CryoEM structures reveal how the bacterial flagellum rotates and switches direction.

Bacterial chemotaxis requires bidirectional flagellar rotation at different rates. Rotation is driven by a flagellar motor, which is a supercomplex containing multiple rings. Architectural uncertainty regarding the cytoplasmic C-ring, or 'switch', limits our understanding of how the motor transmits torque and direction to the flagellar rod. Here we report cryogenic electron microscopy structures for Salmonella enterica serovar typhimurium inner membrane MS-ring and C-ring in a counterclockwise pose (4.0 Å) and isolated C-ring in a clockwise pose alone (4.6 Å) and bound to a regulator (5.9 Å). Conformational differences between rotational poses include a 180° shift in FliF/FliG domains that rotates the outward-facing MotA/B binding site to inward facing. The regulator has specificity for the clockwise pose by bridging elements unique to this conformation. We used these structures to propose how the switch reverses rotation and transmits torque to the flagellum, which advances the understanding of bacterial chemotaxis and bidirectional motor rotation.

Transcriptional responses of cytokines, immunoglobulin A, and nitric oxide genes in 1-day-old chicks post Salmonella typhimurium infection: An experimental study.

Background: Salmonella has become one of the hazards prevalent foodborne pathogens causing different diseases in chickens. However, Salmonella typhimurium (ST), a nonhost-specific serovar, is a major avian agent that causes severe disturbance in young chicken wellness. Aim: The occurrence of Salmonella in chickens and their antimicrobial resistance were explored in this study. In addition, the immune response of 1-day-old broiler chicks, against multidrug resistant (MDR) ST infection, was also assessed at 4 and 24 hours post infection (pi) in the cecum and spleen, representing their mucosal and systemic immune responses, respectively. Methods: A total of 375 samples from 130 diseased and apparently healthy broiler and layer chickens were randomly collected for Salmonella isolation, identification, and resistance profile evaluation, from farms and different clinical laboratories. The immune response of 1-day-old broiler chicks, Ross 308, against in-vivo ST infection was ascertained through the evaluation of heterophile phagocytosis and s expression of cytokines, immunoglobulin A and other immune-regulating genes in the cecum and spleen. Twenty-four, 1-day-old nonvaccinated broiler chicks were used and divided into two groups. The chicks in the infected group were orally inoculated with 0.5 ml of 2 × 108 colony forming units (CFU)/ml of MDR ST suspension, while those in the control group were taken nutrient broth. Results: Seven out of 130 (5.38%) examined chickens were positive for Salmonella. All isolates (100%) were resistant to amoxicillin-clavulanic acid (AMC), cefazolin (CZ), cefoxitin (FOX), ciprofloxacin (CIP), nalidixic acid (NA), tetracycline (TE), fosfomycin (FOS), and colistin (CT) with multiple antimicrobial resistances (MARs) index range of 0.72-0.83, where none of them was resistant to meropenem (MEM). The results of immune response revealed that chicks infected with ST showed significantly different phagocytosis percentages and index values compared to controls. According to the real-time quantitative polymerase chain reaction (RT-qPCR) results, the transcription of IL-8, iNOS, IL-18, IgA, and IFN-γ for chicks infected by ST showed a significantly increased trend (p < 0.01) with increasing chicken age and was higher in the cecum than spleen compared to controls (p < 0.05) during 24 hours after infection. Conclusion: The findings indicated a strong mucosal immune response in the chicks after the ST challenge, which reflects humoral and cellular responses. Our insight recommended the occurrence of a natural immune response stimulator at 1 day age to face the infection, and this can prevent the resistance transfer, with efficient control measures.

Identification of a microbial sub-community from the feral chicken gut that reduces Salmonella colonization and improves gut health in a gnotobiotic chicken model.

A complex microbial community in the gut may prevent the colonization of enteric pathogens such as Salmonella. Some individual or a combination of species in the gut may confer colonization resistance against Salmonella. To gain a better understanding of the colonization resistance against Salmonella enterica, we isolated a library of 1,300 bacterial strains from feral chicken gut microbiota which represented a total of 51 species. Using a co-culture assay, we screened the representative species from this library and identified 30 species that inhibited Salmonella enterica subspecies enterica serovar Typhimurium in vitro. To improve the Salmonella inhibition capacity, from a pool of fast-growing species, we formulated 66 bacterial blends, each of which composed of 10 species. Bacterial blends were more efficient in inhibiting Salmonella as compared to individual species. The blend that showed maximum inhibition (Mix10) also inhibited other serotypes of Salmonella frequently found in poultry. The in vivo effect of Mix10 was examined in a gnotobiotic and conventional chicken model. The Mix10 consortium significantly reduced Salmonella load at day 2 post-infection in gnotobiotic chicken model and decreased intestinal tissue damage and inflammation in both models. Cell-free supernatant of Mix10 did not show Salmonella inhibition, indicating that Mix10 inhibits Salmonella through either nutritional competition, competitive exclusion, or through reinforcement of host immunity. Out of 10 species, 3 species in Mix10 did not colonize, while 3 species constituted more than 70% of the community. Two of these species were previously uncultured bacteria. Our approach could be used as a high-throughput screening system to identify additional bacterial sub-communities that confer colonization resistance against enteric pathogens and its effect on the host.IMPORTANCESalmonella colonization in chicken and human infections originating from Salmonella-contaminated poultry is a significant problem. Poultry has been identified as the most common food linked to enteric pathogen outbreaks in the United States. Since multi-drug-resistant Salmonella often colonize chicken and cause human infections, methods to control Salmonella colonization in poultry are needed. The method we describe here could form the basis of developing gut microbiota-derived bacterial blends as a microbial ecosystem therapeutic against Salmonella.

Microencapsulate Probiotics (MP) Promote Growth Performance and Inhibit Inflammatory Response in Broilers Challenged with Salmonella typhimurium.

Antibiotic-resistant bacteria are prevalent in husbandry around the world due to the abuse of antibiotic growth promoters (AGPs); therefore, it is necessary to find alternatives to AGPs in animal feed. Among all the candidates, probiotics are promising alternatives to AGPs against Salmonella infection. The anti-Salmonella effects of three probiotic strains, namely, Lactobacillus crispatus 7-4, Lactobacillus johnsonii 3-1, and Pediococcus acidilactici 20-1, have been demonstrated in our previous study. In this study, we further obtained the alginate beads containing compound probiotics, namely, microencapsulate probiotics (MP), and evaluated its regulatory effect on the health of broilers. We incubated free and microencapsulate probiotics in simulated gastric and intestinal juice for 2 h, and the results showed that compared to free probiotics, encapsulation increased tolerance of compound probiotics in the simulated gastrointestinal condition. We observed that the application of probiotics, especially MP, conferred protective effects against Salmonella typhimurium (S.Tm) infection in broilers. Compared to the S.Tm group, the MP could promote the growth performance (p < 0.05) and reduce the S.Tm load in intestine and liver (p < 0.05). In detail, MP pretreatment could modulate the cecal microflora and upregulate the relative abundance of Lactobacillus and Enterobacteriaceae. Besides, MP could reduce the inflammation injury of the intestine and liver, reduce the pro-inflammatory cytokines (IL-6, TNF-α, IL-1ß) expression, and induce of anti-inflammatory cytokine (IL-10) expression. Furthermore, MP could inhibit NLRP3 pathway in ileum, thereby attenuating S.Tm-induced inflammation. In conclusion, MP could be a new feeding supplementation strategy to substitute AGPs in poultry feeding.

BolA promotes the generation of multicellular behavior in S. Typhimurium by regulating the c-di-GMP pathway genes yeaJ and yhjH.

The generation of multicellular behavior enhances the stress adaptability, antibiotic resistance, and pathogenic potential of Salmonella enterica serovar Typhimurium (S. Typhimurium), which is challenging for its prevention and control. Therefore, determination of the mechanism of multicellular behavior development is urgently required. Accordingly, this study investigated BolA, a transcription factor that promotes bacterial survival under different stresses. We found that BolA promoted the generation of multicellular behavior. Furthermore, transcriptome analysis revealed that BolA affected the expression of numerous genes, including biofilm formation and motility-related genes. In terms of biofilm formation, compared with the wild-type strain, bolA overexpression (269BolA+) increased the extracellular matrix content (extracellular polysaccharide, extracellular protein, and extracellular DNA (eDNA) by upregulating gene expression, ultimately increasing the biofilm formation ability by 2.56 times. For motility, bolA overexpression inhibited the expression of flagella synthesis genes, resulting in a 91.15 % decrease in motility compared with the wild-type (6 h). Further mechanistic analysis demonstrated that BolA affected the expression of the C-di-GMP pathway genes yeaJ and yhjH, which influenced the generation of multicellular behavior. In terms of biofilms, the extracellular polysaccharide content of 269BolA + ∆Yeaj (bolA overexpression and yeaJ deletion) was reduced by 89.91 % compared with 269BolA+, resulting in a 71.1 % reduction in biofilm forming ability. The motility of the 269∆BolA∆Yhjh (bolA/yhjH double deletion) strain was significantly decreased compared with that of 269∆BolA. Finally, the LacZ gene reporting showed that BolA promoted and inhibited the expression of yeaJ and yhjH, respectively. In conclusion, BolA mainly improves the content of extracellular polysaccharide by promoting the expression of yeaJ, thus enhancing the formation of biofilms. BolA also restricts flagellar synthesis by inhibiting yhjH expression, therefore reducing motility, ultimately promoting multicellular behavior arises. These findings lay a theoretical foundation for the prevention and control of S. Typhimurium.

Gut microbiota of the small intestine as an antimicrobial barrier against foodborne pathogens: Impact of diet on the survival of S. Typhimurium and L. monocytogenes during in vitro digestion.

The human gastrointestinal tract employs an assortment of chemical, enzymatic and immune barriers to impede pathogen colonization. An essential component of these barriers is the gut microbiota, which infers protection against ingested pathogens through its colonization resistance mechanisms. Specifically, the gut microbiota of the distal small intestine (ileum) renders a crucial line of defense, given that this location is regarded as an important interaction site. This study aimed to evaluate the impact of the ileal microbiota on the survival of the foodborne pathogens Salmonella enterica serotype Typhimurium and Listeria monocytogenes, utilizing an in vitro digestion model system. Moreover, the effect of diet on the gut microbiota colonization resistance mechanisms was assessed, by comparing a healthy (high fiber/low sugar) and a western diet (low fiber/high sugar). For S. Typhimurium, the results revealed that the digestion of a healthy diet led to a similar inactivation compared to the western diet, with the values of total log reduction being 0.83 and 0.82 log(CFU), respectively; yet the lack of readily accessible nutrients in the healthy diet combined with the acidic shock during gastric digestion caused the induction of stress tolerance to the pathogen. This resulted in increased pathogen survival in the presence of gut microbiota, with S. Typhimurium proliferating during the ileal phase with a maximum specific growth rate of 0.16 1/h. On the contrary, for L. monocytogenes, the healthy diet was associated with a greater inactivation than the western diet (total log reduction values: 3.08 and 1.30 log(CFU), respectively), which appeared strongly influenced by the encounter of the pathogen with the gut microbiota. Regarding the latter, the species Escherichia coli and Bacteroides thetaiotaomicron appeared to be the most prevalent in most cases. Finally, it was also demonstrated that the ileal microbiota colonization resistance mechanisms largely relied on competitive responses. The obtained knowledge of this research can contribute to the development and/or complementation of defensive strategies against pathogen infection, while also underlining the value of in vitro approaches.

Shift in vacuolar to cytosolic regime of infecting Salmonella from a dual proteome perspective.

By applying dual proteome profiling to Salmonella enterica serovar Typhimurium (S. Typhimurium) encounters with its epithelial host (here, S. Typhimurium infected human HeLa cells), a detailed interdependent and holistic proteomic perspective on host-pathogen interactions over the time course of infection was obtained. Data-independent acquisition (DIA)-based proteomics was found to outperform data-dependent acquisition (DDA) workflows, especially in identifying the downregulated bacterial proteome response during infection progression by permitting quantification of low abundant bacterial proteins at early times of infection when bacterial infection load is low. S. Typhimurium invasion and replication specific proteomic signatures in epithelial cells revealed interdependent host/pathogen specific responses besides pointing to putative novel infection markers and signalling responses, including regulated host proteins associated with Salmonella-modified membranes.

High throughput screening identifies auranofin and pentamidine as potent compounds that lower IFN-γ-induced Nitric Oxide and inflammatory responses in mice: DSS-induced colitis and Salmonella Typhimurium-induced sepsis.

Interferon-gamma (IFN-γ) is a type II interferon produced primarily by T cells and natural killer cells. IFN-γ induces the expression of inducible nitric oxide synthase (NOS2) to catalyze Nitric Oxide (NO) production in various immune and non-immune cells. Excessive IFN-γ-activated NO production is implicated in several inflammatory diseases, including peritonitis and inflammatory bowel diseases. In this study, we screened the LOPAC®1280 library in vitro on the H6 mouse hepatoma cell line to identify novel non-steroidal small molecule inhibitors of IFN-γ-induced NO production. Compounds with the highest inhibitory activity were validated, which led to identifying the lead compounds: pentamidine, azithromycin, rolipram, and auranofin. Auranofin was the most potent compound determined based on IC50 and goodness of fit analyses. Mechanistic investigations revealed that majority of the lead compounds suppress the IFN-γ-induced transcription of Nos2 without negatively affecting NO-independent processes, such as the IFN-γ-induced transcription of Irf1, Socs1 and MHC class 1 surface expression. However, all four compounds lower IFN-γ-induced reactive oxygen species amounts. In addition, auranofin significantly reduced IFN-γ-mediated NO and IL6 production in resident as well as thioglycolate-elicited peritoneal macrophages (PMs). Finally, in vivo testing of the lead compounds in the pre-clinical DSS-induced ulcerative colitis mice model revealed pentamidine and auranofin to be the most potent and protective lead compounds. Also, pentamidine and auranofin greatly increase the survival of mice in another inflammatory model: Salmonella Typhimurium-induced sepsis. Overall, this study identifies novel anti-inflammatory compounds targeting IFN-γ-induced NO-dependent processes to alleviate two distinct inflammatory models of disease.

Differential Salmonella Typhimurium intracellular replication and host cell responses in caecal and ileal organoids derived from chicken.

Chicken infection with Salmonella Typhimurium is an important source of foodborne human diseases. Salmonella colonizes the avian intestinal tract and more particularly the caecum, without causing symptoms. This thus poses a challenge for the prevention of foodborne transmission. Until now, studies on the interaction of Salmonella with the avian gut intestine have been limited by the absence of in vitro intestinal culture models. Here, we established intestinal crypt-derived chicken organoids to better decipher the impact of Salmonella intracellular replication on avian intestinal epithelium. Using a 3D organoid model, we observed a significantly higher replication rate of the intracellular bacteria in caecal organoids than in ileal organoids. Our model thus recreates intracellular environment, allowing Salmonella replication of avian epithelium according to the intestinal segment. Moreover, an inhibition of the cellular proliferation was observed in infected ileal and caecal organoids compared to uninfected organoids. This appears with a higher effect in ileal organoids, as well as a higher cytokine and signaling molecule response in infected ileal organoids at 3 h post-infection (hpi) than in caecal organoids that could explain the lower replication rate of Salmonella observed later at 24 hpi. To conclude, this study demonstrates that the 3D organoid is a model allowing to decipher the intracellular impact of Salmonella on the intestinal epithelium cell response and illustrates the importance of the gut segment used to purify stem cells and derive organoids to specifically study epithelial cell -Salmonella interaction.

Combined Ganoderma lucidum polysaccharide and ciprofloxacin therapy alleviates Salmonella enterica infection, protects the intestinal barrier, and regulates gut microbiota.

Clinical antibiotics used worldwide could diminish the intestinal barrier, enhance contact with microbiota and intestinal immune cells, and induce inflammation. We found that ciprofloxacin treatment of Salmonella enterica serovar Typhimurium infection resulted in the destruction of the intestinal barrier, with decreased concentrations of MUC2, ZO-1, and occludin in the jejunum and colon. Ganoderma lucidum ethanol extracts (GLE), as a prebiotic food extract, significantly decreased inflammation-related enzymes, including COX-2, MPO, and iNOS, and pro-inflammatory cytokines (IL-6, IL-1ß, IL-17, and TNF-α), and protected the intestinal barrier by increasing the concentration of MUC2, ZO-1, and occludin. Meanwhile it significantly increased the abundances of Salmonella, Parabacteroides, Acinetobacter, Enterococcus, and Escherichia-Shigella, which increased the risk of pathogenic bacterial infections. Prebiotic G. lucidum polysaccharide (GLP) provided a significant intestinal barrier, improving the concentration of ZO-1, occludin, and MUC2 in the colon and jejunum. The synergistic effects of GLP and ciprofloxacin were hypothesized to reverse the negative effects resulting from ciprofloxacin alone, as the concentrations of ZO-1, occludin, and MUC2 were significantly increased in the jejunum and colon, especially in the colon. Also, the synergistic effect increased the abundances of probiotic bacteria Lachnospiraceae NK4A136, Ruminococcaceae UGG-014, Lactobacillus, and Parabacteroides. In conclusion, combined GLP and ciprofloxacin therapy against Salmonella infection alleviated the side effects resulting from the clinical application of the antibiotic alone, and increased the probiotic bacterial population.

Salmonella enterica subsp. enterica serovar Typhimurium and Lactobacillus spp. interactions in vitro elicit improved antimicrobial production.

Antimicrobial resistance (AMR) is a global health crisis. Despite the drug discovery efforts, AMR is increasing, and discoveries are nearly nil. It is thus critical to design new strategies. Probiotics are tapped as alternatives to antibiotics for the treatment of gut-associated diseases. Lactobacillus species, common in food products, can inhibit the growth of gut pathogens. Here, we demonstrate the antimicrobial activities of Lactobacillus species - Lactobacillus paracasei, Lactobacillus casei, and Lactobacillus delbrueckii subsp. bulgaricus are enhanced when cocultured with Salmonella enterica subsp. enterica serovar Typhimurium. Cell-free culture supernatants (CFCS) from cocultures of Lactobacillus spp. and Salmonella enterica serovar Typhimurium more potently inhibit pathogen growth than their monoculture counterparts. Interestingly, we discovered that Salmonella enterica serovar Typhimurium could enhance the production of antimicrobials from Lactobacillus spp., most evident in L. delbrueckii subsp. bulgaricus. Also, L. delbrueckii subsp. bulgaricus CFCS upregulates key Salmonella virulence genes, hilA and sipA. Whether this increases Salmonella's pathogenicity in vivo or reduces pathogen fitness and growth inhibition in vitro warrants further investigation. We propose that these probiotic isolates may be utilized for innovative natural food processing and preservation strategies to control Salmonella food contaminations. Importantly, our findings that Salmonella elicits an enhanced antimicrobial activity from Lactobacillus spp. provide evidence of a pathogen-mediated elicitation of antimicrobial production. Therefore, extending this phenomenon to other microbial interactions may help augment the strategies for drug discovery.

Survival of Salmonella Typhimurium in the hemolymph of the German cockroach vector is limited by both humoral immune factors and hemocytes but not by trehalose metabolism.

The German cockroach (Blattella germanica) has been linked to transmission of Salmonella enterica serovar Typhimurium (S. Typhimurium), but infection dynamics within this vector are poorly characterized. Our recent work has focused on S. Typhimurium infection in the cockroach gut. However, microbial dissemination to the hemolymph is an essential aspect of many vector-borne pathogen transmission cycles and could potentially contribute to S. Typhimurium colonization of cockroaches. Therefore, the goal of this study was to examine the ability of S. Typhimurium to disseminate, survive, and proliferate in the hemolymph of cockroaches after oral infection. We detected only low numbers of bacteria in the hemolymph of a minority of insects (~26%) after oral infection. Further, S. Typhimurium was unable to survive overnight in cell-free hemolymph. Several hypotheses to explain the inability of S. Typhimurium to colonize hemolymph were tested. First, we investigated the ability of S. Typhimurium to metabolize trehalose, the primary sugar in hemolymph. S. Typhimurium grew efficiently in vitro using trehalose as a sole carbon source and mutant strains lacking trehalose metabolism genes exhibited no growth deficiencies in media mimicking the composition of hemolymph, suggesting that trehalose metabolism ability is not a factor involved in restricting survival in hemolymph. On the other hand, heat-inactivated cell-free hemolymph was permissive of S. Typhimurium growth, demonstrating that survival in hemolymph is limited specifically by heat-labile humoral factors. The involvement of cellular immune responses was also investigated and cockroach hemocytes in culture were observed to internalize S. Typhimurium within 1 h of exposure. Most hemocytes harbored few to no bacteria after 24 h, indicating that hemocyte responses are additionally involved in clearing infection from the hemolymph. However, dense intracellular clusters of S. Typhimurium were observed sporadically, suggesting a small subset of hemocytes may serve as reservoirs for bacterial replication. Together, our results reveal that a minute proportion of ingested S. Typhimurium is able to escape the cockroach gut and enter the hemolymph, but this systemic population is limited by both humoral effectors and hemocytes. Thus, we conclude that invasion of the hemolymph appears minimally important for colonization of the cockroach vector and that colonization of the gut is the main driver of vector-borne transmission. Our insight into the antimicrobial mechanisms of cockroach hemolymph also highlights the strong ability of these prevalent pests/vectors to cope with frequent infectious challenges in septic habitats.

Identification and Characterization of Probiotic Lactiplantibacillus plantarum BI-59.1 Isolated from tejuino and Its Capacity to Produce Biofilms.

Tejuino is a popular and traditional beverage consumed in north and western of Mexico, due to its biological properties, it is considered a natural source of probiotics. Nevertheless, few studies have been performed on Tejuino microbiota. In this work, the probiotic potential of the tejuino isolated Lactiplantibacillus plantarum BI-59.1 strain was investigated. Its effectiveness was compared with a commercial Lactobacillus spp and identified by 16S rDNA sequence homology. Lactiplantibacillus plantarum BI-59.1 strain showed probiotic properties, i.e., production of antimicrobial compounds (lactic acid and presence of plantaricin A gene), inhibition of entero-pathogens by planktonic cells and metabolites (Salmonella enterica serovar Typhimurium inhibition to HT29-MTX adhesion), biofilm formation, bacterial adhesion (HT29-MTX, 3.96 CFU/cell), and tolerance to stimulated gastrointestinal conditions (tolerance to pH 3 and bile salts). The strain was gamma hemolytic, susceptible to most antibiotics and negative for gelatinase production; thus, the Lactiplantibacillus. plantarum BI-59.1 strain is suitable for its use as a probiotic for nutraceutical or pharmaceutical formulations.

Deceiving the big eaters: Salmonella Typhimurium SopB subverts host cell xenophagy in macrophages via dual mechanisms.

Salmonella, a stealthy facultative intracellular pathogen, utilises an array of host immune evasion strategies. This facilitates successful survival via replicative niche establishment in otherwise hostile environments such as macrophages. Salmonella survives in and utilises macrophages for effective dissemination, ultimately leading to systemic infection. Bacterial xenophagy or macro-autophagy is an important host defense mechanism in macrophages. Here, we report for the first time that the Salmonella pathogenicity island-1 (SPI-1) effector SopB is involved in subverting host autophagy via dual mechanisms. SopB is a phosphoinositide phosphatase capable of altering the phosphoinositide dynamics of the host cell. Here, we demonstrate that SopB mediates escape from autophagy by inhibiting the terminal fusion of Salmonella-containing vacuoles (SCVs) with lysosomes and/or autophagosomes. We also report that SopB downregulates overall lysosomal biogenesis by modulating the Akt-transcription factor EB (TFEB) axis via restricting the latter's nuclear localisation. TFEB is a master regulator of lysosomal biogenesis and autophagy. This reduces the overall lysosome content inside host macrophages, further facilitating the survival of Salmonella in macrophages and systemic dissemination of Salmonella.

The impact of lipid A modification on biofilm and related pathophysiological phenotypes, endotoxicity, immunogenicity, and protection of Salmonella Typhimurium.

This study presents the engineering of a less endotoxic Salmonella Typhimurium strain by manipulating the lipid-A structure of the lipopolysaccharide (LPS) component. Salmonella lipid A was dephosphorylated by using lpxE from Francisella tularensis. The 1-phosphate group from lipid-A was removed selectively, resulting in a close analog of monophosphoryl lipid A. We observed a significant impact of ∆pagL on major virulence factors such as biofilm formation, motility, persistency, and immune evasion. In correlation with biofilm and motility retardation, adhesion and invasion were elevated but with reduced intracellular survival, a favorable phenotype prospect of a vaccine strain. Western blotting and silver staining confirmed the absence of the O-antigen and truncated lipid-A core in the detoxified Salmonella mutant. In vitro and in vivo studies demonstrated that the dephosphorylated Salmonella mutant mediated lower pro-inflammatory cytokine secretion than the wild-type strain. The vaccine strains were present in the spleen and liver for five days and were cleared from the organs by day seven. However, the wild-type strain persisted in the spleen, liver, and brain, leading to sepsis-induced death. Histological evaluations of tissue samples further confirmed the reduced endotoxic activity of the detoxified Salmonella mutant. The detoxification strategy did not compromise the level of protective immunity, as the vaccine strain could enhance humoral and cellular immune responses and protect against the wild-type challenge in immunized mice.

Genotypic and phenotypic characterization of a Salmonella Typhimurium strain resistant to pulsed electric fields.

Pulsed Electric Fields (PEF) technology is regarded as one of the most interesting alternatives to current food preservation methods, due to its capability to inactivate vegetative microorganisms while leaving the product's organoleptic and nutritional properties mostly unchanged. However, many aspects regarding the mechanisms of bacterial inactivation by PEF are still not fully understood. The aim of this study was to obtain further insight into the mechanisms responsible for the increased resistance to PEF of a Salmonella Typhimurium SL1344 variant (SL1344-RS, Sagarzazu et al., 2013), and to quantify the impact that the acquisition of PEF resistance has on other aspects of S. enterica physiology, such as growth fitness, biofilm formation ability, virulence and antibiotic resistance. WGS, RNAseq and qRT-PCR assays indicated that the increased PEF resistance of the SL1344-RS variant is due to a higher RpoS activity caused by a mutation in the hnr gene. This increased RpoS activity also results in higher resistance to multiple stresses (acidic, osmotic, oxidative, ethanol and UV-C, but not to heat and HHP), decreased growth rate in M9-Gluconate (but not in TSB-YE or LB-DPY), increased ability to adhere to Caco-2 cells (but no significant change in invasiveness) and enhanced antibiotic resistance (to six out of eight agents). This study significantly contributes to the understanding of the mechanisms of the development of stress resistance in Salmonellae and underscores the crucial role played by RpoS in this process. Further studies are needed to determine whether this PEF-resistant variant would represent a higher, equal or lower associated hazard than the parental strain.

Dietary protocatechuic acid ameliorates ileal mucosal barrier injury and inflammatory response and improves intestinal microbiota composition in Yellow chickens challenged with Salmonella typhimurium.

Salmonella typhimurium (ST) is a common foodborne pathogen that severely affects the health of humans and livestock. Protocatechuic acid (PCA) has been shown to possess anti-inflammatory and anti-bacterial functions. Chickens were used to investigate the effect of PCA on the gut health infected with ST. A total of one hundred eighty, 1-d-old birds were randomly allocated into 3 treatments, each with 6 replicates per treatment and 10 chicks per replicate. Broiler chicks in the control and ST treatment were fed a basal diet, and birds in the PCA+ST treatment received the basal diet with 600 mg/kg PCA. On d 14 and 16 of the trial, broilers in ST and PCA+ST treatments received an oral dose of ST, while broilers in CON received an equal amount of PBS. The data were analyzed by the one-way ANOVA. Dietary PCA increased (P < 0.05) final body weight, average daily gain, and feed to gain ratio in ST-challenged Yellow broilers. Protocatechuic acid significantly alleviated ST-induced intestinal mucosal injury reflected in the decreased (P < 0.05) plasma activity of diamine oxidase and ileal apoptosis, with increased (P < 0.05) ileal villus height and villus height/crypt depth. Protocatechuic acid treatment significantly decreased (P < 0.05) ST-induced proinflammatory cytokine (Interleukin-1ß, Interleukin-6, Tumor necrosis factor-α, and Interferon-ß) content in ileum. Meanwhile, PCA treatment significantly increased (P < 0.05) the transcript abundances of claudin 1 (CLDN1), zonula occludens-1 (ZO-1), and mucin 2 (MUC2) in ileum, all related to the intestinal barrier in ST-challenged Yellow broilers. Additionally, PCA also increased (P < 0.05) the diversity and richness of the cecal microflora as reflected by reduced (P < 0.05) abundance of Bacteroidota, Proteobacteria and Escherichia-Shigella, and increased (P < 0.05) abundance of Firmicutes and Lactobacillus in ST-challenged Yellow broilers. These findings indicate that PCA relieves ST-induced loss weight, intestinal barrier injury, inflammatory response, and improves intestinal microbiota composition in Yellow broilers.

Effect of culture method on storage, plasma, and dry heat treatment resistance of Salmonella enterica serovar Typhimurium on black pepper.

The purpose of this study was to determine the effect of the culture method on the resistance of Salmonella Typhimurium in low water activity foods to storage, plasma, and dry heat. Whole black peppers were used as the model food. S. Typhimurium cultured in liquid broth (tryptic soy broth) or solid agar (tryptic soy agar) and inoculated on whole black pepper was stored or treated with cold plasma or dry heat. Inactivation of S. Typhimurium cultured in liquid medium was higher in all the treatments. Liquid-cultured S. Typhimurium showed higher DPPP = O (diphenyl-1-pyrenylphosphine oxide) values compared to the solid-cultured S. Typhimurium after plasma or dry heat treatment. Furthermore, the unsaturated fatty acid and saturated fatty acid ratio (USFA/SFA) was significantly (P < 0.05) reduced from 0.41 to 0.29 when S. Typhimurium was cultured on solid agar. These results suggested that the use of food-borne pathogens cultured on solid agar is more suitable for low water activity food pasteurization studies.

Oral GSH Exerts a Therapeutic Effect on Experimental Salmonella Meningitis by Protecting BBB Integrity and Inhibiting Salmonella-induced Apoptosis.

Bacterial meningitis (BM) is the main cause of the central nervous system (CNS) infection and continues to be an important cause of mortality and morbidity. Glutathione (GSH), an endogenous tripeptide antioxidant, has been proved to exert crucial role in reducing superoxide radicals, hydroxyl radicals and peroxynitrites. The purpose of this study is to expand the application scope of GSH via exploring its therapeutic effect on BM caused by Salmonella typhimurium SL1344 and then provide a novel approach for the treatment of BM. The results suggested that intragastric administration of GSH could significantly increase median survival and improve experimental autoimmune encephalomyelitis score of BM model mice. However, exogenous GSH did not affect the adhesion, invasion and cytotoxicity of SL1344 to C6, BV2 and primary microglia. Due to the contradiction between the therapeutic and bactericidal effects of GSH, the effect of GSH on blood-brain barrier (BBB) was investigated to explore its action target for the treatment of meningitis. GSH was found to repair the damage of BBB and then prevent the leakage of SL1344 from the brain to the blood circulation. The repaired BBB could also effectively reduce the entry of macrophages and neutrophils into the brain, and significantly reverse the microglia activation induced by SL1344. More importantly, exogenous GSH was proved to reduce mouse brain cell apoptosis by inhibiting the activation of caspase-8 followed by caspase-3, and reversing the up-regulation of ICAD and PARP-1 caused by SL1344.

Lactiplantibacillus plantarum Regulated Intestinal Microbial Community and Cytokines to Inhibit Salmonella typhimurium Infection.

Lactic acid bacteria (LAB) are recognized as food-grade safe microorganisms and have many beneficial effects. LAB could maintain the host intestinal homeostasis and regulate intestinal microbial community to exert antibacterial effects. In this study, Lactiplantibacillus plantarum (L. plantarum, Lp01) strain isolated from pig intestine was orally administered to C57BL/6 mice, and mice were then infected with Salmonella typhimurium (ATCC14028). The protective effects of L. plantarum were evaluated by monitoring body weight loss, survival rates, bacterial loads in tissue, colon histopathology analysis, and cytokine secretion. 16S rRNA gene sequencing was also utilized to detect the dynamics of the blind gut microbial community in mice. We found that L. plantarum could significantly reduce the body weight loss and improve the survival rates. The survival rate in the L. P-Sty group was up to 67.5%, which was much higher than that in the STY group (25%). Counting of bacterial loads displayed that the colony-forming unit (CFU) of S. typhimurium in the spleen (p < 0.05) and the liver (p < 0.05) from L. P-Sty group both decreased, compared with STY group. Intestinal histopathology showed that it alleviated the intestinal injury caused by Salmonella, inhibited the secretion of pro-inflammatory cytokines, and promoted anti-inflammatory cytokines (p < 0. 01). In addition, L. plantarum also significantly ameliorated the intestinal gut microbiome disturbance caused by Salmonella. It displayed an obvious increase of beneficial bacteria including Lactobacillus and Bacteroidetes and reduction of pathogenic bacteria like Proteobacteria. In conclusion, L. plantarum could regulate microbial community to inhibit Salmonella typhimurium infection.