Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the Gardnerella-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis.

Agnieszka Latka,Leen Van Simaey,Barbara Lebbe,Tess Rogier,Christine Landlinger,Piet Cools,Lorenzo Corsini,Mario Vaneechoutte,Marijke Reynders

doi:10.3390/antibiotics11010111

Agnieszka Latka, Leen Van Simaey + Show 7 more

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https://doi.org/10.3390/antibiotics11010111

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Abstract

Quantification of the number of living cells in biofilm or after eradication treatments of biofilm, is problematic for different reasons. We assessed the performance of pre-treatment of DNA, planktonic cells and ex vivo vaginal biofilms of Gardnerella with propidium monoazide (PMAxx) to prevent qPCR-based amplification of DNA from killed cells (viability-qPCR). Standard PMAxx treatment did not completely inactivate free DNA and did not affect living cells. While culture indicated that killing of planktonic cells by heat or by endolysin was complete, viability-qPCR assessed only log reductions of 1.73 and 0.32, respectively. Therefore, we improved the standard protocol by comparing different (combinations of) parameters, such as concentration of PMAxx, and repetition, duration and incubation conditions of treatment. The optimized PMAxx treatment condition for further experiments consisted of three cycles, each of: 15 min incubation on ice with 50 µM PMAxx, followed by 15 min-long light exposure. This protocol was validated for use in vaginal samples from women with bacterial vaginosis. Up to log2.2 reduction of Gardnerella cells after treatment with PM-477 was documented, despite the complex composition of the samples, which might have hampered the activity of PM-477 as well as the quantification of low loads by viability-qPCR.

Highlights

Clinical microbiology is increasingly confronted with the consequences of biofilm formation, causing problems of chronic infections and chronic dysbiosis
In order to quantify the effect of the endolysin PM-477 on the lysis of Gardnerella cells in bacterial vaginosis (BV) samples, we first optimized the viability-qPCR approach
To set up the method, multiple parameters of PMAxx treatment were tested on planktonic Gardnerella swidsinskii cells and on DNA extracted from these cells

Summary

Introduction

Clinical microbiology is increasingly confronted with the consequences of biofilm formation, causing problems of chronic infections and chronic dysbiosis. This is the case for bacterial vaginosis (BV), which often develops into a chronic and refractory condition and which is generally recognized to be largely due to biofilm formation with Gardnerella as one of the principal bacterial taxa. One option is qPCR, which has the disadvantage that by amplifying DNA from dead cells that is still present in the sample, it provides a false negative result (positive = therapy success). The second option, culturing, may provide false positive results if alive but dormant biofilm cells cannot be cultured under the same conditions as metabolically active planktonic cells

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