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This material is copyright © 2018 by the author/home institution as

specified on the title slide of this presentation. All rights reserved.

This material was prepared for the February 2018 “2nd Annual

Food Science and Microbiology Conference” (February 27, 2018),

presented by the Center for Biofilm Engineering at Montana State

University, Bozeman, Montana 59717-3980, and may represent

information that has not been published, has not been peer

reviewed, or is preliminary. Reproduction or presentation of this

material is prohibited without the express consent of the author(s).Diane K. Walker

Research Engineer

Center for Biofilm Engineering

Montana State University

Center for Biofilm Engineering

FSMC Dallas, TX| Feb 2018

Biofilmsfrom

Formation to

Elimination

©2016 MSU-CBE, L. Lorenz

Center for Biofilm Engineering

Introduction to the

Center for Biofilm Engineering

at Montana State University

Bozeman

Bill Characklis Bill Costerton

Founded 1990

NSF-ERC

Phil Stewart

Matthew Fields

Center for Biofilm Engineering OVERVIEW

Center for Biofilm Engineering

▪ Medical Biofilms

▪ Environmental Biofilms

▪ Microbial Ecology & Biogeochemistry

▪ Biofilm Control & Genetics

▪ Physiology & Ecology

▪ Subsurface Biotechnology, Bioremediation & Souring

▪ Standardized Biofilm Methods

CBE Laboratories:

CBE Industrial Associates (Feb. 2018)

Consumer Products

Church & Dwight

CleanSpot

Masco

Procter & Gamble

SC Johnson & Son

Sherwin Williams

Testing Labs

Accuratus Lab Services

Specialty Chemicals

American Chemet

BASF

Dow Microbial Control

Ecolab/Nalco

Lonza

Medentech

NCH Corp.

PPG Industries

Solvay

Sterilex

US Gov’t Programs/Labs

NASA

Health Care/Biomedical

3M

Baxter Healthcare

Boston Scientific

ICU Medical

Next Science

Sanuwave Health

Sharklet Technologies

Smith & Nephew

STERIS

Zimmer Biomet

Food Safety

DeLaval

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CBE Membership: The Industrial Associates Program

• Industrially relevant research

• Direct sponsorship of

research and testing projects

• Education and training

workshops

• Montana Biofilm Science and

Technology Meetings (MBM)

• Regulatory interactions

Contact: paul_s@montana.edu

Center for Biofilm Engineering

What are biofilms?

Kelli Buckingham-Meyer, SBML

Biofilms are a self-organized, cooperative

community of microorganisms embedded in a

matrix of extracellular polymeric substances (EPS).

Center for Biofilm Engineering

Where are biofilms found?

Natural Environments & Man-Made Structures

Photos by CBE staff and students unless otherwise noted.

The Medical Arena

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Surfaces Around The Home

wjonespainting.com acrylicbath.com

tshirts.com

Broadwaycarpet.co.uk

Food and Food Processing Surfaces

zehabesha.com

imagesfrompo.com

flickr.com

amazon.com

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Center for Biofilm Engineering

How are biofilms formed?

Center for Biofilm Engineering

How are biofilms formed in the lab?

Biofilms are Engineered

Fig 1. CDC reactor lid.

▪ Laboratory reactors are the tools

researchers use to generate

biofilms that exhibit particular

qualities

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▪ Flow regime Re=Dνρ/µ▪ Shear forces▪ Residence time RT=V/Q▪ Material choices▪ Chemical compatibility

Engineering Concepts

acnosite.blogspot.com

Reactor Systems

▪ Batch reactor (closed system)

▪ Plug flow reactor (open system)

▪ Continuously-stirred tank reactor (open system)

▪ Static biofilm

▪ Flask reactor

Batch Reactors

▪ Microtiter plates▪ MBEC device▪ Colony biofilm

▪ Drip flow reactor

▪ Porous media column

Plug Flow Reactors

▪ Flow cell▪ Tubing (i.e., beverage lines)▪ Modified Robbins device

▪ CDC biofilm growth reactor

▪ Rotating disk reactor

CSTR

▪ Annular reactor▪ Constant depth

film fermenter

Static Biofilm Drip Flow Biofilm CDC Biofilm

No Shear Low Shear High Shear

Fluid shear is important

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Center for Biofilm Engineering

Standardized Biofilm Methods Lab

The creation, establishment and transfer

of quantitative biofilm methods for the

benefit of academia, government and

industry.

SBML Mission

▪ The goal when designing or selecting a laboratory system for estimating real world observations is to find the proper balance between field relevancy and practicality while achieving the statistical specifications required of a standard method.

The Goal

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Food and Food Processing Surfaces

zehabesha.com

imagesfrompo.com

flickr.com

amazon.com

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Grow AnalyzeTreat SampleASTM Biofilm Methods

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ASTM Biofilm Methods ASTM Standard Method E2196

▪ Continuous stirred tank reactor

▪ Grows biofilm under moderate shear

Rotating Disk Reactor

Pseudomonas aeruginosa

Polycarbonate coupons

Tryptic soy broth: 300 mg/L INB & BNB,

30 mg/L CFNB – All 24 hr

200 rpm

6.7 mL/min

20oC

ASTM Standard Method E2647

▪ Continuous stirred tank reactor

▪ Grows P.a. biofilm under high shearBacterial air vent

Reactor top

Berzelius Pyrex

beaker

Polypropylene rod

Coupon

Baffled stir bar

Effluent spout

Media inlet/inoculation port

Set screws

L. Lorenz, S. Goeres 2007

CDC Biofilm Reactor

Pseudomonas aeruginosa

Polycarbonate coupons

Tryptic soy broth: 300 mg/L INB & BNB,

100 mg/L CFNB – All 24 hr

125 rpm

11.7 mL/min

20oC

ASTM Standard Method E2647

▪ Plug flow reactor

▪ Grows biofilm under low shear

▪ Air/liquid interfaceDrip Flow Reactor

Pseudomonas aeruginosa

Glass microscope slides

Tryptic soy broth: 3000 mg/L INB – 24 hr

3000 mg/L BNB – 6 hr

270 mg/L CFNB – 48 hr

10° angle

0.8 mL/min/channel

20oC

ASTM Standard Method E2799

▪ Batch reactor with gentle mixing

▪ Grows biofilm under very low shear

▪ Easily implemented in a standard

microbiology laboratory

▪ Useful when only a small volume of the test

compound is available

▪ Suitable for screening multiple bugs and/or

antimicrobials at a range of concentrations

MBEC Assay

P. aeruginosa

Tryptic soy broth:

Full strength INB – 24 hr

Dilute for growth

110 rpm

36oC

GrowTreat

MBEC Assay

1 2 3 4 5 6 7 8 9 10 11 12

A 100 100 100 100 100 50:N N UC SC

B 50 50 50 50 50 50:N N UC SC

C 25 25 25 25 25 50:N N UC SC

D 12.5 12.5 12.5 12.5 12.5 50:N N UC PC

E 6.25 6.25 6.25 6.25 6.25 50:N N UC PC

F 3.125 3.125 3.125 3.125 3.125 50:N N UC PC

G 1.5625 1.5625 1.5625 1.5625 1.5625 50:N N UC PC

H 0.7813 0.7813 0.7813 0.7813 0.7813 50:N N UC PC

▪ # = % Antimicrobial

▪ N = Neutralizer

▪ UC = Untreated controls

▪ SC = Sterility controls

▪ PC = Peg controls

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ASTM Standard Method E2871 ASTM Standard Method E2871

Single Tube Method

P. aeruginosa ATCC 15442

Borosilicate glass coupons

Sonicate/vortex vs

Scrape/homogenize

Methods…with benefits

▪ Teaching tools

▪ Communication

▪ Comparison

▪ Product registration

REGULATORY

INDUSTRYACADEMIA

Industrial

Associates

Collaboration

▪ Reduces/controls biofilm bacteria

(low level of efficacy)

▪ Kills biofilm bacteria (high level of efficacy)

▪ Prevents bacterial biofilm

▪ Removes bacterial biofilm

Biofilm Label Claims Center for Biofilm Engineering

How do you get rid of biofilms?

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MSU Center for Biofilm Engineering

Effect of 65°C - 80°C water on planktonic cells

70°C

80°C

MSU Center for Biofilm Engineering

Effect of 65°C-80°C water on biofilm cells

70°C

80°C

Disinfectant efficacy depends upon how the biofilm was grown

CDC reactor

CDC reactor (dried)

Drip flow reactor

Static biofilm reactor

Dried surface bacteria

Fluid shear is important ASTM Standard Method E2647

▪ Continuous stirred tank reactor

▪ Grows P.a. biofilm under high shearBacterial air vent

Reactor top

Berzelius Pyrex

beaker

Polypropylene rod

Coupon

Baffled stir bar

Effluent spout

Media inlet/inoculation port

Set screws

L. Lorenz, S. Goeres 2007

CDC Biofilm Reactor

Pseudomonas aeruginosa

Polycarbonate coupons

Tryptic soy broth: 300 mg/L INB & BNB,

100 mg/L CFNB – All 24 hr

125 rpm

11.7 mL/min

20oC

Treatment Flow Cell Treatment Flow Cell Under Microscope

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Movie: Alcohol/Quat blend (undiluted) Movie: Phenolic disinfectant (1:16)

Movie: Chlorine (1:20) Center for Biofilm Engineering

Food & Beverage Biofilms

Food Journals Food Growth Conditions

▪ Well plates• mixing• stagnant

▪ Surface deposition• dry• humid

▪ Coupon submersion• mixing• stagnant• fresh media• without transfer• partial (interface)

▪ Other

27%

21%

45%

1%

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Fig. 1. A schematic representation of the rotating disk system (RDS) used in the

present work. Sketch illustrating experimental setup.

Brugnoni et al. (2011) Role of shear stress on biofilm formation of Candida krusei in a

rotating disk system. Journal of Food Engineering 102:266-271.

Food Growth Conditions

FIGURE 1. Experimental system used to model the development of dairy biofilms on SS surfaces. The system consisted of a

continuous flow reactor and a recirculating test loop containing the SS tubes. Milk held at 4oC was heated to 50oC and pumped

into the reactor at a dilution rate that was greater than the growth rate of the bacterial species in the milk. Milk was pumped

around the recirculating test loop at a flow rate of 1.5 m s-1.

Dufour et al. (2004) Development of a laboratory scale clean-in-place system

to test the effectiveness of “natural” antimicrobials against dairy biofilms.

Journal of Food Protection 67:1438-1443.

Food Growth Conditions

Martin et al. (2016) Efficiency of a cleaning protocol for the removal of enterotoxigenic

Staphylococcus aureus strains in dairy plants. International Journal of Food Microbiology

238:295-301.

ASTM Standard Method E2562 Modifications

Pseudomonas aeruginosa ATCC 700888 Staphylococcus aureus (4 dairy isolates)

Polycarbonate coupons Stainless steel & polypropylene coupons

TSB UHT milk; fat content = 3.0%

24 hr 3 hr, 6 hr, 12 hr, C&S

125 rpm 1600 rpm during C&S

11.67 mL/min Not stated

Room Temperature 5oC and 35oC

300 mg/L INB

300 mg/L BNB

100 mg/L CFNB

Food Growth ConditionsMartin et al. (2016) Efficiency of a cleaning protocol for the removal of enterotoxigenic

Staphylococcus aureus strains in dairy plants. International Journal of Food Microbiology

238:295-301.

Food Growth Conditions

Dairy biofilms

ASTM Standard

Method E2562

Modifications

for S. aureus

Modifications

with milkPseudomonas aeruginosa

ATCC 700888

Staphylococcus aureus

ATCC 6538

Staphylococcus aureus

ATCC 6538

Polycarbonate coupons Borosilicate glass coupons Stainless steel coupons

TSB TSB Skim milk

24 hr 24 hr 24 hr

125 rpm 60 rpm 60 rpm

30 min residence time 30 min residence time 30 min residence time

Room Temperature 36oC Room Temperature

300 mg/L INB300 mg/L BNB100 mg/L CFNB

10% INB10% BNB1% CFNB

30 g/L INB30 g/L BNB3 g/L CFNB

ASTM E2562 Modifications

Future modifications for method optimization:

▪ Growth temperature: 36oC

▪ Duration: 48-72 hr

▪ Supplement milk: TSB

▪ Condition system with milk: grow in TSB

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Listeria biofilms

ASTM Standard

Method E2562Modifications

Pseudomonas aeruginosa Listeria monocytogenes

300 mg TSB/L (Inoculum &

Batch)2 g TSB/L (Inoculum & Batch)

100 mg TSB/L (CF) 2 g TSB/L (CF)

11.7 mL/min 5.35 mL/min

125 rpm 60 rpm

24 hour CF 72 hour CF25 x 25 x25 x

Sometimes the reactor you may want to use or

already own is not the best choice for the bacteria

you are trying to grow.

Fluid shear is important

Kelli Buckingham-Meyer

ASTM E2647 Modifications

ASTM Standard

Method E2647Modifications

Pseudomonas aeruginosa Listeria monocytogenes

Polycarbonate coupons Stainless steel coupons

TSB TSBYE or BHI or ?*

48 hr 72 hr?

10o angle 5o angle?

0.8 mL/min/channel 0.4 mL/min/channel?

Room temperature Optimal range 30-37oC

1:100 INB1:100 BNB1:300 CFNB

1:100 INB1:100 BNB1:300 CFNB

*Jarvis et al. (2016) Food Control 66:256-269

Ben Klayman

200 mm

1 mm

Green is gfp P. aeruginosa PA01 Red is dsRed E.coli 0157:H7

T = 72 hrs

Klayman BJ, Volden PA, Stewart PS, Camper AK, "Escherichia coli O157:H7 requires colonizing partner to adhere and persist in a capillary flow cell," Environ Sci Technol 2009; 43(6):2105–2111

Pathogen Survival in Biofilm

Modifications for mixed species including L.p.

Bacterial air vent

Reactor top

Berzelius Pyrex

beaker

Polypropylene rod

Coupon

Baffled stir bar

Effluent spout

Media inlet/inoculation port

Set screws

L. Lorenz, S. Goeres 2007

CDC Biofilm Reactor

Pseudomonas aeruginosa

Klebsiella pneumoniae

Flavobacterium spp.

Legionella pneumophila

Hartmannella vermiformis

Stainless steel coupons

TSB

24 hr (base biofilm)/4 d after L.p. addition

125 rpm

11.7 mL/min

30oC

▪ Selective media

PIAR2A w/ & w/o chloramphenicolBCYE w/GPAV

Biofilms in Beer Draught Lines

L. Lorenz, CBE 2017

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Long Draw Draught System: Clean-In-Place Schematic Overview

Dispense line & faucet

Keg coupler

Beer manifolds

Gas manifolds

CO2 tank & regulator

Dispense line coupler

Kegerator

Credit: Brewers Association & New Belgium Brewing

Beer Lines: 3 Tubing Types

Credit: Brewers Association & New Belgium Brewing

Coupler/Simulated Keg

5/16” vinyl

tubing (5 ft.)

5/16” PET

tubing (15 ft.)

3/16” vinyl

tubing (3 ft.)

Faucet

A B C

Shank and faucet

Pre-conditioning the Draught System

Cleaning canister used to

pre-condition lines with

3% NaOH.

Lines were rinsed with tap

water, then beer.

Draught System: Inoculation Pouring Beer

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Draught System: Sampling Draught System: Sampling

Bacteria enumerated on Universal Beer Agar with

cycloheximide.

Yeast enumerated on WL Nutrient Agar.

▪ Biofilms impact many industries▪ Challenging to eliminate▪ Lab biofilms are engineered▪ Standard biofilm methods help industry and

regulatory agencies

Summary

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Standardized Biofilm Methods Laboratory

Darla Goeres Al Parker Lindsey Lorenz

Diane Walker Paul Sturman Kelli Buckingham-Meyer

Standardized Biofilm Methods Laboratory

Fei San Lee, Maddie Mettler, Jontana Allkja

Lisa Bowersock, Dave Baker

Jennifer Summers

▪ Develop and standardize biofilm methods

▪ Conduct biofilm testing

?

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Sponge ?

Sink Drain ?

Toothbrush ?

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Showerhead Biofilms in Premise Plumbing

L. Lorenz, CBE 2017

Soap Dispenser Biofilms The End