1. BIOFILMS OF PATHOGENS Dr. Işıl VAR Cukurova University,
Department of Food Engineering
Roundtable workshop
Institute of Food Engineering, University of Szeged
June 26-30,2015
Hungary

2. BIOFILMS OF PATHOGENS

3. WHAT is Biofilm?
Biofilms are microbial cellular populations, of no specific shape, that grow up in the extracellular matrix.
Extra polymeric substances (EPS) are generally made of polysaccharide in the biofilm structure.
Biyofilm structure includes 97% water, 2-5% microroganism, 1-2% polysaccharide, 1-2% proteins, 1-2% DNA and various ions.

4. Definition of Biofilm
Biofilms are communities of microorganisms in a matrix that joins them together
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
Over 90% of all bacteria live in biofilms

5. A Biofilm

6. Exopolysaccharides
Extra polymeric substances (EPS) are generally made of polysaccharide in the biofilm structure.
Exopolysaccharides in the glycoclyx contribute to biofilm formation.

7. Extracellular Polymeric Substances(EPS):

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

9. 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

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

11. Biofilm formation

12. Biofilms

13. Biofilms

14. The close relationship of cells provide the ideal conditions for:
BİOFİLM’s Ecology
The smallest unit of biofilm is microcolony.
The close relationship of cells provide the ideal conditions for:
synthesis of food substances
gene changes and
Quorum Sensing (the communication of microbial population).

15. Biofilms

16. Population density
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

17. One of the best studied communication mechanisms in bacteria is quorum sensing which is based on the production of low-molecular mass signalling molecules.
When the bacterial cell density is low, the extracellular concentration of the signals will also be low and remain undetected.
However, as the cell density increases in a growing (biofilm) population, a critical signal concentration will be reached, allowing the signalling molecule to be sensed and enabling the bacteria to respond.

18. Quorum sensing
Quorum sensing is controlled by at least two different quorum sensing signals
N-acyl-homoserine lactones (AHLs) of quorum sensing (QS) signaling molecules are involved in biofilm formation of several bacteria
Acyl-homoserine lactone CAI-1( cholera autoinducer 1) appears to play a significant role in biofilm formation

19. The nature of the signalling molecules is diverse.
While most Gram-negative bacteria use N-acyl-homoserine
lactones (AHL) as signalling molecules (Lazdunski et al. 2004).
Gram-positive bacteria commonly use amino acids and short post translationally processed peptides (Sturme et al. 2002).
Additional families of bacterial signalling molecules have been identified such as Autoinducer- 2 (AI-2) for both Gram-negative and Gram-positive bacteria.

20. Gram Negative

21. Gram Positive

22. Signaling in biofilms

23. Signaling and biofilms

24. Steps in Biofilm Formation
1.Attachment:Formation of adhesion surface
2.Adhesion of pioneer bacteria
3.Mucus(glycocalyx or slime) formation
4.Seconder colonisation
5. Mature biyofilm;
fully functioning biofilm: A cooperative “consortia” of species

25. Formation of Biofilms Form in places with access to water
Attach to a solid surface using several means:
Flagella
Hydrophobic Cell Walls
Sticky Polymers(Exopolysaccharides provide the structural frame for biofilm formation, and determine the biofilm structure).

26. 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

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

28. Staphylococcus aureus adhesins

29. E. coli Adhesins

30. Adhesins Bacterium Adhesins Receptor Attachment Disease Vibrio cholera
N-methylphenylalanine
Fructose and Mannose
phili
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

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

32. Biofilm Development Strategy
For this purpose we can see two Strategies.
Ego (Egoist= high growth rate, instabil structure) Staphylococcus biofilm
Eco (Economic= low growth rate, very strict structure) Listeria monocytogenes and Campylobacter jejuni biofilms

33. Pure biofilms
Ego grows faster initially, during the phase of exponential,unlimited growth. Later, substrate limitation gives Eco the long-term advantage.
Mixed biofilms: Ego and Eco side-by-side
Similar to pure biofilms, Eco wins in the end, independent of density
Mixed biofilms: Ego and Eco alternating
With increasing initial density of cells, Ego’s faster growth during substrate abundance increases its chance to overgrow Eco. Once overgrown, the match is over (truncation selection).(Jan-Ulrich Kreft)

34. Altruism among microorganisms
Biofilms encourage altruism.
Bacteria will often sacrifice their maximum growth rate in an effort to use the available community resources more efficiently. While individuals are disadvantaged, community as a whole benefits.

35. Biofilms like small cities
have many very close neighbors that remain together for extended periods of time
As compared with planktonic bacteria, biofilm
bacteria are more resistant to several antimicrobial agents or other environmental stresses.
It has been postulated that large amounts of biofilm formed by these microorganisms play an important role in the degradation and transformation of pollutants in the increasingly polluted soil and water environment.

36. Biofilms

37. Biofilms: Significance
Detrimental:
Intravenous catheter and implants
Lungs of patients
Peritoneal
Vascular catheter
Implant

38. Biofilms: Significance
Detrimental:
Dental plaque
Contact lenses
Dental Plaque

39. Biofilms: Significance
Detrimental:
Surfaces in food processing plants

40. Where do biofilms form in the food industry?
In the food industry, biofilms can form on any surface (stainless steel, wood, plastic, epoxy resins, etc).
The biofilm protects its resident microorganisms from the effects of sanitisers, and other antimicrobial substances.
Depending on the microorganisms present, biofilms may increase the risk of a food becoming contaminated by pathogens or reduce product shelf-life due to the activity of spoilage organisms.
Biofilms may also lead to corrosion on metal surfaces, blockages of pipes, and reduced heat transfer.

41. In dairy processing plants, biofilms may occur on any wet surface, including in storage tanks, on conveyor belts, in packaging equipment, in drains, and on trolley wheels. Even, for example, L. monocytogenes can make biofilm inside the milk cells.
The challenge to control these films is to hunt down all possible environments and niches.

42. Listeria monocytogenes to adhere stainless steel surface

43. Here we can see the meat fibre.
Some studies showed that the
bacteria use the meat fibre as an
attachment place.
(Schwach and Zottola,1982)

44. When biofilms contaminate a food product they create a hazard to human health, and may cause financial loss in the food industry by disrupting services.

45. Biofilm-forming capabilities of bacteria
Pseudomonas aeruginosa
Listeria monocytogenes
Staphylococcus aureus
Candida albicans
Klepsiella pneumoniae
Salmonella enteritidis
Campylobacter jejuni
Escherichia coli

46. and the other Biofilm-forming capabilities of bacteria
Clostridium perfringens,
Bacillus cereus,
Mycobacterium,
Enterococcus faecalis,
Proteus mirabilis,
Staphylococcus epidermitidis,
Acinetobacter baumannii,
Pseudomonas fluorescens,
Vibrio cholerae,
Vibrio parahaemolyticus,
Vibrio harveyi,
Shewanella putrefaciens,
Lactobacilus casei,

47. Pathogens that have been studied for the formation of biofilms are:
Staphylococcus aureus
Staphylococcus mutans
Salmonella Typhi
Enterococcus faecalis
Pseudomonas aeruginosa

48. Factors affecting the bacterial cell surface
The attachment and biofilm-forming capabilities of bacteria depend on multiple factors including:
the attachment surface,
the presence of other bacteria,
the temperature,
the availability of nutrients and
pH.

49. Implications of biofilm formation
Biofilms formed in food-processing environments are of special importance as they have the potential to act as a
persistent source of microbial contamination that may lead to food spoilage or transmission of diseases.
It is generally accepted and well documented that cells within a biofilm are more resistant to biocides than their planktonic counterparts.

50. Problems Caused by Biofilms
Tend to clog pipes and water filters
Can cause numerous diseases, including many diseases prevalent in hospitals
Extra-resistant to antibiotics
Can form almost anywhere that water is present, including catheters, kitchen counters, etc.

51. Uses of Biofilms
As biofilms are mostly hazardous, they can, to a lesser extent, be useful in various circumstances:
purify water in water treatment plants
break down toxic chemicals
produce useful biological compounds, including medicines

52. Prevention, control, removal and eradication of biofilms in the food industry
Prevention and control
Microbial attachment to (food-processing) surfaces is a
rather fast process, and therefore, it is for most applications not possible to clean and disinfect frequently enough to avoid attachment.
Nevertheless, an adequate frequency of disinfection should be carefully determined to avoid biofilm maturation and build-up of absorbed organic material (product residues), which can influence the hygienic status of the material and the availability of nutrients.

53. Cleaning processes
The primary objective of a cleaning process is the removal of product residues. Indirectly, removal of these residues is also a first critical point in the removal, killing and control of biofilms.
Disinfectants are less effective when food particles or dirt is present on the surfaces
The removal of biofilms is also significantly facilitated by the application of mechanical force (like brushing and scrubbing) to the surface during cleaning (Wirtanen et al. 1996).

54. Various preventative anti-biofilm methods are being developed, which destroy and kill biofilms.
One of the most effective methods known is the use of disinfectants , but the selection of correct materials and hygienic design is important.
Disinfectants such as chlorine, monochloramine, iodoform, hypochlorite, anionic acid, sodium hypochlorite, ozone and UV disinfection, various enzyme complexes, bacteriosides,  fungicides, and organic-based antimicrobial agents are available in the struggle against biofilms.

55. Antimicrobial resistant Mechanisms
Slow penetration
The antibiotic or disinfectant go down layers is very slow in yellow area
Resistant Phenotype
The bacteria remarked green is resistant to the the antibiotic or disinfectant
Changeable Place
The metabolites are seen in pink colour affect ??the antibiotic or disinfectant’s effect ??on biofilms

56. Concluding remarks
Bacterial biofilms are ubiquitous in nature, and the food
industry does not escape from the problems they can
cause.
In particular, biofilms formed on food-processing
equipment and other food contact surfaces act as a persistent source of contamination threatening the microbiological quality and safety of food products, and resulting in food-borne disease and economic losses.
Biofilm prevention and control is therefore a priority in the food industry as well as the health issues

57. References
Michael P. Doyle, Larry R. Beuchat and Thomas J. Montville Food Microbiology: Fundamentals and Frontier. Second Edition. American Society for Microbiology (ASM Press)
Kornacki, J.L Controlling Listeria in the Food Processing Environment. Food Technology. 11(05)
Perl. P Outbreak. The Washington Post Magazine. January. pp
Wirtanen, G., Storgards, E., Saarela, M., Salo, S. and Mattila-Sandholm, T. (2000) Detection of biofilms in the food andbeverage industry. In Industrial Biofouling ed. Walker, J.,Surman, S. and Jass, J. pp. 176–203. Chichester, UK: John Wiley and Sons Ltd.
Goebl, J Biofilms.slights
Houdt, R. Van; Michiels, C.W Biofilm formation and the food industry, a focus on the
bacterial outer surface. Journal of Applied Microbiology ISSN
Zhu, M., M. Du, J. Cordray and D. Uk-Ahn Control of Listeria monocytogenes
Contamination in Ready-to-Eat Meat Products. Comprehensive Reviews in Food Sci.
and Food Safety
Kreft JU.,2001 Biofilms promote altruism. Theoretical Biology, University of Bonn, Germany

58. THANK YOU