Species-specific and ubiquitous-DNA-based assays for rapid identification of Staphylococcus aureus

Abstract

Staphylococcus aureus is the cause of serious infections in humans, including endocarditis, deep-seated abscesses, and bacteremia, which lead to toxic and septic shock syndromes. Rapid and direct identification of this bacterium specifically and ubiquitously directly from clinical specimens would be useful in improving the diagnosis of S. aureus infections in the clinical microbiology laboratory. A wide variety of kits based on biochemical characteristics efficiently identify S. aureus, but the rapidity and the accuracy of each of these methods combined with testing of clinically relevant antibiotic resistance genes need to be improved. On the basis of hybridization assays with randomly selected clones from an S. aureus genomic library, we have identified a chromosomal DNA fragment which is specific for S. aureus and which detected all 82 S. aureus isolates tested. This 442-bp fragment was sequenced and was used to design a set of PCR amplification primers. The PCR assay was also specific and ubiquitous for the identification from bacterial cultures of 195 clinical strains of S. aureus isolated from a variety of anatomical sites and obtained from hospitals throughout the world. The PCR assay that we have developed is simple and can be performed in about 1 h. This DNA-based test provides a novel diagnostic tool for the diagnosis of S. aureus infections.

FIG. 1

Test for specificity by dot blot hybridization by using DNAs from a variety of bacterial species (Table 1) as targets. (A) Test for specificity with the ³²P-labeled, S. aureus-specific, 442-bp DNA fragment as a probe. (B) Example of a test for specificity performed with a nonspecific S. aureus genomic DNA probe. For panels A and B, a variety of gram-positive bacterial species (Table 1) were used as targets (locations 1A to 8D). DNAs from S. aureus isolates are at locations 9D (ATCC 43300), 10D (ATCC 33593), 11D (ATCC 29213), and 12D (ATCC 25923) in panels A and B. (C) Test for specificity with the ³²P-labeled, S. aureus-specific, 442-bp DNA fragment as a probe and DNAs from a variety of gram-negative bacterial species (Table 1) as targets (locations 1A to 11D). Genomic DNA from an S. aureus isolate (ATCC 25923) was spotted at location 12D.

FIG. 2

Ubiquity test by dot blot hybridization by using the ³²P-labeled, S. aureus-specific, 442-bp DNA fragment as a probe and DNAs from 82 clinical isolates of S. aureus as targets. DNAs from the S. aureus strains are at locations 1A (ATCC 43300), 12A (ATCC 25923), and 1B to 8H (80 clinical strains from CHUL). A battery of eight different staphylococcal species including S. epidermidis, S. saprophyticus, S. simulans, S. lugdunensis, S. haemolyticus, S. hominis, S. warneri, and S. schleiferi were used as negative controls (locations 3A to 10A, respectively). Micrococcus luteus was also used as a negative control (location 11A). DNA from transformed E. coli DH5α carrying the recombinant plasmid pGEM-Sa442 was spotted at location 2A. No DNA was spotted at locations 9H to 12H.

FIG. 3

Multiplex PCR amplification with the S. aureus-specific PCR primer pair and the universal primers, which were used to provide an internal control. PCR assays were performed with 1 μl of a bacterial suspension whose turbidity was adjusted to that of a 0.5 McFarland standard prepared from a variety of reference strains or from clinical isolates from CHUL. Lanes: 2, S. aureus ATCC 33591; 3, S. aureus ATCC 33592; 4, S. aureus ATCC 33593; 5, S. aureus ATCC 43300; 6, S. aureus ATCC 25923; 7, S. aureus ATCC 13301; 8, S. aureus ATCC 29213; 9, S. aureus ATCC 27660; 10, S. saprophyticus ATCC 15305; 11, S. epidermidis ATCC 14990; 12, S. haemolyticus ATCC 29970; 13, S. hominis ATCC 27844; 14, S. simulans ATCC 27848; 15, S. lugdunensis ATCC 43809; 16, S. capitis subsp. ureolyticus ATCC 49326; 17, S. schleiferi subsp. coagulans ATCC 49545; 18, S. auricularis ATCC 33753; 19, S. cohnii subsp. urealyticum DSM 20260; 20, S. warneri ATCC 27836; 21, S. sciuri subsp. sciuri ATCC 29060; 22, S. xylosus LSPQ 2517; 23, M. luteus ATCC 9341; 24, S. pneumoniae ATCC 27336; 25, S. pyogenes ATCC 19615; 26, E. faecalis ATCC 29212; 27, E. faecium ATCC 51559. Lanes 1 and 28, controls to which no DNA was added; lanes M, 50-bp molecular size standard ladder.