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.Abbreviations: AHU, air handling unit; BI, biological indicator; CI, chemical indicator; VHP, vaporized hydrogen peroxide

Effects of Corynebacterium bovis on Engraftment of Patient-derived Chronic-Myelomonocytic Leukemia Cells in NSGS Mice

“…7,25,26,29 In addition to the obvious potential for morbidity or mortality, one detrimental drawback of even subclinical infections of immunodefi cient mice is the variability induced into research data, normative physiologic responses, and to study metrics, thereby invalidating the fi ndings. 25 When murine studies involve rare, genetically unique human PDX specimens and when experimental therapeutics are available only in limited aliquots, the tolerance for data variability and study invalidation due to opportunistic microbes becomes substantially less. C. bovis is a known cause of hyperkeratosis, which manifests clinically in a minority of infected immunodefi cient mice, with infection remaining clinically unapparent in many C. bovis PCR-positive mice.…”

“…6,15,27 Herein, we provide the fi rst documentation of research data being altered by the PCRconfi rmed persistence of C. bovis infection of immunodefi cient mice. This present documentation of altered research study data was conducted during the C. bovis outbreak previously described in detail, 18,25 during which C. bovis infection of PCRpositive mice was confi rmed through both aerobic bacterial culture and microbe identifi cation and by direct visualization of gram-positive rods in histopathologic specimens of skin from immunodefi cient mice.…”

“…18 An 'active-closed' method of VHP sterilization of housing equipment and rooms is an important component to successful facility-wide eradication of C. bovis and for mitigating the prevalence of other opportunistic microbes. 1,24,25 Despite C. bovis being among the more egregious of opportunistic outbreaks affecting immunodefi cient mice and although 58% of cancer center respondents confi rm experiencing a case of C. bovis hyperkeratosis in immunodefi cient mice, most facilities (74%) do not test or attempt to characterize the extent of environmental contamination caused by a clinical case of C. bovis hyperkeratosis. Nor do most cancer centers routinely PCR survey the environment or murine inventories for early evidence of C. bovis prior to its clinical manifestation.…”

“…We have also recently reported that the integrity of data derived from immunodeficient mice acquired in a topographically complex research setting relies on enhanced biosecurity measures, including monitoring for opportunistic microbes and the regular implementation of a validated room and equipment sterilization method. 18,24,25 To this end, we developed procedures for sterilizing murine IVC racks and air-handling units by using vaporized hydrogen peroxide (VHP) and PCR monitoring. 24 We then applied this method of PCR environmental and murine monitoring and VHP equipment and room sterilization to the first documented facility-wide eradication of Corynebacterium bovis.…”

“…18 In addition, we documented the PCR prevalence and VHP mitigation of a broad spectrum of murine opportunistic microbes, including Staphylococcus xylosus, Proteus mirabilis, and Pasteurella pneumotropica biotype Heyl. 25 During the development of these PCR testing and C. bovis eradication methods, we surveyed the attending veterinarians of other SPF and viral antibody-free murine facilities based at National Cancer Institute (NCI)-designated Comprehensive Cancer Centers and herein report the C. bovis monitoring and mitigation practices at these institutions.…”