Biofilms: Microbial Life on Surfaces

Rodney M. Donlan

Disclosures

Emerging Infectious Diseases. 2002;8(9) 

In This Article

The Established Community: Biofilm Ecology

The basic structural unit of the biofilm is the microcolony. Proximity of cells within the microcolony (or between microcolonies) (Figure 4A and B) provides an ideal environment for creation of nutrient gradients, exchange of genes, and quorum sensing. Since microcolonies may be composed of multiple species, the cycling of various nutrients (e.g., nitrogen, sulfur, and carbon) through redox reactions can readily occur in aquatic and soil biofilms.

Polymicrobic biofilms grown on stainless steel surfaces in a laboratory potable water biofilm reactor for 7 days, then stained with 4,6-diamidino-2-phenylindole (DAPI) and examined by epifluorescence microscopy. Bar, 20 µm. Photograph by Ricardo Murga and Rodney Donlan, CDC.

Polymicrobic biofilms grown on stainless steel surfaces in a laboratory potable water biofilm reactor for 7 days, then stained with 4,6-diamidino-2-phenylindole (DAPI) and examined by epifluorescence microscopy. Bar, 20 µm. Photograph by Ricardo Murga and Rodney Donlan, CDC.

Biofilms also provide an ideal niche for the exchange of extrachromosomal DNA (plasmids). Conjugation (the mechanism of plasmid transfer) occurs at a greater rate between cells in biofilms than between planktonic cells.[51,52,53] Ghigo[54] has suggested that medically relevant strains of bacteria that contain conjugative plasmids more readily develop biofilms. He showed that the F conjugative pilus (encoded by the tra operon of the F plasmid) acts as an adhesion factor for both cell-surface and cell-cell interactions, resulting in a three-dimensional biofilm of Escherichia coli. Plasmid-carrying strains have also been shown to transfer plasmids to recipient organisms, resulting in biofilm formation; without plasmids these same organisms produce only microcolonies without any further development. The probable reason for enhanced conjugation is that the biofilm environment provides minimal shear and closer cell-to-cell contact. Since plasmids may encode for resistance to multiple antimicrobial agents, biofilm association also provides a mechanism for selecting for, and promoting the spread of, bacterial resistance to antimicrobial agents.

Cell-to-cell signaling has recently been demonstrated to play a role in cell attachment and detachment from biofilms. Xie et al.[55] showed that certain dental plaque bacteria can modulate expression of the genes encoding fimbrial expression (fimA) in Porphyromonas gingivalis. P. gingivalis would not attach to Streptococcus cristatis biofilms grown on glass slides. P. gingivalis, on the other hand, readily attached to S. gordonii. S. cristatus cell-free extract substantially affected expression of fimA in P. gingivalis, as determined by using a reporter system. S. cristatus is able to modulate P. gingivalis fimA expression and prevent its attachment to the biofilm.

Davies et al.[56] showed that two different cell-to-cell signaling systems in P. aeruginosa, lasR-lasI and rhlR-rhlI, were involved in biofilm formation. At sufficient population densities, these signals reach concentrations required for activation of genes involved in biofilm differentiation. Mutants unable to produce both signals (double mutant) were able to produce a biofilm, but unlike the wild type, their biofilms were much thinner, cells were more densely packed, and the typical biofilm architecture was lacking. In addition, these mutant biofilms were much more easily removed from surfaces by a surfactant treatment. Addition of homoserine lactone to the medium containing the mutant biofilms resulted in biofilms similar to the wild type with respect to structure and thickness. Stickler et al.[57] also detected acylated homoserine lactone signals in biofilms of gram-negative bacteria on urethral catheters. Yung-Hua et al.[58] showed that induction of genetic competence (enabling the uptake and incorporation of exogenous DNA by transformation) is also mediated by quorum sensing in S. mutans. Transformational frequencies were 10-600 times higher in biofilms than planktonic cells.

Bacteria within biofilms may be subject to predation by free-living protozoa, Bdellovibrio spp., bacteriophage, and polymorphonuclear leukocytes (PMNL) as a result of localized cell concentration. Murga et al.[59] demonstrated the colonization and subsequent predation of heterotrophic biofilms by Hartmannella vermiformis, a free-living protozoon. Predation has also been demonstrated with Acanthamoeba spp. in contact lens storage case biofilms.[60]

James et al.[46] noted that competition also occurs within biofilms and demonstrated that invasion of a Hyphomicrobium sp. biofilm by P. putida resulted in dominance by the P. putida, even though the biofilm-associated Hyphomicrobium numbers remained relatively constant. Stewart et al.[61] investigated biofilms containing K. pneumoniae and P. aeruginosa and found that both species are able to coexist in a stable community even though P. aeruginosa growth rates are much slower in the mixed culture biofilm than when grown as a pure culture biofilm. P. aeruginosa grow primarily as a base biofilm, whereas K. pneumoniae form localized microcolonies (covering only about 10% of the area) that may have greater access to nutrients and oxygen. Apparently P. aeruginosa can compete because it colonizes the surface rapidly and establishes a long-term competitive advantage. K. pneumoniae apparently survives because of its ability to attach to the P. aeruginosa biofilm, grow more rapidly, and outcompete the P. aeruginosa in the surface layers of the biofilm.

Several frank bacterial pathogens have been shown to associate with, and in some cases, actually grow in biofilms, including Legionella pneumophila,[59]S. aureus,[62]Listeria monocytogenes,[63]Campylobacter spp.,[64]E. coli O157:H7,[65]Salmonella typhimurium,[66]Vibrio cholerae,[67] and Helicobacter pylori.[68] Although all these organisms have the ability to attach to surfaces and existing biofilms, most if not all appear incapable of extensive growth in the biofilm. This may be because of their fastidious growth requirements or because of their inability to compete with indigenous organisms. The mechanism of interaction and growth apparently varies with the pathogen, and at least for L. pneumophila, appears to require the presence of free-living protozoa to grow in the biofilm.[59] Survival and growth of pathogenic organisms within biofilms might also be enhanced by the association and metabolic interactions with indigenous organisms. Camper et al.[65] showed that Salmonella typhimurium persisted in a model distribution system containing undefined heterotrophic bacteria from an unfiltered reverse osmosis water system for >50 days, which suggests that the normal biofilm flora of this water system provided niche conditions capable of supporting the growth of this organism.

The picture of biofilms increasingly is one in which there is both heterogeneity and a constant flux, as this biological community adapts to changing environmental conditions and the composition of the community.

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