Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the <i>Gardnerella</i>-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis
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 <i>Gardnerella</i> with propidium monoazide (PMA...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-01-01
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| Series: | Antibiotics |
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| Online Access: | https://www.mdpi.com/2079-6382/11/1/111 |
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| author | Agnieszka Latka Leen Van Simaey Marijke Reynders Piet Cools Tess Rogier Barbara Lebbe Lorenzo Corsini Christine Landlinger Mario Vaneechoutte |
| author_facet | Agnieszka Latka Leen Van Simaey Marijke Reynders Piet Cools Tess Rogier Barbara Lebbe Lorenzo Corsini Christine Landlinger Mario Vaneechoutte |
| author_sort | Agnieszka Latka |
| collection | DOAJ |
| description | 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 <i>Gardnerella</i> 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 <i>Gardnerella</i> 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. |
| first_indexed | 2024-03-10T03:01:23Z |
| format | Article |
| id | doaj.art-084a7ae6aaa74a41818399d83bb8ec4b |
| institution | Directory Open Access Journal |
| issn | 2079-6382 |
| language | English |
| last_indexed | 2024-03-10T03:01:23Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antibiotics |
| spelling | doaj.art-084a7ae6aaa74a41818399d83bb8ec4b2023-11-23T12:45:35ZengMDPI AGAntibiotics2079-63822022-01-0111111110.3390/antibiotics11010111Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the <i>Gardnerella</i>-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial VaginosisAgnieszka Latka0Leen Van Simaey1Marijke Reynders2Piet Cools3Tess Rogier4Barbara Lebbe5Lorenzo Corsini6Christine Landlinger7Mario Vaneechoutte8Laboratory Bacteriology Research, Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, BelgiumLaboratory Bacteriology Research, Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, BelgiumLaboratory Medicine, Medical Microbiology, AZ St.Jan Brugge-Oostende AV, 8000 Bruges, BelgiumLaboratory Bacteriology Research, Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, BelgiumLaboratory Bacteriology Research, Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, BelgiumDepartment of Gynaecology, AZ St.Jan Brugge-Oostende AV, 8000 Bruges, BelgiumPhagoMed Biopharma GmbH, Vienna Biocenter, 1110 Wien, AustriaPhagoMed Biopharma GmbH, Vienna Biocenter, 1110 Wien, AustriaLaboratory Bacteriology Research, Department of Diagnostic Sciences, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, BelgiumQuantification 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 <i>Gardnerella</i> 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 <i>Gardnerella</i> 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.https://www.mdpi.com/2079-6382/11/1/111endolysinbacterial vaginosisbiofilm<i>Gardnerella</i>viability-qPCR |
| spellingShingle | Agnieszka Latka Leen Van Simaey Marijke Reynders Piet Cools Tess Rogier Barbara Lebbe Lorenzo Corsini Christine Landlinger Mario Vaneechoutte Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the <i>Gardnerella</i>-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis Antibiotics endolysin bacterial vaginosis biofilm <i>Gardnerella</i> viability-qPCR |
| title | Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the <i>Gardnerella</i>-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis |
| title_full | Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the <i>Gardnerella</i>-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis |
| title_fullStr | Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the <i>Gardnerella</i>-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis |
| title_full_unstemmed | Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the <i>Gardnerella</i>-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis |
| title_short | Optimization of Propidium Monoazide qPCR (Viability-qPCR) to Quantify the Killing by the <i>Gardnerella</i>-Specific Endolysin PM-477, Directly in Vaginal Samples from Women with Bacterial Vaginosis |
| title_sort | optimization of propidium monoazide qpcr viability qpcr to quantify the killing by the i gardnerella i specific endolysin pm 477 directly in vaginal samples from women with bacterial vaginosis |
| topic | endolysin bacterial vaginosis biofilm <i>Gardnerella</i> viability-qPCR |
| url | https://www.mdpi.com/2079-6382/11/1/111 |
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