The Gram stain is commonly used in epidemiologic and clinical studies. It involves smearing a sample onto a slide, staining the material using dyes that bind to bacterial cells, and visually inspecting under a microscope. Many epidemiologic and clinical studies of pregnancy, sexually transmitted disease and lower respiratory infections include Gram stains in the study protocol. Clinically, Gram stains are performed routinely to guide diagnosis and treatment.
However, Gram stains are a potential source of bacterial DNA; using quantitative PCR, Gram stains can be interrogated to identify the presence and amount of specific bacterial genera and species. Although qPCR validation is required before testing each organism of interest, archived Gram stains can serve as a valuable resource for initial surveying of specific bacterial organisms and as preliminary resource to study bacterial community composition.
Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies. (2012) PLoS ONE 7(10): e42898. doi:10.1371/journal.pone.0042898
We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 108 cfu/ml of Escherichia coli and 105 cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces.