Lactobacillus, Gram staining, DNA extraction, Polymerase chain reaction, Bacterial vaginosis, Bacterial genomics, Escherichia coli, Bacterial pathogens
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 105cfu/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 ofLactobacillus, 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.
Department of Biology
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UNI ScholarWorks, University of Northern Iowa, Rod Library
©2012 Usha Srinivasan, Steelatha Ponnaluri, Lisa Vilareal, Brenda Gillespie, Ai Wen, Arianna Miles, Brigette Bucholz, Carl F. Marrs, Ram K. Iyer, Dawn Misra, and Betsy Foxman. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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This work is licensed under a Creative Commons Attribution 4.0 License.
Srinivasan, Usha; Ponnalurl, Sreelatha; Villareal, Lisa; Gillespie, Brenda; Wen, Ai; Miles, Arianna; Bucholz, Brigette; Marrs, Carl F.; Iyer, Ram K.; Misra, Dawn; and Foxman, Betsy, "Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies" (2012). Faculty Publications. 9.