Bacterial Biofilms: A Common Cause of Persistent Infections

J. W. Costerton et al.
Science 284, 1318 (1999);
DOI: 10.1126/science.284.5418.1318

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 MICROBES, IMMUNITY, AND DISEASE
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REVIEW

Bacterial Biofilms: A Common Cause of

Persistent Infections
J. W. Costerton,1 Philip S. Stewart,1 E. P. Greenberg2*

Bacteria that attach to surfaces aggregate in a hydrated polymeric matrix with functional heterogeneity.
of their own synthesis to form biofilms. Formation of these sessile Because bacterial biofilms can cause en-
communities and their inherent resistance to antimicrobial agents are at vironmental problems and studies of bio-
the root of many persistent and chronic bacterial infections. Studies of films have required the development of
biofilms have revealed differentiated, structured groups of cells with new analytical tools, many recent advances
community properties. Recent advances in our understanding of the have resulted from collaborations between
genetic and molecular basis of bacterial community behavior point to microbial ecologists, environmental engineers,
therapeutic targets that may provide a means for the control of biofilm and mathematicians. These efforts have led to
infections. our current definition of a bacterial biofilm as
a structured community of bacterial cells en-
For quite some time we have known that scope. However, it was not until the 1970s closed in a self-produced polymeric matrix
bacteria can adhere to solid surfaces and that we began to appreciate that bacteria in and adherent to an inert or living surface.
form a slimy, slippery coat. These bacterial the biofilm mode of existence, sessile bac- Biofilms constitute a protected mode of
biofilms are prevalent on most wet surfaces teria, constitute a major component of the growth that allows survival in a hostile
in nature and can cause environmental bacterial biomass in many environments environment. The structures that form in
problems. Perhaps because many biofilms (1), and it was not until the 1980s and biofilms contain channels in which nutri-
are sufficiently thick to be visible to the 1990s that we began to appreciate that at- ents can circulate (4 ), and cells in different
naked eye, these microbial communities tached bacteria were organized in elaborate regions of a biofilm exhibit different pat-
were among the first to be studied by the ways (2). For example, different bacterial terns of gene expression (5 ). The complex-
late-developing science of microbiology. species specifically attach to different sur- ity of biofilm structure and metabolism has
Anton van Leeuwenhoek scraped the faces or coaggregate with specific partners led to the analogy of biofilms to tissues of
plaque biofilm from his teeth and observed in the mouth (3). Often one species can higher organisms (6 ). These sessile biofilm
the “animalculi” that produced this micro- coaggregate with multiple partners, which communities can give rise to nonsessile
bial community with his primitive micro- themselves can aggregate with other part- individuals, planktonic bacteria that can
ners to form a dense bacterial plaque. Ad- rapidly multiply and disperse. The common
vances in light microscopy coupled with view is that planktonic bacteria must ex-
1
Center for Biofilm Engineering, Montana State Uni-
versity, Bozeman, MT 59717, USA. 2Department of
developments in microelectrode technology pose themselves to deleterious agents in
Microbiology, University of Iowa, Iowa City, IA 52242, have led to an appreciation that bacterial their environment, be they phage or amoe-
USA. biofilms consist of microcolonies on a surface, ba in nature, biocides in industrial settings,
*To whom correspondence should be addressed. E- and that within these microcolonies the bacteria or potent antimicrobial agents in a clinical
mail: [email protected] have developed into organized communities setting. In this light, it is not surprising that

1318 21 MAY 1999 VOL 284 SCIENCE www.sciencemag.org

 MICROBES, IMMUNITY, AND DISEASE
an impressive number of chronic bacterial (6). Therefore, biofilms can act as “niduses” the antimicrobial agent is deactivated in the
infections involve bacterial biofilms, which of acute infection if the mobilized host de- outer layers of the biofilm faster than it
are not easily eradicated by conventional fenses cannot eliminate the planktonic cells diffuses (18). This is true for reactive oxi-
antibiotic therapy. that are released at any one time during the dants such as hypochlorite and hydrogen
infection (12). peroxide (19). These antimicrobial oxidants
Bacterial Biofilm Infections are products of the oxidative burst of
Until the relatively recent development of Bacterial Biofilms Are Inherently phagocytic cells, and poor penetration of
vaccines and antibiotics, human societies Resistant to Antimicrobial Agents reactive oxygen species may partially ac-
have been beset by acute epidemic infec- Biofilms growing in natural and industrial count for the inability of phagocytic cells to
tious diseases caused by the planktonic environments are resistant to bacterio- destroy biofilm microorganisms.
cells of such specialized pathogens as phage, to amoebae, and to the chemically A second hypothesis to explain reduced
Vibrio cholerae and Yersinia pestis. Mod- diverse biocides used to combat biofouling biofilm susceptibility to antibiotics posits that
ern-day acute infections can often be treat- in industrial processes (13). Of importance at least some of the cells in a biofilm expe-
ed effectively with antibiotics (except for with respect to medicine, sessile bacterial rience nutrient limitation and therefore exist
cases of infection by a strain that is antibi- cells can withstand host immune responses, in a slow-growing or starved state (20). Slow-
otic resistant) and are not considered to and they are much less susceptible to anti- growing or nongrowing cells are not very
involve biofilms. However, more than half biotics than their nonattached individual susceptible to many antimicrobial agents.
of the infectious diseases that affect mildly planktonic counterparts (14 ). It is likely Spatial heterogeneity in the physiological
compromised individuals involve bacterial that biofilms evade antimicrobial challeng- state of bacteria within model biofilms has
species that are commensal with the human es by multiple mechanisms. been demonstrated by a variety of microslic-
body or are common in our environments. One mechanism of biofilm resistance to ing and microscopic techniques (21). Such

Downloaded from www.sciencemag.org on June 22, 2012

For example, the skin bacterium Staphylo- antimicrobial agents is the failure of an heterogeneity of biofilms constitutes an im-
coccus epidermidis and the aquatic bacteri- agent to penetrate the full depth of the portant survival strategy because at least some
um Pseudomonas aeruginosa can cause devas- biofilm. Polymeric substances like those of the cells, which represent a wide variety of
tating chronic infections in compromised that make up the matrix of a biofilm are different metabolic states, are almost certain
hosts (6). Electron microscopy of the surfac- known to retard the diffusion of antibiotics to survive any metabolically directed attack.
es of medical devices that have been foci of (15 ), and solutes in general diffuse at slow- A third mechanism of reduced biofilm sus-
device-related infections shows the presence er rates within biofilms than they do in ceptibility, which is more speculative than the
of large numbers of slime-encased bacteria water (16 ). Antibiotics have been shown to preceding hypotheses, is that at least some of
(7). Tissues taken from non– device-related penetrate biofilms readily in some cases the cells in a biofilm adopt a distinct and pro-
chronic infections also show the presence of and poorly in others, depending on the tected biofilm phenotype. This phenotype is not
biofilm bacteria surrounded by an exopo- particular agent and biofilm (17 ). Mathe- a response to nutrient limitation; it is a biolog-
lysaccharide matrix. These biofilm infections matical models predict that a formidable ically programmed response to growth on a
may be caused by a single species or by a penetration barrier should be established if surface.
mixture of species of bacteria or fungi (Table
1).
Biofilm infections share clinical charac- Table 1. Partial list of human infections involving biofilms.
teristics. Biofilms develop preferentially on
inert surfaces, or on dead tissue, and occur Infection or disease Common biofilm bacterial species
commonly on medical devices and frag- Dental caries Acidogenic Gram-positive cocci (e.g., Streptococcus)
ments of dead tissue such as sequestra of Periodontitis Gram-negative anaerobic oral bacteria
dead bone (8); they can also form on living Otitis media Nontypable strains of Haemophilus influenzae
tissues, as in the case of endocarditis. Bio- Musculoskeletal infections Gram-positive cocci (e.g., staphylococci)
films grow slowly, in one or more loca- Necrotizing fasciitis Group A streptococci
tions, and biofilm infections are often slow Biliary tract infection Enteric bacteria (e.g., Escherichia coli)
Osteomyelitis Various bacterial and fungal species—often mixed
to produce overt symptoms (9). Sessile bac- Bacterial prostatitis E. coli and other Gram-negative bacteria
terial cells release antigens and stimulate Native valve endocarditis Viridans group streptococci
the production of antibodies, but the anti- Cystic fibrosis pneumonia P. aeruginosa and Burkholderia cepacia
bodies are not effective in killing bacteria Meloidosis Pseudomonas pseudomallei
within biofilms (Fig. 1) and may cause Nosocomial infections
immune complex damage to surrounding ICU pneumonia Gram-negative rods
Sutures Staphylococcus epidermidis and S. aureus
tissues (10). Even in individuals with excellent Exit sites S. epidermidis and S. aureus
cellular and humoral immune reactions, bio- Arteriovenous shunts S. epidermidis and S. aureus
film infections are rarely resolved by the host Schleral buckles Gram-positive cocci
defense mechanisms (7). Antibiotic therapy Contact lens P. aeruginosa and Gram-positive cocci
typically reverses the symptoms caused by Urinary catheter cystitis E. coli and other Gram-negative rods
planktonic cells released from the biofilm, Peritoneal dialysis (CAPD) peritonitis A variety of bacteria and fungi
IUDs Actinomyces israelii and many others
but fails to kill the biofilm (11). For this Endotracheal tubes A variety of bacteria and fungi
reason biofilm infections typically show re- Hickman catheters S. epidermidis and C. albicans
curring symptoms, after cycles of antibiotic Central venous catheters S. epidermidis and others
therapy, until the sessile population is surgi- Mechanical heart valves S. aureus and S. epidermidis
cally removed from the body (6). Planktonic Vascular grafts Gram-positive cocci
bacterial cells are released from biofilms, and Biliary stent blockage A variety of enteric bacteria and fungi
Orthopedic devices S. aureus and S. epidermidis
evidence supports the notion that there is a Penile prostheses S. aureus and S. epidermidis
natural pattern of programmed detachment

www.sciencemag.org SCIENCE VOL 284 21 MAY 1999 1319

 MICROBES, IMMUNITY, AND DISEASE
A New Era in Biofilm Research: type cells cluster into microcolonies (relatively search area, focused on the ability of bacteria
Molecular Genetic Dissection of small groups of bacteria) on the plastic, the to function in special ways when in groups,
Biofilm Development pilus biogenesis mutants form a monolayer of has provided some interesting clues about
Research in this decade has begun to reveal cells on the surface but are unable to form biofilm maturation. Research on quorum
information about the molecular and genetic microcolonies. Type IV pili in P. aeruginosa sensing in Gram-negative bacteria has shown
basis of biofilm development. Biofilms in- are involved in a type of surface-associated that acylhomoserine lactone signals are pro-
volving several different bacterial species motility called twitching, and this twitching duced by individual bacterial cells. At a crit-
have been studied (6) but perhaps none more might be required for the aggregation of cells ical cell density, these signals can accumulate
intensively than biofilms of Pseudomonas into microcolonies. and trigger the expression of specific sets of
aeruginosa. Here, we use P. aeruginosa and There is evidence that during this attach- genes [for reviews see (26)]. Could a quo-
the chronic lung infections it causes in most ment phase of biofilm development, perhaps rum-sensing signal be required for biofilm
patients afflicted with the recessive genetic after microcolony formation, the transcription development? Two P. aeruginosa quorum-
disease cystic fibrosis (CF) as a model that of specific genes is activated. In particular, sensing systems have been characterized.
exemplifies modern research on biofilm in- studies with P. aeruginosa algC, algD, and One, the LasR-LasI system, controls the ex-
fections. Like other biofilms, P. aeruginosa- algU::lacZ reporter constructs show that the pression of a battery of extracellular viru-
biofilms are developed communities with in- transcription of these genes, which are required lence factors. It also controls the other sys-
dividual bacterial cells embedded in an extra- for synthesis of the extracellular polysaccharide tem, RhlR-RhlI, which in turn controls genes
cellular polysaccharide matrix (22) and are (alginate in this case), is activated after attach- including several required for the production
inherently resistant to antimicrobial treat- ment to a solid surface (24). Thus, it appears of a number of secondary metabolites. RhlI
ment. The pattern of development involves that attachment itself can initiate synthesis of catalyzes the synthesis of butyrylhomoserine
initial attachment to a solid surface, the for- the extracellular matrix in which the sessile lactone, and LasI directs the synthesis of

Downloaded from www.sciencemag.org on June 22, 2012

mation of microcolonies on the surface, and bacteria are embedded. This notion—that bac- 3-oxododecanoylhomoserine lactone. The wild
finally differentiation of microcolonies into teria have a sense of touch that enables detec- type, a lasI mutant, and a rhlI mutant all can
exopolysaccharide-encased, mature biofilms. tion of a surface and the expression of specific colonize a glass surface and form microcolo-
Initial attachment and microcolony forma- genes—is in itself an exciting area that has been nies. Microcolonies of the wild type and the
tion. A recent report by O’Toole and Kolter more thoroughly studied in Vibrio parahaemo- rhlI mutant differentiate into structured,
(23) describes a microtiter dish screen for the lyticus, which causes gastroenteritis in humans thick, biocide-resistant biofilms, whereas the
isolation of P. aeruginosa mutants defective in (25). lasI mutant microcolonies remain thin, undif-
the initial steps of biofilm formation. Two gen- Maturation of attached bacteria into a dif- ferentiated, and sensitive to dispersion by a
eral classes of mutants, called sad (surface at- ferentiated biofilm. At an appropriate time, mi- weak detergent (0.2% sodium dodecyl sul-
tachment defective) mutants, were described. crocolonies differentiate into true biofilms: ex- fate). Addition of the missing signal, 3-oxo-
One class constitutes flagella and motility mu- opolysaccharide-encased communities that are dodecanoylhomoserine lactone, to the lasI
tants and does not adhere well to the plastic resistant to biocides. What is the genetic pro- mutant restores biofilm development (27).
surface used. The other class consists of mu- gram leading to biofilm development? Is there a This indicates that one specific quorum-sens-
tants defective in the biogenesis of hair-like signal that induces differentiation? The conver- ingsignal is required for biofilm differentia-
appendages, Type IV pili. Whereas the wild- gence of biofilm research with another re- tion, at least under the conditions of the ex-

Fig. 1. Diagram of a medical biofilm. (A)

Planktonic bacteria can be cleared by an-
tibodies and phagocytes, and are suscepti-
ble to antibiotics. (B) Adherent bacterial
cells form biofilms preferentially on inert
surfaces, and these sessile communities
are resistant to antibodies, phagocytes,
and antibiotics. (C) Phagocytes are attract-
ed to the biofilms. Phagocytosis is frustrat-
ed but phagocytic enzymes are released.
(D) Phagocytic enzymes damage tissue
around the biofilm, and planktonic bacteria
are released from the biofilm. Release may
cause dissemination and acute infection in
neighboring tissue.

1320 21 MAY 1999 VOL 284 SCIENCE www.sciencemag.org

 MICROBES, IMMUNITY, AND DISEASE
periments. Of some interest, acylhomoserine dispersal of individual cells from community clinical attempts to prevent the initial coloniza-
lactones have been reported to be produced structures [the quorum-sensing genes in R. tion of young patients by P. aeruginosa with
by sessile P. aeruginosa communities on sil- sphaeroides are called cer (community escape prophylactic antibiotics are showing some
icone urethral catheters (28). response) genes (30)]. promise (33).
Thus, a picture of the development of P. Antibiotic therapy in patients colonized
aeruginosa biofilms at a molecular level is Pseudomonas aeruginosa Biofilms in with P. aeruginosa often gives a measure of
emerging (Fig. 2). There are specific cell surface Cystic Fibrosis Lung Infections relief from symptoms but fails to cure the
components required for adhesion to a surface The genetic defect in CF leads to the loss of the basic ongoing infection (19). Our interpreta-
and additional components required for aggre- CF transmembrane regulator (CFTR) chloride tion of this is that the antibiotics act on the
gation of cells into undifferentiated microcolo- channel in the apical membranes of epithelial planktonic cells that are shed by the biofilms.
nies. The generation of a mature P. aeruginosa cells (31). This defect leads to persistent bacte- This can alleviate the acute symptoms of the
biofilm requires an extracellular signaling mol- rial infections of the lungs. Most CF patients are lung infection, but the antibiotic therapy can-
ecule that can be likened to a hormone. With colonized with P. aeruginosa, and eventually not eliminate the antibiotic-resistant sessile
regard to biofilm development, there are a num- they succumb to the lung damage inflicted by biofilm communities.
ber of pressing questions. Are the mechanisms the persistent bacterial infection, with a median The lifelong struggle of CF patients with
of attachment and microcolony formation simi- life expectancy of about 30 years. There are P. aeruginosa pneumonia exemplifies most
lar regardless of the characteristics of the surface several explanations for CF lung pathogenesis, biofilm infections. The causative organisms
involved? Are there conditions where biofilm some of which are not mutually exclusive (32). are ubiquitous and are only pathogenic for a
differentiation can bypass the acylhomoserine One view is that the absence of a chloride particular set of compromised individuals.
lactone signaling step? What acylhomoserine channel leads to an elevated salt content in the The infection develops slowly, except for
lactone–regulated genes are required for biofilm airway surface fluid. The salt inhibits the activ- acute exacerbations, and these acute phases

Downloaded from www.sciencemag.org on June 22, 2012

maturation? Can some of these genes be linked ity of antimicrobial peptides and proteins in- may be responsive to antibiotic therapy. The
directly to the antibiotic resistance of biofilms? volved in the innate immunity of the airways basic deep-seated infection cannot, however,
Are cell-to-cell signals in biofilm formation the (32). This tips the balance of power just enough be cured by conventional antibiotic therapy.
rule among different bacterial groups, or is this so that P. aeruginosa can colonize the epithe- The normal course of the infection produces
a particular characteristic of P. aeruginosa? lium as a biofilm. an antibody response to the infecting patho-
Detachment and dispersal of planktonic The sessile P. aeruginosa communities re- gen, but the antibodies are not effective
cells from biofilms. For bacteria in a sessile lease antigens while growing in microcolonies against sessile bacteria. The microcolonies of
biofilm community to colonize new areas, there in the lung, and very high concentrations of sessile bacteria in the lung act as niduses for
must be some mechanism for dispersion. Pieces antibodies to Pseudomonas are seen in the cir- spread of the infection (32).
of biofilms (Fig. 2) can break off in the flow and culating blood and in the lungs. These antibod- The scientific quandary facing CF patients
may colonize new surfaces. Furthermore, just as ies react with their specific antigens in the outer is that currently available antibiotics were
there are chemical cues for biofilm maturation, reaches of the matrices of the infecting micro- developed against the planktonic phenotype
there may be cues for a program of events colonies, but neither the bactericidal nor the of P. aeruginosa, and therapeutic agents are
leading to the release of planktonic bacteria opsonizing capabilities of these defensive mol- chosen on the basis of their efficacy against
from a biofilm. It has been suggested that escape ecules are realized. In CF patients, a high con- planktonic cells of this pathogen, but direct
of P. aeruginosa cells from the biofilm matrix centration of circulating antibodies to Pseudo- observations have shown that the bacteria
involves the action of an enzyme that digests monas correlates with a negative clinical out- actually grow in the biofilm phenotype in the
alginate (29). It is worth noting that in the come. This has been ascribed to pulmonary lung. Thus, it should come as no surprise that
nonpathogenic, photosynthetic bacterium tissue damage resulting from inflammation. In- current antibiotic therapies are of limited ef-
Rhodobacter sphaeroides, an acylhomoserine deed, immune suppression is a part of the ther- fectiveness in resolving this particular bio-
lactone quorum-sensing signal is required for apeutic arsenal of the CF clinician. Current film infection.

Fig. 2. (A) Models of the develop- A

ment of a mature P. aeruginosa bio-
film from planktonic cells; (B) dis-
persal of bacteria from a biofilm.
Flagella (blue) are involved in at-
tachment, and Type IV pili (black)
are required for twitching motility
on a surface and the formation
of microcolonies in the attached
monolayer that forms on the sur-
face. LasI-dependent quorum sens-
ing serves as a maturation signal
leading to the formation of differ- B
entiated, thick mature biofilm struc-
tures. Two proposed mechanisms for
detachment and dispersal of cells
from a biofilm are depicted. One
pictures a programmed set of events
within the biofilm leading to a local
hydrolysis of the extracellular poly-
saccharide matrix, and conversion
of a subpopulation of cells into mo-
tile planktonic cells, which leave the
biofilm. The other is a physical detachment pathway in which a streamer, or some other fragment of a microcolony, simply detaches from the biofilm
and is carried by the bulk fluid until it lodges in a new location and initiates a new sessile population.

www.sciencemag.org SCIENCE VOL 284 21 MAY 1999 1321

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REVIEW

Type III Secretion Machines: Bacterial

Devices for Protein Delivery into Host Cells
Jorge E. Galán1* and Alan Collmer 2

Several Gram-negative pathogenic bacteria have evolved a complex pro- the next. However, in other cases, fatal disease
tein secretion system termed type III to deliver bacterial effector proteins may occur when these bacterial pathogens en-
into host cells that then modulate host cellular functions. These bacterial counter a host that has been weakened by cir-
devices are present in both plant and animal pathogenic bacteria and are cumstances that alter the delicate balance of the
evolutionarily related to the flagellar apparatus. Although type III secre- microbe-host interaction.
tion systems are substantially conserved, the effector molecules they Recent advances in the fields of immunolo-
deliver are unique for each bacterial species. Understanding the biology of gy and of molecular, cell, and structural biology
these devices may allow the development of novel prevention and ther- are allowing the detailed investigation of the
apeutic approaches for several infectious diseases. interactions between these highly adapted
pathogens and their hosts. This close examina-
A number of bacterial pathogens have evolved of both the pathogen and the host. This is tion is not only helping in the understanding of
the capacity to engage their hosts in complex particularly the case for microbial pathogens microbial pathogenesis but is also providing
intimate interactions aimed not necessarily at that, through the process of host adaptation,
causing disease but rather at securing the mi- have lost the ability to explore other niches. 1
Section of Microbial Pathogenesis, Boyer Center for
crobe’s ability to multiply and move on to a Sometimes, however, these pathogens cause Molecular Medicine, Yale School of Medicine, New
new host. The relationship between bacterial harm to the host. In some instances, disease Haven, CT 06536, USA. 2Department of Plant Pathol-
pathogens and their hosts is most often a peace- symptoms may simply be unpleasant manifes- ogy, Cornell University, Ithaca, NY 14853– 4203, USA.
ful one, because it has been shaped by a coevo- tations of a self-limiting process that leads to *To whom correspondence should be addressed. e-
lutionary process aimed at securing the survival the transmission of the bacteria from one host to mail: [email protected]

1322 21 MAY 1999 VOL 284 SCIENCE www.sciencemag.org