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ANNUAL CONFERENCE :: Abstract Library
Abstract Library
2003 Conference Abstract
| Type of Submission |
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Submission Type: |
Poster Presentation |
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Subject Category: |
Infection and Immunity |
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| Session Information |
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Presentation Date: |
May 27, 2003 |
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Abstract ID: |
D13 |
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Session: |
Poster 2 |
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Time: |
15:00 |
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| Presenting Author |
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| Other Authors |
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G RAMAGE, University of Calgary
D.G. STOREY, University of Calagary
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| Title |
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A rapid method to determine antimicrobial efficacy against Pseudomonas aeruginosa using a semi-quantitative colormetric assay. |
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| Abstract Text |
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Introduction. Pseudomonas aeruginosa contributes to significant morbidity and mortality in patients with CF, and is associated with acute, fulminating, infections of indwelling prosthetic joints. P. aeruginosa utilizes multiple mechanisms of antibiotic resistance thus, selection of an appropriate antibiotic is critical to the treatment of these infections.Aim. It is the aim of this study to demonstrate a rapid technique to determine the efficacy of antibiotics against P. aeruginosa clinical strains. Here we examine the effects of three antibiotics on 6 clinical isolates of P. aeruginosa collected from CF patients, and the reference strains ATCC 27853 and PAO1. Methods. A semi-quantitative colorimetric method based on the use of XTT-tetrazolium salt was optimised by comparing total viable counts of PAO1 with absorbance readings of XTT as it changed color in response to cellular viability. A linear relationship was demonstrated. The 8 P. aeruginosa strains were then grown to an exponential phase at a density equivalent to 108 cfu/ml. Each strain was then challenged with piperacillin, tobramycin and ofloxacin, each at 4, 16 and 64 mg/L, in triplicate, and incubated at 37oC. Macro- and microdilution were performed. Aliquots of culture were removed at selected time intervals (0.5, 1, 2, 4 and 24 h), pelleted and resuspended in XTT. The absorbance at 490nm was measured after 1 h and compared to 0 h triplicate controls. The efficacy of the different antibiotics was determined based on the % reduction of XTT. For ATCC 27853, viable counts were performed simultaneously to demonstrate the validity of XTT as an alternative to viable counting. Result. The metabolic dye, XTT, showed excellent correlation with viable counts for ATCC 27853. We demonstrated a dose dependant effect for the antibiotics tested against all strains. Tobramycin was shown to be the most efficaceous antibiotic against P. aeruginosa, followed by ofloxacin and piperacillin. Piperacillin showed variable killing kinetics between strains. The susceptibility of each strain was shown to corroborate NCCLS testing results. XTT readings were reproducible and showed no significant differences between absorbance readings within experiments. Discussion. We have used a rapid method that negates viable colony counting and overnight incubations to assess the efficacy of various antibiotics against P. aeruginosa isolates. The method yields rapid results and demonstrates the inhibitory/killing dynamics of antibiotics and sensitivity or resistance of the individual strain tested. This method may prove clinically useful in cases where a rapid result are required to administer the best antibiotic. |
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