Download Using bacterial biosensors to understand the genetic basis for

Using Pseudomonas aeruginosa bioluminescent
biosensors to understand the genetic basis for
antimicrobial resistance
Angharad Ellen Green1 ([email protected])
Eshwar Mahenthiralingam1, Thomas Connor1, Yvan Le Marc2 and Alejandro Amézquita2
1Cardiff
2Unilever,
School of Biosciences, Cardiff University, Cardiff, Wales, UK.
Safety and Environment Assurance Centre (SEAC), Bedfordshire, England, UK.
Antibiotic Resistance Mechanisms
Workshop for researchers
27th November 2015
Presentation overview
1. Pseudomonas aeruginosa as a contaminant of
industrial products
Introduction
2. Use of preservatives in home and personal care
products
3. Developing bacterial biosensors and exposing them to
industrial preservatives
Project
Aims
4. Identifying differentially regulated genes after
preservative exposure
5. Optimising industrial preservative formulations
Project
outcomes &
conclusions
Pseudomonas aeruginosa
•
•
Gram-negative opportunistic pathogen
Important clinical microorganism
• Successful contaminant of home and personal care products
• Metabolically and genetically versatile
• Ability to survive with minimal
nutritional requirements
Large 5.2-7.0 Mbp
genome
• High tolerance of antimicrobials
Limited knowledge of industrially isolated populations of
P. aeruginosa
Industrial Preservatives
• Class of biocide with a broad range of activity
•
Globally, the most widely used antimicrobial by weight
• Added to home and personal care
products to control microbial growth
• P. aeruginosa is able to survive in the presence of
industrial preservatives
• Mechanisms of resistance specific to preservatives are
not well characterised
Antibiotics Vs. Preservatives
Preservatives
Antibiotics
Vs.
Lewis, (2013)
Limited number of known
resistance mechanisms specific to
preservatives
Arias and Murray, (2012)
Project Overview
Aim: Map the genetic and metabolic pathways associated with
Pseudomonas aeruginosa preservative insusceptibility
Generate an industrial Pseudomonas bioluminescent
mini-Tn5-luxCDABE biosensor library
Identify differentially expressed
genes on exposure to industry
relevant preservatives
Predict bacterial responses to
industrial preservative
formulations
Bacterial Bioluminescent Biosensors
Construction of mini-Tn5-luxCDABE biosensor library
Industrially isolated
Pseudomonas bacteria
Light emitting reporter transposon
inserts randomly within the genes
(Winson et al, 1998)
Construction of mini-Tn5-luxCDABE biosensor library
Produce light emitting
gene expression reporters
Arrange into a biosensor library
Advantages:
• Direct screening method
• Reusable
• Low cost
• Assess response of numerous
genes to a variety of antimicrobials
Disadvantages:
• Luciferase inactivation
• Derivatives of wild type =
predictive method of analysis
• Lethal mutations
Screening preservatives using bacterial biosensors
Biosensors libraries are exposed to preservatives
Screening preservatives using bacterial biosensors
Identify biosensors with a change in light emission
Biosensor
of interest
Preservative responding biosensor
24 hour exposure to the industrial preservative
Benzisothiazolinone (BIT):
Pigment
overproducer
Preservative responding biosensor
24 hour growth on plates containing the industrial
preservative BIT:
Light emission
increase
Preservative responding biosensor
24 hour exposure to the industrial preservative
Phenoxyethanol (PH):
Preservative responding biosensor
24 hour growth on plates containing the industrial
preservative PH:
Light emission
Decrease
Identifying differentially regulated genes after
preservative exposure
Identifying transposon insertion sites
1. Isolation of biosensor genomic DNA
2. Digestion
3. Ligation of digested product
4. Ligated product amplified by inverse PCR
5. PCR products sequenced with nested primers
x10-6 PCR product amplification
6. Sequence mapped back to the wild type strain/
Pseudomonas genome database
Identifying transposon insertion sites
Identifying transposon insertion sites
Biosensor Screening so far:
2200 biosensors screened in triplicate with BIT:
Various light emission responses observed
750 biosensors screened with PH:
An overall 2.15 fold light emission decrease was observed
when biosensors were exposed to PH
100 biosensors mapped to genes of interest:
Mutations in pigment production pathways, transcriptional
regulators, transporter proteins and efflux pumps identified
Project Outcomes and Conclusions
Project Outcome: Optimising preservative formulations
Responding biosensors linked to metabolic networks and gene pathways will be
used to predict responses to various combinations of preservatives
Conclusions
Project has so far demonstrated:
• Successful construction of a light emitting biosensor library using
an industrially isolated strain of Pseudomonas aeruginosa.
• Screening using sub-MIC preservatives identified biosensors with
altered light expression in both planktonic and agar growth.
• Inverse PCR successfully identified transposon insertion sites;
differential gene expression will be validated by real-time PCR.
Research Outcomes and Next Steps:
• Continue screening to develop a biosensor panel for
optimising preservative formulations.
• Global gene expression to preservatives will also be
mapped by RNA-seq.
Acknowledgements
• Cardiff School of Biosciences, Cardiff University
Supervisors: Professor Eshwar Mahenthiralingam and Dr. Thomas
Connor.
Lab Team: Dr. Matt Bull, Dr. Beky Weiser, Dr. Laura Rushton,
Dr. Cerith Jones and Rachel Rowe.
• Unilever, Safety and Environment Assurance Centre
(SEAC)
Dr. Yvan Le Marc and Dr. Alejandro Amézquita.
Any questions?