PCR detection and identification of bacterial contaminants in ocular samples from post-operative endophthalmitis

Bacterial endophthalmitis is a sight-threatening complication of ocular surgery which requires urgent medical consideration including comprehensive diagnosis. Polymerase chain reaction (PCR) as a sensitive molecular method has been extensively used for detection of microbial species in clinical specimens.

The aim of this study was to identify the causative organisms of endophthalmitis in our patient population using a procedure based on PCR.

Vitreous samples from 32 patients with post-operative endophthalmitis were collected. Total vitreous DNA was extracted and then assessed by agarose gel electrophoresis. Bacterial 16S rRNA gene was amplified from genomic DNA using PCR with a pair of HAD2 universal primers. Library of PCR products from 16S rRNA, cloned into the pTZ57R/T vector. The ligated products were then transformed into E. coli DH5α strain and grown in the LB-ampicillin/X-Gal/IPTG plate.

From the total of 32 vitreous samples, 18 specimens were positive, illustrating the presence of bacterial infection (56.4 %). Twelve species including Escherichia coli, Enterobacter cloacae, Bacillus subtilis, Neisseria gonorrhoeae, Streptococcus pneumoniae, Haemophilus influenzae, Chlamydia trachomatis, Staphylococcus aureus, Neisseria meningitides, Staphylococcus epidermidis, Pseudomonas aeruginosa and Bacillus cereus were identified using BLAST for known 16S rRNA sequences.

Polymerase chain reaction (PCR) accompanied with cloning and sequencing approved to be sensitive and specific. The rapid molecular technique was useful in detection of 12 major microbial species, in infectious endophthalmitis.