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°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.The urinary tract is the most common organ system to experience bacterial infections (17). As a major cause of patient morbidity and health care expenditures for men and women of all age groups, urinary tract infections (UTIs) account for 7 million office visits, more than 1 million emergency room visits, and 100,000 hospitalizations each year (20). The estimated annual cost to the U.S. health care system is approximately $1.6 billion (8). Patients with nosocomial and/or complicated UTIs due to abnormal urinary tract anatomy, the presence of indwelling foreign bodies, and obstructed kidney stones are at risk for life-threatening systemic infections and permanently reduced kidney function. Patients with recurrent communityacquired, simple UTIs may incur significant treatment costs and substantial morbidity due to bothersome symptoms, such as painful urination, frequency, and urgency.The traditional basis for the evaluation of urinary tract pathogens (uropathogens) is urine culture and antibiotic susceptibility testing (13). The major drawback of the current microbiology approach is the time lapse of 2 to 3 days between s...

Development of a Rapid ATP Bioluminescence Assay for Biocidal Susceptibility Testing of Rapidly Growing Mycobacteria

“…Use of a bioluminescence assay based on bacterial ATP has been proposed for assessment of microbial load (10,18) or antimicrobial efficacy (14,19). However, when applied to mycobacteria, a major limitation of this method is the inefficient lysis of the cell wall, resulting in inaccurate measurement of cellular ATP (20,25).…”

A two-hour antibiotic susceptibility test by ATP-bioluminescence

“…In this way, we have a good opportunity to re-examine the current techniques that provide a rapid AST in order to investigate if anyone provides an adequate sensitivity and specificity for clinical practice. In this work we have decided to re-examine the use of ATP-bioluminescence for performing a rapid AST because of its previous published applications in Clinical Microbiology, such as rapid AST in bacteria (including mycobacteria) and yeasts, 4,13-15 screening diagnostic in urinalysis and bacterial detection directly from urine samples 16 and bronchoalveolar lavage. 17 In order to set up the proposed AST based on ATPbiolumeniscence, the RLU obtained from ATCC collection strains incubated with antibiotic were compared to the RLU obtained from the same strains incubated without antibiotic after two hours of incubation.…”

“…In enterococci, seven Enterococcus faecalis and three Enterococcus faecium were tested against Ampicillin, Vancomycin and Ciprofloxacin at concentrations of 32, 16 The results of susceptibility obtained at 35 • C from colonies grown in culture plates by means of the commercial methods VITEK2 (bioMérieux, Marcy l'Etoile, France), MicroScan (Siemens, Tarrytown, NY, USA) and E-test (Liofilchem, Roseto Degli Abruzzi, Italy), applying the criteria published by the EUCAST, 9 were considered as the gold standard. Finally, results obtained by ATPbioluminescence were compared to the gold standard according to FDA criteria 10 ; agreements and disagreements among the susceptibility values obtained were classified as agreements, very major errors (false susceptibility), major errors (false resistance), or minor errors (susceptible/resistant versus intermediate susceptibility).…”

Rapid Antimicrobial Susceptibility Testing by Sensitive Detection of Precursor rRNA Using a Novel Electrochemical Biosensing Platform

“…However, because antibiotics act by widely divergent mechanisms, various approaches may be necessary to achieve comprehensive antibiotic susceptibility testing. For example, we have successfully applied ATP bioluminescence to determine antimicrobial susceptibility of uropathogens within 120 min after inoculation of clinical urine specimens into growth medium with and without antibiotics (18). Application of such assays to bacteria in clinical specimens at the point of care would enable patient-specific antibiotic therapy.…”