Pathogenic and Virulence Factor Detection on Viable but Non-culturable Methicillin-Resistant Staphylococcus aureus.

Hua Jiang,Qian Ye,Tengyi Huang,Xiaojing Lin,Muxia Yan,Kan Wang,Yanrui Ye,Li Zhang,Ling Chen,Junyan Liu

doi:10.3389/fmicb.2021.630053

Abstract

Food safety and foodborne infections and diseases have been a leading hotspot in public health, and methicillin-resistant Staphylococcus aureus (MRSA) has been recently documented to be an important foodborne pathogen, in addition to its recognition to be a leading clinical pathogen for some decades. Standard identification for MRSA has been commonly performed in both clinical settings and food routine detection; however, most of such so-called “standards,” “guidelines,” or “gold standards” are incapable of detecting viable but non-culturable (VBNC) cells. In this study, two major types of staphylococcal food poisoning (SFP), staphylococcal enterotoxins A (sea) and staphylococcal enterotoxins B (seb), as well as the panton-valentine leucocidin (pvl) genes, were selected to develop a cross-priming amplification (CPA) method. Limit of detection (LOD) of CPA for sea, seb, and pvl was 75, 107.5, and 85 ng/μl, indicating that the analytical sensitivity of CPA is significantly higher than that of conventional PCR. In addition, a rapid VBNC cells detection method, designated as PMA-CPA, was developed and further applied. PMA-CPA showed significant advantages when compared with PCR assays, in terms of rapidity, sensitivity, specificity, and accuracy. Compared with conventional VBNC confirmation methods, the PMA-CPA showed 100% accordance, which had demonstrated that the PMA-CPA assays were capable of detecting different toxins in MRSA in VBNC state. In conclusion, three CPA assays were developed on three important toxins for MRSA, and in combination with PMA, the PMA-CPA assay was capable of detecting virulent gene expression in MRSA in the VBNC state. Also, the above assays were further applied to real samples. As concluded, the PMA-CPA assay developed in this study was capable of detecting MRSA toxins in the VBNC state, representing first time the detection of toxins in the VBNC state.

Highlights

In the past few decades, public health has been a major issue for society
It is commonly found in food poisoning cases
Methicillinresistant S. aureus (MRSA) is one of most widely distributed human and animal pathogens that produce a variety of toxins and cause a range of serious illnesses of the skin, soft tissue, bone, and bloodstream (Tacconelli et al, 2009)

Summary

Introduction

In the past few decades, public health has been a major issue for society. From various points of view, food safety and foodborne infections and diseases have been a leading area of concern. Foodborne infections or diseases caused by food pathogens have made up the majority of the cases (Xu et al, 2021). Apart from that, MRSA has seen an increase in numerous countries and emerged as an important pathogen, accounting for up to 40% of all S. aureus isolates in nosocomial infection, and has caused significant morbidity (Graveland et al, 2011). This is due to the effect of β-lactam resistance in S. aureus, which is mediated by the production of penicillin-binding protein 2a PBP2a or PBP2 encoded by the mecA gene (Moellering, 2012). The development of resistance to β-lactam antibiotics has been a cause of concern among the medical community

Methods

Results

Conclusion