CheV enhances the virulence of Salmonella Enteritidis, and the Chev-deleted Salmonella vaccine provides immunity in mice.

BACKGROUND: Salmonella enteritidis (SE) is a major zoonotic pathogen and causes infections in a variety of hosts. The development of novel vaccines for SE is necessary to eradicate this pathogen. Genetically engineered attenuated live vaccines are more immunogenic and safer. Thus, to develop a live attenuated Salmonella vaccine, we constructed a cheV gene deletion strain of SE (named ΔcheV) and investigated the role of cheV in the virulence of SE. First, the ability to resist environmental stress in vitro, biofilm formation capacity, drug resistance and motility of ΔcheV were analyzed. Secondly, the bacterial adhesion, invasion, intracellular survival assays were performed by cell model. Using a mouse infection model, an in vivo virulence assessment was conducted. To further evaluate the mechanisms implicated by the reduced virulence, qPCR analysis was utilized to examine the expression of the strain's major virulence genes. Finally, the immune protection rate of ΔcheV was evaluated using a mouse model. RESULTS: Compared to C50336, the ΔcheV had significantly reduced survival ability under acidic, alkaline and thermal stress conditions, but there was no significant difference in survival under oxidative stress conditions. There was also no significant change in biofilm formation ability, drug resistance and motility. It was found that the adhesion ability of ΔcheV to Caco-2 cells remained unchanged, but the invasion ability and survival rate in RAW264.7 cells were significantly reduced. The challenge assay results showed that the LD50 values of C50336 and ΔcheV were 6.3 × 105 CFU and 1.25 × 107 CFU, respectively. After the deletion of the cheV gene, the expression levels of fimD, flgG, csgA, csgD, hflK, lrp, sipA, sipB, pipB, invH, mgtC, sodC, rfbH, xthA and mrr1 genes were significantly reduced. The live attenuated ΔcheV provided 100% protection in mice against SE infection. CONCLUSION: All the results confirmed that the deletion of the cheV gene reduces the virulence of SE and provides significant immune protection in mice, indicating that ΔcheV could be potential candidates to be explored as live-attenuated vaccines.

Septicemic salmonellosis in suckling piglets resulting from improper intramuscular administration of an oral vaccine.

We describe an unusual outbreak of mortality in suckling piglets following the misadministration of an oral vaccine against Salmonella Typhimurium and Salmonella Choleraesuis. Within 3-48 h of vaccination of a batch of ~700 piglets, ~300 developed marked swelling in the dorsal neck region, respiratory distress, fever, recumbency, and apathy. In total, ~100 died, and 4 were submitted for autopsy. Gross and microscopic lesions consisted of focally extensive areas of purple discoloration in the skin of the cervical region, associated with edema and hemorrhage in the subcutis and muscles. Additionally, there was interstitial pneumonia with marked interlobular edema and mild fibrinous pleuritis. Aerobic bacterial culture identified Salmonella Typhimurium (3 cases) and Salmonella Choleraesuis (1 case) in samples of skeletal muscle and lung and from pleural swab samples. Marked immunostaining against Salmonella spp. was observed in the skeletal muscle of the cervical region, as well as in blood vessels and macrophages from the lung, liver, spleen, and kidney. We concluded that inappropriate intramuscular administration of an oral vaccine against Salmonella resulted in septicemia and death in a batch of piglets.

Molecular characterization and environmental impact of newly isolated lytic phage SLAM_phiST1N3 in the Cornellvirus genus for biocontrol of a multidrug-resistant Salmonella Typhimurium in the swine industry chain.

Salmonella Typhimurium is a highly lethal pathogenic bacterium in weaned piglets, causing significant treatment costs and economic losses in the swine industry. Additionally, due to its ability to induce zoonotic diseases, resulting in harm to humans through the transmission of the pathogen from pork, it presents a serious public health issue. Bacteriophages (phages), viruses that infect specific bacterial strains, have been proposed as an alternative to antibiotics for controlling pathogenic bacteria. In this study, we isolated SLAM_phiST1N3, a phage infecting a multidrug-resistant (MDR) S. Typhimurium wild-type strain isolated from diseased pigs. First, comparative genomics and phylogenetic analysis revealed that SLAM_phiST1N3 belongs to the Cornellvirus genus. Moreover, utilizing a novel classification approach introduced in this study, SLAM_phiST1N3 was classified at the species level. Host range experiments demonstrated that SLAM_phiST1N3 did not infect other pathogenic bacteria or probiotics derived from pigs or other livestock. While complete eradication of Salmonella was not achievable in the liquid inhibition assay, surprisingly, we succeeded in largely eliminating Salmonella in the FIMM analysis, a gut simulation system using weaned piglet feces. Furthermore, using the C. elegans model, we showcased the potential of SLAM_phiST1N3 to prevent S. Typhimurium infection in living organisms. In addition, it was confirmed that bacterial control could be achieved when phage was applied to Salmonella-contaminated pork. pH and temperature stability experiments demonstrated that SLAM_phiST1N3 can endure swine industry processes and digestive conditions. In conclusion, SLAM_phiST1N3 demonstrates potential environmental impact as a substance for Salmonella prevention across various aspects of the swine industry chain.

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.

Unravelling the role of anaerobic metabolism (pta-ackA) and virulence (misL and ssa) genes in Salmonella Heidelberg shedding using chicken infection model.

The mechanism of colonisation of the chicken intestine by Salmonella remains poorly understood, while the severity of infections vary enormously depending on the serovar and the age of the bird. Several metabolism and virulence genes have been identified in Salmonella Heidelberg; however, information on their roles in infection, particularly in the chicken infection model, remains scarce. In the present publication, we investigated three Salmonella Heidelberg mutants containing deletions in misL, ssa, and pta-ackA genes by using signature-tagged mutagenesis. We found that mutations in these genes of S. Heidelberg result in an increase in fitness in the chicken model. The exception was perhaps the pta-ackA mutant where colonisation was slightly reduced (2, 7, 14, and 21 days post-infection) although some birds were still excreting at the end of the experiment. Our results suggest that for intestinal colonisation of the chicken caecum, substrate-level phosphorylation is likely to be more important than the MisL outer membrane protein or even the secretion system apparatus. These findings validate previous work that demonstrated the contribution of ackA and pta mutants to virulence in chickens, suggesting that the anaerobic metabolism genes such as pta-ackA could be a promising mitigation strategy to reduce S. Heidelberg virulence.

Contribution of bacterial and host factors to pathogen "blooming" in a gnotobiotic mouse model for Salmonella enterica serovar Typhimurium-induced enterocolitis.

Inflammation has a pronounced impact on the intestinal ecosystem by driving an expansion of facultative anaerobic bacteria at the cost of obligate anaerobic microbiota. This pathogen "blooming" is also a hallmark of enteric Salmonella enterica serovar Typhimurium (S. Tm) infection. Here, we analyzed the contribution of bacterial and host factors to S. Tm "blooming" in a gnotobiotic mouse model for S. Tm-induced enterocolitis. Mice colonized with the Oligo-Mouse-Microbiota (OMM12), a minimal bacterial community, develop fulminant colitis by day 4 after oral infection with wild-type S. Tm but not with an avirulent mutant. Inflammation leads to a pronounced reduction in overall intestinal bacterial loads, distinct microbial community shifts, and pathogen blooming (relative abundance >50%). S. Tm mutants attenuated in inducing gut inflammation generally elicit less pronounced microbiota shifts and reduction in total bacterial loads. In contrast, S. Tm mutants in nitrate respiration, salmochelin production, and ethanolamine utilization induced strong inflammation and S. Tm "blooming." Therefore, individual Salmonella-specific inflammation-fitness factors seem to be of minor importance for competition against this minimal microbiota in the inflamed gut. Finally, we show that antibody-mediated neutrophil depletion normalized gut microbiota loads but not intestinal inflammation or microbiota shifts. This suggests that neutrophils equally reduce pathogen and commensal bacterial loads in the inflamed gut.

Effects of in ovo injection of bacterial peptides and CpG-ODN on Salmonella enterica serovar Heidelberg infection in specific pathogen-free (SPF) chicks.

RESEARCH HIGHLIGHTS: Peptides + CpG-ODN reduced SH in caeca at the first week post-infection.Administered formulations did not reduce SH-faecal excretion.Levels of intestinal IgA were similar between all groups.CpG-ODN improved some parameters associated with chick intestinal health.

Research Note: Xylooligosaccharide directly attenuates Salmonella Typhimurium colonization and its induction of impairments in intestinal barrier and growth performance of broilers.

Xylooligosaccharide (XOS) is known as a prebiotic, however, it is unknown whether XOS can directly protect against bacterial infection. This study aimed to investigate the direct inhibitory effects of XOS on Salmonella Typhimurium colonization and the inductive impairments in gut health and growth performance in broilers. We first probed the inhibitory effects of XOS on S. Typhimurium adhesion and its induction of intestinal epithelial cell (IPEC-J2) injuries. Afterward, 168 one-day-old yellow-feathered broilers were randomly divided into 3 groups (7 replicates/group): negative control (NC, received a basal diet), positive control (PC, received a basal diet with S. Typhimurium challenge) and XOS group (PC birds + 1,500 mg/kg XOS). All birds except those in NC were orally challenged with S. Typhimurium from 8 to 10 d of age. Parameters were analyzed on d 11. The results showed that XOS inhibited S. Typhimurium adhesion and the inductive injuries of IPEC-J2 cells by lowering (P < 0.05) certain adhesion-related genes expression of this bacterium. It also alleviated S. Typhimurium-induced increase (P < 0.05) in the expression of certain inflammatory cytokines and tight junction (TJ) proteins of IPEC-J2 cells. Supplementing XOS to S. Typhimurium-challenged broilers attenuated the elevations (P < 0.05) in S. Typhimurium colonization of ileal mucosa and its translocation to the liver and spleen, as well as increased (P < 0.05) certain TJ proteins expression of ileum. Besides, XOS addition normalized S. Typhimurium-induced impairments (P < 0.05) in ileal morphology, final body weight and average daily gain in broilers. Collectively, supplemental XOS directly suppressed intestinal colonization of S. Typhimurium by diminishing its adhesiveness and subsequently mitigated destructions in intestinal barriers, thus contributing to weaken growth retardation in challenged broilers. Our findings provide a new insight into the mechanisms of XOS limiting Salmonella infection in chickens.

Differential cytokine profiling and microbial species involved in cecal microbiota modulations in SPF chicks immunized with a dual vaccine against Salmonella Typhimurium infection.

Salmonella Typhimurium (ST) infection in laying hens is a significant threat to public health and food safety. Host resistance against enteric pathogen invasion primarily relies on immunity and gut barrier integrity. This study applied the ST infection model and a dual live vaccine containing Salmonella Enteritidis (SE) strain Sm24/Rif12/Ssq and ST strain Nal2/Rif9/Rtt to investigate the cellular cytokine expression profiles and the differential community structure in the cecal microbiota of specific-pathogen-free (SPF) chicks and field-raised layers. The results showed that ST challenge significantly upregulated expressions of IL-1ß in SPF chicks. Vaccination, on the other hand, led to an elevation in IFNγ expression and restrained IL-1ß levels. In the group where vaccination preceded the ST challenge (S.STvc), heightened expressions of IL-1ß, IL-6, IL-10, and IL-12ß were observed, indicating active involvement of both humoral and cell-mediated immunity in the defense against ST. Regarding the cecal microbiota, the vaccine did not affect alpha diversity nor induce a significant shift in the microbial community. Conversely, ST infection significantly affected the alpha and beta diversity in the cecal microbiota, reducing beneficial commensal genera, such as Blautia and Subdoligranulum. MetagenomeSeq analysis reveals a significant increase in the relative abundance of Faecalibacterium prausnitzii in the groups (S.STvc and STvc) exhibiting protection against ST infection. LEfSe further demonstrated Faecalibacterium prausnitzii as the prominent biomarker within the cecal microbiota of SPF chicks and field layers demonstrating protection. Another biomarker identified in the S.STvc group, Eubacterium coprostanoligenes, displayed an antagonistic relationship with Faecalibacterium prausnitzii, suggesting the limited biological significance of the former in reducing cloacal shedding and tissue invasion. In conclusion, the application of AviPro Salmonella DUO vaccine stimulates host immunity and modulates cecal microbiota to defend against ST infection. Among the microbial modulations observed in SPF chicks and field layers with protection, Faecalibacterium prausnitzii emerges as a significant species in the ceca. Further research is warranted to elucidate its role in protecting layers against ST infection.

Development of recombinant subunit vaccine targeting InvH protein of Salmonella Typhimurium and evaluation of its immunoprotective efficacy against salmonellosis.

Salmonella Typhimurium is the most prevalent non-host specific Salmonella serovars and a major concern for both human and animal health systems worldwide contributing to significant economic loss. Type 3 secretion system (T3SS) of Salmonella plays an important role in bacterial adherence and entry into the host epithelial cells. The product of invH gene of Salmonella is an important component of the needle complex of the type 3 secretion system. Hence, the present study was undertaken to clone and express the 15 kDa InvH surface protein of Salmonella Typhimurium in an E. coli host and to evaluate its immune potency in mice. The purified recombinant InvH (r-InvH) protein provoked a significant (p < 0.01) rise in IgG in the inoculated mice. The immunized mice were completely (100%) protected against the challenge dose of 107.5 LD50, while protection against challenge with the same dose of heterologous serovars was 90%. The bacterin-vaccinated group showed homologous protection of 60% against all three serovars. Findings in this study suggest the potential of the r-InvH protein of S. Typhimurium as an effective vaccine candidate against Salmonella infections.

Limosilactobacillus fermentum IKP 111 reduces pathogen load and improves immunity of broilers when challenged with Salmonella enteritidis.

In this study, we checked the effectiveness of L. fermentum IKP 111 in treating S. enteritidis infection in an in vivo study. Its oral administration to broiler chicks significantly reduced the colonization of S. enteritidis in the gut and there was a lower bacterial count of S. enteritidis in the droppings after infection. The administration of the probiotic L. fermentum IKP 111 also led to increase in weight gain in the broiler chicks as well as their immunomodulation against avian influenza virus (AIV) and Newcastle disease virus (NDV) as compared to the chicks challenged only with S. enteritidis. Our study provides evidence that the probiotic strain L. fermentum IKP 111 could be an alternate for controlling S. enteritidis infection while enhancing the gut health as well as the immune response of broiler chickens against viral infections.

In vitro and in vivo evaluation of tannic acid as an antibacterial agent in broilers infected with Salmonella Typhimurium.

This study was conducted to evaluate tannic acid (TA) as an antibacterial agent against Salmonella Typhimurium in in vitro and in vivo chicken models. The TA formed an inhibitory zone against Salmonella enterica serotypes including S. Typhimurium, S. Enteritidis, and S. Infantis. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of TA against Salmonella Typhimurium nalidixic acid resistant strain (STNR) were determined as 40 and 700 µg/mL, respectively. Sublethal doses of TA (5, 10, and 20 µg/mL) restricted swimming and swarming motility and biofilm formation of STNR compared to the control group (0 µg/mL) (P < 0.05). The TA-bovine serum albumin (BSA) complex formed at simulated gastric pH (pH 3.75) was hydrolyzed at pH 6.75 and 7.25 (P < 0.05), and the hydrolysis of the TA-BSA complex was stronger at pH 7.25 compared to the pH 6.75 (P < 0.05). The inhibitory zone of the TA-BSA complex against STNR at pH 6.75 was lower than TA without BSA at 30 and 60 min (P < 0.05), but not at 120 min (P > 0.1). The inhibitory zone of the TA-BSA complex against STNR at pH 7.25 was not decreased at 0, 30, and 60 min compared to TA without BSA (P > 0.1). The recovery rate of TA was 83, 54.8, 10.5, and 19.6% in the gizzard, jejunum, ileum, and ceca, respectively, in broiler chickens. The STNR-infected broilers fed 0.25 g/kg of TA had significantly lower unweighted beta diversity distance compared to the sham-challenged control (SCC) and challenged controlled (CC) group on D 21. TA supplementation linearly (P < 0.05) and quadratically (tendency; P = 0.071) reduced relative abundance of the family Peptostreptococcaceae in broilers infected with STNR on D 7. TA supplementation linearly (P < 0.05) and quadratically (tendency; P = 0.06) increased the relative abundance of the family Erysipelotrichaceae in broilers infected with STNR on D 21. Therefore, TA has potential to be used as an antibacterial agent against the S. Typhimurium infection in broilers.

Research Note: The effects of a Lactobacillus helveticus ATCC 15009-derived postbiotic mitigating Salmonella Gallinarum colonization in commercial layer chicks.

This study aimed to assess the effects of a Lactobacillus helveticus ATCC 15009-derived postbiotic in mitigating experimental Salmonella Gallinarum infection. For this purpose, a sample of Lactobacillus sp. was inoculated in 2 different media, each containing different postbiotics (sensitized and nonsensitized). Both inocula had their antagonistic effect over S. Gallinarum tested through the spot-on-the-lawn method. It revealed that the sensitized postbiotic had a higher action potential over Lactobacillus sp. than the nonsensitized one (P < 0.05). Then, 48 day of hatch chicks were divided into 4 groups: A = Lactobacillus sp. (109 CFU/mL) inoculum on the 18th day; B = Lactobacillus sp. (109 CFU/mL) inoculum on the 18th day and postbiotic inoculum on the 19th day; C = postbiotic inoculum on the 19th day; and D = sterile saline inoculum on 18th and 19th days. On the 21st day, all chicks were infected with S. Gallinarum (109 CFU/mL). On the 23rd day, the animals were euthanized by cervical dislocation, and the ceca and liver were aseptically removed. Bacterial count of S. Gallinarum with serial decimal dilution was performed with these organs. It revealed that the prophylactic treatment with the postbiotic that modulates the intestinal microbiota was as efficient as the probiotic administration in reducing S. Gallinarum in the cecum and liver of chicks (P < 0.05). These data point to a new range of alternatives for preventing S. Gallinarum, which might help the poultry industry produce safer food for human consumption.

[Influence of breed and herd size on the intra-herd prevalence of Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7) in sheep]. / Einfluss von Rasse und Herdengröße auf die Intraherdenprävalenz von Salmonella enterica subspecies diarizonae Serovar 61: k: 1, 5, (7) bei Schafen.

SUBJECT AND AIM: At present, only little information is available on the within-flock prevalence of Salmonella enterica subspecies diarizonae serovar 61: k: 1, 5, (7) (SASd) in sheep flocks in Germany as well as their possible influencing factors. The aim of the study was to investigate relationships between flock size, breed and within-flock prevalence. MATERIAL AND METHODS: A total of 1610 clinically healthy ewes from 14 sheep flocks of 9 different breeds aged 2 to 12 years were microbiologically tested for SASd by nasal swab and fecal samples. Linear multivariable models were used to analyse the associations between within-flock prevalence and farm factors (flock size, breed) or detection frequencies in fecal or nasal swabs. RESULTS: SASd was detected in all sheep flocks examined, with 75% of adults having at least one positive nasal or fecal result. In comparison to the 11 flocks in which commercial breeds were kept, the 3 flocks of landraces had a lower apparent within-flock prevalence (p=0.01). No association with herd size was evident. With respect to the age of the ewes, there was a negative relationship (p=0.05) with the frequency of detection of SASd in the nasal swab but not in the fecal swab. The health status and fertility performance of the flocks were in line with a normal range for commercial sheep flocks, with lambing losses of 5% to 10% and lambing scores of 130% to 158%. CONCLUSIONS: Despite the high prevalence of SASd infections particularly within commercial breeds, there was no evidence of a relevant risk to sheep health. Compared with commercial breed flocks, a lower spread of SASd within flocks keeping landraces was evident. CLINICAL RELEVANCE: Despite a high prevalence, infections with SASd are very unlikely to lead to clinical symptoms or disease. Regulation and monitoring of SASd in sheep are of low priority for animal health authorities.

Inhibitory effect of resveratrol on swimming motility and adhesion ability against Salmonella enterica serovar Typhimurium infection.

Salmonella enterica serovar Typhimurium (S. typhimurium) is a common Gram-negative foodborne pathogen that threatens public health and hinders the development of livestock industry. Resveratrol, an important component in grape fruits and seeds, has been shown to possess multiple biological activities, but its potential effects on S. typhimurium-mediated virulence have been rarely reported. In this study, we investigated the effect of resveratrol on S. typhimurium flagella -mediated virulence. The results showed that resveratrol significantly reduced the transcription of flagella genes and swimming motility of S. typhimurium, and also inhibited the transcription of T3SS-related virulence genes with varying degrees inhibiting bacterial growth. Simultaneously, resveratrol significantly reduced the adhesion of S. typhimurium to HeLa cells. Unfortunately, resveratrol does not improve the survival rate of S. typhimurium-infected mice, but it reduces the bacterial load in the liver and spleen of infected mice, and it also has a certain degree of anti-inflammatory activity. In summary, these results indicated that resveratrol has the potential to be developed as an alternative drug or antibacterial agent to prevent Salmonella infection.

Isolation and Molecular Identification of Salmonella pullorum from Broiler Chicken in Iraqi Fields.

Pullorum disease (PD) is one of the most common diseases in the world, with devastating consequences. In the chicken sector, there have been financial losses. It is brought on by Salmonella enteric subspecies serovar Gallinarum biovar pullorum; definitive detection requires culture followed by biochemistry analysis and serotyping. This study aimed to verify the presence of bacteria by culture, biochemical characterization, PCR assay, and sequencing. One hundred samples were collected from 12 broiler chicken flocks of different ages for 8districts of Baghdad province, including cloacal swabs (65), visceral organs (15), and dropping (20). Salmonella colonies were identified by selective culture broth and agar with biochemical description for 75% of the total samples, with a higher incidence in visceral organs than dropping and cloacal swabs. ،The Sequencing and phylogenetic tree analysis of 16S rRNA gene for representative Salmonella isolates. The presence of Salmonella pullorum isolates in global genetic strains; was revealed a matching NCBI isolates similarity of 99.02% with (MF445124.1) and 98% with (MH352164.1), respectively. In the current state of molecular and genetic research, phlyogentic research announced the real presence of Salmonella pullorum in Baghdadprovince's broiler chicken, also showing the phylogentic characteristics and links to some global isolates. The detection of Salmonella pullorum in broiler flocks of the current study extent of health risks to other uninfected birds present in the free range.

Trends in Salmonella Dublin over time in Denmark from food and animal related isolates.

Salmonella enterica serovar Dublin is highly adapted to cattle and a relatively rare cause of human infections. In Denmark S. Dublin has been endemic in the cattle population for many years. A national surveillance program in the cattle population was established at herd-level to reduce the occurrence of S. Dublin. In this study, we analyzed 421 S. Dublin genomes from cattle and food in order to determine the trend of S. Dublin's population size over time in Denmark and the impact of intervention in the cattle industry on the bacterial population size. A phylogenetic tree based on SNPs exhibited two major clades and one small cluster. All isolates were ST10. The temporal phylogenetic tree for the S. Dublin isolates showed that the most recent common ancestor was estimated to be in ∼1980 for the two major clades. An effective population size over time based on a Bayesian skyline plot showed that the population size of S. Dublin decreased significantly between 2014 and 2019 in both major clades. This result was concordant with the decrease of infected human cases by S. Dublin in Denmark. The strengthening of a surveillance program in Denmark could be the cause for the reduction of S. Dublin's effective population size. This study showed that whole genome sequencing combined with computer intensive phylogenetic analysis estimating the effective size of the S. Dublin's population over time is a strongly relevant measure with respect to assessing the impact of control measures aiming to reduce the bacterial population in the reservoir and the risk for human infection.

Formate oxidation in the intestinal mucus layer enhances fitness of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium induces intestinal inflammation to create a niche that fosters the outgrowth of the pathogen over the gut microbiota. Under inflammatory conditions, Salmonella utilizes terminal electron acceptors generated as byproducts of intestinal inflammation to generate cellular energy through respiration. However, the electron donating reactions in these electron transport chains are poorly understood. Here, we investigated how formate utilization through the respiratory formate dehydrogenase-N (FdnGHI) and formate dehydrogenase-O (FdoGHI) contribute to gut colonization of Salmonella. Both enzymes fulfilled redundant roles in enhancing fitness in a mouse model of Salmonella-induced colitis, and coupled to tetrathionate, nitrate, and oxygen respiration. The formic acid utilized by Salmonella during infection was generated by its own pyruvate-formate lyase as well as the gut microbiota. Transcription of formate dehydrogenases and pyruvate-formate lyase was significantly higher in bacteria residing in the mucus layer compared to the lumen. Furthermore, formate utilization conferred a more pronounced fitness advantage in the mucus, indicating that formate production and degradation occurred predominantly in the mucus layer. Our results provide new insights into how Salmonella adapts its energy metabolism to the local microenvironment in the gut. IMPORTANCE Bacterial pathogens must not only evade immune responses but also adapt their metabolism to successfully colonize their host. The microenvironments encountered by enteric pathogens differ based on anatomical location, such as small versus large intestine, spatial stratification by host factors, such as mucus layer and antimicrobial peptides, and distinct commensal microbial communities that inhabit these microenvironments. Our understanding of how Salmonella populations adapt its metabolism to different environments in the gut is incomplete. In the current study, we discovered that Salmonella utilizes formate as an electron donor to support respiration, and that formate oxidation predominantly occurs in the mucus layer. Our experiments suggest that spatially distinct Salmonella populations in the mucus layer and the lumen differ in their energy metabolism. Our findings enhance our understanding of the spatial nature of microbial metabolism and may have implications for other enteric pathogens as well as commensal host-associated microbial communities.

Effects of bacteriophage on Salmonella Enteritidis infection in broilers.

Bacteriophages (BP) are viruses that can infect bacteria. The present study evaluated the effect of BP on Salmonella infected broilers. A number of 150 day-old broilers were used in a completely randomized design with five treatments that included: (1) basal diet from day 0 to 28; (2) basal diet + 0.3 g/kg of colistin from day 0 to 28; (3) basal diet from day 1 to 13, and basal diet + 0.4 g/kg of colistin from day 14 to 28; (4) basal diet + 1 g/kg of BP from day 0 to 28; (5) basal diet + 1.5 g/kg of BP from day 0 to 28. On day 13, 15 chickens from each treatment were challenged by Salmonella Enteritidis (SE), while fifteen from each treatment were not; instead, they were kept in the same cage with the challenged chickens (exposed chickens). At 7 and 14 days post-challenge, the number of SE and coliform bacteria in the cecum and liver of colistin and BP-fed birds was lower than the control treatment. In exposed and challenged chickens, the height and surface area of villus were greater in the BP and colistin-supplemented groups. Serum concentrations of aspartate aminotransferase and alanine transaminase were greater, while serum albumin and triglycerides concentrations were lower in the control treatment. The liver of the challenged chickens had more pathological lesions than exposed birds. BP significantly decreased PPARγ gene expression in exposed chickens. In the challenged and exposed chickens, TLR4 gene expression was lower in BP and colistin-treated birds as compared to the control. In conclusion, adding BP to the diet from the day of age prevents the spread of Salmonella.

Unraveling the role of type 1 fimbriae in Salmonella pathogenesis: insights from a comparative analysis of Salmonella Enteritidis and Salmonella Gallinarum.

Significant differences in pathogenicity between Salmonella Enteritidis and Salmonella Gallinarum exist despite the fact that S. Gallinarum is a direct descendant of S. Enteritidis. It was hypothesized that such various properties may be in part the result of differences in structure and functions of type 1 fimbriae (T1Fs). In S. Enteritidis, T1Fs bind to oligomannosidic structures carried by host cell glycoproteins and are called mannose-sensitive T1Fs (MST1F). In S. Gallinarum, T1Fs lost ability to bind such carbohydrate chains, and were named mannose-resistant MRT1Fs (MRT1F). Therefore, the present study was undertaken to evaluate the role of MST1Fs and MRT1Fs in the adhesion, invasion, intracellular survival and cytotoxicity of S. Enteritidis and S. Gallinarum toward chicken intestinal CHIC8-E11cells and macrophage-like HD11 cells. Using mutant strains: S. Enteritidis fimH::kan and S. Gallinarum fimH::kan devoid of T1Fs and in vitro assays the following observations were made. MST1Fs have a significant impact on the chicken cell invasion by S. Enteritidis as MST1F-mediated adhesion facilitates direct and stable contact of bacteria with host cells, in contrast to MRT1Fs expressed by S. Gallinarum. MST1Fs as well as MRT1Fs did not affected intracellular viability of S. Enteritidis and S. Gallinarum. However, absolute numbers of intracellular viable wild-type S. Enteritidis were significantly higher than S. Enteritidis fimH::kan mutant and wild-type S. Gallinarum and S. Gallinarum fimH::kan mutant. These differences, reflecting the numbers of adherent and invading bacteria, underline the importance of MST1Fs in the pathogenicity of S. Enteritidis infections. The cytotoxicity of wild-type S. Enteritidis and its mutant devoid of MST1Fs to HD11 cells was essentially the same, despite the fact that the number of viable intracellular bacteria was significantly lower in the mutated strain. Using HD11 cells with similar number of intracellular wild-type S. Enteritidis and S. Enteritidis fimH::kan mutant, it was found that the lack of MST1Fs did not affect directly the cytotoxicity, suggesting that the increase in cytotoxicity of S. Enteritidis devoid of MST1Fs may be associated with crosstalk between T1Fs and other virulence factors.