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We have investigated the mode of action of synthetic biocides, (2-(thiocyanomethylthio) benzothiazole(TCMTB), dichlorophen, (commonly used in leather industry for preservation) and natural biocides, oregano and eucalyptus oils, on  Aeromonas hydrophila  using Raman spectroscopy in collaboration with multivariate analysis and 2D correlation spectroscopy to evaluate whether Raman spectra acquired contained valuable information to study the action of biocides on bacterial cells. The growth of  A. hydrophila  in clear and outer edge zone of inhibition differ in their reaction with different biocides, which allows us to highlight the differences as a characteristic of two kinds of bacteria. Such classification helps identify oregano oil as the most effective biocide by altering clear and outer edge zone of bacteria. Standard disk diffusion assay method was used for screening biocide bacteria interactions and later analysed by Raman spectroscopy. The paper also presents the introduction of TCMTB and oregano oil into leather processing stages to examine and determine the antimicrobial effect as an application to real-world setting. Therefore, we conclude that Raman spectroscopy with appropriate computational tools constitutes a powerful approach for screening biocides, which provide solutions to all the industries using biocides including leather industry, considering the potentially harmful effect of biocides to humans and the environment. Graphical abstract

Monitoring the mode of action of synthetic and natural biocides against Aeromonas hydrophila by Raman spectroscopy and chemometrics