Lekkala, S. | bioRxiv Science

bioRxiv2024

Investigating the translational value of Periprosthetic Joint Infection (PJI) models to determine the risk and severity of Staphylococcal biofilms.

With the advent of antibiotic-eluting polymeric materials for targeting recalcitrant infections, using preclinical models to study biofilm is crucial for improving the treatment efficacy in periprosthetic joint infections. The stratification of risk and severity of infections is needed to develop an effective clinical dosing framework with better outcomes. Here, using in-vivo and in-vitro implant-associated infection models, we demonstrate that methicillin-sensitive and resistant Staphylococcus aureus (MSSA and MRSA) have model-dependent distinct implant and peri-implant tissue colonization patterns. The maturity of biofilms and the location (implant vs tissue) were found to influence the antibiotic susceptibility evolution profiles of MSSA and MRSA and the models could capture the differing host-microbe interactions in vivo. Gene expression studies revealed the molecular heterogeneity of colonizing bacterial populations. The comparison and stratification of the risk and severity of infection across different preclinical models provided in this study can guide clinical dosing to effectively prevent or treat PJI.

microbiology↗

bioRxiv2023

A semi-automated cell tracking protocol for quantitative analyses of neutrophil swarming to sterile and S. aureus contaminated bone implants in a mouse femur model

Implant-associated osteomyelitis remains a major orthopaedic problem. As neutrophil swarming to the surgical site is a critical host response to prevent infection, visualization and quantification of this dynamic behavior at the native microenvironment of infection will elucidate previously unrecognized mechanisms central to understanding the host response. We recently developed longitudinal intravital imaging of the bone marrow (LIMB) to visualize fluorescent S. aureus on a contaminated transfemoral implant and host cells in live mice, which allows for direct visualization of bacteria colonization of the implant and host cellular responses using two-photon laser scanning microscopy. To the end of rigorous and reproducible quantitative outcomes of neutrophil swarming kinetics in this model, we developed a protocol for robust segmentation, tracking, and quantifications of neutrophil dynamics adapted from Trainable Weka Segmentation and TrackMate, two readily available Fiji/ImageJ plugins. In this work, Catchup mice with tdTomato expressing neutrophils received a transfemoral pin with or without ECFP-expressing USA300 methicillin-resistant Staphylococcus aureus (MRSA) to obtain 30-minute LIMB videos at 2-, 4-, and 6-hours post-implantation. The developed semi-automated neutrophil tracking protocol was executed independently by two users to quantify the distance, displacement, speed, velocity, and directionality of the target cells. The results revealed high inter-reader reliability for all outcomes (ICC > 0.98; p > 0.05). Consistent with the established paradigm on increased neutrophil swarming during active infection, the results also demonstrated increased neutrophil speed and velocity at all measured time points, and increased displacement at later time points (6 hours) in infected versus uninfected mice (p < 0.05). Neutrophils and bacteria also exhibit directionality during migration in the infected mice. The semi-automated cell tracking protocol provides a streamlined approach to robustly identify and track individual cells across diverse experimental settings and eliminates inter-observer variability.

immunology↗