biofilmin washingmachines and howit is assessed · biofilmin washingmachines and howit is assessed...
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Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Biofilm in washing machines and how it is assessedCaroline Amberg
Swissatest testmaterials ag, St. Gallen, Schweiz
Hygiene requirements and measurements in dishwashing1th / 2th of April, 2014, Bonn
1April 2014
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
What is a biofilm? And why is it important in the today’s discussion?
April 2014 2
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
What is a biofilm: ‘Slime City’
• City of microbes with a complex structure• Bacteria, fungi, algae, protozoa• EPS = exopolymere substance consist ofpolysacharides, proteins, lipids, nucleicacids
• Water is the main part of the EPS = Hydrogel
• Diverse compositions depending on attached surface, germs, nutrientsavailable etc.
• Ubiquitär on interfaces with wateravailability
• Quorum sensing = communication withinbiofilm bacteria
Living in a biofilm is the most common wayof life for microbes!
Biofilm = Community of microorganisms embedded in a extracellular polymer matrix (EPS)
Drenkard, E. (2003) in microbes and infection
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Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Quorum Sensing
März 2014 4
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Steps of biofilm formation
University of Colorado, Boulder: http://mathbio.colorado.edu
1. Conditioning and reversible attachment
2. Irreversibles Attachment
3. young Biofilm
4. mature Biofilm
5. Detachment, steady state biofilm
April 2014 5
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
• Surfaces / materials in contact with water or > 90 % relative humidity
• Temperatures between 4° and 60°C
• Organic or inorganic nutrients in water, air or material
• Materials: • Metals e.g. iron, stainless steal, aluminium
even copper!• Concrete, sand stone, glass• Plastics, colors, glues• Wood, leather, textiles• Oils, fats, emulsions
Biofilm is expected on…
Source: Flemming, H.C., Heitz, E., and Sand, W. (1996). Microbially influenced Corrosion of Material. Springer Verlag, Berlin.
Every surface is sooner or later colonized!
April 2014 6
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Biofilm sampling and analysis
Sampling:Swabbing, material coupons
Direct investigation: e.g. Microscopy, FTIR‐Spectrometry
Analysis:• Amount of cells• Microbial species• Distribution of species within biofilm• Biofilm structure• Biofilm amount (biomass)• EPS‐Amount• Viability• Presence of specific microorganisms
Analysis method depends on what we want to measure One parameter alone is often not sufficient and meaningful
April 2014 7
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Biofilm sampling and analysis
Analysis:• Amount of cells: Culturing, staining and microscopy, staining and
microplate reader (Crystal violet, fluorescent dyes) • Microbial species: Culturing, PCR, etc.• Distribution of species within biofilm: Microscopy (CSLM)• Biofilm structure: Microscopy (CSLM, SEM, FM)• Biofilm amount (biomass): Total organic carbon• EPS‐Amount: Proteine amount and polysaccharide amount, staining of
EPS• Viability: Live/Dead staining (Syto 9 / Propidium iodid), Tetrazolium salts
etc., ATP‐measurements• Presence of specific microorganisms: PCR, realtime PCR,
Immunoassays, etc.
A natural biofilm is a highly complex structure. The choice ofanalysis method(s) and its interpretation has to be done carefullyand fitting to the research question!
April 2014 8
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Is it likely to have Biofilm in our household devices?
April 2014 9
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
• Water / humidity• Drain, pump, tubings, outer drum, drum, detergent
drawer case• consumer: handling and cleaning of washing machine
• Temperatures between 4°C and 60°C• Low temperatures: 20°C to 40°C
• Organic / Inorganic nutrients• Soil load in the laundry• Rubber and tubings• Metals / allows (crossbar)
• Insufficient sanitization• Detergent without bleach• Short washing cycles• Low water consumption
Biofilm in washing machines must be expected and isnot totally avoidable
Biofilm growth in washing machines
April 2014 10
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Is Biofilm in washing machines a problems?• Biofilm formation odor customer complaints• Microbial colonization in washing / dishwashingmachinesmicrobes on laundry items (odor offreshly‐washed textiles
• Biocorrosion
• Resistence against disinfectants / Cleaners / Bleachis higher in microbial biofilms than in microbialsuspensions
• Potential health risk: infections and silent spread ofresistant strains
• Disinfection (killing of the microbes) alone is not THE solution Biofilm regrows very fast on deadorganic material
Biofilm in washing machines is a problem! Biofilm control is the aim
April 2014 11
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
What about dishwashers and Biofilm formation?
April 2014 12
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
• Water / humidity• Drain, ion exchanger, pump, tubings, detergent dosing
case, rubber seal, door• consumer: handling and cleaning of dishwasher
• Temperatures between 4°C and 60°C• Higher temperatures 50 to 75°C
• Organic / Inorganic nutrients• High soil load in the drain / filter• Rubber and tubings
• Sanitization • Trends toward lower temperature• Short dishwashing cycles• water consumption
Due to the humid environment and the soil load biofilm formation must be expected as well in dishwashers
Biofilm growth in dishwasher
April 2014 13
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Is Biofilm in dishwashing machines a problems?• Biofilm formation odor• Microbial colonization in dishwashing machinesContamination from machine to dish items
• Biocorrosion
• Potential health risk: infections and silent spread ofresistant strains
Dishwashing process is more efficient againstmicroorganisms than a washing process(temperature, chemistry)
Soil level in the drain, filter is higher in thedishwasher, biofilm formation is there very likelyOdour
Machine hygiene is more relevant than thedishwashing process hygiene
Trends: lower T!!
April 2014 14
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Biofilm control
Machine cleaning cycle: Chemistry (detergent with bleach, additives) weakens biofilm matrix, high temperature kills germs, intense mechanical action detaches the live / dead biofilm material
Surfaceproperties: hydrophilic, smooth
Chemistry: Chlorine, Ozone, H2O2,
Tensides, Enzymes, Complexing agent
Physical methods: Ultrasound, UV, T, mechanical action /
Shear force
Biofilm prevention Weaken biofilm matrix, kill biofilm cells
Kill cells, remove organic matter
April 2014 15
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
How can the efficiency of biofilm control be tested?
April 2014 16
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Testing the efficiency of the biofilm control strategy
In situ Test in the washing machine• Biofilm growth test (over several weeks):
• Most consumer‐relevant, very specific
• Biofilm removal in a single cycle:• Less consumer‐relevant, model biofilm
Simulation / Labscale test system• Labscale test that simulates a washing cycle
• Model system for general investigations, not so consumer‐relevant
April 2014 17
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
In situ tests in the washing machine
Biofilm growth test• Over 12 weeks• e.g. 2 washing cycles per week, mimicking worst conditions
and bad consumer habits• Testing a frequent use of a product / cleaning cycle in
comparison with a washing machine run without the productor cleaning cycle
• After the test, washing machines are dissassembled andbiofilm on different washing machine parts characterized
• Visual: clean / organic or inorganic incrustinations• Biofilm amount: Protein‐, polysaccharide amounts,
microbial count and present microorganisms
Prevention of biofilm formation over time, biofilm controlstrategy
Most consumer‐relevant, very specific
April 2014 18
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
In situ tests in the washing machine
Biofilm removal in a single washing cycle• Biofilm ports to implementation standardized biofilms• Biofilms are grown in a bioreactor under high shear
forces• Biofilms are available of Pseudomonas aeruginosa,
Pseudomonas putida, Rhodotorula mucilaginosa andCandida albicans
• Biofilm amount before and after the washing cycle isassessed• Proteine, Polysaccharide amount• Microbial counts
Biofilm removal of single cycles like cleaning cycles orproducts
Less consumer‐relevant, Model biofilm
April 2014 19
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Labscale testing method (Impact on biofilm formation)Pseudomonas aeruginosa
Microbial growth in the solution OD595
Simulated washing test, e. g.30 min: 30°C, 100 U/min, Detergent2 x 10 min rinsing
24 h incubation at 30°C in growth medium
4 h Incubation at 30°C in medium
Microbial growth in the solution OD595
Crystal violet assay
E. coli
Candida albicans
Staphylcoccusaureus
Mixed biofilm
Quantification of growth in the suspension
Quantification of biofilm amount
Rinsing to remove planctonic cells
April 2014 20
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Biofilm tests in washing machines
Biofilm test Application Advantage Limitations result
Biofilm growthtest
Test of a specificproduct / cycle overa longer test period(min. 12 weeks)
Consumer‐relevant, veryspecific
Very specific, duration, costs, comparativetesting
‐ Long termefficiency of a product / procedure‐ Critical locations in a device
Biofilm removaltest (single cycle)
Test of a specificcycle in a machineequipped withmodel biofilms
Fast, still consumer‐relevant
Model biofilm,costs
‐ How is theefficiency of a cycle / procedure againstbiofilm in situ
Lab scale test Generalinvestigations, optimization of testconditions like productconcentration, temperature, waterhardness, soil etc
Fast, cost‐saving, high‐throughputsystem, singlespecies biofilmor multispeciesbiofilm
Model, onlylimited simulationof mechanicalaction
‐optimal concentrations ortest conditions‐general investigation
April 2014 21
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Test proposals for Dishwashers
April 2014 22
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Testing the efficiency of the biofilm control strategy
In situ Test in the Dishwasher• Contamination of the dishwasher and/or mimicking bad
consumer habits (most consumer relevant, very specific)• Aim 1: Assessment of the machine design from a
hygienic point of view• Aim 2: Long term biofilm build‐up / prevention with a
specific program / detergent / procedure• Less knowledge about critical points in a dishwasher
(drain, rubber ?)• Relevance of the results
• What does it mean e.g. if the drain is highlycontaminated relevant for crosscontaminationof dishes or surfaces that are touched or odourformation
• What is measured?
April 2014 23
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Test possibilities in Dishwashers
In situ Test in the dishwasher• Biofilm removal with a model biofilm on a
carrier• Aim 1: Assessment of the biofilm removal
efficiency of single cycles (comparativetesting!)
• Aim 2: Assessment of carry‐over rates frommodel biofilm to dishes
• Key Microorganisms in a dishwasher for a model biofilm (P. aeruginosa!)
• Where to implement the carriers with themodel biofilm (filter, drain, dish items) and withthe sterile surface
• Model versus real situation
April 2014 24
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Test possibilities in Dishwashers
Simulation / Labscale test system• Testing of dishwashing cleaners under
simulated test conditions• Simulated washing test system can be easely
adapted to dishwashing processes• Screening of products or product
concentrations• Screening of differt test conditions
• Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus as some representativesof dishwasher biofilms
April 2014 25
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Some final questions
Most crucial question: What shall be detected?• General contamination of a household device• Dangerous / dirty spots due to construction or function of the
device
• Presence and amount of potentially pathogenic germs?• Crosscontamination on items / surfaces where the consumer is
likely to get into contact with
• Efficiency of a cleaning / disinfecting procedure short term• Efficiency of a cleaning / disinfecting procedure long term
• How shall the results be used, comparative, any requirements?
April 2014 26
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Summary
• A biofilm is a highly complex community and can be diverse in household devices depending on the household, climate, culture etc.
• Biofilm formation in household devices like washing machines and dishwasher has to be expected
• Odor formation might be the most import issue
• A biofilm control strategy is needed including:• material / adhesion• combination of chemistry, T and mechanical action• Monitoring of the strategie’s efficiency
• Different methods to investigate the devices or cleaning procedures can be used
• In situ tests in the dishwasher• Lab scale tests
• Important to define what should be assessed and what the results really say
April 2014 27
Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch
Thank you for your attention!
April 2014 28