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Duodenoscopy-associated infections and outbreaks are reported globally despite strict adherence to duodenoscope reprocessing protocols. Therefore, new developments in the reprocessing procedure are needed.
We evaluated a novel dynamic flow model for an additional cleaning step between precleaning and manual cleaning in the reprocessing procedure.
A parallel plate flow chamber with a fluorinated ethylene propylene bottom plate was used to mimic the duodenoscope channels. The flow chamber was inoculated with a suspension containing Klebsiella pneumoniae to simulate bacterial contamination during a duodenoscopic procedure. After inoculation the flow chamber was flushed with a detergent mimicking precleaning. Subsequently the flow chamber was subjected to different interventions: flow with phosphate-buffered saline (PBS), flow with 2 commercial detergents, flow with sodium dodecyl sulfate with 3 different concentrations, and flow with microbubbles. Adhering bacteria were counted using phase-contrast microscopy throughout the experiment, and finally, bacterial viability was assessed.
During precleaning both PBS and 1% (v/v) Neodisher Mediclean Forte were able to desorb bacteria, but neither proved superior. After precleaning only sodium dodecyl sulfate could desorb bacteria.
Flushing during precleaning is an essential step for reducing adhering luminal bacteria, and sodium dodecyl sulfate is a promising detergent for bacterial desorption from duodenoscope channels after precleaning.
Imaging of cellular layers in a gut-on-a-chip system has been confined to two-dimensional (2D)-imaging through conventional light microscopy and confocal laser scanning microscopy (CLSM) yielding three-dimensional- and 2D-cross-sectional reconstructions. However, CLSM requires staining and is unsuitable for longitudinal visualization. Here, we compare merits of optical coherence tomography (OCT) with those of CLSM and light microscopy for visualization of intestinal epithelial layers during protection by a probiotic Bifidobacterium breve strain and a simultaneous pathogen challenge by an Escherichia coli strain. OCT cross-sectional images yielded film thicknesses that coincided with end-point thicknesses derived from cross-sectional CLSM images. Light microscopy on histological sections of epithelial layers at the end-point yielded smaller layer thicknesses than OCT and CLSM. Protective effects of B. breve adhering to an epithelial layer against an E. coli challenge included the preservation of layer thickness and membrane surface coverage by epithelial cells. OCT does not require staining or sectioning, making OCT suitable for longitudinal visualization of biological films, but as a drawback, OCT does not allow an epithelial layer to be distinguished from bacterial biofilms adhering to it. Thus, OCT is ideal to longitudinally evaluate epithelial layers under probiotic protection and pathogen challenges, but proper image interpretation requires the application of a second method at the end-point to distinguish bacterial and epithelial films.
Bacterial biofilms relieve themselves from external stresses through internal rearrangement, as mathematically modeled in many studies, but never microscopically visualized for their underlying microbiological processes. The aim of this study was to visualize rearrangement processes occurring in mechanically deformed biofilms using confocal-laser-scanning-microscopy after SYTO9 (green-fluorescent) and calcofluor-white (blue-fluorescent) staining to visualize bacteria and extracellular-polymeric matrix substances, respectively. We apply 20% uniaxial deformation to Pseudomonas aeruginosa biofilms and fix deformed biofilms prior to staining, after allowing different time-periods for relaxation. Two isogenic P. aeruginosa strains with different abilities to produce extracellular polymeric substances (EPS) were used. By confocal-laser-scanning-microscopy all biofilms showed intensity distributions for fluorescence from which rearrangement of EPS and bacteria in deformed biofilms were derived. For the P. aeruginosa strain producing EPS, bacteria could not find new, stable positions within 100 s after deformation, while EPS moved toward deeper layers within 20 s. Bacterial rearrangement was not seen in P. aeruginosa biofilms deficient in production of EPS. Thus, EPS is required to stimulate bacterial rearrangement in mechanically deformed biofilms within the time-scale of our experiments, and the mere presence of water is insufficient to induce bacterial movement, likely due to its looser association with the bacteria.
Henk J. Busscher, Department of Biomedical Engineering, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands,
Henny C. van der Mei, Department of Biomedical Engineering, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
In most textbooks, biofilm formation on surfaces has been depicted as a sequence of events. Depending on the particular interest of the author, these events are split up into four or more steps (Escher & Characklis, 1990; Van Loosdrecht et al., 1990). The sequence is presented in Fig. 1. Traditionally, biofilm formation is said to begin with mass transport of micro-organisms towards a substratum surface (Fig. 1b), but in almost all environments, the mass transport step is preceded by the adsorption of conditioning film components (Fig. 1a), such as: an adsorbed tear film on a contact lens (Baguet et al., 1995; Landa et al., 1998); the salivary pellicle on surfaces in the oral cavity (Busscher et al., 1989; Bradshaw et al., 1997); a film of adsorbed urinary components on urogenital surfaces (Reid et al., 1998); and adsorbed macromolecules on marine surfaces (Schneider & Marshall, 1994). Many more examples of the formation of a film conditioning a surface as the first step in biofilm formation can be given. Once transported to a substratum surface, organisms may or may not adhere, depending on the interaction forces (Rutter & Vincent, 1980). This initial adhesion (Fig. 1c) is generally reversible (Norde & Lyklema, 1989), but even in the absence of exopolymer production, becomes less reversible within minutes due to the progressive removal of water from in-between the interacting surfaces (Meinders et al., 1995). The unfolding of binding molecules and other non-metabolic mechanisms eventually lead to the strong anchoring of the initially adhering organisms (Fig. 1e). At this stage, there is often a neglected step which occurs almost simultaneously, that is, coadhesion (Fig. 1d).
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