A rapid and simple measurement method for biofilm formation inhibitory activity using 96-pin microtiter plate lids
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(2020) 36:189
ORIGINAL PAPER
A rapid and simple measurement method for biofilm formation inhibitory activity using 96‑pin microtiter plate lids Tadayuki Tsukatani1 · Fumihiko Sakata1 · Rieko Kuroda1 Received: 10 July 2020 / Accepted: 16 November 2020 © Springer Nature B.V. 2020
Abstract The purpose of this study was to develop a rapid and simple measurement method for biofilm formation inhibitory activity, and to screen food additives and foodstuffs that inhibit biofilm formation. The measurement method for biofilm formation inhibitory activity was developed by combining biofilm formation on pins of microtiter plate lids and staining using crystal violet. The optimum conditions for biofilm formation on the pins were established for seven Gram-positive and six Gramnegative bacteria by investigations of media, incubation time, and pin materials. Minimum concentrations of food additives required to inhibit biofilm formation were determined using the proposed method. The values obtained by the proposed and conventional methods agreed well. In addition, by sequential measurements of minimum inhibitory concentrations and minimum bactericidal concentrations using the proposed method, mechanisms of inhibition of biofilm formation were assessed. Furthermore, inhibitory activities of the water extracts of 498 different plant foodstuffs on biofilm formation by Streptococcus mutans were measured; five of the extracts showed potent inhibitory activities. The method proposed here circumvents the tedious and time-consuming conventional method in which biofilms are cultivated on the bottom of wells of microtiter plates. Keywords Biofilm formation inhibition · 96-pins · Microtiter plate lid · Crystal violet · Staining assay
Introduction Biofilms are formed on solid surfaces by microbial communities that are embedded in a self-produced matrix of extracellular polysaccharides and proteins (Flemming et al. 2016). Biofilms can be extremely difficult to eradicate (Olsen 2015). Biofilm formation is thought to be closely associated with various chronic tissue infections, such as periodontitis, lung infection in cystic fibrosis, wound infection, and medical device-related infections (Mara et al. 2015; Ciofu et al. 2017). Therefore, treatment to inhibit formation of biofilms by pathogenic microorganisms is important to prevent these diseases. However, the use of antibiotics has resulted in the evolution of multidrug resistant bacteria. Foodstuffs and their components are thought to be acceptable for use in the control of pathogenic biofilms because they are safe for humans. A number of studies have reported * Tadayuki Tsukatani [email protected] 1
Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, 1465‑5 Aikawamachi, Kurume 839‑0861, Japan
on inhibition of biofilm formation by foodstuffs such as essential oils and cranberry, or food components such as eugenol, epigallocatechin-gallate, gallic acid, and rutin (Kim et al. 2015; Shao et al. 2015; Sun et al. 2015; Al-Shabib et al. 2017; Du e
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