title: Anti-adhesive and anti-biofilm potential of extra-cellular biosurfactants produced by mycolate actinobacteria of the suborder Corynebacterineae against biofilm-forming human pathogens
Creators: name: Hodar, Janki Hiren
Publisher: name: Edinburgh Napier University
publisherIdentifier: https://ror.org/03zjvnn91
publisherIdentifierScheme: ROR
Year: 2024

description: Microbial biosurfactants, amphipathic surface-active compounds, are promising candidates for the prevention and control of clinical biofilms and unlike their chemical counterparts exhibit low toxicity and are biodegradable. Members of mycolic acid-containing actinobacteria (MACA), notably of suborder Corynebacterineae produce various types of biosurfactants, some of which have anti-adhesion and anti-biofilm properties but investigations to date are limited, perhaps because some species produce mycolates that play a role in pathogenicity. Yet, the MACA group encompasses a great number of species and genera with diverse metabolic capabilities and more extensive study may therefore yield biosurfactants, both known and novel, with effective anti-biofilm properties. Thus, this work aimed to establish how widespread biosurfactant production is amongst diverse MACA, investigate the anti-adhesion and anti-biofilm properties of extracellular biosurfactants from representative species, establish the relationship between biosurfactant production and growth during batch cultivation, and investigate the chemical properties of biosurfactants with anti-biofilm properties. Production of both cell-associated and extra-cellular biosurfactants, determined by various physico-chemical assays, was found to be widely distributed in species across the nine genera investigated, including genera not previously reported for biosurfactant production (Millisia and Williamsia). Reduction in surface tension (ST) to ≤40mN/m using the de Noüy ring method was reported in 90/94 strains when screened as whole cell broths (WCB) and 12 strains in cell-free supernatants (CFS) during growth on hydrophobic hexadecane (2% v/v) as the sole carbon source. Only weak negative correlations were observed between de Noüy ST and other surface tension-based screening assays: drop collapse (DC) (rs = -0.087) and oil spreading (OS) (rs = -0.125). Weak to moderate correlations were observed between all other assays revealing that the physico-chemical properties of biosurfactants can vary between MACA strains, and that screening assays vary in their predictability of biosurfactant production. There was no correlation between hydrophobicity and mycolic acid chain length (based on genus assessment).
Extracellular crude biosurfactants extract from eleven MACA were screened for the ability to prevent cell adhesion, inhibit biofilm formation, and disrupt mature biofilms formed by various bacterial strains. All crude biosurfactant extracts inhibited Klebsiella pneumonia NCIMB 8865 (24-45%) biofilms and distrupt pre-formed biofilms of Pseudomonas aeruginosa ENU 18 (20-53%), and some demonstrated antibiofilm properties against other pathogens. Further study of glycolipid-containing extracts from three strains, Rhodococcus ruber IEGM 231T, Rhodococcus yunnanensis DSM 44837T and Williamsia muralis N1261T, revealed ≤55% anti-adhesion and ≤80% inhibition of K. pneumonia NCIMB 8865 biofilms and ≤28% anti-adhesion, ≤57% inhibition and ≤20% disruption of preformed biofilms with P. aeruginosa ENU 18. Glycolipid-containing extract from these strains showed similar and in some cases, higher potency confirming the glycolipid were the bioactive components in the glycolipid-containing extracts. Biosurfactant synthesis was cell-associated until the end of exponential growth (~96 h) after which the compounds were released extra-cellularly during stationary phase. The critical cell micelle dilution of cell-free supernatants ranged from 1:100 to 1:500. Glycolipid-containing extract were identified as rhamnolipids by thin MALDI-TOF-MS. This is the first report of anti-biofilm activity by glycolipid biosurfactants produced by R. yunnanensis DSM 44837T and W. muralis N1261Tsp and paves the way for further investigation. This study demonstrates that extra-cellular glycolipids produced by diverse MACA possess antibiofilm activities that could be applied to the development of novel bio-based coatings for clinical surfaces and devices. This work also highlights the MACA group as a potentially rich source for further novel biosurfactants, which may have anti-biofilm properties.