Rapid antimicrobial susceptibility determination of uropathogens in clinical urine specimens by use of ATP bioluminescence

Abstract

We describe the first direct testing of the antimicrobial susceptibilities of bacterial pathogens in human clinical fluid samples by the use of ATP bioluminescence. We developed an ATP bioluminescence assay that eliminates somatic sources of ATP to selectively quantify the bacterial load in clinical urine specimens with a sensitivity of <1,000 CFU per milliliter. There was a log-log relationship between light emission and the numbers of CFU in clinical urine specimens. A clinical study was performed to evaluate the accuracy of the ATP bioluminescence assay for determination of the antimicrobial susceptibilities of uropathogens in clinical urine specimens tested in a blinded manner. ATP bioluminescent bacterial density quantitation was used to determine the inoculation volume in growth medium with and without antibiotics. After incubation at 37 degrees C for 120 min, the ATP bioluminescence assay was repeated to evaluate the uropathogen response to antibiotics. The ability of the ATP bioluminescence assay to discriminate between antimicrobial susceptibility and resistance was determined by comparison of the results obtained by the ATP bioluminescence assay with the results obtained by standard clinical microbiology methods. Receiver operator characteristic curves were used to determine the optimal threshold for discriminating between susceptibility and resistance. Susceptibility and resistance were correctly predicted in 87% and 95% of cases, respectively, for an overall unweighted accuracy of 91%, when the results were stratified by antibiotic. For samples in which the pathogen was susceptible, the accuracy improved to 95% when the results for samples with less than a 25-fold increase in the amount of bacterial ATP in the medium without antibiotics were excluded. These data indicate that a rapid bioluminescent antimicrobial susceptibility assay may be useful for the management of urinary tract infections.

FIG. 1.

Relationship between bioluminescence and the concentration of uropathogens in clinical urine cultures. The bioluminescence assay results for 50 clinical urine specimens from patients with UTIs show the relationship between the log of the RLU measurements and the log of the number of CFU present in the specimen. The roughly linear log RLU-log CFU relationship was maintained, despite the inclusion of specimens containing 11 different species of uropathogens.

FIG. 2.

Bioluminescent response to antibiotics of pathogens in clinical urine specimens. The time course of two representative clinical urine specimens from patients with UTIs after inoculation in TSB with and without antibiotics is shown. (A) A urine specimen containing an E. coli isolate sensitive to cephalothin and gentamicin but resistant to ciprofloxacin. (B) A urine specimen containing a Klebsiella oxytoca isolate sensitive to cephalothin, ciprofloxacin, and gentamicin. In each case (A and B), susceptibility could be differentiated from resistance at 150 and 90 min after inoculation, respectively.