点击显示 收起
Department of Microbiology, Royal Prince Alfred Hospital, Missenden Rd., Camperdown 2050, NSW, Australia
ABSTRACT
PCR allows rapid determination of both the presence of Staphylococcus aureus and of methicillin resistance. I present here a simple method for direct extraction from blood cultures or colonies for use in real-time PCR, eliminating tedious, time-consuming methods of DNA extraction which may otherwise deter microbiology labs from offering PCR.
TEXT
Bacteremia with Staphylococcus aureus results in significant mortality in both nosocomial and community-acquired infection (3). There are many published assays for the detection of S. aureus and methicillin resistance by PCR (6, 9, 10, 11-14). PCR enables an earlier determination of methicillin resistance by detecting mecA, which has been called a "gold standard" for the detection of the presence of methicillin resistance in staphylococci (4, 7), and confirming the presence of S. aureus. In this study, the presence of S. aureus was determined by detecting femASA, an S. aureus-specific gene (4). These methods allow earlier commencement of correct treatment, as glycopeptide therapy is thought less effective for methicillin-sensitive S. aureus than penicillinase-resistant -lactam therapy (8). Earlier and appropriate therapy for S. aureus bacteremia reduces mortality (15). Non-culture-based determination of the organism in blood cultures which have tested positive with gram-positive organisms in clusters would enable appropriate therapy to be ensured at least 18 h earlier. PCR from colonies allows earlier determination of methicillin resistance than determinations by culture.
For effective PCR, DNA has to be extracted from inhibitors of the polymerase, and in real-time applications, opaque material, which affects the reading of fluorescence, must be extracted. DNA extraction can be tedious and labor-intensive and may prevent the implementation of PCR in a microbiology laboratory. In our laboratory, we use charcoal and sodium polyanetholesulfonic acid (SPS)-containing blood culture bottles (BacT/ALERT FA, FN, and PF). With a Rotorgene RG3000 (Corbett Research, Mortlake, NSW, Australia) real-time thermocycler, which has a rotating carousel containing the reaction tubes inclined outward, small amounts of charcoal spin to the bottom just below where the fluorescence is read, allowing diluted blood culture material to be processed without charcoal removal.
After demonstrating no inhibition with PCR directly performed on 0.1-μl samples of acetone-washed, dried blood culture streaks of 98 blood cultures of staphylococci on chocolate agar plates (data not shown), the following extraction method was undertaken. DNA was extracted from 17 blood culture bottles where the Gram stain suggested staphylococci as follows. Four drops of blood culture fluid were diluted into 4 ml of peptone water; a 1-μl loop of this material was placed into 20 to 25 μl of thawed prealiquoted master mix in a 200-μl tube. This was placed into the thermocycler, with the first step consisting of 15 min at 95°C, serving to both extract the DNA and activate the polymerase. This was followed by 35 cycles of 15 seconds at 95°C and 30 seconds at 60°C. Suspensions with a 0.5 McFarland standard in peptone water were also made from 102 colonies from clinical isolates, which were processed as described above. The master mix was made of Immomix (Bioline, London, United Kingdom), a 250 nM concentration of primers and a 100 nM concentration of 6-carboxytetramethylrhodamine-quenched dual-labeled probes for the detection of femASA (6-carboxyfluorescein) and mecA (JOE [6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein]) as used by Francois et al. (4). Controls were used for each assay. A comparison was made between the culture identification of S. aureus and a method using DNase and mannitol salt agar plates and BactiStaph latex (Remel, Lenexa, KS), and a comparison was made between the culture identification of methicillin resistance and a method using microdilution on Mueller-Hinton agar with 2% NaCl testing with oxacillin at 0.5 and 2.0 mg/liter.
Of the 119 specimens, the results for 5 were indeterminate due to low starting fluorescence readings on acquisition of data prior to cycling; these results were noted for underfilled tubes for which an air bubble at the tip of the tube was thought to be the cause.
Table 1 shows the comparison of PCR results with culture results. S. aureus was correctly detected for all but one isolate, and the result for the one isolate occurred as the result of a specimen interchange. Methicillin resistance was correctly detected for all but one S. aureus isolate, for which femASA was detected, indicating that there was no extraction problem, and for which resistance was still not detected when the mecA assay was run alone, indicating that this was not due to competition with the femASA PCR.
In the interpretation of blood cultures, mixed cultures of S. aureus and methicillin-resistant coagulase-negative staphylococci (CoNS) carry both genes and seem to be methicillin-resistant S. aureus (MRSA) strains. This is uncommon; in our microbiology laboratory over the last 4 years, only 4 of 340 blood culture sets with methicillin-sensitive S. aureus were mixed with CoNS (the majority of which are methicillin resistant).
The PCR assay used detects products by cleavage of target annealed dual-labeled probes by polymerase 5'-exonuclease activity during extension. Although S. aureus produces a thermonuclease, hydrolysis of probe was not observed, likely due to the elevated cycling temperatures and prompt loading of samples on the thermocycler once they were mixed into the master mix.
Although many inhibitors of PCR, including immunoglobulin G, heme, lactoferrin, SPS, and leukocyte DNA, are known, the degrees of inhibition differ with the polymerase used (1, 2). Al-Soud et al. demonstrated that the polymerase rTth had 50 times more tolerance to immunoglobulin G than some of the other tested polymerases (1). They also reported that rTth could still amplify in the presence of 20% (vol/vol) blood (1). Only one of the polymerases they tested was a hot-start, and so it is not clear how the newer polymerases are affected by these inhibitors (2).
Peptone water dilution was used, as this dilution is used in the laboratory for organism identification by using replicator plates. The PCR amplification of blood cultures diluted in peptone water demonstrates that SPS and inhibitors from the patient's blood are not major inhibitors of the polymerase used. The dilution of the blood cultures would be about 400 times, significantly less diluted than the dilution of 5,000 times that Fredericks and Relman showed would be needed to prevent inhibition with the polymerase they tested (5).
This study shows simple extraction methods are possible where an organism is plentiful, such as in colonies and signaled blood cultures. In this study, it was the presence of Staphylococcus aureus and of methicillin resistance that was sought, but the presence of other genes could likewise be determined.
ACKNOWLEDGMENTS
This study was done as part of the evaluation of PCR in rapid assessment of staphylococci. No funding was received from any of the companies listed above; funding was solely from the microbiology department.
I thank the laboratory scientific and technical staff in making specimens available for processing.
Mailing address: Department of Microbiology, Royal Prince Alfred Hospital, Missenden Rd., Camperdown 2050, NSW, Australia. Phone: 61 2 95158278. Fax: 61-2 95155235. E-mail: nick.adams@email.cs.nsw.gov.au.
REFERENCES
Al-Soud, W. A., L. J. Jnsson, and P. Rdstrm. 2000. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. J. Clin. Microbiol. 38:345-350.
Al-Soud, W. A., and P. Rdstrm. 2001. Purification and characterization of PCR-inhibitory components in blood cells. J. Clin. Microbiol. 39:485-493.
Cosgrove, S. E., G. Sakuolas, E. N. Perencevich, M. J. Schwaber, A. W. Karchmer, and Y. Carmeli. 2003. Comparison of mortality associated with methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteremia: a meta-analysis. Clin. Infect. Dis. 36:53-59.
Francois, P., D. Pittet, M. Bento, B. Pepey, P. Vandaux, D. Lew, and J. Schrenzel. 2003. Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or nonsterile clinical samples by a new molecular assay. J. Clin. Microbiol. 41:254-260.
Fredricks, D. N., and D. A. Relman. 1998. Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate. J. Clin. Microbiol. 36:2810-2816.
Grisold, A. J., E. Leitner, G. Mühlbauer, E. Marth, and H. H. Kessler. 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.
Hardy, K. J., P. M. Hawkey, F. Gao, and B. A. Oppenheim. 2004. Methicillin resistant Staphylococcus aureus in the critically ill. Br. J. Anaesth. 92:121-130.
Hussain, Z., L. Stoakes, M. A. John, S. Garrow, and V. Fitzgerald. 2002. Detection of methicillin resistance in primary blood culture isolates of coagulase-negative staphylococci by PCR, slide agglutination, disk diffusion, and a commercial method. J. Clin. Microbiol. 40:2251-2253.
Jaffe, R. I., J. D. Lane, S. V. Albury, and D. M. Niemeyer. 2000. Rapid extraction from and direct identification in clinical samples of methicillin-resistant staphylococci using the PCR. J. Clin. Microbiol. 38:3407-3412.
Lem, P., J. Spiegelman, B. Toye, and K. Ramotar. 2001. Direct detection of mecA, nuc and 16S rRNA genes in BacT/Alert blood culture bottles. Diagn. Microbiol. Infect. Dis. 41:165-168.
Louie, L., J. Goodfellow, P. Mathieu, A. Glatt, M. Louie, and A. E. Simor. 2002. Rapid detection of methicillin-resistant staphylococci from blood culture bottles by using a multiplex PCR assay. J. Clin. Microbiol. 40:2786-2790.
Maes, N., J. Magdalena, S. Rottiers, Y. De Gheldre, and M. J. Struelens. 2002. Evaluation of a triplex PCR assay to discriminate Staphylococcus aureus from coagulase-negative staphylococci and determine methicillin resistance from blood cultures. J. Clin. Microbiol. 40:1514-1517.
Reischl, U., H. Lind, M. Metz, B. Leppmeier, and N. Lehn. 2000. Rapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR. J. Clin. Microbiol. 38:2429-2433.
Shrestha, N. K., M. J. Tuohy, G. S. Hall, C. M. Isada, and G. W. Procop. 2002. Rapid identification of Staphylococcus aureus and the mecA gene from BacT/ALERT blood culture bottles using the LightCycler system. J. Clin. Microbiol. 40:2659-2661.
Tan, T. Y., S. Corden, R. Barnes, and B. Cookson. 2001. Rapid identification of methicillin-resistant Staphylococcus aureus from positive blood cultures by real-time fluorescence PCR. J. Clin. Microbiol. 39:4529-4531.