Effects of imipenem on Escherichia coli studied using bioluminescence, viable counting and microscopy

Abstract

The effects of imipenem on the growth of Escherichia coli ATCC 25922 were studied using a bioluminescence assay of bacterial ATP, microscopy and viable counting in iso-osmotic Mueller-Hinton broth (MHB) and hypo-osmotic nutrient broth (NB). Imipenem showed a post-antibiotic effect (PAE) of > 2 h for E. coli in both MHB and NB after 2 h exposure to 1 and 8 mg/L of imipenem when determined by bioluminescence and microscopy. The intracellular ATP level increased after 2 h exposure of E. coli to 1 mg/L of imipenem in MHB. In this culture there was a predominance of spheroplasts. These spheroplasts were large and osmotically fragile and a 10 min treatment in water-diluted MHB (hypo-osmotic) prior to the assays lysed the large spheroplasts. This reduced the intracellular ATP level and shortened the PAE when determined by bioluminescence, and caused more rapid initial killing and a negative PAE when determined by viable counting. At 8 mg/L imipenem in MHB and at all concentrations in NB there was a predominance of rods and only a small number of spheroplasts which all disappeared when the cultures resumed logarithmic growth. In these cultures there was a significant initial decrease in intracellular ATP. This study showed reasonable agreement between microscopy and bioluminescence, which are direct methods, for determining the initial killing and PAE of imipenem on E. coli. More rapid initial killing and shorter or no PAEs, were in general, obtained in both MHB and NB when determined by viable counting. However, the effective regrowth time, defined as the time for the bacterial density to increase 1 log10 from the pre-exposure inoculum, was independent of the method used for measuring regrowth in both MHB and NB.