April, 2013

Biofilm formation in washing machines

A novel labscale method to assess the impact of washing parameters on biofilm formation

Caroline Amberg,

empa testmaterials ag, St. Gallen, Switzerland

Düsseldorf, 9th of April 2013 46th International Detergency Conference

April, 2013

What is a biofilm: ‘Slime City’

• 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

Biofilm = Community of microorganisms embedded in a extracellular polymer matrix (EPS)

Drenkard, E. (2003) in microbes and infection

April, 2013

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, 2013

• 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

• Light (growth of algae or cyanobacteria)

• 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, 2013

• 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

Biofilm growth in washing machines

April, 2013

Is Biofilm in washing machines a problems?

• Biofilm formation odor customer complaints

• Microbial colonization in washing 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 (IFH Report 2011)

• Disinfection (killing of the microbes) alone is not THE solution Biofilm regrows very fast on dead organic material Biofilm in washing machines is a problem! Biofilm control is the aim Source: IFH report (2011).The infection risks associated with clothing and

household linens in home and everyday life settings, and the role of laundry

April, 2013

Solution / Ideas for Biofilm control

1. Prevention: surface properties • Hydrophilic, smooth surfaces • Dynamic surfaces (hydrophobic / hydrophilic,

negative/positive charge, …) 2. Weakening of biofilm matrix

• Chlorine, Ozone, H2O2 • Tensides • Enzymes • Komplexing agent

3. Physical methods of biofilm removal • Ultrasound • UV • Temperature • Mechanical action / shear force: Rinsing / Spinning

Washing machine cleaning cycle: Chemistry (detergent with bleach, additives) weakens biofilm matrix, high temperature kills germs, intense mechanical action detaches the live / dead biofilm material

A. Rumpf Ph.D. thesis 2009

April, 2013

Testing the efficiency of the biofilm control strategy

Biofilm growth test: • Over 12 weeks • 2 washing cycles per week • Mimicking 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 were dissassembled and biofilm on different washing machine parts characterized

• Visual:clean / organic or inorganic incrustinations

• Biofilm amount: Protein, polysaccharide amounts, microbial count and microbial community

Prevention of biofilm formation over time, biofilm control strategy

April, 2013

Testing the efficiency of the biofilm control strategy

Testing of 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

April, 2013

Testing the efficiency of the biofilm control strategy

Lab scale method for evaluating the efficiency of biofilm removal or prevention: • Growth of a young biofilm (24 h) • Chemical treatment or simulated washing tests • Fast screening of biofilm amount with the Crystal

violet assay (O’Toole, 2011)

Applications: • Chemical coatings • Washing machine cleaners • Disinfectants • Evaluation of the best concentration • Evaluation of the best washing condition • Testing for synergistic or antagonistic effects with

e.g. the detergent, water hardness etc.

April, 2013

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, Detergent 2 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

Staphylcoccus aureus

Mixed biofilm

Quantification of growth in the suspension

Quantification of biofilm amount

Rinsing to remove planctonic cells

April, 2013 Microplate reader

Biofilm quantification - Crystal violet assay (CV) (O’Toole, 2011)

15 min at RT

150 ul 0.1% CV 100 µl Suspension OD 595 nm

Wash biofilm with 3x sterile H2O hard / entmineralized water

Wash with 3x sterile H2Odest

125 µl in a new plate OD 560 nm or 550 nm

air dry

Incubation 15 min at RT

150 µl 30% acetic acid

Beaten on a stack of paper, Dried upside down

April, 2013

Example: Test approaches Washing machine cleaner A

Positive controls No washing tests

Negative Controls: no test germs washing test no washing tests

A concen-tration x

A concen-tration y

Concen-tration x + detergent

Concen-tration y + detergent

A concen-tration x

A concen-tration y

Concen-tration x + detergent

Concen-tration y + detergent

Example for testing different product concentrations

April, 2013

Example for testing different product concentrations and applications (single washing test)

-0.5

0

0.5

1

1.5

2

2.5

3

3.5

cleaner A, concentration 5%

cleaner A, concentration10 %

cleaner A, concentration 5% + detergent

cleaner A, concentration10 % + detergent

positive control, nowashing tests and

treatment

OD

at w

avel

enth

550

nm

• The example showed that a mixed biofilm was fully removed in the approach with 10 % concentrated washing machine cleaner A in combination with an added detergent.

• 5 % concentrated cleaner A was already able to lower the biofilm

amount

April, 2013

Advantages / Limitations of labscale protocol

Advantages Limitations The system is very flexible: • Testmicroorganisms (single strains or mixed

biofilm), Pseudomonas aeruginosa, E. coli, Staphylococcus aureus, Candida albicans and mixed biofilm

• Single washing cycles can be simulated • Repeated washing cycles can be simulated • Regrowth of biofilm after a treatment can be

simulated

Model-biofilms, natural biofilm are complexe and changing structures

A lot of different conditions can be tested in one test approach like washing temperatures, products, concentrations, combination, water hardness, soil etc.

Mechanical action can not fully be simulated

Fast and cost saving screening approach It can not fully replace in situ washing tests

April, 2013

Summary • Biofilm formation is a direct consequence of insufficient

process hygiene • Odor formation due to biofilm in washing machines is a

problem

• Biofilm formation can not be completely prevented, a biofilm control strategy is needed including:

• material / adhesion • combination of chemistry, T and mechanical action • Monitoring of the strategie’s efficiency

• A biofilm control strategy should be monitored for its

efficiency • No test protocols are available for testing the efficiency of

washing machine cleaners / disinfectants against biofilms • EMPA Testmaterials has developed a new screening

method to test biofilm removal / preventation in a labscale test.

• In situ tests are needed to finally control the strategy from a systemic point of view

April, 2013

Thank you for your attention!