Altered Schaedler flora

The altered Schaedler flora (ASF) is a community of eight bacterial species: two Lactobacilli, one Bacteroides, one spiral bacteria of the Flexistipes genus, and four extremely oxygen sensitive (EOS) Fusobacterium species. The bacteria are selected for their dominance and persistence in the normal microflora of mice, and for their ability to be isolated and grown in laboratory settings. Germ-free animals, mainly mice, are infected with ASF for the purpose of studying the gastrointestinal (GI) tract. Intestinal commensal bacteria play an important role in affecting gene expression of the GI tract, immune responses, nutrient absorption, and pathogen resistance. The standardized microbial cocktail enabled the controlled study of microbe and host interactions, role of microbes, pathogen effects, and intestinal immunity and disease association, such as cancer, inflammatory bowel disease, diabetes, and other inflammatory or autoimmune diseases. Also, compared to germfree animals, ASF mice have fully developed immune system, resistance to opportunistic pathogens, and normal GI function and health, and are a great representation of normal mice The altered Schaedler flora (ASF) is a community of eight bacterial species: two Lactobacilli, one Bacteroides, one spiral bacteria of the Flexistipes genus, and four extremely oxygen sensitive (EOS) Fusobacterium species. The bacteria are selected for their dominance and persistence in the normal microflora of mice, and for their ability to be isolated and grown in laboratory settings. Germ-free animals, mainly mice, are infected with ASF for the purpose of studying the gastrointestinal (GI) tract. Intestinal commensal bacteria play an important role in affecting gene expression of the GI tract, immune responses, nutrient absorption, and pathogen resistance. The standardized microbial cocktail enabled the controlled study of microbe and host interactions, role of microbes, pathogen effects, and intestinal immunity and disease association, such as cancer, inflammatory bowel disease, diabetes, and other inflammatory or autoimmune diseases. Also, compared to germfree animals, ASF mice have fully developed immune system, resistance to opportunistic pathogens, and normal GI function and health, and are a great representation of normal mice The GI tract is particularly difficult to study due to its complex host-pathogen interaction. With 107-1012 bacteria, 400-plus species, and variations between individuals, there are many complications in the study of a normal gastrointestinal system. For example, it is problematic to assign biological function to specific microbes and community structure, and to investigate the respective immune responses. Furthermore, the varying mice microbiome need to be under controlled conditions for repetitions of the experiments. Germfree mice and specific pathogen free (SPF) mice are helpful in addressing some of the issues, but inadequate in many areas. Germfree mice are not a good representation of normal mice, with issues of enlarged cecum, low reproductive rates, poorly developed immune system, and reduced health. SPF mice still contain varying microbiota, just without certain known pathogen species. There is a need in the scientific field for a known bacterial mixture that is necessary and sufficient for healthy mice. In the mid-1960s, Russell W. Schaedler isolated and grew bacteria from normal and SPF mice. Aerobic and less oxygen-sensitive anaerobic bacteria are easy to culture. Fusiform and EOS bacteria are difficult to culture, even though they represent the majority of the normal rodent microbiota. He selected for the bacteria that dominated and can be isolated in culture, and then colonized germfree mice with different bacteria combinations. For example, one combination could include Escherichia coli, Streptococcus fecalis, Lactobacillus acidophilus, L. salivarius, Bacteroides distasonis, Clostridium spp., and some anaerobic fusiform bacteria. Certain defined microflora are able to restore germfree mice to resemble normal mice with reduced cecal volume, restored reproductive ability, colonization resistance, and well developed immune system. So named Schaedler flora, the defined microflora combinations was widely used in gnotobiotic studies. In 1978, the National Cancer Institute revised and standardized the Schaedler flora, with the help of Roger Orcutt. In what was named altered Schaedler flora, four bacteria of the original mixture were kept: the two Lactobacilli, the Bacteroides, and the EOS fusiform bacterium. Four more bacteria from the microbiome isolates were added: a spirochete bacterium and three new EOS fusiform bacteria. Due to the limited technology of the time, not much was known of the specific bacterial genus and species. These bacteria are persistent and dominant in normal and SPF mice GI tract. Confirmation of the correct microbiota presence was limited to looking at the cell Morphology (biology), biochemical traits and growth characteristics With the recent advancement in biotechnology, researchers were able to determine the precise genus and species of the ASF bacteria using sequence analysis of 16S rRNA. The strains identified are different from the presumptive identities. The distribution of the bacteria species in the gut depends on their need of and aversion to oxygen, flow rate, and substrate abundance, with variability based on age, gender and other pathogens present in the mice.ASF 360 and ASF 361 are Lactobacilli. Lactobacilli are filamentous, Gram-positive, aerotolerant bacteria, and common colonizers of the GI mucosa and squamous epithelia of mice. ASF 360 was thought to be L. acidophilus. However, 16SrRNA results showed that it is closely related to but distinct from L. acidophilus. ASF 360 is a novel lactobacillus species; clustered with L. acidophilus and L. lactis. ASF 361 has nearly identical 16S rRNA sequences to L. murinus and L. animalis. Both species are routinely found in GI tracts of mice and rats. A thorough examination of the two species and strains is necessary to determine the identity of ASF 361 with more confidence. ASF 361 is completely distinct from the L. salivarius that it was believed to be. ASF 360 and ASF 361 colonize in high numbers in the esophagus, the stomach, the small intestine, and the cecum due to their aerotolerance. ASF 519 is related to B. distasonis, the species it was mistaken to be. However, like the previous bacteria, it is a distinct species by 16S rRNA evidence. Bacteroides species are often found in GI tracts of mammals, and included non-motile, Gram-negative, anaerobic, rod-shaped bacteria. Recently, many of Bacteroides species are being recognized as actually belonging to other genera, like Porphyromonas and Prevotella. In the case of ASF 519, it belongs to the newly named Parabacteroides genus, along with the bacteria formerly known as distasonis, merdae, CDC group DF-3, and forsythus. The spiral-shaped obligate anaerobe ASF 457 can be found in small amounts in the small intestine, and in high concentration in the large intestine. This bacterium is related to G. ferrireducens, Deferribacter thermophilus, and Flexistipes sinusarabici. ASF 457 is later named Mucispirillum schaedleri. The species is related to the Flexistipes phylum with iron-reducing environmental isolates. EOS fusiform bacteria make up the majority of the intestinal microbiota, and are mainly found in the large intestine. They vastly outnumber facultative anaerobic and aerobic bacteria. All four fusiform belong to the low G+C content, Gram-positive bacteria group. ASF 356 is of the Clostridium species, closely related to Clostridium propionicum. ASF 502 is most related to Ruminococcus gnavus. ASF 492 is confirmed by 16S rRNA sequences as Eubacterium pexicaudatum, and is closely related to Roseburia ceciola. ASF 356, ASF 492, and ASF 502 are all part of the low G+C, Gram-positive bacteria of the Clostridium cluster XIV. ASF 500 is a deeper branch into the low G+C, Gram-positive bacteria of Firmicutes, Bacillus-Clostridium group, but not much can be found in the GenBank database on this branch of Clostridium cluster Only mice have been colonized with ASF in experiments, since ASF bacteria originate from mice intestinal microbiome. Germfree mice are colonized by ASF through one of two methods. Pure culture of each living ASF bacteria can be grown in anaerobic conditions in laboratory setting. Lactobacilli and Bacteroides are given by gavage to germfree mice first to establish a microbial environment in the GI tract, which then supports the colonization of the spirochete and fusiform bacteria that are given later. An alternative way is to inoculate the drinking water of germfree mice with fresh feces from cecum and colon of gnotobiotic mice (ASF mice), over a period of four days. The establishment and concentration of each bacteria species vary slightly depending on the age, gender, and environmental conditions of the mice.