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Antimicrobial Agents and Chemotherapy, July 2004, p. 2659-2664, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2659-2664.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Oxygen Limitation Contributes to Antibiotic Tolerance of Pseudomonas aeruginosa in Biofilms

Giorgia Borriello,1 Erin Werner,1 Frank Roe,1 Aana M. Kim,1 Garth D. Ehrlich,2 and Philip S. Stewart1*

Center for Biofilm Engineering and Department of Chemical Engineering, Montana State University—Bozeman, Bozeman, Montana 59717-3980,1 Center for Genomic Sciences, Allegheny Singer Research Institute, Pittsburgh, Pennsylvania 152122

Received 3 October 2003/ Returned for modification 2 January 2004/ Accepted 6 March 2004

The role of oxygen limitation in protecting Pseudomonas aeruginosa strains growing in biofilms from killing by antibiotics was investigated in vitro. Bacteria in mature (48-h-old) colony biofilms were poorly killed when they were exposed to tobramycin, ciprofloxacin, carbenicillin, ceftazidime, chloramphenicol, or tetracycline for 12 h. It was shown with oxygen microelectrodes that these biofilms contain large anoxic regions. Oxygen penetrated about 50 µm into the biofilms, which averaged 210 µm thick. The region of active protein synthesis was visualized by using an inducible green fluorescent protein. This zone was also limited to a narrow band , approximately 30 µm wide, adjacent to the air interface of the biofilm. The bacteria in mature biofilms exhibited a specific growth rate of only 0.02 h–1. These results show that 48-h-old colony biofilms are physiologically heterogeneous and that most of the cells in the biofilm occupy an oxygen-limited, stationary-phase state. In contrast, bacteria in 4-h-old colony biofilms were still growing, active, and susceptible to antibiotics when they were challenged in air. When 4-h-old colony biofilms were challenged under anaerobic conditions, the level of killing by antibiotics was reduced compared to that for the controls grown aerobically. Oxygen limitation could explain 70% or more of the protection afforded to 48-h-old colony biofilms for all antibiotics tested. Nitrate amendment stimulated the growth of untreated control P. aeruginosa isolates grown under anaerobic conditions but decreased the susceptibilities of the organisms to antibiotics. Local oxygen limitation and the presence of nitrate may contribute to the reduced susceptibilities of P. aeruginosa biofilms causing infections in vivo.

* Corresponding author. Mailing address: Center for Biofilm Engineering and Department of Chemical Engineering, Montana State University—Bozeman, Bozeman, Montana 59717-3980. Phone: (406) 994-2890. Fax: (406) 994-6098. E-mail: phil_s{at}erc.montana.edu.

Antimicrobial Agents and Chemotherapy, July 2004, p. 2659-2664, Vol. 48, No. 7
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.7.2659-2664.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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