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Antimicrobial Agents and Chemotherapy, May 2004, p . 1879-1881, Vol . 48, No . 5 Rapid Colorimetric Assay for Antimicrobial Susceptibility Testing of Pseudomonas aeruginosa
Michael M . Tunney,1* Gordon Ramage,2, The Clinical and Practice Research Group, School of Pharmacy, The Queen's University of Belfast, Belfast BT9 7BL, United Kingdom,1 Department of Biological Sciences, University of Calgary, Calgary, Canada T2N 1N42 Received 23 September 2003/ Returned for modification 17 December 2003/ Accepted 18 January 2004
Promising alternative colorimetric methods have been proposed as rapid methods for antifungal susceptibility testing of yeasts (8, 13-15) and Aspergillus species (9-11) and for detection of drug resistance in Mycobacterium tuberculosis (4-6) . These methods utilize the reduction of a tetrazolium salt {2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-car-boxanilide (XTT)} by metabolically active cells to a colored-water-soluble formazan derivative that can be easily quantified colorimetrically . In the study described here, we developed an XTT-based assay for antimicrobial susceptibility testing which provided results within 5 h . The assay was used to determine the susceptibility of clinical P . aeruginosa isolates to a range of antibiotics, and the drug MICs obtained after 5 h using this method were compared with the MICs obtained after 18 h using the conventional NCCLS and BSAC broth microdilution methods . An initial set of experiments were performed with 4A, a clinical P . aeruginosa isolate, to optimize the quantities of XTT (Sigma Chemical Co, Dorset, United Kingdom) and menadione (Aldrich, Dorset, United Kingdom) required to quantify bacterial metabolic activity . XTT was prepared as saturated solutions at 0.5 and 1 mg/ml in phosphate-buffered saline, filter sterilized, and stored at –70°C . Menadione was prepared as a 10 mM stock solution in acetone and stored at –70°C . Prior to each assay, aliquots of XTT were thawed and menadione was added to give the desired final concentration . The planktonic growth of strain 4A over 24 h in Isosensitest broth was measured with the XTT assay . In brief, at each time point six 1-ml samples were removed and centrifuged and 1-ml aliquots of XTT with menadione were added to the resultant pellet to obtain final concentrations of 0.5 and 1.0 mg of XTT/ml and 50, 100, and 200 µM menadione . The samples were incubated in the dark for 1 h at 37°C, after which a colorimetric change in the XTT reduction assay was measured using a microtiter plate reader (Tecan Sunrise absorbance reader; Tecan UK, Reading, United Kingdom) at 492 nm . To determine the relationship between XTT reduction and viable counts, total viable counts were also performed at each time point . We demonstrated that XTT at a concentration of 0.5 mg/ml and menadione at a concentration of 50 µM gave the optimal change in absorbance after 1 h of incubation over the 24-h period of the planktonic growth curve . There was a direct correlation between the viable count results (ranging from 1 x 106 to 2 x 109 CFU/ml) determined by plating and XTT colorimetric readings (Fig . 1), with a correlation coefficient (R2) of 0.9943 . The excellent correlation between XTT readings and total viable counts was also demonstrated for a number of additional clinical strains (R2 > 0.98 for all strains tested; results not shown) .
After incubation, the MIC was read as the lowest concentration of each antimicrobial agent that inhibited the visible growth of the isolate . Determinations of the MIC of each antibiotic for each isolate were carried out in triplicate, and the results were taken in cases in which there was agreement between the results obtained with at least two out of three wells . The microtiter plates were also scanned in the microtiter plate reader at 540 nm; for all isolates tested, a 90% reduction in optical density (compared with control growth results) led to the determination of the same drug MIC . Therefore, a 90% reduction in optical density compared with control growth results was used to determine the drug MIC in the XTT assay for a number of isolates with which visual determination of the MIC by the unaided eye was difficult . The MICs were also determined colorimetrically using XTT in a modified broth microdilution method . The susceptibility trays were prepared as for the NCCLS and BSAC methods, with 50 µl of serially diluted antibiotic added to each well of the tray . Initial experiments were then performed using strain 4A and tobramycin to determine the inoculum density to be used and the earliest time at which an MIC reading could be determined . Inoculum densities of 105 (standard BSAC inoculum) and 108 CFU/ml were prepared in Isosensitest broth, and 50 µl was added to the wells of the microtiter trays . After 2, 4, and 6 h of incubation at 37°C, the trays were removed and 100 µl of fresh XTT was added . After incubation for 1 h at 37°C, the MIC was read as the lowest concentration of antimicrobial agent at which no color change occurred . For isolates with which visual determination of the drug MIC by the unaided eye was difficult, the plates were scanned in the microtiter plate reader at 492 nm and the MIC was determined as the drug concentration that gave a 90% reduction in optical density compared with control growth results . The MIC obtained was then compared with the MIC obtained using the BSAC broth microdilution method following 18 h of incubation . Results from these experiments demonstrated that no drug MIC readings could be determined (using a standard inoculum of 105 CFU/ml) after 2, 4, and 6 h . When the higher inoculum density of 108 CFU/ml was used, however, drug MICs determined at 4 and 6 h corresponded to the MIC obtained using the BSAC broth microdilution method at 18 h . Therefore, subsequent MIC determinations for all antibiotics and isolates were performed with an inoculum density of 108 CFU/ml, and the results were read after addition of XTT to trays which had been incubated for 4 h . The MICs of piperacillin, meropenem, and ceftazidime could not be determined using the XTT assay at 4 h, as there was no difference in growth results between test and control wells (results not shown) . No comparison could therefore be made with the MICs determined by the broth microdilution method at 18 h . The MICs of tobramycin and ofloxacin were determined using the XTT assay at 4 h, however, and there was good correlation between these MICs and the MICs determined using the NCCLS and BSAC broth microdilution methods after 18 h (Table 1) . The inability of the XTT assay to differentiate between growth levels in control and test wells for piperacillin, meropenem, and ceftazidime at 4 h is probably related to the mode of action of these ß-lactam antibiotics . ß-Lactams exhibit time-dependent killing of pathogens . For such antibiotics, killing is concentration independent and proceeds relatively slowly with time; the length of time that the concentration of the antibiotic at the site of infection is maintained above the antibiotic MIC for the pathogen is critical to bacterial eradication .
In conclusion, we have developed a simple and inexpensive assay that allows more rapid determination of the susceptibility of P . aeruginosa isolates to bactericidal antibiotics . Although the XTT assay developed employs a higher inoculum density than the conventional NCCLS and BSAC broth microdilution methods, results obtained are comparable . The XTT assay could therefore be used to rapidly determine the susceptibility of P . aeruginosa to bactericidal antibiotics, thereby ensuring the earlier commencement of appropriate antibiotic therapy . However, further studies with a number of different antibiotics and with additional P . aeruginosa isolates and isolates from other bacterial species would be required to confirm the applicability of the XTT assay for rapidly determining antimicrobial susceptibility in routine clinical practice .
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