The anaerobic bacterium designated as M5a3 was originally isolated in 1996 from human fecal samples (Bernalier et al., 1996, doi: 10.1007/s002849900081). However, since its initial isolation, M5a3 has received almost no attention in subsequent research despite its unusual property to produce lactate, along with acetate from H₂ + CO₂ by employing the Wood-Ljungdahl pathway. Lactate production is an uncommon metabolic feature within acetogens.

Heterotrophic reactivation of the lyophilized cells from the original 1996 stock was followed by amplifying and sequencing the 16S rDNA and the bacterium was identified as Intestinibacter bartlettii. Intestinibacter is often found to be a member of the human gut microbiota community. To further investigate the metabolic capabilities of I. bartlettii, whole-genome sequencing was performed using Illumina technology. Gram-staining was performed with cells obtained in its exponential and stationary growth phase. Gram-staining indicated that the bacterium showed a Gram-positive staining during the exponential phase, whereas in the stationary phase, a mixed staining pattern was observed. This staining variation may suggest Gram variability in this species. Also, a marked decrease in cell size was observed from the exponential phase (approx. 14 µm) to the stationary phase (approx. 3 µm). Furthermore, detailed morphological analysis using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were performed and respective micrographs recorded. The heterotrophic substrate spectrum was started to be investigated by growth experiments, which demonstrated its inability to utilize lactose and glycerol as carbon sources. In contrast, fructose served as a favorable substrate and enabled the production of 2.4 mM lactate, 17 mM ethanol and 27 mM acetate, which resulted in a pH decrease to 4.9 of the medium. Growth experiments utilizing H₂ + CO₂ as substrates were performed. The natural antibiotic resistance profile was analyzed using a selection of antibiotics, showing natural resistance against spectinomycin, kanamycin and clarithromycin. In contrast, thiamphenicol and tetracycline consistently inhibited bacterial growth.

In summary, cells are still viable after being stored for 28 years and it could be shown that the heterotrophic metabolic profile fits the original description. Further analysis will be done to investigate the strains characteristics with elucidate its acetogenic properties.