Nitrosomonas eutropha D23 - an in vitro, evaluation of its metabolic phenotype and evidence for its bio-activity

Abstract

Contemporary lifestyle and hygienic behaviours have led to cutaneous microbial imbalances, including the loss of ammonia-oxidising bacteria (AOB) from the human skin. Ubiquitous in soil, AOB perform the nitritation step within the global nitrogen cycle. Their sole source of energy originates from the oxidation of ammonia (NH3) to nitrite (NO2 - ), a reaction during which also nitric oxide (NO) is produced. Although believed to have been a constituent of the normal skin microbiome in the past, their role when present on the skin remains unknown. Topical application of Nitrosomonas eutropha D23 (AO+ Mist, MotherDirt™, AOBiome LLC) has been proposed to restore absent AOB on the skin. It may also serve as a biological tool to modulate the bioavailability of NO and its metabolites via oxidation of NH3 present in sweat. There is limited information on the metabolic phenotype available, and often this is inferred from the model organism, Nitrosomonas europaea. It was hypothesised that the metabolic products NO and/or NO2 - produced by N. eutropha D23 are of sufficient quantities to elicit in vitro and/or in vivo bioactivity. Experiments were carried out using an array of analytical instrumentation. The biochemical assessment was carried out in pure, live cultures of N. eutropha D23, and revealed the ability of energy-starved N. eutropha D23 to produce high levels of NO2 - over time, immediately upon addition of substrate. The production of NO by N. eutropha D23 was quantified by gas-phase chemiluminescence in dependence of temperature, pH, concentration of substrate, and bacterial density. Maximal in vitro production of NO was observed at 25 mmol L-1 NH4 + , at pH 8, and at 37-40 °C. Despite the apparent low yields of NO (fentomoles per N. eutropha D23 cell), these were found to be of sufficient quantities to trigger dispersal of Pseudomonas aeruginosa (PAO1) biofilms. NO was found to be the principal agent underpinning the reduction in P. aeruginosa biofilm biomass, and this effect was dependent on live N. eutropha D23. Furthermore, significant reductions in biofilm biomass were also demonstrated for the majority of clinical isolates of P. aeruginosa tested. Further biological activity of N. eutropha D23 was explored in healthy human volunteers, who applied the commercial product of these bacteria on the skin for 14 days. Although the study was underpowered and only exploratory, local and systemic changes were observed upon application of N. eutropha D23 (e.g. transepidermal water loss, blood pressure). Unexpectedly, circulatory NO2 - concentrations were not found to be elevated. Instead, biomarker analysis in plasma samples suggested the existence of a potential oxidation process. This was indicated by a slight increase in oxidative stress markers, a decrease in total free thiols, and a generalised decrease in circulatory free thiols in the majority of participants, including reduced glutathione and cysteine. This systemic signature warrants further investigations in order to mechanistically characterise the action of N. eutropha D23 on the skin, which may ultimately offer opportunities for its medicinal use beyond the treatment of inflammatory skin diseases.

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Identifiers

ORCID for Diogo Goncalves daSilva: orcid.org/0000-0001-7430-3037

ORCID for Martin Feelisch: orcid.org/0000-0003-2320-1158

ORCID for Michael Ardern-Jones: orcid.org/0000-0003-1466-2016

ORCID for Jeremy Webb: orcid.org/0000-0003-2068-8589

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