Optimized bacterial absolute quantification method by qPCR using an exogenous bacterial culture as a normalization strategy in triple-species BV-like biofilms

Abstract

Quantitative Polymerase Chain Reaction (qPCR) is a widely used method in molecular biology to quantify target DNA sequences. Despite its accuracy, there are important experimental controls that should be considered to avoid biased results. One of them is gDNA loss during extraction, which is higher among samples with lower bacterial concentrations. Improvement in qPCR quantification procedures is mandatory to obtain reproducible and accurate results. Herein, we report an improved qPCR method for bacterial quantification of Gardnerella vaginalis, Prevotella bivia, and Fannyhessea vaginae, three key-bacterial vaginosis (BV)-associated bacteria (BVAB) thought to play important roles in the pathogenesis of this common vaginal infection. The formation of a mature biofilm on vaginal epithelial cells is an unique feature of BV and, despite over 60 years of research, the exact etiology of BV remains unknown. Here, we optimized a qPCR method that accurately quantified triple-species biofilms containing these key BVAB, after the addition of an exogenous bacterial control containing a fixed concentration of Escherichia coli, prior to gDNA extraction. This improved method minimized and normalized the inherent losses associated with bacterial centrifugation, which allows better sensitivity at lower bacterial concentrations.

Keywords: Bacterial quantification; Bacterial vaginosis; Calibration curves; Centrifugation loss; Exogenous control; qPCR.

Figure 1 |. Bacterial losses from pure cultures of G. vaginalis, P. bivia, and F. vaginae after 10, 20, and 30 min of centrifugation.

The bacterial concentrations present in the supernatant after 10, 20, and 30 min centrifugating 1 × 10⁸ CFU/mL of Gardnerella vaginalis (A), Prevotella bivia (B) and Fannyhessea vaginae (C) are shown in this figure. The bars represent the mean and the standard deviation of at least 3 independent experimental replicates. Statistical analysis was performed using a t-test, p<0.05.

Figure 2 |. Calibration curves to relate cycle threshold detection (Δ Ct) and bacterial load quantification.

Calibration curves for G. vaginalis (A), P. bivia (B), and F. vaginae (C) were assessed by adding 1 × 10⁸ CFU/ mL of E. coli, as an exogenous control, to each sample for each gDNA extraction. Each dot represents an independent biological extraction, with triplicate qPCR quantification.

Figure 3 |. Accuracy of bacterial quantification of mock cultures of G. vaginalis, P. bivia, and F. vaginae.

The quantification of each single species in the mock cultures was determined using the 3 calibration curves shown above. Mock culture A is constituted by 1 × 10⁸ CFU/ mL of G. vaginalis, 1 × 10⁸ CFU/ mL of P. bivia and 1 × 10⁸ CFU/ mL of F. vaginae. Mock culture B is constituted by 1 × 10³ CFU/ mL of G. vaginalis, 1 × 10³ CFU/ mL of P. bivia and 1 × 10³ CFU/ mL of F. vaginae. Mock culture C is constituted by 1 × 10⁵ CFU/ mL of G. vaginalis, 1 × 10³ CFU/ mL of P. bivia and 1 × 10⁵ CFU/ mL of F. vaginae. To each mock culture, 1 × 10⁸ CFU/ mL of E. coli was added as exogenous control, before extracting gDNA. The bars represent the mean and the standard deviation of at least 3 experimental replicates. Statistical analysis was performed using Two-way ANOVA with Tukey’s multiple comparisons test. No significant differences were denoted. Abbreviations: Gv – Gardnerella vaginalis; Pb – Prevotella bivia; Fv – Fannyhessea vaginae.

Figure 4 |. Triple-species quantification of 24 h, 48 h, and 72 h biofilms of G. vaginalis, P. bivia, and F. vaginae.

(A) Assessment of bacterial concentrations comprising the triple-species biofilms by qPCR, using calibration curves previously performed. (B) The biomass growth was analyzed by measuring the optical density (OD) at 620 nm. The bars represent the mean and the standard deviation of three independent experiments. Statistical analysis was performed using a t-test, *p <0.05. Abbreviations: Gv – Gardnerella vaginalis; Pb – Prevotella bivia; Fv – Fannyhessea vaginae.