By David N. Leff
The spreading pandemic of bacterial biofilms ¿ befouling medical and industrial targets ¿ almost makes one wonder whether the whole world of microorganisms has declared biowar against the human race.
Some pathogens pass their existence as free-floating cells ¿ planktonically ¿ but many germs club tightly together into self-spawned slimy sheets of living microorganisms called biofilms. These ubiquitous coatings devastate biological and industrial surfaces. They¿re populated by a motley collection of infectious bacteria and fungi that not only cause diseases but also practice their own special strategy of antibiotic resistance. (See BioWorld Today, April 10, 1998, p. 1.)
One of the most virulent of these germs is the life-threatening Pseudomonas aeruginosa, which menaces children and adults ¿ but not alike. In the very young, P. aeruginosa colonizes the lungs of patients with cystic fibrosis (CF), and eventually causes their death. But the ubiquitous microbe doesn¿t stop there in its no-holds-barred attack on Homo sapiens. It¿s one of the prime perpetrators of hospital-acquired infections, and the leading cause of respirator-associated pneumonia. Respirators offer tempting surfaces for bacterial biofilms.
Just over a year ago. P. aeruginosa became the largest bacterial genome to be totally sequenced. It weighed in at 6,284,403 base pairs, totaling 5,570 genes. (See BioWorld Today, Aug. 31, 2000, p. 1.)
A member of that international sequencing consortium, bacteriologist Stephen Lory at Harvard Medical School, told BioWorld Today: ¿We have now carried out the first functional genomic application of the P. aeruginosa sequence ¿ with very clinical relevance.¿ Lory is a co-author of a report in today¿s Nature, dated Oct. 25, 2001. Its title: ¿Gene expression in Pseudomonas aeruginosa biofilms.¿ The paper¿s senior author is microbiologist E. Peter Greenberg, at the University of Iowa College of Medicine in Iowa City.
¿What we found,¿ Greenberg told BioWorld Today, ¿was that a limited number of genes were differentially regulated when the Pseudomonas is in a mature biofilm, rather than planktonic. Of its 5,570 genes, we identified 73 that were either activated or repressed by the biofilm state. So this is the first big step in being able to identify specific genes required for biofilm development, or biofilm resistance to antibiotics. We couldn¿t have done this study without the complete genome sequence of Pseudomonas.¿
DNA Chip Analysis Scoped 73 Genes
¿That enabled our colleague, Stephen Lory, to make one of those microarrays,¿ Greenberg recalled, ¿so we could probe every single gene in the bacterium. Most of them are differentially regulated. To be able to sort through like that and get a small group of genes that may be the critical ones is a huge advance. Just from a bioinformatics point of view,¿ he continued, ¿knowing what we know about genes and gene products, we are particularly excited by a handful of other genes. Some of them look as if they¿re involved in pumping antibiotics out of cells, for example. We¿re going to pursue those in our academic lab, and if we find any of critical importance to antibiotic resistance, then strategies for drug development will be defined.
¿When Pseudomonas enters a biofilm,¿ Greenberg continued, ¿the entire microbial group becomes resistant to antibiotics. This is not caused by a mutation, as in planktonic pathogens. And it¿s not a permanent effect. When we take bacterial cells out of a biofilm and grow them as individuals again, they become sensitive to antibiotics once more. We don¿t know what it is about the biofilm lifestyle that makes them tolerant of antibiotics. We need to know what that mechanism of resistance is, so we can interfere with it, and then treat biofilm successfully.
¿We have no idea as to the molecular mechanism of biofilm resistance,¿ he reiterated. ¿No one in the field has a clue. This Nature paper carries the first report that begins to get at the genetic basis of resistance in biofilms. Among the genes we found that were activated, for example, is one called tolA. People who study non-biofilm bacteria have made mutants that express less tolA than the wild type. And those mutants,¿ Greenberg went on, ¿are hypersensitive to antibiotics. So when we grew biofilms, tolA went up higher than when it¿s not in the biofilm. It would be easy to imagine that that might elicit extra antibiotic resistance.
¿When we added to biofilms an antibiotic called tobramycin ¿ the antibiotic of choice in treating CF infection ¿ it caused differential expression of 20 genes,¿ Greenberg recounted. ¿So we imagine that that gene product could be very important in biofilm resistance. Those are just two examples among about a dozen we¿re particularly interested in.
¿Through our work and some other academic labs,¿ Greenberg observed, ¿we¿re now giving the biotech community the tool to develop high-throughput screening of things that can bust up biofilm. The more companies that want to have at it, the better.¿
War Declared On Bioweapons
Then he turned to a broader question: ¿Because anthrax and bioweaponeering are so much in the news, I¿m not saying bin Laden is responsible for biofilms. But can any analogy be made? Whether you can weaponize biofilms or not,¿ Greenberg ruminated, ¿I don¿t even like to ponder. Nobody¿s really done it yet. Rather, I think we may be able to use biofilms in ways you might imagine as defensive against bioweapons.
¿Biofilms are persistent,¿ he pointed out. ¿You can make a biofilm, put it away somewhere, and it will last. We can make it so that it works as a detector, let¿s say, of anthrax. Biofilms also tend to absorb things that come into them,¿ he added, ¿so we might be able to make biofilms that entrap and disinfect contaminated biological samples.
¿With bacteria, there¿s always two sides to the coin,¿ Greenberg observed. ¿If you want to pick out one and make it into a weapon, you can. If you want to pick out some other ones and try to make them something good, you can. Just as there are naturally pathogenic bacteria and bacteria that are by nature useful, I don¿t want people to lose sight of the useful bacteria. The idea of making biofilms that could be used as weapons is pretty far off,¿ Greenberg concluded, ¿but not so far off as using biofilms in some beneficial way to help us in the fight against bacterial warfare.¿