Characterisation of the influence of Nitrosomonas eutropha on the skin regulation of NO metabolism and the microbiome

Abstract

The skin is the largest organ and demonstrates a wide spectrum of physiological functions which facilitate its various biological characteristics including host defence, water regulation, thermoregulation and micronutrient synthesis. However, in settings of cutaneous inflammation, these processes are significantly disrupted and can result in systemic inflammation and altered biochemistry. Previous studies using microbiological methodology based on plate culture identification, were unable to fully characterise the skin microbiome. However, with the advent of culture independent techniques such as ribosomal RNA 16s analysis, the true diverse structure of human microbiome systems on epithelial surfaces has been uncovered. In this thesis I describe the optimal methodology for 16s rRNA skin microbiome sampling which has not been investigated comprehensively.

Nitric oxide (NO) has recently been shown to be a key regulator of biofilm function in respiratory mucosa, and has been shown to regulate the microbiome at epithelial surfaces. In this study of healthy human volunteers (n=23), I set out to characterise the skin NO physiology in different body sites and after exercise.
I show that NO emanation from the skin is enhanced by sweating and exercise. In contrast to previous work, I found no evidence that skin pH provided significant regulation of NO emanation.

The common soil bacteria, Nitrosomonas eutropha, which is not found on human skin, facilitates the oxidation of ammonia to form nitrite (hence ammonia oxidising bacteria, AOB). Nitrite is a key component of the pathway to NO formation by further reduction in the epithelial surface. Recently Nitrosomonas eutropha has become commercially available as a spray for skin application. However, the question of whether the microbe can survive on human skin, or whether it has any physiological effect has not been addressed.

I show for the first time that Nitrosomonas can survive on the skin surface and become part of the skin microbiome. In addition, enhanced skin NO emanation and increased skin pH (p=0.011) were noted following Nitrosomonas application. Furthermore, the impact of perturbation of the skin microbiome with Nitrosomonas was examined using 16s rRNA analysis. I show that the presence of Nitrosomonas in the skin microbiome did not lead to a significantly altered diversity or microbiome structure at a genus or phylum level. Despite recent work suggesting loss of skin microbiome diversity following S. aureus infection in atopic dermatitis, these findings point to a robustness of the skin microbiome. Whilst no significant differences pre and post AOB application were seen in an extensive panel of markers of systemic NO physiology, a significant elevation of total free thiols (TFTs) was detected in plasma (p=0.006). This is of note, because TFT elevation has been associated with reduced cardiovascular morbidity and mortality which may suggest that investigation of AOB application to the skin should be further explored.

The findings presented here demonstrate that sweating, pH, and microbiome are likely to be important in regulating skin NO emanation and potentially systemic inflammation. The delivery of Nitrosomonas eutropha to the skin in a human clinical study as reported here, offers a unique opportunity to study the skin microbiome and demonstrates a resilience in the microbiome community to new pathogens. Overall this study suggests that application of AOBs as a cutaneous ‘probiotic’ is safe and offers significant clinical potential.

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Identifiers

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

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

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