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

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.

Microfluidics for Biofilm Studies.

Biofilms are multicellular communities held together by a self-produced extracellular matrix and exhibit a set of properties that distinguish them from free-living bacteria. Biofilms are exposed to a variety of mechanical and chemical cues resulting from fluid motion and mass transport. Microfluidics provides the precise control of hydrodynamic and physicochemical microenvironments to study biofilms in general. In this review, we summarize the recent progress made in microfluidics-based biofilm research, including understanding the mechanism of bacterial adhesion and biofilm development, assessment of antifouling and antimicrobial properties, development of advanced in vitro infection models, and advancement in methods to characterize biofilms. Finally, we provide a perspective on the future direction of microfluidics-assisted biofilm research.

In Situ Investigation of Pseudomonas aeruginosa Biofilm Development: Interplay between Flow, Growth Medium, and Mechanical Properties of Substrate.

To better understand the impact of biomaterial mechanical properties and growth medium on bacterial adhesion and biofilm formation under flow, we investigated the biofilm formation ability of Pseudomonas aeruginosa in different media on polydimethylsiloxane (PDMS) of different stiffness in real time using a microfluidic platform. P. aeruginosa colonization was recorded with optical microscopy and automated image analysis. The bacterial intracellular level of cyclic diguanylate (c-di-GMP), which regulates biofilm formation, was monitored using the transcription of the putative adhesin gene (cdrA) as a proxy. Contrary to the previous supposition, we revealed that PDMS material stiffness within the tested range has negligible impact on biofilm development and biofilm structures, whereas culture media not only influence the kinetics of biofilm development but also affect the biofilm morphology and structure dramatically. Interestingly, magnesium rather than previously reported calcium was identified here to play a decisive role in the formation of dense P. aeruginosa aggregates and high levels of c-di-GMP. These results demonstrate that although short-term adhesion assays bring valuable insight into bacterial and material interactions, long-term evaluations are essential to better predict overall biofilm outcome. The microfluidic system developed here presents a valuable application potential for studying biofilm development in situ. .

Uncoupling bacterial attachment on and detachment from polydimethylsiloxane surfaces through empirical and simulation studies.

Bacterial infections related to medical devices can cause severe problems, whose solution requires in-depth understanding of the interactions between bacteria and surfaces. This work investigates the influence of surface physicochemistry on bacterial attachment and detachment under flow through both empirical and simulation studies. We employed polydimethylsiloxane (PDMS) substrates having different degrees of crosslinking as the model material and the extended Derjaguin - Landau - Verwey - Overbeek model as the simulation method. Experimentally, the different PDMS materials led to similar numbers of attached bacteria, which can be rationalized by the identical energy barriers simulated between bacteria and the different materials. However, different numbers of residual bacteria after detachment were observed, which was suggested by simulation that the detachment process is determined by the interfacial physicochemistry rather than the mechanical property of a material. This finding is further supported by analyzing the bacteria detachment from PDMS substrates from which non-crosslinked polymer chains had been removed: similar numbers of residual bacteria were found on the extracted PDMS substrates. The knowledge gained in this work can facilitate the projection of bacterial colonization on a given surface.

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.

Bacterial Adhesion on Soft Materials: Passive Physicochemical Interactions or Active Bacterial Mechanosensing?

The influence of mechanical stiffness of biomaterials on bacterial adhesion is only sparsely studied and the mechanism behind this influence remains unclear. Here, bacterial adhesion on polydimethylsiloxane (PDMS) samples, having four different degrees of stiffness with Young's modulus ranging from 0.06 to 4.52 MPa, is investigated. Escherichia coli and Pseudomonas aeruginosa are found to adhere in greater numbers on soft PDMS (7- and 27-fold increase, respectively) than on stiff PDMS, whereas Staphylococcus aureus adheres in similar numbers on the four tested surfaces. To determine whether the observed adhesion behavior is caused by bacteria-specific mechanisms, abiotic polystyrene (PS) beads are employed as bacteria substitutes. Carboxylate-modified PS (PS-COOH) beads exhibit the same adhesion pattern as E. coli and P. aeruginosa with four times more adhered beads on soft PDMS than on stiff PDMS. In contrast, amine-modified PS (PS-NH2 ) beads adhere in similar numbers on all tested samples, reminiscent of S. aureus adhesion. This work demonstrates for the first time that the intrinsic physicochemical properties associated with PDMS substrates of different stiffness strongly influence bacterial adhesion and challenge the previously reported theory on active bacterial mechanosensing, which provides new insights into the design of antifouling surfaces.

Substrate viscosity plays an important role in bacterial adhesion under fluid flow.

Many materials used in the medical settings such as catheters and contact lenses as well as most biological tissues are not purely elastic, but rather viscoelastic. While substrate elasticity has been investigated for its influence on bacterial adhesion, the impact of substrate viscosity has not been explored. Here, the importance of considering substrate viscosity is explored by using polydimethylsiloxane (PDMS) as the substrate material, whose mechanical properties can be tuned from predominantly elastic to viscous by varying cross-linking degree. Interfacial rheology and atomic force microscopy analysis prove that PDMS with a low cross-linking degree exhibits both low stiffness and high viscosity. This degree of viscoelasticity confers to PDMS a remarkable stress relaxation, a good capability to deform and an increased adhesive force. Bacterial adhesion assays were conducted under flow conditions to study the impact of substrate viscosity on Escherichia coli adhesion. The viscous PDMS not only enhanced E. coli adhesion but also conferred greater resistance to desorption against shear stress at air/liquid interface, compared to the PDMS with high crosslinking degree. These findings highlight the importance to consider substrate viscosity while studying bacterial adhesion. The current work provides new insights to an improved understanding of how bacteria interact with complex viscoelastic environments.