Mode-of-action studies concluded that alkyldimethylbenzylammonium chloride (ADBAC) (a blend of C 12 , C 14 and C 16 alkyl homologues) and didecyldimethylammonium chloride (DDAC) are both membrane-active agents, possessing subtly different modes of action reflecting early cell interactions against Staphylococcus aureus. ADBAC and DDAC exhibited similar MIC behaviors from 0.4 ppm to 1.8 ppm over an inoculum range of 1 ؋ 10 5 to 1 ؋ 10 9 CFU/ml at 35°C. For ADBAC and DDAC, an increased rapidity of killing against S. aureus (final concentration, 2 ؋ 10 9 CFU/ml) was observed at 35°C compared to 25°C. Concentration exponents () for killing were <2.5 for both agents, and temperature influenced the value. Examination of leakage and kill data suggested that a single leakage marker was not indicative of cell death. ADBAC and DDAC possessed Langmuir (L4) and high-affinity (H3/4) uptake isotherms, respectively. ADBAC molecules formed a single monolayer of coverage of cells at the end of primary uptake, and DDAC formed a double monolayer. Rapid cell leakage occurred at bactericidal concentrations, with total depletion of the intracellular potassium and 260-nm-absorbing pools released in this strict order. Autolysis was observed for ADBAC and DDAC at concentrations of 9 g/ml (0.0278 mM and 0.0276 mM, respectively) and above, together with the depletion of approximately 30% of the internal potassium pool. Autolysis contributed to ADBAC and DDAC lethality, although high biocide concentrations may have inhibited autolytic enzyme activity.

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To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n ‫؍‬ 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 ؋ 10 8 purified phage per ml for 24 h at 37°C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10 10 and 10 12 phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time-and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.

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Aims: To demonstrate that the nonlinear concentration-dependent inhibition of Pseudomonas aeruginosa to EDTA can be used to successfully model and predict the potentiation of antimicrobials by EDTA. Methods and Results: A model used successfully to describe the concentration-dependent inhibition of bacterial growth caused by many antimicrobials was unable to describe the inhibition of P. aeruginosa by EDTA. Examination of the inhibition profiles for EDTA against P. aeruginosa revealed a biphasic inhibitory pattern suggesting different mechanisms of action at different concentrations. A modelled, two-stage inhibitory process was shown to fit the observations. This model was then used to examine the effect of combining EDTA with other antimicrobials. The apparent synergy of mixtures of EDTA with quaternary ammonium surfactants (QAC) and specific antibiotics was successfully modelled. Minimum inhibitory concentrations (MIC) of the QAC and that of oxacillin and cefamandole were reduced by a factor of 3-10, whereas ampicillin was reduced by a factor of 70 from an MIC of 1524 to 21 mg l À1 in the presence of 500 mg l À1 of EDTA. Conclusions: A nonlinear concentration-dependent inhibition of P. aeruginosa by EDTA gives rise to apparent observation of synergy with other antimicrobials. Significance and Impact of the Study: This is a further example where the current methodology for the examination of antimicrobial synergy (the summed fractional inhibitory concentrations) leads to false conclusions.