Raman spectroscopic signatures of carotenoids and polyenes enable label-free visualization of microbial distributions within pink biofilms

Hiroto Horiue,Shinsuke Shigeto,Mai Sasaki,Masanori Toyofuku,Yuki Yoshikawa

doi:10.1038/s41598-020-64737-3

Hiroto Horiue, Shinsuke Shigeto + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-64737-3

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Abstract

Pink biofilms are multispecies microbial communities that are commonly found in moist household environments. The development of this pink stain is problematic from an aesthetic point of view, but more importantly, it raises hygienic concerns because they may serve as a potential reservoir of opportunistic pathogens. Although there have been several studies of pink biofilms using molecular analysis and confocal laser scanning microscopy, little is known about the spatial distributions of constituent microorganisms within pink biofilms, a crucial factor associated with the characteristics of pink biofilms. Here we show that Raman spectroscopic signatures of intracellular carotenoids and polyenes enable us to visualize pigmented microorganisms within pink biofilms in a label-free manner. We measured space-resolved Raman spectra of a pink biofilm collected from a bathroom, which clearly show resonance Raman bands of carotenoids. Multivariate analysis of the Raman hyperspectral imaging data revealed the presence of typical carotenoids and structurally similar but different polyenes, whose spatial distributions within the pink biofilm were found to be mutually exclusive. Raman measurements on individual microbial cells isolated from the pink biofilm confirmed that these distributions probed by carotenoid/polyene Raman signatures are attributable to different pigmented microorganisms. The present results suggest that Raman microspectroscopy with a focus on microbial pigments such as carotenoids is a powerful nondestructive method for studying multispecies biofilms in various environments.

Highlights

Pink biofilms are multispecies microbial communities that are commonly found in moist household environments
We present a new approach based on Raman microspectroscopy to visualizing the distributions of microorganisms within multispecies biofilms without the need of molecular techniques used for Confocal laser scanning microscopy (CLSM)
We carried out two Raman experiments: Raman imaging of a pink biofilm empowered by multivariate curve resolution–alternating least-squares[21,22] (MCR–ALS) and Raman microspectroscopy of isolates from the same pink biofilm

Summary

Introduction

Pink biofilms are multispecies microbial communities that are commonly found in moist household environments. We show that Raman spectroscopic signatures of intracellular carotenoids and polyenes enable us to visualize pigmented microorganisms within pink biofilms in a label-free manner. Raman measurements on individual microbial cells isolated from the pink biofilm confirmed that these distributions probed by carotenoid/polyene Raman signatures are attributable to different pigmented microorganisms. The present results suggest that Raman microspectroscopy with a focus on microbial pigments such as carotenoids is a powerful nondestructive method for studying multispecies biofilms in various environments. We utilized characteristic Raman signatures of a specific class of intracellular biomolecules, that is, polyenes including carotenoids They are commonly found in pigmented microorganisms and serve as an excellent endogenous probe for visualization purposes. Our results demonstrate that the Raman signatures of microbe-produced polyene compounds could be a facile and powerful probe for label-free visualization of microbial distributions within complex multispecies biofilms

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