The antimicrobial resistance of Helicobacter pylori (H. pylori) in most countries and regions has increased significantly. It has not been fully confirmed whether the detection of H. pylori resistance gene mutation can replace antibiotic drug sensitivity test to guide the clinical personalized treatment. The objective of this study was to assess and compare the efficacy of different antimicrobial resistance-guided quadruple therapies in refractory H. pylori-infected individuals who had undergone unsuccessful prior eradication treatments.

Group B streptococcus (GBS) is the major pathogen causing diseases in neonates, pregnant/puerperal women, cows and fish. Recent studies have shown that GBS may be infectious across hosts and some fish GBS strain might originate from human. The purpose of this study is to investigate the genetic relationship of CC103 strains that recently emerged in cows and humans, and explore the pathogenicity of clinical GBS isolates from human to tilapia. Ninety-two pathogenic GBS isolates were identified from 19 patients with different diseases and their evolution and pathogenicity to tilapia were analyzed. The multilocus sequence typing revealed that clonal complex (CC) 103 strain was isolated from 21.74% (20/92) of patients and ST485 strain was from 14.13% (13/92) patients with multiple diseases including neonates. Genomic evolution analysis showed that both bovine and human CC103 strains alternately form independent evolutionary branches. Three CC67 isolates carried gbs2018-C gene and formed one evolutionary branch with ST61 and ST67 strains that specifically infect dairy cows. Studies of interspecies transmission to tilapia found that 21/92 (22.83%) isolates including all ST23 isolates were highly pathogenic to tilapia and demonstrated that streptococci could break through the blood-brain barrier into brain tissue. In conclusions, CC103 strains are highly prevalent among pathogenic GBS from humans and have evolved into the highly pathogenic ST485 strains specifically infecting humans. The CC67 strains isolated from cows are able to infect humans through evolutionary events of acquiring CC17-specific type C gbs2018 gene and others. Human-derived ST23 pathogenic GBS strains are highly pathogenic to tilapia.

Biosurfactant plays an important role in bioremediation of crude oil contamination and microbial enhanced oil recovery (MEOR). In the present study, a salt-tolerant, biosurfactant-producing bacterium, designated A-8, was isolated from wastewater contaminated with petroleum collected from the Changqing reservoir in China. A phylogenetic analysis based on the 16S rRNA sequence suggests that strain A-8 belongs to the genus Achromobacter. The optimal growth conditions for strain A-8 in mineral salt (MS) medium were 30°C, pH 7, and 10 g/L NaCl, while the optimal conditions for biosurfactant production in a fermentation medium were 40-45°C, pH 7, and more than 70 g/L NaCl. Better biosurfactant production was obtained from strain A-8 when edible oil and liquid paraffin were used as carbon sources and when (NH4)2SO4 was used as an inorganic nitrogen source compared with other tested carbon and nitrogen sources. The biodegradation of petroleum in MS medium in different optimized conditions reached 56.23-73.87% for 20 days. The biodegradation of petroleum, together with the production of organic acid and biosurfactant, decreased the viscosity of petroleum by about 45%. The decrease in petroleum viscosity and the biodegradation of petroleum suggest the potential use of strain A-8 for MEOR and bioremediation of petroleum-contaminated environments.

Interferons (IFNs) are proteins produced by a variety of cells during the process of virus infection. It can activate the transcription of multiple functional genes in cells, regulate the synergistic effect of multiple signaling pathways, and mediate a variety of biological functions such as antiviral activity and immune regulation. The symptoms of hosts infected with African swine fever virus (ASFV) depend on the combined interaction between viruses and the host. However, it is unclear whether IFNs can be used as an emergency preventive treatment for ASFV. This study focused on the use of recombinant porcine IFNs, produced by Escherichia coli, to inhibit the replication of ASFV. The activity of IFN against ASFV was detected using primary alveolar macrophages at different doses through immunofluorescence assays and quantitative real-time PCR. We found that both 1000 and 100 U/mL doses significantly inhibited the replication of ASFV. Meanwhile, we found that IFNs could significantly trigger the production of a variety of IFN-induced genes (IFIT1, IFITM3, Mx-1, OASL, ISG15, PKR, GBP1, Viperin, BST2, IRF-1, and CXCL10) and MHC molecules, which play key roles in resistance to virus infection. Peripheral blood samples were also obtained from surviving pigs treated with IFNs, and the viral load was determined. Consistent with in vitro tests, low-dose (105 U/kg) recombinant porcine IFNs (PoIFN-α and PoIFN-γ) significantly reduced viral load compared to that with high-dose (106 U/kg) treatment. Our results suggest that recombinant porcine IFNs have high antiviral activity against ASFV, providing a new strategy for the prevention of African swine fever.

Fusarium wilt is a devastating soil-borne fungal disease caused by Fusarium oxysporum f.sp. cubense (Foc). In recent years, some antifungal bacteria have been applied for the prevention and biocontrol of pathogenic fungi. In our study, a bacterial strain HQB-1, isolated from banana rhizosphere soil, was cultured for investigation. It showed broad-spectrum antifungal activities against representative phytopathogenic fungi including Fusarium oxysporum, Colletotrichum gloeosporioides, Botrytis cinerea, and Curvularia fallax. The strain HQB-1 was identified as Burkholderia sp. by morphological, physiological, and biochemical examinations, confirmed by 16S rRNA gene sequence analysis. Among the metabolites produced by the strain, we identified an antifungal compound which was identified phenazine-1-carboxylic acid (PCA) (C13H8N2O2) through ultraviolet, liquid chromatography quadrupole-time of flight mass spectrometer, and nuclear magnetic response. Furthermore, PCA exhibited the lowest minimum inhibitory concentration (MIC) against F. oxysporum (1.56 μg/ml) and yielded the highest MIC against C. gloeosporioides. Pot experiments showed that application of 5 μg/ml or more of PCA efficiently controlled banana wilt and promoted the growth of banana plants. These results suggested that Burkholderia sp. HQB-1, as an important microbial resource of PCA, could be a promising biological agent against wilt diseases and promoting banana growth.

Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of enveloped DNA viruses. Recent studies have found that host factors can suppress HBV replication. HBV envelope proteins are reported to be degraded by the endoplasmic reticulum-associated degradation (ERAD) pathway. As a component of the ERAD pathway, suppressor of lin-12-like 1 (SEL1L) was earlier found to be upregulated in the inactive carrier phase of chronic HBV infection relative to that in the immune tolerant phase. However, the role of SEL1L in regulating HBV replication remains largely unknown. In this study, we found the levels of HBV RNA, DNA, and core and envelope proteins to be significantly downregulated by SEL1L overexpression and upregulated by SEL1L silencing in Huh7 cells transiently transfected with an overlength HBV genome. Similar upregulation was observed in HepG2.2.15 cells as well. SEL1L co-localized with HBV surface antigen (HBsAg), which changed its staining pattern. Treatment with an inhibitor of ERAD pathway remarkably increased intracellular S protein. Surprisingly, silencing SEL1L to block the ERAD pathway activated an alternative ER quality control (ERQC)-autophagy pathway, which might account for the increased HBV RNAs and core protein. Together, our results demonstrate that SEL1L is a host restriction factor that exerts anti-HBV effect through ERAD and alternative ERQC-autophagy pathway.

Streptococcus agalactiae, or Group B Streptococcus (GBS), is a major pathogen causing neonatal sepsis and meningitis, bovine mastitis, and fish meningoencephalitis. CC23, including its namesake ST23, is not only the predominant GBS strain derived from human and cattle, but also can infect a variety of homeothermic and poikilothermic species. However, it has never been characterized in fish. This study aimed to determine the pathogenicity of ST23 GBS to fish and explore the mechanisms causing the difference in the pathogenicity of ST23 GBS based on the genome analysis. Infection of tilapia with 10 human-derived ST23 GBS isolates caused tissue damage and the distribution of pathogens within tissues. The mortality rate of infection was ranged from 76 to 100%, and it was shown that the mortality rate caused by only three human isolates had statistically significant difference compared with fish-derived ST7 strain (P < 0.05), whereas the mortality caused by other seven human isolates did not show significant difference compared with fish-derived ST7 strain. The genome comparison and prophage analysis showed that the major genome difference between virulent and non-virulent ST23 GBS was attributed to the different prophage sequences. The prophage in the P1 region contained about 43% GC and encoded 28-39 proteins, which can mediate the acquisition of YafQ/DinJ structure for GBS by phage recombination. YafQ/DinJ belongs to one of the bacterial toxin-antitoxin (TA) systems and allows cells to cope with stress. The ST23 GBS strains carrying this prophage were not pathogenic to tilapia, but the strains without the prophage or carrying the pophage that had gene mutation or deletion, especially the deletion of YafQ/DinJ structure, were highly pathogenic to tilapia. In conclusion, human ST23 GBS is highly pathogenic to fish, which may be related to the phage recombination.

Composition of the gut microbiota has been linked with human immunedeficiency virus (HIV)-infected patients on antiretroviral therapy (ART). Evidence suggests that ART-treated patients with poor CD4+ T-cell recovery have higher levels of microbial translocation and immune activation. However, the association of the gut microbiota and immune recovery remains unclear. We performed a cross-sectional study on 30 healthy controls (HC) and 61 HIV-infected individuals, including 15 immunological ART responders (IRs), 20 immunological ART non-responders (INRs) and 26 untreated individuals (VU). IR and INR groups were classified by CD4+ T-cell counts of ≥350 cells/mm3 and <350 cells/mm3 after 2 years of ART, respectively. Each subject's gut microbiota composition was analyzed by metagenomics sequencing. Levels of CD4+ T cells, CD8+HLA-DR+ T cells and CD8+CD38+ T cells were measured by flow cytometry. We identified more Prevotella and fewer Bacteroides in HIV-infected individuals than in HC. Patients in INR group were enriched with Faecalibacterium prausnitzii, unclassified Subdoligranulum sp. and Coprococcus comes when compared with those in IR group. F. prausnitzii and unclassified Subdoligranulum sp. were overrepresented in individuals in VU group with CD4+ T-cell counts <350 cells/mm3. Moreover, we found that the relative abundance of unclassified Subdoligranulum sp. and C. comes were positively correlated with CD8+HLA-DR+ T-cell count and CD8+HLA-DR+/CD8+ percentage. Our study has shown that gut microbiota changes were associated with CD4+ T-cell counts and immune activation in HIV-infected subjects. Interventions to reverse gut dysbiosis and inhibit immune activation could be a new strategy for improving immune reconstitution of HIV-1-infected individuals.

Nuclear protein LaeA is known as the global regulator of secondary metabolism in Aspergillus. LaeA connects with VeA and VelB to form a heterotrimeric complex, which coordinates fungal development and secondary metabolism. Here, we describe a new interaction partner of LaeA, the kinetochore protein Spc105, from the aflatoxin-producing fungus Aspergillus flavus. We showed that in addition to involvement in nuclear division, Spc105 is required for normal conidiophore development and sclerotia production of A. flavus. Moreover, Spc105 positively regulates the production of secondary metabolites such as aflatoxin and kojic acid, and negatively regulates the production of cyclopiazonic acid. Transcriptome analysis of the Δspc105 strain revealed that 23 backbone genes were differentially expressed, corresponding to 19 of the predicted 56 secondary metabolite gene clusters, suggesting a broad regulatory role of Spc105 in secondary metabolism. Notably, the reduced expression of laeA in our transcriptome data led to the discovery of the correlation between Spc105 and LaeA, and double mutant analysis indicated a functional interdependence between Spc105 and LaeA. Further, in vitro and in vivo protein interaction assays revealed that Spc105 interacts directly with the S-adenosylmethionine (SAM)-binding domain of LaeA, and that the leucine zipper motif in Spc105 is required for this interaction. The Spc105-LaeA interaction identified in our study indicates a cooperative interplay of distinct regulators in A. flavus, providing new insights into fungal secondary metabolism regulation networks.

Pseudomonas aeruginosa is the most relevant pathogen to the severe exacerbations of patients with chronic obstructive pulmonary disease (COPD). However, the genetic and functional characteristics of P. aeruginosa isolates from COPD airways still remain less understood. In this study, the genetic, phylogenetic, phenotypic, and transcriptional features of P. aeruginosa isolates from COPD sputa were comprehensively explored by susceptibility testing, comparative-genomic analysis, phylogenetic analysis, phenotypic profiling, and comparative-transcriptomic analysis. We found that P. aeruginosa was prevalent in elder COPD patients and highly resisted to many commonly used antibiotics. P. aeruginosa COPD isolates harbored a substantial number of variant sites that might influence the primary metabolism and substance transport system. These isolates were discretely distributed in the phylogenetic tree and clustered with internationally collected P. aeruginosa in two major groups, and could be classified into three groups according to their differences in virulence-related phenotypes. Furthermore, the transcriptional patterns of COPD isolates could be classified into PAO1-like group with reduced protein secretion and motility and PAO1-distinct group with decreased substance transport but enhanced primary metabolism. In conclusion, this study demonstrates that P. aeruginosa isolates from COPD patients have abundant genetic and phenotypic diversity, and provides an important reference for further exploring the survival strategy of P. aeruginosa in COPD airways and the development of anti-pseudomonal therapy.

The spread of antimicrobial resistance in environment is promoted at least in part by the inappropriate use of antibiotics in animals and humans. The present study was designed to investigate the impact of different concentrations of ciprofloxacin in soil containing manure on the development of plasmid-mediated quinolone resistance (PMQR) - encoding genes and the abundance of soil bacterial communities. For these studies, high-throughput next-generation sequencing of 16S rRNA, real-time polymerase chain reaction and standard microbiologic culture methods were utilized. We demonstrated that the dissipate rate of relative abundances of some of PMQR-encoding genes, such as qnrS, oqxA and aac(6')-Ib-cr, were significantly lower with ciprofloxacin 0.04 and 0.4 mg/kg exposure as compared to no-ciprofloxacin control and ciprofloxacin 4 mg/kg exposure during 2 month. Also, the number of ciprofloxacin resistant bacteria was significantly greater in ciprofloxacin 0.04 and 0.4 mg/kg exposure as compared with no-ciprofloxacin control and the ciprofloxacin 4 mg/kg exposure. In addition, lower ciprofloxacin concentration provided a selective advantage for the populations of Xanthomonadales and Bacillales in orders while Agrobacterium, Bacillus, Enterococcus, and Burkholderia in genera. These findings suggest that lower concentration of ciprofloxacin resulted in a slower rate of PMQR-encoding genes dissipation and selected development of ciprofloxacin-resistant bacteria in soil amended with manure.

FosB, a Mg2+ dependent thioltransferase, confers antibiotic resistance to fosfomycin through enzymatic drug inactivation. Among all antibiotic resistant proteins in the Antibiotic Resistance Genes Database and the Comprehensive Antibiotic Resistance Database, FosB is within 5% of the most number of ARPs identified in Human Microbiome Project reference database but mainly distributed in limited genera, i.e., 122 of total 133 FosB homologues are found from Bacillus and Staphylococcus. Furthermore, these FosB sequences could be divided into three clusters based on their phylogenetic relationship, i.e., two groups of FosB were mainly from Bacillus, and another was mainly from Staphylococcus. Finally, we confirmed that FosB from the group of Staphylococcus presented the highest resistance ability to fosfomycin by in silico and in vitro comparisons. In summary, this study elaborates the specific taxonomic characteristics and resistant abilities of FosB in human microbiota, which might help in developing more promising fosfomycin-like antibiotics.

β-Carotene is the precursor of vitamin A, and also exhibits multiple pharmaceutical functions by itself. In comparison to chemical synthesis, the production of β-carotene in microbes by metabolic engineering strategy is relatively inexpensive. Identifying genes enhancing β-carotene production in microbes is important for engineering a strain of producing higher yields of β-carotene. Most of previous efforts in identifying the gene targets have focused on the isoprenoid pathway where the β-carotene biosynthesis belongs. However, due to the complex interactions between metabolic fluxes, seemingly irrelevant genes that are outside the isoprenoid pathway might also affect β-carotene biosynthesis. To this end, here we provided an example that several novel gene targets, which are outside the isoprenoid pathway, have improving effects on β-carotene synthesis in yeast cells, when they were over-expressed. Among these targets, the class E protein of the vacuolar protein-sorting pathway (Did2) led to the highest improvement in β-carotene yields, which was 2.1-fold to that of the corresponding control. This improvement was further explained by the observation that the overexpression of the DID2 gene generally boosted the transcriptions of β-carotene pathway genes. The mechanism by which the other targets improve the production of β-carotene is discussed.

Myasthenia gravis (MG) is an autoimmune-mediated disorder, the etiology of which involves both environmental factors and genetics. While the exact factors responsible for predisposition to MG remain elusive, it is hypothesized that gut microbiota play a critical role in the pathogenesis of MG. This study investigated whether gut microbiota are altered in MG patients by comparing the fecal microbiota profiles of MG patients to those of age- and sex-matched healthy controls. Phylotype profiles of gut microbial populations were generated using hypervariable tag sequencing of the V4 region of the 16S ribosomal RNA gene. Fecal short-chain fatty acids (SCFAs) were assessed by gas chromatographic analyses. The results demonstrated that, compared to the healthy cohort, the gut microbiota of the MG group was changed in terms of the relative abundances of bacterial taxa, with sharply reduced microbial richness, particularly in the genus Clostridium. The fecal SCFA content was significantly lower in the MG group. Furthermore, microbial dysbiosis was closely related to the levels of inflammatory biomarkers in the sera of MG patients.

Microbiota in the pit mud (PM) plays a crucial role in the production of Chinese strong-flavor liquor (CSFL), the most popular distilled liquor in China. However, previous studies used total microbes, instead of viable ones, for the characterization of the microbial community in this environment. In this study, we used propidium monoazide (PMA) combined with quantitative polymerase chain reaction (qPCR) and 16S rRNA gene sequencing to verify the effect of non-viablee bacteria on the characterization of PM bacteria. After PMA concentration optimization, 50 μM PMA was chosen to pretreat 5 and 20 years PMs. The qPCR results showed that there were 50.78 and 71.84% of non-viable bacteria in the 5-year PM and 20-year PM, respectively. Both copy numbers of total bacteria and viable bacteria were significantly higher in 20-year PM than those in 5-year PM. Nevertheless, in terms of bacterial diversity and composition analyses at the operational taxonomic unit (OTU), phylum, class, and genus levels, 16S rRNA gene sequencing results displayed no significant differences between total bacteria and viable bacteria in both PM types. In conclusion, it is necessary for non-viable bacteria to be considered in determining absolute biomass of bacteria in PM, but not necessary in the analysis of diversity and composition of PM bacteria. To the best of our knowledge, our study is the first attempt to analyze viable bacteria in the PM of CSFL and provides useful information on how to accurately characterize a microbial community in a PM environment.

Alcoholic fermentation is fundamentally an adaptation process, in which the yeast Saccharomyces cerevisiae outperforms its competitors and takes over the fermentation process itself. Although wine yeast strains appear to be adapted to the stressful conditions of alcoholic fermentation, nitrogen limitations in grape must cause stuck or slow fermentations, generating significant economic losses for the wine industry. One way to discover the genetic bases that promote yeast adaptation to nitrogen-deficient environments are selection experiments, where a yeast population undergoes selection under conditions of nitrogen restriction for a number of generations, to then identify by sequencing the molecular characteristics that promote this adaptation. In this work, we carried out selection experiments in bioreactors imitating wine fermentation under nitrogen-limited fermentation conditions (SM60), using the heterogeneous SGRP-4X yeast population, to then sequence the transcriptome and the genome of the population at different time points of the selection process. The transcriptomic results showed an overexpression of genes from the NA strain (North American/YPS128), a wild, non-domesticated isolate. In addition, genome sequencing and allele frequency results allowed several QTLs to be mapped for adaptation to nitrogen-limited fermentation. Finally, we validated the ECM38 allele of NA strain as responsible for higher growth efficiency under nitrogen-limited conditions. Taken together, our results revealed a complex pattern of molecular signatures favouring adaptation of the yeast population to nitrogen-limited fermentations, including differential gene expression, allele frequency changes and loss of the mitochondrial genome. Finally, the results suggest that wild alleles from a non-domesticated isolate (NA) may have a relevant role in the adaptation to the assayed fermentation conditions, with the consequent potential of these alleles for the genetic improvement of wine yeast strains.

Necrotic enteritis (NE) caused by Clostridium perfringens is responsible for huge financial losses in the poultry industry annually. A diet highly supplemented with fishmeal is one factor predisposing chickens to the development of clinical NE. However, the effects of fishmeal-rich diets on the gut microbiota and immune response in chickens with C. perfringens challenge over the long-term are not well-understood. Here, a chicken NE model was established in which chickens were fed high fishmeal diet and subsequently infected with C. perfringens (FM/CP). Two control groups of chickens, one that was not infected and had a high fishmeal feeding (FM) and another group only infected with C. perfringens with basic diets (CP), were used as comparators. We analyzed the gut microbiota and immune response of the three groups at the age of 20, 24 [1 day post-infection (dpi)] and 30 days (7 dpi) using 16S rDNA sequencing and real-time PCR, respectively. We found that the composition of the gut microbiota had significant shifted in both the CP and FM/CP groups, although the CP group did not have intestinal lesions. The structure of the gut microbiota in C. perfringens-challenged chickens, independent of a high fishmeal diet, had the tendency to return to their non-infection state if the chickens no longer received C. perfringens challenge. Gut microbiota variation with time in challenged chickens with high fishmeal diet feeding was superimposed upon that of non-infected chickens with high fishmeal feeding. For the immune response, the relative expression of IL-8 in the ileum was significantly higher in infected chickens independent of high fishmeal feeding than in non-infected chickens. However, the expression of alpha 1-acid glycoprotein (AGP) and serum amyloid A (SAA) genes in chicken liver were significantly increased in FM/CP compared to the other groups. In conclusion, high fishmeal feeding induced significant changes to the structure of chicken gut microbiota over time and such changes provided an opening for C. perfringens infection to progress to NE. The relative expression of AGP and SAA in liver tissue may be used as diagnostic biomarkers for poultry NE but such an indication requires further investigation.

Influenza A virus (IAV) has developed elegant strategies to utilize cellular proteins and pathways to promote replication and evade the host antiviral response. Identification of these sabotaged host factors could increase the number of potential antiviral drug targets. Here, IAV A/PR/8/34 (PR8)- and A/California/04/2009-infected A549 and 293T cells displayed differential virus replication. To determine the host cellular responses of A549 and 293T cells to IAV infection, RNA-seq was used to identify differentially expressed genes. Our data revealed that IAV-infected A549 cells activated stronger virus-sensing signals and highly expressed cytokines, which play significant roles in initiating the innate immune and inflammatory responses. In addition, IAV-infected 293T cells displayed weak immune signaling and cytokine production. Remarkably, IL-17A and associated genes were highly enriched in IAV-infected 293T cells. Furthermore, IL-17A can partially facilitate A549 cell infection by the PR8 strain and PR8-infected IL-17A knock-out mice consistently exhibited decreased weight loss and reduced lung immunopathology, as compared to controls. This work uncovered the differential responses of cells infected with two H1N1 IAV strains and the potential roles of IL-17A in modulating virus infection.

Phosphorylation and dephosphorylation of nucleoprotein (NP) play significant roles in the life cycle of influenza A virus (IAV), and the biological functions of each phosphorylation site on NP are not exactly the same in controlling viral replication. Here, we identified tyrosine 78 residue (Y78) of NP as a novel phosphorylation site by mass spectrometry. Y78 is highly conserved, and the constant NP phosphorylation mimicked by Y78E delayed NP nuclear export through reducing the binding of NP to the cellular export receptor CRM1, and impaired virus growth. Furthermore, the tyrosine kinase inhibitors Dasatinib and AG490 reduced Y78 phosphorylation and accelerated NP nuclear export, suggesting that the Janus and Src kinases-catalyzed Y78 phosphorylation regulated NP nuclear export during viral replication. More importantly, we found that the NP phosphorylation could suppress NP ubiquitination via weakening the interaction between NP and E3 ubiquitin ligase TRIM22, which demonstrated a cross-talk between the phosphorylation and ubiquitination of NP. This study suggests that the phosphorylation status of Y78 regulates IAV replication by inhibiting the nuclear export and ubiquitination of NP. Overall, these findings shed new light on the biological roles of NP phosphorylation, especially its negative role in NP ubiquitination.

Listeria monocytogenes (Lm) is a foodborne zoonotic pathogen that causes listeriosis with a mortality rate of 20-30%. Serovar 4b and 1/2b isolates account for most of listeriosis outbreaks, however, no listeriosis vaccine is available for either prophylactic or therapeutic use. Here, we developed a triple-virulence-genes deletion vaccine strain, and evaluated its safety, immunogenicity, and cross-protective efficiency. The virulence of NTSNΔactA/plcB/orfX was reduced 794-folds compared with the parental strain. Additionally, it was completely eliminated in mice at day 7 post infection and no obvious pathological changes were observed in the organs of mice after prime-boost immunization for 23 days. These results proved that the safety of the Lm vaccine strain remarkably increased. More importantly, the NTSNΔactA/plcB/orfX strain stimulated higher anti-Listeriolysin O (LLO) antibodies, induced significantly higher expression of IFN-γ, TNF-α, IL-17, and IL-6 than the control group, and afforded 100% protection against serovar 4b and 1/2b challenges. Taken together, our research demonstrates that the triple-genes-deletion vaccine has high safety, can elicit strong Th1 type immune response, and affords efficient cross-protection against two serovar Lm strains. It is a promising vaccine for prevention of listeriosis.