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[Ionic binding of 3H-gentamicin and short-term bactericidal activity of gentamicin against Pseudomonas aeruginosa
T Saika1, M Hasegawa, I Kobayashi
1Chemotherapy Division, Mitsubishi Kagaku Bio Clinical Laboratories, Inc.
Abstract:
The clinical isolates of Pseudomonas aeruginosa can be roughly classified into long- and short-lipopolysaccharide (LPS) strains and LPS-deficient strains, based on the silver-stained patterns of their LPSs after SDS-PAGE. The ionic binding of 3H-gentamicin, a polycationic antibiotic, to the negatively charged sites on the surface structures of P. aeruginosa strains, often differing in LPS structure, was the highest in the long-LPS strains followed in descending order by the short-LPS strains and LPS-deficient strains. It was presumed that a clinical isolate of P. aeruginosa No. 45 is lacking in the O-polysaccharide chains and some structures of the core-regions consisting of its LPS-structure after SDS-PAGE. On the other hand, the binding of 3H-gentamicin to this strain was quite. high, i.e., similar to that to of the long-LPS strains. To clarify this finding, P. aeruginosa PAC1R and its LPS-deficient mutants were used as reference strains because the chemical structures of their LPSs containing the repeated units of O-polysaccharides and the neutral sugar contents in the core-regions were previously confirmed. The PAC605 strain of the LPS-mutants of the PAC series, was completely lacking in the repeated units of O-polysaccharide and also lacking in some neutral sugar residues of the core-oligosaccharide region. However, this strain was highly bound to 3H-gentamicin, suggesting that the negatively charged sites on the deep core-oligosaccharide region and/or on lipid A participated in the binding of 3H-gentamicin. This manner of binding may be also applied to P. aeruginosa No. 45. When P. aeruginosa PAC1R, PAC605 and No. 45 strains were each exposed to gentamicin (20 micrograms/ml) for 10 minutes, the viable cell counts of PAC1R decreased to about 70% of the initial count, whereas the viable cell counts of PAC605 and No. 45 strains decreased to 3.6 and 11.0% respectively, indicating the vulnerability of both types of the strains to be enhanced by the bactericidal action of gentamicin with short-term incubation.
Insights
Pseudomonas aeruginosa strains with altered lipopolysaccharide (LPS) structures, particularly those lacking O-polysaccharide chains, show increased binding of the antibiotic gentamicin. This enhanced binding correlates with increased gentamicin
Area of Science:
- Microbiology
- Bacterial Pathogenesis
- Antibiotic Resistance
Background:
- Pseudomonas aeruginosa clinical isolates are classified by lipopolysaccharide (LPS) structure: long-LPS, short-LPS, and LPS-deficient.
- Gentamicin binding varies with LPS structure, being highest in long-LPS strains.
- Strain No. 45 exhibits high gentamicin binding despite presumed LPS structural defects.
Purpose of the Study:
- To investigate the mechanism of high gentamicin binding in Pseudomonas aeruginosa strain No. 45.
- To elucidate the role of LPS structure, specifically O-polysaccharide and core regions, in gentamicin interaction.
- To assess the susceptibility of different LPS mutant strains to gentamicin.
Main Methods:
- Classification of P. aeruginosa strains based on LPS structure via SDS-PAGE and silver staining.
- Quantification of 3H-gentamicin binding to various P. aeruginosa strains, including LPS-deficient mutants (e.g., PAC605).
- Assessment of bacterial viability after short-term exposure to gentamicin (20 µg/ml).
Main Results:
- Strain PAC605, lacking O-polysaccharide and core sugars, demonstrated high 3H-gentamicin binding, implicating deep core or lipid A.
- Strain No. 45 also showed high gentamicin binding, similar to long-LPS strains, supporting the involvement of deep core/lipid A.
- Short-term gentamicin exposure (10 min) significantly reduced viability in PAC605 (96.4%) and No. 45 (89.0%) compared to PAC1R (30%).
Conclusions:
- Negatively charged sites in the deep core-oligosaccharide region and/or lipid A are crucial for high gentamicin binding in LPS-deficient P. aeruginosa.
- LPS-deficient strains, like PAC605 and No. 45, exhibit enhanced vulnerability to gentamicin's bactericidal action.
- Understanding LPS structure-gentamicin interactions is vital for predicting antibiotic efficacy against P. aeruginosa clinical isolates.