Transcript 05_Microb_biofilm_I_2014

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 of General Medicine
March 17, 2014
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 – revision
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
5
4
3
2
1
D = decimal reduction time =
= the time required to reduce
the No of microbes to 1/10 =
= the time required to kill 90 % of
microbes present (at the
specific temperature)
D
1
2
3
4
5
6
(min)
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
inner polysaccharide
lipid A
lipopolysaccharide
(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
1. Oxidizing agents (peracetic acid, H2O2, O3)
2. Halogens (hypochlorite, sol. iodi)
3. Alkylating agents (aldehydes)
4. Cyclic compounds (cresol, chlorophenols)
5. Biguanides (chlorhexidine)
6. Strong acids and alkali (e.g. slaked lime)
7. Heavy metal compounds (Hg, Ag, Cu, Sn)
8. Alcohols (ethanol, propanols)
9. Surface active agents (QAS; e.g. cetrimid)
10.Others (e.g. crystal violet & other dyes)
Relative resistance of different
agents to biocides – revision
Enveloped viruses
Some protozoa
Gram-positive bacteria
Gram-negative bacteria
Yeasts
Moulds
Naked viruses
Protozoal cysts
Acidoresistant rods
Helminth eggs
Bacterial spores
Coccidia
Prions
herpesviruses
very susceptible
Trichomonas
Streptococcus
Salmonella
susceptible
Candida
Trichophyton
enteroviruses
relatively resistant Giardia
Mycobacterium
Ascaris
very resistant
Clostridium
Cryptosporidium
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
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
a) 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
Importance of biofilm for the life
of microorganisms – I
More favourable environment for the life of
microorganisms
Possibility of effective cooperation and specialization of
cells
because of stable mutual position of cells of different
species in the intercellular matrix and corresponding
presence of various enzymes
Considerably easier transfer of genes
Effective homeostasis
Inside the aerobically established biofilm anaerobic
places may occur
Primitive circulation system
brings in and carries away nutrients, waste products as
well as signal molecules
Importance of biofilm for the life
of microorganisms – II
Protection against harmful influences
in environment: against amoebae, phages,
dessication, washing away,
toxic substances
in macroorganism: against phagocytes,
washing away,
complement,
antibodies,
antibiotics
Resistance of biofilm towards
toxic substances
MICROBES IN THE BIOFILM FORM ARE ALWAYS
MORE RESISTANT THAN IN THE PLANKTONIC
FORM
• Higher resistance applies also to disinfectants and
antibiotics
• Differences in sensitivity sometimes amount up to 3 orders
• General mechanism of the higher resistance is not known
• In each microbe-antimicrobial combination the mechanism
can be different
Possible causes of higher
resistance of biofilm
1. More difficult penetration of toxic matter
through the biofilm
2. Character of environment in the biofilm is
altered
3. Also the microbial population in the biofilm is
altered
Ad 1. Difficult penetration
EXTRACELLULAR MATRIX SERVES AS THE
PENETRATION BARRIER
•
difficult diffusion of the antimicrobial:
logical explanation
not always demonstrable
•
influence of surface charge seems more important
e.g. aminoglycosides (+) combine with
alginate (-) of P. aeruginosa
Ad 2. Altered environment
LOCAL ALTERATION IN THE BIOFILM
ENVIRONMENT
(consumption of O2 in certain areas,
increase in osmotic pressure,
accumulation of acidic products of metabolism)
AFFECTS THE ACTION OF ANTIBIOTICS
directly – suppression of the action of
antibiotics
chinolones, aminoglycosides
indirectly – reducing the growth rate of microbes
beta-lactams, glycopeptides
Ad 3. Altered microbial
population
• common dosage of a toxic substance kills 99 % microbes
in the biofilm
• a subpopulation of cells remains that is highly resistant to
the particular substance
it is not obvious if the subpopulation existed from the
beginning,
or if it has evolved by the action of the toxic
substance
• the subpopulation is responsible for the increased
resistance of biofilm
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:
[email protected]
Thank you for your attention