In Vitro Simulated Hemoperfusion on Seraph®-100 as a Promising Strategy to Counteract Sepsis

Abstract

Blood purification represents a treatment option for sepsis, improving inflammation and the hyper-activated immune system. This study investigates the binding efficacy of Seraph^®-100 against 10⁸ CFU/mL of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli) during a simulated hemoperfusion treatment. The fluorescence-activated cell sorting (FACS) technique was used to evaluate the bacteria reduction, whereas kinetic analysis and cultures revealed bacterial detection and counting at established time points. At the end of the experiment, the filter was cut at three different levels, obtaining suspensions for cultures and scanning electron microscopy (SEM) analyses. The FACS technique revealed a 78.77% reduction of the total bacterial load at the end of the treatment, with maximum filter sequestration occurring in the first 30 min of the treatment. Non-linear regression analysis of kinetic experiments (T_{0-240 min}) highlighted a lower growth rate of S. aureus than the other two Gram bacteria, demonstrating a greater affinity without influencing a reduction rate of 99% for all three bacteria. The analyses of the suspension aliquots of the filter sections confirmed these data, revealing 1 × 10⁸ CFU/mL, equal to the initial bacterial charge. Furthermore, the filter head adsorbed approximately 50% of bacteria, whereas the remaining amount was equally distributed between the body and the tail, as corroborated by SEM analysis. In conclusion, Seraph^®-100 adsorbed 10⁸ CFU/mL of S. aureus, E. coli, and P. aeruginosa during an in vitro simulated hemoperfusion session.

Keywords: Seraph®-100; acute kidney injury; adsorption; renal replacement therapy; sepsis.

Figure 1

Strategies, actions, and challenges in septic patients.

Figure 2

Schematic overview of the experimental set-up.

Figure 3

Two-dimensional dot-plot and one-dimensional cytogram of bacterial cell counting obtained by FACS analyses at the starting point (T₀, (A) and (B), respectively) and at the end of the simulated in vitro perfusion session (T_{240 min}, (C) and (D), respectively) with Seraph^®-100.

Figure 4

Non-linear regression analysis of kinetic experiments (T_{0 – 240 min}). Results, which represent the average of three independent experiments in triplicate (n = 3), were expressed as the n. of colony-forming units (CFUs) with respect to time (min), as detected by selective culture media.

Figure 5

Representative SEM micrograph of the blank stationary phase (heparin/heparan sulfate-coated beads) of Seraph^®-100 (A) in comparison with the Seraph^®-100 stationary phase at the end of perfusion time (4 h). (B,C) show some bacteria (red rectangle) attached to the filter stationary phase; (D) shows a magnification of (C), in which the bacterial size is highlighted.

Figure 6

Adsorption processes in Seraph^®-100 heparin-coated microspheres, which mimic the heparin sulfate of the endothelial glycocalyx. (A) The endothelial glycocalyx and bindings of bacteria. (B) Seraph structure miming the endothelial glycocalyx.