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 (ϳ1.5 h) of staphylococci in positive blood culture bottles, but also for strain delineation of S. aureus.The coagulase-positive species Staphylococcus aureus has been well documented as an opportunistic human pathogen. Infections produced by S. aureus are often acute and pyogenic and, if left untreated, can spread to surrounding tissue or other organs. Identification of S. aureus in blood cultures begins with presumptive identification of gram-positive cocci in clusters (GPCC) in gram-stained smears of blood culture bottles, signaling a positive result, whereas final identification must await subculture and overnight incubation. Direct identification of methicillin-resistant S. aureus (MRSA) from GPCC-positive blood culture bottles may provide important diagnostic information, which would allow the selection of an appropriate course of treatment in a timely manner. A variety of rapid identification systems for direct identification of S. aureus from positive blood culture bottles have been evaluated, and although the overall specificity is excellent, a wide range of sensitivities have been reported (2, 32). Molecular techniques, such as gene probe hybridization assay (16), DNA sequencing of rRNA genes (25), fluorescence in situ hybridization (23), and PCR (18,19,35), have also been reported for the identification of S. aureus directly from positive blood cultures. Although these methods provide same-da...

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