Biofilms That Foul CF Patients' Lungs, Factory Piping Yield To Microbial Gene Knockouts

By David N. Leff

Some folks are by nature loners. Others are joiners. It's the same with most bacteria.

Some germs pass their existence as free-floating cells in blood or saliva. But many tend to club together into vast, intricate sheets of living organisms called biofilms.

As microbiologist Peter Greenberg pointed out, "Antibiotics were really discovered based on their ability to kill bacteria growing in a test tube." And that is where most bacteriologists today are studying the mechanism by which individual microbes develop antibiotic resistance.

But like bachelors opting for matrimony, many, if not most, pathogens tend to seek the clubbiness of belonging to a biofilm.

The first step in a microbe's transition from solitary to social existence is finding a solid surface and sticking to it. Then it multiplies and colonizes that infrastructure.

"At some point in this development," Greenberg recounted, "the microorganisms change their coat and start making lots of extracellular polysaccharide. In this thickening slimy substrate, the bacteria distance themselves from each other, and become antibiotic-resistant."

In an experimental biofilm generated by Greenberg and collaborators in Montana and New York, "the fully developed biofilms are about 100 micrometers [one-tenth of a millimeter] thick," he told BioWorld Today. "So if a microbe is one or two micrometers in length, you can imagine there's a big pile-up of cells."

But those bugs aren't just piling on to each other. Rather, each finds its separate space embedded in that polysaccharide, and goes on dividing. Meanwhile, that collegial microbial community forms structures that enable nutrients to penetrate to the depths of the biofilm.

Besides strongly resisting antibiotic drugs, these virtually ubiquitous biofilms wreak havoc on biological and industrial solid surfaces.

Pathogens Stick Together Inside Body

Greenberg's own vendetta is against biofilms created in the lungs of patients with cystic fibrosis (CF) by the pathogen Pseudomonas aeruginosa. He is associate director of the University of Iowa's Cystic Fibrosis Research Center, in Iowa City.

"This particular bacterium that we work with, Pseudomonas aeruginosa," Greenberg told BioWorld Today, "colonizes the lungs of almost everyone who has cystic fibrosis, and CF patients live with this colonization for years and years. "This progressive pulmonary damage," Greenberg pointed out, "ultimately leads to their death."

That scenario is just one of the many medical sins of which biofilms are guilty.

"Any time there are bacteria growing where they're not supposed to be," Grenberg observed, "that's essentially an infection. So if you implant a device in a human — like a catheter, an intravenous line or an artificial heart valve — it often becomes colonized by bacteria. Then chronic infection ensues, the device fails and has to be replaced. And that's why it's so difficult to treat endocarditis with traditional antibiotics."

The biofilm menace, Greenberg surmised, "is probably an under-appreciated problem." He has begun a three-way collaboration with long-time biofilm researchers at the Montana State University Center for Biofilm Engineering, in Bozeman, and with microbiologists at the University of Rochester, in N.Y.

Greenberg is senior author of a paper by this mini-consortium in today's issue of Science, dated April 10, 1998. Its title: "The involvement of cell-to-cell signals in the development of a bacterial biofilm."

Chemical signaling is presumably what warns bacteria, for example, when the biofilm is getting too densely congested with abutting microorganisms, or, conversely, lets it know when the environment has lightened up. More basically, Greenberg pointed out, "we concluded that the signal must be triggering the developmental process that leads to these thick biofilms."

That chemical messenger, he explained, "the auto-inducer, consists of an unusual amino acid, homoserine lactone, which is not one of the 20 essential amino acids. It's stitched onto a 12-carbon fatty acid synthesized by the cell."

The signal transmits its messages by diffusing through the bacterial cell membrane. "So when there aren't very many neighboring cells around," Greenberg pointed out, "the signal diffuses away. When lots of cells are there, the auto-transducer takes a census of the critical density and starts to accumulate.

"We know a lot about the genes that that enzyme and protein control," he added. "Our discovery, reported in Science, shows how they control biofilm development, but we don't know what genes they're controlling. We have work on that under way now."

In their experiment just reported, one of the co-authors, James Pearson at the University of Rochester, made knockout mutants of Pseudomonas bacteria that couldn't express the signals, but were otherwise identical to the wild type.

"Then we asked," Greenberg recalled, "'Can it develop into this thick, biocide-resistant biofilm?' And the answer was 'No, it couldn't' — until we added back the missing signal. Then the rescued mutant developed biofilms just like those of the normal, non-mutant strain."

The Iowa and Rochester groups hold a joint issued patent on the chemistry of the signaling auto-inducer, and they plus the Montana co-authors have a pending patent on using this technology to treat biofilms.

That technology will not be limited to coping with matters medical.

"Biofilms are a serious problem in industrial settings too," Greenberg observed, "because they grow on the surface of equipment used to manufacture all sorts of things." He cited as examples paper industry equipment and filters used in desalinization plants. The films clog piping and ship bottoms.

"It's difficult to eradicate such biofilms, once they start to grow," he pointed out. "Today they are treated with so-called biocides, such as chlorine, which is highly toxic, but they don't quite do the job. There's a federal government mandate to come up with gentler biocides.

"I'm told," Greenberg added, "that the biocide effort against biofilms is a billion-dollar industry."

A company intent on a piece of this action, Quorum Sciences Inc., filed papers of incorporation in Delaware Monday, Greenberg revealed.

"That new company," he concluded, " with which I and another co-author if this Science paper, Barbara Iglewski in Rochester, are involved, is dedicated to finding such compounds as can block this kind of communication." *

Oncor CEO Backing Quorum

Pioneer bio-entrepreneur Steven Turner, CEO of Gaithersburg, Md.-based Oncor Inc., is bankrolling Quorum Sciences Inc.'s start-up as a personal venture.

"The mission of Quorum Sciences," Turner told BioWorld Today, "is multicellular-based antibacterial therapeutics. The idea is that current antibiotics address the killing of individual pathogenic cells, and that maybe we've gone as far with that as we can."

"With Greenberg's work [see ajacent story]," Turner went on, "antibacterial therapeutics in the future are going to be more appropriate if they address that emerging picture of sophisticated bacterial colony-formation — these biofilms. So the signaling pathways that help maintain these biofilms are clearly good targets for future therapeutic development.

"Quorum has licensed Greenberg's patents from the University of Iowa," Turner said, "and intends to offer screening services for companies that want to look for auto-inducer blockers. We're going to base the initial operations in Iowa City, commencing this summer, and we are now entering the fund-raising stage."