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 dir ected towards intact biofilms. Methods and Results: Escherichia coli and Staphylococcus epidermidis were c ultured for up to 48 h within 96-well microtitre plates. The planktonic pha se was removed and the wells rinsed. Residual biofilms were exposed to vari ous concentrations of chloroxylenol, peracetic acid, polyhexamethylene bigu anide (PHMB), cetrimide or phenoxyethanol for 1 h. At 15-min intervals, bio cide was removed, and the wells washed in neutraliser and filled with volum es of fresh medium. Re-growth of the cultures was monitored during incubati on 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 survi ving cells. Time-survival curves were constructed and the survival of the a ttached bacteria, following exposure to the agents for 30 min, interpolated for each biocide concentration. Log-log plots of these survival data and b iocide concentration were constructed, and linear regression analysis perfo rmed in order to (i) calculate concentration exponents and (ii) compare the effectiveness of the biocides between variously aged biofilm and planktoni c cells. From such analyses iso-effective concentrations of biocide (95% ki ll in 30 min) were calculated and expressed as planktonic : biofilm indices (PBI). Conclusions: PBI varied between 1.02 and 0.02, were relatively unaffected b y age of the biofilms but differed significantly between organism and bioci de. Notably those compounds with the higher activity against planktonic bac teria (PHMB and peracetic acid) were most prone to a biofilm effect but rem ained the most effective of the agents selected. Significance and Impact of the Study: The endpoint method proved robust, en abled the bactericidal effects of the biocides to be assessed against in-si tu biofilms, and was suitable for routine screening applications.