IOWA CITY, Iowa -- Bacteria can exist in a free-floating (planktonic) form or as a biofilm where the cells form organized communities encased in a self-produced slime. One important difference between these two bacterial lifestyles is that biofilms are highly resistant to antibiotics. This poses serious health problems for individuals with biofilm infections, such as Pseudomonas aeruginosa (P. aeruginosa) infections in the lungs of individuals with cystic fibrosis.
Using a technology known as microarray analysis, University of Iowa researchers and their collaborators at Harvard University, Northwestern University and the University of Washington, Seattle, have identified a subset of genes in a bacterium that behave differently when the organism exists as a biofilm. These findings may lead scientists to the genetic causes of increased antibiotic resistance in biofilms. The study results appear in the October 25 issue of the journal Nature.
The P. aeruginosa genome was recently sequenced and appears to contain around 5,500 genes. This sequence information was critical for making the microarray, which the researchers used to examine differences in gene activation for P. aeruginosa when that organism is growing free or growing as a biofilm. Contrary to expectations, only 73 of the bacterium's genes have different activation patterns when they exist as biofilms.
"The hypothesis was there would be hundreds of genetic differences," said E. Peter Greenberg, Ph.D., the Virgil L. and Evalyn Shepperd Endowed Professor of Molecular Pathogenisis and UI professor of microbiology, and principal investigator on the study. "We were surprised that there were only 73 significant differences. The hypothesis that they were completely different was put to rest by these experiments."
In addition, Greenberg noted that these results validate their approach of studying biofilms as a whole despite the fact that biofilm are made up of non-identical cells.
"You can study these heterogeneous biofilm populations and get useful information," Greenberg said. "That's really important; people have argued that because the cells within biofilms are not uniform you can't analyze the biofilm as a whole."
The researchers reasoned that if the genes found by the microarray analysis were critical for biofilm lifestyle, then mutations in those genes should cause changes in biofilm growth and maintenance. To test this, the team investigated a P. aeruginosa strain that contained a mutation in one gene identified by the microarray assay. The mutation was in a gene called rpoS, which was repressed in biofilms.
The researchers found that biofilms formed by the mutant bacteria grew faster and thicker than those of normal strains and were even more resistant to tobramycin, a frontline antibiotic used to treat P. aeruginosa infections.
"You get the idea that rpoS is a gene whose expression has to be turned down to be able to get antibiotic resistance in biofilms," Greenberg said.
The microarray studies also provided other insights into antibiotic resistance mechanisms used by biofilms.
The genome-sequencing project had previously identified several gene clusters for proteins that allow bacteria to pump antibiotics out of their cells. These genes were not expressed in planktonic cells, suggesting that they might be turned on in biofilms and account for antibiotic resistance. In fact, the microarray experiments showed that none of these genes were activated in biofilms.
However, by examining the list of genes that were differentially activated or repressed in biofilms, the researchers have found other genes that have also been linked to the ability of non-biofilm cells to withstand lower concentrations of antibiotics. These include genes for proteins that hinder the entry into cells of aminoglycosides, a particular class of antibiotics. The ability to hinder antibiotic entry to cells is a major resistance mechanism used by P. aeruginosa.
In addition to these, the list contains genes that code for proteins with unknown functions. The researchers are particularly interested in discovering what these proteins do and whether they play a role in antibiotic resistance. Proteins shown to contribute to antibiotic resistance could prove to be important targets for therapies aimed at treating biofilms.
Because cells in biofilms are spatially fixed, antibiotics applied to the biofilm, even at very high concentrations, only gradually diffuse to distant parts of the biofilm. Thus the UI researchers believe that biofilm cells are exposed to a gradually increasing concentration of antibiotic and, rather than being killed outright, the cells have time to respond.
The researchers investigated the genetic nature of this response by treating a biofilm with tobramycin. They found that 20 genes change their activation pattern when compared to gene expression in non-treated biofilms. Greenberg and his team are particularly excited about some of these genes as potential resistance candidates.
One gene activated by antibiotic treatment codes for a protein pump that makes P. aeruginosa resistant to quaternary ammonium compounds. These positively charged molecules are used in disinfectants – beer glasses in bars are usually cleaned with a disinfectant containing some type of quaternary ammonium compound.
"This pump that is turned on in tobramycin-treated biofilms is already known to be a resistance mechanism for other charged antimicrobials," Greenberg said. "We are really excited that maybe this protein has something to do with increased antibiotic resistance in biofilms."
Greenberg noted that microarrays are really good tools for generating hypotheses. Microarrays don't necessarily prove theories but they raise interesting questions, which can then be tested using other biochemical and molecular biology techniques.
"We now have sets of genes that we can really begin to probe to try to understand why biofilms are resistant to antibiotics and once we know that we may be on the home stretch for creating new drugs," Greenberg said.
In addition to Greenberg, the research team included Marvin Whiteley, Ph.D., who recently completed his thesis in Greenbergs' lab and is now at Stanford University, Stephen Lory, Ph.D., professor of microbiology at Harvard University and M. Gita Bangera, Ph.D., also at Harvard, Roger E. Bumgarner, Ph.D., research assistant professor of microbiology at the University of Washington, Matthew R. Parsek, Ph.D., the Louis Berger Junior Professor of Civil Engineering at Northwestern University, and Gail M. Teltzel, a graduate student in Parsek's lab.
The study was funded by grants from the National Institutes of Health, the Cystic Fibrosis Foundation, the National Science Foundation (NSF) and the Proctor and Gamble Company. Whiteley received training grants from NSF and the U.S. Public Health Service.
Journal
Nature