Facility-wide Eradication of Corynebacterium bovis by using PCR-validated Vaporized Hydrogen Peroxide

Abstract

Facility-wide Corynebacterium bovis eradication was established using vaporized hydrogen peroxide (VHP) decontamination guided by C. bovis PCR surveillance. Prior attempts limited to culling PCR-positive mice and decontaminating affected rooms were ineffective in preventing recurrence. Because research aims often require trafficking to and use of procedural cores, a 12-mo facility-wide C. bovis PCR surveillance of 2064 specimens was performed and documented that, despite the presence of few clinically hyperkeratotic mice, 35% of the murine housing and use space was contaminated by C. bovis. The airways of IVC racks and air-handling units (AHU) provided a substantive niche for C. bovis survival, comparable to the primary enclosure, with 26% of murine and 22% of airway specimens PCR-positive for C. bovis. Equipment airway VHP sterilization in a 'flex room' required an 'active-closed' setting with the IVC rack connected to the AHU set to the VHP cycle, because 12% of specimens from 'static-open' VHP-exposed airways remained PCR-positive for C. bovis, whereas 0% of specimens from active-closed VHP exposures were positive. VHP decontamination of the 29,931-ft2 facility was completed in 2 mo. C. bovis PCR testing of IVC exhaust plenums for 200 d in previously C. bovis-affected rooms confirmed that none of the 259 specimens tested were PCR-positive for the organism. Monthly surveillance identified a single recurrence during June 2017 (month 9), ensuring rapid culling of C. bovis PCR-positive mice and acute VHP decontamination of equipment and rooms. Molecular persistence of C. bovis was resolved in procedural and personnel areas, and no murine or housing specimens tested C. bovis PCR-positive during study months 11 and 12. Furthermore, since the conclusion of the 12-mo study, none of the 452 additional murine, cell biologic, environmental, and monthly equipment surveillance specimens tested were C. bovis PCR-positive, documenting an 11-mo period of facility-wide C. bovis eradication to date. Study invalidation due to C. bovis can be avoided through PCR surveillance for the organism, immediate culling of PCR-positive mice, and acute VHP decontamination of affected areas.

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

“…At the time of the present report, no standard procedures to exclude or to monitor and not tolerate C. bovis were in place. 18 Mice. NSGS mice (stock no.…”

“…An outbreak of C. bovis in the Stabile Research Building was first recognized in October 2016. Prior to the depopulation of all C. bovis PCR-positive immunodeficient mice, and the VHP decontamination of all facility rooms and equipment, 18 we assessed murine recipients of CMML PDX for CMML engraftment and PCR-monitored these mice, IVC rack exhaust plenums, and IVC primary enclosures used to house these mice for C. bovis until the completion of study at clinical endpoints. Mice were housed in IVC microisolation caging (Blueline, Tecniplast, Buguggiate, Italy) in a doubled-sided IVC rack configuration, with HEPA-filtered air supplied and exhausted by separate, mobile air-handling units (SmartFlow, Tecniplast).…”

“…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.…”

“…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.…”

“…Furthermore, although C. bovis is known to cause hyperkeratotic acanthotic dermatitis in nude mice and skin disease in haired Pkrdc scid mice and hairless immunocompetent SKH1-Hrhr mice, can be transmitted in resected PDX or allograft tumors, results in broad facility contamination, and can be eradicated by PCR testing and VHP sterilization, [3][4][5][16][17][18]27 only anecdotal comments have thus far supported the notion that research data may be altered due to C. bovis infection of immunodeficient mice. 6,15,27 Consequently, during the development of methods resulting in the first facility-wide eradication of C. bovis, 18 we also monitored cohorts of CMML PDX-recipient NSGS mice, some of which exhibited periocular alopecia, for evidence of CMML engraftment and for C. bovis status. Murine and environmental specimens representative of these CMML PDX recipients were PCR-evaluated for C. bovis, and mice were monitored to IACUC-approved clinical endpoints for CMML PDX engraftment.…”