Abstract
Germ-Free (GF) research has required highly technical pressurized HEPA-ventilation anchored systems for decades. Herein, we validated a GF system that can be easily implemented and portable using Nested Isolation (NesTiso). GF-standards can be achieved housing mice in non-HEPA-static cages, which only need to be nested ‘one-cage-inside-another’ resembling ‘Russian dolls’. After 2 years of monitoring ~100,000 GF-mouse-days, NesTiso showed mice can be maintained GF for life (>1.3 years), with low animal daily-contamination-probability risk (1 every 867 days), allowing the expansion of GF research with unprecedented freedom and mobility. At the cage level, with 23,360 GF cage-days, the probability of having a cage contamination in NesTiso cages opened in biosafety hoods was statistically identical to that of opening cages inside (the ‘gold standard’) multi-cage pressurized GF isolators. When validating the benefits of using NesTiso in mouse microbiome research, our experiments unexpectedly revealed that the mouse fecal microbiota composition within the ‘bedding material’ of conventional SPF-cages suffers cyclical selection bias as moist/feces/diet/organic content (‘soiledness’) increases over time (e.g., favoring microbiome abundances of Bacillales, Burkholderiales, Pseudomonadales; and cultivable Enterococcus faecalis over Lactobacillus murinus and Escherichia coli), which in turn cyclically influences the gut microbiome dynamics of caged mice. Culture ‘co-streaking’ assays showed that cohoused mice exhibiting different fecal microbiota/hemolytic profiles in clean bedding (high-within-cage individual diversity) ‘cyclically and transiently appear identical’ (less diverse) as bedding soiledness increases, and recurs. Strategies are proposed to minimize this novel functional form of cyclical bedding-dependent microbiome selection bias.
Highlights
Elevators or stairs due to their large footprint and combined weight with their anchored ventilation systems
With a total of 23,360 GF cage-days, we determined that the cumulative probability of having a cage contamination event for each cage-opening of Nested Isolation (NesTiso) sets inside biosafety hoods can be identical to the probability of cage contamination inside multi-cage pressurized GF isolators (‘gold standard’ in this study)
Implemented using commercially available static cages, we housed cohorts of GF mice born in HEPA-pressurized isolators by placing the mice (SAMP1/YitFc [SAMP]25,26, C57BL/6 [B6], and Swiss Webster [SW]) inside mouse cages, and nesting such cages inside larger rat cages
Summary
Elevators or stairs due to their large footprint and combined weight with their anchored ventilation systems. To expand the current GF experimental efficiency of available systems[19,20], we propose to use static ventilation for housing GF mice in individual GF cages that require no mechanical ventilation, pressurization, or HEPA filtration. Our NesTiso cages are portable since one person can transport and maintain them indefinitely without the need of positive ventilation, allowing the establishment and movement of GF animals or colonies outside of GF facilities for unprecedented experimental purposes. Microbiome experiments showed (i) that soiled (i.e., mixed with mouse excrements) corncob bedding material remarkably favors the enrichment of fecal murine Bacillales, Burkholderiales and Pseudomonadales, and (ii) that two different levels of bedding ‘soiledness’ can result in different fecal colonization patterns in GF mice, which combined represent a novel source of data variability and bias not currently accounted for in mouse research. Strategies are here proposed to minimize this novel functional form of Cyclical Bedding-dependent (CyBeD) Microbiome Bias in animal microbiome studies
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