in-vitro assessment of anti-biofilm technologies · center for biofilm engineering garth james...
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Center for Biofilm Engineering
Garth JamesAssociate Research Professor
Department of Chemical & Biological EngineeringMontana State University
FDA/CBEBiofilmWorkshop|February2014
In-vitro assessment ofanti-biofilm technologies
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In‐vitroBiofilmModels96-well Plate MBEC
CDC Reactor
Drip Flow Reactor
Colony Model
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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Samuelson, Pulcini, and James – unpublished data
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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
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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.
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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.
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DripFlowReactor(DFR)
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6‐ChannelDFR
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Hydroxyapatite‐coatedglassslides
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Titaniumslides
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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
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Antibiotictreatments– MRSA DFRbiofilm
Treatmenttime
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S.aureus(MRSA)DFRbiofilm
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Biofilmkill
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Biofilmkillandremoval
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CDCBiofilmReactor
Single species Pseudomonas aeruginosa Staphylococcus aureus Streptococcus mutans Gardnerella vaginalis Candida albicans
Mixed species Saliva
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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
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Mouthrinsetesting
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TreatmentC(salinecontrol)
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TreatmentY
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Catheterlumentesting
Artificial urine
Flow breakValve
Foley catheterPump
Valve
Vent
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CentralvenouscathetertestingExtraluminal surface
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CaseStudy
Antimicrobial central venous catheterCHSS11st generation Introduced in 1990Chlorhexidine and silver sulfadiazineOuter surface of catheter treated
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In‐vitrocathetertestingStaphylococus aureus
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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%
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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
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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
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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