Characterization of growth inhibition of oral bacteria by sophorolipid using a microplate-format assay

Authors: Solaiman, Daniel; Ashby, Richard - Rick; Uknalis, Joseph

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/24/2017
Publication Date: 3/6/2017
Citation: Solaiman, D., Ashby, R.D., Uknalis, J. 2017. Characterization of growth inhibition of oral bacteria by sophorolipid using a microplate-format assay. Journal of Microbiological Methods. 136:21-29. doi: 10.1016/j.mimet.2017.02.012.

Interpretive Summary: Sophorolipid (SL) is a class of sugar-lipid molecules (called glycolipids) produced by yeast and could be manufactured by growing the yeast on less expensive renewable agriculture byproducts. SL possesses good surfactant property and is henced used as "green" substitute for the detergents in some niche-market washing/cleaning products. SL has also been known to have antimicrobial activity which could add to its commercial attractiveness. In this report, we described a systematic characterization of SL's antimicrobial activity against representative strains of two groups of tooth decay- (caries)-causing bacteria, namely the so-called Lactobacilli (rod-shape) and the so-called Streptococci ("twisted berry" shape). The results showed for the first time that low levels (less than 50 microgram per ml) of SL could cause growth-delay (called bacteriostatic) of the Streptococci, and a complete killing (called bacteriocidal) of these bacteria occurred at 50 or higher microgram per ml of SL. Towards the Lactobacilli, however, SL exerts bacteriocidal activity in a concentration-dependent manner, with a complete killing occurred at 1 mg/ml concentration. Scanning electron microscopy study revealed that SL caused the appearance of protruding features on the cell surface of Streptococci, but induced cell membrance damage and cell lysis in Lactobacilli. The outcome of the research is valuable for the understanding of SL's antimicrobial activity against tooth caries-causing bacteria, thus providing important groundwork toward development of oral care and hygience products.

Technical Abstract: Sophorolipid (SL) is a class of glycolipid biosurfactant produced by yeast and has potent antimicrobial activity against many microorganisms. In this paper, a microplate-based method was developed to characterize the growth inhibition by SL on five representative species of caries-causing oral bacteria. Bacterial growth on microplate in the absence and presence of varying concentrations of SL was continuously monitored by recording the absorbance at 600 nm of the cultures using a microplate reader. The results showed that SL completely inhibited the growth of the Lactobacilli at =1 mg/ml and the Streptococci at much lower concentrations of =50 ug/ml. More importantly, we further defined the mechanism of antimicrobial activity of SL by analyzing the pattern of the cell growth curves. SL at sub-lethal concentrations (<1 mg/ml) is bacteriocidal towards the Lactococci; it lengthens the apparent cell-doubling time (Td) and decreases the final cell density (as indicated by A600nm) in a concentration-dependent manner. Against the oral Streptococci, on the other hand, SL at sub-lethal concentrations (<50 ug/ml) is bacteristatic; it delays the onset of cell growth in a concentration-dependent fashion, but once the cell growth is commenced there is no noticeable adverse effect on Td and the final A600nm. Scanning electron microscopic study of L. acidophilus grown in sub-lethal concentration of SL reveals extensive structural damage to the cells. S. mutans grown in sub-lethal level of SL did not show morphological damage to the cells, but numerous protruding structures could be seen on the cell surface. In summary, this paper presents the first report on a detailed analysis of the effects of SL on Lactobacilli and Streptococci important to oral health and hygiene.