Search Results (338)

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen capable of surviving and replicating within macrophages, which causes foodborne diseases such as gastroenteritis. To develop a strategy against intracellular bacteria in macrophages, we designed silver-loaded biodegradable polymer nanoparticles functionalized with S. Typhimurium membrane vesicles (MVs). Silver nanoparticles (Ag NPs) were initially encapsulated within biodegradable poly(lactic-co-glycolic) nanoparticles (Ag-P NPs), which were then surface-modified with polyethyleneimine to form Ag-PP NPs. These were subsequently fused with S. Typhimurium MVs via a sonication method to generate Ag-PP@MV NPs. The resulting MV-coated nanoparticles displayed a similar protein profile to that of native MVs and exhibited antibacterial activity against intracellular S. Typhimurium. Notably, the enhanced cellular uptake of the MV-modified NPs contributed to their intracellular bactericidal efficacy. This study highlights MV modification as a promising strategy to improve NP delivery to macrophages for treating persistent intracellular infections. Full article

Background/Objective: The present study focused on the analysis of the Spanish clone belonging to the successful 4,[5],12:i:- monophasic variant of Salmonella enterica serovar Typhimurium. Methods: All isolates of the clone recovered in a Spanish region from human clinical samples between 2008 and 2018 (N = 14) were investigated using microbiological approaches and genome sequence analysis. In addition, they were compared with isolates from the years 2000 to 2003 (N = 21), which were previously characterized but had not yet been sequenced. Results: Phylogenetic analyses indicate that all isolates are closely related (differing by 1 to 103 SNPs) but belong to two clades termed A and B. With few exceptions, clade A comprised isolates of the first period, also including two “older” control strains, LSP 389/97 and LSP 272/98. Clade B only contained isolates from the second period. Isolates from both periods were resistant to antibiotics and biocides, with almost all resistance genes located on large IncC plasmids, additionally carrying pSLT-derived virulence genes. The number of resistance genes was highly variable, resulting in a total of 22 ABR (antibiotic biocide resistance) profiles. The number of antibiotic resistance genes, but not that of biocide resistance genes, was considerably lower in isolates from the second than from the first period (with averages of 5.5 versus 9.6 genes). Importantly, IS26, which resides in multiple copies within these plasmids, appears to be playing a crucial role in the evolution of resistance, and it was also responsible for the monophasic phenotype, which was associated with four different deletions eliminating the fljAB region. Conclusions: the observed reduction in the number of antibiotic resistance genes could correlate with the loss of adaptive advantage originating from the ban on the use of antibiotics as feed additives implemented in the European Union since 2006, facilitated by the intrinsic instability of the IncC plasmids. Two consecutive IS26 transposition events, which can explain both the clonal relationship of the isolates and their variability, may account for the observed fljAB deletions. Full article

Anti-infectives include molecules that target microbes in the context of infection but lack antimicrobial activity under conventional growth conditions. We previously described D66, a small molecule that kills the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) within cultured macrophages and murine tissues, with low host toxicity. While D66 fails to inhibit bacterial growth in standard media, the compound is bacteriostatic and disrupts the cell membrane voltage gradient without lysis under growth conditions that permeabilize the outer membrane or reduce efflux pump activity. To gain insights into specific bacterial targets of D66, we pursued two genetic approaches. Selection for resistance to D66 revealed spontaneous point mutations that mapped within the gmhB gene, which encodes a protein involved in the biosynthesis of the lipopolysaccharide core molecule. E. coli and S. Typhimurium gmhB mutants exhibited increased resistance to antibiotics, indicating a more robust barrier to entry. Conversely, S. Typhimurium transposon insertions in genes involved in outer membrane permeability or efflux pump activity reduced fitness in the presence of D66. Together, these observations underscore the significance of the bacterial cell envelope in safeguarding Gram-negative bacteria from small molecules. Full article

Background: Antimicrobial resistance (AMR) is a critical global health threat, with AMR Salmonella enterica serovar Typhimurium strains being a major foodborne pathogen. Integrons, a type of mobile genetic element, capture and transfer resistance genes, thereby playing a role in the spread of AMR. Objectives: This study aimed to characterize the locations of integrons carrying AMR genes within the whole genomes of 32 Salmonella Typhimurium isolates collected from dairy cattle by two U.S. Veterinary Diagnostic Laboratories between 2009 and 2012. Methods: Class I integrons were sequenced from PCR-amplified products. DNA was extracted, quantified, barcoded, and sequenced on the Illumina MiSeq platform. Whole genome sequences were trimmed and assembled using the SPAdes assembler in Geneious Prime^®, and plasmids were identified with the PlasmidFinder pipeline in Linux. Integron locations were determined by aligning their sequences with whole genome contigs and plasmids, while AMR genes were identified through BLAST with the MEGARes 3.0 database and confirmed by alignment with isolate, plasmid, and integron sequences. Statistical analysis was applied to compare the proportions of isolates harboring integrons on their chromosome versus plasmids and also to examine the associations between integron presence and AMR gene presence. Results: Seven plasmid types were identified from all isolates: IncFII(S) (n = 14), IncFIB(S) (n = 13), IncC (n = 7), Inc1-I(Alpha) (n = 3), and ColpVC, Col(pAHAD28), and Col8282 (1 isolate each). Of the 32 isolates, 16 (50%) carried at least one size of integron. Twelve of them carried both 1000 and 1200 bp; 3 carried only 1000 bp and 1 carried 1800 bp integrons. Of the 15 isolates that carried 1000 bp integron, 12 harbored it on IncFIB(S) plasmids, 2 on IncC plasmids, and 1 on the chromosome. The 1200 bp integrons from all 12 isolates were located on chromosomes. There were significant positive associations between the presence of integrons and the presence of several AMR genes including sul1, aadA2, blaCARB-2, qacEdelta1, tet(G), and floR (p < 0.05). AMR genes were located as follows: aadA2 on IncFIB(S) and IncC plasmids; bla_CMY-2 on IncC plasmid; qacEdelta1 on IncFIB(S), IncC, and chromosome; bla_CARB-2, floR, tet(A) and tet(G) on the chromosome. Conclusions: The findings highlight the genomic and plasmid complexity of Salmonella Typhimurium which is impacted by the presence and location of integrons, and this study provides genomic insights that can inform efforts to enhance food safety and protect both animal and public health. Full article

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that survives and replicates within host cells. Macrophages, key immune cells in infection defense, play a vital role in pathogen clearance through polarization (M1/M2) and NLRP3 inflammasome activation. While TRIM67 regulates macrophage recruitment in the liver, its role in S. Typhimurium infection remains unclear. In this study, a S. Typhimurium infection model was established by orally infecting streptomycin-pretreated TRIM67 WT and KO mice with 1 × 10⁹ CFU of S. Typhimurium. TRIM67 expression in the ileum, colon, mesenteric lymph nodes (MLNs), and peritoneal macrophages (PMs) was assessed via qRT-PCR and Western blotting. Histopathological changes were analyzed using HE and PAS staining. IHC staining, flow cytometry (FCM), qRT-PCR, and Western blotting were used to evaluate TRIM67 knockout effects on macrophage recruitment, polarization, and NLRP3 inflammasome activation. In vitro, PMs were infected with S. Typhimurium (MOI 1:20), and TRIM67’s role in macrophage polarization and NLRP3 activation was validated. S. Typhimurium infection significantly upregulated TRIM67 in the ileum, colon, and MLN. TRIM67 knockout reduced intestinal inflammatory cell infiltration but worsened goblet cell loss and impaired digestion. Bacterial load assays revealed weakened pathogen clearance, leading to weight loss and increased mortality. TRIM67 knockout inhibited intestinal macrophage recruitment, M1 polarization in MLN, and NLRP3 activation. In vitro, TRIM67 knockout increased PMs’ intracellular bacterial load and suppressed NLRP3, caspase-1, and IL-1β expression. TRIM67 knockout impairs the host’s ability to clear S. Typhimurium by inhibiting M1 macrophage polarization and NLRP3 inflammasome activation. Full article

In poultry production, antibiotics have been excessively used as growth promoters to support well-being and decrease mortality caused by pathogenic microorganisms. The overuse of antibiotics has led to the emergence of antibiotic-resistant bacteria and the presence of antibiotic residues in poultry products. To counteract this problem, probiotics could be used as adjuncts or as substitutes for preserving a diverse and balanced microflora to prevent the colonization and multiplication of pathogenic bacteria in the GI tract. This study aimed to isolate and characterize the potential probiotic properties of lactic acid bacteria from the small intestine of 23-week-old broiler breeders, with the goal of identifying potential probiotic candidates. Four phenotypically healthy broiler breeders were selected, and intestinal contents were aseptically collected and cultured on MRS agar. From the initial pool of 39 colonies, six isolates were identified based on Gram-positive and catalase-negative characteristics and further classified using 16S rRNA sequencing as Levilactobacillus brevis (n = 3), Pediococcus pentosaceus (n = 2), and Streptococcus salivarius (n = 1). These strains were further evaluated for probiotic properties such as transit resistance to simulated upper gastrointestinal conditions, antagonist activity, haemolytic activity, and cell surface properties such as autoaggregation, co-aggregation and hydrophobicity, in vitro. L. brevis NKFS8 showed good tolerance to pH 3, while P. pentosaceus NKSF10 exhibited good tolerance to pH 4 acidic conditions. All isolates demonstrated good survivability in bile salt concentration of 3% (w/v), with P. pentosaceus NKSF10 exhibiting the highest tolerance. The isolates showed a wide range of antagonistic activity against the test pathogens Pseudomonas aeruginosa (ATCC 27853), Salmonella typhimurium, Salmonella enterica (ATCC 13314), Staphylococcus aureus (ATCC 29213), and Listeria monocytogenes (ATCC 7644). Furthermore, these strains exhibited good auto-aggregation, co-aggregation, and hydrophobicity properties. In conclusion, lactic acid bacteria from the small intestine of broiler breeders present a valuable prospect for the development of effective probiotics. These probiotics can be utilized as a supplementary inclusion in poultry feed, obviating the need for antibiotics as growth promoters. Nevertheless, additional in vivo studies are required to closely monitor and assess the effects of probiotics on the gastrointestinal system of chickens. Full article

Background: Outer Membrane Vesicles (OMVs), nanosized particles derived from Gram-negative bacteria, are promising vaccine carriers due to innate immunogenicity and self-adjuvant properties. Yet the systematic evaluations of OMV-associated toxicity remain limited. Methods: We developed a CRISPR/Cas9-engineered Salmonella enterica serovar Typhimurium ΔmsbB mutant (Mut4_STM) to produce detoxified OMVs (Mut4_OMVs) with enhanced yield. Subcutaneous immunization of BALB/c mice with Mut4_OMVs to evaluate safety, and the adjuvant efficacy was also determined by injecting Mut4_OMVs with Yersinia pestis F1Vmut or Bacillus anthracis PA_D4 antigens, mixing formulation, respectively. Results: Mut4_OMVs showed a 9-fold protein yield increase over wild-type OMVs. While all mice injected with wild-type OMVs died, 100% survival was observed in those receiving Mut4_OMVs. However, dose-dependent pathological alterations became evident in specific organs as the administration dose escalated, such as induced splenic extramedullary hematopoiesis and renal edema. Despite residual toxicity, 2–3 doses of 10 μg Mut4_OMVs elicited antigen-specific antibody titers comparable to aluminum adjuvant controls and superior T-cell immune responses. Conclusion: While Mut4_OMVs retain potent adjuvant activity, their residual toxicity necessitates further biocompatibility optimization for safe vaccine applications. Full article

Detection of Salmonella, a highly diverse foodborne pathogen, is paramount to ensure safety and protection of the animal industry and its consumers. Salmonella enterica serovar Typhimurium is among the most important non-typhoidal serovars causing gastroenteritis worldwide. However, traditional serovar identification is labor- and resource-intensive, while typical molecular tools require expensive reagents and equipment. Hence, this study developed and optimized a calcein-based and closed-tube loop-mediated isothermal amplification (LAMP)-based assay to detect S. Typhimurium following enrichment steps compared with an optimized PCR assay. The PCR assay showed 100% specificity in silico confirmed through DNA sequencing. For actual specificity testing, both PCR and LAMP showed 100% specificity to S. Typhimurium. For DNA sensitivity, while PCR showed a limit of detection of 22 pg/μL, LAMP showed a 100-fold higher sensitivity at 220 fg/μL. Meanwhile, for pure culture sensitivity, both assays detected at least 4.98 × 10⁴ CFU/mL. Parallel testing of 208 raw meat samples from wet markets in Metro Manila, Philippines, showed corroboration and statistical association of the optimized PCR and LAMP with 89.42% and 90.87% positivity rates for S. Typhimurium, respectively. Hence, the developed closed-tube and calcein-based LAMP assay is potentially a powerful yet simple, sensitive, and fast method for S. Typhimurium detection. Full article

Graphene oxide (GO), owing to its extraordinary application prospects in biomedicine, is attracting growing research attention. However, the biosafety and blood compatibility of GO required for its clearance for use in clinical trials remain elusive. Therefore, we studied the mutagenic properties of GO as well as its cell toxicity and blood compatibility. Prior to biological experiments, we assessed the structural organization of GO using dynamic light scattering and microscopic visualization methods. The results of both the Ames mutagenicity test performed on Salmonella enterica serovar Typhimurium TA98 and TA102 strains and the cytotoxicity test on noncancerous, immortalized human keratinocytes revealed no mutagenic or toxic effects of GO. Simultaneously, GO reduced the viability of the MelJuSo human melanoma cell line. A blood compatibility assay revealed that a concentration of 10 μg/mL was critical for GO biosafety, as greater concentrations induced diverse side effects. Specifically, GO disrupts erythrocytes’ membranes in the dose-dependent manner. Moreover, GO at higher concentrations both inhibited the process of ADP (a physiological platelet agonist)-induced cell aggregation and affected their disaggregation process in platelet-rich plasma. However, in the blood clotting assessment, GO showed no effects on the activated partial thromboplastin time, prothrombin time, or thrombin time of the platelet-poor plasma. The obtained results clearly indicate that the relationship between the GO preparation method, its size, and concentration and biosafety must be cautiously monitored in the context of further possible biomedical applications. Full article

Foodborne illnesses, particularly those caused by Salmonella enterica subsp. enterica Serovar Typhimurium, present a significant challenge to public health, especially within the seafood industry due to biofilm formation on foods. This study investigated the antibiofilm potential of fucoidan, a sulfated polysaccharide, against Salmonella enterica subsp. enterica Serovar Typhimurium biofilm on crab and shrimp surfaces. Fucoidan’s minimum inhibitory concentration (MIC) was determined to be 150 µg/mL. Sub-MIC (1/8, 1/4, 1/2, and MIC) were evaluated for their impact on inhibition of biofilm formation. Fucoidan treatment resulted in significant, dose-dependent inhibition in biofilm formation, achieving 2.61 log CFU/cm² and 2.45 log CFU/cm² reductions on crab and shrimp surfaces, respectively. FE-SEM analysis confirmed biofilm disruption and cell membrane damage. Real-time PCR showed the downregulation of quorum-sensing (luxS) and virulence (rpoS, avrA, and hilA) genes. These results propose that fucoidan has the ability as a natural antibacterial agent for controlling Salmonella enterica subsp. enterica Serovar Typhimurium biofilms in seafood processing, thereby enhancing food safety and minimizing contamination. 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

The objective of this study was to determine the frequency of Salmonella in pig samples analyzed at the Veterinary Diagnostic Unit of the Faculty of Agricultural Sciences of the University of Antioquia, Colombia, between 2022 and 2023. Out of 5820 serum samples analyzed using indirect enzyme-linked immunosorbent assay, 63.76% were positive. Additionally, Salmonella was isolated and identified in a separate group of 848 samples (feces, tissues, etc.) with a positivity rate of 23.47%. Eight serotypes were identified, with the most common being Salmonella enterica subsp. enterica serotype Typhimurium (49.2%), followed by its monophasic variant I 4,[5],12:i:- (23%), and serotype Choleraesuis (18%). Antimicrobial susceptibility profiles of 105 isolates were evaluated using the Kirby–Bauer method, which demonstrated higher resistance (100%) to ampicillin, tiamulin, penicillin, tylosin, and erythromycin (these were the least tested), followed by florfenicol (44/54), doxycycline (4/5), spectinomycin (18/25), amoxicillin (32/46), chloramphenicol (2/3), tetracycline (2/3), and enrofloxacin (34/64), with lower resistance observed for fosfomycin (2/38) and ceftiofur (5/35). Multi-drug resistance was observed in 59% (62/105) of the isolates. The high proportion of Salmonella and the levels of resistance to various drugs raise significant concerns, indicating potential deficiencies in responsible antimicrobial use and management practices on pig farms in the region. Full article

Antibacterial resistance has become a major problem where new promising drugs are needed. The extracts obtained from marine invertebrates Mytilus galloprovincialis, Patella sp., Gibbula sp. and Arbacia lixula were tested against bacteria using the disc diffusion method. Citrobacter sp. from seawater and Paenibacillus sp., Bacillus sp. and Geobacillus sp. from soil were used as well as the reference bacterial strains Staphylococcus aureus NCTC 12981, S. aureus subsp. aureus Rosenbach ATCC 6538, Salmonella enterica subsp. enterica serovar Enteritidis ATCC 13076, Salmonella enterica subsp. enterica serotype Typhimurium NCTC 12023, Listeria monocytogenes ATCC 19111, Klebsiella aerogenes ATCC 13048 and Escherichia coli NCTC 12241. The most successful bacterial inhibitors, inhibiting 8 of 13 strains were extracts of M. galloprovincialis, Patella sp., Gibbula sp., Enteromorpha sp., C. sinuosa and U. lactuca, extract of A. lixula showed antibacterial activity against five bacteria, while extract of C. officinalis showed no antibacterial activity. These results indicate the potential of these marine organisms as a source of antibacterial compounds and may serve as a basis for further research and development of new antibacterial agents. Full article

This study aims to highlight the therapeutic potential of some Lamiacea essential oils (EOs). For this purpose, eight EOs, including two from Lavandula angustifolia Mill. cultivated in Romania and Spain (LA1 and LA2), Salvia officinalis L. (SO), Lavandula hybrida Balb. ex Ging (LH), Salvia sclarea L. (SS), Mentha smithiana L. (MS), Perovskia atriplicifolia Benth. (PA), and Mentha x piperita L. (MP), were evaluated in vitro in terms of antioxidant, cytotoxic, antimicrobial, and anti-migratory activities. As regards the antioxidant capacity, expressed as the EO concentration that produces 50% of the maximum effect (IC₅₀ value), the EOs obtained from the cultivated plants of the Lamiaceae family are ordered as follows: LA2 ˃ LA1 ˃ LH > MP > MS > SO > SS > PA. For the determination of antimicrobial activity, the reference strains used for testing were Salmonella enterica serotype typhimurium, Shigella flexneri serotype 2b, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Candida parapsilosis. The most intense inhibitory effect was observed in EOs of MS and MP on all tested microbial strains. The cytotoxic and anti-migratory activity of EOs was tested on two melanoma cell lines (A375 and B164A5) and on a healthy keratinocyte line (HaCaT). EOs LA1 and MP manifested the highest selectivity on the analysed tumoural cells, by reducing their migration in comparison with the control, proving to have therapeutic potential. 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