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Antimicrobial Agents and Chemotherapy, April 2003, p. 1251-1256, Vol. 47, No. 4
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.4.1251-1256.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Role of Nutrient Limitation and Stationary-Phase Existence in Klebsiella pneumoniae Biofilm Resistance to Ampicillin and Ciprofloxacin

Jeff N. Anderl,1,2,{dagger} Jeff Zahller,1,2 Frank Roe,1 and Philip S. Stewart1,2*

Center for Biofilm Engineering,1 Department of Chemical Engineering, Montana State University, Bozeman, Montana 59717-39802

Received 2 August 2002/ Returned for modification 29 October 2002/ Accepted 30 December 2002

Biofilms formed by Klebsiella pneumoniae resisted killing during prolonged exposure to ampicillin or ciprofloxacin even though these agents have been shown to penetrate bacterial aggregates. Bacteria dispersed from biofilms into medium quickly regained most of their susceptibility. Experiments with free-floating bacteria showed that stationary-phase bacteria were protected from killing by either antibiotic, especially when the test was performed in medium lacking carbon and nitrogen sources. These results suggested that the antibiotic tolerance of biofilm bacteria could be explained by nutrient limitation in the biofilm leading to stationary-phase existence of at least some of the cells in the biofilm. This mechanism was supported by experimental characterization of nutrient availability and growth status in biofilms. The average specific growth rate of bacteria in biofilms was only 0.032 h-1 compared to the specific growth rate of planktonic bacteria of 0.59 h-1 measured in the same medium. Glucose did not penetrate all the way through the biofilm, and oxygen was shown to penetrate only into the upper 100 µm. The specific catalase activity was elevated in biofilm bacteria to a level similar to that of stationary-phase planktonic cells. Transmission electron microscopy revealed that bacteria were affected by ampicillin near the periphery of the biofilm but were not affected in the interior. Taken together, these results indicate that K. pneumoniae in this system experience nutrient limitation locally within the biofilm, leading to zones in which the bacteria enter stationary phase and are growing slowly or not at all. In these inactive regions, bacteria are less susceptible to killing by antibiotics.

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

{dagger} Present address: University of Washington, Seattle, WA 98195.

Antimicrobial Agents and Chemotherapy, April 2003, p. 1251-1256, Vol. 47, No. 4
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.4.1251-1256.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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