MacGowan, A. | Search bioRxiv

bioRxiv2026

Development of low cost, robust and reproducible biofilm static and pharmacodynamic assays

SynopsisO_ST_ABSBackgroundC_ST_ABSThe complexity of diagnosing and treating biofilm-associated infections necessitates a comprehensive strategy to mitigate the rising rates of antimicrobial resistance (AMR). Microtiter plate methods are used globally for determination of biofilm eradication concentrations (MBEC) but few have been adapted to observe pharmacodynamic observations. Here, we describe a method which allows for both static and pharmacodynamic assays of biofilm evaluation. MethodsA total of 150 clinical isolates from Southmead Hospital were assessed, representing five bacterial species (N=30 per bacterial species): Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae, Staphylococcus aureus and Klebsiella pneumoniae. MBECs were determined using a developed method using 96 well plates and glass beads. MBECs of seven different antibiotics were compared to those determined using the established Calgary biofilm device (CBD). Dynamic pharmacodynamic evaluations to produce Biofilm Time Kill curve (BTKC) based on published planktonic time kill curve (TKC) data and ISO recommendations were carried out using the glass bead model for K. pneumoniae and ciprofloxacin, S. aureus and levofloxacin and S. pneumoniae and vancomycin. Quantification of biofilm biomass was assessed at 0, 2, 4, 8 and 24 hours and compared to planktonic culture survival under comparable challenge conditions. ResultsComparing MBEC results for all bacterial strains and antibiotic challenges showed no statistical difference between the glass bead and CBD methods (P <0.05). Biofilm BTKC AUBKC were inferior to planktonic equivalents but demonstrated specific pharmacodynamic patterns of biofilm reduction efficacy. MBEC correlated with biofilm BTKC penetration in line with clinical observations for S. aureus vs vancomycin and S. pneumoniae vs levofloxacin. ConclusionsThe glass bead biofilm models provide robust, reproducible alternatives to the traditional methods of determining MBEC and bridge the gap with biofilm pharmacodynamic evaluations. These methods also provide a low-cost option to current methods as only standard laboratory equipment is required, allowing for the generation of comprehensive data sets. This ensures greater translatability to complex in vitro models and clinical scenarios.

microbiology↗

bioRxiv2020

Modelling the impact of clot fragmentation on the microcirculation after thrombectomy

1Many ischaemic stroke patients who have a mechanical removal of their clot (thrombectomy) do not get reperfusion of tissue despite the thrombus being removed. One hypothesis for this no-reperfusion phenomenon is micro-emboli fragmenting off the large clot during thrombectomy and occluding smaller blood vessels downstream of the clot location. This is impossible to observe in-vivo and so we here develop an in-silico model based on in-vitro experiments to model the effect of micro-emboli on brain tissue. Through in-vitro experiments we obtain, under a variety of clot consistencies and thrombectomy techniques, micro-emboli distributions post-thrombectomy. Blood flow through the microcirculation is modelled for statistically accurate voxels of brain microvasculature including penetrating arterioles and capillary beds. A novel micro-emboli algorithm, informed by the experimental data, is used to simulate the impact of micro-emboli successively entering the penetrating arterioles and the capillary bed. Scaled-up blood flow parameters - permeability and coupling coefficients - are calculated under various conditions. We find that capillary beds are more susceptible to occlusions than the penetrating arterioles with a 4x greater drop in permeability per volume of vessel occluded. Individual microvascular geometries determine robustness to micro-emboli. Hard clot fragmentation leads to larger micro-emboli and larger drops in blood flow for a given number of micro-emboli. Thrombectomy technique has a large impact on clot fragmentation and hence occlusions in the microvasculature. As such, in-silico modelling of mechanical thrombectomy predicts that clot specific factors, interventional technique, and microvascular geometry strongly influence reperfusion of the brain. Micro-emboli are likely contributory to the phenomenon of no-reperfusion following successful removal of a major clot. 2 Author summaryAfter an ischaemic stroke - one where a clot blocks a major artery in the brain - patients can undergo a procedure where the clot is removed mechanically with a stent - a thrombectomy. This reopens the blocked vessel, yet some patients dont achieve blood flow returning to their tissue downstream. One hypothesis for this phenomenon is that the clot fragments into smaller clots (called micro-emboli) which block smaller vessels downstream. However, this cant be measured in patients due to the inability of clinical imaging resolving the micro-scale. We therefore develop a computational model here, based on experimental thrombectomy data, to quantify the impact of micro-emboli on blood flow in the brain after the removal of a clot. With this model, we found that micro-emboli are a likely contributor to the no-reflow phenomenon after a thrombectomy. Individual blood vessel geometries, clot composition, and thrombectomy technique all impacted the effect of micro-emboli on blood flow and should be taken into consideration to minimise the impact of micro-emboli in the brain. Furthermore, the computational model developed here allows us to now build large-scale models of blood flow in the brain, and hence simulate stroke and the impact of micro-emboli on the entire brain.

neuroscience↗