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Phages provide a potential therapy for multi-drug-resistant (MDR) bacteria. However, a significant portion of viral genes often remains unknown, posing potential dangers. The identification of non-essential genes helps dissect and simplify phage genomes, but current methods have various limitations. In this study, we present an in vivo two-plasmid transposon insertion system to assess the importance of phage genes, which is based on the V. cholerae transposon Tn6677, encoding a nuclease-deficient type I-F CRISPR–Cas system. We first validated the system in Pseudomonas aeruginosa PAO1 and its phage S1. We then used the selection marker AcrVA1 to protect transposon-inserted phages from CRISPR-Cas12a and enriched the transposon-inserted phages. For a pool of selected 10 open-reading frames (2 known functional protein genes and 8 hypothetical protein genes) of phage S1, we identified 5 (2 known functional protein genes and 3 hypothetical protein genes) as indispensable genes and the remaining 5 (all hypothetical protein genes) as dispensable genes. This approach offers a convenient, site-specific method that does not depend on homologous arms and double-strand breaks (DSBs), holding promise for future applications across a broader range of phages and facilitating the identification of the importance of phage genes and the insertion of genetic cargos.

It has been reported that about a quarter of the world’s agriculture products is unable to be consumed each year because of mold contamination, resulting in incalculable economic losses. Despite modern food technology and the various preservation techniques available, the problem of mold contamination of food is still not adequately controlled. In this study, we simulated the biofilm formed by Aspergillus niger and Penicillium glaucum in liquid and solid food in 96 well cell culture plates and polycarbonate membrane models, respectively, and investigated the fungicidal effect of IPL on planktonic and biofilm molds at three different capacitance parameters at room and refrigerator temperatures. The results show that IPL can achieve fungicidal rates of over 99% for planktonic molds and over 90% for biofilm molds, and that the smaller the capacitance, the more frequent the irradiation required to achieve the same fungicidal rate. In addition, temperature, A. niger or Penicillium glaucum have no effect on the fungicidal effect of IPL. We believe that IPL is a promising non-thermal physical sterilization technique for fungal inhibition on food surfaces.

ABSTRACT Lactiplantibacillus plantarum and Saccharomyces cerevisiae are frequently co-isolated in food, although playing different roles. This study aimed at investigating the microbial interaction between L. plantarum and S. cerevisiae, especially cell-cell direct interaction and their mechanism. Cell-cell and supernatant-cell coculture models were set up, with CFU counting, OD600 measurement, optical and atomic force microscopy performed to examine the growth and morphology of L. plantarum and S. cerevisiae cells. In cell-cell coculture model, L. plantarum cells inhibited S. cerevisiae growth (inhibition rate ~80%) with its own growth pattern unaffected. Cell-cell aggregation happened during coculture with surface roughness changed and partial S. cerevisiae cell lysis. Mature (24 h) L. plantarum cell-free culture supernatant showed inhibition (35%-75%) on S. cerevisiae growth independent of pH level, while supernatant from L. plantarum-S. cerevisiae coculture showed relatively stronger inhibition. Upon transcriptomics analysis, hypothesis on the mechanism of microbial interaction between L. plantarum and S. cerevisiae was demonstrated. When L. plantarum cell density reached threshold at 24 h, all genes in lamBDCA quorum sensing (QS) system was upregulated to potentially increase adhesion capability, leading to the aggregation to S. cerevisiae cell. The downregulation of whole basic physiological activity from DNA to RNA to protein, cell cycle, meiosis, and mitogen-activated protein kinase (MAPK) signaling pathways, as well as growth maintenance essential genes ari1, skg6, and kex2/gas1 might induce the decreased growth and proliferation rate and partial death of S. cerevisiae cells in coculture. IMPORTANCE L. plantarum and S. cerevisiae are frequently co-isolated in food, although playing different roles. The co-existence of L. plantarum and S. cerevisiae could result in variable effects, raising economic benefits and safety concerns in food industry. Previous research has reported the microbial interaction between L. plantarum and S. cerevisiae mainly rely on the signaling through extracellular metabolites. However, cell-cell aggregation has been observed with mechanism remain unknown. In the current study, the microbial interaction between L. plantarum and S. cerevisiae was investigated with emphasis on cell-cell direct interaction and further in-depth transcriptome level study showed the key role of lamBDCA quorum sensing system in L. plantarum. The results yield from this study demonstrated the antagonistic effect between L. plantarum and S. cerevisiae.

Pediococcus acidilactici may significantly reduce the pH-value, and thus has different influence, including serving as a probiotic in human microbiota but a spoilage in human food as it could change the flavor. Pediococcus acidilactici is also capable of entering into the viable but non-culturable (VBNC) state causing false negative results of standard culture-based detection method. Thus, development of detection method for VBNC state P. acidilactici is of great significance. In this study, propidium monoazide (PMA) combined with cross priming amplification (CPA) was developed to detect the VBNC cells of P. acidilactici and applied on the detection in different systems. With detection limit of 104 cells/ml, high sensitivity, and 100% specificity, PMA-CPA can successfully detect VBNC cells of P. acidilactici and be applied in with high robustness.

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.

Salmonella enterica is a typical foodborne pathogen with multiple toxic effects, including invasiveness, endotoxins, and enterotoxins. Viable but nonculturable (VBNC) is a type of dormant form preserving the vitality of microorganisms, but it cannot be cultured by traditional laboratory techniques. The aim of this study is to develop a propidium monoazide-crossing priming amplification (PMA-CPA) method that can successfully detect S. enterica rapidly with high sensitivity and can identify VBNC cells in food samples. Five primers (4s, 5a, 2a/1s, 2a, and 3a) were specially designed for recognizing the specific invA gene. The specificity of the CPA assay was tested by 20 different bacterial strains, including 2 standard S. enterica and 18 non-S. enterica bacteria strains covering Gram-negative and Gram-positive isolates. Except for the two standard S. enterica ATCC14028 and ATCC29629, all strains showed negative results. Moreover, PMA-CPA can detect the VBNC cells both in pure culture and three types of food samples with significant color change. In conclusion, the PMA-CPA assay was successfully applied on detecting S. enterica in VBNC state from food samples.

Formation of viable but non-culturable (VBNC) status in methicillin-resistant Staphylococcus aureus (MRSA) has never been reported, and it poses a significant concern for food safety. Thus, this study aimed to firstly develop a rapid, cost-effective, and efficient testing method to detect and differentiate MRSA strains in the VBNC state and further apply this in real food samples. Two targets were selected for detection of MRSA and toxin, and rapid isothermal amplification detection assays were developed based on cross-priming amplification methodology. VBNC formation was performed for MRSA strain in both pure culture and in artificially contaminated samples, then propidium monoazide (PMA) treatment was further conducted. Development, optimization, and evaluation of PMA-crossing priming amplification (CPA) were further performed on detection of MRSA in the VBNC state. Finally, application of PMA-CPA was further applied for detection on MRSA in the VBNC state in contaminated food samples. As concluded in this study, formation of the VBNC state in MRSA strains has been verified, then two PMA-CPA assays have been developed and applied to detect MRSA in the VBNC state from pure culture and food samples.