Variability in oxygen tolerance among bacterial strains associated with the normal intestinal microbiota

Anaerobic incubation methods are widely used to cultivate pathogenic anaerobes, but recent years have seen increased interest in potentially therapeutic species originating from the normal intestinal microbiota. We compared the abilities of selected anaerobic pathogens, “normal microbiota” and more recently characterized potentially therapeutic strains to grow on agar at 37°C in the presence of increasing oxygen concentrations, using a variable atmosphere workstation to control oxygen concentration in increments of 0.1%. In initial screening with a high inoculum of 105 to 106 cfu on streak plates, Bacteroides fragilis and Clostridioides difficile strains grew in the presence of up to 2.4% v/v oxygen. Bifidobacterium, Fusobacterium and Finegoldia strains tolerated 0.5 – 1.0% and Eggerthella lenta tolerated 0.1%. Strains of Roseburia, Alistipes, Blautia and Faecalibacterium grew only in strictly anaerobic conditions (<0.01% oxygen) and not in 0.1% oxygen. For strains tolerating >0.1% oxygen in initial experiments, percentage recoveries of smaller inocula (100 – 300 cfu on surface spread plates) were determined in atmospheric oxygen concentrations increasing by 0.1% increments, in comparison with strictly anaerobic colony counts. In 2.0% v/v oxygen, inoculum recovery for 2 × B. fragilis and 1 × C. difficile, was >90%, while recovery of a second C. difficile strain was 25%. For F. magna, inoculum recovery ranged from approximately 100% in 0.1% oxygen to <1% in 0.5% oxygen. These findings demonstrate the variable oxygen tolerance of obligately anaerobic bacteria and emphasize the need for stringent anaerobiosis when culturing the more recently characterized strains currently being developed as live biotherapeutic products.