Debugging Biofilms and Superbugs

Transcription

1 Debugging Biofilms and Superbugs Timothy Lu, M.D., Ph.D. Assistant Professor Synthetic Biology Group Research Lab of Electronics Department of Electrical Engineering and Computer Science Massachusetts Institute of Technology March 15, 2011

2 Biofilms are the Natural Shelter for Bacteria Biofilms are composed of bacterial cells (the fruit) and extracellular slime matrix (the Jello) Microbe 2(7):350,

3 Biofilms Infect Clinical and Industrial Surfaces Dental infections Cystic fibrosis Corrosion and biofouling Food contamination Catheter/medical device infections & & &

4 Imperfect Approaches Against Biofilms Mechanical disruption Chemical treatment Sonication Antibiotics & &

5 Losing the Antimicrobial Arms Race

6 History, Problems, and Solutions for Bacteriophage Therapy Poor efficacy against biofilms Synthetic modules expressing biofilm-degrading enzymes Bacteriophage resistance Synthetic modules to potentiate co-administered antimicrobials Host specificity Historically, many poorly controlled trials Biocontrol recently completed Phase I/IIb against P. aeruginosa Neurophage is in pre-clinical i l trials against Alzheimer s disease Products made by Omnilytics (Agriphage) and Intralytix (ListShield, EcoShield) Generally Recognized as Safe (GRAS) by FDA/EPA Immune clearance Phage capsid evolution, PEGylation to avoid immune clearance Topical, gastrointestinal, environmental applications

7 What are in Biofilms? Biofilms contain bacteria enclosed in a 3D matrix of: β-1,6-n-acetyl-d-glucosamine 16N d Cellulose Alginate Colanic acid Functional amyloid fibers Extracellular DNA Whitchurch et al., Science 2002

8 Pre-Grown Biofilm is Dispersed by Treatment with DspB Itoh et al., J. Bacteriol. 2005

9 Biofilm-Degrading Enzymes and Bacteriophage Biofilm-Degrading Enzymes Natural Bacteriophage Degrade biofilm slime Viruses that kill bacterial cells Do not infect human cells Self-replicating in bacteria Easy to produce and deliver Do not kill bacterial cells Expensive to produce and purify Difficult to deliver Do not penetrate biofilms well Lu, Collins PNAS 2007

10 Combining Enzymes and Bacteriophage DNA module expresses biofilmdegrading enzyme + Natural bacteriophage Enzymatic bacteriophage Kill bacterial cells Self-replicating Easy to produce and deliver Degrade biofilm slime Penetrate into biofilms Lu, Collins PNAS 2007

11 Engineered Enzymatically-Active Phage Initial infection Bacteriophage replication + Biofilm-degrading enzyme expression Degradation of biofilm slime Continued infection Trojan Horse with positive feedback loop Lu, Collins PNAS

12 Untreated Biofilms Lu, Collins PNAS 2007

13 Treatment with Unmodified Phage Lu, Collins PNAS 2007

14 Treatment with Engineered Enzymatic Phage Lu, Collins PNAS 2007

15 Efficient Biofilm Removal by Enzymatic Phage >99.997% killing Initial bacteriophage dose = 1000 and shown to replicate rapidly Lu, Collins PNAS 2007

16 Enzymatic Phage Effective at Low Doses Lu, Collins PNAS 2007

17 Diverse Antibiotic Resistance Mechanisms DNA Damage Mutations Kohanski et al. Cell 130:797, 2007.

18 Targeting Bacterial Defense Networks A One-Two Punch : attack bacteria from multiple angles with phage as antibiotic potentiators Lu, Collins PNAS 2009

19 Potentiating Phage Enhance Killing for 3 Antibiotic Classes Ofloxacin Gentamicin i Ampicillin illi Lu, Collins PNAS 2009

20 Potentiating Phage Revive Antibiotics That Don t Work Ofloxacin-Resistant E. coli Lu, Collins PNAS 2009

21 Potentiating Phage Suppress Antibiotic Resistance Assay: Generate antibiotic-resistant mutants for 12 h with subinhibitory oflox, then treat for 12 h with phage + oflox. Plate on 100 ng/ml oflox. Lu, Collins PNAS 2009

22 Potentiating Phage Rescue Mice from Death Lu, Collins PNAS 2009

23 Potentiating Phage are Effective at Many Doses Lu, Collins PNAS 2009

24 Targeting the Superoxide Regulatory Gene Network Lu, Collins PNAS 2009

25 Targeting Biofilm Regulatory Gene Networks Alone Lu, Collins PNAS 2009

26 Targeting Porins Expression Alone Lu, Collins PNAS 2009

27 Targeting Biofilm Networks and Porins in Combination Lu, Collins PNAS 2009

28 Paradigm Shift for Next-Generation Antimicrobial Agents Old Paradigm New Paradigm Antibio otic Resist tance Time Antibio otic Resist tance Time New New Rationally Designed Antibiotic Antibiotic Antimicrobial Agents Lu, Collins PNAS 2009

29 Target Identification and Selection with Systems Biology Di Bernardo, Nature Biotechnology, 2007.

30 Acknowledgements Lu Lab Members Yuyin Chen (MD/PhD HMS) Ramez Danial (Postdoc EECS) Allen Cheng (MD/PhD HMS) Samuel David Perli (PhD EECS) Jacob Rubens (PhD Microbiology) Chris Yoon (UROP ChemE) Jessica Perez (UROP BE) Many CSBi and Microbiology rotation students Collaborators Susan Lindquist (WI/MIT) Rajaraman Krishnan (WI/Neurophage) Jessica Goodman (WI/MIT) Charles O Donnell (MIT) Srini Devadas (MIT) Bonnie Berger (MIT) James Collins (BU) MIT Center for Integrative Synthetic Biology

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