title: Validation of constitutively expressed bioluminescent Pseudomonas aeruginosa as a rapid microbiological quantification tool.
abstract: Whole cell biosensors have been extensively used for monitoring toxicity and contamination of various compounds and xenobiotics in environmental biology and microbial ecology; their application in the pharmaceutical and cosmetics industries has been limited. According to several pharmacopoeias, pharmaceutical products must be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. However there is a lack of a validated bioluminescence method. Prototype whole cell microbial biosensors have already been developed in Pseudomonas aeruginosa ATCC 9027. Validation of the bioluminescent strains was performed in accordance with the pharmacopoeia, Parenteral Drug Association and International Organisation of Standardisation. These strains demonstrated that the bioluminescent method was accurate, precise and equivalent, as compared with plate counting at a range of 10(3)-10(7) CFU/mL. Percentage recoveries using the bioluminescent method were between 70% and 130% for all bioluminescent strains and therefore the bioluminescent method was accurate according to the criteria set in PDA technical report 33. The method was also more precise (relative standard deviation less than 15%) than the traditional plate counting method or the ATP bioluminescent method. The lower limit of detection was 10(3) CFU/mL. Two-way ANOVA showed no significant difference between the traditional plate counting and the novel bioluminescent method for all bioluminescent strains. The bioluminescent constructs passed/exceeded pharmacopoeia-specified criteria for range, limit of detection, accuracy, precision and equivalence.
authors.affiliation: Microbiology, Molecular Biology and Biotechnology Research Group, School of Life and Medical Sciences, University of Hertfordshire, Hatfield Al10 9AB, Hertfordshire, UK.
authors.forename: N
authors.initials: N
authors.lastname: Shah
authors.affiliation: Microbiology, Molecular Biology and Biotechnology Research Group, School of Life and Medical Sciences, University of Hertfordshire, Hatfield Al10 9AB, Hertfordshire, UK. Electronic address: d.c.naseby@herts.ac.uk.
authors.forename: D C
authors.initials: DC
authors.lastname: Naseby
mesh_terms: D015374:Biosensing Techniques; D057230:Limit of Detection; D008163:Luminescent Measurements; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000302:isolation & purification*
keywords: Accuracy; Bioluminescence; Equivalence; Precision; Pseudomonas aeruginosa; Rapid microbiological test with real-time monitoring