Sensitivity of Staphylococcus aureus to the antibiotics tetracycline, benzylpenicillin and vancomycin was found to decrease by 2-10-fold when cells were grown adherent to silicone catheter surfaces. Sensitivity to rifampicin and fusidic acid was not significantly altered in adherent cells. Susceptibility further decreased with increased adherence time prior to antibiotic challenge. The resistance observed was not genotypic, or due to the presence of a specialized subpopulation of bacteria, as it disappeared when the bacteria were removed from the catheter, subcultured and retested. Also, adherent bacteria were found to grow more slowly than bacteria growing planktonically. It is concluded that the decrease in antibiotic susceptibility of adherent bacteria is a function of the physiological status of the individual cells rather than a function of biofilm formation or slime production. The decrease in growth rate of the adherent bacteria is a result of the adherence process rather than a result of nutrient depletion. The decrease in growth rate is implicated, but is not the sole factor, in the decreased antibiotic susceptibility of adherent bacteria.
E. coli K12 was found to utilise both D-and L-stereoisomers of alanine as sole sources of carbon, nitrogen and energy for growth. This capability was absolutely dependent upon the possession of an active membrane-bound D-alanine dehydrogenase, and was lost by mutants in which the enzyme was defective. The Michaelis constant for the enzyme with D-alanine as substrate was 30 mM, and the pH optimum about 8.9. D-alanine was the most active substrate, L-alanine was inactive and several other D-amino acids were 10--50% as active as D-alanine. Oxidation of D-alanine was linked to oxygen via a cytochrome-containing respiratory chain. Synthesis of the dehydrogenase was induced 16 to 23-fold by incubation with D- or L-alanine, but only D-alanine was intrinsically active as an inducer. L-alanine was active either as a substrate or inducer only in t he presence of an uninhibited alanine racemase which converted it to the D-isomer. The map-location of their structural genes between ara and leu, together with other similarities, indicate that D-alanine dehydrogenase and the "alaninase" of Wijsman (1972a) are the same enzyme. Both D- and L-alanine were intrinsically active as inducers of alanine racemase synthesis. The synthesis of both D-alanine dehydrogenase and alanine racemase was found to be regulated by catabolite repression.
The techniques of flow cytometry, scanning and transmission electron microscopy, and confocal scanning laser microscopy were used to study the physiology of Staphy/ococcus aureus in the early stages of surface-attached culture, and to make direct comparisons with planktonic bacteria grown under the same conditions. Attached bacteria growing in nutrient-rich batch culture were found to go through the same growth phases as equivalent planktonic cultures, but with an exponential growth rate of about half that of the planktonic bacteria. Viability of attached bacteria was very high (around 100°/~) throughout the first 24 h of growth. The size and protein content of attached bacteria varied with growth phase, and both measurements were always smaller than in planktonic bacteria at equivalent growth phases. Respiratory activity per bacterium, as measured by flow cytofluorimetry, and corrected for cell volume, peaked very early in attached cultures (before the first cell division) and declined from then on, whereas in planktonic bacteria it peaked in late exponential phase. Attached and planktonic bacteria showed thicker cell walls in stationary phase than in exponential phase. Membrane potentials of planktonic and attached bacteria were similar in stationary phase, but were much lower in exponential-phase attached cells than in the equivalent planktonic cells. It is apparent that a range of significant physiological adaptations occur during the early phases of attached growth.
Pseudomonas putida mt-2 (ATCC 33015) carrying the TOL plasmid pWW0 could adapt to growth on the aromatic amines aniline and m- and p-toluidine. In strain UCC2, a derivative adapted to rapid growth on these compounds, they were oxidatively deaminated to catechol or 4-methylcatechol, which in turn were dissimilated by a meta-cleavage pathway. The aniline/toluidine oxygenase and the meta-cleavage pathway enzymes were inducible by aromatic amines. Evidence is presented that in strain UCC2, plasmid pWW0 has undergone deletion of its catabolic genes, and that it is a novel plasmid, pTDN1, which is involved in the catabolism of aniline and m- and p-toluidine. The meta-cleavage pathway genes which are carried by pTDN1 were shown not to have originated in pWW0.
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