Chronic Wound Microbiome Colonization on Mouse Model Following Cryogenic Preservation
Craig D. Tipton1, Nick Sanford2, Jake Everett3, Randall D. Wolcott2, Kendra P. Rumbaugh3, Caleb D. Phillips1.
1Texas Tech University, Lubbock, TX, USA, 2Southwest Regional Wound Care Center, Lubbock, TX, USA, 3Texas Tech University Health Sciences Center, Lubbock, TX, USA.
It was recently shown that slough isolated from chronic wounds could re-establish polymicrobial biofilm infections in the mouse model, providing an experimental design to study patient wound biofilm using an in vivo model. However, there are practical limitations of sample collection, timing and transportation for wounding procedures that complicate such experiments. The purpose of this study was to investigate cryogenic preservation on the ability of polymicrobial biofilms to re-establish in a mouse wound model. Slough from five patients was homogenized and divided into three preservation strategies which included refrigeration until infection as previously reported, being frozen in liquid nitrogen, or being placed in glycerol solution before freezing in liquid nitrogen. Individual mice were subsequently infected with slough treatments and were matched with controls. Four days following inoculation, wound microbiota were characterized by 16s rDNA community profiling and bacterial load by quantitative PCR. Analyses were conducted to understand the effect of patient origin and preservation strategy on microbiome community composition. It was found that patient origin explained a significantly greater amount of variation than treatment, which indicated original patient wound microbiome could be partially re-established in a mouse model following preservation. The prior relative abundances of individual species in slough from patients had a significant positive relationship with colonization success in mice. Wound microbiome diversity was also found to be negatively associated with bacterial load, indicating a relationship between microbiome community diversity and bioburden. Cell viability comparisons among preservation treatments were also made with samples from an additional 11 patients where it was found that freezing did not present a significant reduction in viability. Although it was known that metagenomic studies can be enhanced by cryogenic archives, results of the current study indicate an expanded utility in biofilm and microbiome research.
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