Rapid Extraction from and Direct Identification in Clinical Samples of Methicillin-Resistant Staphylococci Using the PCR

Abstract

Methicillin-resistant staphylococci (MRS) are one of the most common causes of nosocomial infections and bacteremia. Standard bacterial identification and susceptibility testing frequently require as long as 72 h to report results, and there may be difficulty in rapidly and accurately identifying methicillin resistance. The use of the PCR is a rapid and simple process for the amplification of target DNA sequences, which can be used to identify and test bacteria for antimicrobial resistance. However, many sample preparation methods are unsuitable for PCR utilization in the clinical laboratory because they either are not cost-effective, take too long to perform, or do not provide a satisfactory DNA template for PCR. Our goal was to provide same-day results to facilitate rapid diagnosis and therapy. In this report, we describe a rapid method for extraction of bacterial DNA directly from blood culture bottles that gave quality DNA for PCR in as little as 20 min. We compared this extraction method to the standard QIAGEN method for turnaround time (TAT), cost, purity, and use of template in PCR. Specific identification of MRS was determined using intragenic primer sets for bacterial and Staphylococcus 16S rRNA and mecA gene sequences. The PCR primer sets were validated with 416 isolates of staphylococci, including methicillin-resistant Staphylococcus aureus (n ‫؍‬ 106), methicillin-sensitive S. aureus (n ‫؍‬ 134), and coagulase-negative Staphylococcus (n ‫؍‬ 176). The total supply cost of our extraction method and PCR was $2.15 per sample with a result TAT of less than 4 h. The methods described herein represent a rapid and accurate DNA extraction and PCR-based identification system, which makes the system an ideal candidate for use under austere field conditions and one that may have utility in the clinical laboratory.

Evaluation of methods for detecting oxacillin resistance in coagulase-negative staphylococci including cefoxitin disc diffusion

“…As the majority of methicillin-resistant staphylococci harbour the mecA gene, tests based on amplification, such as PCR or DNA hybridization, have been expected to identify even the most heterogeneous strains correctly (Jaffe et al, 2000). Although PCR is considered the best method for detecting oxacillin-resistant staphylococci, several studies have reported discrepancies showing that some strains do not harbour the mecA gene, but show phenotypic resistance to oxacillin.…”

Real‐time identification of Pseudomonas aeruginosa direct from clinical samples using a rapid extraction method and polymerase chain reaction (PCR)

“…This level of sensitivity for our BB+C method is lower than with our previous work with Methicillin-resistant staphylococci and in the range of other published extraction methods (6). Previ-ously, we determined that a 1 × 10 4 CFU/ml concentration of bacteria requires a 10.1 hour incubation to test positive in the BACTEC 9240 (13). In our experience, once the blood-culture bottle becomes positive (bacterial density 1.25-4.0 × 10 9 CFU/ml), we can detect P. aeruginosa with our system.…”

“…We have developed a procedure for rapid extraction of microorganism DNA directly from select clinical samples for molecular testing in our laboratory (13). Our mechanical lysis procedure generated DNA from the bacterial agent directly from the clinical sample within 20 minutes of sample submission.…”

“…The rapid extraction method, Bead Beating Plus CHELEX (BB+C), has been previously described (13). To test the method with Pseudomonas, each bacterial isolate was taken from an overnight subculture and resuspended into 1 ml of 1X phosphate buffered saline (PBS).…”

“…We have previously described a simple and rapid extraction method of bacteria DNA direct from clinical samples as well as a simple PCR procedure for the direct identification of Methicillin-Resistant Staphylococci (13). The entire procedure can be performed with a results TAT within 4 hours following the detection of positive blood-culture bottles or urine samples.…”