Center for Biofilm Engineering

Garth JamesAssociate Research Professor

Department of Chemical & Biological EngineeringMontana State University

FDA/CBEBiofilmWorkshop|February2014

In-vitro assessment ofanti-biofilm technologies

In‐vitroBiofilmModels96-well Plate MBEC

CDC Reactor

Drip Flow Reactor

Colony Model

S.aureusproteinexpressionModel Proteins

Up ExpressedProteins Down Expressed

Percent Difference*

96 Well 3 10 17%

Colony 13 16 37%

DFR 22 11 44%

Total 38 37

*Percent difference = (Up expressed + Down expressed) x 100Total number of proteins for all models

Samuelson, Pulcini, and James – unpublished data

LogReduction(LR) Plate count technique Colony forming units (CFU, ~ # bacteria) Viable (culturable) bacteria Log (10) transformationLog CFU control - Log CFU test = LR(e.g. saline treated – antibiotic treated)

1 LR = 90% kill 2 LR = 99% kill 3 LR = 99.9% kill 4 LR = 99.99% kill

BiofilmMaturity– DFR

Wolcott RD, Rumbaugh KP, James G, Schultz G, Phillips P, Yang Q, Watters C, Stewart PS, and SE Dowd (2010) J. Wound Care 19:320–328.

BiofilmMaturity– DFR

Wolcott RD, Rumbaugh KP, James G, Schultz G, Phillips P, Yang Q, Watters C, Stewart PS, and SE Dowd (2010) J. Wound Care 19:320–328.

DripFlowReactor(DFR)

6‐ChannelDFR

Hydroxyapatite‐coatedglassslides

Titaniumslides

DFRBiofilmsSingle species Pseudomonas aeruginosa Staphylococcus aureus Staphylococcus epidermidis Propionibacterium acnes Enterococcus faecium group B Streptococcus

Mixed species 9 species oral biofilm model Saliva Wound sample homogenate

Antibiotictreatments– MRSA DFRbiofilm

Treatmenttime

S.aureus(MRSA)DFRbiofilm

Biofilmkill

Biofilmkillandremoval

CDCBiofilmReactor

Single species Pseudomonas aeruginosa Staphylococcus aureus Streptococcus mutans Gardnerella vaginalis Candida albicans

Mixed species Saliva

CDCBiofilmReactor– Oralmodel

Hydroxyapatite coupons Saliva inoculum 10% Strength tryptic soy

broth+ 0.5% sucrose+ 7.5 mg/l amphotericin B

37°C 6 hours batch 18 hours flow

Mouthrinsetesting

TreatmentC(salinecontrol)

TreatmentY

Catheterlumentesting

Artificial urine

Flow breakValve

Foley catheterPump

Valve

Vent

CentralvenouscathetertestingExtraluminal surface

CaseStudy

Antimicrobial central venous catheterCHSS11st generation Introduced in 1990Chlorhexidine and silver sulfadiazineOuter surface of catheter treated

In‐vitrocathetertestingStaphylococus aureus

AnimalTestingofCHSS1Catheters

Study Model Colonization

Bach et al 1993 Rat - subcutaneous Standard – 100% (3 & 7 days)CHSS1 – 90% (3 days)CHSS1 – 40% (7 days)

Bach et al 1994 Rat - intravenous Standard – 100% (3 & 7 days)CHSS1 – 30-100%

Sampath et al 1995 Rat - subcutaneous Standard – 100%CHSS1 – 19%

ClinicalTestingofCHSS1CathetersStudy Number of CVC % Colonized Odds ratio

CHSS1 CVC Standard CVC

Bach et al (1996) 116 vs 117 18.1 30.8 0.51Ciresi et al (1996) 124 vs 127 10.9 12.1 0.84Collin (1999) 98 vs 139 2.0 16.5 0.21Hannan et al (1999) 174 vs 177 27.2 40.2 0.56Heard et al (1998) 151 vs 157 39.7 51.6 0.62Maki et al (1997) 209 vs 195 13.5 24.1 0.50Osma et al (2006) 64 vs 69 21.9 20.3 1.10Jaeger et al (2005) 51 vs 55 9.8 16.4 0.57Sheng et al (2000) 113 vs 122 7.1 20.5 0.36Tennenberg et al (1997) 137 vs 145 5.8 22.1 0.26Van Heerden et al (1996) 28 vs 26 14.3 38.5 0.29Marik et al (1999) 36 vs 39 19.4 25.2 0.62Overall 0.51

Meta-analysis: Casey et al. (2008) Lancet Infect Dis 8:763-776

ClinicalTestingofCHSS1CathetersStudy Number of CVC % CRBSI Odds ratio

CHSS1 CVC Standard CVCBach et al (1996) 116 vs 117 0 2.6 0.13Ciresi et al (1996) 124 vs 127 8.7 8.1 1.03Collin (1999) 98 vs 139 1.0 2.9 0.41Hannan et al (1999) 174 vs 177 0.6 1.7 0.37Heard et al (1998) 151 vs 157 3.3 3.8 0.86Logghe et al (1997) 338 vs 342 5.0 4.4 1.15Maki et al (1997) 209 vs 195 1.0 4.6 0.25Osma et al (2006) 64 vs 69 6.3 1.4 3.73Pemberton et al (1996) 32 vs 40 6.3 7.5 0.83Jaeger et al (2005) 51 vs 55 2.0 14.5 0.20Sheng et al (2000) 113 vs 122 0.8 1.6 0.55Tennenberg et al (1997) 137 vs 145 3.6 6.2 0.58Marik et al (1999) 36 vs 39 2.8 5.1 0.56Overall 0.68

Meta-analysis: Casey et al. (2008) Lancet Infect Dis 8:763-776

Conclusions

A variety of in vitro biofilm models are available Models differ with respect to protein

expression by the biofilms Antimicrobial tolerance increases with age of

the biofilm An example device has shown anti-biofilm

efficacy in vitro, in vivo (animal studies) and in randomized clinical trials