A Facile High-Throughput Model of Surface-Independent Staphylococcus aureus Biofilms by Spontaneous Aggregation

Abstract

The canonical model of biofilm formation begins with the attachment and growth of microbial cells on a surface. While these in vitro models reasonably mimic biofilms formed on foreign bodies such as catheters and implants, this is not the case for biofilms formed in cystic fibrosis and chronic wound infections, which appear to present as aggregates not attached to a surface.

Biofilm Formation by Staphylococcus aureus in the Specific Context of Cystic Fibrosis

“…Studying the expression of three representative genes that are known to be upregulated in surface-attached S. aureus biofilms: sdrC (Ser-Asp-Arg-rich fibrinogen-binding protein) encoding proteins for fibrinogen mediated cell adhesion, arcB (ornithine transcarbamylase) involved in extraction and catabolism of arginine, and ureC (urease accessory protein C) involved in metabolism of urea, showed that these three genes were differentially upregulated over time in hanging-drop S. aureus biofilms compared with surface-attached ones supporting that hanging-drop biofilm maturation occurred earlier than that of surfaceattached biofilm. In addition, sdrC expression levels were unchanged in hanging-drop model but dramatically upregulated in surface-attached biofilm model suggesting that the biofilm formation in the hanging-drop formation may not depend on fibrinogen-mediated adhesion [4]. Again, these observations highlighted the importance of using clinical isolates and testing methods that mimic CF lung conditions as closely as possible.…”

“…However, more recently, biofilms have also been found unattached to any surface. presenting as three-dimensional aggregates, a form that may be closer to the in vivo situation, particularly in mucosal infections like CF [4,5]. Biofilm formation is a dynamic, coordinated, cyclic process, classically described as involving several stages.…”

“…The importance of the patient's colonization history was also highlighted as two MRSA strains, isolated three years apart in a chronically colonized patient, showed distinct patterns of biofilm formation (increased biofilm formation for the latest isolated strain) in the BioFlux™ 200 assay. More recently, Cheng et al, considering that in vivo biofilm may be formed in the absence of any surface for attachment, developed a surface-independent method to study biofilm formation based on a hanging-drop biofilm culture model with the aim of reducing the gap between in vitro predictions and in vivo responses [4]. The method appeared highly attractive for studying CF clinical isolates as, in CF, biofilm consists of unattached aggregates dispersed in the mucus [49].…”

“…The method appeared highly attractive for studying CF clinical isolates as, in CF, biofilm consists of unattached aggregates dispersed in the mucus [49]. Using this original, 96-well plate hanging-drop technology coupled with gene expression quantification and CLSM with differential fluorescent staining of biofilm components, the authors showed that the biofilm formed by a CF MRSA isolate displayed specific characteristics compared with that of an MRSA strain from a central catheter-related infection [4]. Indeed, this biofilm was less rich in matrix and had a lower viable cell count but was formed with double-sized micro-colonies and had a 50% higher metabolic activity.…”

Biofilm Formation of Helicobacter pylori in Both Static and Microfluidic Conditions Is Associated With Resistance to Clarithromycin

“…This stage of the experiment was carried out at 37°C and microaerophilic conditions (Pecon Incubator XL S1, Carl Zeiss, Jena, Germany). After the designated incubation period the flow of the medium was stopped, the inlet wells were emptied from the remaining medium and then filled with a solution containing three fluorescent dyes: 0.3 µL of SYTO9 (L10316, ThermoFisher, Waltham, MA, USA), 1 µL of DAPI (62248, ThermoFisher, Waltham, MA, USA) and 100 µL of SYPRO RUBY (F10318, ThermoFisher, Waltham, MA, USA), enabling for determination of the bacterial biomass, extracellular DNA (eDNA) and extracellular proteins of the biofilm matrix, respectively (Cheng et al, 2021). The medium flow was switched from the inlet to the outlet wells at a rate of 0.1 dyne/cm 2 and a period of 1 h. Photographs from the experiments were taken using an inverted fluorescence microscope (GmbH, Jena, Germany) and were analyzed using integrated with this system the Bioflux Montage software (Fluxion, San Francisco, CA, USA).…”