Biofilm in washing machines and how it is assessed
Caroline Amberg Swissatest testmaterials ag, St. Gallen, Schweiz
Hygiene requirements and measurements in dishwashing 1th / 2th of April, 2014, Bonn
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 1 What is a biofilm? And why is it important in the today’s discussion?
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 2 What is a biofilm: ‘Slime City’
Biofilm = Community of microorganisms embedded in a extracellular polymer matrix (EPS) • City of microbes with a complex structure • Bacteria, fungi, algae, protozoa • EPS = exopolymere substance consist of polysacharides, proteins, lipids, nucleic acids • Water is the main part of the EPS = Hydrogel • Diverse compositions depending on attached surface, germs, nutrients available etc. • Ubiquitär on interfaces with water availability • Quorum sensing = communication within biofilm bacteria
Drenkard, E. (2003) in microbes and infection Living in a biofilm is the most common way of life for microbes!
März 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 3 Quorum Sensing
März 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 4 Steps of biofilm formation
1. Conditioning and reversible attachment
2. Irreversibles Attachment
3. young Biofilm
4. mature Biofilm
5. Detachment, steady state biofilm
University of Colorado, Boulder: http://mathbio.colorado.edu
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 5 Biofilm is expected on…
• 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
Every surface is sooner or later colonized!
Source: Flemming, H.C., Heitz, E., and Sand, W. (1996). Microbially influenced Corrosion of Material. Springer Verlag, Berlin.
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 6 Biofilm sampling and analysis
Sampling: Direct investigation: Swabbing, material coupons 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 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 7 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 of analysis method(s) and its interpretation has to be done carefully and fitting to the research question!
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 8 Is it likely to have Biofilm in our household devices?
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 9 Biofilm growth in washing machines
• 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 is not totally avoidable
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 10 Is Biofilm in washing machines a problems? • Biofilm formation odor customer complaints • Microbial colonization in washing / dishwashing machines microbes on laundry items (odor of freshly‐washed textiles • Biocorrosion • Resistence against disinfectants / Cleaners / Bleach is higher in microbial biofilms than in microbial suspensions • Potential health risk: infections and silent spread of resistant strains • Disinfection (killing of the microbes) alone is not THE solution Biofilm regrows very fast on dead organic material
Biofilm in washingmachinesisa problem! Biofilm control is the aim
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 11 What about dishwashers and Biofilm formation?
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 12 Biofilm growth in dishwasher
• 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
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 13 Is Biofilm in dishwashing machines a problems? • Biofilm formation odor • Microbial colonization in dishwashing machines Contamination from machine to dish items • Biocorrosion • Potential health risk: infections and silent spread of resistant strains
Dishwashing process is more efficient against microorganisms than a washing process (temperature, chemistry) Soil level in the drain, filter is higher in the dishwasher, biofilm formation is there very likely Odour Machine hygiene is more relevant than the dishwashing process hygiene Trends: lower T!!
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 14 Biofilm control
Surface Chemistry: Chlorine, Physical methods: properties: Ozone, H2O2, Ultrasound, UV, T, hydrophilic, Tensides, Enzymes, mechanical action / smooth Complexing agent Shear force
Biofilm prevention Weaken biofilm matrix, kill Kill cells, remove biofilm cells organic matter
Machine cleaning cycle: Chemistry (detergent with bleach, additives) weakens biofilm matrix, high temperature kills germs, intense mechanical action detaches the live / dead biofilm material
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 15 How can the efficiency of biofilm control be tested?
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 16 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 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 17 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 product or cleaning cycle • After the test, washing machines are dissassembled and biofilm 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 control strategy Most consumer‐relevant, very specific
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 18 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 and Candida albicans • Biofilm amount before and after the washing cycle is assessed • Proteine, Polysaccharide amount • Microbial counts
Biofilm removal of single cycles like cleaning cycles or products Less consumer‐relevant, Model biofilm
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 19 Labscale testing method (Impact on biofilm formation) Mixed biofilm Pseudomona s aeruginosa 24 h incubation at 30°C in growth medium E. coli Staphylcoccus aureus Candida albicans Microbial growth in
the solution OD595 Simulated washing test, e. g. 30 min: 30°C, 100 U/min, Detergent 2 x 10 min rinsing Quantification of Microbial growth in growth in the the solution OD595 suspension 4 h Incubation at 30°C in medium
Rinsing to Quantification of remove biofilm amount planctonic cells
Crystal violet assay
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 20 Biofilm tests in washing machines
Biofilm test Application Advantage Limitations result
Biofilm growth Test of a specific Consumer‐ Very specific, ‐ Long term test product / cycle over relevant, very duration, costs, efficiency of a a longer test period specific comparative product / procedure (min. 12 weeks) testing ‐ Critical locations in a device Biofilm removal Test of a specific Fast, still Model biofilm, ‐ How is the test (single cycle) cycle in a machine consumer‐ costs efficiency of a cycle / equipped with relevant procedure against model biofilms biofilm in situ Lab scale test General Fast, cost‐ Model, only ‐optimal investigations, saving, high‐ limited simulation concentrations or optimization of test throughput of mechanical test conditions conditions like system, single action ‐general product species biofilm investigation concentration, or multispecies temperature, water biofilm hardness, soil etc
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 21 Test proposals for Dishwashers
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 22 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 highly contaminated relevant for crosscontamination of dishes or surfaces that are touched or odour formation • What is measured?
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 23 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 (comparative testing!) • Aim 2: Assessment of carry‐over rates from model biofilm to dishes • Key Microorganisms in a dishwasher for a model biofilm (P. aeruginosa!) • Where to implement the carriers with the model biofilm (filter, drain, dish items) and with the sterile surface • Model versus real situation
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 24 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 representatives of dishwasher biofilms
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 25 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 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 26 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 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 27 Thank you for your attention!
April 2014 Swissatest Testmaterialien AG, Mövenstrasse 12, CH‐9015 St. Gallen, swissatest.ch 28