The following new series of in vitro experiments were developed and carried out in our laboratoryto study the effect of β-lactam antibiotic inactivating substance (β-lactam-AIS) in the therapy of mixedinfectious diseases caused by gram negative rods (GNRs):
1) Agar plates containing CB-PC and P. aeruginosa were prepared by inoculation of heart infusionagar with overnight broth culture of P. aeruginosa (MIC, CB-PC 50μg per ml.) and addition ofserial 2 fold dilutions of CB-PC. Then, discs 6.5mm. in diameter which were dipped in overnight brothculture of GNRs isolated from clinical materials were put on the plates. After incubation at 37°C for 48 hours, the growth zone of P.aeruginosa around the disc was observed. From the experiments, itwas found that the growth zone did not appear when the concentration of CB-PC was higher than acertain value; the minimal concentration was defined as “MIC and/or MBC around the GNR-containingdisc” in this paper. This phenomenon can be explained from the inactivation of CB-PC by β-lactam-AIS produced by GNRs within the discs and from the bactericidal effects of CB-PC on P.aeruginosa. Increasesin “MIC around the GNR-containing disc” were observed in 4 out of 8 strains of E. coli, 40 of 44Klebsiella, 10 of 14 Enterobacter, and 3 of 15 P.aeruginosa. Especially high values of “MIC around the GNR-containing disc”, i.e., from 1600 to ≥3200μg per ml., were observed in some E.coli, Klebsiellaand Enterobacter strains. Similar results were obtained in the experiments using plates containingE.coli, Klebsiella or Enterococcus, or CET antibiotics.
2) β-lactam-AIS released in the filtrate of heart infusion broth culture of GNRs was assayed. Plateswere made by adding a certain amount of filtrate and serial 2 fold dilutions of CB-PC to heart infusionagar. Then, the plates were streaked with indicator bacteria according to the MIC assay method.From these experiments, it was confirmed that β-lactam-AIS accumulates in broth culture of bacteriawith time and that a dose-response relationship exists between the amount of filtrate and the changesof the MIC value.
It was supposed that the activity of β-lactam-AIS consists mainly of that of β-lactamase.