A rapid method for post-antibiotic bacterial susceptibility testing.

Abstract

Antibiotic susceptibility testing is often performed to determine the most effective antibiotic treatment for a bacterial infection, or perhaps to determine if a particular strain of bacteria is becoming drug resistant. Such tests, and others used to determine efficacy of candidate antibiotics during the drug discovery process, have resulted in a demand for more rapid susceptibility testing methods. Here, we have developed a susceptibility test that utilizes chemiluminescent determination of ATP release from bacteria and the overall optical density (OD600) of the bacterial solution. Bacteria release ATP during a growth phase or when they are lysed in the presence of an effective antibiotic. Because optical density increases during growth phase, but does not change during bacterial lysing, an increase in the ATP:optical density ratio after the bacteria have reached the log phase of growth (which is steady) would indicate antibiotic efficacy. Specifically, after allowing a kanamycin-resistant strain of Escherichia coli (E.coli) to pass through the growth phase and reach steady state, the addition of levofloxacin, an antibiotic to which E. coli is susceptible, resulted in a significant increase in the ATP:OD600 ratio in comparison to the use of kanamycin alone (1.80 +/- 0.50 vs. 1.12 +/- 0.28). This difference could be measured 20 minutes after the addition of the antibiotic, to which the bacteria are susceptible, to the bacterial sample. Furthermore, this method also proved useful with gram positive bacteria, as the addition of kanamycin to a chloramphenicol-resistant strain of Bacillus subtilis (B. subtilis) resulted in an ATP:OD600 ratio of 2.14 +/- 0.26 in comparison to 0.62 +/- 0.05 for bacteria not subjected to the antibiotic to which the bacteria are susceptible. Collectively, these results suggest that measurement of the ATP:OD600 ratio may provide a susceptibility test for antibiotic efficacy that is more rapid and quantitative than currently accepted techniques.