Effect of arginine-based surfactants on Pseudomonas aeruginosa biofilm

DI SANTO MEZTLER, P.; AN, S-Q; FAIT, M.E.; HERMET, M.; WEBB, J.; MORCELLE, S. R.

Southampton

Simposio; Young Microbiologists Symposium on Microbe Signalling, Organisation and Pathogenesis; 2020

University of Southampton

Antiseptics and disinfectants can be considered as the first line agents with the ability of interrupting the spread of potential pathogens, thus reducing the incidence of infection by eliminating surface contamination. A plethora of chemical agents with such characteristics are available to be used alone or as part of a formulation. Among them, surfactants consist in a group of molecules with diverse chemical structures that share in common their amphiphilic nature, i.e., a polar group (or head) and hydrophobic moiety (or tail). This characteristic is the main responsible of the ability of surfactants, which is their interfacial activity. Consequently, this kind of compounds are involved in the composition of many formulations. However, the antimicrobial activity of the arginine-based surfactants has been mainly reported against planktonic microbial cultures, whereas information about their antibiofilm activity is in general scarce. It is important to point out that life in biofilms is the most common mode of growth of microorganisms in the natural environment. Biofilms are composed by an extracellular matrix that provides structure and protection to the community like an increased resistance to a range of antimicrobial agents including clinically relevant antibiotics. The biofilm effect on bacterial resistance is thought to be related to the role of the exopolymeric matrix as a diffusion barrier, to a chemical reaction of some chemicals with the biofilm matrix and to physiological differences between fixed and suspended organisms. In this circumstance, the antibiofilm activity assay of biocide agents is crucial as they are meant to be applied as disinfectants and/or antiseptics.We have developed two arginine-based surfactants having the general formula Bz-Arg-NHCnX (n=10 or 12; X= Cl- or Br-). Previous results showed antimicrobial activity against planktonic fungi and bacteria. Recent results obtained at Southampton University showed the ability of these surfactants disrupting preformed Pseudomonas aeruginosa biofilms. For these assays we used PA14 (wild type) and PA5017 strains. To test the disruption of biofilms we used as concentrations the minimum inhibitory concentration (MIC) for all the assays. Chlorhexidine (CHX) and cetyl trimethyl ammonium bromide (CTAB) were included as controls. To study the antimicrobial activity of these surfactants, growth kinetics curves and biofilm disruption tests in two different surfaces were performed. For growth kinetics assays the behavior of both strains was similar. In the case of the culture in M63, for all the surfactants there was no sign of bacterial growth but for the culture in LB, there was a lower growth regarding control for incubation with Bz-Arg-NHC10Br and CTAB. For the biofilm disruption two assays were made on two different surfaces: 0.2 ml tubes (eppendorf style) and 8-well chamber slide. After biofilm formation (16 hours incubation), 5 hours of treatment was applied by removing medium and adding the same volume of the surfactants solutions prepared in the same medium at the corresponding MIC. For testing with crystal violet in eppendorf-type tubes, results showed that the wild type strain (PA14) was more resistant than the mutant (PA5017) for all the surfactants including the controls. In fact for PA14 biofilm formation was promoted with CTAB and Bz-Arg-NHC12Br incubation. Unexpectedly, for biofilm disruption assay in an 8-well chamber slide, fluorescence analysis showed an increase of biofilm formation for both strains. Ratio between dead/alive was similar for all the treatments. For Bz-Arg-NHC10Br, Bz-Arg-NHC12Cl and Bz-Arg-NHC12Br a clear disruption and change of morphology of the biofilm was observed. Surface type may influence biofilm development, for the assay in an 8-well chamber slide concentrations higher than MIC should be tested.