Methodologies for Studying <em>B. subtilis</em> Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

Abstract

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Eradication of Enterococcus faecalis Biofilms on Human Dentin

“…D -amino acids compete with D -Alanine for the fifth position in the B. subtilis pentapeptide, and interfere with transpeptidation (Lam et al, 2009; Cava et al, 2011; Lupoli et al, 2011) and transglycosylation (Lam et al, 2009). D -amino acids were found to inhibit and disperse biofilms without affecting planktonic growth in various model organisms (Kolodkin-Gal et al, 2010; Hochbaum et al, 2011; Yu et al, 2012; Sanchez et al, 2013; Li and Wang, 2014; Bucher et al, 2015, 2016; She et al, 2015), but to the best of our knowledge their efficacy on endodontic biofilms was never evaluated.…”

“…Recently, flavomycin, an antibiotic that inhibits transglycosylation directly by binding the transglycosylation domain of PBP enzymes (Dengler et al, 2011) was found to antagonize biofilm formation but not planktonic growth in the soil bacterium, Bacillus subtilis (Bucher et al, 2015). An additional group of cell-wall interfering agents that promote dispersal are non-canonical D -amino acids (Bucher et al, 2015, 2016). D -amino acids compete with D -Alanine for the fifth position in the B. subtilis pentapeptide, and interfere with transpeptidation (Lam et al, 2009; Cava et al, 2011; Lupoli et al, 2011) and transglycosylation (Lam et al, 2009).…”

Beyond the Wall: can D-Amino Acids and Small Molecule Inhibitors Eliminate Infections?

“…The specific mechanisms that link cell-wall interference and biofilm formation remain to be determined. Intriguingly, macro-analysis of B. subtilis biofilms indicated that DAAs induced a malfunction of the anchoring of the extracellular matrix to the cell wall [10,11]. DAAs can also directly interfere with protein translation, leading to production of defective proteins.…”

“…Then, a handful of conflicting publications emerged, generating a discussion within the field [11,13]. Variation in results and conclusions at an early stage is not a unique to the study of DAAs.…”

“…Higher resolution single cell imaging techniques, as well as systemic transcriptional and translational profiling of the biofilm cells [10] are necessary for detection of functional differences in biofilms grown under laboratory conditions. Furthermore, it is clear that establishing a consistent and robust framework relying on multiple independent criteria for biofilm inhibition is essential, and several experimental conditions (e.g., growth media, temperature, inoculum condition) should be used [11]. If only a single condition is tested and the kinetics of biofilm development is not taken under consideration, false-negative results are likely.…”