Search Results (52)

The growing threat of antibiotic-resistant Gram-negative bacteria highlights the urgent need for innovative, non-bactericidal therapeutic strategies. Quorum-sensing (QS) inhibition has emerged as a promising approach to attenuate bacterial virulence without exerting selective pressure. This study evaluated the antimicrobial, anti-QS, and antibiofilm properties of aqueous extracts from five medicinal plants native to northeastern Mexico: Gymnosperma glutinosum, Ibervillea sonorae, Larrea tridentata, Olea europaea, and Tecoma stans. Disk diffusion and violacein quantification assays using Chromobacterium violaceum demonstrated significant QS inhibition by G. glutinosum and T. stans, with violacein reductions of 60.02% and 52.72%, respectively, at 40 mg/mL. While L. tridentata and O. europaea exhibited antibacterial activity, I. sonorae showed no growth or pigment inhibition but achieved the highest biofilm disruption (89.89%) against Salmonella typhimurium. UPLC-MS analysis identified chlorogenic acid, kaempferol, and D-(−)-quinic acid as major constituents, compounds previously associated with QS modulation. These findings highlight the potential of traditional Mexican plant species as sources of QS inhibitors and bio-film-disrupting agents, supporting their further development as alternatives to conventional antibiotics. Full article

Background:Salmonella Typhimurium is a major zoonotic pathogen, in which type 1 fimbriae play a crucial role in intestinal colonization and immune modulation. This study aimed to improve the protective immunity of a previously developed growth-deficient strain—a double auxotroph for D-glutamate and D-alanine—by engineering the inducible expression of type 1 fimbriae. Methods: P_tetA-driven expression of the fim operon was achieved by λ-Red mutagenesis. fimA expression was quantified by qRT-PCR, and fimbriation visualized by transmission electron microscopy. Adhesive properties were evaluated through FimH sequence analysis, yeast agglutination, mannose-binding/inhibition assays, and HT-29 cell adherence. BALB/c mice were immunized orogastrically with IRTA ΔΔΔ or IRTA ΔΔΔ P_tetA::fim. Safety and immunogenicity were assessed by clinical monitoring, bacterial load, fecal shedding, ELISA tests, and adhesion/blocking assays using fecal extracts. Protection was evaluated after challenging with wild-type and heterologous strains. Results: IRTA ΔΔΔ P_tetA::fim showed robust fimA expression, dense fimbrial coverage, a marked mannose-sensitive adhesive phenotype and enhanced HT-29 attachment. Fimbrial overexpression did not alter intestinal colonization or translocation to mesenteric lymph nodes (mLNs). Immunization elicited a mixed IgG1/IgG2a, significantly increased IgA and IgG against type 1 fimbriae-expressing Salmonella, and enhanced the ability of fecal extracts to inhibit the adherence of wild-type strains. Upon challenge (IRTA wild-type/20220258), IRTA ΔΔΔ P_tetA::fim reduced infection burden in the cecum (−1.46/1.47-log), large intestine (−1.35/2.17-log), mLNs (−1.32/0.98-log) and systemic organs more effectively than IRTA ΔΔΔ. Conclusions: Inducible expression of type 1 fimbriae enhances mucosal immunity and protection, supporting their inclusion in next-generation Salmonella vaccines. Future work should assess cross-protection and optimize FimH-mediated targeting for mucosal delivery. Full article

Bacterial cancer therapy (BCT) is emerging as an important option for the treatment of solid tumours, with promising outcomes in preclinical trials. Further progress is hampered by an incomplete understanding of how oncotropic bacteria, such as attenuated strains of Salmonella enterica serovar Typhimurium, colonise tumours and the responses of both the bacteria and tumour cells to this colonisation. To model this, we developed organoids that are permissive for bacterial colonisation, replacing the conventional commercially available extracellular matrix (e.g., Matrigel) with a type I collagen matrix scaffold. A comparison of the two extracellular matrices indicated that type 1 collagen permitted an initial infection efficiency more than 5-times greater than with Matrigel. In addition, subsequent growth within type 1 collagen expanded bacterial cell numbers by over 10-fold within 4 days of infection. These organoids allow for the visualisation of bacterial chemoattraction, cell invasion and subsequent population of the interior lumen, and will permit the future optimisation of BCT. In addition, by establishing patient-derived organoids, we demonstrate a platform for developing future personalised treatments exploiting BCT. Full article

Salmonella enterica subsp. enterica serovar Typhimurium is an important foodborne pathogen, and poultry products are a major source of human infection. Live attenuated vaccines for poultry are an effective tool for reducing the prevalence of infection, but vaccine strains must be differentiated from wild strains to ensure effective disease surveillance and control. This study reports the validation of the SalTypm&PriSal-T qPCR Duplex kit, a DIVA qPCR assay for the differentiation of the Primun Salmonella T vaccine from wild strains using DNA extracted from isolated colonies. Analytical specificity and sensitivity, as well as diagnostic specificity and sensitivity, were evaluated with optimal results. This qPCR assay significantly reduces the time required to obtain a diagnostic result compared to reference methods based on antibiogram differentiation. Notably, this is the first qPCR test available worldwide for distinguishing this vaccine from wild strains, providing a valuable tool for improving the efficiency and accuracy of Salmonella surveillance programs in poultry production systems. Full article

The cytokines IFN-γ and CD154 have been well established, and they play pivotal roles in immune protection against Salmonella in mice, but their effects and specific mechanisms in Salmonella-infected chickens are less understood. In this study, we conducted animal experiments to screen the highly immunoprotective chIFN-γ-chCD154 fusion protein compared with single protein chIFN-γ or chCD154 in white Leghorn chickens. The results showed that compared with separate pretreatments with chIFN-γ and chCD154, the fusion protein, chIFN-γ-chCD154, synergistically increased survival of infected chickens, reduced bacterial load in feces and organs, and attenuated pathological damage to the liver and cecum. Pretreatment with chIFN-γ-chCD154 also increased humoral immune responses, expression of the tight junction proteins zo-1, occludin, and claudin-1, and the relative abundance of Enterococcus_cecorum, Lactobacillus_helveticus, and Lactobacillus_agilis, which protect against intestinal inflammation. Compared with single protein pretreatment, chIFN-γ-chCD154 significantly upregulated STAT1, IRF1, and GBP1 in infected chickens while decreasing mRNA expression of TLR4, MyD88, NF-κB, TNF-α, IL-6, and IL-1β. In summary, damage to the cecal epithelial barrier and the inflammation induced by S. typhimurium infection was alleviated by chIFN-γ-chCD154 pretreatment through a mechanism involving the TLR4/MyD88/NF-κB and IFN-γ/STAT/IRF1/GBP1 pathways. Full article

Background: Salmonella Typhimurium poses a substantial health risk to both humans and animals. This study evaluated the potential of using the Salmonella Typhimurium ΔsptP mutant as a live-attenuated vaccine candidate by constructing it through homologous recombination and assessing its key biological properties, including growth characteristics, immunogenicity, and protective efficacy. Methods: We generated the ΔsptP mutant through targeted gene deletion, ensuring the preservation of the bacterial strain’s growth and stability. In vitro and in vivo assays were performed to compare the invasive capabilities between the mutant and the wild-type strains. Specifically, we examined the invasion into RAW264.7 murine macrophages and mice. Furthermore, the virulence of the mutant was evaluated by determining the median lethal dose (LD₅₀). To evaluate immunogenicity and protection, mice were immunized with 2 × 10⁴ CFUs of the ΔsptP mutant, followed by a booster immunization, and then challenged with a virulent strain. Results: The ΔsptP mutant exhibited no significant changes in growth characteristics or genetic stability compared to the wild-type strain. However, it demonstrated a significantly diminished capacity for invasion in both murine macrophages and mice. The LD₅₀ for the mutant was 39.92-fold higher than that of the wild-type, indicating a marked reduction in virulence. Mice immunized with the ΔsptP mutant and administered a booster immunization exhibited 87.5% protection against challenge with a virulent strain, as compared to the PBS control group. Moreover, the mutant induced IgG antibody levels comparable to those induced by the wild-type strain. Conclusions: The ΔsptP mutant of Salmonella Typhimurium exhibits markedly reduced virulence while retaining robust immunogenicity and protective efficacy. These findings suggest that the ΔsptP mutant is a promising candidate for a live-attenuated vaccine, potentially providing an effective strategy to prevent Salmonella Typhimurium infections. Full article

Salmonella is considered the major zoonotic and foodborne pathogen responsible for human infections. It includes the serovars causing typhoid fever (S. typhi and S. paratyphi) and the non-typhoidal salmonella (NTS) serovars (S. enteritidis and S. typhimurium), causing enteric infections known as “Salmonellosis”. NTS represents a major public health burden worldwide. The consumption of S. enteritidis-contaminated animal foods is the main source of this disease in humans, and eradicating bacteria from animals remains a challenge. NTS causes various clinical manifestations, depending on the quantity of bacteria present in the food and the immune status of the infected individual, ranging from localized, self-limiting gastroenteritis to more serious systemic infections. Salmonellosis prevention is based on hygienic and behavioral rules related to food handling that aim to reduce the risk of infection. However, no vaccine against NTS is available for human use. This aspect, in addition to the increase in multidrug-resistant strains and the high morbidity, mortality, and socioeconomic costs of NTS-related diseases, makes the development of new prevention and control strategies urgently needed. The success of the vaccines used to protect against S. typhi encouraged the development of NTS vaccine candidates, including live attenuated, subunit-based, and recombinant-protein-based vaccines. In this review, we discuss the epidemiological burden of Salmonellosis and its primary prevention, focusing on the current status and future perspectives of the vaccines against NTS. Full article

Indole-3-acetic acid (IAA), a metabolite derived from microbial tryptophan metabolism, plays a crucial role in regulating intestinal homeostasis. However, the influence and potential applications of IAA in the context of animal pathogen infections remain underexplored. This study investigates the prophylactic effects of IAA pretreatment against Salmonella typhimurium (ST) SL1344 infection, focusing on its ability to attenuate inflammatory responses, enhance intestinal barrier integrity, inhibit bacterial colonization, and restore colonic microbiota dysbiosis. The results demonstrated that IAA ameliorated the clinical symptoms in mice, as evidenced by reduced weight loss and histopathological damage. Furthermore, IAA inhibited the inflammatory response by downregulating the gene expression of pro-inflammatory cytokines IL-17A, TNF-α, IL-1β, and IL-6 in colon, ileum, and liver. IAA also preserved the integrity of the intestinal mucosal barrier and promoted the expression of tight junction proteins. Additionally, 16S rRNA gene sequencing revealed significant alterations in intestinal microbiota structure induced by ST infection following IAA treatment. Notable changes in β diversity and species richness were characterized by the enrichment of beneficial bacteria including Bacteroideaceae, Spirillaceae, and Bacillus. The proliferation of Salmonella enterica subspecies enterica serovar Typhi was significantly inhibited, thereby enhancing the intestinal health of the host. In summary, the oral administration of IAA contributes to the alleviation of inflammation, restoration of the intestinal barrier, and correction of colonic microbiota disturbance in mice challenged with ST. Full article

From the first report in 1891 by Dr. Coley of the effective treatment of tumors in 1000 patients with Streptococcus and the first successful use of bacterial vectors for transferring therapeutic genes in 1980 by Dr. Schnaffer, bactofection has been shown to be a promising strategy in the fields of vaccination, gene therapy, and cancer therapy. This review describes the general theory of bactofection and its advantages, disadvantages, challenges, and expectations, compiling the most notable advances in 14 vaccination studies, 27 cancer therapy studies, and 13 clinical trials. It also describes the current scope of bactofection and promising results. The extensive knowledge of Salmonella biology, as well as the multiple adequacies of the Ty21a vaccination platform, has allowed notable developments worldwide that have mainly been reflected in therapeutic efforts against cancer. In this regard, we strongly recommend the creation of a recombinant Ty21a model that constitutively expresses the GtgE protease from S. typhimurium, allowing this vector to be used in animal trials, thus enhancing the likelihood of favorable results that could quickly transition to clinical trials. From the current perspective, it is necessary to explore a greater diversity of bacterial vectors and find the best combination of implemented attenuations, generating personalized models that guarantee the maximum effectiveness in cancer therapy and vaccination. Full article

In the current study, two Salmonella Typhimurium strains, JOL 912 and JOL 1800, were engineered from the wild-type JOL 401 strain through in-frame deletions of the lon and cpxR genes, with JOL 1800 also lacking rfaL. These deletions significantly attenuated the strains, impairing their intracellular survival and creating unique immunological profiles. This study investigates the response of these strains to various abiotic stress conditions commonly experienced in vivo, including temperature, acidity, osmotic, and oxidative stress. Notably, cold stress induced a non-significant trend towards increased invasion by Salmonella compared to other stressors. Despite the observed attenuation, no significant alterations in entry mechanisms (trigger vs. zipper) were noted between these strains, although variations were evident depending on the host cell type. Both strains effectively localized within the cytoplasm, demonstrating their ability to invade and interact with the intracellular environment. Immunologically, JOL 912 elicited a robust response, marked by substantial activation of nuclear factor kappa B (NF-kB), and chemokines, interleukin 8 (CXCL 8) and interleukin 10 (CXCL 10), comparable to the wild-type JOL 401 (over a fourfold increase compared to JOL 1800). In contrast, JOL 1800 exhibited a minimal immune response. Additionally, these attenuations influenced the expression of cyclins D1 and B1 and caspases 3 and 7, indicating cell cycle arrest at the G2/M phase and promotion of the G0/G1 to S phase transition, alongside apoptosis in infected cells. These findings provide valuable insights into the mechanisms governing the association, internalization, and survival of Salmonella mutants, enhancing our understanding of their regulatory effects on host cell physiology. Full article

Salmonella species are causal pathogens instrumental in human food-borne diseases. The pandemic survey related to multidrug resistant (MDR) Salmonella genomics enables the prevention and control of their dissemination. Currently, serotype Mbandaka is notorious as a multiple host-adapted non-typhoid Salmonella. However, its epidemic and MDR properties are still obscure, especially its genetic determinants accounting for virulence and MD resistance. Here, we aim to characterize the genetic features of a strain SMEH pertaining to Salmonella Mbandaka (S. Mbandaka), isolated from the patient’s hydropericardium, using cell infections, a mouse model, antibiotic susceptibility test and comparative genomics. The antibiotic susceptibility testing showed that it could tolerate four antibiotics, including chloramphenicol, tetracycline, fisiopen and doxycycline by Kirby–Bauer (K-B) testing interpreted according to the Clinical and Laboratory Standards Institute (CLSI). Both the reproducibility in RAW 264.7 macrophages and invasion ability to infect HeLa cells with strain SMEH were higher than those of S. Typhimurium strain 14028S. In contrast, its attenuated virulence was determined in the survival assay using a mouse model. As a result, the candidate genetic determinants responsible for antimicrobial resistance, colonization/adaptability and their transferability were comparatively investigated, such as bacterial secretion systems and pathogenicity islands (SPI-1, SPI-2 and SPI-6). Moreover, collective efforts were made to reveal a potential role of the plasmid architectures in S. Mbandaka as the genetic reservoir to transfer or accommodate drug-resistance genes. Our findings highlight the essentiality of antibiotic resistance and risk assessment in S. Mbandaka. In addition, genomic surveillance is an efficient method to detect pathogens and monitor drug resistance. The genetic determinants accounting for virulence and antimicrobial resistance underscore the increasing clinical challenge of emerging MDR Mbandaka isolates, and provide insights into their prevention and treatment. Full article

Somatostatin (SS) plays crucial regulatory roles in animal growth and reproduction by affecting the synthesis and secretion of growth hormone (GH). However, the mechanism by which SS regulates growth and development in goats is still unclear. In order to investigate the regulatory networks of the hypothalamus and pituitary in goats affected by SS DNA vaccines, in this study, we used a previously established oral attenuated Salmonella typhimurium SS DNA vaccine, X9241 (ptCS/2SS-asd), to treat wethers. We analyzed the protein changes in hypothalamic and pituitary tissues using a TMT-based proteomics approach. Additionally, we examined the metabolic profiles of the serum of control and immunized wethers through untargeted metabolomics using liquid chromatography–mass spectrometry (LC–MS). Key signaling pathways were identified based on differentially expressed metabolites (DEMs) and differentially expressed proteins (DEPs). Furthermore, the effect of critical DEPs on signaling pathways was confirmed through Western blotting (WB) experiments, which elucidated the mechanism of active SS immunization in wethers. A proteomics analysis revealed that the expression of 58 proteins in the hypothalamus and 124 in the pituitary gland was significantly altered following SS vaccine treatment (fold change > 1.2 or < 0.83, p < 0.05). In the hypothalamus, many DEPs were associated with gene ontology (GO) terms related to neuronal signaling. In contrast, most DEPs were associated with metabolic pathways. In the pituitary gland, the DEPs were largely related to immune and nutrient metabolism functions, with significant enrichment in KEGG pathways, particularly those involving the metabolic pathway, sphingolipid signaling, and the cGMP-PKG signaling pathway. A metabolomic analysis further showed that active SS immunization in wethers led to significant alterations in seven serum metabolites. Notably, the sphingolipid signaling pathway, secondary bile acid synthesis, sphingolipid metabolism, and lysine synthesis were significantly disrupted. SS vaccines induced marked changes in hypothalamic–pituitary proteins in wethers, facilitating alterations in their growth processes. This study not only provides insights into the mechanism of the SS gene in regulating GH secretion in wethers but also establishes a basis for hormone immunoregulation technology to enhance livestock production performance. Full article

Bacterial surface display platforms have been developed for applications such as vaccine delivery and peptide library screening. The type V secretion system is an attractive anchoring motif for the surface expression of foreign proteins in gram-negative bacteria. SadA belongs to subtype C of the type V secretion system derived from Salmonella spp. and promotes biofilm formation and host cell adherence. The inner membrane lipoprotein SadB is important for SadA translocation. In this study, SadA was used as an anchoring motif to expose heterologous proteins in Salmonella typhimurium using SadB. The ability of SadA to display heterologous proteins on the S. typhimurium surface in the presence of SadB was approximately three-fold higher than that in its absence of SadB. Compared to full-length SadA, truncated SadAs (SadA⁸⁷⁷ and SadA²⁶⁹) showed similar display capacities when exposing the B-cell epitopes of urease B from Helicobacter pylori (UreB158–172aa and UreB349–363aa). We grafted different protein domains, including mScarlet (red fluorescent protein), the urease B fragment (UreBm) from H. pylori SS1, and/or protective antigen domain 4 from Bacillus anthracis A16R (PAD4), onto SadA⁸⁷⁷ or SadA¹²⁹². Whole-cell dot blotting, immunofluorescence, and flow cytometric analyses confirmed the localization of Flag×3-mScarlet (~30 kDa) and Flag×3-UreBm-mScarlet (~58 kDa) to the S. typhimurium surface using truncated SadA⁸⁷⁷ or SadA¹²⁹² as an anchoring motif. However, Flag×3-UreBm-PAD4-mScarlet (~75 kDa) was displayed on S. typhimurium using SadA¹²⁹². The oral administrated pSadBA¹²⁹²-FUM/StmΔygeAΔmurI and pSadBA⁸⁷⁷-FUM/StmΔygeAΔmurI could elicit a significant mucosal and humoral immunity response. SadA could thus be used as an anchoring motif for the surface expression of large heterologous proteins as a potential strategy for attenuated bacterial vaccine development. Full article

The lipopolysaccharide (LPS) that resides on the outermost surface and protects Gram-negative bacteria from host defenses is one of the key components leading to Salmonella infection, particularly the endotoxic lipid A domain of LPS. Lipid A modifications have been associated with several genes such as the arnT that encodes 4-amino-4-deoxy-L-arabinose transferase, which can be critical for bacteria to resist cationic antimicrobial peptides and interfere with host immune recognition. However, the association of arnT with virulence is not completely understood. Thus, this study aimed to elucidate the interrelationship of the major lipid A modification gene arnT with Salmonella Typhimurium virulence. We observed that the arnT-deficient S. Typhimurium (JOL2943), compared to the wild type (JOL401), displayed a significant decrease in several virulence phenotypes such as polymyxin B resistance, intracellular survival, swarming, and biofilm and extracellular polymeric substance (EPS) production. Interestingly, the cell-surface hydrophobicity, adhesion, and invasion characteristics remained unaffected. Additionally, LPS isolated from the mutant induced notably lower levels of endotoxicity-related cytokines in RAW and Hela cells and mice, particularly IL-1β with a nine-fold decrease, than WT. In terms of in vivo colonization, JOL2943 showed diminished presence in internal organs such as the spleen and liver by more than 60%, while ileal infectivity remained similar to JOL401. Overall, the arnT deletion rendered the strain less virulent, with low endotoxicity, maintained gut infectivity, and reduced colonization in internal organs. With these ideal characteristics, it can be further explored as a potential attenuated Salmonella strain for therapeutics or vaccine delivery systems. Full article

Neutrophils are innate immune cells that respond quickly to sites of bacterial infection and play an essential role in host defense. Interestingly, some bacterial pathogens benefit from exuberant neutrophil inflammation. Salmonella is one such pathogen that can utilize the toxic mediators released by neutrophils to colonize the intestine and cause enterocolitis. Because neutrophils can aid gut colonization during Salmonella infection, neutrophils represent a potential host-directed therapeutic target. Myristoylated alanine-rich C-kinase substrate (MARCKS) is an actin-binding protein that plays an essential role in many neutrophil effector responses. We hypothesized that inhibition of MARCKS protein would alter bovine neutrophil responses to Salmonella Typhimurium (STm) ex vivo. We used a MARCKS inhibitor peptide to investigate the role of MARCKS in neutrophil responses to STm. This study demonstrates that MARCKS inhibition attenuated STm-induced neutrophil adhesion and chemotaxis. Interestingly, MARCKS inhibition also enhanced neutrophil phagocytosis and respiratory burst in response to STm. This is the first report describing the role of MARCKS protein in neutrophil antibacterial responses. Full article