Cellular impermeability and uptake of biocides and antibiotics in Gram-negative bacteria

Uptake of the fluorescent probe 1-N-phenylnaphthylamine (NPN), as adapted to an automated spectrofluorometer enabling multiwell reading of microtitre plates, was applied to determine permeability changes in Gram-negative bacteria. An intact outer membrane is a permeability barrier, and excludes hydrophobic substances such as NPN but, once damaged, it can allow the entry of NPN to the phospholipid layer, resulting in prominent fluorescence. With Escherichia coli O157, Pseudomonas aeruginosa, and Salmonella typhimurium as test organisms and ethylenediaminetetraacetic acid and sodium hexametaphosphate as the model permeabilizers, quantitative and highly reproducible NPN uptake levels were obtained that differed characteristically between the test bacteria. Furthermore, citric acid was shown to be a potent permeabilizer at millimolar concentrations, its effect being partly (Ps. aeruginosa, Salm. typhimurium) or almost totally (E. coli O157) abolished by MgCl2, suggesting that part of the action occurs by chelation. Sodium citrate induced weak NPN uptake, which was totally abolished by MgCl2. In conclusion, the NPN uptake assay with the automated spectrofluorometer serves as a convenient method in analysing and quantifying the effects of external agents, including potential food preservatives, on Gram-negative bacteria.

Silver-resistant mutants were selected by stepwise exposure of silver-susceptible clinical strains of Escherichia coli, two of which did not contain any plasmids, to either silver nitrate or silver sulfadiazine. These mutants showed complete cross-resistance to both compounds. They showed low-level cross-resistance to cephalosporins and HgCl2 but not to other heavy metals. The Ag-resistant mutants had decreased outer membrane (OM) permeability to cephalosporins, and all five resistant mutants tested were deficient in major porins, either OmpF or OmpF plus OmpC. However, the well-studied OmpF- and/or OmpC-deficient mutants of laboratory strains K-12 and B/r were not resistant to either silver compound. Resistant strains accumulated up to fourfold less (110m)AgNO3 than the parental strains. The treatment of cells with carbonyl cyanide m-chlorophenylhydrazone increased Ag accumulation in Ag-susceptible and -resistant strains, suggesting that even the wild-type Ag-susceptible strains had an endogenous Ag efflux activity, which occurred at higher levels in Ag-resistant mutants. The addition of glucose as an energy source to starved cells activated the efflux of Ag. The results suggest that active efflux, presumably coded by a chromosomal gene(s), may play a major role in silver resistance, which is likely to be enhanced synergistically by decreases in OM permeability.