Optimizing the use of low-frequency ultrasound for bacterial detachment of in vivo biofilms in dental research-a methodological study

Cassandra Rux; Annette Wittmer; Anja Stork; Kirstin Vach; Elmar Hellwig; Fabian Cieplik; Ali Al-Ahmad

doi:10.1007/s00784-023-05397-1

Optimizing the use of low-frequency ultrasound for bacterial detachment of in vivo biofilms in dental research-a methodological study

Clin Oral Investig. 2023 Dec 23;28(1):19. doi: 10.1007/s00784-023-05397-1.

Authors

Cassandra Rux¹, Annette Wittmer², Anja Stork¹, Kirstin Vach³, Elmar Hellwig¹, Fabian Cieplik⁴, Ali Al-Ahmad⁵

Affiliations

¹ Department of Operative Dentistry and Periodontology, Medical Center, Faculty of Medicine, University of Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany.
² Institute of Medical Microbiology and Hygiene, Faculty of Medicine, University of Freiburg, Hermann-Herder- Str. 11, 79104, Freiburg, Germany.
³ Institute for Medical Biometry and Statistics, Faculty of Medicine, University of Freiburg, Stefan-Meier-Str. 26, 79104, Freiburg, Germany.
⁴ Department of Conservative Dentistry and Periodontology, University Hospital Regensburg, Franz-Josef-Strauß-Allee 11, 93053, Regensburg, Germany.
⁵ Department of Operative Dentistry and Periodontology, Medical Center, Faculty of Medicine, University of Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany. ali.al-ahmad@uniklinik-freiburg.de.

Abstract

Objectives: Low-frequency, low-intensity ultrasound is commonly utilized in various dental research fields to remove biofilms from surfaces, but no clear recommendation exists in dental studies so far. Therefore, this study aims to optimize the sonication procedure for the dental field to efficiently detach bacteria while preserving viability.

Materials and methods: Initial biofilm was formed in vivo on bovine enamel slabs (n = 6) which were worn by four healthy participants for 4 h and 24 h. The enamel slabs covered with biofilm were then ultrasonicated ex vivo for various time periods (0, 1, 2, 4, 6 min). Colony-forming units were determined for quantification, and bacteria were identified using MALDI-TOF. Scanning electron microscopic images were taken to also examine the efficiency of ultrasonications for different time periods.

Results: Ultrasonication for 1 min resulted in the highest bacterial counts, with at least 4.5-fold number compared to the non-sonicated control (p < 0.05). Most bacteria were detached within the first 2 min of sonication, but there were still bacteria detached afterwards, although significantly fewer (p < 0.0001). The highest bacterial diversity was observed after 1 and 2 min of sonication (p < 0.03). Longer sonication periods negatively affected bacterial counts of anaerobes, Gram-negative bacteria, and bacilli. Scanning electron microscopic images demonstrated the ability of ultrasound to desorb microorganisms, as well as revealing cell damage and remaining bacteria.

Conclusions: With the use of low-frequency, low-intensity ultrasound, significantly higher bacterial counts and diversity can be reached. A shorter sonication time of 1 min shows the best results overall.

Clinical relevance: This standardization is recommended to study initial oral biofilms aged up to 24 h to maximize the outcome of experiments and lead to better comparability of studies.

Keywords: Biofilm removal; Bovine enamel slabs; Desorption; Initial biofilm; Low-frequency ultrasound; Sonication.

MeSH terms

Aged
Animals
Bacteria
Bacterial Load
Biofilms*
Cattle
Dental Enamel / diagnostic imaging
Dental Research*
Humans