The 5th lecture for 2nd-year students Institute for Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospital in Brno Miroslav Votava MICROBIAL BIOFILM – I The 5th lecture for 2nd-year students March 21, 2011

Resistance of microbes to drying up – revision Very sensitive: agents of STD – gonococci, treponemes Less sensitive: all Gram-negative bacteria A bit more resistant: skin flora – staphylococci, corynebacteria acidoresistant rods – mycobacteria Rather resistant: xerophiles – actinomycetes, nocardiae, moulds parasite cysts, helminth eggs Highly resistant: bacterial spores

Practical application of water shortage Lowering water activity stops action of most microbes → we use it for food preservation Examples: drying – meat, mushroom, fruit (prunes) concentration – plum jam salting – meat, fish, butter sugaring – sirups, jams, candied fruit

The influence of heat – revision The temperature higher than optimum → heat shock and gradual dying of cells The number of killed cells depends on the duration of the exposure to higher temperature The relation between the number of surviving cells and the duration of heating is inversely logarithmic one The time needed for exterminating the whole microbial population depends on its size (on the initial number of microbes)

Decimal reduction time – revision The relation between the duration of heating and the number of surviving microbes: Log10 number of survivors 6 D = decimal reduction time = 5 = the time required to reduce 4 the No of microbes to 1/10 = 3 = the time required to kill 90 % of 2 D microbes present (at the 1 specific temperature) 1 2 3 4 5 6 (min)

pH – revision Alkalophiles: e.g. Vibrio cholerae (pH 7.4-9.6) alkalotolerant: Proteus (it splits urea) Acidophiles: yeasts, lactobacilli (>3), coxiellae Some microbes are sensitive to extremes of pH: e.g. gonococci; others tolerate them, e.g Enterococcus (broad range of pH 4.8-11) Low pH hinders most bacteria, namely streptococci, vibrios, putrefactive bacteria Why sparkling water lasts longer? Because its pH is lower Low pH keeps spores from germinating → botulism can be obtained from oil-preserved mushrooms or preserved strawberries, not from pickled gherkins or mixed pickles

Redox potential (rH) – revision Aerobes – need high rH levels (>200 mV) Anaerobes – need low rH levels (≤0 mV) Anaerobes are killed by O2, aerobes without O2 will live Even so, anaerobes can prosper both in nature and in our bodies – thanks to the cooperation with aerobes and facultative anaerobes (e.g. in biofilms) Anaerobes in the body: large intestine (99 % of bowel microorganisms) vagina oral cavity (sulci gingivales)

Radiation – revision UV radiation Artificially: UV radiation is used for disinfection of surfaces, water, air; in PCR laboratories for destroying residues of DNA Ionizing radiation (X and gamma radiation) For sterilizing disposable syringes, infusion sets, materials for dressing and sewing, tissue grafts, some drugs, even waste and food (not in EU) Bacterial spores are quite resistant to ionizing radiation

Toxic substances – revision Their influence depends on the concentration and duration of exposure Various microbes markedly differ in relative resistance to different types of toxic substances In general (and contrary to drying): G– bacteria are more resistant to toxic substances than G+ bacteria (because of different structure of bacterial cell wall → presence of enzymes in periplasmatic space of G– bacteria) For application it is vital to know the effects of the particular substances used for disinfection

Bacterial cell wall – revision G+ G– lipoteichoic acid O-antigen lipopoly- inner polysaccharide saccharide lipid A (endotoxin) murein porin outer membrane lipoprotein ENZYMES periplasmatic space inner membrane (G–) cytoplasmatic membrane (G+)

Sterilization versus disinfection – revision Sterilization = removal of all microorganisms from objects or environment Disinfection = removal of infectious agents from objects and environment or from the body surface Disinfection aims at breaking the chain of infection transmission Biocides = a new general term including also disinfectants

Types of disinfectants – revision Oxidizing agents (peracetic acid, H2O2, O3) Halogens (hypochlorite, sol. iodi) Alkylating agents (aldehydes) Cyclic compounds (cresol, chlorophenols) Biguanides (chlorhexidine) Strong acids and alkali (e.g. slaked lime) Heavy metal compounds (Hg, Ag, Cu, Sn) Alcohols (ethanol, propanols) Surface active agents (QAS; e.g. cetrimid) Others (e.g. crystal violet & other dyes)

Relative resistance of different agents to biocides – revision Enveloped viruses herpesviruses Some protozoa very susceptible Trichomonas Gram-positive bacteria Streptococcus Gram-negative bacteria Salmonella Yeasts susceptible Candida Moulds Trichophyton Naked viruses enteroviruses Protozoal cysts relatively resistant Giardia Acidoresistant rods Mycobacterium Helminth eggs Ascaris Bacterial spores very resistant Clostridium Coccidia Cryptosporidium Prions extremely resistant agent of CJD - - -

Two forms of microbial growth Growth in planktonic form Isolated microbial cells float freely in a fluid environment Growth in biofilm form Result of the natural tendency of microbial cells to stick to one another and to a solid surface and to form a community connected by an extracellular matter

Which form is more frequent? Planktonic form fairly common in the laboratory (e.g. in nutrient broth) relatively scarce in a natural environment Biofilm form standard and crucial in the natural environment more advantageous for microbes

Definition of biofilm Microbial biofilm is a community of microorganisms that forms at the boundary of phases (usually of the solid and fluid phase) sticks to inert as well as to live surfaces is surrounded by an extracellular matter, in which a complex system of channels may form

Three examples of biofilm Have you ever slipped on a wet stone in a creek? Certainly – and in was biofilm that you slipped on Have you an aquarium and do you clean its walls? If you do, what you wipe from them is the biofilm formed by algae Do you clean your teeth regularly? I hope so and by doing this you remove the biofilm called dental plaque

History of biofilm 1676 Antony van Leeuwehoek 1935 C. E. Zobel bacteria in dental plaque 1935 C. E. Zobel the first description of biofilm in marine bacteria 1950 – 1960 first information about problems with the biofilm 1978 J. W. Costerton drawing attention to the ubiquity of biofilm 1999 Costerton, Stewart  Greenberg biofilm involvement in persistent infections

Microbiology lead astray – 1 For 100 years since Pasteur and Koch times it never occurred to anybody that in nature bacteria grow in other ways than as a freely floating plankton in seas or as colonies on agar From the half of the 19th to the half of the 20th century, throughout the whole „golden age of bacteriology“, the only subject of study were planktonic forms If signs of the biofilm growth appeared the experiment was quickly „sewered“

Microbiology lead astray – 2 The whole microbiology has been mislead by efforts to examine and investigate pure cultures of planktonically growing cells only, whereas the natural microbial growth is in the form of biofilm The last area of microbiology that started to be concerned with the biofilm is regrettably the medical microbiology, proud of its achievements with planktonic forms

How does the biofilm develop? Development of biofilm = cyclic process 1. Attraction of planktonic cells to a surface 2. Adhesion of planktonic cells to the surface 3. Aggregation of cells and the development of colonies – quorum-sensing phenomenon 4. Accumulation of exopolysaccharide matrix (slime) – development of typical architecture 5. Dispersal of cells from the surface of biofilm

Development of biofilm – attraction Attraction does not concern solid surfaces only but in general the boundaries of phases Prominent in mobile bacteria with flagella How does the bacterium know the proximity of a surface? It sends out chemical signals that diffuse more quickly into free areas while they concentrate in the vicinity of boundaries of phases

Development of biofilm – adhesion Bacterial adhesins fimbriae (pilli) colonization factors of enteropathogenic E. coli proteins and lipopolysaccharides of outer membrane generally in most of Gram-negative bacteria slime both coagulase-negative and golden staphylococci curli E. coli

Development of biofilm – aggregation I 1. Movement by means of flagella (E. coli, Vibrio cholerae) by means of fimbriae (type IV pilli of Pseudomonas aeruginosa) divergent – continuous layer of cells forms convergent – aggregates develop, even of different species (e.g. coaggregation of Streptococcus gordonii + Fusobacterium nucleatum in dental plaque) 2. Multiplication both aggregation and cell division in aggregates lead to the development of microcolonies

Development of biofilm – aggregation II 3. Quorum sensing During division individual cells emit chemical signals (homoserinlactones in P. aeruginosa) After reaching a particular number of cells (quorum) the elevated concentration of signals causes the change of cellular properties: - switching off some so far functioning genes (e.g. a gene for the production of flagellin) - expression of other genes, and from this ensuing - production of new molecules (in particular exopolysaccharides)

Development of biofilm – accumulation Production of exopolysaccharides colanic acid (E. coli) alginate (P. aeruginosa) polysaccharide intercellular adhesin (Staph. epidermidis) leads to the development of typical biofilm architecture Its appearance depends mainly on the nature of the environment

Development of biofilm – dispersal After reaching the critical amount of biomass or after the reduction of the amount of nutrients in the environment the character of cells at the surface of biofilm changes e.g. in P. aeruginosa the superficial cells - cease producing alginate - begin producing lyase and flagellin superficial layer of biofilm starts to disintegrate cells grow flagella and get loose of biofilm The cells as a planktonic population drift away to look for more suitable environment and to colonize new surfaces The cycle closes…

Architecture of biofilm – I Depends above all on the concentration of nutrients <10 mg/L (mountain streams, lakes, open sea) heterogeneous mosaic (a thin layer + columns of microcolonies) 10-1000 mg/L (majority of our rivers and ponds) complex system with channels (created by mushroom-like, partially merging microcolonies) 1000 mg/L (in the environment of macroorganism) compact biofilm (almost without traces of channels)

Architecture of biofilm – II Low concentrations of nutrients (0.1 – 10 mg/L – mountain streams, lakes, open sea) Heterogeneous mosaic = thin layer of individual cells above which columned microcolonies rise here and there

Architecture of biofilm – III Medium concentration of nutrients (10 – 1000 mg/L – eutrophic water environment) System with channels = mushroom-shaped microcolonies partially merging together, interwoven with water channels

Architecture of biofilm – IV

Architecture of biofilm – V High concentrations of nutrients (>1000 mg/L – in the macroorganism) compact biofilm = closely interconnected numerous microcolonies almost without traces of possible channels polymicrobial = e.g. dental plaque, normal microflora of mucous membranes

Architecture of biofilm – VI High concentrations of nutrients (>1000 mg/L – in the macroorganism) compact biofilm = closely interconnected numerous microcolonies almost without traces of possible channels b) monomicrobial = e.g. chronic osteomyelitis biofilm on inert surfaces of medical devices

Architecture of biofilm – VII Candida albicans biofilm. Alcian blue has coloured extracellular polysaccharides. Photo: Veronika Holá

Architecture of biofilm – VIII Candida albicans biofilm. Toluidin blue. At the photo mushroom-like structure of the biofilm is obvious. Photo: Veronika Holá

Properties of biofilm Biofilm is a higher and more complex form of microbial growth It utilizes the opportunity of mutual cooperation of cells It enables the easier transfer of genes It is characterized by an effective homeostasis It shows features of a primitive circulation system It provides a high protection against antimicrobial factors It plays an important part in many significant occasions including medically important conditions

Properties of microbes in biofilm Summary: The properties of microbes growing in the biofilm form are fundamentally different from the properties of microbes growing in the planktonic form; the microbes express different genes produce different products (extracellular matrix  flagella) enjoy a higher degree of protection

Recommended reading material Paul de Kruif: Microbe Hunters Paul de Kruif: Men against Death Axel Munthe: The Story of San Michele Sinclair Lewis: Arrowsmith André Maurois: La vie de Sir Alexander Fleming Could you kindly supply me with another work in connection with microbes or at least medicine? Please mail me your suggestions at: mvotava@med.muni.cz Thank you for your attention