Rapid determination of spore germinability of Clostridium perfringens based on microscopic hyperspectral imaging technology and chemometrics

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Highlights

  • The effects of different AGFK concentrations (0, 50, 100, 200 mM/mL) on C. perfringens spore germination.

  • A novel rapid method for the measurement of spore germination rate based on microscopic hyperspectral imaging technology (HSIT) was proposed.

  • Multivariate analyses (GA-siPLS and GA-PLS) and the gray symbiotic matrix (GLCM) were used to extract highly correlated spectral and spatial descriptors from the time-series data from microscopic HSIT, respectively.

  • Single spectral, spatial signals and the data fusion of spectral and spatial information were used to predict the Srate, the OD600% and Ca2+-DPA % by GA-PLS during spore germination process.

Abstract

The Gram-positive, anaerobic, spore-forming bacterium, Clostridium perfringens (C. perfringens) causes a variety of diseases in humans and other animals. Spore germination is thought to be the first stage of infection by C. perfringens. AGFK, a mixture of l-asparagine, d-glucose, d-fructose, and potassium ions, is an effective nutrient germinant. The objective of this study was to investigate the effects of different AGFK concentrations (0, 50, 100, 200 mM/mL) on C. perfringens spore germination. This paper proposes a novel rapid method for the measurement of spore germinability based on microscopic hyperspectral imaging technology (HSIT). The spore germination rate (Srate), the OD600% and Ca2+-DPA% of C. perfringens were determined by chemical methods under different concentrations of AGFK. The results showed that spores have a maximum germination rate of 94.59% after 80 min with 100 mM/mL AGFK. Microscopic HSIT revealed that the spectral and spatial characteristics of spores varied during the spore germination process. Multivariate analyses (GA-siPLS and GA-PLS) and the gray symbiotic matrix (GLCM) were used to extract highly correlated spectral and spatial descriptors from the time-series data from microscopic HSIT, respectively. Single spectral, spatial signals and data fusion of spectral and spatial information were then used to predict the Srate, the OD600% and Ca2+-DPA % by GA-PLS, respectively. The result show that the Srate calibration was built by GA-PLS using data fusion variables and yielded acceptable results (Rc = 0.96, RMSEC = 0.64, Rcv = 0.93, RMSEP = 0.87, Rp = 0.94). The OD600% optimal model was built by GA-PLS using image variables and yielded acceptable results (Rc = 0.93, RMSEC = 19.36, Rcv = 0.91, RMSEP = 24.36, Rp = 0.89). For Ca2+-DPA %, the model based on the fusion of spectral and imaging data was optimal. The Ca2+-DPA % calibration yielded acceptable results (Rc = 0.95, RMSEC = 49.83, Rcv = 0.93, RMSEP = 58.98, Rp = 0.92). This work demonstrates the potential of microscopic HSIT for the non-destructive detection of spore germinability. The data fusion models also significantly improved the prediction of spore germinability. In conclusion, microscopic HSIT exhibits considerable promise for nondestructive diagnostics of spore germination.

Introduction

Clostridium perfringens (C. perfringens) is a Gram-positive, anaerobic, spore-forming pathogenic bacterium causing gastrointestinal (GI) diseases in humans and animals (Grass and Gould, 2013, Banawas and Paredes-Sabja, 2013). The spores of C. perfringens, which are extremely resistant to environmental stresses, such as heat, radiation, and toxic chemicals, can survive food preservation processes and upon germination outgrowth to be converted into vegetative cells that can breed and produce enterotoxins, cause food spoilage and safety risks (Setlow, P., and E. A. Johnson, 2007; Monma and Hatakeyama, 2015). Thus, C. perfringens spores are important morphotypes for infection (Grass and Gould, 2013; May et al., 2016).

Spores in nature germinate only in response to nutrients, termed germinants (Komatsu and Inui, 2012, Paredes-Sabja et al., 2008). Once spore germination, they lost the extreme resistance of dormant spores and are thus relatively easy to kill (Komatsu and Inui, 2012). A mixture of l-asparagine, d-glucose, d-fructose, and potassium ions (AGFK) is an effective nutrient germinant (Setlow, 2003). Within minutes or hours of mixing spores, different concentration of AGFK can cause the different effect on the spores germination (McClane, Robertson and Li and Xue, 2008; Wang and Li, 2018). Spore germination rate (Srate), which is routinely calculated by plant count, is a very important significance to the meat industry and the research field on the prevention and control of food spoilage and foodborne disease of C. perfringens. The release of Ca2+-DPA (Ca2+-DPA%) varies with the duration of germination and can be used to explain the spore germination process. The completion of Ca2+-DPA release is a hallmark of the completion of spore germination (Kawarizadeh and Tabatabaei, 2019). The progress of a spore's germination can be determined based on the transformation of spores from phase-bright to dark-phase on microscopic images. Changes in spore refractivity can be measured by phase-contrast microscopy due to the release and spore cortex hydrolysis of Ca2+-DPA (Setlow and Wang, 2017). Thus, changes in the brightness of time-series images provide a better understanding of the overall germination process. Besides, monitoring the optical density at 600 nm (OD600) of spore cultures, which drops 60% upon complete spore germination (Paredes-Sabja and Setlow et al., 2011). The relationship between OD600 and the level of spore germination was confirmed by phase-contrast microscopy. Thus, the Srate, the loss of C. perfringens spore refractivity (OD600%) and the release of Ca2+-DPA (Ca2+-DPA%) are key indicators of the spore germination and can be used to assess the rate of spore germination or the optimal concentration of germination agents.

At present, various traditional physical and chemical methods are used to calculate Srate, OD600% and Ca2+-DPA % during spore germination (Rao and Feeherry, 2018). Such methods can achieve high accuracies, but they are tedious, expensive, and time-consuming, which makes them unsuitable for rapid assessments (Korel and Luzuriaga, 2001). Raman spectroscopy (Wang and Doona, 2016), near-infrared spectroscopy (NIRS) (Eady and Setia, 2019) and hyperspectral imaging technology (HSIT) (Tao and Peng, 2012; Schultz and Nielsen, 2001) are rapid, noninvasive, and chemical-free techniques that have been widely developed for measurements of chemical data and microorganisms in the food industry. Such spectroscopic techniques have unique advantages, but remain limited due to the inherently weak scattering signals and the strong interference of biological fluorescence background in Raman spectroscopy, the limited “single spectrum” without spatial information in NIR analysis, and the low resolution for microorganisms in the HSI approach. Microscopic hyperspectral imaging technology (HSIT) is an emerging technique that integrates microscopic imaging and spectroscopy to obtain 2-D spatial and 1-D spectral information from analytes (Monma and Hatakeyama, 2015). In recent years, microscopic HSIT has become known as a promising method that integrates hyperspectral data with microscopic imaging, which has been successfully used to capture spectral and spatial information of tissue sections (Li and Xue, 2008; Gao and Smith, 2015).

In this study, microscopic HSIT was used to rapidly predict Srate, OD600% and Ca2+-DPA % over time during C. perfringens spore germination under different AGFK concentrations. We found that spectral at wavelength regions of 484.2–610.4 nm, 442.3–654.1 nm and 463.2–588.7 nm were significantly correlated with the Srate, OD600%, and Ca2+-DPA % of C. perfringens spore during germination process. In addition, microscopic hyperspectral images of spore had the potential of determine spore germination according color, texture and form during spore germination process. Therefore microscopic hyperspectral was used to predict the spore germinability under different concentration germinents to control C. perfringens. The objectives of this study were to: (1) measured the Srate, acquire time-lapse phase-contrast images to quantifying the loss of C. perfringens spore optical density (OD600%) and Ca2+-DPA%; (2) compare and analyze the effects different AGKF concentrations on Srate, OD600%, and Ca2+-DPA % during spore germination; (3) acquire microscopic hyperspectral images, and extract and preprocess spectral and image data; and (4) build the calibration models of Srate, as well as the OD600% and Ca2+-DPA% based on the single spectral, spatial and data fusion signals under different AGFK concentrations, respectively. The optimum model was then selected and verified.

Section snippets

Preparation of strains and spores

Wild-type C. perfringens (strain C1) was directly isolated from vacuum-packaged cooked meat by the Microbiology Laboratory of the College of Food Science and Technology at the He Nan Agricultural University (Zhengzhou, China), and identified by Sangon Biotech Co., Ltd. (Shanghai, China). Spores were prepared using our previously-described method (Daniel et al., 2008). The spore crop containing ~107 CFU/mL was stored at −80 °C until use.

The spore crop was prepared separately from each strain of

The spore germination rate of C. perfringens

The concentration of germinant solution plays a critical role in initiating spore germination in C. perfringens. Thus, the rate of AGFK-induced spore germination was dependent on the AGFK concentration. To determine the maximum germination at specific concentrations of AGFK, the Srate was examined at 0, 50, 100, and 200 mM/mL of AGFK. Different AGFK concentrations had significant effects on the Srate of C. perfringens spores. The Srate of the AGFK-induced spores increased with increasing

Conclusions

C. perfringens spores are important factors causing food spoilage and the swelling of food bags. Following spore germination, the exotoxin is one of the foodborne pathogens with strong pathogenicity. According to Srate, OD600%, and Ca2+-DPA %, spore germination was analyzed under different AGFK concentrations. Furthermore, this paper presents a novel method based on microscopic HSIT to successfully predict the Srate, OD600%, and Ca2+-DPA % of spores at different AGFK concentrations. The method

Author contributions

Yao-Di Zhu designed the study, finished the experiment, and drafted the manuscript; Jia-Ye-Zhang assisted the experiment and collected test data; Miao-Yun Li improve the research plan and revise the manuscript; Li-Jun Zhao revised the manuscript; Hong-rong Ren and Long-Gang Yan helped the experiment and collected test data; Gai-Ming Zhao helped improve the research plan; and Chao-Zhi Zhu guided the experiment.

Declaration of competing interest

The authors have no relevant financial interests or conflicts of interest to disclose.

Acknowledgements

This work was financially supported by the National key r&d projects in the 13th five-year plan of China (No. 2018YFD0401200), the National Natural Science Foundation of China (No. 31571856), the Henan Science and Technology Major Project (No. 161100110800), the key science and technology Support Program of Henan province (No. 192102110216), National modern agriculture (beef yak) industrial technology system construction special (CARS-37), and Science and technology innovation talent support

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    The authors contributed equally to this study.

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