3D Bioprinting of mature bacterial biofilms for antimicrobial resistance drug testing

Research output: Contribution to journalArticle

Abstract

The potential to bioprint and study 3D bacterial biofilm constructs could have great clinical significance at a time when antimicrobial resistance (AMR) is rising to dangerously high levels worldwide. In this study, clinically relevant bacterial species including Escherichia coli, Staphylococcus aureus (MSSA), Methicillin-resistant staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted to form mature bacteria biofilms, characterized by Confocal laser scanning microscopy (CLSM) and fluorescent staining. Solid and porous bacteria-laden constructs were reproducibly bioprinted with thicknesses ranging from 0.25 to 4 mm. We demonstrated the 3D bioprinting thicker biofilms (>4mm) than found in currently available in vitro models. Bacterial viability was excellent in the bioprinted constructs, with CLSM observation of bacterial biofilm production and maturation possible for at least 28 days in culture. Importantly, we first time observed the complete five-step biofilm life cycle in vitro, suggesting the formation of mature 3D bioprinted biofilms. Bacterial growth was faster in thinner, more porous constructs whilst constructs crosslinked with BaCl2 concentrations of above 10 mM had superior biofilm formation. 3D MRSA and MSSA biofilm constructs were found to show greater resistance to antimicrobial drugs than corresponding two-dimensional (2D) cultures. Thicker 3D E.coli biofilms had greater resistance to tetracycline than thinner constructs over 7 days of treatment. Our methodology allowed for the precise 3D bioprinting of self-supporting 3D bacterial biofilm structures that developed biofilms during extended culture. 3D biofilm constructs containing bacterial biofilms produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing.
LanguageEnglish
Number of pages23
JournalBiofabrication
Publication statusAccepted/In press - 5 Jul 2019

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Bioprinting
Biofilms
Microbial Drug Resistance
Testing
Pharmaceutical Preparations
Methicillin
Methicillin-Resistant Staphylococcus aureus
Confocal Microscopy
Escherichia coli
Bacteria
Microscopic examination
Microbial Viability
Scanning
Bacterial Structures
Tetracycline Resistance

Keywords

  • bioprinting
  • 3D bacterial biofilm
  • antimicrobial resistance

Cite this

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title = "3D Bioprinting of mature bacterial biofilms for antimicrobial resistance drug testing",
abstract = "The potential to bioprint and study 3D bacterial biofilm constructs could have great clinical significance at a time when antimicrobial resistance (AMR) is rising to dangerously high levels worldwide. In this study, clinically relevant bacterial species including Escherichia coli, Staphylococcus aureus (MSSA), Methicillin-resistant staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted to form mature bacteria biofilms, characterized by Confocal laser scanning microscopy (CLSM) and fluorescent staining. Solid and porous bacteria-laden constructs were reproducibly bioprinted with thicknesses ranging from 0.25 to 4 mm. We demonstrated the 3D bioprinting thicker biofilms (>4mm) than found in currently available in vitro models. Bacterial viability was excellent in the bioprinted constructs, with CLSM observation of bacterial biofilm production and maturation possible for at least 28 days in culture. Importantly, we first time observed the complete five-step biofilm life cycle in vitro, suggesting the formation of mature 3D bioprinted biofilms. Bacterial growth was faster in thinner, more porous constructs whilst constructs crosslinked with BaCl2 concentrations of above 10 mM had superior biofilm formation. 3D MRSA and MSSA biofilm constructs were found to show greater resistance to antimicrobial drugs than corresponding two-dimensional (2D) cultures. Thicker 3D E.coli biofilms had greater resistance to tetracycline than thinner constructs over 7 days of treatment. Our methodology allowed for the precise 3D bioprinting of self-supporting 3D bacterial biofilm structures that developed biofilms during extended culture. 3D biofilm constructs containing bacterial biofilms produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing.",
keywords = "bioprinting, 3D bacterial biofilm, antimicrobial resistance",
author = "Evita Ning and Gareth Turnbull and Jon Clarke and Frederic Picard and Philip Riches and Marc Vendrell and Duncan Graham and Wark, {Alastair W.} and Karen Faulds and Wenmiao Shu",
year = "2019",
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journal = "Biofabrication",
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T1 - 3D Bioprinting of mature bacterial biofilms for antimicrobial resistance drug testing

AU - Ning, Evita

AU - Turnbull, Gareth

AU - Clarke, Jon

AU - Picard, Frederic

AU - Riches, Philip

AU - Vendrell, Marc

AU - Graham, Duncan

AU - Wark, Alastair W.

AU - Faulds, Karen

AU - Shu, Wenmiao

PY - 2019/7/5

Y1 - 2019/7/5

N2 - The potential to bioprint and study 3D bacterial biofilm constructs could have great clinical significance at a time when antimicrobial resistance (AMR) is rising to dangerously high levels worldwide. In this study, clinically relevant bacterial species including Escherichia coli, Staphylococcus aureus (MSSA), Methicillin-resistant staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted to form mature bacteria biofilms, characterized by Confocal laser scanning microscopy (CLSM) and fluorescent staining. Solid and porous bacteria-laden constructs were reproducibly bioprinted with thicknesses ranging from 0.25 to 4 mm. We demonstrated the 3D bioprinting thicker biofilms (>4mm) than found in currently available in vitro models. Bacterial viability was excellent in the bioprinted constructs, with CLSM observation of bacterial biofilm production and maturation possible for at least 28 days in culture. Importantly, we first time observed the complete five-step biofilm life cycle in vitro, suggesting the formation of mature 3D bioprinted biofilms. Bacterial growth was faster in thinner, more porous constructs whilst constructs crosslinked with BaCl2 concentrations of above 10 mM had superior biofilm formation. 3D MRSA and MSSA biofilm constructs were found to show greater resistance to antimicrobial drugs than corresponding two-dimensional (2D) cultures. Thicker 3D E.coli biofilms had greater resistance to tetracycline than thinner constructs over 7 days of treatment. Our methodology allowed for the precise 3D bioprinting of self-supporting 3D bacterial biofilm structures that developed biofilms during extended culture. 3D biofilm constructs containing bacterial biofilms produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing.

AB - The potential to bioprint and study 3D bacterial biofilm constructs could have great clinical significance at a time when antimicrobial resistance (AMR) is rising to dangerously high levels worldwide. In this study, clinically relevant bacterial species including Escherichia coli, Staphylococcus aureus (MSSA), Methicillin-resistant staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted to form mature bacteria biofilms, characterized by Confocal laser scanning microscopy (CLSM) and fluorescent staining. Solid and porous bacteria-laden constructs were reproducibly bioprinted with thicknesses ranging from 0.25 to 4 mm. We demonstrated the 3D bioprinting thicker biofilms (>4mm) than found in currently available in vitro models. Bacterial viability was excellent in the bioprinted constructs, with CLSM observation of bacterial biofilm production and maturation possible for at least 28 days in culture. Importantly, we first time observed the complete five-step biofilm life cycle in vitro, suggesting the formation of mature 3D bioprinted biofilms. Bacterial growth was faster in thinner, more porous constructs whilst constructs crosslinked with BaCl2 concentrations of above 10 mM had superior biofilm formation. 3D MRSA and MSSA biofilm constructs were found to show greater resistance to antimicrobial drugs than corresponding two-dimensional (2D) cultures. Thicker 3D E.coli biofilms had greater resistance to tetracycline than thinner constructs over 7 days of treatment. Our methodology allowed for the precise 3D bioprinting of self-supporting 3D bacterial biofilm structures that developed biofilms during extended culture. 3D biofilm constructs containing bacterial biofilms produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing.

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SN - 1758-5082

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