Rapid detection of the staphylococcal mecA gene from BACTEC blood culture bottles by the polymerase chain reaction.

A rapid polymerase chain reaction (PCR) method for the direct detection of the staphylococcal mecA gene from BACTEC blood culture bottles (Becton Dickinson, Sparks, MD) was developed. Published primer sequences and sample preparation using Achromopeptidase for cell lysis were adapted to the use of the Idaho Technology Air Thermocycler 1605 (Idaho Technologies, Idaho Falls, ID). The method was validated with 80 strains of coagulase-positive and coagulase-negative geographically diverse methicillin-resistant and susceptible isolates of staphylococci. There was a 100% correlation between the PCR results and the results of standard susceptibility testing methods. From BACTEC 9240 blood cultures, mixed aliquots of blood and broth containing gram-positive cocci in clusters were centrifuged at low speed to sediment red blood cells. After additional centrifugation and wash steps, PCR was performed on the resuspended pellet. The turnaround time from initial Gram stain detection of positive BACTEC bottles to PCR amplicon detection by agarose gel electrophoresis is less than 3 hours. In a clinical evaluation of 181 blood culture isolates, there was a 99% correlation with standard susceptibility results for Staphylococcus aureus. Discrepant results for Staphylococcus aureus isolates were verified by a Mueller Hinton plate supplemented with 6 microg/mL of oxacillin and 2% sodium chloride. For coagulase-negative staphylococci, the PCR method detected an additional seven resistant isolates that were reported by the Vitek as susceptible. Coagulase-negative staphylococcal susceptibility results that were in disagreement with the PCR assay were confirmed by the disk-diffusion method. This procedure is accurate, rapid and fits well into laboratory work flow. Rapid detection of the mecA gene on positive blood culture vials has become a routine test in the authors' clinical microbiology laboratory.

Comparison of MIDI Sherlock system and pulsed-field gel electrophoresis in characterizing strains of methicillin-resistant Staphylococcus aureus from a recent hospital outbreak.

An outbreak of methicillin-resistant Staphylococcus aureus infections at the University of Utah Health Sciences Center occurred over a 7-month period. While the isolates phenotypically appeared to be similar in gross morphology and have similar Vitek antibiotic susceptibility patterns, two additional methods of strain characterization were evaluated to enhance the epidemiological investigation: pulsed-field gel electrophoresis and gas chromatography with the MIDI Sherlock system. Sherlock uses gas chromatography to qualitatively and quantitatively analyze the cellular fatty acid composition of organisms and creates two-dimensional plots based on principal-component analysis to define groups of closely related organisms. All isolates were also evaluated by digesting their chromosomal DNAs with the low-frequency-cutting enzyme SmaI and separating the restriction fragments by contour-clamped homogeneous electric field gel electrophoresis. Sample preparation for this pulsed-field gel electrophoresis included a novel cell lysis procedure involving achromopeptidase, greatly reducing the turnaround time. Isolates tested were recovered from the following: 45 suspected outbreak patients, 6 hospitalized patients believed to be unrelated to the outbreak, 6 patients from outside the hospital, and one health care practitioner implicated in the outbreak. Of 45 phenotypically similar suspect strains, 43 clustered tightly on the Sherlock two-dimensional plot. All outbreak patient isolates were also identical by pulsed-field gel electrophoresis with the exception of the same two outliers identified by Sherlock. In this epidemiologic investigation, we found an excellent correlation between the Sherlock and pulsed-field gel electrophoresis results for strain characterization of methicillin-resistant S. aureus.

Comparison of "gram-sure" with vancomycin disks in distinguishing between gram-negative and gram-positive rods.

A study was performed to compare Gram-Sure (JRGA Diagnostics, Rancho Dominguez, CA) with vancomycin disk susceptibility (5 micrograms and 30 micrograms) for clarification of the Gram reaction. Eighty-eight isolates representing 14 gram-negative and gram-positive genera were tested. Gram-Sure was superior to vancomycin susceptibility as a predictor of the Gram reaction in a select group of difficult organisms.