Direct Observation of the Sporicidal Action of Hydrogen Peroxide on Bacillus atrophaeus Spores By Optical Trapping Raman Microscopy

Abstract

Hydrogen peroxide (H2O2), a strong oxidizer, is frequently used for chemical sterilization in aseptic food processing. Due to their inherent robustness, bacterial spores, i.e. spores of Bacillus atrophaeus, are commonly employed to evaluate the efficacy of the sterilization procedure [1]. Despite its widespread use, however, the details of the sporicidal action of H2O2 are not yet fully understood, with various mechanisms being discussed in the literature [2,3]. Here, we employ optical trapping Raman microscopy to elucidate the sporicidal mechanism of H2O2. Optical trapping Raman microscopy allows us to isolate, investigate, and manipulate individual spores from a sample (Fig. 1A). Combining optical microscopy with the real-time, label-free chemical information obtained from Raman spectroscopy directly reveals the sequence of steps that lead to oxidative spore death after H2O2 exposure (Fig. 1B).We find that spore degradation begins with the fast, cooperative release of dipicolinic acid (DPA), a key spore biomarker and a major component of spore’s core. This indicates a breach of the inner membrane surrounding the core. The onset of DPA release depends on the concentration of H2O2. DPA outflux is accompanied by the formation of oxidation products (mostly carbonyl compounds) and oxidation continues after exchange of DPA with the surrounding medium is complete. The remaining spore shell then slowly disintegrates and continues to shrink on a timescale of hundreds of seconds. Simultaneous observation of spore morphology and size by optical microscopy and particle tracking corroborates this three-step mechanism.The presented assay allows on-line monitoring of spore composition and morphology, which directly reveals the mechanism underlying the sporicidal action of H2O2. In addition, trapped spores can be exposed to different chemical or physical stimuli (temperature, light, chemical composition of the surrounding medium, etc.) to investigate a variety of industrially relevant sterilization conditions. Jildeh, Z. B., Wagner, P. H. & Schöning, M. J. Sterilization of Objects, Products, and Packaging Surfaces and Their Characterization in Different Fields of Industry: The Status in 2020. physica status solidi (a) 218, 2000732 (2021). Setlow, P. & Christie, G. What’s new and notable in bacterial spore killing. World J Microbiol Biotechnol 37, 144 (2021). Setlow, P. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol 101, 514-525 (2006). Figure 1