We have developed a procedure for rapid extraction of microorganism DNA directly from select clinical samples for molecular testing in our laboratory. Our mechanical lysis procedure generated DNA from the bacterial agent directly from the clinical sample within 20 min of sample submission. Significant progress has been made in the development of commercial extraction kits that can be used for rapid nucleic acid extraction from microbial cultures for PCR. However, they require multiple steps (5 to 40) and extended times (15 to 150 min). They may require a pure culture and may be cost-prohibitive for large numbers of samples (Dion et al., Abstr. 99th Gen. Meet. Am. Soc. Microbiol.). The QIAGEN procedure for DNA extraction, against which our preparation method was compared, requires the use of these lysozyme, lysostaphin, and proteinase K enzymes specifically with gram-positive organisms (33
). The most important component of an extraction method is the ability to obtain quality DNA for PCR. Even with the wide range of concentrations from the five S. aureus
strains, the DNA was easily amplified by standard PCR and the target sites selected were readily amplified using a battery of primer sets that specifically identified MRSA from different sample sources (Fig. ). The results were available in less than 4 h, confirming the identification of the microorganism as well as determining the presence of antibiotic resistance markers.
Our BB+C method does have one significant limitation. The minimum amount of organism determined to be necessary for extraction of the DNA and detection of the target sequences by PCR was 109 CFU/ml. However, when E. coli ATCC 25922 was tested, the target sequences could be amplified utilizing only 103 CFU/ml. The QIAGEN procedure states that its lower limit of detection is 103 CFU/ml; however, we could not duplicate this with our S. aureus strain. Nevertheless, our results indicate that a 104-CFU/ml concentration of bacteria requires a 10.1-h incubation to test positive in the BACTEC 9240 system. In our experience, once the blood culture bottle becomes positive (bacterial density, 1.25 × 109 to 4.0 × 109 CFU/ml), we can detect MRS with our system. However, in our clinical microbiology laboratory, the blood culture bottles are monitored continuously but the positives are not worked up until the next morning. When we tested our positive blood culture bottles the next day, all had more than 109 CFU/ml. Therefore, there will be a sufficient concentration of bacteria for extraction and detection by our method.
The cost of the rapid extraction and PCR-based method is affordable, and setup is readily applicable to the clinical laboratory. Standard identification methods, which include VITEK cards, media, inoculating loops, antibiotic disks, and reagents, can cost more than $7.00 per sample for confirmation. If it is determined that the identity of a clinical isolate with a positive blood culture bottle result needs to be confirmed using the MRS primer set (bacterial and Staphylococcus 16S rRNA gene and mecA gene), the cost could be less than that of standard identification. The cost per test for the RTG tubes is $1.50/tube plus $0.10 for the set of primers, totaling $1.60/PCR for each primer set. If a panel consisting of three primer sets were to be used, then the cost would be $1.60 × 3 (for three different primer sets for identification), or $4.80. Adding the cost of the BB+C extraction method ($0.55/extraction) makes the total cost per test $5.35 (Table ). Technician time can also be calculated to include exact sample processing time. One sample takes approximately 30 min for DNA extraction and PCR setup. Electrophoresis setup is about 10 min. If five samples were to be tested the DNA extraction and PCR setup time would be under 40 min for all five samples. Therefore, the reported method was determined to be both time- and cost-effective compared to standard clinical procedures.
TABLE 3 Cost comparison of rapid extractionmethods
The presence of the mecA
gene, as detected by our PCR procedures, had a 99% agreement with clinical findings of methicillin resistance in the 416 Staphylococcus
isolates tested. Interestingly, during the validation process we determined that there were two isolates that were classified as MSSA and two isolates classified as OxSCoNS by the MicroScan system in which we were able to detect the presence of the mecA
gene by PCR. Upon further testing of these four isolates by broth microdilution, it was shown that all of these samples were phenotypically methicillin and oxacillin resistant. There were four additional isolates classified as OxRCoNS in which we were not able to detect the presence of the mecA
gene, but all of these isolates were positive by broth microdilution for methicillin and oxacillin resistance (MIC, 8 μg/ml). Further investigation of these four OxRCoNS isolates lacking mecA
revealed that they produced β-lactamase and were all susceptible to amoxicillin-clavulanate. In addition, one isolate classified as OxSCoNS also lacked mecA
but was broth microdilution positive. Borderline resistant strains that do not contain the mecA
gene have been hypothesized to result from modification of normal PBP genes or overproduction of staphylococcal β-lactamase (8
). However, the role of β-lactamase overproduction in borderline resistance is less clear, and no clinical data have suggested that the level of resistance expressed by borderline resistant strains lacking mecA
leads to treatment failure (8
). Further testing revealed that two of the four OxRCoNS isolates lacking mecA
were species of the genus Micrococcus
, which is known to be more closely related to Arthrobacter
, a genus of environmental coryneforms, than to Staphylococcus
). Therefore, the PCR method did discriminate between high-level β-lactam (methicillin) resistance by the presence of the mecA
gene in Staphylococcus
isolates and related strains that were β-lactamase producers only.
PCR detection of mecA
should be considered as the potential “gold standard” for staphylococcal methicillin resistance. Previous studies have reported discrepancies, noting that some strains lacking mecA
displayed phenotypic resistance to methicillin while others containing mecA
showed phenotypic susceptibility (1
). Additionally, mecA
transcriptional activity does not correlate with phenotypic methicillin resistance (31
). Until new sets of recommendations are established, a combination of methods should be used routinely in detecting MRSA and OxRCoNS (8
A rapid PCR method that utilizes capillary air thermal cyclers to improve TAT has been published (7
). Air-thermocycling–real-time PCR holds great promise as a rapid diagnostic tool, but instrumentation cost may be prohibitive, being 10 times that of regular thermocyclers. We further investigated this approach and recently presented research data utilizing this new technology with our rapid extraction method (D. M. Niemeyer, G. Veltri, and R. I. Jaffe, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 876, 1999). Our procedure took less than 40 min from DNA extraction to confirmation with our method specific for methicillin resistance and had a 100% correlation with methicillin sensitivity testing and mecA
determination by standard PCR.
The sensitivity of PCR coupled with the speed of our procedure can assist the provider in making a prudent and timely selection of chemotherapeutic agents. This approach for identifying antibiotic resistance markers has great potential for augmenting standard microbiological methods. The primary value of the rapid testing approach is that it will work well where standard microbiological testing capabilities are limited but agent identification is critical (remote, deployed medical facilities), although it may find good utility in clinical laboratories in the near future as these laboratories incorporate PCR into their normal work flow. We evaluated a field PCR laboratory set up in both a deployable medical system at an army reserve training facility in Dublin, Calif., and an air force field hospital (30
; D. M. Niemeyer, M. Dempsey, W. Hamilton, J. McAvy, J. Benjack, J. Ruiz, L. Lim, and K. Lohman, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1560, 1999). Additionally, to continue evaluation of real-time PCR use, an air force laboratory has been set up in Southwest Asia (D. M. Niemeyer, M. Corkern, W. J. Barnes, D. White, W. Johnson, M. Dempsey, and K. Lohman, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., 2000). In many military field hospitals, microbiological testing capabilities are limited and may not include standard culture and identification capabilities. As such, routine samples are shipped to regional or stateside reference laboratories for testing, which increases result TAT to a week or longer. PCR would provide select on-site preliminary test capabilities to assist the health care provider in patient treatment decisions under work conditions that normally may not afford standard microbiological testing.