Managing the bacterial contamination risk in an axenic mice animal facility

Abstract

A gap exists between good laboratory practises with axenic animals and procedures applied. This work aimed at choosing the appropriate disinfectant between sodium dichloroisocyanurate (MB-10) and potassium peroxymonosulfate (Virkon<sup>™</sup>) disinfectants and to adjust the soaking time of the material used with ISOcage biosafety stations. Another aim was to compare the microbial load on cage systems hosting mice since two weeks in axenic (AR) rooms and typical specific-pathogen-free (SPF) non-axenic rooms (NAR) to identify resistant microorganisms targeted for longer soaking disinfection as well as evaluate microbial concentration reduction procedures in AR. <i>Staphylococcus</i> was the most frequently isolated genus (AR and NAR). An average of three spore-forming microorganisms per cage were counted from AR. The disinfection time to reach 1 log reduction for <i>Bacillus atrophaeus</i> spores varied from 138 (100 ppm MB-10) to 290 (Virkon<sup>™</sup>) seconds and below 20 seconds for <i>S. epidermidis</i> (100 ppm MB-10). AR management protocols lead to 1000 times lower microorganisms burden compared to NAR. Data comparing microbial load on SPF and axenic facilities can be used as comparison for facilities aiming at improving the effectiveness of their microbial control procedures.

Contaminants and Where to Find Them: Microbiological Quality Control in Axenic Animal Facilities

“…BHI being a non-selective medium allowing fastidious and non-fastidious bacteria, yeast, and fungi, it represents a good polyvalent compromise between several of the media used by other axenic users. However, knowing that a great part of axenic environment are colonizers of the human, other mice hosted in axenic facility and environmental microorganisms (Basic and Bleich, 2019;Lebeuf et al, 2021), the choice of media and incubation conditions are adapted to most probable contaminant of axenic cages. Consequently, knowing that some microorganisms might not growth in this media, the use of other methods of detection is recommended to detect them.…”

“…The frequency at which quality control is conducted for a certain microorganism should be representative of its likelihood of entering into the cages. The literature suggests that most of the contaminants detected in axenic animal facilities are human skin colonizer and environmental microorganisms (Basic and Bleich, 2019;Lebeuf et al, 2021). According to researchers, the risk of viruses and parasites entering into the cages is very low (Nicklas et al, 2015;Basic and Bleich, 2019), and consequently, they require quality control detection less often than bacteria and environmental microorganisms and Archaea, which might also colonize axenic animals (Samuel and Gordon, 2006;Ramezani et al, 2018).…”

“…The axenic animal facility used in this report consisted of two rooms that are exclusively used for axenic or gnotobiotic purposes, as previously described by Lebeuf et al (2021). Each room contained a full rack of 30 Isocages and an Isocage Biosafety Station (IBS, Tecniplast R ).…”

“…All the mice were handled according to the CPAUL and the CCPA guidelines and as described in the ethics authorization request # 2016-109-4A before the protocol started. Sets of three axenic mice were artificially contaminated with only one of the following non-pathogenic microbial strains: Staphyloccocus epidermidis (SE, 37 • C aerobic, isolated from a previously contaminated axenic Isocage, GenBank sequence ID: MT585538); Lactobacillus reuteri (LR, 37 • C anaerobic, isolated from a non-axenic mouse feces, GenBank sequence ID: CP054657.1); Bacillus atrophaeus (BA, 25 • C aerobic, from the author's bacterial collection and previously used as a model for the management of contaminants in axenic mice facilities (Lebeuf et al, 2021), GenBank sequence ID: MN826517.1); Clostridium sporogenes (CS, spore forming anaerobic bacterium, mice gut colonizer, often used in germfree mice researches (Itoh and Mitsuoka, 1985;Furusawa et al, 2013), from Luc Trudel at Université Laval, GenBank sequence ID: MT356160.1); or Saccharomyces cerevisiae (SC, to act as a negative bacterial control, common mice gut colonizer (Clark, 2017), also to provide data about yeast detection in axenic context, isolated from Fleichmann's instant yeast, GenBank sequence ID: NR_111007.1). Three axenic mice were maintained as negative controls.…”

“…It has thus been documented that the presence of other microorganisms than bacteria is sometimes sought by molecular method i.e., for Archaea and molds (Packey et al, 2013) and certain specific viruses (Nicklas et al, 2015). However, a recent publication has demonstrated that bacteria from mammal skin and gut, as well as environmental microorganisms can be found on axenic room cages (Lebeuf et al, 2021).…”