Studies on the development of antimicrobial tolerance in monospecies and binary biofilms

  • Nisreen Al-Hmoud

    Student thesis: Doctoral Thesis


    Biofilms are formed by a spectrum of microorganisms, including Pseudomonas  aeruginosa PAOl and Escherichia coli ATCC 10000, and provide a means for these organisms to protect themselves against antimicrobial agents. Several mechanisms have been proposed to explain the phenomenon of tolerance within biofilms, including delayed penetration of the antimicrobial into the biofilm extracellular matrix, slow growth rate   of organisms within the biofilm, other physiologic changes brought about by interactions of the organisms with a surface and the biofilm phenotype. However, none of these mechanisms on its own can explain the development of resistance in biofilm cells. The practical implications of biofilm formation are that alternative control strategies must be devised both for testing the susceptibility of the organisms within the biofilm and treating the established biofilm to alter its structure.

    The primary objective of this study was to induce tolerance in monospecies biofilms towards selected antimicrobial agents (including Zinc Pyrithione (ZnPT), Sodium Pyrithione (NaPT), Cetrimide, Benzisothiazolone (BIT) and Thiomersal) and investigating the possibility of cross-resistance between these biocides. This was followed by observation of the action of BIT in binary biofilms and subsequent susceptibility of component species this biocide. Monoculture of Ps. aeruginosa PAOl biofilms grown on the Sorbarod filters were passaged in the presence of sub-minimum inhibitory concentrations (MIC/4) of the biocides in a chemically defined medium (CDM). During 10 passages in increasing MIC/4, a gradual increase was observed in the MIC value of up to 3-fold and 4-fold from the original value (for ZnPT and Cetrimide). In the case of BIT, there was an overall increase of 17-fold for the adherent cells and 10-fold for the eluate cells. The difference in MIC values between biofilm and eluate cells was partially explained by the presence of thiol groups in the EPS that surrounds the biofilm, which quenched the effect of BIT. Once the resistant cells were passaged in biocide-free media, the MIC started to decrease. The results from cross-resistance studies in the case of ZnPT and BIT exhibited a marked increase in the MIC when compared to the other three biocides for both types of cells. On exposure of binary biofilms (two species biofilms, composed of E. coli and Ps. aeruginosa) to BIT, the original, pre-exposure value for Ps. aeruginosa was 5 pg mL’1 for both biofilm and eluate cells. After 5 passages in increasing the concentration of the biocide, there was an overall increase in MIC of 13.2-fold and12.4-fold for biofilm and eluate cells, respectively. For E. coli cells growing as binary biofilms, the original MIC was 5 pg mL"1 for biofilm and eluate cells. The MIC increased in a step-wise fashion until Passage 5, at which point there were increases of 12.4-fold for biofilm cells and 10.5-fold for eluate cells. Once, the cells were cultured in the absence of biocide, a significant decrease in the MIC was observed, indicating that the mechanism of resistance was dependent upon the exposure of the biocide. Analysing the outer membrane profiles of both adherent and eluate (mono- and binary species) cells illustrated marked differences between sensitive and resistant biofilm and eluate cells. 

    This project yielded novel information and techniques regarding the use of passage approaches to develop antimicrobials tolerance and resistance in both monospecies and binary species biofilms of medically important bacteria. The results from these experiments suggest that it is possible to use these models to investigate the results of environmental exposure of bacteria to sub-MICs of biocides and develop an understanding of their subsequent tolerance and resistance characteristics.
    Date of AwardApr 2002
    Original languageEnglish
    SupervisorPhillip J. Collier (Supervisor)

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