Penicillin-Resistant Mechanisms in Pseudomonas aeruginosa : Effects of Penicillin G and Carbenicillin on Transpeptidase and d -Alanine Carboxypeptidase Activities

Abstract

A membrane fraction from Pseudomonas aeruginosa KM 338 was shown to catalyze in vitro peptidoglycan synthesis from uridine 5'-diphosphate-N-acetylmuramyl-L-alanyl-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine and uridine 5'-diphosphate-N-acetylglucosamine. Synthesized peptidoglycan was partially cross-linked by transpeptidation, which was accompanied by the release of D-alanine. This reaction was strongly inhibited by 25 and 50 ,ug of penicillin G and carbenicillin per ml respectively, whereas the intact cells were relatively resistant to penicillins (minimal inhibitory concentration of penicillin G and carbenicillin, 30 and 0.125 mg/ml, respectively). Soluble D-alanine carboxypeptidase present in P. aeruginosa KM 338 was studied as well, which was found almost completely inhibited by penicillin G and carbenicillin (10

Penicillin-Resistant Mechanisms in Pseudomonas aeruginosa : Binding of Penicillin to Pseudomonas aeruginosa KM 338

“…In a previous paper (27) it was reported that the membrane fraction derived from P. aeruginosa KM 338 catalyzes peptidoglycan synthesis, including the cross-linking reaction. This transpeptidase is inhibited by a low concentration of penicillin G or carbenicillin in a similar manner (27), whereas the intact cells are unsusceptible to penicillin G and are relatively susceptible to carbenicillin (22).…”

“…,B-Lactamase activities were estimated by a modification of the bioassay method of Humphrey et al (13) on May 11, 2018 by guest http://aac.asm.org/ Downloaded from G was irreversibly bound to the membrane fraction which catalyzes peptidoglycan synthesis as previously described (27) (Fig. 1) (Fig.…”

“…The organism used was Pseudomonas aeruginosa KM 338, derived from ATCC 17653. This strain has been used in the previous study (27) Assays for the binding of radioactive penicillin G to whole cells and the membrane fraction of P. aeruginosa KM 338. The methods used were almost the same as described in the previous study (26).…”

“…The methods used were almost the same as described in the previous study (26). The organism was routinely grown at 37 C to mid-or late-log phase in Trypticase soy broth (BBL) as described previously (27). The membrane fraction (particulate fraction) was prepared by differential centrifugation from disrupted cells after alumina grinding (27) In the second assay, the membrane fraction-bound [14Cjpenicillin G was pelleted by centrifugation at 45,000 x g for 20 min, and the pellet was then washed four times by centrifugation with 8 ml of 0.05 M Tris-hydrochloride (pH 7.5) containing 0.001 M MgCl2.…”

“…3) Works, Ltd., Tokyo, Japan), and the broken cell preparation was fractionated by differential centrifugations as described previously (27).…”