PCR-RFLP-mediated detection and speciation of bacterial species causing endophthalmitis.
OBJECTIVE
To determine the usefulness of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in the identification and speciation of bacteria causing endophthalmitis.


METHODS
PCR-RFLP was performed on 53 strains of 14 bacterial species (eight Gram positive and five Gram negative) collected from both keratitis and endophthalmitis patients. Two pairs of oligonucleotide primers based on the 16S rDNA gene were used to PCR-amplify 1.2- and 1.0-kb fragments of bacterial genomic DNA. RFLPs within the PCR product were used to speciate the organisms.


RESULTS
The sensitivity of the nested PCR amplification reaction was one organism. All bacteria tested could be identified and speciated using RFLP analysis except for Escherichia coli and Serratia marcescens, which could not be interdifferentiated using RFLP. Molecular analysis of two vitreous samples from two eyes with typical signs of bacterial endophthalmitis confirmed the presence of E. coli in the vitreous from a culture-positive case with E. coli endophthalmitis and revealed the presence of Staphylococcus epidermidis in the vitreous of a culture-negative case.


CONCLUSIONS
It is expected that this technique will provide a useful laboratory tool for future microbiologic diagnosis of patients presenting with endophthalmitis, especially for those eyes that prove culture negative.
Okhravi
N
N
Department of Clinical Ophthalmology, The Institute of Ophthalmology and University College London, United Kingdom. nokhravi@hgmp.mrc.ac.uk
Adamson
P
P
Matheson
M M
MM
Towler
H M
HM
Lightman
S
S
D015373:Bacterial Typing Techniques; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000032:analysis; D004271:DNA, Fungal / Q000032:analysis; D004275:DNA, Ribosomal / Q000235:genetics; D009877:Endophthalmitis / Q000175:diagnosis / Q000382:microbiology*; D015818:Eye Infections, Bacterial / Q000175:diagnosis / Q000382:microbiology*; D015821:Eye Infections, Fungal / Q000175:diagnosis / Q000382:microbiology; D005658:Fungi / Q000145:classification / Q000235:genetics; D016680:Genome, Bacterial; D006090:Gram-Negative Bacteria / Q000145:classification* / Q000235:genetics; D006094:Gram-Positive Bacteria / Q000145:classification* / Q000235:genetics; D006801:Humans; D007634:Keratitis / Q000175:diagnosis / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D004271:DNA, Fungal; D004275:DNA, Ribosomal