Direct ferrous sulfate exposure facilitates the VBNC state formation rather than ferroptosis in Listeria monocytogenes.

Listeria monocytogenes frequently causes Listeriosis in humans and animals. In present study, we discovered that in the presence of FeSO4, L. monocytogenes became viable but non-culturable (VBNC), and remained virulent to Caenorhabditis elegans. The killing assay indicated that these VBNC cells kept sensitive to tetracycline, differing from dormant cells. Transcriptomic analysis revealed more gene transcription occurrence in the VBNC cells compared to dormant cells, involving stress response and ribosome binding. No ferroptosis hallmarks were observed in the VBNC cells, whereas the application of either intracellular Fe2+ chelator or the ferroptosis inhibitor arrested the formation of VBNC state by FeSO4, as well as by Benzakonium chloride or Haz-Tab. This implicated the universal involvement of intracellular Fe2+ and other cascades related to ferroptosis in the formation of VBNC state in L. monocytogenes. Taken together, we discovered an iron-induced VBNC state in L. monocytogenes, and provided clues to further understanding their potential risks.

A Broad-Spectrum Phage Endolysin (LysCP28) Able to Remove Biofilms and Inactivate Clostridium perfringens Strains.

Clostridium perfringens is a gram-positive, anaerobic, spore-forming bacterium capable of producing four major toxins which cause disease symptoms and pathogenesis in humans and animals. C. perfringens strains carrying enterotoxins can cause food poisoning in humans and are associated with meat consumption. An endolysin, named LysCP28, is encoded by orf28 from C. perfringens bacteriophage BG3P. This protein has an N-terminal glycosyl-hydrolase domain (lysozyme) and a C-terminal SH3 domain. Purified LysCP28 (38.8 kDa) exhibited a broad spectrum of lytic activity against C. perfringens strains (77 of 96 or 80.21%), including A, B, C, and D types, isolated from different sources. Moreover, LysCP28 (10 µg/mL) showed high antimicrobial activity and was able to lyse 2 × 107 CFU/mL C. perfringens ATCC 13124 and C. perfringens J21 (animal origin) within 2 h. Necessary due to this pathogenic bacterium's ability to form biofilms, LysCP28 (18.7 µg/mL) was successfully evaluated as an antibiofilm agent in both biofilm removal and formation inhibition. Finally, to confirm the efficacy of LysCP28 in a food matrix, duck meat was contaminated with C. perfringens and treated with endolysin (100 µg/mL and 50 µg/mL), which reduced viable bacteria by 3.2 and 3.08 units-log, respectively, in 48 h at 4 °C. Overall, the endolysin LysCP28 could potentially be used as a biopreservative to reduce C. perfringens contamination during food processing.

The Broad Host Range Phage vB_CpeS_BG3P Is Able to Inhibit Clostridium perfringens Growth.

Clostridium perfringens is an important pathogen for both humans and animals, causing human foodborne disease and necrotic enteritis in poultry. In the present study, a C. perfringens-specific phage, vB_CpeS_BG3P (designated as BG3P hereafter), was isolated from chicken farm sewage. Both electron microscopy and phylogenetic analysis suggested that phage BG3P is a novel phage belonging to Siphoviridae family. Phage BG3P exhibited a broad host range against different C. perfringens isolates (90.63% of strains were infected). Sequencing of the complete genome revealed a linear double-stranded DNA (43,528 bp) with 28.65% GC content. After sequence analysis, 73 open reading frames (orfs) were predicted, of which only 13 were annotated with known functions. No tRNA and virulence encoding genes were detected. It should be noted that the protein of orf 15 has 97.92% homology to C. perfringens-specific chloramphenicol resistance protein, which has not been reported for any C. perfringens phage. Phylogenetic analysis of the ssDNA binding protein demonstrated that this phage is closely related to C. perfringens phages phiSM101 and phi3626. In considering future use as an antimicrobial agent, some biological characteristics were observed, such as a good pH (3−11) stability and moderate temperature tolerance (<60 °C). Moreover, bacteriophage BG3P showed a good antimicrobial effect against C. perfringens liquid cultures. Thus, phage treatment with MOI ≥ 100 completely inhibited bacterial growth compared to untreated cultures. Although phage BG3P shows good lytic efficiency and broad host range in vitro, future development and application may need to consider removal of the chloramphenicol-like resistance gene or exploring its lysin for future antibacterial applications.