ABSTRACT
In this study we used strains of Stenotrophomonas maltophilia grown at 30 degrees C and 37 degrees C to investigate the role of lipopolysaccharide (LPS) in temperature-dependent variations in sensitivity (TDVS) to gentamicin. TDVS was scored as 'good' if a four-fold or greater difference in minimum inhibitory concentration (MIC) was found between the two incubation temperatures (good TDVS strains; n = 23), and otherwise as 'poor' (poor TDVS strains; n = 15). Phosphate content of isolated LPS in the strains exhibiting good TDVS grown at 37 degrees C was significantly (P < 0.001) higher than those grown at 30 degrees C. However, the phosphate content from LPS of strains exhibiting poor TDVS did not alter significantly with growth temperature. There was no significant difference in 3-deoxy-D-manno-octulosonic acid (KDO) content between the strains grown at the different incubation temperatures. Fluorescence-activated cell sorting analysis showed significant differences in binding of fluorescein Isothiocyanate conjugated gentamicin to cells grown at 30 degrees C or 37 degrees C. We conclude that the temperature-dependent variation in the aminoglycoside susceptibility of this species was not correlated with any detectable change in KDO content, but correlated well with phosphate content of LPS and that LPS phosphate is the major site of ionic interaction for aminoglycosides in S. maltophilia.
Subject(s)
Anti-Bacterial Agents/metabolism , Pseudomonas Infections/metabolism , Pseudomonas/metabolism , Binding Sites/drug effects , Fluorescein-5-isothiocyanate , Fluorescence , Fluorescent Dyes , Gentamicins/metabolism , Lipopolysaccharides/chemistry , Lipopolysaccharides/metabolism , Microbial Sensitivity Tests , Phosphates/chemistry , Pseudomonas/drug effects , Pseudomonas Infections/microbiology , Scattering, Radiation , Sugar Acids/metabolism , TemperatureABSTRACT
Clinical strains of Stenotrophomonas (Xanthomonas) maltophilia often show large, growth temperature-dependent, variations in their susceptibility (TDVS) to aminoglycoside antibiotics. Strains showing more than a fourfold increase in susceptibility between 30 degrees and 37 degrees C (TDVS+ strains; n = 23) were contrasted with those showing lesser variation (TDVS- strains; n = 15) in studies of growth temperature-dependent variation in protein and cell-wall lipopolysaccharide (LPS) electrophoresis patterns in an attempt to determine the mechanism of TDVS. Several proteins showed increased intensity with increasing growth temperature. These comprised bands at c. 65, 55, 42.5, 26 and 21.5 kDa in the whole cell proteins, an outer membrane protein band at c. 21.5 kDa, and cytoplasmic membrane protein bands at c. 42.5 and 27 kDa. Two whole cell protein bands at c. 30 and 24 kDa and three outer membrane protein bands at c. 45, 30 and 24 kDa decreased in intensity with increasing growth temperature. However, there was no correlation with the extent of variation in susceptibility, either in the extent of temperature dependent changes in protein banding patterns, or the presence or absence of specific protein bands. By contrast, temperature-dependent variation in LPS patterns correlated well with TDVS. TDVS+ strains yielded intense ladder patterns of more than 30 discrete bands, and the mean molecular weight of the ladder pattern was markedly higher at growth temperatures < or = 30 degrees C, than at > or = 37 degrees C. TDVS- strains gave a clearly distinct high mol. wt LPS banding pattern showing fewer, less intense bands and a smaller and less consistent shift in mean molecular weight with temperature. Strains which were clearly resistant at 30 degrees and 37 degrees C, had a high mol. wt. polysaccharide component but an absence of the typical LPS-ladder pattern. We conclude that the temperature-dependent variation in the aminoglycoside susceptibility of this species was not correlated with any detectable change in protein composition, but correlated well with changes in LPS structure.
Subject(s)
Anti-Bacterial Agents/pharmacology , Xanthomonas/drug effects , Aminoglycosides , Bacterial Proteins/analysis , Drug Resistance, Microbial , Electrophoresis, Polyacrylamide Gel , Humans , Lipopolysaccharides/analysis , Membrane Proteins/analysis , Microbial Sensitivity Tests , Temperature , Xanthomonas/chemistry , Xanthomonas/growth & developmentABSTRACT
Clinical isolates of Stenotrophomonas (Xanthomonas) maltophilia showed growth temperature-dependent variation in susceptibility (TDVS) to aminoglycoside antibiotics between 30 degrees C and 37 degrees C, but little or no TDVS effect for polymixin B, colistin, ceftazidime, chloramphenicol and piperacillin. When phenylethanol was added at sub-inhibitory concentrations, the TDVS effect was eliminated. Gas liquid chromatography showed that 13-methyl tetradecanoate (i-15;0), was the predominant fatty acid, and was present in lower proportions in cells grown at 30 degrees C than 37 degrees C, by contrast to the unsaturated acids, which were found in increased proportions in cells grown at 30 degrees C. However, the extent of these shifts in composition did not correlate with the extent of the TDVS effect in individual strains. Membrane analysis by spin label-electron spin resonance spectroscopy showed that strains exhibiting TDVS had significantly decreased membrane fluidity compared with susceptible strains at 30 degrees C. Furthermore, analysis of the outer and cytoplasmic membranes from the strains with TDVS revealed that in organisms grown at 30 degrees C, the outer membrane remained in a more rigid conformation than the cytoplasmic membrane. We conclude that resistance of S. maltophilia to aminoglycoside antibiotics at 30 degrees C correlates with changes in the conformation of the outer membrane so that binding and/or uptake of the antibiotic is inhibited.
