Biofilms and

disease
MIC610, Fall 2007
 Biofilms
 Biofilms
 A community of
microorganisms attached
to a surface (biotic or
abiotic)
 Provide a haven from low Contact
nutrient and hostile lens
environmental conditions
 Most environmental
biofilms contain multiple
species
 Not uncommon to find
single species biofilms in
human and animal Medical
infections implant
Biofilms in the environment
 Some are beneficial
 Sewage treatment plants rely on
biofilms to remove contaminants from
water
 Some are harmful
 Cause pipe corrosion
 Clog water filters

 Cause rejection of medical implants

 Contamination of drinking water

Development of a biofilm
 Attachment to the surface
 Cell to cell interaction
 Production of extracellular
polysaccharide (EPS)
 Growth, maturation and detachment
 Factors involved in biofilm
formation

flagella
pili EPS production
quorum sensing
EPS production
 The extracellular matrix
 Usually composed primarily of polysaccharide
 Although proteins and even DNA may play important roles in
matrix assembly and maintenance
 Much of the extracellular polysaccharide is insoluble and
not easily separated from cells
 Often the matrix is composed of polysaccharide that
would form the capsule during planktonic growth
 The meeting of two matrices
 Often in biofilm mediated infections the matrix is composed not
only of bacterial products but also host products including
portions of the host extracellular matrix
 Biofilms are very different to
planktonic bacteria
 Several microarray experiments suggests
that a significant number of genes are
differentially regulated under biofilm
conditions vs. planktonic growth
 In Vibrio cholerae, 138 (early biofilm) and 383
(mature biofilm) genes were found to be
differentially expressed
 In Staphylococcus aureus, 25 (early biofilm)
and 35 (late biofilm) genes were found to be
increased in expression >10x
 Probably underestimates because of averaging
of gene expression over a biofilm population
(which is heterogeneous compared to
planktonic culture)
 Is Biofilm formation a
developmental process?
 Has been proposed that biofilm formation is a
developmental process similar to fruiting body
formation in Myxococcus xanthus and sporulation in
Bacillus subtilis
 Biofilm formation does follow a series of discrete and
well-regulated steps
 Genes not unique to biofilm formation, some genes
that are expressed during stationary phase are also
expressed in biofilms
 Biofilm induced genes are not necessarily common
between species
 Some are, like flagella, fimbriae and respiratory enzymes
 Why do we need to study
biofilms?
 It is estimated that 90-99% of bacteria in
the environment live in biofilm
communities
 The CDC implicates biofilms in 65% of all
human bacterial infections
 Problem
 Microbiologists have traditionally focused on
planktonic bacteria floating freely in lab
culture
 Planktonic bacteria and biofilm (sessile)
bacteria behave differently
 Advantages to biofilm
bacteria
 Quorum sensing (operating as a
multicellular organism)

 Resistance to antimicrobial agents

 Resistance to phagocytic killing

 Quorum sensing
 Mechanism of cell to cell signaling or
density sensing in bacteria
 Small diffusible molecules secreted by
bacterial cells which when cell density
becomes sufficient can be detected by
other cells in the population
 In Gram negative bacteria these molecules are
usually derivatives of acylated homoserine
lactone (AHL)
 In Gram positives they are often small
peptides
 A “universal” autoinducer AI-2 is produced by
a number of bacteria and may mediate cross-
talk between species
 Quorum sensing in
Pseudomonas aeruginosa
 Two systems, Las
and Rhl, each
produce AHL
derivatives
 AHL are freely
diffusible into and
out of cells
 When eternal
concentrations are
high enough  Las - pili, capsule production,
diffusion back into secretion apparatus and secreted
proteins
cells activates the  Rhl - rhamnolipid, elastase, LasA
respective protease, hydrogen cyanide,
regulator pyocyanin, siderophores and the
cytotoxic lectins PA-I and PA-II
 Quorum sensing in Gram
positive bacteria
 Similar to Gram
negative bacteria
 Signal is a peptide
 Peptide is detected
at the cell surface by
a two component
regulatory system
Quorum sensing in a
biofilm
 Antimicrobial resistance
 Difficulty of eradicating biofilm bacteria with
antibiotic treatment is a prime concern
 Bacterial biofilms can survive treatment with
antibiotics at concentrations hundreds or even a
thousand times the MIC of planktonic bacteria
 Antibiotics may suppress infection from detached
bacteria, but not eradicate biofilms
 Leads to reoccurrence following termination of
antibiotic therapy
Infectious kidney stone and
recurrent urinary tract
infection

 Infectious kidney stones are a biofilm between bacteria

(Proteus, Klebsiella, Pseudomonas sp.) and mineral
substrates from urine
 Urinary tract infection relapses when antibiotics are
removed. Eradicated only after stone was removed
 Mechanism of antibiotic
resistance
 Normal modes of resistance (efflux,
modifying enzymes, target
mutations) are not responsible
 However, conventional resistance
mechanisms can develop in
biofilms treated for a long period of
time and contribute to persistence
Mechanisms of biofilm
antibiotic resistance
Most antibiotics can
freely diffuse
through the biofilm
matrix

Most of the biofilm

bacteria are killed
by the antibiotic,
survivors persist
despite continued
exposure

Anaerobic:
aminoglycosides
Non-growing cells:
All depend beta-lactams
on multicellular
behavior of biofilms
 Genetic changes in biofilm
cells lead to antibiotic

resistance
Recent evidence suggest that it is possible to obtain
mutants of Pseudomonas aeruginosa that can still form
biofilms but don’t develop full biofilm resistance to
tobramycin
 One gene isolated from this screen was ndvB, known to
be upregulated in biofilm cultures
 NdvB is responsible for the production of cyclic glycans
in the periplasm which are postulated to slow the
diffusion of tobramycin into the cell
 So, biofilm related antibiotic resistance may have more
to do with genes turned on during biofilm formation
than the structure of the biofilm itself
 Biofilms and the host
immune system
 Biofilm bacteria are resistant to host defenses
 Introduction of preformed biofilms into animals with
circulating antibodies and normal neutrophils does
not prevent survival of the biofilm
 Exclusion of antibodies and neutrophils from the
biofilm matrix?

 Neutrophils can enter biofilms

through open water channels
 But, appear to be paralyzed and
not internalizing bacteria
 Phagocyte resistance and
quorum sensing
 Quorum sensing mutants cannot block
activation of phagocytes, but are also usually
defective (in some way) in biofilm formation
 If inhibitors of quorum sensing molecules are
added to pre-formed biofilms, phagocytes can
then be activated
 Direct evidence that AHL molecules can act as
immune modulators
 Inhibition of leucocyte proliferation
 Modulaton of cytokine production
 Induction of apoptosis in macrophages/neutrophils
 Inhibition of phagocyte activation
 Biofilms and medical
devices
 Indwelling medical devices
 contact lenses, central venous catheters, endotracheal
tubes, intrauterine devices, mechanical heart valves,
pacemakers, peritoneal dialysis catheters, prosthetic
joints, urinary catheters, and voice prostheses
 Staphylococcus, Streptococcus, Enterococcus
(Gram positive), E. coli, Klebsiella, Proteus,
Pseudomonas (Gram negative)
 Bacteria may originate from the skin of the
patient, or a heathcare worker, tap water or
other environmental sources
 Biofilm contaminated
medical devices and

infection
Urinary tract infections
 10-50% of short-term urinary
catheterization (7 days) become infected
 Virtually all patients receiving long-term
catheterization (>28 days) become
infected
 Biofilms forming on the surface of
indwelling devices act as a source of
acute infection
 However, although biofilm formation
on vascular catheters is almost
universal, a resultant blood stream Staphylococcus
infection is rare
epidermidis biofilm
 Few infections from contaminated on a catheter
indwelling medical devices are cured surface
by antibiotic treatment without
removal of the device
 Biofilms as initiators of
infections
 Since most bacteria in natural ecosystems
exist as biofilms, challenge to humans
often comes from this source
 Natural aquatic biofilms can attract and
retain pathogens such as E. coli and
Salmonella sp. OR naturally aquatic
bacteria such as Vibrio cholerae may form
biofilms
 One of the papers deals with biofilms of
conditionally viable environmental cells (CVEC)
of V. cholerae which reactivate in vivo
 Challenge from biofilms may serve as a
means of ensuring an infective dose
Pulmonary infections with
biofilms
 While the GI tract may be colonized by
environmental biofilms, the lungs are
most susceptible
 The trachea and lungs are efficient at
clearing planktonic bacteria
 However, when biofilm fragments are
introduced into the lung, they resist
phagocytosis by resident phagocytes
and killing by both innate and acquired
immune factors, and can persist for
weeks and even months
Examples of biofilm derived
infections of the lung
 Mannheimia haemolytica
 normal oropharyngeal flora of cattle
 proliferates when animals are shipped to feedlots
 aspiration of fragments of biofilms into the lungs of stressed
animals results in respiratory infections that kill as many as 2%
of these calves
 Legionella pneumophila
 legionellosis (Legionnaires' Disease)
 lives in warm water, forms biofilms, and resists phagocytosis
 survives very well in the condensate trays of air conditioners,
hot water systems or in human lungs
 Biofilm formation and
the plaque
 Biofilms of Yersinia pestis
form on the proventricular
valve of the flea
 Partially or fully blocks the flea
taking a blood meal (making the
flea hungrier and more likely to
attempt feeding from several
individuals)
 Fleas secrete anticoagulants and
other factors into the host during
feeding thus depositing biofilm
bacteria into the host
bloodstream
A role for biofilms in chronic
infections
 Chronic infections are like “disease stalemates”
between the virulence of the bacterium and the
host immune system
 Many organisms that cause chronic infections
also produce acute infections
 Pseudomonas aeruginosa
 blood infections, death within hours
 In cystic fibrosis, can survive for decades at high numbers
(108-1010 cfu/ml) in the lungs. Never become invasive.
 Bacteria become less invasive and more
persistent
Chronic infections with clear
biofilm associations
 Infectious Bacterial Endocarditis
 Cystic Fibrosis
 Staphylococcus Osteomyelitis
 Middle Ear Infections (Otitis media)
 Chronic Prostatitis
 Infectious Kidney Stones
 Chronic sinusitis
 Bacterial endocarditis
 Primary lesion is a complex biofilm (vegetation) of
bacterial and host components
 Physically disrupts value function (leakage)
 Source of near continuous infection of the bloodstream
(recurrent fever, chronic systemic inflammation)
 Pieces of biofilm can break off form emboli (brain, kidneys,
extremities)
 Treatment
 Prolonged intravenous antibiotic treatment
 Surgical excision and replacement of the infected valve
The cardiac valve biofilm
 Valve biofilm is composed of
bacteria, their exoproducts,
platelets, and fibrin, with damaged
endothelial surface acting as the
substratum
 Pathogens
 Streptococcus sanguis (glucans)
 Streptococcus mutans
 Staphylococcus aureus (fibrinogen,
fibronectin binding)
Staphylococcus aureus cells
of a cardiac biofilm
surrounded by a matrix of
fibrin strands
 Periodontal disease and
endocarditis

 In some populations, the risk of

infectious endocarditis is increased
2-5 times by preexisting peridontal
disease
 Dental plaque
 Dental plaque is
probably the
most complex
biofilm found in
humans
 Colonization of a
clean tooth
surface is a
highly specific
and complex
process
 Relationship between
periodontal disease
bacterial endocarditis
 Significant correlation between periodontal
Significant correlation between periodontal
disease and cardiovascular disease
 Periodontal disease results from bacterial
colonization of the periodontal pocket. The
continual host inflammatory response results
in breakdown of periodontal tissue
 Oral flora (e.g.; the Streptococci) gain access
to the blood stream and can adhere to the
platelets and fibrin of the injured valve
Cystic Fibrosis P. aeruginosa
infections
 Genetic defect in CFTR chloride channel
(1/3500 births)
 Become permanently infected with P.
aeruginosa
 Biofilm formation
 Same strain can persist in the airway for years or
decades
 coincides with lung function decline, increased
respiratory symptoms
 High levels of antibodies are produced
against P. aeruginosa, but
elimination of infection is not accomplished
 Chronic immune-complex-mediated
inflammation eventually destroys the lung
and causes respiratory failure
 Mucoid CF associated P.
aeruginosa
 P. aeruginosa with a mucoid
phenotype emerge in the CF
lung
 Overproduction of alginate (EPS)
 Mucoid strains rare in
environment and other diseases
(not involved in environmental
biofilm growth)
 Mucoid isolates arise from non-
mucoid isolates by specific
mutation (e.g., mucA mutations)
 Emergence of mucoid P. aeruginosa in sputum
phenotype may be facilitated of a CF patient
by biofilm growth in vivo, and surrounded by matrix
augment biofilm-mediated
resistance
 Antibiotic therapy in CF
patients
 Antibiotic therapy does not really
work because this is a biofilm disease
 In addition, because of the high
levels of antibiotic used and
prolonged treatment, traditional
antibiotic resistance mechanisms
arise frequently among CF P.
aeruginosa isolates
 Therapy for biofilm
infections
 As antibiotics and host immunity are
ineffective, what options are available for
biofilm infections
 CF patients-
 early aggressive antibiotic therapy (inhalation
of tobramycin, gentamycin)
 Intravenous antibiotics are used as a
maintenance therapy
 Anti-quorum sensing
 therapy
Since quorum sensing plays an integral role in biofilm
formation and maintenance, could quorum sensor
inhibitors be used for therapy
 Inhibitors of specific quorum sensors have been
discovered in plants and molds, and chemically
synthesized.
 Mostly act as antagonistics for AHL binding to regulator
 Experiments have shown that the inhibitor furanone
C30 can prevent lung infection of mice with P.
aeruginosa (uncertain if therapy or prophylaxis)
 But, inhibition of quorum sensing in Staphylococcus sp.
has been shown to enhance biofilm formation and
increase persistence in device related infections
 However, use of competing autoinducing peptide from other
Staphylococcal species has recently been shown to reduce
bacterial numbers on indwelling devices