The Fe-S cluster biosynthesis in Enterococcus faecium is essential for anaerobic growth and gastrointestinal colonization.

The facultative anaerobic Gram-positive bacterium Enterococcus faecium is a ubiquitous member of the human gut microbiota. However, it has gradually evolved into a pathogenic and multidrug resistant lineage that causes nosocomial infections. The establishment of high-level intestinal colonization by enterococci represents a critical step of infection. The majority of current research on Enterococcus has been conducted under aerobic conditions, while limited attention has been given to its physiological characteristics in anaerobic environments, which reflects its natural colonization niche in the gut. In this study, a high-density transposon mutant library containing 26,620 distinct insertion sites was constructed. Tn-seq analysis identified six genes that significantly contribute to growth under anaerobic conditions. Under anaerobic conditions, deletion of sufB (encoding Fe-S cluster assembly protein B) results in more extensive and significant impairments on carbohydrate metabolism compared to aerobic conditions. Consistently, the pathways involved in this utilization-restricted carbohydrates were mostly expressed at significantly lower levels in mutant compared to wild-type under anaerobic conditions. Moreover, deletion of sufB or pflA (encoding pyruvate formate lyase-activating protein A) led to failure of gastrointestinal colonization in mice. These findings contribute to our understanding of the mechanisms by which E. faecium maintains proliferation under anaerobic conditions and establishes colonization in the gut.

New Insights into the Lactic Acid Resistance Determinants of Listeria monocytogenes Based on Transposon Sequencing and Transcriptome Sequencing Analyses.

Listeria monocytogenes is a foodborne pathogen that can tolerate a variety of extreme environments. In particular, its acid resistance (AR) capability is considered one of the key factors threating food safety. Here, we employed a microbial functional genomic technology termed transposon sequencing (Tn-seq), leading to the identification of two genes involved in cell wall peptidoglycan biosynthesis (murF) and phosphate transport (lmo2248) that play key roles in lactic acid resistance (LAR) of L. monocytogenes. Deletion of lmo2248 significantly impaired the ability of LAR in L. monocytogenes, demonstrating the accuracy of the Tn-seq results. Transcriptome analysis revealed that 31.7% of the L. monocytogenes genes on the genome were differentially expressed under lactic acid (LA) treatment, in which genes involved in phosphate transport were influenced most significantly. These findings shed light on the LAR mechanisms of L. monocytogenes, which may contribute to the development of novel strategies against foodborne pathogens. IMPORTANCE Listeria monocytogenes is a Gram-positive foodborne pathogen with high lethality and strong stress resistance, and its strong acid tolerance leads to many foodborne illnesses occurring in low-pH foods. Lactic acid is a generally recognized as safe (GRAS) food additive approved for use by the FDA. However, the genetic determinants of lactic acid resistance in L. monocytogenes have not been fully identified. In this study, the lactic acid resistance determinants of L. monocytogenes were comprehensively identified by Tn-seq on a genome-wide scale. Two genes, murF (cell wall peptidoglycan biosynthesis) and lmo2248 (phosphate transport), were identified to play an important role in the lactic acid resistance. Moreover, genome-wide transcriptomic analysis showed that phosphotransferase system (PTS)-related genes play a key role at the transcriptional level. These findings contribute to a better understanding of the lactic acid resistance mechanism of L. monocytogenes and may provide unique targets for the development of other novel antimicrobial agents.

Synergistic Antibacterial Mechanism of Mannosylerythritol Lipid-A and Lactic Acid on Listeria monocytogenes Based on Transcriptomic Analysis.

Mannosylerythritol lipids-A (MEL-A) is a novel biosurfactant with multiple biological effects. The synergistic antibacterial activity and mechanism of MEL-A and lactic acid (LA) against Listeria monocytogenes were investigated. The synergistic effect resulted in a significant increase in the antibacterial rate compared to LA treatment alone. Genome-wide transcriptomic analysis was applied to deeply investigate the synergistic antibacterial mechanism. Gene Ontology (GO) enrichment analysis showed that the synergy between MEL-A and LA affected many potential cellular responses, including the sugar phosphotransferase system, carbohydrate transport, and ribosomes. KEGG enrichment analysis showed that the PTS system and ribosome-related pathways were significantly enriched. In addition, synergistic treatment affected locomotion and membrane-related cellular responses in GO enrichment analysis and carbohydrate metabolism and amino acid metabolism pathways in KEGG enrichment analysis compared to LA treatment alone. The accuracy of the transcriptome analysis results was verified by qPCR (R2 = 0.9903). This study will provide new insights for the prevention and control of L. monocytogenes.

Functional Genomics Identified Novel Genes Involved in Growth at Low Temperatures in Listeria monocytogenes.

Listeria monocytogenes (Lm) is a foodborne pathogen that can cause severe human illness. Standard control measures for restricting bacterial growth, such as refrigeration, are often inadequate as Lm grows well at low temperatures. To identify genes involved in growth at low temperatures, a powerful functional genomics method Tn-seq was performed in this study. This genome-wide screening comprehensively identified the known and novel genetic determinants involved in low-temperature growth. A novel gene lmo1366, encoding rRNA methyltransferase, was identified to play an essential role in Lm growth at 16°C. In contrast, the inactivation of lmo2301, a gene encoding the terminase of phage A118, significantly enhanced the growth of Lm at 16°C. The deletion of lmo1366 or lmo2301 resulted in cell morphology alterations and impaired the growth rate in milk and other conditions at low temperatures. Transcriptomic analysis revealed that the Δlmo1366 and Δlmo2301 mutants exhibited altered transcriptional patterns compared to the wild-type strain at 16°C with significant differences in genes involved in ribosome structural stability and function, and membrane biogenesis, respectively. This work uncovered novel genetic determinants involved in Lm growth at 16°C, which could lead to a better understanding of how bacteria survive and multiply at low temperatures. Furthermore, these findings could potentially contribute to developing novel antibacterial strategies under low-temperature conditions. IMPORTANCE Listeria monocytogenes is a Gram-positive pathogen that contributes to foodborne outbreaks due to its ability to survive at low temperatures. However, the genetic determinants of Lm involved in growth at low temperatures have not been fully defined. Here, the genetic determinants involved in the low-temperature growth of Lm were comprehensively identified on a genome-wide scale by Tn-seq. The gene lmo1366, encoding rRNA methyltransferase, was identified essential for growth under low-temperature conditions. On the other hand, the gene lmo2301, encoding terminase of phage A118, plays a negative role in bacterial growth at low temperatures. The transcriptomic analysis revealed the potential mechanisms. These findings lead to a better understanding of how bacteria survive and multiply at low temperatures and could provide unique targets for novel antibacterial strategies under low-temperature conditions.

Enterocins: Classification, Synthesis, Antibacterial Mechanisms and Food Applications.

Enterococci, a type of lactic acid bacteria, are widely distributed in various environments and are part of the normal flora in the intestinal tract of humans and animals. Although enterococci have gradually evolved pathogenic strains causing nosocomial infections in recent years, the non-pathogenic strains have still been widely used as probiotics and feed additives. Enterococcus can produce enterocin, which are bacteriocins considered as ribosomal peptides that kill or inhibit the growth of other microorganisms. This paper reviews the classification, synthesis, antibacterial mechanisms and applications of enterocins, and discusses the prospects for future research.

The Lipoteichoic Acid-Related Proteins YqgS and LafA Contribute to the Resistance of Listeria monocytogenes to Nisin.

Listeria monocytogenes is a major pathogen contributing to foodborne outbreaks with high mortality. Nisin, a natural antimicrobial, has been widely used as a food preservative. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. A mariner transposon library was constructed in L. monocytogenes, leading to the identification of 99 genes associated with the innate resistance to nisin via Transposon sequencing (Tn-seq) analysis. To validate the accuracy of the Tn-seq results, we constructed five mutants (ΔyqgS, ΔlafA, ΔvirR, ΔgtcA, and Δlmo1464) in L. monocytogenes. The results revealed that yqgS and lafA, the lipoteichoic acid-related genes, were essential for resistance to nisin, while the gtcA and lmo1464 mutants showed substantially enhanced nisin resistance. Densely wrinkled, collapsed surface and membrane breakdown were shown on ΔyqgS and ΔlafA mutants under nisin treatment. Deletion of yqgS and lafA altered the surface charge, and decreased the resistance to general stress conditions and cell envelope-acting antimicrobials. Furthermore, YqgS and LafA are required for biofilm formation and cell invasion of L. monocytogenes. Collectively, these results reveal novel mechanisms of nisin resistance in L. monocytogenes and may provide unique targets for the development of food-grade inhibitors for nisin-resistant foodborne pathogens. IMPORTANCE Listeria monocytogenes is an opportunistic Gram-positive pathogen responsible for listeriosis, and is widely present in a variety of foods including ready-to-eat foods, meat, and dairy products. Nisin is the only licensed lantibiotic by the FDA for use as a food-grade inhibitor in over 50 countries. A prior study suggests that L. monocytogenes are more resistant than other Gram-positive pathogens in nisin-mediated bactericidal effects. However, the mechanisms of L. monocytogenes involved in nisin resistance have not yet to be fully defined. Here, we used a mariner transposon library to identify nisin-resistance-related genes on a genome-wide scale via transposon sequencing. We found, for the first time, that YqgS and LafA (Lipoteichoic acid-related proteins) are required for resistance to nisin. Subsequently, we investigated the roles of YqgS and LafA in L. monocytogenes stress resistance, antimicrobial resistance, biofilm formation, and virulence in mammalian cells.

Antibacterial Efficacy and Mechanism of Mannosylerythritol Lipids-A on Listeria monocytogenes.

Mannosylerythritol lipids-A (MEL-A) is a novel biosurfactant with excellent surface activity and potential biomedical applications. In this study, we explored the antibacterial activity and the underlying mechanisms of MEL-A against the important food-borne pathogen Listeria monocytogenes. The bacterial growth and survival assays revealed a remarkable antibacterial activity of MEL-A. Since MEL-A is a biosurfactant, we examined the cell membrane integrity and morphological changes of MEL-A-treated bacteria by biochemical assays and flow cytometry analysis and electron microscopes. The results showed obvious damaging effects of MEL-A on the cell membrane and morphology. To further explore the antibacterial mechanism of MEL-A, a transcriptome analysis was performed, which identified 528 differentially expressed genes (DEGs). Gene ontology (GO) analysis revealed that the gene categories of membrane, localization and transport were enriched among the DEGs, and the analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways demonstrated significant changes in the maltodextrin ABC transporter system and stress response system. Furthermore, the growth of L. monocytogenes could also be significantly inhibited by MEL-A in milk, a model of a real food system, suggesting that MEL-A could be potentially applied as an natural antimicrobial agent to control food-borne pathogens in the food industry.