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NEW 3D PRINTED BIOFILM MODELS FOR STUDYING MULTISPECIES BACTERIAL COMMUNITIES

Mitch Sanders, PHD and Lindsay Poland, MS Drug & Device Discovery Lab

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INTRODUCTION

Unlike free-floating planktonic bacteria that are quite

resistant to antibiotics and antimicrobials (such as

chlorhexidine (CHG) and nano crystalline silver), biofilms

are polymicrobial bacterial communities that are more

resistant to mechanical shear, antibiotics, and

antimicrobials.

Biofilms consist of a close network of bacteria that are

tethered together with a slime-like matrix mostly

consisting of exopolysaccharides, proteins, and nucleic acids (referred to as the EPS). This

dense community of bacteria has multiple layers with the top layer shedding active

planktonic-like bacteria while the deeper layers are more senescent (no longer capable of

dividing but still alive, from Latin: senescere, meaning "to grow old”).

3D BIOFILM MODELS

The challenge with testing antimicrobials with planktonic bacteria is that most culture

models do not reflect the complex molecular determinants that mediate quorum

sensing, sporulation, and other adaptive phenotypes that are representative of a true

biofilm. Several labs have made great strides in creating biofilm models using

bioreactors and cartridge-like drip models. In our experience, these models fail to set up

robust biofilms that are as durable as those developed in vivo. Imagine just for a

moment the biofilms and plaque that build up on our teeth while sleeping overnight or

the biofilm that grows on your pet’s water dish in less than 24 hours when you forget to

change the dish. Another example is the biofilm in a chronic wound that is resistant to

most antibiotics and antimicrobials including bleach solution.

In 2013, Connell and colleagues at the University of Texas demonstrated that they could

use a 3D printer to study bacterial communities. We have used this approach at 3DL

with an experimental 3D printer to establish polymicrobial biofilms that are more robust

and reproducible that can be tested both in vitro and in vivo in a modified mouse model.

The 3D printed biofilm models are much more consistent in terms of the amount of

protein and bacteria dispensed that can provide for more uniform replicates that have

less standard deviation of error than those established from the other well-

characterized biofilm models described below.

KEY POINTS

Every biofilm model

has it pitfalls and

strengths.

Use at least two

models to validate

the efficacy of your

antimicrobial

therapy.

3DL can provide

robust biofilm

models to accelerate

your pre-clinical

development.

Figure 1 Atomic Force Microscopy (AFM): This is an image of a bacterial biofilm of Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pyogenes

Figure 2 This 3D Printer is used in printing complex bacterial communities. A separate white paper will be submitted to Wound Repair and Regeneration demonstrating the validity of the model. We use a Makerbot 2x replicator configured with a high precision (NE 1000) syringe pump configured with a thermo-kinetic heat clamp to form bacterial biofilms.

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CDC BIOREACTOR MODEL

The CDC bioreactor model is a well-established continuous flow model

for forming multi-colony biofilms that was developed by Donlan et al.,

(2004) in the CDC Biofilm Lab. This model is well suited for microscopy

because the coupon material can be punched out of the slides so that

several replicates can be obtained for each sample condition. A typical

reactor has multiple polypropylene coupon holders suspended from

the support lid. Liquid growth media/biocide/etc. is circulated through

the chamber, while the liquid is mixed by a magnetic stir bar to

generate mechanical shear. More recent studies indicate that

coupons made of polyetheretherketone (PEEK) material can set up

more durable bio-films (Williams et al., 2011).

STIRRED CELLS AND DRIP CELLS

Stirred and drip cells are other examples of continuous biofilm models that

were developed to account for the mechanical shear forces that drive the

formation of more stable biofilms developed at Montana State University,

(MSU, Bozeman, MT: Herigstad et al., 2001). The stirred cells have the

benefit of a well-established EPS matrix and biofilm while removing the

preponderance of planktonic bacteria. The Center for Biofilm Engineering

(CBE) at MSU continues to be one of the leading institutes in studying

biofilm models. CBE hosts a variety of symposia and workshops on

biofilms both for industry and academia alike.

SKIN EXPLANT MODELS

UV sterilized porcine skin was developed as a biofilm model by Greg Schultz’s lab demonstrating

that a saline rinse retained 109 bacteria but hydro debridement reduced the bio-burden to 104

CFU/g (Yang et al., 2013). The explant model has shown to be a useful model to set up biofilms

in under 7 days for P. aeruginosa but > 7 days for S. aureus. The porcine skin is a reasonable

surrogate for the mouse model described below given that the data is comparable to the mouse

model but less expensive. Critics of this porcine skin model

suggest that it does not reflect human skin and there is no

immune response. However, Schultz and colleagues have

demonstrated that this model can produce biofilms that

are quite robust and even resistant to treatment with high

concentrations of antimicrobials and even chlorine bleach.

Figure 3 CDC Type Bioreactor: This continuous flow vessel has room for 6 channels that can be processed simultaneously and can be used with a plethora of material types, including plastics, metals, and ceramics.

Figure 4 A stirred cell bioreactor allows for the formation of robust biofilms. This model system has the ability to run multiple coupons ille tempore. Other variations include a rotating disk that uses centrifugal shear forces to set up robust biofilms.

Figure 5 Porcine skin is ideal for establishing host pathogen binding studies, less variable than the in vivid model.

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Biofilm Models

Figure 6 Contact Mitchell.sanders@cmc-co.net for pricing inquiries on Biofilm Models.

MOUSE MODELS

Mouse biofilm models allow researchers to study how

biofilms can stall wound healing in normal and diabetic

animals (Zao et al., 2010). However, if you are not

studying wound healing and only studying biofilm

formation, the in vitro models are probably more than

sufficient because they have less variability than the

mouse model system. Because of the inherent variability of

the mouse model it takes 9 mice per group to get statistical

significance. Many of our colleagues feel that this mouse

model is less favorable than the in vitro models because of this variability. Our hypothesis is

that 3D printed biofilms will make the mouse model more robust and more applicable by

reducing the variability and therefore the number of replicates required for this model. We plan

to present these new results at the next Symposium on Advanced Wound Care (SAWC) in the

Fall of 2015.

SUMMARY

There are several models to study biofilms. However, each model has its own pitfalls and strengths. We

recommend that researchers use more than one model to validate their testing protocol with the

antibacterial or antimicrobial combination product. When you think about which lab you should use for

biofilm studies, consider a lab that has at least 20+ years of experience in studying biofilms and

determine if they are capable of generating timely, statistically significant, and high publication quality

data.

Figure 7 The balb/c mouse is commonly used in biofilm studies. This model is far less expensive than the partial thickness porcine infection model or the rabbit urinary catheter model (not shown), but is more variable.

Biofilm Models Multispecies Advantages Pitfalls Measurments

CDC Bioreacter ++ Moderate Throughput STD Model/Cumbersome CFU Plating

Rotary Disk ++ Measures Shear Force Cumbersome CFU Plating

Drip Module ++ Robust Biofilms Old Model/Cumbersome CFU Plating/Fl Confocal Microscopy

New 3D Bioprinting +++ Versatile for in vitro & in vivo models New System CFU Plating/Fl Confocal Microscopy

Porcine Skin + Direct interaction with host protiens No Host Response, oversimplified CFU Plating/Fl Confocal Microscopy

Mouse Chronic Infection ++ Closest to Chronic Wound Infection Higher error requires 9 replicates CFU Plating

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AUTHORS

Mitchell Sanders MS, PhD, is the Managing Director of the Drug and Device Discovery Lab at CMC Consulting. Mitch has 30+ years of experience in studying bacterial biofilms and chronic wound infections. With ECI Biotech, Mitchell has produced over 12 peer-reviewed publications and 24 worldwide patents in medical device and in vitro diagnostics. Mitchell is an expert in clinical and translational research and is a reviewer for the Wound Healing Society, CIMIT, MassVentures, MIT, WPI, Tech Sandbox, Piranha Pond, SBANE and the Venture Forum. Mitchell has an MS and PhD from WPI in molecular biology and biomedical sciences with 2 Postdocs (biochemistry and pathogen genetics) at the Whitehead Institute/MIT.

Lindsay Poland is a scientist at 3DL who has 10+ years of experience in studying clinical microbiology and protein biochemistry. Lindsay is an expert in molecular biology and protein biochemistry of chronic wounds. She has 14 years of experience with almost 11 of them being in the industry with Mitch Sanders at ECI Biotech (Worcester MA) studying wound repair and regeneration and chronic wound infection.

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REFERENCES

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Pseudomonas aeruginosa biofilm.

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PMID:25959709

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for understanding permeability and mechanics. Rep Prog Phys. 2015 Feb;78(3):036601. doi:

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13. Zhao G1, Hochwalt PC, Usui ML, Underwood RA, Singh PK, James GA, Stewart PS, Fleckman P, Olerud JE.

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475X.2010.00608.x. Epub 2010 Aug 19.

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