Transcript LE BIOFILM - Endo

BIOTECH
GERMANDE
BIOFILMS AND ENDOSCOPES
Dr. Lionel PINEAU
BIOTECH-GERMANDE
Marseille - FRANCE
BIOTECH
GERMANDE
BIOFILMS AND ENDOSCOPES
1.
GENERAL INFORMATION ON BIOFILMS :
significance, definition, characteristics, etc…
2.
BIOFILM AND ENDOSCOPES
3.
WHAT CAN BE DONE ?
BIOTECH
GERMANDE
BIOFILM SIGNIFICANCE
Planktonic bacteria : 0.02 à 0.04%
Sessile bacteria : 99.96 à 99.98%
BIOTECH
GERMANDE
DEFINITION
J.W. Costerton
Biofilms are defined as matrix-enclosed bacterial populations
adherent to each other and/or to surfaces or interfaces.
Biofilm consists of single cells and microcolonies of sister cells
all embedded in a highly hydrated, predominantly anionic
matrix of bacterial exopolymers and trapped extraneous
macromolecules.
BIOTECH
GERMANDE
BIOFILM FORMATION
Bacterial colonization of surfaces can be divided
into three principal stages:
•the transport of the bacteria from the aqueous phase to the
boundary layer
•the adhesion, initially in a reversible association and eventually
in an irreversible adhesion.
•Colonization leading to the biofilm formation.
BIOTECH
GERMANDE
STAGE 1
THE TRANSPORT OF BACTERIA FROM THE AQUEOUS
PHASE TO THE BOUNDARY LAYER
Hydrodynamic forces
Chimiotactism
Brownien movement
Boundary layer
Surface
GERMANDE
BIOTECH
ETAPE 2
INITIAL ADHESION
Reversible adhesion
d > 50 nm
+ + + + + 10 < d < 20 nm
Surface
Van der Waals interactions
Surface
Van der Waals and
electrostatic interactions
Irreversible adhesion
Electrostatic
interactions
Specific
interactions
+
Surface
Van der Waals, electrostatic
and specific interactions
10 < d < 20 nm
GERMANDE
BIOTECH
ETAPE 3
COLONISATION
Surface
Microcolonies
GERMANDE
BIOTECH
SUMMARY
Bacterial microcolonyy delineated by their exopolysaccharide matrix
Network of water channel (convective flow)
Surface
Within biofilms bacterial replication and exopolysaccharide
production are regulated (quorum sensing or complex cell-cell
communication) so that an open system of microcolonies and water
channels is produced and maintained.
BIOTECH
GERMANDE
GLYCOCALYX
DEFINITION
Glycocalyx, EPS (extracellular polymeric substance)
=
water (99%) + polysaccharide-containing stuctures, of
bacterial origin, lying outside the integral elements of the
outer membranes of Gram-negative cells and the
peptidoglycan of Gram-positive cells. (homopolymères
simples ou hétéropolymères complexes de glucose,
mannose, galactose, xylose, ...
GLYCOCALYX
BIOTECH
GERMANDE
The polysaccharides produced vary depending on the species but
are typically made up of repeating oligosaccharides, such as
glucose, mannose, galactose, xylose, and others.
GERMANDE
BIOTECH
RESISTANCE OF
BIOFILM BACTERIA
Martin EXNER
« Influence of Biofilms by chemical disinfectants and mechanical cleaning »
1987.
Disinfection parameters
(time/concentration)
to kill the biofilm cells (2)
Biofilm removal
efficacy
Chemicals
MBC (1)
Aldehydes
0.007%
P.A.A
0.0004%
1h / 1%
very low
H2O2
0.3%
1h / 3%
important
Chlorine
0.0001%
(1)
1h / 3%
10 min / 10%
1 h / 10 mg/l Cl2
none
low
: Minimum batericidal concentration : 5 log reduction within 5 minutes et 20°C against
Pseudomonas aeruginosa
(2) : 106 UFC/cm2
GERMANDE
BIOTECH
RESISTANCE OF
BIOFILM BACTERIA
Antibody
Antiseptics and
disinfectants
Antibiotics
Surfactants
2
1
3
Surface
1 : glycocalyx provides a penetration barriers to antimicrobial agent
2 : Interaction of antimicrobial agent with charged biding sites on the matrix polymers
3 : resistance which derives from phenotypic changes in sessile organisms
BIOTECH
GERMANDE
BIOFILM AND
MICROBIOLOGICAL CORROSION
Microbial corrosion induces by iron oxidizing organisms
BIOTECH
GERMANDE
CONCLUSION
« the highly structured biofilm mode of growth provides
bacteria with a primitive circulatory system, a
framework for the development of cooperative and
specialized cell functions and a large measure of
protection from antibacterial agents. These advantages
led to its functional predominance in most natural
aquatic ecosystems compare to planktonic cells»
Costerton JW
BIOTECH
GERMANDE
BIOFILM AND
ENDOSCOPES
BIOTECH
GERMANDE
BIOFILM AND
ENDOSCOPES
Somes references
•Glutaraldehydes-resistant Mycobacterium chelonae from
endoscope washer-disinfector. Griffiths PA, Babb JR, Bradley CR,
Fraise AP. J Appl Microbiol 1997, Apr;82(4):519-26
•Audit of bronchoscope disinfection : a survey of procedures in
England and Wales and incidents of mycobacterial contamination. J
Hosp Infect 1994 Apr;(4):301-8
•Pseudo-outbreak of Mycobacterium chelonae and
Methylobacterium mesophilicum caused by contamination of an
automated endoscopy washer. Infect Control Hosp Epidemiol 2001
Jul;22(7):414-8
•…
BIOTECH
GERMANDE
BIOFILM AND
ENDOSCOPES
Internal structure of an endoscope
BIOTECH
GERMANDE
BIOFILM AND
ENDOSCOPES
Internal structure of an endoscope
BIOTECH
GERMANDE
BIOFILM AND
ENDOSCOPES
Evaluation of the contamination level and surface conditions of two
different type of endoscope biopsy channels (Teflon vs Goa®)
according to the number of exams performed with the endoscope
BIOTECH
GERMANDE
BIOFILM AND
ENDOSCOPES
Internal surface of an endoscope biopsy channel after 300 exams (Teflon)
20µm
BIOTECH
GERMANDE
BIOFILM AND
ENDOSCOPES
Internal surface of an endoscope biopsy channel after 300 exams (Teflon)
4µm
BIOTECH
GERMANDE
BIOFILM AND
ENDOSCOPES
Internal surface of an endoscope biopsy channel after 600 exams (Teflon)
4µm
BIOTECH
GERMANDE
BIOFILM AND
ENDOSCOPES
Internal surface of an endoscope biopsy channel after 600 exams (Goa®)
4µm
BIOTECH
GERMANDE
BIOFILM AND
ENDOSCOPES
Internal surface of an endoscope biopsy channel after 600 exams (Goa®)
4µm
GERMANDE
BIOTECH
BIOFILM AND
ENDOSCOPES
Internal surface of a brand new endoscope biopsy channel (Goa®)
20µm
GERMANDE
BIOTECH
BIOFILM AND
ENDOSCOPES
Internal surface of a brand new endoscope biopsy channel (Teflon )
4µm
GERMANDE
BIOTECH
BIOFILM
Influence of surface condition
5µm
10µm
Control
T = 24 hours
GERMANDE
BIOTECH
BIOFILM
Influence of surface condition
4µm
4µm
Control
T = 24 hours
BIOTECH
GERMANDE
CONCLUSION
• Endoscopes are not designed to be easily cleaned and
disinfected
•Some medical biomaterials facilitate attachment and
colonization of microorganisms
•The use of fixative agent during the disinfection stage
may contribute to biofilm formation
There is a real risk of biofilm formation in endoscope channels
which may constitute uncontrolled foci but biofilm bacteria
may be difficult to recover with standard sampling procedures
BIOTECH
GERMANDE
WHAT CAN BE DONE?
Select chemicals and/or cleaning and disinfecting
procedures which are able to remove and/or kill biofilm
bacteria.
BIOFILM FORMATION
BIOTECH
GERMANDE
A
B
P2
P1
Fiole de garde
Réserve de substrat
Evacuation
Bain marie à 30°C
P1 : Pump for the adhesion broth supply (2,5 to 3 ml/min).
P2 : Agitation pump (100 ml / min).
B : Zone d'inoculation de la boucle.
A-B : Zone de prélèvement des portions de tube. (90 cm).
BIOTECH
GERMANDE
BIOFILM FORMATION
4µm
Biofilm formed experimentally on Tygon tube
CHEMICALS EVALUATION
BIOTECH
GERMANDE
Counting of the number of
viable bacteria still fixed to
the tube
P
Determination of the amount of residual
exopolysaccharide
After each contact
time
Determination of the amount of residual
proteins
SEM
Tested solution
Counting of the number of viable
bacteria removed from the surface
Tygon® Tube + biofilm
BIOTECH
GERMANDE
THEORITICAL RESULTS
Log(CFU/cm2)
([proteins] µg/cm2)
Log(Nb. of released bacteria/cm2)
Log(Nb. of fixed bacteria/cm2)
Proteins
Contact time (min.)
Theoretical evolutions of the number of viable bacteria (log.) still adherent or removed
from the internal surface of the Tygon® tube and of the amount of residual proteins on
the support according to the contact time with the tested solution.
BIOTECH
GERMANDE
EXPERIMENTAL DATA
Comparaison of the activity of several detergent solutions against
Pseudomonas aeruginosa biofilm
Log (Nb. bacteria/cm2)
9,0
8,0
Control (SDW)
F
7,0
E
6,0
5,0
4,0
3,0
D
2,0
1,0
B
0,0
0,0
2,0
4,0
C
6,0
8,0
10,0
Contact time (min.)
A
12,0
14,0
16,0
ENDOSCOPE WD
Log. CFU/ cm2
9,0
Evaluation of the efficacy of
an endoscope
cleaning/disinfection cycle
against biofilm
8,0
7,0
6,0
5,0
4,0
3,0
2,0
1,0
Drying
Rinsing
Rinsing
Disinfection
Rinsing
Rinsing
Washing
0,0
Control
BIOTECH
GERMANDE
Log CFU/ cm2 of tygon tube (E. coli biofilm).
Log CFU/ cm2 of tygon tube (S. aureusbiofilm).
Log CFU/ cm2 of tygon tube (P. aeruginosa mucoïd biofilm).
BIOTECH
GERMANDE
ENDOSCOPE WD
HTM 2030
Washer-disinfectors
Validation and verification
« The piping used to convey rinse water to the endoscope, if contaminated, may
easily develop a layer of biofilm containing many micro-organisms in a state
which is highly resistant to chemical disinfection »
« for chemical self disinfection cycle the test required is designed to ensure that
the self disinfection cycle will disinfect contaminated tubing by evaluating the
effect of the cycle against a biofilm containing Pseudomonas aeruginosa »
BIOTECH
GERMANDE
CONCLUSION
Biofilms are universal, occurring in aquatic and industrial water systems as
well as a large number of environments and medical devices relevant for
public health (biomedical implant infection, hemodialysis, catheter
associated infection, …).
Effective strategies to prevent or control biofilms on medical devices need to
be developed :
•Medical biomaterials which minimize microbial cell attachment
•Chemicals and/or process which penetrate the biofilm and kill and/or
remove the associated cells.
•New treatment based on inhibition of genes involved in cell attachment and
biofilm formation (Donlan RM, 2002)
BIOTECH
GERMANDE
CONCLUSION
The last one!!!
“Performing antimicrobial susceptibility tests using pure
cultures and in planktonic growth mode should be questioned”
(Dunne WM, 2002)