A microfluidic platform for in situ investigation of biofilm formation and its treatment under controlled conditions

Abstract Background Studying bacterial adhesion and early biofilm development is crucial for understanding the physiology of sessile bacteria and forms the basis for the development of novel antimicrobial biomaterials. Microfluidics technologies can be applied in such studies since they permit dynamic real-time analysis and a more precise control of relevant parameters compared to traditional static and flow chamber assays. In this work, we aimed to establish a microfluidic platform that permits real-time observation of bacterial adhesion and biofilm formation under precisely controlled homogeneous laminar flow conditions. Results Using Escherichia coli as the model bacterial strain, a microfluidic platform was developed to overcome several limitations of conventional microfluidics such as the lack of spatial control over bacterial colonization and allow label-free observation of bacterial proliferation at single-cell resolution. This platform was applied to demonstrate the influence of culture media on bacterial colonization and the consequent eradication of sessile bacteria by antibiotic. As expected, the nutrient-poor medium (modified M9 minimal medium) was found to promote bacterial adhesion and to enable a higher adhesion rate compared to the nutrient-rich medium (tryptic soy broth rich medium ). However, in rich medium the adhered cells colonized the glass surface faster than those in poor medium under otherwise identical conditions. For the first time, this effect was demonstrated to be caused by a higher retention of newly generated bacteria in the rich medium, rather than faster growth especially during the initial adhesion phase. These results also indicate that higher adhesion rate does not necessarily lead to faster biofilm formation. Antibiotic treatment of sessile bacteria with colistin was further monitored by fluorescence microscopy at single-cell resolution, allowing in situ analysis of killing efficacy of antimicrobials. Conclusion The platform established here represents a powerful and versatile tool for studying environmental effects such as medium composition on bacterial adhesion and biofilm formation. Our microfluidic setup shows great potential for the in vitro assessment of new antimicrobials and antifouling agents under flow conditions.

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A Microfluidic Platform for in Situ Investigation of Biofilm Formation and Its Treatment Under Controlled Conditions

10.21203/rs.3.rs-59824/v1 ◽

2020 ◽

Author(s):

Hervé Straub ◽

Leo Eberl ◽

Manfred Zinn ◽

René M Rossi ◽

Katharina Maniura-Weber ◽

...

Keyword(s):

Single Cell ◽

Real Time ◽

Bacterial Adhesion ◽

Biofilm Formation ◽

Bacterial Colonization ◽

Rich Medium ◽

Flow Conditions ◽

Microfluidic Platform ◽

Adhesion Rate

Abstract Background Studying bacterial adhesion and early biofilm development is crucial for understanding the physiology of sessile bacteria and forms the basis for the development of novel antimicrobial biomaterials. Microfluidics technologies can be applied in such studies since they permit dynamic real-time analysis and a more precise control of relevant parameters compared to traditional static and flow chamber assays. In this work, we aimed to establish a microfluidic platform that permits real-time observation of bacterial adhesion and biofilm formation under precisely controlled homogeneous laminar flow conditions. Results Using Escherichia coli as the model bacterial strain, a microfluidic platform was developed to overcome several limitations of conventional microfluidics such as the lack of spatial control over bacterial colonization and allow label-free observation of bacterial proliferation at single-cell resolution. This platform was applied to demonstrate the influence of culture media on bacterial colonization and the consequent eradication of sessile bacteria by antibiotic. As expected, the nutrient-poor medium was found to promote bacterial adhesion and to enable a higher adhesion rate compared to the nutrient-rich medium. However, in rich medium the adhered cells colonized the glass surface faster than those in poor medium under otherwise identical conditions. For the first time, this effect was demonstrated to be caused by a higher retention of newly generated bacteria in the rich medium, rather than faster growth especially during the initial adhesion phase. These results also indicate that higher adhesion rate does not necessarily lead to faster biofilm formation. Antibiotic treatment of sessile bacteria with colistin was further monitored by fluorescence microscopy at single-cell resolution, allowing in situ analysis of killing efficacy of antimicrobials.Conclusion The platform established here represents a powerful and versatile tool for studying environmental effects such as medium composition on bacterial adhesion and biofilm formation. Our microfluidic setup shows great potential for the in vitro assessment of new antimicrobials and antifouling agents under flow conditions.

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A Microfluidic Platform for in situ Investigation of Biofilm Formation and its Treatment under Controlled Conditions

10.21203/rs.3.rs-59824/v2 ◽

2020 ◽

Author(s):

Hervé Straub ◽

Leo Eberl ◽

Manfred Zinn ◽

René M Rossi ◽

Katharina Maniura-Weber ◽

...

Keyword(s):

Single Cell ◽

Bacterial Adhesion ◽

Biofilm Formation ◽

Organic Nitrogen ◽

Bacterial Colonization ◽

Rich Medium ◽

Flow Conditions ◽

Microfluidic Platform ◽

Adhesion Rate

Abstract Background Studying bacterial adhesion and early biofilm development is crucial for understanding the physiology of sessile bacteria and forms the basis for the development of novel antimicrobial biomaterials. Microfluidics technologies can be applied in such studies since they permit dynamic real-time analysis and a more precise control of relevant parameters compared to traditional static and flow chamber assays. In this work, we aimed to establish a microfluidic platform that permits real-time observation of bacterial adhesion and biofilm formation under precisely controlled homogeneous laminar flow conditions. Results Using Escherichia coli as the model bacterial strain, a microfluidic platform was developed to overcome several limitations of conventional microfluidics such as the lack of spatial control over bacterial colonization and allow label-free observation of bacterial proliferation at single-cell resolution. This platform was applied to demonstrate the influence of culture media on bacterial colonization and the consequent eradication of sessile bacteria by antibiotic. As expected, the nutrient-poor medium (modified M9 medium containing 1 g/l of organic nitrogen) was found to promote bacterial adhesion and to enable a higher adhesion rate compared to the nutrient-rich medium (tryptic soy broth containing 20 g/l of organic nitrogen). However, in rich medium the adhered cells colonized the glass surface faster than those in poor medium under otherwise identical conditions. For the first time, this effect was demonstrated to be caused by a higher retention of newly generated bacteria in the rich medium, rather than faster growth especially during the initial adhesion phase. These results also indicate that higher adhesion rate does not necessarily lead to faster biofilm formation. Antibiotic treatment of sessile bacteria with colistin was further monitored by fluorescence microscopy at single-cell resolution, allowing in situ analysis of killing efficacy of antimicrobials.Conclusion The platform established here represents a powerful and versatile tool for studying environmental effects such as medium composition on bacterial adhesion and biofilm formation. Our microfluidic setup shows great potential for the in vitro assessment of new antimicrobials and antifouling agents under flow conditions.

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A microfluidic platform for characterizing the structure and rheology of biofilm streamers

10.5194/biofilms9-76 ◽

2020 ◽

Author(s):

Giovanni Savorana ◽

Roberto Rusconi ◽

Roman Stocker ◽

Eleonora Secchi

Keyword(s):

Flow Rate ◽

Material Properties ◽

Extracellular Polymeric Substances ◽

Fluid Shear Stress ◽

Bacterial Colonization ◽

Secondary Flows ◽

Bacterial Suspension ◽

Flow Conditions ◽

Microfluidic Platform

In many environmental or medical settings, biofilm formation is the most successful strategy for bacterial colonization1,2. In the biofilm lifestyle, bacteria embed themselves in a self-secreted matrix of extracellular polymeric substances (EPS), acting as a shield against mechanical and chemical insults3. When ambient flow is present, this viscoelastic EPS scaffold can take a streamlined shape, forming biofilm threads suspended in flow, called streamers4. In many situations, the streamer architecture can enhance the harmful effects of biofilms, bridging the spaces between obstacles in the flow path5. Despite their importance for biofilm survival, little is known about the material properties of the matrix. In particular, these are really hard to probe with traditional rheological techniques when the biofilm grows into the thread-like streamer shape. In this work we present a microfluidic platform that allows to reproducibly grow biofilm streamers in controlled chemical and flow conditions and to characterize their structure and mechanical properties in situ6.This platform overcomes the main sources of error and variability of the experiments performed with traditional flow-chambers: the randomness in the location and shape of the streamers. Our device consists of a straight channel with isolated micropillars, where a bacterial suspension is injected at a constant flow rate. The micropillars act as nucleation points for the growth of a pair of biofilm filaments, developing on the midplane of the channel under the action of secondary flows. The microfluidic technology allows to control the chemical and flow conditions and to perform live imaging of the growth process. By controlling the flow rate, we are also able to perform in situ stress tests on the streamers by inducing controlled variations of the fluid shear stress exerted on them. We developed a theoretical framework to estimate the material properties of biofilm streamers from the flow-induced deformation measured in our experiment. Thanks to this platform, we are able to investigate the role of the different EPS components7and the physico-chemical microenvironment in determining biofilm structure and rheology. References 1Flemming and Wingender, Nat. Rev. Microbiol. 8, 623 (2010). 2Flemming et al., Nat. Rev. Microbiol. 14, 563 (2016). 3Peterson et al., FEMS Microbiol. Rev. 39, 2 (2015). 4Rusconi et al., J. R. Soc. Interface 7, 1293 (2010). 5Drescher et al., Proc. Natl. Acad. Sci. 112, 11353 (2015). 6Savorana et al., paper in preparation 7Secchi et al., paper in preparation

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A Real-Time Thermal Sensor System for Quantifying the Inhibitory Effect of Antimicrobial Peptides on Bacterial Adhesion and Biofilm Formation

Sensors ◽

10.3390/s21082771 ◽

2021 ◽

Vol 21 (8) ◽

pp. 2771

Author(s):

Tobias Wieland ◽

Julia Assmann ◽

Astrid Bethe ◽

Christian Fidelak ◽

Helena Gmoser ◽

...

Keyword(s):

Antimicrobial Peptides ◽

Real Time ◽

Bacterial Adhesion ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Bovine Mastitis ◽

Inhibitory Effect ◽

Thermal Sensor ◽

Bacterial Cells ◽

Static Conditions

The increasing rate of antimicrobial resistance (AMR) in pathogenic bacteria is a global threat to human and veterinary medicine. Beyond antibiotics, antimicrobial peptides (AMPs) might be an alternative to inhibit the growth of bacteria, including AMR pathogens, on different surfaces. Biofilm formation, which starts out as bacterial adhesion, poses additional challenges for antibiotics targeting bacterial cells. The objective of this study was to establish a real-time method for the monitoring of the inhibition of (a) bacterial adhesion to a defined substrate and (b) biofilm formation by AMPs using an innovative thermal sensor. We provide evidence that the thermal sensor enables continuous monitoring of the effect of two potent AMPs, protamine and OH-CATH-30, on surface colonization of bovine mastitis-associated Escherichia (E.) coli and Staphylococcus (S.) aureus. The bacteria were grown under static conditions on the surface of the sensor membrane, on which temperature oscillations generated by a heater structure were detected by an amorphous germanium thermistor. Bacterial adhesion, which was confirmed by white light interferometry, caused a detectable amplitude change and phase shift. To our knowledge, the thermal measurement system has never been used to assess the effect of AMPs on bacterial adhesion in real time before. The system could be used to screen and evaluate bacterial adhesion inhibition of both known and novel AMPs.

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Real-time and in Situ Drug Release Monitoring from Nanoporous Implants under Dynamic Flow Conditions by Reflectometric Interference Spectroscopy

ACS Applied Materials & Interfaces ◽

10.1021/am4013984 ◽

2013 ◽

Vol 5 (12) ◽

pp. 5436-5442 ◽

Cited By ~ 25

Author(s):

Tushar Kumeria ◽

Karan Gulati ◽

Abel Santos ◽

Dusan Losic

Keyword(s):

Drug Release ◽

Real Time ◽

Flow Conditions ◽

Dynamic Flow ◽

Reflectometric Interference Spectroscopy

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Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization

Journal of Medical Microbiology ◽

10.1099/jmm.0.011213-0 ◽

2009 ◽

Vol 58 (10) ◽

pp. 1359-1366 ◽

Cited By ~ 48

Author(s):

Ali Al-Ahmad ◽

Marie Follo ◽

Ann-Carina Selzer ◽

Elmar Hellwig ◽

Matthias Hannig ◽

...

Keyword(s):

Biofilm Formation ◽

Laser Scanning ◽

Bacterial Colonization ◽

Bacterial Species ◽

Fusobacterium Nucleatum ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Actinomyces Naeslundii ◽

Oral Biofilms

Oral biofilms are one of the greatest challenges in dental research. The present study aimed to investigate initial bacterial colonization of enamel surfaces in situ using fluorescence in situ hybridization (FISH) over a 12 h period. For this purpose, bovine enamel slabs were fixed on buccal sites of individual splints worn by six subjects for 2, 6 and 12 h to allow biofilm formation. Specimens were processed for FISH and evaluated with confocal laser-scanning microscopy, using probes for eubacteria, Streptococcus species, Veillonella species, Fusobacterium nucleatum and Actinomyces naeslundii. The number of adherent bacteria increased with time and all tested bacterial species were detected in the biofilm formed in situ. The general percentage composition of the eubacteria did not change over the investigated period, but the number of streptococci, the most frequently detected species, increased significantly with time (2 h: 17.7±13.8 %; 6 h: 20.0±16.6 %; 12 h: 24.7±16.1 %). However, ≤1 % of the surface was covered with bacteria after 12 h of biofilm formation in situ. In conclusion, FISH is an appropriate method for quantifying initial biofilm formation in situ, and the proportion of streptococci increases during the first 12 h of bacterial adherence.

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Bacterial adhesion and biofilm formation on yttria-stabilized, tetragonal zirconia and titanium oral implant materials with low surface roughness - an in situ study

Journal of Medical Microbiology ◽

10.1099/jmm.0.000267 ◽

2016 ◽

Vol 65 (7) ◽

pp. 596-604 ◽

Cited By ~ 13

Author(s):

Ali Al-Ahmad ◽

Lamprini Karygianni ◽

Max Schulze Wartenhorst ◽

Maria Bächle ◽

Elmar Hellwig ◽

...

Keyword(s):

Surface Roughness ◽

Bacterial Adhesion ◽

Biofilm Formation ◽

Tetragonal Zirconia ◽

Implant Materials ◽

In Situ Study ◽

Oral Implant

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In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

Journal of Visualized Experiments ◽

10.3791/57823 ◽

2019 ◽

Author(s):

Anna Maria Marbà-Ardébol ◽

Joern Emmerich ◽

Michael Muthig ◽

Peter Neubauer ◽

Stefan Junne

Keyword(s):

Single Cell ◽

Real Time ◽

Cell Morphology ◽

Time Determination

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Integration of Microresonant Sensor into a Microfluidic Platform for the Real Time Analysis of Platelets-Collagen Interaction in Flow Condition

Proceedings ◽

10.3390/proceedings2130940 ◽

2018 ◽

Vol 2 (13) ◽

pp. 940

Author(s):

Aleksandr Oseev ◽

Benoît Le Roy de Boiseaumarié ◽

Fabien Remy-Martin ◽

Jean-François Manceau ◽

Alain Rouleau ◽

...

Keyword(s):

Real Time ◽

Flow Condition ◽

Flow Conditions ◽

Time Analysis ◽

Flow Rates ◽

Microfluidic Platform ◽

Real Time Analysis ◽

Controlled Flow ◽

The One

The contribution focuses on the development of microresonant sensor solution integrated in microfluidic platform for the haemostasis assessment at realistic rheological flow conditions similar to the one in blood vessels. A multi-parameter sensor performs real time analysis of interactions between immobilized collagen and platelets. The detection and characterization of such interactions at controlled flow rates provide information to evaluate the dynamic of each step of primary haemostasis. The microresonant sensor concept was developed and is described in the contribution.

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Biofilm dynamics: linking in situ biofilm biomass and metabolic activity measurements in real-time under continuous flow conditions

npj Biofilms and Microbiomes ◽

10.1038/s41522-020-00153-9 ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Kyle B. Klopper ◽

Riaan N. de Witt ◽

Elanna Bester ◽

Leon M. T. Dicks ◽

Gideon M. Wolfaardt

Keyword(s):

Real Time ◽

Metabolic Activity ◽

Continuous Flow ◽

Control Strategies ◽

Mass Function ◽

Flow Conditions ◽

Carbon Dioxide Evolution ◽

Activity Measurements ◽

The Internet Of Things

Abstract The tools used to study biofilms generally involve either destructive, end-point analyses or periodic measurements. The advent of the internet of things (IoT) era allows circumvention of these limitations. Here we introduce and detail the development of the BioSpec; a modular, nondestructive, real-time monitoring system, which accurately and reliably track changes in biofilm biomass over time. The performance of the system was validated using a commercial spectrophotometer and produced comparable results for variations in planktonic and sessile biomass. BioSpec was combined with the previously developed carbon dioxide evolution measurement system (CEMS) to allow simultaneous measurement of biofilm biomass and metabolic activity and revealed a differential response of these interrelated parameters to changing environmental conditions. The application of this system can facilitate a greater understanding of biofilm mass–function relationships and aid in the development of biofilm control strategies.

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