In vitro development of Staphylococcus aureus biofilms using slime-producing variants and ATP-bioluminescence for automated bacterial quantification

In this work, a method was developed to establish Staphylococcus aureus bio films on 96-well plates and automatically quantify viable cells within thes e biofilms by ATP-bioluminescence. Different strains were compared for biof ilm formation. Cells from slime producing (SP) strain variants were more ad herent (p < 0.001) and therefore more suitable for biofilm formation than n on-slime producing original isolates. To compare biofilm support surfaces, SP biofilms were formed for 6, 24 and 48 h on 96-well polystyrene plates, c ontaining wells coated with gelatin, poly-L-lysine or pre-treated for tissu e culture and uncoated wells. Tissue culture-treated wells enhanced biofilm formation, allowing the highest growth (p < 0.001) in well-established bio films (24 or 48 h old). For ATP quantification, the efficacy of different A TP extractants was compared: dimethyl sulphoxide (DMSO), trichloroacetic ac id (TCA), a commercially available releasing reagent(R) (RR) and lysostaphi n. A greater inhibitory effect on the ATP detection (p < 0.01), a more vari able light emission (variation coefficient greater than or equal to 50% vs. < 19%, respectively) and a lower extraction efficiency (p < 0.05) were fou nd in the case of TCA or lysostaphin in relation to RR or DMSO. DMSO was fo und preferable in relation to RR (upper detection limits 2.3 x 10(9) and 2 x 10(8) CFU/mL respectively) for bacterial ATP extraction from biofilms wit h high bacterial density. DMSO extracted ATP within seconds, light emission being stable for 6 h. The method developed allows automated viability dete rmination of biofilm cells using bioluminescence and simultaneous Study of factors affecting this viability (culture media, antibiotic types, antimicr obial concentrations, support surfaces and biofilm ages). It may be of use in bacteriological and antimicrobial research. Copyright (C) 1999 John Wile y & Sons, Ltd.