Invited review: Role of Bacillus licheniformis in the dairy industry- friends or foes?

Bacillus licheniformis is one of the major spore-forming bacteria with great genotypic diversity in raw milk, dairy ingredients, final dairy products, and is found throughout the dairy processing continuum. Though being widely used as a probiotic strain, this species also serves as a potential risk in the dairy industry based on its roles in foodborne illness and dairy spoilage. Biofilm formation of B. licheniformis in combined with the heat resistance of its spores, make it impossible to prevent the presence of B. licheniformis in final dairy products by traditional cleaning and disinfection procedures. Despite the extensive efforts on the identification of B. licheniformis from various dairy samples, no reviews have been reported on both hazard and benefits of this spore-former. This review discusses the prevalence of B. licheniformis from raw milk to commercial dairy products, biofilm formation and spoilage potential of B. licheniformis, and its potential prevention methods. In addition, the potential benefits of B. licheniformis in the dairy industry were also summarized.

Development and evaluation of rapid and accurate one-tube RPA-CRISPR-Cas12b-based detection of mcr-1 and tet(X4).

The emergence and quick spread of the plasmid-mediated tigecycline resistance gene tet(X4) and colistin resistance gene mcr-1 have posed a great threat to public health and raised global concerns. It is imperative to develop rapid and accurate detection systems for the onsite surveillance of mcr-1 and tet(X4). In this study, we developed one-tube recombinase polymerase amplification (RPA) and CRISPR-Cas12b integrated mcr-1 and tet(X4) detection systems. We identified mcr-1- and tet(X4)-conserved and -specific protospacers through a comprehensive BLAST search based on the NCBI nt database and used them for assembling the detection systems. Our developed one-tube RPA-CRISPR-Cas12b-based detection systems enabled the specific detection of mcr-1 and tet(X4) with a sensitivity of 6.25 and 9 copies within a detection time of ~ 55 and ~ 40 min, respectively. The detection results using pork and associated environmental samples collected from retail markets demonstrated that our developed mcr-1 and tet(X4) detection systems could successfully monitor mcr-1 and tet(X4), respectively. Notably, mcr-1- and tet(X4)-positive strains were isolated from the positive samples, as revealed using the developed detection systems. Whole-genome sequencing of representative strains identified an mcr-1-carrying IncI2 plasmid and a tet(X4)-carrying IncFII plasmid, which are known as important vectors for mcr-1 and tet(X4) transmission, respectively. Taken together, our developed one-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems show promising potential for the onsite detection of mcr-1 and tet(X4). KEY POINTS: • One-tube RPA-CRISPR-Cas12b-based mcr-1 and tet(X4) detection systems were developed based on identified novel protospacers. • Both detection systems exhibited high sensitivity and specification with a sample-to-answer time of less than 1 h. • The detection systems show promising potential for onsite detection of mcr-1 and tet(X4).

The haemagglutinin-neuraminidase protein of velogenic Newcastle disease virus enhances viral infection through NF-κB-mediated programmed cell death.

The haemagglutinin-neuraminidase (HN) protein, a vital membrane glycoprotein, plays a pivotal role in the pathogenesis of Newcastle disease virus (NDV). Previously, we demonstrated that a mutation in the HN protein is essential for the enhanced virulence of JS/7/05/Ch, a velogenic variant NDV strain originating from the mesogenic vaccine strain Mukteswar. Here, we explored the effects of the HN protein during viral infection in vitro using three viruses: JS/7/05/Ch, Mukteswar, and an HN-replacement chimeric NDV, JS/MukHN. Through microscopic observation, CCK-8, and LDH release assays, we demonstrated that compared with Mukteswar and JS/MukHN, JS/7/05/Ch intensified the cellular damage and mortality attributed to the mutant HN protein. Furthermore, JS/7/05/Ch induced greater levels of apoptosis, as evidenced by the activation of caspase-3/8/9. Moreover, JS/7/05/Ch promoted autophagy, leading to increased autophagosome formation and autophagic flux. Subsequent pharmacological experiments revealed that inhibition of apoptosis and autophagy significantly impacted virus replication and cell viability in the JS/7/05/Ch-infected group, whereas less significant effects were observed in the other two infected groups. Notably, the mutant HN protein enhanced JS/7/05/Ch-induced apoptosis and autophagy by suppressing NF-κB activation, while it mitigated the effects of NF-κB on NDV infection. Overall, our study offers novel insights into the mechanisms underlying the increased virulence of NDV and serves as a reference for the development of vaccines.

Mobile Colistin Resistance and Plasmid-Mediated Quinolone Resistance Genes in Escherichia coli from China, 1993-2019.

Plasmid-mediated quinolone resistance (PMQR) genes and mobile colistin resistance (MCR) genes in Escherichia coli (E. coli) have been widely identified, which is considered a global threat to public health. In the present study, we conducted an analysis of MCR genes (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5) and PMQR genes [qnrA, qnrB, qnrC, qnrD, qnrE1, qnrVC, qnrS, aac(6')-Ib-cr, qepA, and oqxAB] in E. coli from China, 1993-2019. From the 3,663 E. coli isolates examined, 1,613 (44.0%) tested positive for PMQR genes, either individually or in combination. Meanwhile, 262 isolates (7.0%) carried the MCR genes. Minimum inhibitory concentration (MIC) analyses of 17 antibiotics for the MCR gene-carrying strains revealed universal multidrug resistance. Resistance to polymyxin varied between 4 µg/mL and 64 µg/mL, with MIC50 and MIC90 at 8 µg/mL and 16 µg/mL, respectively. In addition, fluctuations in the detection rates of these resistant genes correlated with the introduction of antibiotic policies, host origin, temporal trends, and geographical distribution. Continuous surveillance of PMQR and MCR variants in bacteria is required to implement control and prevention strategies.

Characterization of Probiotic Properties and Whole-Genome Analysis of Lactobacillus johnsonii N5 and N7 Isolated from Swine.

In our previous microbiome profiling analysis, Lactobacillus (L.) johnsonii was suggested to contribute to resistance against chronic heat stress-induced diarrhea in weaned piglets. Forty-nine L. johnsonii strains were isolated from these heat stress-resistant piglets, and their probiotic properties were assessed. Strains N5 and N7 exhibited a high survival rate in acidic and bile environments, along with an antagonistic effect against Salmonella. To identify genes potentially involved in these observed probiotic properties, the complete genome sequences of N5 and N7 were determined using a combination of Illumina and nanopore sequencing. The genomes of strains N5 and N7 were found to be highly conserved, with two N5-specific and four N7-specific genes identified. Multiple genes involved in gastrointestinal environment adaptation and probiotic properties, including acidic and bile stress tolerance, anti-inflammation, CAZymes, and utilization and biosynthesis of carbohydrate compounds, were identified in both genomes. Comparative genome analysis of the two genomes and 17 available complete L. johnsonii genomes revealed 101 genes specifically harbored by strains N5 and N7, several of which were implicated in potential probiotic properties. Overall, this study provides novel insights into the genetic basis of niche adaptation and probiotic properties, as well as the genome diversity of L. johnsonii.

Unraveling the significance of calcium as a biofilm promotion signal for Bacillus licheniformis strains isolated from dairy products.

Bacillus licheniformis, a quick and strong biofilm former, is served as a persistent microbial contamination in the dairy industry. Its biofilm formation process is usually regulated by environmental factors including the divalent cation Ca2+. This work aims to investigate how different concentrations of Ca2+ change biofilm-related phenotypes (bacterial motility, biofilm-forming capacity, biofilm structures, and EPS production) of dairy B. licheniformis strains. The Ca2+ ions dependent regulation mechanism for B. licheniformis biofilm formation was further investigated by RNA-sequencing analysis. Results revealed that supplementation of Ca2+ increased B. licheniformis biofilm formation in a dose-dependent way, and enhanced average coverage and thickness of biofilms with complex structures were observed by confocal laser scanning microscopy. Bacterial mobility of B. licheniformis was increased by the supplementation of Ca2+ except the swarming ability at 20 mM of Ca2+. The addition of Ca2+ decreased the contents of polysaccharides but promoted proteins production in EPS, and the ratio of proteins/polysaccharides content was significantly enhanced with increasing Ca2+ concentrations. RNA-sequencing results clearly indicated the variation in regulating biofilm formation under different Ca2+ concentrations, as 939 (671 upregulated and 268 downregulated) and 951 genes (581 upregulated and 370 downregulated) in B. licheniformis BL2-11 were induced by 10 and 20 mM of Ca2+, respectively. Differential genes were annotated in various KEGG pathways, including flagellar assembly, two-component system, quorum sensing, ABC transporters, and related carbohydrate and amino acid metabolism pathways. Collectively, the results unravel the significance of Ca2+ as a biofilm-promoting signal for B. licheniformis in the dairy industry.

Genotype-phenotype evaluation of the heterogeneity in biofilm formation by diverse Bacillus licheniformis strains isolated from dairy products.

The spoilage bacterium Bacillus licheniformis has been identified as a quick and strong biofilm former in the dairy industry. In our previous study, intra-species variation in bacterial biofilms has been observed in diverse B. licheniformis strains from different genetic backgrounds; however, the mechanisms driving the observed heterogeneity of biofilms remain to be determined. In this study, the genotype-phenotype evaluation of the heterogeneity in biofilm formation of four B. licheniformis strains were examined. The heterogeneity in biofilm phenotype was accessed in aspects of bacterial growth and motility, cell viability, biofilm matrix production, and biofilm architectures. The underlying mechanisms of the intra-species variability in biofilms were also explored by whole genome resequencing (WGR). Results from bacterial motility tests showed a diverse motility among the strains, but there was no clear correlation between bacterial motility and biofilm formation. The cell viability results showed a different number of live cells in biofilms at the intra-species level. Analysis of chemical components in biofilm matrix demonstrated the great intra-species differences regarding extracellular matrix composition, and a negative correlation between biofilm formation on stainless steel and the protein: carbohydrate ratio in biofilm matrix was observed. Confocal laser scanning microscopy analysis also revealed the intra-species variability by showing great differences in general properties of B. licheniformis biofilms. WGR results identified important pathways involved in biofilm formation, such as two-component systems, quorum sensing, starch and sucrose metabolism, ABC transporters, glyoxylate and dicarboxylate metabolism, purine metabolism, and a phosphotransferase system. Overall, the above results emphasize the necessity of exploring the intra-species variation in biofilms, and would provide in-depth knowledge for designing efficient biofilm control strategies in the dairy industry.

Genomic epidemiology of hypervirulent Listeria monocytogenes CC619: Population structure, phylodynamics and virulence.

Listeria monocytogenes is a ubiquitous foodborne pathogen causing human and animal listeriosis with high mortality. Neurological and maternal-neonatal listeriosis outbreaks in Europe and the United States were frequently associated with clonal complexes CC1, CC2 and CC6 harboring Listeria Pathogenicity Island-1 (LIPI-1), as well as CC4 carrying both LIPI-1 and LIPI-4. However, human listeriosis in China was predominantly linked to CC87 and CC619 from serotype 1/2b. To understand the genetic evolution and distribution patterns of CC619, we characterized the epidemic history, population structure, and transmission feature of CC619 strains through analysis of 49,421 L. monocytogenes genomes globally. We found that CC619 was uniquely distributed in China, and closely related with perinatal infection. As CC619 strains were being mainly isolated from livestock and poultry products, we hypothesized that pigs and live chicken were the reservoirs of CC619. Importantly, all CC619 strains not only harbored the intact LIPI-1 and LIPI-4, but these also carried LIPI-3 that could facilitate host colonization and invasion. The deficiency of LIPI-3 or LIPI-4 markedly decreased L. monocytogenes colonization capacity in a model of intragastric infection in the mouse. Altogether, our findings suggest that the hypervirulent CC619 harboring three pathogenicity islands LIPI-1, LIPI-3 and LIPI-4 is a putatively persistent population in various foods, environment, and human population, warranting the further research for deciphering its pathogenicity and strengthening epidemiological surveillance.

Activation dynamics of antigen presenting cells in vivo against Mycobacterium bovis BCG in different immunized route.

BACKGROUND: Control of Tuberculosis (TB) infection is mainly the result of productive teamwork between T-cell populations and antigen presenting cells (APCs). However, APCs activation at the site of initiating cellular immune response during BCG early infection is not completely understood. METHODS: In this study, we injected C57BL/6 mice in intravenous (i.v) or subcutaneous (s.c) route, then splenic or inguinal lymph node (LN) DCs and MΦs were sorted, and mycobacteria uptake, cytokine production, antigen presentation activity, and cell phenotype were investigated and compared, respectively. RESULTS: Ag85A-specific T-cell immune response began at 6 days post BCG infection, when BCG was delivered in s.c route, Th17 immune response could be induced in inguinal LN. BCG could induce high level of activation phenotype in inguinal LN MΦs, while the MHC II presentation of mycobacteria-derived peptides by DCs was more efficient than MΦs. CONCLUSIONS: The results showed that BCG immunized route can decide the main tissue of T-cell immune response. Compared with s.c injected route, APCs undergo more rapid cell activation in spleen post BCG i.v infection.

Assessment of beneficial effects and identification of host adaptation-associated genes of Ligilactobacillus salivarius isolated from badgers.

BACKGROUND: Ligilactobacillus salivarius has been frequently isolated from the gut microbiota of humans and domesticated animals and has been studied as a candidate probiotic. Badger (Meles meles) is known as a "generalist" species that consumes complex foods and exhibits tolerance and resistance to certain pathogens, which can be partly attributed to the beneficial microbes such as L. salivarius in the gut microbiota. However, our understanding of the beneficial traits and genomic features of badger-originated L. salivarius remains elusive. RESULTS: In this study, nine L. salivarius strains were isolated from wild badgers' feces, one of which exhibited good probiotic properties. Complete genomes of the nine L. salivarius strains were generated, and comparative genomic analysis was performed with the publicly available complete genomes of L. salivarius obtained from humans and domesticated animals. The strains originating from badgers harbored a larger genome, a higher number of protein-coding sequences, and functionally annotated genes than those originating from humans and chickens. The pan-genome phylogenetic tree demonstrated that the strains originating from badgers formed a separate clade, and totally 412 gene families (12.6% of the total gene families in the pan-genome) were identified as genes gained by the last common ancestor of the badger group. The badger group harbored significantly more gene families responsible for the degradation of complex carbohydrate substrates and production of polysaccharides than strains from other hosts; many of these were acquired by gene gain events. CONCLUSIONS: A candidate probiotic and nine L. salivarius complete genomes were obtained from the badgers' gut microbiome, and several beneficial genes were identified to be specifically present in the badger-originated strains that were gained in the evolution. Our study provides novel insights into the adaptation of L. salivarius to the intestinal habitat of wild badgers and provides valuable strain and genome resources for the development of L. salivarius as a probiotic.

Evaluation of Hygiene Practice for Reducing Campylobacter Contamination on Cutting Boards and Risks Associated with Chicken Handling in Kitchen Environment.

Cutting boards can serve as potential carriers for the cross-contamination of pathogens from chicken to other surfaces. This study aimed to assess chefs' handling practices of cutting boards across five provinces in China and identify the key factors contributing to unsafe cutting board usage, including cleaning methods and handling practices. Handling practices associated with cutting boards were examined through a web-based survey (N = 154), while kitchen environment tests were conducted to investigate the splashing or survival of Campylobacter, inoculated in chicken or on cutting boards, to mimic the practices of chefs. Among chefs in the five provinces of China, wood and plastic cutting boards were the most commonly used for preparing chicken meat. Approximately 33.7% of chefs washed boards with running tap water, 31.17% of chefs washed boards with detergent, and 24.03% of chefs cleaned boards by scraping them with a knife after preparing other meats or chicken. The study tested 23 cutting boards from commercial kitchens for Campylobacter presence before and after chicken preparation and cleaning. Among these, 17 were cleaned with a knife, 5 with running tap water, and only 1 with disinfectant. Results showed that cleaning with a knife significantly reduced Campylobacter presence on cutting boards (p < 0.05), while the three main cleaning methods were inadequate in eliminating contamination to a safe level. In kitchen environment tests, contaminated chicken was chopped on cutting boards, with a maximum distance of 60 cm for low contamination, and 120 cm for medium and high contamination levels. This suggested a contamination risk exposure area ranging from 60 cm to 120 cm. Campylobacter survival on surfaces of wood, plastic, and stainless steel was also tested, with plastic surfaces showing the longest survival time (4.5 h at 15 °C and 3.5 h at 25 °C) In comparison, survival time on stainless steel or wood surfaces was only 3 h, implying a cross-contamination risk exposure period of 3 to 4.5 h after chicken preparation. In conclusion, based on the current study data, the practices employed by chefs play an important role in Campylobacter transfer in the kitchen environment. The presence of Campylobacter on cutting boards even after wiping or droplet splashing highlights its potential as a source of cross-contamination in the kitchen environment. So, chefs in China should reinforce their hygiene culture and adopt effective cutting board cleaning practices to prevent pathogen contamination.

Efficient screening of adsorbed receptors for Salmonella phage LP31 and identification of receptor-binding protein.

The adsorption process is the first step in the lifecycle of phages and plays a decisive role in the entire infection process. Identifying the adsorption mechanism of phages not only makes phage therapy more precise and efficient but also enables the exploration of other potential applications and modifications of phages. Phage LP31 can lyse multiple Salmonella serotypes, efficiently clearing biofilms formed by Salmonella enterica serovar Enteritidis (S. Enteritidis) and significantly reducing the concentration of S. Enteritidis in chicken feces. Therefore, LP31 has great potential for many practical applications. In this study, we established an efficient screening method for phage infection-related genes and identified a total of 10 genes related to the adsorption process of phage LP31. After the construction of strain C50041ΔrfaL 58-358, it was found that the knockout strain had a rough phenotype as an O-antigen-deficient strain. Adsorption rate and transmission electron microscopy experiments showed that the receptor for phage LP31 was the O9 antigen of S. Enteritidis. Homology comparison and adsorption experiments confirmed that the tail fiber protein Lp35 of phage LP31 participated in the adsorption process as a receptor-binding protein. IMPORTANCE A full understanding of the interaction between phages and their receptors can help with the development of phage-related products. Phages like LP31 with the tail fiber protein Lp35, or a closely related protein, have been reported to effectively recognize and infect multiple Salmonella serotypes. However, the role of these proteins in phage infection has not been previously described. In this study, we established an efficient screening method to detect phage adsorption to host receptors. We found that phage LP31 can utilize its tail fiber protein Lp35 to adsorb to the O9 antigen of S. Enteritidis, initiating the infection process. This study provides a great model system for further studies of how a phage-encoded receptor-binding protein (RBP) interacts with its host's RBP binding target, and this new model offers opportunities for further theoretical and experimental studies to understand the infection mechanism of phages.

[Characterization of the cellular immune response induced by Mycobacterium tuberculosis Rv2626c].

Nearly a quarter of the world's population is infected with Mycobacterium tuberculosis and remains long-term asymptomatic infection. Rv2626c is a latent infection-related protein regulated by DosR of M. tuberculosis. In this study, the Rv2626c protein was prokaryotically expressed and purified, and its immunobiological characteristics were analyzed using RAW264.7 cells and mice as infection models. SDS-PAGE and Western blotting analysis showed that the Rv2626c-His fusion protein was mainly expressed in soluble form and specifically reacted with the rabbit anti-H37RV polyclonal serum. In addition, we found that the Rv2626c protein bound to the surface of RAW264.7 macrophages and up-regulated the production of NO. Moreover, the Rv2626c protein significantly induced the production of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6 and MCP-1, and induced strong Th1-tendency immune response. These results may help to reveal the pathogenic mechanism of M. tuberculosis and facilitate the development of new tuberculosis vaccine.

Emergence of a new designated clade 16 with significant antigenic drift in hemagglutinin gene of H9N2 subtype avian influenza virus in eastern China.

H9N2 avian influenza viruses (AIVs) pose an increasing threat to the poultry industry worldwide and have pandemic potential. Vaccination has been principal prevention strategy to control H9N2 in China since 1998, but vaccine effectiveness is persistently challenged by the emergence of the genetic and/or antigenic variants. Here, we analysed the genetic and antigenic characteristics of H9N2 viruses in China, including 70 HA sequences of H9N2 isolates from poultry, 7358 from online databases during 2010-2020, and 15 from the early reference strains. Bayesian analyses based on hemagglutinin (HA) gene revealed that a new designated clade16 emerged in April 2012, and was prevalent and co-circulated with clade 15 since 2013 in China. Clade 16 viruses exhibited decreased cross-reactivity with those from clade 15. Antigenic Cartography analyses showed represent strains were classified into three antigenic groups named as Group1, Group2 and Group3, and most of the strains in Group 3 (15/17, 88.2%) were from Clade 16 while most of the strains in Group2 (26/29, 89.7%) were from Clade 15. The mean distance between Group 3 and Group 2 was 4.079 (95%CI 3.605-4.554), revealing that major switches to antigenic properties were observed over the emergence of clade 16. Genetic analysis indicated that 11 coevolving amino acid substitutions primarily at antigenic sites were associated with the antigenic differences between clade 15 and clade 16. These data highlight complexities of the genetic evolution and provide a framework for the genetic basis and antigenic characterization of emerging clade 16 of H9N2 subtype avian influenza virus.

Multi-omics reveals the increased biofilm formation of Salmonella Typhimurium M3 by the induction of tetracycline at sub-inhibitory concentrations.

Exposure to sub-inhibitory concentrations (sub-MICs) of antibiotics could induce the biofilm formation of microorganisms, but its underlying mechanisms still remain elusive. In the present work, biofilm formation by Salmonella Typhimurium M3 was increased when in the presence of tetracycline at sub-MIC, and the highest induction was observed with tetracycline at 1/8 MIC. The integration of RNA-sequencing and untargeted metabolomics was applied in order to further decipher the potential mechanisms for this observation. In total, 439 genes and 144 metabolites of S. Typhimurium M3 were significantly expressed after its exposure to 1/8 MIC of tetracycline. In addition, the co-expression analysis revealed that 6 genes and 8 metabolites play a key role in response to 1/8 MIC of tetracycline. The differential genes and metabolites were represented in 12 KEGG pathways, including five pathways of amino acid metabolism (beta-alanine metabolism, tryptophan metabolism, arginine and proline metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, and glutathione metabolism), three lipid metabolism pathways (biosynthesis of unsaturated fatty acids, fatty acid degradation, and fatty acid biosynthesis), two nucleotide metabolism pathways (purine metabolism, and pyrimidine metabolism), pantothenate and CoA biosynthesis, and ABC transporters. Metabolites (anthranilate, indole, and putrescine) from amino acid metabolism may act as signaling molecules to promote the biofilm formation of S. Typhimurium M3. The results of this work highlight the importance of low antimicrobial concentrations on foodborne pathogens of environmental origin.

First Detection of blaNDM-1-Haboring IncHI2 Plasmid in Escherichia coli Strain Isolated from Goose in China.

Carbapenem-resistant Enterobacteriaceae infections are among the most serious threats to human and animal health worldwide. Of the 1013 strains of Escherichia coli isolated and identified in 14 regions of China from 2007 to 2018, seven strains were resistant to meropenem and all were positive for blaNDM. The seven New Delhi metallo-ß-lactamase (NDM)-positive strains belonged to five different sequence types, indicating that most of the NDM-positive strains were nonclonal. An IncHI2 plasmid carrying the blaNDM-1 element was identified in the C1147 strain from a goose source and reported for the first time, showing a specific structure. Conjugation experiments revealed that the IncHI2 plasmid was conjugatable, and the horizontal propagation of the plasmid led to the rapid propagation of NDM in the same and different strains. This study revealed that waterfowl, as a potential transmission factor for carbapenem-resistant blaNDM-1, poses a threat to human health.

A live attenuated DIVA vaccine affords protection against Listeria monocytogenes challenge in sheep.

Listeria monocytogenes (Lm) is a deadly foodborne pathogen that comprises 14 serotypes, among which, serotype 4b Lm is the primary cause of listeriosis outbreaks in humans and animals. Here, we evaluated the safety, immunogenicity, and protective efficacy of a serotype 4b vaccine candidate Lm NTSNΔactA/plcB/orfX in sheep. The infection dynamics, clinical features, and pathological observation verified that the triple genes deletion strain has adequate safety for sheep. Moreover, NTSNΔactA/plcB/orfX significantly stimulated humoral immune response and provided 78% immune protection to sheep against lethal wild-type strain challenge. Notably, the attenuated vaccine candidate could differentiate infected and vaccinated animals (DIVA) via serology determination of the antibody against listeriolysin O (LLO, encoded by hly) and phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB). These data suggest that the serotype 4b vaccine candidate has high efficacy, safety, and DIVA characteristics, and may be used to prevent Lm infection in sheep. Our study provides a theoretical basis for its future application in livestock and poultry breeding.

LygA retention on the surface of Listeria monocytogenes via its interaction with wall teichoic acid modulates bacterial homeostasis and virulence.

Wall teichoic acid (WTA) is the abundant cell wall-associated glycopolymer in Gram-positive bacteria, playing crucial roles in surface proteins retention, bacterial homeostasis, and virulence. The WTA glycosylation of Listeria monocytogenes is essential for surface anchoring of virulence factors, whereas the nature and function of the noncovalent interactions between cell wall-associated proteins and WTA are less unknown. In this study, we found that galactosylated WTA (Gal-WTA) of serovar (SV) 4h L. monocytogenes plays a key role in modulating the novel glycine-tryptophan (GW) domain-containing autolysin protein LygA through direct interactions. Gal-deficient WTA of Lm XYSN (ΔgalT) showed a dramatic reduction of LygA on the cell surface. We demonstrated that LygA binds to Gal-WTA through the GW domains, and the binding affinity is associated with the number of GW motifs. Moreover, we confirmed the direct Gal-dependent binding of the GW protein Auto from the type I WTA strain, which has no interaction with rhamnosylated WTA, indicating that the complexity of both WTA and GW proteins affect the coordination patterns. Importantly, we revealed the crucial roles of LygA in facilitating bacterial homeostasis as well as crossing the intestinal and blood-brain barriers. Altogether, our findings suggest that both the glycosylation patterns of WTA and a fixed numbers of GW domains are closely associated with the retention of LygA on the cell surface, which promotes the pathogenesis of L. monocytogenes within the host.

Environmental factors and gut microbiota: Toward better conservation of deer species.

Thousands of microbial species inhabiting the animal gut, collectively known as the gut microbiota, play many specific roles related to host nutrient metabolism and absorption, immune regulation, and protection from pathogenic bacteria. Gut microbiota composition is affected by several internal and external factors, such as the host genotype, dietary intake, breeding environment, and antibiotic exposure. As deer species are important members for maintaining ecosystem balance, understanding the effects of multiple factors on the gut microbiota of deer species, particularly endangered ones, is crucial. In this review, we summarize and discuss the factors that significantly affect the gut microbiota of deer and present the impacts of these factors on microbial composition. In particular, we focused on the changes in gut microbiota due to dietary differences under different conditions, including seasonal changes, different geographical locations, and captivity, as well as weaning and pathogen disturbance. Understanding the correlations between gut microbiota composition and its driving factors is important for evaluating and improving the captive breeding environment for better conservation of endangered deer species, and reintroducing wild deer populations in the future.

Characterization and comparative genomic analysis of novel lytic bacteriophages targeting Cronobacter sakazakii.

Cronobacter sakazakii, a foodborne pathogen, can contaminate powdered infant formula (PIF) and cause life-threatening meningitis, necrotizing colitis and meningoencephalitis in infants. Bacteriophages are increasingly considered an efficient approach to target pathogenic microorganisms. In the current study, four virulent phages that can infect C. sakazakii were isolated from sewage samples, and their biological and complete genomic characteristics were analyzed. A comparative genomic analysis was performed to investigate the functional genes and phylogenetic evolution of the four phages. The results revealed that all four phages belonged to the Ackermannviridae family. Notably, the viral burst size of the phages ranged from 10 to 250 PFU/cell, following a latent period of 5 min to 20 min. Moreover, phages were stable over a pH range of 4 to 10 and a temperature range of 50 ℃ to 60 ℃. The full length of the complete genomes of the four phages ranged from 41,929 bp to 146,806 bp, containing lysis genes but no virulence genes. Phylogenetic tree analysis showed that the four phages were members of two distinct genetic groups with a significant genetic evolutionary distance between each C. sakazakii phage. Furthermore, the antibacterial assay revealed that all phages could inhibit the growth of C. sakazakii for up to 24 h. Taken together, the four phages have huge prospects as additives in dairy products to counter C. sakazakii.