Biofilms
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Biofilms. Microbial Biochemistry. Definition of a Biofilm. Biofilms are communities of microorganisms in a matrix that joins them together and to living or inert substrates

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Biofilms

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Biofilms

Biofilms

Microbial Biochemistry


Definition of a biofilm

Definition of a Biofilm

  • Biofilms are communities of microorganisms in a matrix that joins them together and to living or inert substrates

  • Biofilms are surface-attached communities of bacteria, encased in an extracellular matrix of secreted proteins, carbohydrates, and/or DNA, that assume phenotypes distinct from those of planktonic cells


Formation of biofilms in nature

Formation of biofilms in nature

  • Biofilms offer their member cells several benefits

  • Biofilms are diverse from their formation on teeth as plaques and submerged rocks in a stream


Biofilms1

Biofilms

BIOFILMS may form:

  • On solid substratums in contact with moisture

  • On soft tissue surfaces in living organisms 

  • At liquid-air interfaces. 


Study of biofilms

Study of biofilms

  • Formation of multicellular communities depends on the production of extracellular substances( matrix)

  • Diversity in the formation of the matrix


Pathogens that have been studied for the formation of biofilms

Pathogens that have been studied for the formation of biofilms

  • Staphylococcus aureus

  • Staphylococcus mutans

  • Salmonella typhi

  • Enterococcus faecalis

  • Pseudomonas aeruginosa


Biofilms now a universal feature

Biofilms – Now a Universal Feature

  • Now scientists believe biofilm formation is a universal feature of all bacteria


Biofilm characteristics

Biofilm characteristics

  • Submerged biofilms seems to form columns and mushroom like projections that are separated by water-filled channels

  • Floating biofilms form a skin or pellicle at the air- liquid interface – shows organization of cells with the matrix at the outside

  • Films that form on the surface of solid media such as agar or other surfaces


Steps in biofilm formation

Steps in biofilm formation

  • Initiation of biofilm formation – interaction of cells with a surface or with each other

  • Films aggregate

  • Then the cells form an extracellular matrix

  • Structure of biofilms are dramatically different due to the specific organisms in the film and environmental conditions


Figure from kolter r and r losick 1998 one for all and all for one science 280 226 227

Figure from: Kolter, R. and R. Losick. 1998. One for all and all for one. Science 280:226-227.


Steps in biofilm formation1

Steps in Biofilm Formation


Exopolysaccharides

Exopolysaccharides

  • Exopolysaccharides

  • In the glycoclyx contribute to biofilm formation


Matrix

Matrix

  • Key components of the matrix are extracellular polysaccharides and proteins

  • Dead cells have also been identified in biofilms

  • Extracellular DNA is also important


Biofilm formation

Biofilm formation


Matrix constituents

Matrix constituents


Polysaccharides

Polysaccharides

  • Carbohydrates significantly impact bacterial virulence

  • Bacteria have capsular polysaccharides and exopolysaccharides

  • The polysaccharides are not soluble and do not disassociate with the bacterial cells


Polysaccahrides

Polysaccahrides

  • Many bacteria have been found to produce cellulose

  • This is a novel finding in the case of Salmonella typhimurium and E. coli

  • The bacterium Gluconacetobacter xylinus has been recognized as a cellulose producer

  • Many other bacteria have genes homologous to the bcs, bacterial cellulose synthesis genes

  • Vibrio cholera does not appear to have a gene which encodes a cellulose – but the bacterium has two domains homologous to Gluconeacetobacter.


Biofilm genetics initiation

Biofilm Genetics - initiation

  • Staphylococcal polysaccharide intercellular adhesin( PIA)

  • PIA – poly-N-acetyl glucosamine (PNAG) polymer

  • PIA polymers present in E. coli

  • The pga gene is involved with the formation of biofilms

  • This gene is similar to the staphylococcal gene ica

  • Now Yersinia pestis has been shown to make a gene homologous to this


Bap protein

Bap Protein

  • The biofilm associated protein

  • Structurally similiar to the surface proteins

    Esp of Enterococcus faecalis

    mus20 of Pseudomonas aeruginosa

    sty2875 of Salmonella typhi


Pseudomonas aeruginosa

Pseudomonas aeruginosa

  • Pseudomonas strains were thought to produce alginate

  • However alg mutants did not produce changes in the biofilm formation

  • Two new polymers have been found in Pseudomonas polymers

  • pelA-G gene produces a glucose rich polymer

  • pslA-O genes produce a mannose rich polymer


Population density and polysaccharide production

Population density and polysaccharide production

  • The connection between quorum sensing and biofilm architecture

  • Biofilm thickness seems to affect communication

  • Quorum sensing mechanism is not clearly defined but seems to be essential in the formation of the film and its channels


Vibrio cholera

Vibrio cholera

Major extracellular polysaccharide in the biofilm are VPS and the genes, vps

VPS is negatively regulated by hapR. Hap R mutants produce rugose colonies and narrow channels in the biofilm architecture

hapR gene encodes a transcription factor

hapR is repressed by LuxO


Conjugative pili

Conjugative pili

  • Conjugative pili greatly accelerates initial adhesion and biofilm development by E. coli

  • Gram negative bacteria have adhesins at the tip of its fimbriae

  • E. coli responds to levels of nutrients and osmolarity


Adhesins

Adhesins

  • Adhesins are molecules that are attached to bacterial fimbriae


Staphylococcus aureus

Staphylococcus aureus


E coli adhesins

E. Coli Adhesins


Adhesins1

Adhesins


Adhesins and vaccines

Adhesins and vaccines


Bacterial cell characteristics

Bacterial Cell Characteristics

  • The phenotypes of the cells include:

  • a slower growth rate

  • increased antibiotic resistance

  • elevated frequency of lateral gene transfer


Quorum sensing

Quorum sensing

  • Is controlled by at least two different quorum sensing signals

  • Acyl-homoserine lactone CAI-1( cholera autoinducer 1) appears to play a significant role in biofilm formation

  • Under low cell density CAI levels are lose enough to permit LuxO mediated repression of hapR resulting in VPS production

  • This bacterium appears to initiate production of an extracellular matrix under conditions of low population density presumably before the establishment of a multicellular community


Signaling in biofilms

Signaling in biofilms


Signaling and biofilms

Signaling and biofilms


Gram negative

Gram Negative


Gram positive

Gram Positive


Antibiotic resistance

Antibiotic Resistance

  • In the center of the biofilm the bacteria have greater antibiotic resistance

  • Opsonisation of antibiotics

  • To resistance to phagocytosis


E faecalis

E. faecalis

  • E. faecalis biofilms on dental root canals, urethral catheters, uretheral stents ,and heart valves have been observed.

  • While it is not clear that the ability of E. faecalis to form biofilms is essential for virulence, it appears that a majority of clinical isolates do possess the ability to form a biofilm in vitro


Dental plaque

Dental plaque

  • Genome – genome interactions:bacterial communities in initial dental plaque – Paul Kolenbrander et al – Trends in Microbiology. January 2005.

  • Found on the enamel of teeth

  • Epithelial cells of the oral mucosa

  • Participate in coaggregation which occurs between different species of bacteria


Dental plaque colonization

Plaque smear

400 different bacterial species can be found in plaque

1010bacteria/mg

High levels of Ca++ and P

Matrix forms with cells

Dental Plaque - colonization


Definition

Supra gingival – above gums

Subgingival –below the gums

Also related to the tooth surface

Definition


Primary colonizers

Primary Colonizers

  • The pellicle-coated tooth surface is colonized by Gram-positive bacteria such as Streptococcus sanguis, Streptococcus mutans, and Actinomyces viscosus

  • These organisms are examples of the "primary colonizers" of dental plaque. Bacterial surface molecules interact with components of the dental pellicle to enable the bacteria to attach or adhere to the pellicle-coated tooth surface.


Primary colonization

Primary colonization

  • For example, specific protein molecules found as part of the bacterial fimbria (hair-like protein extensions from the bacterial cell surface) on both Streptococcus sanguis and Actinomyces viscosus interact with specific proteins of the pellicle (the proline-rich proteins) with a "lock and key" mechanism

  • This results in the bacteria firmly sticking to the pellicle-coating on the tooth surface (Mergenhagen et al. 1987). Within a short time after cleaning a tooth, these Gram-positive species may be found on the tooth surface.


Mechanisms for plaque formation

Mechanisms for plaque formation

  • two distinct mechanisms: 1 ) the multiplication of bacteria already attached to the tooth surface, and 2) the subsequent attachment and multiplication of new bacterial species to cells of bacteria already present in the plaque mass.


Mechanisms

Mechanisms

  • Two distinct mechanisms:

  • 1 ) the multiplication of bacteria already attached to the tooth surface

  • 2) the subsequent attachment and multiplication of new bacterial species to cells of bacteria already present in the plaque mass.


Complexity increases

Complexity increases

  • The secondary colonizers include Gram-negative species such as Fusobacterium nucleatum, Prevotella intermedia, and Capnocytophaga species.


Tertiary colonizers

Tertiary colonizers

  • After one week of plaque accumulation, other Gram-negative species may also be present in plaque. These species represent what is considered to be the "tertiary colonizers", and include Porphyromonas gingivalis, Campylobacter rectus, Eikenella corrodens, Actinobacillus actinomycetemcomitans, and the oral spirochetes (Treponema species).


Characteristics

Characteristics

  • The structural characteristics of dental plaque in this time period reveal complex patterns of bacterial cells of cocci, rods, fusiform, filaments, and spirochetes.

  • In particular, specific associations of different bacterial forms have been observed. For example, the adherence of cocci to filaments results in a typical form referred to as "test-tube brushes" or "corn-cob" arrays

  • these structures can be seen in The structural interactions of the bacteria probably are a reflection of the complex metabolic interactions that are known to occur between different plaque microorganisms.


Coaggregation

Coaggregation

  • Express components that mediate cell to cell binding

  • One cell type in a coaggregation partnerships, one cell type expresses a heat-inactivated protease sensitive surface adhesion

  • The other partner expresses a complementary heat stable protein


Biofilm biochemistry

Biofilm Biochemistry

  • The production of succinic acid from Campylobacter species that is known to be used as a growth factor by Porphyromonas gingivalis. Streptococcus and Actinomyces species produce formate, which may then be used by Campylobacter species.

  • Fusobacterium species produce both thiamine and isobutyrate that may be used by spirochetes to support their growth. The metabolic and structural interactions between different plaque microorganisms are a reflection of the incredible complexity of this ecological niche.


Genes and biofilms november 2005

Genes and BiofilmsNovember 2005

  • Biologist Alejandro Toledo Arana has identified two genes that regulate the formation of biofilms in Staphylococcus aureus


Chronic infections

Chronic Infections

  • The study has been boosted on discovering their relation to chronic infections associated to medical implants

  • These include those tissues involving infections of the middle ear, of the prostate gland, pneumonia in patients with cystic fibrosis, osteomyelitis


Antibiotic resistance in biofilms

Antibiotic resistance in biofilms

  • In patients such as those that suffer from Cystic fibrosis and infections of Pseudomonas, there is a weighted response in the CD4 T helper cells( Th1/Th2)

  • Th1 might improve prognosis for elimination and management of infections because they are associated with an influx of phagocytes and the ingestion of sessile bacteria.

  • They also ingest biofilm fragments


Survival strategies of infectious biofilms c a fux j w costerton et al trends in microbiology

Survival Strategies of Infectious BiofilmsC.A. Fux, J. W.Costerton et al Trends in Microbiology

  • There is a growing concern for antibiotic resistance in bacteria growing in surface-adherent biofilms

  • Many antibiotic assays for susceptibility and resistance are based upon planktonic or free cells rather than attached

  • Chronic and device related infections go unresolved even when the organisms do indeed test for antibiotic sensitivity


Biofilm characteristics1

Biofilm characteristics

  • Top to bottom gradient of decreasing antibiotic susceptibility

  • The gradient originates in the surface layers of the biofilms where there is complete consumption of oxygen and glucose

  • There are patches of antibiotic resistance at the surface

  • Proximity of cells lead to horizontal transfer of genes for resistance


Biofilms and antibiotics

Biofilms and Antibiotics

  • The diffusion of antibiotics in biofilms has been studied

  • Beta lactamase producing bacteria increase enzyme production in response to antibiotic treatment

  • The enzyme accumulates in the matrix of the biofilm thereby inactivating the antibiotic


Mar operon

Mar Operon

  • Multiple Antibiotic Resistance operon (Mar) is chromosomal, and encodes for permease proteins (AcrB) which actively export a wide range of xenobiotics from bacterial cells.

  • Mar is widely distributed. Recent reports show that Mar can be regulated not only by exposure to sub-MIC levels of antibiotic, but also through slow growth rate, the stringent response and a number of other unrelated stimuli.


Mar ii

Mar II

  • It is not regulated through homoserinelactone but does appear to be part of a global regulatory system that also controls exopolymer biosynthesis.

  • This operon is of major interesdt since it is likely that this would be switched on in biofilms and might be a major factor in the high level antibiotic resistance observed in biofilms.


Endocarditis

Endocarditis

  • Biofilm of bacteria + host components on valve = vegetation

  • Requires prior valve injury

  • 200X increase in antibiotic resistance

  • Rabbit model: block biofilm formation --> acute virulent infection


Infectious kidney stones

Infectious Kidney Stones

  • 15-20% involve urinary tract infection

  • Bacterium --> biofilm --> mineralization

  • Causative organisms have urease

  • urea --> NH4 + H2CO3

  • Biofilm concentrates urease --> crystal formation


Biofilms

CF

  • Mutation in chloride channel in epithelial cells

  • 1st stage: intermittent infections

  • 2nd stage: permanent infection with Pseudomonas aeruginosa

  • Mucoid type - overproduce alginate

  • Antibiotic resistance


Microarrays

Microarrays

  • Used to assess the genes present in different stages of biofilm formation

  • In Staphylococcal biofilms the same genes are active the sar A staphylococcal accessory regulator and the ica ADBC regulator


Microarrays1

Microarrays

  • One of the best ways to evaluate gene expression

  • DNA chips are used for a solid support

  • These are made of silicon or glass

  • They have DNA attached in orderly arrays


Microarrays 1

Microarrays( 1)

  • The DNA is deliverd to specific position on the chip using tiny pins to apply a solution

  • The spots are treated and dried in order to bind the DNA

  • Usually cDNA

  • cDNA is prepared from mRNA

  • These pieces of DNA are usually 500-5000 nucleotides long


Commercial chips ii

Commercial chips( II)

  • Oligonucleotides about 25 bases in length can be synthesized and placed directly on the chip

  • The chip is 1.3 cm on a side and can have over 200,000 addressable positions

  • The probes are often expressed sequence tags( ESTs)

  • The nucleic acids to be analyzed are isolated and labeled with fluorescent reporter groups

  • The DNA or target nucleotides are incubated with the fluorescent groups and then washed

  • The chip is scanned with lasers


Preparation of microarrays

Preparation of Microarrays


Interpretation of microarrays

Interpretation of microarrays


Agents for the destruction of biofilms industrial biocides

Agents for the destruction of biofilms( Industrial biocides)

  • (alexidine, chlorhexidine, polyhexamethylene biguanides), monophenylethers (phenoxyethanol) and quaternary amonium compounds (cetrimide, benzalkoniums) and have demonstrated biochemical bases for the activities and associated mammalian cell toxicities of thiol interactive agents (bronopol, isothiazolones).


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