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with traditional molecular microbiological techniques difficult. Bacteria in these mature biofilms are phenotypically resistant to microbicidal agents, including antibiotics. Thus biofilms cause many different types of chronic or persistent bacterial infections (for a recent review of biofilm infections and biofilm physiology see ref.2).

Recent studies have linked quorum sensing and biofilm maturation (48). This is a particularly gratifying finding because quorum sensing functions to control gene expression in groups of bacteria, and biofilms are just that, organized groups of bacteria. A mutation in lasI has a dramatic affect on biofilm maturation. LasI mutants are incapable of 3OC12-HSL synthesis, and the development of LasI mutant biofilms is arrested after microcolony formation but before maturation of the microcolonies into thick structured assemblages. Thus LasI mutant biofilms appear flat and undifferentiated. The normal biofilms architecture can be restored to the mutant by addition of the LasI-generated quorum-sensing signal 3OC12-HSL. A RhlI mutant exhibits normal biofilm development and architecture. The 3OC12-HSL-responsive qsc genes involved in biofilm maturation remain unknown. Of interest, the LasI mutant biofilms were susceptible to treatment with the detergent SDS, whereas wild-type biofilms were resistant. LasR mutants have biofilm phenotypes similar to that of LasI mutants (Fig. 4). These observations suggests that antimicrobial therapies targeting the quorum-sensing mechanism of P. aeruginosa may result in the formation of abnormal biofilms that are more amenable to treatment. Another study showed that acyl-HSLs could be detected in clinical biofilm isolates and on catheters colonized by P. aeruginosa in mice (49).

Some critical questions remain to be answered regarding the quorum-sensing mechanism in biofilms: What constitutes a quorum in a biofilm? Does a LasR–LasI, RhlR–RhlI regulatory cascade exist within a biofilm? Do acyl-HSL synthesis patterns change within a biofilm? Biofilm and quorum-sensing research has led us to appreciate the fact that P. aeruginosa can behave as a community. Because evidence supports the hypothesis that both biofilms and quorum sensing play integral roles in P. aeruginosa pathogenesis, more studies of the community activities of this bacterium are needed.

Future Challenges

It is now generally appreciated that bacteria possess specific communication systems and that they are capable of organizing into functional communities. We have described one type of signaling system, an acyl-HSL system, in some detail in this article. Other signaling systems are known, but it seems apparent that many more remain to be discovered. Aside from the discovery of different types of chemical communication systems for intraspecies signaling, a challenge for the future is to begin to address the possibility that there is significant interspecies communication. The idea of interspecies communication is supported by a limited body of information. For example, we know that many different bacterial species make a signal to which Vibrio harveyi responds; however, we do not yet know the nature of the signal (for a recent review see ref.9). Because quorum sensing is required for virulence of P. aeruginosa and other bacteria, the quorum-sensing system is a target for development of new types of therapeutics, antipathogenic agents, agents that do not kill bacterial pathogens but that do interfere with their ability to cause infections. A challenge that faces us is to identify inhibitors of quorum sensing and test their effectiveness in the treatment of infections, particularly persistent biofilm infections. Another challenge is to better understand the network of genes regulated by quorum sensing, and to identify qsc genes involved in normal biofilm maturation and infection by organisms such as P. aeruginosa.

Fig. 4. Scanning confocal microscope images of a mature P. aeruginosa wild-type biofilm (Upper) and a quorum-sensing mutant biofilm (Lower). In this case the quorum-sensing mutant was a lasR, rhlR double mutant. The perspective is from above the biofilm on a glass surface. The glass surface is red, and the green is from the green fluorescent protein encoded by the gfp gene in the recombinant P. aeruginosa. The wild-type biofilm consists of thick microcolonies. The immature mutant biofilm appears thinner, and more of the glass surface is exposed. With the lasR, rhlR mutant shown here (but not with lasI, rhlI mutants) zones of clearing around microcolony towers are often observed. Other experiments have shown that these zones are filled with extracellular polysaccharide (M.R.P., unpublished data). The biofilms are in flow-through reaction vessels similar to those described in ref. 48. The colors were applied to the image by computer enhancement with Adobe PHOTOSHOP 5.0. The black marker bar is 100 µm in length.

We thank M. Hentzer, A. Heydorn, M. Givskov, and S. Mølin for help with the Biofilm experiments. Work by the authors was supported by grants from the National Institutes of Health (GM59026), the National Science Foundation (MCB 9808308), and the Cystic Fibrosis Foundation.

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