Rapid and Early Detection of Salmonella Serotypes with Hyperspectral Microscopy and Multivariate Data Analysis

Abstract

This study was designed to evaluate hyperspectral microscope images for early and rapid detection of Salmonella serotypes Enteritidis, Heidelberg, Infantis, Kentucky, and Typhimurium at incubation times of 6, 8, 10, 12, and 24 h. Images were collected by an acousto-optical tunable filter hyperspectral microscope imaging system with a metal halide light source measuring 89 contiguous wavelengths every 4 nm between 450 and 800 nm. Pearson correlation values were calculated for incubation times of 8, 10, and 12 h and compared with data for 24 h to evaluate the change in spectral signatures from bacterial cells over time. Regions of interest were analyzed at 30% of the pixels in an average cell size. Spectral data were preprocessed by applying a global data transformation algorithm and then subjected to principal component analysis (PCA). The Mahalanobis distance was calculated from PCA score plots for analyzing serotype cluster separation. Partial least-squares regression was applied for calibration and validation of the model, and soft independent modeling of class analogy was utilized to classify serotype clusters in the training set. Pearson correlation values indicate very similar spectral patterns for reduced incubation times ranging from 0.9869 to 0.9990. PCA score plots indicated cluster separation at all incubation times, with incubation time Mahalanobis distances of 2.146 to 27.071. Partial least-squares regression had a maximum root mean squared error of calibration of 0.0025 and a root mean squared error of validation of 0.0030. Soft independent modeling of class analogy correctly classified values at 8 h (98.32%), 10 h (96.67%), 12 h (88.33%), and 24 h (98.67%) with the optimal number of principal components (four or five). The results of this study suggest that Salmonella serotypes can be classified by applying a PCA to hyperspectral microscope imaging data from samples after only 8 h of incubation.