Properties of Pseudomonas aeruginosa resistant to quaternary ammonium compound Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/rf55zc548

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  • Pseudomonas aeruginosa cells were selected for their ability to grow in the presence of 750 ppm alkyldimethylethylbenzyl ammonium chloride (QAC). These cells were found to retain their resistance to the germicide throughout tri-weekly transfers for 7 months in tryptone glucose yeast extract (TGY) broth containing no QAC. Comparisons of the resistant and sensitive cells were made in an attempt to define the mechanism of resistance and, in addition, to provide some information as to the mode of action of QAC. The germicidal activity of QAC solutions against both sensitive and resistant cells in TGY broth was shown to be greatly affected by the concentration of tryptone and yeast extract, but not by the amount of sugar. The pH of the broth also influenced the germicidal activity; both strains were more susceptible under slightly . acid conditions. A comparison of the pH range of growth of sensitive and resistant cells demonstrated the ability of the former to grow in TGY broth at pH 4.5 while the latter could not achieve growth at pH 5.0. A study of the effects of 50 ppm QAC, buffered to various pH levels, indicated that the susceptibility of resistant cells was nearly the same as that of sensitive cells below pH 3.0, at pH 6.0 and above pH 9.0. The greatest difference between the two cell types occurred from pH 3.5 to pH 4.5 with a second peak of resistance being observed from pH 7.0 to pH 8.5. Electron microscopy revealed many dense inclusion bodies in the resistant cells, some being near 0.2 μ in diameter. Furthermore, the resistant cells were found to be much smaller (0.35 X 1.0 μ) than the sensitive cells (0.75 X 3.0 μ). Sensitive cells possessed single polar flagella while resistant cells were completely devoid of flagella. Micrographs of sensitive cells exposed to 100 ppm QAC exhibited no visible signs of lysis and the flagella did not appear to be disrupted. In contrast, 10,000 ppm QAC caused abrupt lysis of sensitive cells. A concentration of 1,000 ppm QAC had no visible effect on resistant cells. Broth cultures of the resistant strain displayed a distinct fruity odor. Gas chromatographic analysis showed the QAC-resistant cells, unlike the sensitive, produced large quantities of ethyl acetate and ethyl valerate. Gel electrophoresis of cell-free extracts revealed a difference in total protein and esterase patterns between the two cell types. Two bands of esterase activity were demonstrated in sensitive cell extracts while only one band was detected in the resistant cell extracts when alpha napthyl acetate was used as the substrate. Biochemical tests disclosed numerous differences between the two cell types, many of which appeared to be interrelated. The most significant differences were the losses in the ability of resistant cells to synthesize extra-cellular lipase and protease enzymes. Many other biochemical tests on resistant cells were negative or became positive only after prolonged incubation. Permeability studies indicated a greatly reduced rate of glucose uptake by resistant cells. Furthermore, growth curve studies indicated a slower rate of growth by resistant cells and a 15 minute longer generation time.
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