1. Microbial Biochemistry
Biofilms
Microbial Biochemistry

2. 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

3. 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

4. Biofilms BIOFILMS may form:
On solid substratums in contact with moisture
On soft tissue surfaces in living organisms 
At liquid-air interfaces. 

5. Study of biofilms
Formation of multicellular communities depends on the production of extracellular substances( matrix)
Diversity in the formation of the matrix

6. Pathogens that have been studied for the formation of biofilms
Staphylococcus aureus
Staphylococcus mutans
Salmonella typhi
Enterococcus faecalis
Pseudomonas aeruginosa

7. Biofilms – Now a Universal Feature
Now scientists believe biofilm formation is a universal feature of all bacteria

8. 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

9. 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

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

11. Steps in Biofilm Formation

12. Exopolysaccharides Exopolysaccharides
In the glycoclyx contribute to biofilm formation

13. Matrix
Key components of the matrix are extracellular polysaccharides and proteins
Dead cells have also been identified in biofilms
Extracellular DNA is also important

14. Biofilm formation

15. Matrix constituents

16. 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

17. 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.

18. 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

19. Bap Protein The biofilm associated protein
Structurally similiar to the surface proteins
Esp of Enterococcus faecalis
mus20 of Pseudomonas aeruginosa
sty2875 of Salmonella typhi

20. 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

21. 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

22. 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

23. 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

24. Adhesins
Adhesins are molecules that are attached to bacterial fimbriae

25. Staphylococcus aureus

26. E. Coli Adhesins

27. Adhesins Bacterium Adhesins Receptor Attachment Disease Vibrio cholera
N-methylphenylalanine
Fucose and Mannose
pili
Intestinal epithelium
Cholera
Bordetella pertussis
Fimbriae ("filamentous hemagglutinin")
Galactose on sulfated glycolipids
Respiratory epithelium
Whooping cough
Streptococcus pyogenes
Protein F
amino terminus of fibronectin
Pharyngeal epithelium
Sore throat
E. coli
Type 1 Fimbriae
Species specific carboydrates
Diarrhea

28. Adhesins and vaccines

29. Bacterial Cell Characteristics
The phenotypes of the cells include:
a slower growth rate
increased antibiotic resistance
elevated frequency of lateral gene transfer

30. 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

31. Signaling in biofilms

32. Signaling and biofilms

33. Gram Negative

34. Gram Positive

35. Antibiotic Resistance
In the center of the biofilm the bacteria have greater antibiotic resistance
Opsonisation of antibiotics
To resistance to phagocytosis

36. 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

37. 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

38. 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

39. Definition Supra gingival – above gums Subgingival –below the gums
Also related to the tooth surface

40. 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.

41. 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.

42. 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.

43. 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.

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

45. 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).

46. 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.

48. Facultative Anaerobic
SELECTED BACTERIAL SPECIES FOUND IN DENTAL PLAQUE
Facultative
Anaerobic
Gram-Positive
Streptococcus mutans Streptococcus sanguis Actinomyces viscosus
Gram-negative
Actinobacillus actinomycetemcomitans Capnocytophypa species Eikenella corrodens
Porphyromonas gingivalis Fusobacterium nucleatum Prevotella intermedia Bacteroides forsythus Campylobacter rectus
Spirochetes
Treponema denticola (Other Treponema species)

49. 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

50. 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.

51. Genes and Biofilms November 2005
Biologist Alejandro Toledo Arana has identified two genes that regulate the formation of biofilms in Staphylococcus aureus

52. 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

53. 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

54. Survival Strategies of Infectious Biofilms C. A. Fux, J. W
Survival Strategies of Infectious Biofilms C.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

55. 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

56. 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

57. 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.

58. 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.

59. 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

60. Infectious Kidney Stones
15-20% involve urinary tract infection
Bacterium --> biofilm --> mineralization
Causative organisms have urease
urea --> NH4 + H2CO3
Biofilm concentrates urease --> crystal formation

61. 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

62. 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

63. 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

64. 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 nucleotides long

65. 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

66. Preparation of Microarrays

67. Interpretation of microarrays

68. 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).