Multiplex PCR protocol for the diagnosis of staphylococcal infection

Abstract

We report the development of a multiplex PCR protocol for the diagnosis of staphylococcal infection. The protocol was designed to (i) detect any staphylococcal species to the exclusion of other bacterial pathogens (based on primers corresponding to Staphylococcus-specific regions of the 16S rRNA genes), (ii) distinguish between S. aureus and the coagulase-negative staphylococci (CNS) (based on amplification of the S. aureus-specific clfA gene), and (iii) provide an indication of the likelihood that the staphylococci present in the specimen are resistant to oxacillin (based on amplification of the mecA gene). The expected fragments were amplified from each of 60 staphylococcal isolates (13 oxacillin-resistant S. aureus isolates, 23 oxacillin-sensitive S. aureus isolates, 17 oxacillin-resistant CNS, and 7 oxacillin-sensitive CNS). No amplification products were observed with template DNA from nonstaphylococcal species, and the efficiency of amplification of staphylococcal targets was not adversely affected by the presence of DNA from other bacterial species in the same sample. The utility of the protocol for the analysis of clinical samples was verified by analysis of aliquots taken directly from BacT/Alert blood culture bottles. Of 77 blood cultures tested, only 7 yielded results inconsistent with those of conventional methods of diagnosis and susceptibility testing. Of those, one was identified as a CNS species by PCR and S. aureus by conventional methods. We also identified two isolates that were mecA positive but were oxacillin sensitive according to conventional methods. The other four samples failed to yield any amplification product even with a control set of primers corresponding to a conserved region of the eubacterial rRNA genes.

FIG. 1

Specificity of multiplex PCR. Template DNA was isolated from TSB cultures of the indicated bacteria and subjected to PCR as described in the text. The approximate number of bacteria in the starting sample was 5 × 10⁸ CFU. MT refers to cases in which template DNA was derived from mixed cultures of bacteria containing all of the nonstaphylococcal species with or without the ORSA strain UAMS-601. MS refers to those cases in which template DNA was derived from pure cultures of each nonstaphylococcal species and then mixed prior to analysis with or without template DNA from UAMS-601. (Top) Multiplex PCR utilizing primers for staphylococcal rRNA, clfA, and mecA; (bottom) PCR using primers for conserved regions of eubacterial rRNA genes. Lane M, molecular size markers. Molecular sizes (in kilobases) are on the right.

FIG. 2

Specificity of blood culture PCR. Blood culture bottles were inoculated with approximately 10 OSSA, ORSA, OSCNS, or ORCNS isolates. Aliquots were processed for template DNA after the culture was identified as positive as described in the text. Lane M, molecular size markers. Approximate sizes (in kilobases) of the amplification products are on the left.

FIG. 3

Sensitivity of blood culture PCR. A blood culture bottle containing 10 ml of venous blood was inoculated with the ORSA strain UAMS-601 and incubated for 15 h at 37°C. Serial dilutions were prepared using medium from a sterile blood culture as a diluent. DNA isolated from a 1-ml aliquot of cultures containing the indicated number of viable bacteria was subjected to PCR. Lane C, positive control with template DNA derived from a TSB culture of UAMS-601; lane M, molecular size markers. Molecular sizes (in kilobases) are on the left.

FIG. 4

Analysis of blood cultures by PCR. Template DNA was isolated from positive blood cultures obtained from the clinical laboratory prior to phenotypic characterization of the bacteria present in the sample. The results shown were chosen because they include all four classes of staphylococci (ORSA, OSSA, ORCNS, and OSCNS) and representative nonstaphylococcal species (SV, viridans group streptococci; PA, P. aeruginosa). (Top) Results obtained with the multiplex protocol; (bottom) results obtained with the eubacterial primers. Lane M, molecular size markers. Molecular sizes (in kilobases) are on the left.