Assessment of resistance towards biocides following the attachment of micro-organisms to, and growth on, surfaces

Aims: To develop a rapid method for the assessment of biocidal activity directed towards intact biofilms. Methods and Results: Escherichia coli and Staphylococcus epidermidis were cultured for up to 48 h within 96-well microtitre plates. The planktonic phase was removed and the wells rinsed. Residual biofilms were exposed to various concentrations of chloroxylenol, peracetic acid, polyhexamethylene biguanide (PHMB), cetrimide or phenoxyethanol for 1 h. At 15-min intervals, biocide was removed, and the wells washed in neutraliser and filled with volumes of fresh medium. Re-growth of the cultures was monitored during incubation at 35 degreesC in the plate reader. Times taken for the treated wells to re-grow to fixed endpoints were determined and related to numbers of surviving cells. Time-survival curves were constructed and the survival of the attached bacteria, following exposure to the agents for 30 min, interpolated for each biocide concentration. Log-log plots of these survival data and biocide concentration were constructed, and linear regression analysis performed in order to (i) calculate concentration exponents and (ii) compare the effectiveness of the biocides between variously aged biofilm and planktonic cells. From such analyses iso-effective concentrations of biocide (95% kill in 30 min) were calculated and expressed as planktonic : biofilm indices (PBI). Conclusions: PBI varied between 1.02 and 0.02, were relatively unaffected by age of the biofilms but differed significantly between organism and biocide. Notably those compounds with the higher activity against planktonic bacteria (PHMB and peracetic acid) were most prone to a biofilm effect but remained the most effective of the agents selected. Significance and Impact of the Study: The endpoint method proved robust, enabled the bactericidal effects of the biocides to be assessed against in-situ biofilms, and was suitable for routine screening applications.