The Germ-Free Factories (Video)
In order to model the human microbiome in animals, it’s best to begin with a blank slate.
The gut microbiome—the total DNA content of microbes inhabiting our bodies—carries a wellspring of information about the role and function of the 30-plus trillion bacteria, fungi, protozoa and viruses that live on and in us.
Chinese scientists estimate more than 3 million microbial genes reside in the gut microbiota, and the Human Microbiome Project, created in 2007 by the US National Institutes of Health to characterize the human microbiome and analyze its role in human health and disease, have found that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin, oral cavity and vagina.
With evidence mounting that the gut microbiome can significantly affect the progression of disease, particularly metabolic disorders such as diabetes and autoimmune diseases such as lupus, inflammatory bowel disease and arthritis, demand is growing for specific kinds of mice that can be used to examine interactions between the host and the microbiota.
One such mouse is the germ-free mouse, which lacks all microorganisms (as determined within the limitations of the detection methods available). Another type of mouse is the gnotobiotic mouse, which is intentionally inoculated with a cocktail of one or more non-pathogenic microorganisms, all of which are known.
A valuable tool for studying our guts
Germ-free mice, in particular, are uniquely valuable to companies developing microbiome-based therapeutics. Germ-free mice are also a pillar of many academic research experiments looking at how disruptions in this ecosystem influence diseases as diverse as irritable bowel syndrome and cancer, and how we might be able to reverse these diseases by altering the microbiota. “One way to think about germ-free mice is as a kind of a clean slate or even better a blank notebook where you can start to develop a consortium or group of bacteria within a germ free animal and then study the mechanisms of that bacteria in the host.”
To conduct meaningful research, the living conditions of the germ-free and gnotobiotic mice must remain as pristine as the mice. The mice must be maintained in isolators and under strict protocols that prevent exposure to foreign organisms that could colonize the gut and change the animal’s microbiomes.
In addition to preserving mice in these bubble-like environments, animal research facilities also need to ensure the quality of the germ-free experiments by regularly analyzing animals for microbes that shouldn’t be there. Charles River’s Microbiome Diagnostics Services (MDS), launched in 2017 in response to the growing demand for germ-free mice, offers a repertoire of such services that extend from standard-practice aerobic and anaerobic culturing, and motility testing to monitor germ-free mice to next-generation sequencing methods that identify the microbial diversity and abundance in colonized mice, says Christina Parkinson, Manager of Health Monitoring and Bacteriology at Charles River.
Aerobic bacteria, such as Staphyloccus sp., use the oxygen present in the air for energy metabolism. In contrast, anaerobic bacteria, such as Clostridium sp., do not need oxygen from the air for energy metabolism. Labs like Charles River screen for both types by growing aerobic and anaerobic bacteria in a liquid culture medium. This medium creates an environment which is oxygen rich at the top to support aerobic bacteria and oxygen deficient at the bottom supporting anaerobic growth. Additionally Charles River has the ability to plate samples directly onto agar in their selected environments. This can be of particular importance for the detection of Anaerobic bacteria that are more sensitive to oxygen. This is achieved through the use of an anaerobic chamber limiting the samples exposure to oxygen prior to inoculation onto selective media.
Parkinson says her site also conducts an additional procedure, the motility test, using microscopy. A small sample of the material submitted for culture is placed on a glass slide and observed under a light microscope to see if any rods or cocci are present. “Whether or not they grow we should be able to determine if bacteria are present based upon whether they are visible doing light microscopy,” Parkinson.
Molecular methods for studying the gut microbiome
Other strategies to determine if mice are germ free are more molecular-based. One can sequence the gene for 16S ribosomal DNA, which is widely used in studies of bacterial evolutionary relationships, to identify bacteria and determine if the microbe has been seen before or is something that is entirely new.
“The other molecular tool we have been developing, and it’s really more a larger picture tool for us, is next generation sequencing,” says Henderson. “Let’s say someone has added a defined bacterial culture set to an animal, we can then monitor that animal before, during or after an experiment and see if the treatment has had an impact on the change in that bacterial flora.”
But Henderson says the high specificity of next-generation sequencing also exposes one of the major dilemmas confronting microbiome researchers today. “It is very evident that the phenotype of a transgenic animal can be greatly influenced by the bacteria that are present. If you did a fecal transplant from one colony you might get different results then if you took bacteria from another colony.”
Henderson says what we really need is a stable bacterial microbiome that is going to give us consistent results. “Just like we see with the process of genetic drift [when events change the gene frequencies in a population for no reason whatsoever] there is also a microbiome drift,” he says. “The sex of the animal, the feed, lighting conditions can all influence the microbiome.”
“Because the microbiome has become pretty popular, a lot of research facilities are acquiring germ-free isolators. A lot of them are really good at doing the research but not that great at providing the QC. That’s where we step in and provide a service. Our clients can submit samples from their animals and we can evaluate them to make sure they are really germ-free or that they contain the microbes intended by the researcher.”