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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

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 biofilms

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


Biofilm characteristics
Biofilm characteristics biofilms

  • 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 biofilms

  • 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



Steps in biofilm formation1
Steps in Biofilm Formation all for one. Science 280:226-227


Exopolysaccharides
Exopolysaccharides all for one. Science 280:226-227

  • Exopolysaccharides

  • In the glycoclyx contribute to biofilm formation


Matrix
Matrix all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227


Matrix constituents
Matrix constituents all for one. Science 280:226-227


Polysaccharides
Polysaccharides all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • Adhesins are molecules that are attached to bacterial fimbriae


Staphylococcus aureus
Staphylococcus aureus all for one. Science 280:226-227


E coli adhesins
E. Coli Adhesins all for one. Science 280:226-227


Adhesins1
Adhesins all for one. Science 280:226-227


Adhesins and vaccines
Adhesins and vaccines all for one. Science 280:226-227


Bacterial cell characteristics
Bacterial Cell Characteristics all for one. Science 280:226-227

  • The phenotypes of the cells include:

  • a slower growth rate

  • increased antibiotic resistance

  • elevated frequency of lateral gene transfer


Quorum sensing
Quorum sensing all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227


Signaling and biofilms
Signaling and biofilms all for one. Science 280:226-227


Gram negative
Gram Negative all for one. Science 280:226-227


Gram positive
Gram Positive all for one. Science 280:226-227


Antibiotic resistance
Antibiotic Resistance all for one. Science 280:226-227

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

  • Opsonisation of antibiotics

  • To resistance to phagocytosis


E faecalis
E. faecalis all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

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 all for one. Science 280:226-227

Subgingival –below the gums

Also related to the tooth surface

Definition


Primary colonizers
Primary Colonizers all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

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


Tertiary colonizers
Tertiary colonizers all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 Biofilms all for one. Science 280:226-227November 2005

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


Chronic infections
Chronic Infections all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 Biofilms all for one. Science 280:226-227C.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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 15-20% involve urinary tract infection

  • Bacterium --> biofilm --> mineralization

  • Causative organisms have urease

  • urea --> NH4 + H2CO3

  • Biofilm concentrates urease --> crystal formation


CF all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227

  • 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) all for one. Science 280:226-227

  • 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) all for one. Science 280:226-227

  • 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 all for one. Science 280:226-227


Interpretation of microarrays
Interpretation of microarrays all for one. Science 280:226-227


Agents for the destruction of biofilms industrial biocides
Agents for the destruction of biofilms( Industrial biocides) all for one. Science 280:226-227

  • (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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