Biosynthesis of peptidoglycan in Pseudomonas aeruginosa: comparison of the inhibitory effects of cefotaxime, its anti isomer, and the syn S-oxide compound

Abstract

The intrinsic effect of three novel methoxyimino derivatives of cephalosporin (cefotaxime [syn HR 756]; its anti isomer, R 02 5328 A; and the syn S-oxide derivative, HR 109) on the biosynthesis of peptidoglycan ofPseudomonas aeruginosa X-48 was investigated. Cefotaxime at very low concentrations (50% inhibition at 0.025 ,ug/ml) inhibited the transpeptidase reaction which catalyzes the incorporation and attachment of newly synthesized peptidoglycan to the preexisting cell wall sacculus. The S-oxide compound, HR 109, was a much less efficient inhibitor of this reaction (50% inhibition at 0.55 ,ug/ml), whereas the anti isomer of cefotaxime, R 02 5328 A, had no inhibitory effect. All three compounds were quite similar in being relatively poor inhibitors of D-alanine carboxypeptidase.As previously described (3,4), the biosynthesis of peptidoglycan of Pseudomonas aeruginosa and the enzyme complex which cross-links the peptidoglycan appear to be somewhat similar to those found in Escherichia coli. They consist mainly of a transpeptidase and a DD-carboxypeptidase-endopeptidase system (1, 3, 5). A considerable number of f?-lactam antibiotics have been shown to inhibit these reactions to various degrees. In the present communication, we describe the effects of three novel compounds which are oxime derivatives of cephalosporins. One of these compounds, cefotaxime (HR 756), has been found to have excellent in vivo activity (2, 6) against a wide variety ofbacteria, including P. aeruginosa, and was therefore studied in greater detail. To try to better understand any possible relationship between the structure and the activity of this promising compound, we carried out a comparison of its intrinsic activity at the enzymatic level with two compounds which have closely related structures (Fig. 1), the anti isomer, R 02 5328 A, and the syn Soxide derivative, HR 109. cpmn'/pmol1'), with or without the addition of UDPMurNAc-pentapeptide (50 nmol), as previously described (3, 5). Incubations were for 60 min at 37°C, and determination of peptidoglycan insoluble in trichloroacetic acid and sodium dodecyl sulfate, as well as peptide cross-linkage formations, were as described (3). Determinations of DD-carboxypeptidase and transpeptidase activity were done in assays containing UDP-MurNAc-L-Ala-D-Glu-ms-A2pm-D-Ala-D-["C]-Ala (100,000 cpm, 15 cpm-1/pmol['), with or without UDP-GlcNAc (50 nmol), as described (3, 5). MATERIALS AND METHODSGrowth inhibition. A known amount of antibiotic solutions was placed in a filter paper disk and allowed to dry. The disk was added to a plate (antibiotic no. 3 medium; Difco Laboratories), inoculated with P.aeruginosa X-48, and incubated overnight at 370C.

Des-, syn- and anti-oxyimino-Δ3-cephalosporins. Intrinsic reactivity and reaction with RTEM-2 serine β-lactamase and d-alanyl-d-alanine-cleaving serine and Zn2+-containing peptidases

“…The first observation is in good agreement with that reported by Labia et al (1980). The second, however, does not apply to the DD-carboxypeptidase and transpeptidase activities of ether-treated Pseudomonas aeruginosa cells, for which Mirelman & Nuchamowitz (1980) reported a much decreased effect on the anti isomer.…”

The Combination Effect of Korean Red Ginseng Saponins with Kanamycin and Cefotaxime against Methicillin-Resistant Staphylococcus aureus

“…21,22) Some studies have demonstrated that impermeability of the bacterial plasma membrane plays an important role in resistance to kanamycin. 23) Therefore, if the entry of kanamycin into the cytoplasm is increased by changes in the permeability of the plasma membrane, the antibacterial activity of kanamycin, against kanamycin-resistant bacterial strains, could be increased.…”

Insensitivity of peptidoglycan biosynthetic reactions to beta-lactam antibiotics in a clinical isolate of Pseudomonas aeruginosa

“…In the clinical strain, the only antibiotics tested that affected this reaction were cefotaxime and its analog LY 97962. The reason for this remarkable and apparently exclusive mode of action of cefotaxime is extremely intriguing, and it certainly warrants an in-depth investigation and comparison at the molecular level of the mode of interaction between different 8-lactams and the transpeptidase systems of the resistant and sensitive strains (14).…”

“…The enzymatic reactions which catalyze the attachment of newly synthesized peptidoglycan to the preexisting cell wall material (transpeptidases) are very sensitive to most ,B-lactam antibiotics (13,14,16).…”