Detection of and discrimination between gram-positive and gram-negative bacteria in intraocular samples by using nested PCR

Abstract

A nested PCR protocol has been developed for the detection of and discrimination between 14 species of gram-positive and -negative bacteria in samples of ocular fluids. First-round PCR with pan-bacterial oligonucleotide primers, based on conserved sequences of the 16S ribosomal gene, was followed by a gram-negative-organism-specific PCR, which resulted in a single 985-bp amplification product, and a multiplex PCR which resulted in two PCR products: a 1,025 bp amplicon (all bacteria) and a 355 bp amplicon (gram-positive bacteria only). All products were detected by gel electrophoresis. The sensitivity of the assay was between 10 fg and 1 pg of bacterial DNA, depending on the species tested, equivalent to between 24 and 4 live bacteria spiked in water. The identification was complete in 3.5 h. The molecular techniques were subsequently applied to four samples of intraocular fluid, (three vitreous and one aqueous) from three patients with clinical signs of bacterial endophthalmitis (test samples) and two samples of vitreous from a patient with chronic intraocular inflammation (control samples). In all culture-positive samples (two of three vitreous and one of one aqueous), a complete concordance was observed between molecular methods and culture results. PCR correctly identified the gram stain classification of the organisms. The bacterial etiology was also identified in a culture-negative patient with clinical history and signs highly suggestive of bacterial endophthalmitis. Furthermore, control samples from a patient with chronic intraocular inflammation remained PCR negative. In summary, this protocol has demonstrated potential as a rapid diagnostic test in confirming the diagnosis of infection and also determining the Gram status of bacteria with high specificity and sensitivity.

FIG. 1

Sensitivities of the primer sets were evaluated using dilutions of DNA in water. (A) Multiplex PCR in which the 1,025-bp amplicon is the product of the NF-NR primers (universal bacterial primers) and the 355-bp amplicon is the product of the P2F-NR primers (specific for gram-positive bacteria). The template DNA was S. aureus NCTC 8532. Lane 1, 10 ng of DNA; lane 2, 1 ng of DNA; lane 3, 100 pg of DNA; lane 4, 10 pg of DNA; lane 5, 1 pg of DNA; lane 6, 100 fg of DNA; lane 7, 10 fg of DNA; lane 8, DNA ladder; lane 9, negative control. (B) Gram-negative-organism-specific PCR in which the 985-bp amplicon is the product of the primers NF-N6R. The template DNA was E. coli NCTC 10418. Lane 1, 10 ng of DNA; lane 2, 1 ng of DNA; lane 3, 100 pg of DNA; lane 4, 10 pg of DNA; lane 5, 1 pg of DNA; lane 6, 100 fg of DNA; lane 7, 10 fg of DNA; lane 8, DNA ladder; lane 9, negative control.

FIG. 2

Results of PCR from a clinical case of culture-positive bacterial endophthalmitis secondary to gram-positive bacteria. The vitreous sample was subjected to PCR as described in the text and electrophoresed on a 1% agarose–TAE gel. Patient sample PCR results appear in duplicate (lanes 3 to 6). Lane 1, multiplex PCR of gram-negative DNA in water (positive control); lane 2, multiplex PCR of gram-positive DNA in neat vitreous (positive control); lanes 3 and 4, multiplex PCR of patient sample (vitreous, diluted 1/10); lanes 5 and 6, multiplex PCR of patient sample (neat vitreous); lane 7, gram-negative-organism-specific PCR of patient sample (vitreous, diluted 1/10); lane 8, Gram negative PCR of gram-negative DNA in water (positive control); lane 9, multiplex PCR of gram-negative DNA in vitreous (positive control); lane 10, multiplex PCR of gram-negative DNA in water (positive control); lane 11, gram-negative-organism-specific PCR of gram-negative DNA in vitreous; lane 12, gram-negative-organism-specific PCR of gram-negative DNA in water; lane 13, DNA ladder; lane 14, negative control (water only, no vitreous); lane 15, negative control (vitreous and water).