BACKGROUND AND OBJECTIVES: Differentiation of live and dead cells is an important challenge when using molecular diagnosis for microbial identification. This is particularly relevant when bacteria have been exposed to antimicrobial agents. The objective of this study was to test a method using quantitative real-time polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), developed for the selective quantification of viable P. gingivalis, A. actinomycetemcomitans, F. nucleatum and total bacteria in an in vitro biofilm model after antimicrobial treatment. MATERIAL AND METHODS: PMA-qPCR method was tested in an in vitro biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30 min to isopropyl alcohol by immersion. Biofilms were disrupted and PMA added (final concentration of 100 μm). After DNA isolation, qPCR was carried out using specific primers and probes for the target bacteria. The differentiation of live and dead cells was tested by analysis of variance. RESULTS: When PMA was used in the presence of viable target bacterial cells, no statistically significant inhibition of qPCR amplification was detected (p > 0.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, PMA resulted in a significant total reduction of qPCR amplification of about 4 log10 . P. gingivalis showed a vitality reduction in the biofilm of 3 log10 , while A. actinomycetemcomitans and F. nucleatum showed a 2 log10 reduction. CONCLUSION: These results demonstrate the efficiency of PMA for differentiating viable and dead P. gingivalis, A. actinomycetemcomitans and F. nucleatum cells, as well as total bacteria, in an in vitro biofilm model, after being exposed to an antimicrobial agent. Hence, this PMA-qPCR method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms.
BACKGROUND AND OBJECTIVES: Differentiation of live and dead cells is an important challenge when using molecular diagnosis for microbial identification. This is particularly relevant when bacteria have been exposed to antimicrobial agents. The objective of this study was to test a method using quantitative real-time polymerase chain reaction (qPCR) combined with propidium monoazide (PMA), developed for the selective quantification of viable P. gingivalis, A. actinomycetemcomitans, F. nucleatum and total bacteria in an in vitro biofilm model after antimicrobial treatment. MATERIAL AND METHODS:PMA-qPCR method was tested in an in vitro biofilm model, using isopropyl alcohol as the antimicrobial agent. Matured biofilms were exposed for 1, 5, 10 and 30 min to isopropyl alcohol by immersion. Biofilms were disrupted and PMA added (final concentration of 100 μm). After DNA isolation, qPCR was carried out using specific primers and probes for the target bacteria. The differentiation of live and dead cells was tested by analysis of variance. RESULTS: When PMA was used in the presence of viable target bacterial cells, no statistically significant inhibition of qPCR amplification was detected (p > 0.05 in all cases). Conversely, after immersion in isopropyl alcohol of the biofilm, PMA resulted in a significant total reduction of qPCR amplification of about 4 log10 . P. gingivalis showed a vitality reduction in the biofilm of 3 log10 , while A. actinomycetemcomitans and F. nucleatum showed a 2 log10 reduction. CONCLUSION: These results demonstrate the efficiency of PMA for differentiating viable and dead P. gingivalis, A. actinomycetemcomitans and F. nucleatum cells, as well as total bacteria, in an in vitro biofilm model, after being exposed to an antimicrobial agent. Hence, this PMA-qPCR method may be useful for studying the effect of antimicrobial agents aimed at oral biofilms.
Authors: Eleanor M Townsend; Leighann Sherry; Ryan Kean; Donald Hansom; William G Mackay; Craig Williams; John Butcher; Gordon Ramage Journal: Antimicrob Agents Chemother Date: 2017-08-24 Impact factor: 5.191
Authors: Marian Kacerovsky; Roberto Romero; Martin Stepan; Jaroslav Stranik; Jan Maly; Lenka Pliskova; Radka Bolehovska; Vladimir Palicka; Helena Zemlickova; Helena Hornychova; Jiri Spacek; Bo Jacobsson; Percy Pacora; Ivana Musilova Journal: Am J Obstet Gynecol Date: 2020-07 Impact factor: 10.693
Authors: Abish S Stephen; Emma Millhouse; Leighann Sherry; Joseph Aduse-Opoku; Shauna Culshaw; Gordon Ramage; David J Bradshaw; Gary R Burnett; Robert P Allaker Journal: PLoS One Date: 2016-12-29 Impact factor: 3.240
Authors: Aina E Fossum Moen; Jonas Christoffer Lindstrøm; Tone Møller Tannæs; Simen Vatn; Petr Ricanek; Morten H Vatn; Jørgen Jahnsen Journal: Sci Rep Date: 2018-11-22 Impact factor: 4.996
Authors: Emma Millhouse; Anto Jose; Leighann Sherry; David F Lappin; Nisha Patel; Andrew M Middleton; Jonathan Pratten; Shauna Culshaw; Gordon Ramage Journal: BMC Oral Health Date: 2014-06-28 Impact factor: 2.757
Authors: Leighann Sherry; Gillian Lappin; Lindsay E O'Donnell; Emma Millhouse; Owain R Millington; David J Bradshaw; Alyson S Axe; Craig Williams; Christopher J Nile; Gordon Ramage Journal: Front Microbiol Date: 2016-06-10 Impact factor: 5.640
Authors: Honorato Ribeiro-Vidal; María Carmen Sánchez; Andrea Alonso-Español; Elena Figuero; Maria José Ciudad; Luís Collado; David Herrera; Mariano Sanz Journal: Nutrients Date: 2020-09-14 Impact factor: 5.717