2 to d11, LZ on d12 to d26
    ID33 S, VA, TE; I, MI; R, SXT, OX, EM, CC, GM VA on d0 to d3, TE on d3 to d21
    ID40 S, VA, TE, SXT; R, MI, OX, EM, CC, GM TE on d0 to d14
    ID41 S, VA, TE, MI, SXT; R, OX, EM, CC, GM TE on d2 to d14
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a

S, susceptible; R, resistant; I, intermediate; VA, vancomycin; TE, teicoplanin; MI, minocycline; SXT, trimethoprim-sulfamethoxazole; OX, oxacillin; EM, erythromycin; CC, clindamycin; GM, gentamicin; LZ, linezolid.

b

Day 0 (d0) was the day on which the first blood sample positive for MRSA by culture was drawn.

Higher levels of mecA DNA in the nonsurvivors than the survivors.

We next compared the levels of mecA DNA, the WBC counts, and the positive blood culture rates between the nonsurvivors and survivors during the course of infection. The WBC counts from days 3 to 5 were higher in the nonsurvivor group than in the survivor group (means, 15.06 and 9.14 cells/mm3, respectively; P = 0.03, two-tailed Mann-Whitney U test), whereas they were not significantly different between the two groups during the first (days 0 to 2) and third (days 6 to 8) periods examined. The positive blood culture rates were higher in the nonsurvivors than in the survivors during the three periods, but the differences were not statistically significant (89% and 55%, 100% and 33%, and 67% and 18% during the three periods, respectively; P > 0.1, P > 0.05, and P > 0.1, respectively, Fisher's exact test). Interestingly, the mean mecA DNA levels had a trend of an initial increase followed by a decrease, which generally paralleled the change in the mean WBC counts and the positive blood culture rates, in the nonsurvivors and a trend of a continuous decline in the survivors (Fig. 2E and F). The levels of mecA DNA were significantly higher in the nonsurvivors than in the survivors during the three periods (means during the three periods, 5.48 and 4.58 log copies/ml, 5.73 and 4.39 log copies/ml, and 5.16 and 3.89 log copies/ml, respectively; P = 0.003, two-tailed Mann-Whitney U test).

We also investigated whether the mecA DNA levels at specific time points after anti-MRSA therapy can be predictive of a poor prognosis. For convenience in the clinical evaluation, times of 3 days and 7 days posttherapy were chosen and data from those times were analyzed. We found that the levels of mecA DNA were significantly higher in the nonsurvivors than in the survivors after 3 days and 7 days of anti-MRSA therapy (medians for nonsurvivors and survivors at 3 days, 5.86 and 4.30 log copies/ml, respectively; medians for nonsurvivors and survivors at 7 days, 5.21 and 4.36 log copies/ml, respectively; P = 0.02 and P = 0.04, respectively, two-tailed Mann-Whitney U test). In comparison, the WBC counts as well as the CRP levels and the rates of positive blood cultures at these two time points were not significantly different between the two groups, although relatively few samples were available for the analysis of CRP levels and blood culture results (data not shown).

Comparison of mecA DNA levels and blood culture results.

To further examine the relationship between blood culture positivity and the mecA DNA level, we focused on the 87 blood samples for which cultures were concomitantly performed with the PCR assay. As shown in Fig. 3A, the levels of mecA DNA in the blood culture-positive samples were significantly higher than those in the blood culture-negative samples (medians, 5.22 and 4.54 log copies/ml, respectively; P = 0.00002, two-tailed Mann-Whitney U test). Of note, mecA DNA was detectable in the blood of culture-negative samples (interquartile range, 3.74 to 4.88 log copies/ml).

FIG. 3.

FIG. 3.

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Relationship between the levels of mecA DNA and blood culture positivity in patients with MRSA bacteremia. The levels of mecA DNA in 87 samples with which concomitant blood cultures were performed were analyzed for blood culture-positive and -negative samples altogether (A) and for samples subgrouped on the basis of the duration of anti-MRSA antibiotic therapy (0 to 2 days, 3 to 5 days, 6 to 8 days, and ≥9 days) (B). The levels of mecA DNA in blood were determined as described in Materials and Methods. The limit of detection of the assay was 100 copies mecA DNA per ml whole blood.

Since blood culture positivity is believed to be related to the amounts of bacteria in blood, the presence of antibiotic, and its level in serum, which is affected by the timing and duration of therapy, we analyzed the mecA DNA levels in blood culture-positive and -negative samples subgrouped by the duration of anti-MRSA therapy (0 to 2 days, 3 to 5 days, 6 to 8 days, and ≥9 days). As shown in Fig. 3B, higher levels of mecA DNA were found in blood culture-positive samples than in blood culture-negative samples for the four subgroups (P = 0.05, 0.04, 0.03, and 0.002, respectively, two-tailed Mann-Whitney U test). Interestingly, the lowest mecA DNA level that yielded a positive blood culture result increased from 2.76 log copies/ml at 0 to 2 days of therapy to 4.41, 4.62, and 5.02 log copies/ml with durations of therapy of 3 to 5 days, 6 to 8 days, and ≥9 days, respectively.

DISCUSSION

Although several risk factors and high clinical severity scores based on different systems have been identified to be associated with a high rate of mortality from MRSA bacteremia, including age, underlying illness, the presence of a cardiovascular prosthesis, the presence of metastatic infections, and certain initial conditions (6, 18, 21, 39), they have provided little information that clinicians may use to monitor treatment responses during the course of infection. In light of the high rate of mortality from MRSA bacteremia, it is crucial that new methods and good parameters for the monitoring of MRSA bacteremia be developed (3, 6, 11, 13, 14, 27). A recent study has shown that an increased vancomycin MIC and decreased killing by vancomycin in vitro are associated with prolonged MRSA bacteremia; however, the time-consuming processes of blood culture and bactericidal assay may limit its clinical application (32, 36). In this study, we developed a quantitative real-time PCR assay for the mecA gene and successfully employed this assay to quantify the mecA DNA in blood samples from patients with culture-proven MRSA bacteremia. Compared with the traditional quantitative culture method, this is a simpler, more rapid, and more convenient quantitative method. The levels of mecA DNA were significantly higher in the nonsurvivors than in the survivors during the three periods of bacteremia examined. To our knowledge, this is the first report demonstrating that mecA DNA levels determined by a quantitative real-time PCR assay could potentially be used to monitor MRSA bacteremia.

In our mecA real-time PCR assay, we employed previously described mecA gene-specific primers and a mecA gene-specific probe tested by examining 79 strains of different bacteria and fungi (16). The mecA primer pair covered a highly conserved 99-bp region in the mecA gene, which may contribute to the good sensitivity (100 copies of mecA DNA/ml whole blood) of this assay. The standard curve of our assay was based on increasing copy numbers of the plasmid with mecA DNA. Plasmid DNA is stable, convenient to use, and easy to handle and has been shown to be a reliable standard for quantitative real-time PCR assays for other bacteria, such as Streptococcus pneumoniae and Brucella melitensis (34, 44). On the other hand, a real-time PCR assay for femASe was also developed to rule out the possibility of contamination by a common contaminant, S. epidermidis, more than 80% of the strains of which have been reported to be methicillin resistant and to have a mecA gene identical to that of MRSA (1, 12). While our mecA and femASe real-time PCR assays could distinguish the reference MRSA strain (mecA negative and femASe negative), the MSSA strain (mecA positive and femASe negative), and the S. epidermidis strain (femASe positive and mecA negative) tested, the possibility that some coagulase-negative staphylococci, such as methicillin-resistant strains of S. haemolyticus, S. hominis, S. capitis, and S. sciuri, or coagulase-positive staphylococci, such as methicillin-resistant strains of S. intermedius, were present cannot be ruled out (2). Moreover, the possibility that cases of polymicrobial bacteremia caused by MSSA and S. epidermidis were present cannot be excluded. Therefore, confirmation of the presence of MRSA by standard blood culture according to the guidelines of the CLSI is essential to interpret the results of our real-time PCR assays.

Of note, mecA DNA remained detectable in blood culture-negative samples (Fig. 3A). This could be due to a drastic decrease in the sensitivity of blood culture after antibiotic therapy (17, 19, 31). To investigate the relationship of mecA DNA levels to blood culture positivity and antibiotic usage, we analyzed the mecA DNA levels in blood culture-positive and -negative samples subgrouped by the duration of anti-MRSA therapy. We found that the lowest mecA DNA level that yielded a positive blood culture result increased as the duration of therapy increased (Fig. 3B). If it is assumed that the anti-MRSA antibiotic concentration reaches steady state in serum after 3 days of therapy, the results of our analysis suggest that a mecA DNA level of 4.41 log copies/ml (range of mecA DNA level 3 to 5 days posttherapy in the blood culture-positive subgroup, 4.41 to 6.32 log copies/ml) is the lowest level that yields a positive blood culture result once the level of the anti-MRSA antibiotic in serum stabilizes. Further study with the simultaneous measurement of serum antibiotic levels is needed to elucidate the relationship of the serum antibiotic level to blood culture positivity and the mecA DNA level.

The levels of mecA DNA were significantly higher in the nonsurvivors than in the survivors during the three periods of bacteremia examined (Fig. 2E and F), and during these periods, the concomitant cultures of many blood samples might have become negative. Thus, the possibility that the mecA DNA detected in these samples was derived from dead or degraded bacteria rather than viable bacteria cannot be completely ruled out. A study comparing the amounts of Neisseria meningitidis in blood by quantitative PCR and culture simultaneously revealed that the DNA copy numbers were more than 2 log units higher than the numbers of CFU, suggesting the presence of significant amounts of nonviable bacteria (35). Therefore, interpretation of the mecA DNA level should be done with care and other clinical parameters should be taken into consideration. Nonetheless, it is worth noting that several recent studies have shown a good correlation between the bacterial DNA load and disease severity and/or outcome in patients with meningococcal and pneumococcal diseases, suggesting that the bacterial DNA load, although it is derived from both viable and dead bacteria, can be a clinically useful marker for the prediction of disease severity (4, 22, 35).

A recent study of the nosocomial MRSA strains in our hospital revealed that the predominant clone after 2003 was staphylococcal cassette chromosome mec type III and multilocus sequence type 239 (46). The MRSA strains from the study participants were mostly multiresistant to non-β-lactam antibiotics, a feature typical of the nosocomial MRSA strains in our hospital (45, 46). Analysis of the antimicrobial susceptibility profiles of the MRSA strains from nonsurvivors and those from survivors revealed no significant differences (Table 2). Although a shift in the predominant clone from 2003 to 2006 (the period of our study) and strain variation between the two groups cannot be completely ruled out, these differences would not affect our major findings that higher bacterial loads measured according to the mecA DNA levels correlated with a poor outcome. Specifically, higher levels of mecA DNA were found in the nonsurvivors than in the survivors after 3 days and 7 days of anti-MRSA therapy, suggesting that the levels of mecA DNA in blood posttherapy could potentially be used to evaluate the response to therapy and predict the outcome, especially in patients with persistent fever and/or slowly resolving symptoms. Future studies involving a larger number of patients with different clinical outcomes would validate and extend the findings of this study.

Acknowledgments

We thank Hui-Ling Chen, Mei-Ling Chen, Duckling Chen, and Chia-Ming Weng for technical assistance.

This work was supported by the National Science Council (grant NSC95-2745-B-002-007) of Taiwan.

Footnotes

▿

Published ahead of print on 11 March 2009.

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