N Shah1, D C Naseby. 1. Microbiology, Molecular Biology and Biotechnology Research Group, School of Life and Medical Sciences, University of Hertfordshire, Hatfield, Hertfordshire, UK.
Abstract
AIMS: Detection of microbial contamination in pharmaceuticals, food and cosmetics has been problematic for several decades. Numerous investigations highlight the urgency for novel methods; development of bioluminescent constructs allows real-time monitoring, rapid analysis and high-throughput screening of products. Microbial growth can be studied by measuring constitutive gene expression. The aim is to develop whole-cell microbial biosensors with Pseudomonas aeruginosa and quantify their growth rate by measuring constitutive expression of lux. METHODS AND RESULTS: Pseudomonas aeruginosa cells were genetically modified to produce bioluminescence constitutively. Strains were characterized by assessing their growth kinetics, plasmid stability and gene expression with bacterial replication. Furthermore, cell viability was measured by fluorescence quantification. Promoter strengths were evaluated by comparing bioluminescence (RLU) per colony-forming units (CFU) at various growth stages and related to promoter sequences. Promoter strength decreased in the order of P(lpp) > P(tat) > P(lysS) > P(ldcC) > P(spc) during exponential phase whilst P(tat) was stronger than P(lpp) during stationary phase. Good correlations between RLU and CFU at 24 h indicated a strong relationship for all bioluminescent strains; however, weaker correlations between RLU and CFU and between fluorescence (RFU) and CFU beyond 24 h indicated that a proportion of cells had lost the ability to culture. CONCLUSIONS: Equivalence analysis showed no significant difference between bioluminescence and plate counting for all five bioluminescent strains. Pseudomonas aeruginosa-containing P(tat) had steady bioluminescence when correlated to CFU (R > 0·9), and together with fluorescence data, it can be concluded that Ps. aeruginosa ATCC 9027 tat-pMElux is preferred for testing microbial viability. SIGNIFICANCE AND IMPACT OF THE STUDY: These whole-cell bioluminescent strains provide a platform for utilization in monitoring toxicity and contamination of compounds in environmental biology and microbial ecology, preservative efficacy testing (PET) in the pharmaceutical cosmetics and food industries; the use of such biosensors provides an alternative, fast and efficient method to traditional methods.
AIMS: Detection of microbial contamination in pharmaceuticals, food and cosmetics has been problematic for several decades. Numerous investigations highlight the urgency for novel methods; development of bioluminescent constructs allows real-time monitoring, rapid analysis and high-throughput screening of products. Microbial growth can be studied by measuring constitutive gene expression. The aim is to develop whole-cell microbial biosensors with Pseudomonas aeruginosa and quantify their growth rate by measuring constitutive expression of lux. METHODS AND RESULTS:Pseudomonas aeruginosa cells were genetically modified to produce bioluminescence constitutively. Strains were characterized by assessing their growth kinetics, plasmid stability and gene expression with bacterial replication. Furthermore, cell viability was measured by fluorescence quantification. Promoter strengths were evaluated by comparing bioluminescence (RLU) per colony-forming units (CFU) at various growth stages and related to promoter sequences. Promoter strength decreased in the order of P(lpp) > P(tat) > P(lysS) > P(ldcC) > P(spc) during exponential phase whilst P(tat) was stronger than P(lpp) during stationary phase. Good correlations between RLU and CFU at 24 h indicated a strong relationship for all bioluminescent strains; however, weaker correlations between RLU and CFU and between fluorescence (RFU) and CFU beyond 24 h indicated that a proportion of cells had lost the ability to culture. CONCLUSIONS: Equivalence analysis showed no significant difference between bioluminescence and plate counting for all five bioluminescent strains. Pseudomonas aeruginosa-containing P(tat) had steady bioluminescence when correlated to CFU (R > 0·9), and together with fluorescence data, it can be concluded that Ps. aeruginosa ATCC 9027 tat-pMElux is preferred for testing microbial viability. SIGNIFICANCE AND IMPACT OF THE STUDY: These whole-cell bioluminescent strains provide a platform for utilization in monitoring toxicity and contamination of compounds in environmental biology and microbial ecology, preservative efficacy testing (PET) in the pharmaceutical cosmetics and food industries; the use of such biosensors provides an alternative, fast and efficient method to traditional methods.