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JAC Advance Access published online on August 16, 2005
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dki285
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© The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org
Received April 28, 2005
Revised July 8, 2005
Accepted July 24, 2005

Original article

Discrepancy between viable counts and light output as viability measurements, following ciprofloxacin challenge of self-bioluminescent Pseudomonas aeruginosa biofilms

Cláudia N. H. Marques 1 {dagger}, Vyvyan C. Salisbury 1, John Greenman 1, Karen E. Bowker 2, and Shona M. Nelson 1*

1 Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England, Bristol, Coldharbour Lane, Bristol BS16 1QY, UK
2 Bristol Centre for Antimicrobial Research and Evaluation, Department of Medical Microbiology, Southmead Hospital, Bristol BS10 5NB, UK

* To whom correspondence should be addressed.
Shona M. Nelson, E-mail: Shona.Nelson{at}uwe.ac.uk


  Abstract

Objectives: To utilize bioluminescence to follow the effect of ciprofloxacin challenge on Pseudomonas aeruginosa biofilms.

Methods: The Sorbarod continuous perfusion culture system was used for the cultivation of biofilms of a self-bioluminescent strain of P. aeruginosa PAO1. Biofilms were challenged with ciprofloxacin (5 mg/L) in the perfusing medium for 3 h and allowed to recover to pre-challenge population levels before initiation of a second 3 h challenge. In addition to determining eluate and biofilm cell survival by conventional viable plate counts, light output was monitored via a luminometer and a low-light-level ICCD camera, to give an indication of metabolism. The effect of drug challenge on biofilm structure was investigated using an environmental scanning electron microscope, which allowed discernment of changes to the three-dimensional biofilm architecture.

Results: On challenge with ciprofloxacin, eluate light output measurements declined to a lesser extent than viable counts for the same samples and also indicated that post-challenge recovery of the biofilm metabolism did not occur as rapidly as suggested by viable count data. Photon detection by ICCD camera allowed real-time, non-invasive imaging of metabolic activity within intact biofilms.

Conclusions: The application of a bioluminescent reporter strain to biofilm research provides valuable real-time positional data on the efficacy of anti-biofilm treatment strategies.

Keywords: P. aeruginosa; biofilms; bioluminescence.
{dagger}Present address. Department of Biological Sciences, PO Box 6000, SUNY at Binghamton, Binghamton, NY 13902-6000, USA.
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