Our in-house laboratory team recently examined the variability in oxygen tolerance among anaerobic bacteria associated with the human intestinal tract, with the aim of comparing potential pathogens with the normal microbiota. Their findings were presented in a poster at “Anaerobe 2021: the microbiota and beyond”, a meeting organized by the Society for Anaerobic Microbiology.
Although anaerobic incubation methods, such as anaerobic jars and workstations, have been used for many years in clinical laboratories to isolate pathogenic anaerobes, recent years have seen increased interest in potentially therapeutic bacterial species originating from the normal intestinal microbiota. To better understand the growth requirements of such species, we compared the abilities of selected anaerobic pathogens, “normal microbiota” and more recently characterized potentially therapeutic strains to grow on agar at 35°C in the presence of increasing oxygen concentrations.
The effects of oxygen were studied using two different Whitley Workstations: an A35 model to provide strictly anaerobic “reference” conditions and an H35 to provide precisely controlled atmospheric oxygen concentrations in increments of 0.1%.
In initial experiments with high bacterial inoculum densities, the potential pathogens Bacteroides fragilis and Clostridioides difficile grew in the presence of up to 2.4% v/v oxygen, while common “normal microbiota” species of Bifidobacterium, Fusobacterium and Finegoldia tolerated 0.5 – 1.0% and Eggerthella lenta tolerated 0.1%. Potentially therapeutic species of Roseburia, Alistipes, Blautia and Faecalibacterium grew only in strictly anaerobic conditions and were unable to grow when incubated in 0.1% oxygen.
Using the bacterial strains that tolerated at least 0.1% oxygen, we then performed quantitative experiments to determine the percentage recoveries of smaller inocula (100 – 300 cfu on surface spread plates) in the presence of increasing oxygen, in comparison with strictly anaerobic colony counts. In 2.0% v/v oxygen, inoculum recovery for two B. fragilis strains and one C. difficile was >80%. In contrast, recovery of an F. magna strain decreased from 83% in 0.1% oxygen to <1% in 0.5% oxygen.
Our results suggest that variability in oxygen tolerance between anaerobe species may not be evident when high numbers of bacterial cells are present, such as when working with heavily colonized clinical specimens or pure cultures but can be significant at low inoculum levels. We also demonstrated that the normal human microbiota, in particular the potentially therapeutic strains, can be highly intolerant of oxygen. Strict anaerobic conditions and careful techniques are required when working with such strains and are, of course, recommended when working with any anaerobes.