Rapid identification of staphylococcal strains from positive-testing blood culture bottles by internal transcribed spacer PCR followed by microchip gel electrophoresis

Abstract

PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) followed by microchip gel electrophoresis (MGE) was evaluated for its usefulness in identification of staphylococci. Forty ITS PCR patterns were demonstrated among 228 isolated colonies of Staphylococcus aureus: 26 patterns for methicillin-susceptible S. aureus (MSSA; 91 strains), 11 patterns for methicillin-resistant S. aureus (MRSA; 99 strains), and 3 patterns for both MSSA and MRSA (38 strains). Thirty-seven control strains of coagulase-negative staphylococci (CNS) representing 16 species showed unique ITS PCR patterns (24 patterns) at the species and subspecies levels: two patterns for S. caprae, S. cohnii, S. haemolyticus, and S. saprophyticus; three patterns for S. lugdunensis; four patterns for S. capitis; and one pattern for each of the other CNS species. The combined PCR-MGE method was prospectively adapted to the positive blood culture bottles, and this method correctly identified MSSA and MRSA in 102 (89%) of 114 blood cultures positive for S. aureus on the basis of the ITS PCR patterns. Eight ITS PCR patterns were demonstrated from 166 blood culture bottles positive for CNS. The most frequent CNS species isolated from blood cultures were S. epidermidis (76%), S. capitis (11%), and S. hominis (8%). Overall, all 280 blood culture bottles shown to contain a single Staphylococcus species by routine phenotypic methods were correctly identified by the PCR-MGE method at the species level, whereas the organism failed to be identified in 8 culture bottles (3%) with mixed flora. The PCR-MGE method is useful not only for rapid identification ( approximately 1.5 h) of staphylococci in positive blood culture bottles, but also for strain delineation of S. aureus.

FIG. 1.

Representative internal transcribed spacer PCR patterns of S. aureus strains. (A) MSSA K12614 (ITS PCR type, aur6); (B) MSSA K8912 (aur24); (C) MRSA K1456 (aur11); (D) MRSA K6783 (aur14); (E) MRSA K14137 (aur19); (F) MRSA K15719 (aur22). Asterisks indicate DNA size markers of 100 (left) and 1,000 (right) bp. Int., intensity.

FIG. 2.

Representative internal transcribed spacer PCR patterns of non-aureus staphylococcal strains. (A) S. capitis K12464 (ITS PCR type, cap3); (B) S. capitis K332 (cap2); (C) S. caprae K469 (car2); (D) S. epidermidis ATCC14990^T (epi1); (E) S. haemolyticus GTC290 (hae1); (F) S. hominis GTC485 (hom1). Asterisks indicate DNA size markers of 100 (left) and 1,000 (right) bp. Int., intensity.