An Optimized Method to Assess Viable Escherichia coli O157:H7 in Agricultural Soil Using Combined Propidium Monoazide Staining and Quantitative PCR.

Yulong Fu,Yangyang Jia,Zhe Ye,Muhammad Zaffar Hashmi,Jiahui Fan,Chaofeng Shen

doi:10.3389/fmicb.2020.01809

Yulong Fu, Yangyang Jia + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2020.01809

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Abstract

Agricultural soil contaminated by manure is becoming an important source for the transmission of foodborne pathogens. There is an urgent need for a rapid and accurate method for viable pathogen detection in agricultural soil samples. Propidium monoazide (PMA) is a DNA-binding dye that can inhibit the amplification of DNA from dead cells through subsequent quantitative polymerase chain reaction (qPCR), thus allowing for viable cells detection and quantification. The objective of this study was to detect viable Escherichia coli O157:H7 in the agricultural soils by PMA-qPCR. In this study, cell extraction and gradient density centrifugation were incorporated before PMA-qPCR to reduce the interference of soil particle including turbidity and a high ratio of dead cells. The optimized treatment conditions were determined as follows, the maximum removal of DNA from dead cells was achieved by 1.067 g/mL Percoll of centrifugation and 50 μM PMA treatment. Under these conditions, the turbidity of paddy soil suspensions decreased from 3500 to 28.4 nephelometric turbidity units (NTU), and the ratio of viable cells to dead cells increased from 0.001 to 1.025%. For typical agricultural soils collected in China, as low as 102colony-forming units (CFU)/g of viable cells could be accurately detected in the presence of a large number of dead cells (107 CFU/g) by the optimized PMA-qPCR. Significantly, with comparable accuracy, the optimized PMA-qPCR assay was more sensitive, accessible and rapid than conventional culture methods. In addition, the viable but non-culturable (VBNC) state of E. coli O157:H7 cells in paddy soils, which often escaped the detection by conventional culture methods, could be quantitatively characterized by the optimized PMA-qPCR method. Potentially, the optimized PMA-qPCR can be further applied for viable pathogens detection and give insight into the prevalence of VBNC E. coli O157:H7 in agricultural soil.

Highlights

Fresh produces have frequently been associated with foodborne disease outbreaks due to Escherichia coli O157:H7 (Yeni et al, 2016; Wadamori et al, 2017)
The Ct value obtained from viable E. coli O157:H7 is accurate compared to the positive control (Supplementary Figure S2)
The results suggested that the optimized PMAqPCR method is more effective methods as compared to other methods for detection of viable cells in the presence of a large number of dead cells. (Herold et al, 2004), 100 CFU/10 g of E. coli O157:H7 was inoculated into soils

Summary

Introduction

Fresh produces have frequently been associated with foodborne disease outbreaks due to Escherichia coli O157:H7 (Yeni et al, 2016; Wadamori et al, 2017). E. coli O157:H7 poses a significant threat to public health because of its low infectious dose and severe pathogenicity, leading to complications such as hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (Pennington, 2010). Agricultural soils are an important reservoir for pathogens, and plays an important role in the fecal-oral route (Alegbeleye et al, 2018). To better protect public health, there is a great demand for rapid, sensitive, specific, and accurate quantification of pathogens in agricultural soil

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