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Examining the environmental and genetic control of Escherichia coli biofilm formation.

Accumulation of bacteria on various interfaces leads to the production of biofilms. Biofilm-associated bacteria are embedded in an extracellular matrix secreted by the bacteria themselves. According to Centers for Disease Control and Prevention, biofilms are estimated to be involved in about 65% of human bacterial infections (1). Biofilm formation is dependent on the environmental conditions, sensed by varies regulators. In this high-throughput study, we are focused on determining the environmental and genetic factors responsible for biofilm formation in Escherichia coli.

For this study, we took combinations of four environmental conditions (i.e. variable temperatures, medium, cell dilutions and incubation times) and nineteen strains of E. coli which includes the wild-type strain, AJW678, and a set of isogenic mutants in varies cell surface organelle or regulator genes. In order to reduce the number of experiments but at the same time cover as many conditions as possible, we used the D-Optimal response surface design algorithm (Design-Expert version 6.0.7, Stat-Ease Inc., Minneapolis MN and JMP 6, SAS Institute Inc., Cary NC).

The bacterial strains were first grown on Luria Bertani plates (LB; 1.5% agar, 1% tryptone, 0.5% NaCl, 0.5% yeast extract) and incubated overnight at 34[degrees]C. They were then grown in appropriate liquid media from which they were transferred to 96 well plates in appropriate dilutions. Plates were incubated without shaking at the designated temperature. In order to quantify the biofilms, an established crystal violet (CV) assay was used (2). Briefly, the biofilms were stained with 0.1% CV solution; the CV was solubilized with 80% ethanol/20% acetone. Optical densities were determined at 600 nm.

From the data collected we can say conclusively that out of the four environmental conditions, temperature and media cause the highest variation in biofilm formation among the strains (Fig. 1). At 37[degrees]C, all the E. coli strains show better biofilm formation compared to that at 27[degrees]C. In the nutrien rich tryptone soy broth (TSB), all strains produced more biofilm than in the nutrient poor tryptone broth (TB).


Using a novel algorithm that follows the vector-item pattern mining concept, an additional interesting observation was made. We related functional annotations (Gene Ontology, GO) of the proteins that are encoded by the mutated genes to quantitative amounts and found that the GO that relates to 'pyruvate catabolic process' was significant if GOs that related to the type I fimbrium were omitted. Mutants in any of the components of the type I fimbrium did not produce any biofilm under any of the conditions tested. Apparently, attachment is crucial to bioiflm formation. Mutants relating to pyruvate catabolism were in acetate kinase (Ack) and phosphotransacetylase (Pta) which are part of acetate metabolism. We conclude that acetate or one of its intermediates are benefitial to biofilm formation. Another mutant that was consistently low in biofilm production was the rcsD mutant that affects the production of the capsule

(1) Dartmouth Medical School (2003, November 20). Biofilm Antibiotic Resistance May Be Susceptible To Genetic Approach.

(2) O'Toole et al. 1999. Genetic approaches to study of biofilms. Meth. Enzymol. 310:91-109.

The authors thank Shane Stafslien and David Christianson (Center for Nanoscale Science and Engineering, NDSU, Fargo ND) for their help with selecting the environmental conditions that were used for this study. The work was funded by the ND Agricultural Experiment Station

Karan Verma [1] *, Anne Denton [2], Birgit Pruss [1]

[1] Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND

[2] Department of Computer Sciences, North Dakota State University, Fargo, ND
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Author:Verma, Karan; Denton, Anne; Pruss, Birgit
Publication:Proceedings of the North Dakota Academy of Science
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2009
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