Identification and characterization of bacterial pathogens causing bloodstream infections by DNA microarray

Abstract

Bloodstream infections are potentially life-threatening and require rapid identification and antibiotic susceptibility testing of the causative pathogen in order to facilitate specific antimicrobial therapy. We developed a prototype DNA microarray for the identification and characterization of three important bacteremia-causing species: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The array consisted of 120 species-specific gene probes 200 to 800 bp in length that were amplified from recombinant plasmids. These probes represented genes encoding housekeeping proteins, virulence factors, and antibiotic resistance determinants. Evaluation with 42 clinical isolates, 3 reference strains, and 13 positive blood cultures revealed that the DNA microarray was highly specific in identifying S. aureus, E. coli, and P. aeruginosa strains and in discriminating them from closely related gram-positive and gram-negative bacterial strains also known to be etiological agents of bacteremia. We found a nearly perfect correlation between phenotypic antibiotic resistance determined by conventional susceptibility testing and genotypic antibiotic resistance by hybridization to the S. aureus resistance gene probes mecA (oxacillin-methicillin resistance), aacA-aphD (gentamicin resistance), ermA (erythromycin resistance), and blaZ (penicillin resistance) and the E. coli resistance gene probes blaTEM-106 (penicillin resistance) and aacC2 (aminoglycoside resistance). Furthermore, antibiotic resistance and virulence gene probes permitted genotypic discrimination within a species. This novel DNA microarray demonstrates the feasibility of simultaneously identifying and characterizing bacteria in blood cultures without prior amplification of target DNA or preidentification of the pathogen.

FIG. 1.

Workflow for hybridization assay used with the prototype microarray. (A) For DNA-chip construction, capture probes were produced by PCR amplification of plasmid-cloned gene segments, followed by ethanol precipitation. Purified probes were deposited onto glass slides by robotic printing. (B) For hybridization assays, bacterial target DNA was extracted from positive blood cultures, clinical isolates, or reference strains and then labeled with fluorescent dyes and hybridized to the spotted DNA capture probes. Images of fluorescent, hybridized probes were acquired by using a laser scanner and processed by computer analysis.

FIG.2.

DNA microarray analyses of 42 clinical isolates, 3 reference strains, and 13 blood cultures. Each column shows the results of an individual hybridization with target DNA prepared from: S. aureus ATCC 29213 (column 1), MW2 (column 2), clinical isolates (columns 3 to 7), and positive blood cultures (columns 8 to 11); P. aeruginosa ATCC 27853 (column 12), clinical isolates (columns 13 to 17), and positive blood cultures (column 18); E. coli ATCC 25922 (column 19), clinical isolates (columns 20 to 25), and positive blood cultures (columns 26 and 27); S. epidermidis clinical isolates (columns 28 to 32) and blood cultures (columns 33 to 35); and clinical isolates of CoNS S. auricularis (column 36), S. capitis (column 37), S. haemolyticus (column 38), S. hominis (column 39), and S. warneri (column 40). Other gram-negative species included a Proteus mirabilis positive blood culture (column 41), clinical isolates of Proteus mirabilis (columns 42 and 43), Serratia marcescens (columns 44 and 45), Klebsiella pneumonia (columns 46 to 48), Stenotrophomonas maltophilia (column 49), Acinetobacter baumannii (column 50), Enterobacter cloacae (column 51), and Enterobacter aerogenes (column 52). Other gram-positive species included clinical isolates of Micrococcus spp. (column 53), Enterococcus spp. (column 54), Enterococcus faecalis (column 55), and Streptococcus pneumoniae (column 56) and two positive blood cultures of S. pneumoniae (columns 57 and 58). (A) Hybridization of DNA prepared from bacterial isolates, reference strains, and blood cultures with E. coli gene probes; (B) hybridization with P. aeruginosa gene probes; (C) hybridization with S. aureus gene probes. Gray boxes represent gene probes that hybridized with the respective target DNA; white boxes represent gene probes that showed no hybridization with the respective target DNA. Experiments performed with positive blood cultures are indicated (BC).

FIG.2.

DNA microarray analyses of 42 clinical isolates, 3 reference strains, and 13 blood cultures. Each column shows the results of an individual hybridization with target DNA prepared from: S. aureus ATCC 29213 (column 1), MW2 (column 2), clinical isolates (columns 3 to 7), and positive blood cultures (columns 8 to 11); P. aeruginosa ATCC 27853 (column 12), clinical isolates (columns 13 to 17), and positive blood cultures (column 18); E. coli ATCC 25922 (column 19), clinical isolates (columns 20 to 25), and positive blood cultures (columns 26 and 27); S. epidermidis clinical isolates (columns 28 to 32) and blood cultures (columns 33 to 35); and clinical isolates of CoNS S. auricularis (column 36), S. capitis (column 37), S. haemolyticus (column 38), S. hominis (column 39), and S. warneri (column 40). Other gram-negative species included a Proteus mirabilis positive blood culture (column 41), clinical isolates of Proteus mirabilis (columns 42 and 43), Serratia marcescens (columns 44 and 45), Klebsiella pneumonia (columns 46 to 48), Stenotrophomonas maltophilia (column 49), Acinetobacter baumannii (column 50), Enterobacter cloacae (column 51), and Enterobacter aerogenes (column 52). Other gram-positive species included clinical isolates of Micrococcus spp. (column 53), Enterococcus spp. (column 54), Enterococcus faecalis (column 55), and Streptococcus pneumoniae (column 56) and two positive blood cultures of S. pneumoniae (columns 57 and 58). (A) Hybridization of DNA prepared from bacterial isolates, reference strains, and blood cultures with E. coli gene probes; (B) hybridization with P. aeruginosa gene probes; (C) hybridization with S. aureus gene probes. Gray boxes represent gene probes that hybridized with the respective target DNA; white boxes represent gene probes that showed no hybridization with the respective target DNA. Experiments performed with positive blood cultures are indicated (BC).