PCR Prevalence of Murine Opportunistic Microbes and their Mitigation by Using Vaporized Hydrogen Peroxide

Abstract

Exposing immunodeficient mice to opportunistic microbes introduces risks of data variability, morbidity, mortality, and the invalidation of studies involving unique human reagents, including the loss of primary human hematopoietic cells, patient-derived xenografts, and experimental therapeutics. The prevalence of 15 opportunistic microbes in a murine research facility was determined by yearlong PCR-based murine and IVC equipment surveillance comprising 1738 specimens. Of the 8 microbes detected, 3 organisms- Staphylococcus xylosus, Proteus mirabilis, and Pasteurella pneumotropica biotype Heyl-were most prevalent in both murine and IVC exhaust plenum specimens. Overall, the 8 detectable microbes were more readily PCR-detectable in IVC exhaust airways than in murine specimens, supporting the utility of PCR testing of IVC exhaust airways as a component of immunodeficient murine health surveillance. Vaporized hydrogen peroxide (VHP) exposure of IVC equipment left unassembled (that is, in a 'static-open' configuration) did not eliminate PCR detectable evidence of microbes. In contrast, VHP exposure of IVC equipment assembled 'active-closed' eliminated PCR-detectable evidence of all microbes. Ensuring data integrity and maintaining a topographically complex immunodeficient murine research environment is facilitated by knowing the prevalent opportunistic microbes to be monitored and by implementing a PCR-validated method of facility decontamination that mitigates opportunistic microbes and the risk of invalidation of studies involving immunodeficient mice.

Figure 1.

Boxplot depiction of the median percentage of PCR-positive murine specimens for each microbe that was PCR-detected during the 12-mo surveillance. The median percentage-positive value is represented by the thick horizontal line inside each box, and the lower and upper limits of each box portray the interquantile range (that is, the 2nd and 4th quantiles). Murine specimens were collected monthly for 12 mo. Blue dots indicate the percentage of tests that were PCR-positive during each month for each microbe.

Figure 2.

Boxplot depiction of the median percentage of PCR-positive exhaust plenum specimens for each microbe that was PCR-detected during the 12-mo surveillance. The median percentage-positive value is represented by the thick horizontal line inside each box, and the lower and upper limits of each box portray the interquantile range (that is, the 2nd and 4th quantiles). Exhaust plenum specimens were collected monthly from each mouse-occupied IVC rack for 12 mo. Blue dots indicate the percentage of tests that were PCR-positive during each month for each microbe.

Figure 3.

Monthly mean percentage of PCR-positive tests for Staphylococcus xylosus in soiled, washed, and VHP-exposed exhaust plenums during the 12-mo surveillance. Staphylococcus xylosus was the most prevalent microbe detected but became PCR-undetectable in exhaust plenums after active-closed VHP exposure during months 8 through 10.