Extracytoplasmic stress responses induced by antimicrobial cationic polyethylenimines.

The ability of an antimicrobial, cationic polyethylenimine (PEI+) to induce the three known extracytoplasmic stress responses of Escherichia coli was quantified. Exposure of E. coli to PEI+ in solution revealed specific, concentration-dependent induction of the Cpx extracytoplasmic cellular stress response, ~2.0-2.5-fold at 320 µg/mL after 1.5 h without significant induction of the σ(E) or Bae stress responses. In comparison, exposure of E. coli to a non-antimicrobial polymer, poly(ethylene oxide) (PEO), resulted in no induction of the three stress responses. The antimicrobial small molecule vanillin, a known membrane pore-forming compound, was observed to cause specific, concentration-dependent induction of the σ(E) stress response, ~6-fold at 640 µg/mL after 1.5 h, without significant induction of the Cpx or Bae stress responses. The different stress response induction profiles of PEI+ and vanillin suggest that although both are antimicrobial compounds, they interact with the bacterial membrane and extracytoplasmic area by unique mechanisms. EPR studies of liposomes containing spin-labeled lipids exposed to PEI+, vanillin, and PEO reveal that PEI+ and PEO increased membrane stability, whereas vanillin was found to have no effect.

Evaluation of the antimicrobial activity of cationic polyethylenimines on dry surfaces.

The antimicrobial activity of cationic N-dodecyl-N-methylpolyethylenimine (PEI+) against S. aureus, A. baumannii, and E. coli was evaluated when the polymer was applied as a coating to various surfaces using a paint-like procedure. Antimicrobial activity of PEI+ as a function of time was determined using an assay for long-term survival involving placement of single drops of various bacterial concentrations on dry surfaces. These data were compared with an assay method where bacteria were applied by spraying and surfaces were incubated overnight under agar. PEI+-coated surfaces were found to be highly bactericidal after 30 min when bacteria were sprayed onto surfaces. However, when bacteria were applied as single drops, PEI+-coated surfaces were less biocidal at short contact times particularly for A. baumannii and E. coli. The observations are explained in the context of the difference in drying time between drops deposited on uncoated surfaces and PEI+-coated surfaces and the sensitivity of bacterial survival to dehydration. These results demonstrate that PEI+-coated surfaces are not effectively biocidal for some types of bacteria under certain conditions and that the method of assaying bactericidal efficiency can greatly affect the results obtained.

Midazolam oxidation by cytochrome P450 3A4 and active-site mutants: an evaluation of multiple binding sites and of the metabolic pathway that leads to enzyme inactivation.

Midazolam (MDZ) oxidation by recombinant CYP3A4 purified from Escherichia coli and 30 mutants generated at 15 different substrate recognition site positions has been studied to determine the role of individual residues in regioselectivity and to investigate the possible existence of multiple binding sites. Initial results showed that oxidation of MDZ by CYP3A4 causes time- and concentration-dependent enzyme inactivation with K(I) and k(inact) values of 5.8 microM and 0.15 min(-1), respectively. The different time courses of MDZ hydroxylation by mutants that predominantly formed 1'-OH MDZ as opposed to 4-OH MDZ provided strong evidence that the 1'-OH MDZ pathway leads to CYP3A4 inactivation. Correlational analysis of 1'-OH formation versus 4-OH formation by the mutants supports the inference that the two metabolites result from the binding of MDZ at two separate sites. Thus, substitution of residues Phe-108, Ile-120, Ile-301, Phe-304, and Thr-309 with a larger amino acid caused an increase in the ratio of 1'-OH/4-OH MDZ formation, whereas substitution of residues Ser-119, Ile-120, Leu-210, Phe-304, Ala-305, Tyr-307, and Thr-309 with a smaller amino acid decreased this ratio. Kinetic analyses of nine key mutants revealed that the alteration in regioselectivity is caused by a change in kinetic parameters (V(max) and K(M)) for the formation of both metabolites in most cases. The study revealed the role of various active-site residues in the regioselectivity of MDZ oxidation, identified the metabolic pathway that leads to enzyme inactivation, and provided an indication that the two proposed MDZ binding sites in CYP3A4 may be partially overlapping.