Development of a DNA microarray assay for rapid detection of fifteen bacterial pathogens in pneumonia

Abstract

Background: The rapid identification of pathogenic bacteria is important for determining an appropriate antimicrobial therapy for pneumonia, but traditional bacterial culture is time-consuming and labourious. The aim of this study was to develop and evaluate a DNA microarray assay for the simultaneous detection of fifteen bacterial species directly from respiratory tract specimens in patients with pneumonia. These species included Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Mycoplasma pneumoniae, Enterococcus faecalis, Enterococcus faecium, Enterobacter cloacae, Stenotrophomonas maltophilia, Burkholderia cepacia, Legionella pneumophila and Chlamydia pneumoniae. The 16S rDNA genes and other specific genes of each pathogen were chosen as the amplification targets, amplified via multiplex polymerase chain reaction (PCR), and hybridized to oligonucleotide probes in a microarray.

Results: The DNA microarray detection limit was 10³ copies/μL. Nineteen standard strains and 119 clinical isolates were correctly detected with our microarray, and 3 nontarget species from 4 clinical isolates were not detected. Additionally, bacterial pathogens were accurately identified when two or three bacterial targets were mixed together. Furthermore, the results for 99.4% (156/157) of clinical specimens were the same as those from a conventional assay.

Conclusions: We developed a DNA microarray that could simultaneously detect various bacterial pathogens in pneumonia. The method described here has the potential to provide considerable labour and time savings due to its ability to screen for 15 bacterial pathogens simultaneously.

Keywords: Bacteria; DNA microarray; Pneumonia.

Fig. 1

a. The layout of the hybridization capture-chip. The probe 20 T is the QC probe. The probe N1, N2, N3 are the negative control probes. The probe P is the universal 16S rDNA probe. Each probe was spotted as two. The sequences of probe 1–13 all come from 16S rDNA and their corresponding target pathogen were: 1 Acinetobacter baumannii; 2 Streptococcus pneumoniae; 3 Haemophilus influenzae; 4 Pseudomonas aeruginosa; 5 Mycoplasma pneumoniae; 6 Staphylococcus aureus; 7 Burkholderia cepacia; 8 Stenotrophomonas maltophilia; 9 Enterococcus faecalis; 10 Chlamydia pneumoniae; 11 Klebsiella pneumoniae or Enterobacter cloacae or Escherichia coli; 12 Enterococcus faecium; 13 Legionella pneumophila, respectively. b. The typical hybridization results of fifteen species of bacterial pathogens in pneumonia, non-target bacteria from pure bacterial cultures and ddH2O

Fig. 2

The sensitivity of the pathogen probes. Microarray hybridized with PCR products which diluted for concentration gradient. 10 μL dilution used in each well, and the concentration of probes were 50 μM

Fig. 3

The specificity of the pathogen probes. a Microarray hybridized with PCR products amplified from mixed plasmid DNAs. b The hybridization results of clinical samples which contains two or more target pathogens