Bacterial Biofilms, Other StructuresSeen as Mainstream ConceptsGet used to it: bacterial microcolonies form regular shapes, such asnanowires or honeycomb-like structures

Christoph Schaudinn, Paul Stoodley, Aleksandra Kainovic, Teresa O’Keeffe,

Bill Costerton, Douglas Robinson, Marc Baum, Garth Ehrlich, and Paul Webster

As the notion that some bacteria livein structurally complex, multicellu-lar communities gains momentum,let us pause to collect our thoughts.Many microbiologists consign or-

ganisms with complex structures or behaviors as“weird” and outside the mainstream. Thus, mi-crobiologists who focus on Escherichia coliK-12 acknowledge the complexities of Myxo-bacteria and Beggiatoa but may not spend muchtime thinking about them!

When we see a particular structure or behav-ior in one organism, we really should look forthis structure or behavior throughout the do-main. Woody Hastings and Ken Nealson de-scribed signal-controlled luminescence in ma-rine vibrios in 1977. However, another twodecades elapsed before Peter Greenberg, now at

the University of Washington in Seattle, andBarbara Iglewski at the University of Rochesterin Rochester, N.Y., established that cell-cell sig-naling is critical throughout the bacterial do-main, and even longer for our group to recog-nize that such signals help to control bacterialcommunity development. Now we can searchfor genetic homologies “in silico,” enabling usto search more efficiently for common molecu-lar mechanisms anywhere within microbiology.

Structured Microbial Communities

Come in Many Forms

When individual cells of a single species such asMyxobacteria aggregate, they may produce mac-roscopic communities (Microbe, January 2007, p.

18). However, many natural biofilms typi-cally are featureless until viewed with lightmicroscopes. These magnified views revealmicrocolonies in an English garden of topiarySummary

• Microbial species reproducibly form regularstructures, including “honeycombs” and“veils” that can grow to macroscopic sizes.

• These structures, which are not artifacts, occurboth in cultures and ecosystems, and they con-stitute a genetically determined, heretofore un-recognized structural component of many mi-crobial communities.

• These structures are associated with large num-bers of bacterial cells when they are first formed,but may be devoid of cells once the structuresmature.

• The structures are not composed of a singleextracellular constituent, but appear to containmany components of the cells that form them.

Christoph Schaudinn is a Research Associate and Bill Costerton is Directorof the Center for Biofilms, School of Dentistry, University of Southern Cali-fornia, Los Angeles; Paul Stoodley is Associate Professor at the Center forGenomic Sciences, Allegheny Singer Research Institute, and at the Depart-ment of Microbiology and Immunology, Drexel University College of Medi-cine, Allegheny Campus, Pittsburgh, Pa., and Garth Ehrlich is Executive Di-rector of the former and Professor at the latter; Aleksandra Kainovic andTeresa O’Keeffe are Research Fellows and Marc Baum is an Associate Fac-ulty Member at the OakCrest Institute of Science, Pasadena, Calif.; DouglasRobinson is President and Chief Scientific Officer at deNovo Biologic LLC,Arlington, Va.; and Paul Webster is Director of the Ahmanson Advanced EMand Imaging Center at the House Ear Institute, Los Angeles, Calif.

Perspective

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delights, taking shapes that resemble mushrooms,towers, and arboreal structures that sometimesbear spores at their apices.

From viewing an extensive variety of suchstructures, we conclude that phenotypically dis-tinct sessile bacterial cells surround themselveswith extracellular polymeric substances (EPS) toform microcolonies whose shape and structureare determined by cell-cell signals and influ-

enced by environmental conditions.Perhaps the epitome of community or-ganization is found in the very exten-sive (more than 10 cm2) “veil” com-munities formed by Thiovulum on thesurfaces of marine sulfide deposits,with some cells retaining their flagellawith which they “ventilate” the wholecommunity while suspended from acommon scaffolding.

We can see these highly structuredbacterial communities change shapeunder microscopes. Indeed, we nowrealize that matrix components formthe structures and carry out many ofthose behaviors. Because self-assem-bling protein structures are well de-fined and because activities such as mo-tility and conjugation are measureable,the pili in the intracellular spaces ofbiofilms can be deduced and in somecases detected. Moreover, nanowireshave been discovered, helping to ex-plain how energy is shared among bac-teria in a community, how individualcells can be brought together to conju-gate, how cells and whole communitiescan move by twitching or gliding, andperhaps how particular cells can be“placed” within a biofilm.

Are we ready to think of pathogenicbacteria in periodontitis or in the lungsof patients with cystic fibrosis (CF) asmembers of highly organized commu-nities? If so, we can begin to developstrategies for disrupting those commu-nities by jamming their signals ordraining their energy supplies.

Discoveries of Nanowires and

Honeycomb-Like Structures at

First Prompted Skepticism

Describing microbial communities asproducing nanowires and honeycomb-likestructures is to assert a major leap in their com-plexity. Because both those discoveries camethrough scanning electron microscopy (SEM),they were met with skepticism. SEM is a useful,high-resolution imaging method. However, speci-mens for SEM must be dehydrated, either in airor by solvents, which leaves dissolved organiccomponents behind.

F I G U R E 1

(A) Unmagnified view of a 3-day liquid culture of the MH strain of S. epidermidis showingthe white “nodes” that are suspended in a network that fills the whole test tube andgradually forms a dense white pellet. (B) Confocal micrograph of hexagonal honeycombstructures in a living liquid culture of the MH strain of S. epidermidis. (C) The extensivehoneycomb-like structures formed, as the result of eutectic formation, when concen-trated protein solutions are frozen by the liquid propane method. (D) TEM of the cells andhoneycomb elements of a honeycomb produced by the # 355547 ATCC strain of S.epidermidis, in a preparation that had been frozen at high pressure to preclude eutecticformation.

232 Y Microbe / Volume 2, Number 5, 2007

These “sticky” residues coat the sur-faces of solid components such as cellsand pili, sometimes generating bogus“overcoats” and bridges. Simple freez-ing also removes water and leaves dis-solved organic molecules behind, inthis case in an equally sticky “eutectic”that can appear as artifacts of baroquecomplexity when the eutectic freezes.Hence, to rule out artifacts, microsco-pists insist that novel structures be seenusing at least two independent meth-ods before they are considered credible.

For instance, after Yuri Gorby of thePacific Northwest National Labora-tory in Richland, Washington, de-scribed nanowires, skeptics told himthat they might be artifacts consistingof simple eutectic bridges. These argu-ments led him to use transmission elec-tron microscopy (TEM) and othermethods to prove that nanowires aregenuine structures that carry electricalcurrents for hundreds of micronsthrough microbial communities, layingsuch doubts to rest.

Meanwhile, we visualized the honey-comb-like structures formed by theMH strain of Staphylococcus epider-midis by direct observation (Fig. 1A)and confocal microscopy (Fig. 1B) ofliving, fully hydrated preparations.However, when we examined the hon-eycomb-like structures by SEM (Fig. 3and Fig. 4), we suspected an artifactbecause Paul Webster could produce ahoneycomb-like image by freezingconcentrated protein solutions to form a eutec-tic (Fig 1C). When he next used high-pressurefreezing to prepare sections of the MHcommunity for TEM, this more reliable meansfor preparing specimens showed that the cellsproduce an extensive honeycomb-like structure(Fig. 1D). Because the honeycomb-like struc-tures of the MH strain are seen in unfixed fullyhydrated preparations and by using the rapidhigh-pressure freezing method, we are confidentin using SEM to study them.

Biofilms and Other Structures Can Be

Regular and Reproducible

Because EPS generally lacks tensile strength, weat first assumed that the characteristic shapes of

microcolonies in biofilms result from the rigidityof the cells and the equally rigid pili and nano-wires. EPS was thought to account for the over-all viscoelastic properties of biofilms and for theelastic deformations seen when these communi-ties are subjected to shear forces.

However, Doug Robinson helped to refinethis sense of what underlies the size and com-plexity of microbial communities when he de-scribed the honeycomb-like structures that canfill test tubes in liquid cultures of the MH strainof S. epidermidis (Fig. 1A). Similarly, PaulStoodley and Marc Baum noted that their PAO1and EvS4-B1 strains of Pseudomonas aerugi-nosa and Pseudomonas sp. TM7_1 form com-parable macroscopic networks (Fig. 2). These

F I G U R E 2

(A) Confocal micrograph of the honeycomb (green) formed by the cells (red) in a 3-dayliquid culture of the PAO 1 strain of P. aeruginosa. (B) SEM of a collapsed and foldedstreamer formed in a shaken liquid culture of the EvS4-B1 strain of Pseudomonas sp.TM7_1. (C) Detail of the membrane (arrow 1) of a streamer which is cleaved to show thedistribution of cells (arrow 2) in an amorphous matrix inside this structure. (D) Detail of theinterior of another area of the same streamer in which the bacterial cells are integratedinto a honeycomb structure with a very fine periodicity of �1 �m.

Volume 2, Number 5, 2007 / Microbe Y 233

large networks can be lifted from test tubes andlaid on microscope slides. Once magnified, thecells are seen as associated with a flexible, three-dimensional, honeycomb-like structure.

Many strains of S. epidermidis from dogs withlymphomas produce these huge honeycomb-likestructures for one or two serial transfers, andthen lose this ability. However, the MH strainretains this capability, and several ATCC strains

of S. epidermidis and the PAO 1 andEvS4-B1 Pseudomonas strains also re-tain this community-building capacitythrough many transfers. The EvS4-B1strain of Pseudomonas sp. TM7_1 thatMarc Baum’s group isolated from soilforms very extensive honeycomb-likestructures when cultivated in a shakenfluid culture (Fig. 2B-D), while thePAO 1 strain of P. aeruginosa formssimilar structures (Fig. 2A) when culti-vated in flowing liquid culture withperiodic nutrient replacement.

We speculate that these networkstructures help biofilms survive whenthey are subject to fluid forces. A rigidsheet of honeycomb-like structuresmay provide mechanical stability thatcould serve as an important virulencefactor, helping to wall off host de-fenses. The elastic honeycomb sheetmay allow deformation in response tostress applied along any one of the sixaxes of symmetry. Flexible networkscould deform yet allow the biofilm toreturn to its original structure once astress is removed.

A honeycomb arrangement may alsoprovide a “rip-stop” function, limitingtears by distributing the force over sixvertices. This attribute would be a use-ful when a biofilm is exposed to a mul-tidirectional flow field, such as those ofstreambeds and ocean sediments. Thefluttering and stretching observedwhen these structures are subjected toshear forces supports this hypothesis.In addition, honeycomb-like structuresmay lower the energy costs of individ-ual cells faced with limited nutrients.Moreover, the tertiary structure ofhoneycombs may maximize the surfacearea available for absorbing nutrients.

Regularly Structured Matrices Can Form

within Microbial Communities

In flowing systems, the PAO 1 strain of P.aeruginosa makes honeycomb-like structures(Fig. 2A) that form discrete membrane-enclosedstreamers very similar to those that the EvS4-B1strain forms in shaken cultures (Fig. 2B). After 3

F I G U R E 3

SEMs of the honeycomb structures produced by the MH strain of S. epidermidis showing(A) the development of plate-like structures that extend for as far as 100 microns throughthe liquid culture, and (B) the alignment of the plates at intervals of �/� 8 �m and thedevelopment of partitions at similar intervals. Note that the coccoid bacterial cells arealigned with the plates and partitions, and appear to be intimately associated with thesehoneycomb structures.

234 Y Microbe / Volume 2, Number 5, 2007

days, the flow-based PAO1 culture iscomposed of a mixture of hexagonalsheets (Fig. 2A) and discrete streamers,while the shaken culture of theEvRS-B1 strain is composed predomi-nantly (Fig. 2B) of streamers (at 7days). Those streamers contain numer-ous rod-shaped cells encased in a struc-tured matrix material (Fig. 2C, arrow2, and Fig. 2D).

In both strains the streamers con-taining the bacterial cells are sur-rounded by a coherent membrane (Fig.2B and Fig. 2C, arrow 1) that enclosesthe cells in a manner that would pre-clude their escape into the fluid. Insome areas within the streamers thecells of EvS4-B1 strain of Pseudomo-nas sp. TM7_1 are embedded in anamorphous material (Fig. 2C). How-ever, elsewhere, they are enmeshed in ahoneycomb-like structure with a veryfine periodicity of less than 1 �m (Fig.2D).

Liquid cultures of the MH strain ofS. epidermidis grow as a suspension ofindividual planktonic cells until day 2,when white macroscopic “nodes” be-gin to form. With SEM, we can recon-struct the process of honeycomb build-ing (Fig. 3 and 4). Simple plate-likestructures continue to appear in theculture until thin planar “walls” ex-tend for more than 100 �m (Fig. 3A).In other locations the walls align atdistances of about 8 �m, and individ-ual cells intimately associate with theseplanar structures, forming sites thatappear clear and smooth in someplaces, while others are studded withcoccoid bacteria.

When the walls are structurally co-herent and almost devoid of adherentcells, cells gather into rows at approximately 8-�m intervals on the wall surfaces. These aggre-gates of coccoid cells appear to form “parti-tions” joining the walls (Fig. 3B). Ultimately,mature honeycomb-like structures have walls ofabout 150 nm (Fig. 4B, arrows) and partitionsof about 100 nm. These honeycomb-like struc-tures are highly organized and no longer areassociated with individual cells (Fig. 4B andcover).

Ruminations on the Ramifications of

These Microbial Ramparts

These honeycomb-like structures in liquid cul-tures of the MH and other strains of S. epider-midis and of the PAO 1 and EvS4-B1 Pseudo-monas strains are made from pure cultures ofbacteria, all of which were transferred by stan-dard techniques in serial cycles. They contain noeukaryotic cells or extraneous DNA.

F I G U R E 4

SEMs of mature honeycomb structures produced by the MH strain of S. epidermidis inwhich (A) some cells are still associated with the walls and partitions and (B) in whichsome areas of these very regular structures are devoid of the bacterial cells that formedthem. The very regular dimensions (arrows) of the walls (�/� 150 nm) and partitions (�/�100 nm) can be seen, where they are cross-fractured, and each element of these complexstructures is seen to be very deep (�30 �m).

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These honeycomb-like structures fill thegreater part of culture vessels, and each has anarchitecture peculiar to the organism and thegenome concerned, indicating that their tertiarystructures are firmly under genetic control. Thedevelopmental cycle of each community is re-peated when the culture is transferred, meaningthe ontogeny of the honeycomb-like structuresis as reproducible as the embryology of higherorganisms. Thus, we can formulate several “em-bryological” questions for these bacteria. Howdo the cells consistently produce a plate shape,not a blob or star? How do the plates align atsuch regular 8-�m intervals? How do these bac-teria construct partitions at 8-�m intervals onthe face of each wall? What stops the bacteriawhen a wall or a partition reaches its “pro-grammed” thickness? Perhaps the networksfunction in other than structural capacities. Forexample, they might serve as communicationroutes for chemical signals, “roadways” alongwhich bacteria glide, or, extending the nanowireconcept, electrical conduits for solid-phase elec-tron acceptors.

It is striking to us that the tertiary honey-comb-producing microorganisms include S. epi-dermidis, a human skin commensal species thatis ubiquitous in our environment, and P. aerugi-nosa, the predominant aquatic organism onearth. These are not rare or unusual bacteria.Further, the ability to construct honeycomb-likestructures is widespread among ATCC strains ofS. epidermidis, and it is retained through multi-ple serial transfers. For instance, the EvS4-B1strain of Pseudomonas sp. TM7_1, which wasisolated from soil at Sulphur Mountain in Ven-tura County, Calif., has retained its ability toconstruct wall-enclosed honeycomb-like struc-tures through at least 20 serial transfers, whilethe PAO 1 strain has been carried in various labsfor 30 years. Meanwhile, the common occur-

rence of microbial veils on the surfaces of sul-fidic deposits in marine environments indicatesthat Thiovulum species form complex biofilms,with some cells retaining their flagella whileothers adopt the biofilm phenotype.

These recent discoveries embarrass some of usfor having overlooked them for so long. Thus,3-day-old cultures of S. epidermidis typicallycontain visible white foci and complex honey-comb-like structures. Some of them likely satunnoticed in test tubes on lab benches since the1860s. But it is time to set such regrets aside. Wenow know the genomic sequences of both S.epidermidis and P. aeruginosa, and we can in-troduce mutations that will impair the ability ofboth these organisms to produce or control thedetailed structures of their respective honey-comb architectures to determine what genes areresponsible.

As we fill blank spaces in the genomes ofbacteria, we will identify the genes that con-trol biofilm formation, interspecies interac-tions, and the architecture of structures thatconstitute multispecies communities in whichmost bacteria live. We will also discover thegenes that control the acquisition and struc-ture of commensal biofilms on which much ofhuman health depends, enabling us to culti-vate our microbial friends and confound ourmicrobiological enemies. Perhaps, microbialendocrinologists will develop signals that willmake lactobacilli grow faster and Streptococ-cus pyogenes grow more slowly, while micro-bial neurologists will learn to short-circuitnanowires running within mixed-species com-munities in periodontal pockets. The bordersbetween eukaryotic and prokaryotic biologyare blurring, making microbiology even moreexciting as we begin to apply general biologi-cal concepts to bacteria.

ACKNOWLEDGMENTS

Funding by NIH (DC04173) and the Amado Foundation. Technical assistance by Laura Nistico, Duc Nguyen, Bethany Dice,Siva Wu, Amita Gorur, and Nalinia Mehta and collaboration with Fen Ze Hu.

SUGGESTED READING

Beveridge, T. J. 2006. Visualizing bacterial cell walls and biofilms. Microbe 1:279–284.Costerton, J. W. 2007. The biofilm primer. Springer, in press.Davies, D. G., M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, and E. P. Greenberg. 1998. The involvement ofcell-to-cell signals in the development of a bacterial biofilm. Science 280:295–298.Gorby, Y. A., S. Yanina, J. S. McLean, K. M. Russo, D. Moyles, A. Dohnalkova, T. J. Beveridge, I. S. Chang, B. H. Kim, K. S.Kim, D. E. Culley, S. B. Reed, M. F. Romine, D. A. Saffarini, E. A. Hill, L. Shi, D. A. Elias, D. W. Kennedy, G. Pinchuck, K.

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Watanabe, S. I. Iishi, B. Logan, K. H. Nealson, and J. K. Fredrickson. 2006. Electrically conductive bacterial nanowiresproduced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc. Natl. Acad. Sci. USA 103:11358–11363.Moscoso, M., E. Garcia, and R. Lopez. 2006. Biofilm formation by Streptococcus pneumoniae: role of choline, extracellularDNA, and capsular polysaccharide in microbial accretion. J. Bacteriol. 188:7785–7795.Robinson, D. H. 2005. Pleomorphic mammalian tumor-derived bacteria self-organize as multicellular mammalian eukaryotic-like organisms : morphogenetic properties in vitro, possible origins, and possible roles in mammalian ‘tumor ecologies.’ Med.Hypotheses 64:177–185.Stoodley, P., K. Sauer, D. G. Davies, and J. W. Costerton. 2002. Biofilms as complex differentiated communities. Annu. Rev.Microbiol. 56:187–209.Thar, R., and M. Kuhl. 2002. Conspicuous veils formed by vibroid bacteria in sulfidic mine sediment. Appl. Environ.Microbiol. 68:6310–6320.Tolker-Nielsen, T., U. C. Brinch, P. C. Ragas, J. B. Andersen, C. S. Jacobsen, and S. Molin. 2000. Development and dynamicsof Pseudomonas sp. biofilms. J. Bacteriol. 182:6482–6489.Webster, P., S. Wu, S. Webster, K. A. Rich, and K. McDonald. 2004. Ultrastructural preservation of biofilms formed bynon-typeable Haemophilus influenzae. Biofilms 1:165–182.

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