biofilmin washingmachines and howit is assessed · biofilmin washingmachines and howit is assessed...

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


What is a biofilm? And why is it important in the today’s discussion?

April 2014 2


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

März 2014 3


Quorum Sensing

März 2014 4


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


• 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


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


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


Is it likely to have Biofilm in our household devices?

April 2014 9


• 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


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


What about dishwashers and Biofilm formation?

April 2014 12


• 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


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


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


How can the efficiency of biofilm control be tested?

April 2014 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 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 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


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


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


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


Test proposals for Dishwashers

April 2014 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 highlycontaminated relevant for crosscontaminationof dishes or surfaces that are touched or odourformation

• What is measured?

April 2014 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 (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


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


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


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


Thank you for your attention!

April 2014 28

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