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Urinary tract infections (UTI) by antibiotic resistant and virulent K. pneumoniae are a growing concern. Understanding the genome and validating the genomic profile along with pangenome analysis will facilitate surveillance of high-risk clones of K. pneumoniae to underpin management strategies toward early detection. The present study aims to correlate resistome with phenotypic antimicrobial resistance and virulome with pathogenicity in Klebsiella spp. The present study aimed to perform complete genome sequences of Klebsiella spp. and to analyse the correlation of resistome with phenotypic antimicrobial resistance and virulome with pathogenicity. To understand the resistome, pangenome and virulome in the Klebsiella spp, the ResFinder, CARD, IS Finder, PlasmidFinder, PHASTER, Roary, VFDB were used. The phenotypic susceptibility profiling identified the uropathogenic kp3 to exhibit multi drug resistance. The resistome and in vitro antimicrobial profiling showed concordance with all the tested antibiotics against the study strains. Hypermucoviscosity was not observed for any of the test isolates; this phenotypic character matches perfectly with the absence of rmpA and magA genes. To the best of our knowledge, this is the first report on the presence of ste, stf, stc and sti major fimbrial operons of Salmonella enterica serotype Typhimurium in K. pneumoniae genome. The study identifies the discordance of virulome and virulence in Klebsiella spp. The complete genome analysis and phenotypic correlation identify uropathogenic K. pneumoniae kp3 as a carbapenem-resistant and virulent pathogen. The Pangenome of K. pneumoniae was open suggesting high genetic diversity. Diverse K serotypes were observed. Sequence typing reveals the prevalence of K. pneumoniae high-risk clones in UTI catheterised patients. The study also highlights the concordance of resistome and in vitro susceptibility tests. Importantly, the study identifies the necessity of virulome and phenotypic virulence markers for timely diagnosis and immediate treatment for the management of high-risk K. pneumoniae clones. [ABSTRACT FROM AUTHOR]

Salmonella enterica Serovar Typhimurium (Salmonella) and its bacteriophage P22 are a model system for the study of horizontal gene transfer by generalized transduction. Typically, the P22 DNA packaging machinery initiates packaging when a short sequence of DNA, known as the pac site, is recognized on the P22 genome. However, sequences similar to the pac site in the host genome, called pseudo-pac sites, lead to erroneous packaging and subsequent generalized transduction of Salmonella DNA. While the general genomic locations of the Salmonella pseudo-pac sites are known, the sequences themselves have not been determined. We used visualization of P22 sequencing reads mapped to host Salmonella genomes to define regions of generalized transduction initiation and the likely locations of pseudo-pac sites. We searched each genome region for the sequence with the highest similarity to the P22 pac site and aligned the resulting sequences. We built a regular expression (sequence match pattern) from the alignment and used it to search the genomes of two P22-susceptible Salmonella strains—LT2 and 14028S—for sequence matches. The final regular expression successfully identified pseudo-pac sites in both LT2 and 14028S that correspond with generalized transduction initiation sites in mapped read coverages. The pseudo-pac site sequences identified in this study can be used to predict locations of generalized transduction in other P22-susceptible hosts or to initiate generalized transduction at specific locations in P22-susceptible hosts with genetic engineering. Furthermore, the bioinformatics approach used to identify the Salmonella pseudo-pac sites in this study could be applied to other phage—host systems. Author summary: Bacteriophage P22 has been a genetic tool and a key model for the study of generalized transduction in Salmonella since the 1950s, yet certain components of the generalized transduction molecular mechanism remain unknown. Specifically, the locations and sequences of pseudo-pac sites, hypothesized to facilitate packaging of Salmonella DNA by P22, have not been determined to date. In this study, we identified the specific locations and sequences of the pseudo-pac sites frequently recognized by P22 in Salmonella genomes. The identification of highly efficient pseudo-pac sites in Salmonella helps us understand the sequence specificity necessary for P22 pac site recognition and paves the way for more targeted use of generalized transduction with P22. [ABSTRACT FROM AUTHOR]

The gastrointestinal tract is densely colonized by a polymicrobial community known as the microbiota which serves as primary line of defence against pathogen invasion. The microbiota can limit gut-luminal pathogen growth at different stages of infection. This can be traced to specific commensal strains exhibiting direct or indirect protective functions. Although these mechanisms hold the potential to develop new approaches to combat enteric pathogens, they remain far from being completely described. In this study, we investigated how a mouse commensal Escherichia coli can outcompete Salmonella enterica serovar Typhimurium (S. Tm). Using a salmonellosis mouse model, we found that the commensal E. coli 8178 strain relies on a trojan horse trap strategy to limit S. Tm expansion in the inflamed gut. Combining mutants and reporter tools, we demonstrated that inflammation triggers the expression of the E. coli 8178 antimicrobial microcin H47 toxin which, when fused to salmochelin siderophores, can specifically alter S. Tm growth. This protective function was compromised upon disruption of the E. coli 8178 tonB-dependent catecholate siderophore uptake system, highlighting a previously unappreciated crosstalk between iron intake and microcin H47 activity. By identifying the genetic determinants mediating S. Tm competition, our work not only provides a better mechanistic understanding of the protective function displayed by members of the gut microbiota but also further expands the general contribution of microcins in bacterial antagonistic relationships. Ultimately, such insights can open new avenues for developing microbiota-based approaches to better control intestinal infections. The microbiota serves as the first line of defence against pathogen invasion in the gut. This study shows that, in a mouse model of salmonellosis, commensal E. coli 8178 uses a trojan horse strategy to limit the expansion of Salmonella in the inflamed gut, using microcin H47 fused to salmochelin siderophores. [ABSTRACT FROM AUTHOR]

The application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell‐free glycosylation assay that offers the speed and simplicity of a 'yes' or 'no' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell‐free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity. [ABSTRACT FROM AUTHOR]

Major histocompatibility complex class II (MHC-II) molecules are involved in immune responses against pathogens and vaccine candidates' immunogenicity. Immunopeptidomics for identifying cancer and infection-related antigens and epitopes have benefited from advances in immunopurification methods and mass spectrometry analysis. The mouse anti-MHC-II-DR monoclonal antibody L243 (mAb-L243) has been effective in recognising MHC-II-DR in both human and non-human primates. It has also been shown to cross-react with other animal species, although it has not been tested in livestock. This study used mAb-L243 to identify Staphylococcus aureus and Salmonella enterica serovar Typhimurium peptides binding to cattle and swine macrophage MHC-II-DR molecules using flow cytometry, mass spectrometry and two immunopurification techniques. Antibody cross-reactivity led to identifying expressed MHC-II-DR molecules, together with 10 Staphylococcus aureus peptides in cattle and 13 S. enterica serovar Typhimurium peptides in swine. Such data demonstrates that MHC-II-DR expression and immunocapture approaches using L243 mAb represents a viable strategy for flow cytometry and immunopeptidomics analysis of bovine and swine antigen-presenting cells. [ABSTRACT FROM AUTHOR]

Background: The world faces a major infectious disease challenge. Interest in the discovery, design, or development of antimicrobial peptides (AMPs) as an alternative approach for the treatment of bacterial infections has increased. Insects are a good source of AMPs which are the main effector molecules of their innate immune system. Black Soldier Fly Larvae (BSFL) are being developed for large-scale rearing for food sustainability, waste reduction and as sustainable animal and fish feed. Bioinformatic studies have suggested that BSFL have the largest number of AMPs identified in insects. However, most AMPs identified in BSF have not yet undergone antimicrobial evaluation but are promising leads to treat critical infections. Results: Jg7197.t1, Jg7902.t1 and Jg7904.t1 were expressed into the haemolymph of larvae following infection with Salmonella enterica serovar Typhimurium and were predicted to be AMPs using the computational tool ampir. The genes encoding these proteins were within 2 distinct clusters in chromosome 1 of the BSF genome. Following removal of signal peptides, predicted structures of the mature proteins were superimposed, highlighting a high degree of structural conservation. The 3 AMPs share primary sequences with proteins that contain a Kunitz-binding domain; characterised for inhibitory action against proteases, and antimicrobial activities. An in vitro antimicrobial screen indicated that heterologously expressed SUMO-Jg7197.t1 and SUMO-Jg7902.t1 did not show activity against 12 bacterial strains. While recombinant SUMO-Jg7904.t1 had antimicrobial activity against a range of Gram-negative and Gram-positive bacteria, including the serious pathogen Pseudomonas aeruginosa. Conclusions: We have cloned and purified putative AMPs from BSFL and performed initial in vitro experiments to evaluate their antimicrobial activity. In doing so, we have identified a putative novel defensin-like AMP, Jg7904.t1, encoded in a paralogous gene cluster, with antimicrobial activity against P. aeruginosa. [ABSTRACT FROM AUTHOR]

Bacterial pathogens have remained a major public health concern for several decades. This study investigated the antibacterial activities of Miang extracts (at non-neutral and neutral pH) against Bacillus cereus TISTR 747, Escherichia coli ATCC 22595, Salmonella enterica serovar Typhimurium TISTR 292 and Streptococcus mutans DMST 18777. The potential of Polyvinylpolypyrrolidone (PVPP)-precipitated tannin-free Miang extracts in growth-inhibition of the cariogenic Streptococcus mutans DMST 18777 and its biofilms was also evaluated. The tannin-rich fermented extracts had the best bacterial growth inhibition against S. mutans DMST 18777 with an MIC of 0.29 and 0.72 mg/mL for nonfilamentous fungi (NFP) Miang and filamentous-fungi-processed (FFP) Miang respectively. This observed anti-streptococcal activity still remained after PVPP-mediated precipitation of bioactive tannins especially, in NFP and FFP Miang. Characterization of the PVPP-treated extracts using High performance liquid chromatography quadrupole-time of flight-mass spectrometry (HPLC-QToF-MS) analysis, also offered an insight into probable compound classes responsible for the activities. In addition, Crystal violet-staining also showed better IC50 values for NFP Miang (4.30 ± 0.66 mg/mL) and FFP Miang (12.73 ± 0.11 mg/mL) against S. mutans DMST 18777 biofilms in vitro. Homology modeling and molecular docking analysis using HPLC-MS identified ligands in tannin-free Miang supernatants, was performed against modelled S. mutans DMST 18777 sortase A enzyme. The in silico analysis suggested that the inhibition by NFP and FFP Miang might be attributed to the presence of ellagic acid, flavonoid aglycones, and glycosides. Thus, these Miang extracts could be optimized and explored as natural active pharmaceutical ingredients (NAPIs) for applications in oral hygienic products. [ABSTRACT FROM AUTHOR]

Background: The most prevalent probiotic bacterium employed in the food industry is Lactobacillus because it can produce metabolites with antibacterial capabilities and exhibits hostility towards infections and microorganisms that cause spoilage. Aim: This study set out to identify naturally occurring Lactobacillus and plantaricin (pln EF) coding genes in raw cow milk and to assess the antibacterial potency of isolated Lactobacillus isolates. Methods: Following enrichment in De Man, Rogosa and Sharpe (MRS) broth, single colonies were isolated, and pure colonies were obtained by streaking on MRS agar. The 16S rRNA gene was amplified using polymerase chain reaction (PCR) to confirm the cultural positivity of all isolates. Additionally, the presence of plantaricin was verified by targeting the pln EF gene through PCR. Outcome: Out of the 166 raw milk specimens acquired from cows, 153 (91.17%; CI: 86.98–95.76) were identified as positive for Lactobacillus through both culture and biochemical screening. Subsequently, 121 (72.89%; CI: 65.46–79.49) of the isolates were affirmed to harbour Lactobacillus through PCR analysis. Within this subset, 6 isolates (4.96%; CI: 1.84–10.48) were found to possess the plnEF gene. When exposed to Lactobacillus isolates, Salmonella Typhimurium and Salmonella enterica displayed an average maximum zone of inhibition with a diameter measuring 24 mm. In contrast, Escherichia coli exhibited an average minimum zone of inhibition, featuring a diameter of 11 mm. Additionally, the Lactobacillus isolates demonstrated inhibitory zones against Staphylococcus aureus, Klebsiella pneumoniae and Klebsiella oxytoca, measuring 14, 22 and 19 mm, respectively. Clinical significance: Lactic acid bacteria, particularly Lactobacilli, are plentiful in cow milk and possess broad‐spectrum antibacterial properties. [ABSTRACT FROM AUTHOR]

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen–host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host–pathogen interactions in the cytosol with high spatiotemporal resolution. A previous study in PLOS Biology developed a FluidFM-based approach to inject organelles into living cells. Using this technology, this study shows that establishment of a Salmonella Typhimurium replicative niche in the cytosol of enteroid cells, when uncoupled from invasion, does not require flagellin or type III secretion system effectors. [ABSTRACT FROM AUTHOR]

In all domains of life, Hsp70 chaperones preserve protein homeostasis by promoting protein folding and degradation and preventing protein aggregation. We now report that the Hsp70 from the bacterial pathogen Salmonella enterica serovar Typhimurium—termed DnaK—independently reduces protein synthesis in vitro and in S. Typhimurium facing cytoplasmic Mg2+ starvation, a condition encountered during infection. This reduction reflects a 3-fold increase in ribosome association with DnaK and a 30-fold decrease in ribosome association with trigger factor, the chaperone normally associated with translating ribosomes. Surprisingly, this reduction does not involve J-domain cochaperones, unlike previously known functions of DnaK. Removing the 74 C-terminal amino acids of the 638-residue long DnaK impeded DnaK association with ribosomes and reduction of protein synthesis, rendering S. Typhimurium defective in protein homeostasis during cytoplasmic Mg2+ starvation. DnaK-dependent reduction in protein synthesis is critical for survival against Mg2+ starvation because inhibiting protein synthesis in a dnaK-independent manner overcame the 10,000-fold loss in viability resulting from DnaK truncation. Our results indicate that DnaK protects bacteria from infection-relevant stresses by coordinating protein synthesis with protein folding capacity. Hsp70 chaperones preserve protein synthesis by promoting protein folding and preventing its aggregation. This study reports a new role of Hsp70 in Salmonella enterica survival during low Mg2+ conditions by binding to the ribosome and reducing protein synthesis. [ABSTRACT FROM AUTHOR]

This study aimed to isolate Bacillusspecies from indigenous non-salted fermented soybean of Northeast India to select a potential novel probiotic candidate. A total of 150 isolates were obtained and identified as Bacillussp. from hawaijar (109) and kinema (41) based on phenotypic characterization. Antimicrobial activity was evaluated against the five pathogens such as Salmonella enterica typhimurium MTCC 98, Klebsiella pneumoniae MTCC 109, Shigella flexneri MTCC 1457, Pseudomonas aeruginosa MTCC 741, and Escherichia coli (clinical isolates). Forty five Bacillus isolates showed antagonism to at least 3 pathogens. In the antioxidant assay, the Bacillus isolates displayed varying degrees of scavenging activity which were in the range of 37.5–89.6%. In the hydrophobicity and autoaggregation tests, marked differences in cell hydrophobicity to xylene and auto-aggregation, ranging from 3.2 to 79.2% and from 34 to 97%, respectively, were noted among the tested strains. The most of the isolates revealed susceptibility to antibiotics except for nalidixic acid. In the hemolysis test, hemolytic strains were discarded and non-haemolytic strains were used for the in vitro test. Twenty five strains produced exopolysaccharides. In adhesion to Caco-2 cells, all the tested strains showed adhesion but with variation, Bacillus subtilis strain Kak2a showed the highest adhesion (2.34%). Based on the in vitro test, the best three isolates such as Pis3, Kak2a, and, Kw3 were selected as potential probiotics candidates. 16S rRNA sequence analyses were performed to confirm the identity of the selected three Bacillus isolates. [ABSTRACT FROM AUTHOR]

The purpose of this study was to identify the effect of amalgam UV lamp-activated gallic acid on the inactivation of foodborne pathogens (Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes). When the bactericidal effect of amalgam UV lamp-activated gallic acid was compared with that of conventional low-pressure (LP) Hg UV lamp-activated gallic acid, the reduction levels of all three foodborne pathogens were significantly higher by amalgam UV lamp-activated gallic acid than LP lamp-activated gallic acid. Bactericidal mechanism was investigated using four reactive oxygen species (ROS) scavengers (mannitol, sodium pyruvate, trion, and sodium azide) and a wider range of ROS was produced by amalgam lamp-activated gallic acid than LP lamp-activated gallic acid. It is assumed that dual-wavelength (185 and 254 nm) emission characteristics of amalgam lamp generate a wider range of ROS. When applied to cherry tomato samples, amalgam lamp-activated gallic acid reduced levels of pathogens to a significantly greater level than LP lamp-activated gallic acid. These results indicate that amalgam lamp-activated gallic acid can effectively control foodborne pathogens in the food processing industry. [ABSTRACT FROM AUTHOR]

In food preparation and manufacturing environments, surfaces contaminated with Salmonella can lead to outbreaks of Salmonellosis. We hypothesise that Salmonella resides on dry surfaces in a biofilm form leading to potential environmental persistence and transfer following contact. This is the first study reporting that Salmonella Typhimurium can form dry surface biofilm (DSB). Six disinfectants commonly used in the food industry were evaluated for their efficacy against the DSB. The two most efficacious formulations reduced bacterial viability in DSB by >99.99% when combined with mechanical removal (5 sec wiping; 300 g weight). Five out of six formulations significantly reduced bacterial transfer when combined with wiping. Complete eradication of Salmonella Typhimurium DSB was challenging, and mechanical removal was essential to produce a >99.99% reduction in bacterial viability within DSB. This study highlights a potential mode of survival of Salmonella Typhimurium on food-contact surfaces and DSB challenges for disinfection. [ABSTRACT FROM AUTHOR]

The incidence of gastrointestinal illness attributable to Salmonella enterica serovar Typhimurium (ST) remains a concern for public health worldwide, as it can progress into systemic infections mediated by the type-three secretion system (T3SS), which allows for adherence and invasion to intestinal epithelial cells. The current study evaluates the ability of gallic acid (GA), protocatechuic acid (PA), and vanillic acid (VA) to impair the adhesion and invasion abilities of ST to a human epithelial (INT-407) cell monolayer while also assessing their cytotoxicity. GA, PA, and VA inhibited detectable ST growth at specific concentrations but showed cytotoxicity against INT-407 cells (>20% reduction in viability) after 3 h of treatments. Adjusting the pH of the solutions had a neutralizing effect on cytotoxicity, though it did reduce their antimicrobial potency. Adhesion of ST was reduced significantly when the cells were treated with 4.0 mg/mL of VA, whereas invasion was reduced in all treatments, with GA requiring the lowest concentration (0.5 mg/mL). Relative gene expression of virulence genes after treatment with GA showed downregulation in the T3SS regulator and effector hilA and sipA, respectively. These findings suggest further use of phenolic acids in reducing the activity of key virulence factors critical during ST infection. [ABSTRACT FROM AUTHOR]

Succinate is a potent immune signalling molecule that is present in the mammalian gut and within macrophages. Both of these infection niches are colonised by the pathogenic bacterium Salmonella enterica serovar Typhimurium during infection. Succinate is a C4-dicarboyxlate that can serve as a source of carbon for bacteria. When succinate is provided as the sole carbon source for in vitro cultivation, Salmonella and other enteric bacteria exhibit a slow growth rate and a long lag phase. This growth inhibition phenomenon was known to involve the sigma factor RpoS, but the genetic basis of the repression of bacterial succinate utilisation was poorly understood. Here, we use an experimental evolution approach to isolate fast-growing mutants during growth of S. Typhimurium on succinate containing minimal medium. Our approach reveals novel RpoS-independent systems that inhibit succinate utilisation. The CspC RNA binding protein restricts succinate utilisation, an inhibition that is antagonised by high levels of the small regulatory RNA (sRNA) OxyS. We discovered that the Fe-S cluster regulatory protein IscR inhibits succinate utilisation by repressing the C4-dicarboyxlate transporter DctA. Furthermore, the ribose operon repressor RbsR is required for the complete RpoS-driven repression of succinate utilisation, suggesting a novel mechanism of RpoS regulation. Our discoveries shed light on the redundant regulatory systems that tightly regulate the utilisation of succinate. We speculate that the control of central carbon metabolism by multiple regulatory systems in Salmonella governs the infection niche-specific utilisation of succinate. Author summary: During infection, Salmonella enterica serovar Typhimurium must efficiently utilise the nutrients provided by the host and the microbiota. Within host niches colonised by Salmonella, key carbon source is succinate, which is abundant in both the gut and within macrophages. Although S. Typhimurium possesses all the molecular machinery to import and catabolise succinate, the utilisation of this carbon source is strongly inhibited under laboratory conditions. Here, we have used diverse genetic approaches to discover that several distinct genetic regulatory systems act in concert to repress succinate utilisation by Salmonella. Our study sheds new light on how carbon source utilisation is managed by this important model enteropathogenic bacterium. [ABSTRACT FROM AUTHOR]

Genetic screening of pools of mutants can reveal genetic determinants involved in complex biological interactions, processes, and systems. We previously constructed two single-gene deletion resources for Salmonella enterica serovar Typhimurium 14028s in which kanamycin (KanR) and chloramphenicol (CamR) cassettes were used to replace non-essential genes. We have now used lambda-red recombination to convert the antibiotic cassettes in these resources into a tetracycline-resistant (TetR) version where each mutant contains a different 21-base barcode flanked by Illumina Read1 and Read2 primer sequences. A motility assay of a pool of the entire library, followed by a single-tube processing of the bacterial pellet, PCR, and sequencing, was used to verify the performance of the barcoded TetR collection. The new resource is useful for experiments with defined subsets of barcoded mutant strains where biological bottlenecks preclude high numbers of founder bacteria, such as in animal infections. The TetR version of the library will also facilitate the construction of triple mutants by transduction. The resource of 6197 mutants covering 3490 genes is deposited at Biological and Emerging Infections Resources (beiresources.org). [ABSTRACT FROM AUTHOR]

The Salmonella enterica serovars Typhimurium (S. Typhimurium), Heidelberg (S. Heidelberg), and their monophasic variants (S. 1,4,[5],12:i:-, S. 1,4,[5],12:r:- and S. 1,4,[5],12:-:1,2) are highly disseminated in poultry farming and can contaminate chicken meat, eggs, and other foods of avian origin. A time-consuming bacteriological and serological analysis is usually required to identify serovars by traditional methods. Incomplete and inconclusive serological results are frequent in routine analysis, mainly due to the occurrence of bacterial isolates presenting similar antigenic profiles. Molecular biology assays have been developed to improve the detection of specific Salmonella serovars and strains. This study aimed to develop a multiplex real-time PCR (SHTAmp) for the rapid DNA detection of S. Typhimurium, S. Heidelberg, and their monophasic variants from poultry samples. The methodology was used in the analysis of 147 field isolates from Brazilian poultry flocks previously evaluated with serological analysis. The results demonstrated that it was able to specifically and rapidly detect 21 S. Typhimurium and 57 S. Heidelberg isolates with complete antigenic formulae. Furthermore, SHTAmp was able to differentiate nine S. Typhimurium and 44 S. Heidelberg isolates with incomplete serological formulae (monophasic and aphasic variants). The complete methodology was also successfully used to detect these bacteria directly from 34 poultry samples after pre-enrichment in buffered peptone water (BPW). In conclusion, SHTAmp is a fast and accurate method to detect the two frequent and concerning serovars S. Typhimurium and S. Heidelberg directly from poultry samples. [ABSTRACT FROM AUTHOR]

Tellimagrandin-I (TL) and camptothin A (CA) are ellagitannins widely found in diverse plant species. Numerous studies demonstrated their significant biological activities, which include antitumor, antioxidant, and hepatoprotective properties. Despite this protective profile, the effects of TL and CA on DNA have not been comprehensively investigated. Thus, the aim of this study was to determine the mutagenic and antimutagenic effects attributed to TL and CA exposure on Salmonella enterica serovar Typhimurium strains using the Ames test. In addition, the cytotoxic and genotoxic effects were examined on human lymphocytes, employing both trypan blue exclusion and CometChip assay. The antigenotoxic effect was determined following TL and CA exposure in the presence of co-treatment with doxorubicin (DXR). Our results from the Ames test indicated that TL or CA did not display marked mutagenic activity. However, TL or CA demonstrated an ability to protect DNA against the damaging effects of the mutagens 4-nitroquinoline-1-oxide and sodium azide, thereby exhibiting antimutagenic properties. In relation to human lymphocytes, TL or CA did not induce significant cytotoxic or genotoxic actions on these cells. Further, these ellagitannins exhibited an ability to protect DNA from damage induced by DOX during co-treatment, indicating their potential beneficial usefulness as antigenotoxic agents. In conclusion, the protective effects of TL or CA against mutagens, coupled with their absence of genotoxic and cytotoxic effects on human lymphocytes, emphasize their potential therapeutic value in chemopreventive strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To ensure a robust immune response to pathogens without risking immunopathology, the kinetics and amplitude of inflammatory gene expression in macrophages need to be exquisitely well controlled. There is a growing appreciation for stress-responsive membraneless organelles (MLOs) regulating various steps of eukaryotic gene expression in response to extrinsic cues. Here, we implicate the nuclear paraspeckle, a highly ordered biomolecular condensate that nucleates on the Neat1 lncRNA, in tuning innate immune gene expression in murine macrophages. In response to a variety of innate agonists, macrophage paraspeckles rapidly aggregate (0.5 h poststimulation) and disaggregate (2 h poststimulation). Paraspeckle maintenance and aggregation require active transcription and MAPK signaling, whereas paraspeckle disaggregation requires degradation of Neat1 via the nuclear RNA exosome. In response to lipopolysaccharide treatment, Neat1 KO macrophages fail to properly express a large cohort of proinflammatory cytokines, chemokines, and antimicrobial mediators. Consequently, Neat1 KO macrophages cannot control replication of Salmonella enterica serovar Typhimurium or vesicular stomatitis virus. These findings highlight a prominent role for MLOs in orchestrating the macrophage response to pathogens and support a model whereby dynamic assembly and disassembly of paraspeckles reorganizes the nuclear landscape to enable inflammatory gene expression following innate stimuli. [ABSTRACT FROM AUTHOR]

Essential oils (EOs) and plant extracts have demonstrated inhibitory activity against a wide range of pathogenic bacteria. In this study, the chemical composition of manuka, kanuka, peppermint, thyme, lavender, and feijoa leaf and peel EOs and feijoa peel and leaf extracts were analyzed, and their antimicrobial activity against Escherichia coli, Salmonella enterica Typhimurium, Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes were determined. The results showed that the major compounds varied among different EOs and extracts, with menthol in peppermint EO, thymol and carvacrol in thyme EO, linalool in lavender EO, β‐caryophyllene in feijoa EO, and flavones in feijoa extract being the most prevalent. The study found that while EOs/extracts had antimicrobial activity alone, no individual EO/extract was highly effective against all tested species. Therefore, their combinations were tested to identify those that could broaden the spectrum of activity and act synergistically. The checkerboard method was applied to assess the possible synergism between the paired combinations of EOs/extract. The peppermint/thyme, peppermint/lavender, and peppermint/feijoa peel extract combinations exhibited a synergistic effect against E. coli and L. monocytogenes, with the peppermint/thyme and peppermint/feijoa peel extract combinations being the most effective against all five pathogens. Time‐to‐kill kinetics assays demonstrated that peppermint/thyme and peppermint/feijoa peel extract combinations achieved complete eradication of E. coli within 10–30 min and L. monocytogenes within 4–6 h. This study provides a promising approach to developing a natural alternative for food preservation using synergistic combinations of EOs/extracts, which could potentially reduce the required dosage and broaden their application in food products as natural preservatives. [ABSTRACT FROM AUTHOR]

Despite the significant presence of plant-derived tricarboxylic acids in some environments, few studies detail the bacterial metabolism of trans-aconitic acid (Taa) and tricarballylic acid (Tcb). In a soil bacterium, Acinetobacter baylyi ADP1, we discovered interrelated pathways for the consumption of Taa and Tcb. An intricate regulatory scheme tightly controls the transport and catabolism of both compounds and may reflect that they can be toxic inhibitors of the tricarboxylic acid cycle. The genes encoding two similar LysR-type transcriptional regulators, TcuR and TclR, were clustered on the chromosome with tcuA and tcuB, genes required for Tcb consumption. The genetic organization differed from that in Salmonella enterica serovar Typhimurium, in which tcuA and tcuB form an operon with a transporter gene, tcuC. In A. baylyi, tcuC was not cotranscribed with tcuAB. Rather, tcuC was cotranscribed with a gene, designated pacI, encoding an isomerase needed for Taa consumption. TcuC appears to transport Tcb and cis-aconitic acid (Caa), the presumed product of PacI-mediated periplasmic isomerization of Taa. Two operons, tcuC-pacI and tcuAB, were transcriptionally controlled by both TcuR and TclR, which have overlapping functions. We investigated the roles of the two regulators in activating transcription of both operons in response to multiple effector compounds, including Taa, Tcb, and Caa. [ABSTRACT FROM AUTHOR]

The intensive workload associated with the preparation of high-quality DNA libraries remains a key obstacle toward widespread deployment of sequencing technologies in remote and resource-limited areas. We describe the development of single-use microfluidic devices driven by an advanced pneumatic centrifugal microfluidic platform, the PowerBlade, to automate the preparation of Illumina-compatible libraries based on adaptor ligation methodology. The developed on-chip workflow includes enzymatic DNA fragmentation coupled to end-repair, adaptor ligation, first DNA cleanup, PCR amplification, and second DNA cleanup. This complex workflow was successfully integrated into simple thermoplastic microfluidic devices that are amenable to mass production with injection molding. The system was validated by preparing, on chip, libraries from a mixture of genomic DNA extracted from three common foodborne pathogens (Listeria monocytogenes, Escherichia coli and Salmonella enterica serovar Typhimurium) and comparing them with libraries made via a manual procedure. The two types of libraries were found to exhibit similar quality control metrics (including genome coverage, assembly, and relative abundances) and led to nearly uniform coverage independent of GC content. This microfluidic technology offers a time-saving and cost-effective alternative to manual procedures and robotic-based automation, making it suitable for deployment in remote environments where technical expertise and resources might be scarce. Specifically, it facilitates field practices that involve mid- to low-throughput sequencing, such as tasks related to foodborne pathogen detection, characterization, and microbial profiling. [ABSTRACT FROM AUTHOR]

In this study, a accurate, rapid quantitative PCR method for the simultaneous detection of 4 kinds of pathogenic bacteria in water was established, and the distribution of pathogenic bacteria in surface waters with different levels of pollution (Yulin region, China) was detected. The results showed that the detection accuracy was 94%; the detection limit was 2.7 in bacterial cells. Salmonella enterica subsp. enterica serovar typhimurium and Salmonella dysenteria were always present in water when the universal primer for pathogenic bacteria abundance detection was greater than 104 copies 100 mL−1. When the detection value is lower than 104 copy 100 mL−1, the bacteria in the water are rarely pathogenic bacteria, so the detection value of 104 copy 100 mL−1 can be used as a new indicator of waterborne pathogen pollution. [ABSTRACT FROM AUTHOR]

As human spaceflight increases in duration, cultivation of crops in spaceflight is crucial to protecting human health under microgravity and elevated oxidative stress. Foodborne pathogens (e.g., Salmonella enterica) carried by leafy green vegetables are a significant cause of human disease. Our previous work showed that Salmonella enterica serovar Typhimurium suppresses defensive closure of foliar stomata in lettuce (Lactuca sativa L.) to ingress interior tissues of leaves. While there are no reported occurrences of foodborne disease in spaceflight to date, known foodborne pathogens persist aboard the International Space Station and space-grown lettuce has been colonized by a diverse microbiome including bacterial genera known to contain human pathogens. Interactions between leafy green vegetables and human bacterial pathogens under microgravity conditions present in spaceflight are unknown. Additionally, stomatal dynamics under microgravity conditions need further elucidation. Here, we employ a slow-rotating 2-D clinostat to simulate microgravity upon in-vitro lettuce plants following a foliar inoculation with S. enterica Typhimurium and use confocal microscopy to measure stomatal width in fixed leaf tissue. Our results reveal significant differences in average stomatal aperture width between an unrotated vertical control, plants rotated at 2 revolutions per minute (2 RPM), and 4 RPM, with and without the presence of S. typhimurium. Interestingly, we found stomatal aperture width in the presence of S. typhimurium to be increased under rotation as compared to unrotated inoculated plants. Using confocal Z-stacking, we observed greater average depth of stomatal ingression by S. typhimurium in lettuce under rotation at 4 RPM compared to unrotated and inoculated plants, along with greater in planta populations of S. typhimurium in lettuce rotated at 4 RPM using serial dilution plating of homogenized surface sterilized leaves. Given these findings, we tested the ability of the plant growth-promoting rhizobacteria (PGPR) Bacillus subtilis strain UD1022 to transiently restrict stomatal apertures of lettuce both alone and co-inoculated with S. typhimurium under rotated and unrotated conditions as a means of potentially reducing stomatal ingression by S. typhimurium under simulated microgravity. Surprisingly, rotation at 4 RPM strongly inhibited the ability of UD1022 alone to restrict stomatal apertures and attenuated its efficacy as a biocontrol following co-inoculation with S. typhimurium. Our results highlight potential spaceflight food safety issues unique to production of crops in microgravity conditions and suggest microgravity may dramatically reduce the ability of PGPRs to restrict stomatal apertures. [ABSTRACT FROM AUTHOR]

Modern requirements for 'green label' meat products have led to the design of novel antimicrobial innovations which prioritise quality, safety and longevity. Plasma-functionalised water (PFW), ultraviolet light and natural antimicrobial compositions have been investigated and optimised for control of foodborne pathogens like Campylobacter jejuni and Salmonella enterica serovar Typhimurium. However, given the adaptive mechanisms present in bacteria under external stresses, it is imperative to understand the effect that sublethal treatment may have on the bacterial transcriptome. In this study, Salmonella Typhimurium and C. jejuni were treated with sublethal doses of ultraviolet light, a citrus juice/essential oil marinade, and 'spark' or 'glow' cold plasma generation system-produced PFW. Immediately after treatment, cells were lysed and RNA was extracted and purified. mRNA was converted to cDNA by reverse transcription-PCR and sequenced by an Illumina MiSeq® system. Sequences were filtered and analysed using the Tuxedo workflow. Sublethal treatment of Campylobacter jejuni and Salmonella Typhimurium led to increased immediate cellular and metabolic activity, as well as diversification in protein and metabolic functioning. There was further expression of pathogenesis and virulence-associated traits associated with spark PFW and marinade treatment of Salmonella Typhimurium. However, similar concerns were not raised with glow PFW or UV-treated samples. This study provides science-based evidence of the efficacy of multi-hurdle antimicrobial system using green-label marinades and PFW or UV to inactivate pathogens without upregulating virulence traits in surviving cells. This study will inform policymakers and food industry stakeholders and reinforces the need to incorporate in-line novel technologies to ensure consumer safety. Key points: • Salmonella and C. jejuni showed increased cell activity in immediate response to stress. • Virulence genes showed increased expression when treated with natural antimicrobials and sPFW. • Reduced immediate transcriptomic response to gPFW and UV treatment indicates lower risk. [ABSTRACT FROM AUTHOR]

Emergence of genetic variants with increased resistance/tolerance to natural antimicrobials, such as essential oils, has been previously evidenced; however, it is unknown whether mutagenesis follows a general or a specific pattern. For this purpose, we carried out four adaptive laboratory evolutions (ALE) in parallel of Salmonella enterica Typhimurium with carvacrol. After 10 evolution steps, we selected and characterized one colony from each lineage (SeCarA, SeCarB, SeCarC, and SeCarD). Phenotypic characterization of the four evolved strains revealed enhanced survival to lethal treatments; two of them (SeCarA and SeCarB) showed an increase of minimum inhibitory concentration of carvacrol and a better growth fitness in the presence of carvacrol compared to wild-type strain. Whole genome sequencing revealed 10 mutations, of which four (rrsH, sseG, wbaV, and flhA) were present in more than one strain, whereas six (nirC, fliH, lon, rob, upstream yfhP, and upstream argR) were unique to individual strains. Single-mutation genetic constructs in SeWT confirmed lon and rob as responsible for the increased resistance to carvacrol as well as to antibiotics (ampicillin, ciprofloxacin, chloramphenicol, nalidixic acid, rifampicin, tetracycline, and trimethoprim). wbaV played an important role in increased tolerance against carvacrol and chloramphenicol, and flhA in cross-tolerance to heat treatments. As a conclusion, no common phenotypical or genotypical pattern was observed in the isolated resistant variants of Salmonella Typhimurium emerged under carvacrol stress. Furthermore, the demonstration of cross-resistance against heat and antibiotics exhibited by resistant variants raises concerns regarding food safety. Key points: • Stable resistant variants of Salmonella Typhimurium emerged under carvacrol stress • No common pattern of mutagenesis after cyclic exposures to carvacrol was observed • Resistant variants to carvacrol showed cross-resistance to heat and to antibiotics [ABSTRACT FROM AUTHOR]

Several bioactive compounds, such as polyphenols, demonstrate low toxicity and prominent effects on cancer cells with antioxidant, anti-inflammatory, and antitumor activities. Such compounds can be found in Amazon mosses Leucobryum martianum (Hornsch.) Hampe ex Müll. Hal. (Hornsch.) and Leucobryum laevifolium (Broth). Antimutagenic assay with Salmonella enterica serovar Typhimurium and cytotoxicity with different eukaryotic cell lines were carried out to screen aqueous, hydroalcoholic, and ethanolic extracts of those Amazon mosses for anticancer potential. The results indicate the capacity of all extracts of both mosses to exert chemopreventive effects against 4-nitroquinoline- N -oxide (4NQO) and 2-aminoanthracene (2-AA), which are direct or indirect mutagens. In particular, the ethanolic and aqueous extract from L. martianum. The ethanolic extract from L. martianum induces significant cytotoxicity by mitochondrial metabolism and cell membrane disruption pathways to tumor or non-tumor cells. The aqueous extract from L. martianum showed a mainly cytotoxic response in the HepG2 cells, a human liver carcinoma, reaching ~90% cytotoxicity. The same extract did not induce significant damage to normal liver cells (F C3H cells) by membrane interaction pathway. The selective cytotoxicity in the aqueous extract of L. martianum makes it a candidate against liver cancer. Further studies, including in vivo models, are necessary to validate the efficacy and safety of the aqueous extract of L. martianum. [ABSTRACT FROM AUTHOR]

Objective: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a representative model organism for investigating host–pathogen interactions. It was reported that S. Typhimurium spvC gene alleviated intestinal inflammation to aggravate systemic infection, while the precise mechanisms remain unclear. In this study, the influence of spvC on the antibacterial defense of macrophage/neutrophil mediated by gasdermin D (GSDMD) was investigated. Methods: Mouse macrophage-like cell lines J774A.1 and RAW264.7, neutrophil-like cells derived from HL-60 cells (human promyletic leukemia cell lines) were infected with S. Typhimurium wild type, spvC deletion and complemented strains. Cell death was evaluated by LDH release and Annexin V-FITC/PI staining. Macrophage pyroptosis and neutrophil NETosis were detected by western blotting, live cell imaging and ELISA. Flow cytometry was used to assess the impact of spvC on macrophage-neutrophil cooperation in macrophage (dTHP-1)-neutrophil (dHL-60) co-culture model pretreated with GSDMD inhibitor disulfiram. Wild-type and Gsdmd−/− C57BL/6J mice were utilized for in vivo assay. The degree of phagocytes infiltration and inflammation were analyzed by immunofluorescence and transmission electron microscopy. Results: Here we find that spvC inhibits pyroptosis in macrophages via Caspase-1/Caspase-11 dependent canonical and non-canonical pathways, and restrains neutrophil extracellular traps extrusion in GSDMD-dependent manner. Moreover, spvC could ameliorate macrophages/neutrophils infiltration and cooperation in the inflammatory response mediated by GSDMD to combat Salmonella infection. Conclusions: Our findings highlight the antibacterial activity of GSDMD in phagocytes and reveal a novel pathogenic mechanism employed by spvC to counteract this host defense, which may shed new light on designing effective therapeutics to control S. Typhimurium infection. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Typhimurium (S. Typhimurium), an important foodborne pathogen, causes diarrheal illness and gastrointestinal diseases. S. Typhimurium survives and replicates in phagocytic and non-phagocytic cells for acute or chronic infections. In these cells, S. Typhimurium resides within Salmonella-containing vacuoles (SCVs), in which the phosphate (Pi) concentration is low. S. Typhimurium senses low Pi and expresses virulence factors to modify host cells. However, the mechanism by which host cells reduce the Pi concentration in SCVs is not clear. In this study, we show that through the TLR4-MyD88-NF-κB signaling pathway, S. Typhimurium upregulates PIT1, which in turn transports Pi from SCVs into the cytosol and results in Pi starvation in SCVs. Immunofluorescence and western blotting analysis reveal that after the internalization of S. Typhimurium, PIT1 is located on SCV membranes. Silencing or overexpressing PIT1 inhibits or promotes Pi starvation, Salmonella pathogenicity island-2 (SPI-2) gene expression, and replication in SCVs. The S. Typhimurium ΔmsbB mutant or silenced TLR4-MyD88-NF-κB pathway suppresses the expression of the SPI-2 genes and promotes the fusion of SCVs with lysosomes. Our results illustrate that S. Typhimurium exploits the host innate immune responses as signals to promote intracellular replication, and they provide new insights for the development of broad-spectrum therapeutics to combat bacterial infections. [ABSTRACT FROM AUTHOR]

Background: Disinfectants are important in the food industry to prevent the transmission of pathogens. Excessive use of disinfectants may increase the probability of bacteria experiencing long-term exposure and consequently resistance and cross-resistance to antibiotics. This study aims to investigate the cross-resistance of multidrug-resistant, drug-resistant, and drug-susceptible isolates of Salmonella enterica serovar Typhimurium (S. Typhimurium) with different sequence types (STs) to a group of antibiotics after exposure to different food-grade disinfectants. Methods: A panel of 27 S. Typhimurium strains with different antibiograms and STs were exposed to increasing concentrations of five food-grade disinfectants, including hydrogen peroxide (H2O2), benzalkonium chloride (BAC), chlorine dioxide (ClO2), sodium hypochlorite (NaClO), and ethanol. Recovered evolved strains were analyzed using genomic tools and phenotypic tests. Genetic mutations were screened using breseq pipeline and changes in resistance to antibiotics and to the same disinfectant were determined. The relative fitness of evolved strains was also determined. Results: Following exposure to disinfectants, 22 out of 135 evolved strains increased their resistance to antibiotics from a group of 14 clinically important antibiotics. The results also showed that 9 out of 135 evolved strains had decreased resistance to some antibiotics. Genetic mutations were found in evolved strains. A total of 77.78% of ST34, 58.33% of ST19, and 66.67% of the other STs strains exhibited changes in antibiotic resistance. BAC was the disinfectant that induced the highest number of strains to cross-resistance to antibiotics. Besides, H2O2 induced the highest number of strains with decreased resistance to antibiotics. Conclusions: These findings provide a basis for understanding the effect of disinfectants on the antibiotic resistance of S. Typhimurium. This work highlights the link between long-term exposure to disinfectants and the evolution of resistance to antibiotics and provides evidence to promote the regulated use of disinfectants. [ABSTRACT FROM AUTHOR]

Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. When a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we engineered S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche but failed to clear the bacteria from the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. To clear an infection, cells may have specific tasks that they must complete before they die; different modes of cell death could initiate these 'bucket lists' in either convergent or divergent ways. [ABSTRACT FROM AUTHOR]

Edema disease (ED) is a severe and lethal infectious ailment in swine, stemming from Shiga-toxin-producing Escherichia coli (STEC). An efficient, user-friendly, and safe vaccine against ED is urgently required to improve animal welfare and decrease antibiotic consumption. Recombinant attenuated Salmonella vaccines (RASV) administered orally induce both humoral and mucosal immune responses to the immunizing antigen. Their potential for inducing protective immunity against ED is significant through the delivery of STEC antigens. rSC0016 represents an enhanced recombinant attenuated vaccine vector designed for Salmonella enterica serotype Choleraesuis. It combines sopB mutations with a regulated delay system to strike a well-balanced equilibrium between host safety and immunogenicity. We generated recombinant vaccine strains, namely rSC0016 (pS-FedF) and rSC0016 (pS-rStx2eA), and assessed their safety and immunogenicity in vivo. The findings demonstrated that the mouse models immunized with rSC0016 (pS-FedF) and rSC0016 (pS-rStx2eA) generated substantial IgG antibody responses to FedF and rStx2eA, while also provoking robust mucosal and cellular immune responses against both FedF and rStx2eA. The protective impact of rSC0016 (pS-FedF) against Shiga-toxin-producing Escherichia coli surpassed that of rSC0016 (pS-rStx2eA), with percentages of 83.3%. These findings underscore that FedF has greater suitability for vaccine delivery via recombinant attenuated Salmonella vaccines (RASVs). Overall, this study provides a promising candidate vaccine for infection with STEC. [ABSTRACT FROM AUTHOR]

The sale and distribution of small turtles (shell length <4 inches) as pets has been banned in the United States since 1975 because of the risk of Salmonella transmission, especially to children. Despite this 48‐year‐old ban, salmonellosis outbreaks continue to be linked to contact with small turtles. During investigations of turtle‐associated outbreaks, information regarding the turtle farm of origin is difficult to obtain because turtles are commonly sold by transient vendors. During 2020–2021, public health officials investigated a multistate illness outbreak caused by Salmonella enterica serotype Typhimurium linked to pet small turtles. Cases were defined as a laboratory‐confirmed Salmonella Typhimurium infection highly related (within 0–6 allele differences) to the outbreak strain based on whole‐genome sequencing analysis by core‐genome multilocus sequence typing with illness onset occurring during 27 August 2020–14 May 2021. Forty‐three patients were identified from 12 states; of these, 35% (15/43) were children <5 years old. Among patients with available information, 37% (14/38) were hospitalized, and one death was reported. Seventy‐four percent (25/34) of patients reported turtle exposure in the week before illness onset, and 84% (16/19) specified exposure to small turtles. The outbreak strain was isolated from samples collected from a Pennsylvania patient's small turtle tank. Two patients reported purchasing their small turtles from pet stores. Salmonella Braenderup was isolated from samples collected from small turtles and their habitat at one of these stores; however, at that time, this strain was not associated with any human illnesses. This investigation was notable because of the documented sale of small turtles from several pet stores combined with the identification of a single small turtle supplier to these pet stores. The high proportion of children involved in this outbreak highlights the continued need to educate the pet industry as well as parents and caregivers about the risk of turtle‐associated salmonellosis especially in children. Understanding and addressing the persisting challenges related to the illegal sale and distribution of small turtles could reduce the burden of turtle‐associated salmonellosis. [ABSTRACT FROM AUTHOR]

Low‐energy X‐rays can be used to reduce the number of pathogenic microorganisms in fresh produce, but the efficacy of this process against internalized bacteria in leafy greens has not yet been reported. The leaves of iceberg lettuce were cut into pieces and subjected to vacuum perfusion to force the foodborne pathogen cells into the intercellular spaces within the leaves. Sodium hypochlorite (200–400 ppm) washes were not effective in inactivating internalized bacterial cells from lettuce leaves. In contrast, treatment with 1.5 kGy low‐energy X‐rays reduced Escherichia coli O157: H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes levels by 6.89, 4.48, and 3.22 log CFU/g, respectively. Additionally, the maximum dose of X‐rays did not adversely affect the color or texture of lettuce. These results suggest that low‐energy X‐ray treatment can be used to control internalized and surface‐adhering pathogens in leafy vegetables without affecting product quality. [ABSTRACT FROM AUTHOR]

Background Adjuvant addition of approved drugs or foodborne additives to colistin might be a cost-effective strategy to overcome the challenge of plasmid-mediated mobile colistin resistance gene emergence, which poses a threat in the clinic and in livestock caused by infections with Gram-negative bacteria, especially carbapenem-resistant Enterobacteriaceae. Methods Chequerboard assay was applied to screen the colistin adjuvants from natural compounds. The killing–time curve, combined disc test and membrane permeation assay were conducted to identify the synergy efficacy of thymol and colistin in vitro. Thin-layer chromatography (TLC), LC-MS and fluorescence spectra were used to indicate the interaction of thymol and MCR-1. The potential binding sites were then investigated by molecular simulation dynamics. Finally, a thymol nanoemulsion was prepared with high-pressure homogenization as the clinical dosage form. Results Thymol presented an excellent synergistic effect in vitro with colistin against Salmonella enterica serovar Typhimurium and Escherichia coli bacteria. Thymol addition, forming a complex with MCR-1, might interfere with the efficacy of MCR-1. Moreover, thymol strengthened colistin activity associated with potentiating membrane damage, destroying the biofilm and enhancing reactive oxygen species-mediated oxidative damage. Thymol nanoemulsion combined with colistin remarkably prevented the intestinal damage caused by S. Typhimurium infection, resulting in a survival rate higher than 60%. Conclusions This study achieved a promising thymol oral formulation as colistin adjuvant to combat S. Typhimurium infection, which could be used to extend the lifespan of colistin in clinical veterinary medicine. [ABSTRACT FROM AUTHOR]

While emerging evidence highlights the significance of gut microbiome in gastrointestinal infectious diseases, treatments like Fecal Microbiota Transplantation (FMT) and probiotics are gaining popularity, especially for diarrhea patients. However, the specific role of the gut microbiome in different gastrointestinal infectious diseases remains uncertain. There is no consensus on whether gut modulation therapy is universally effective for all such infections. In this comprehensive review, we examine recent developments of the gut microbiome's involvement in several gastrointestinal infectious diseases, including infection of Helicobacter pylori, Clostridium difficile, Vibrio cholerae, enteric viruses, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa Staphylococcus aureus, Candida albicans, and Giardia duodenalis. We have also incorporated information about fungi and engineered bacteria in gastrointestinal infectious diseases, aiming for a more comprehensive overview of the role of the gut microbiome. This review will provide insights into the pathogenic mechanisms of the gut microbiome while exploring the microbiome's potential in the prevention, diagnosis, prediction, and treatment of gastrointestinal infections. [ABSTRACT FROM AUTHOR]

Salmonella infection causes epidemic death in wild songbirds, with potential to spread to humans. In February 2021, public health officials in Oregon and Washington, USA, isolated a strain of Salmonella enterica serovar Typhimurium from humans and a wild songbird. Investigation by public health partners ultimately identified 30 illnesses in 12 states linked to an epidemic of Salmonella Typhimurium in songbirds. We report a multistate outbreak of human salmonellosis associated with songbirds, resulting from direct handling of sick and dead birds or indirect contact with contaminated birdfeeders. Companion animals might have contributed to the spread of Salmonella between songbirds and patients; the outbreak strain was detected in 1 ill dog, and a cat became ill after contact with a wild bird. This outbreak highlights a One Health issue where actions like regular cleaning of birdfeeders might reduce the health risk to wildlife, companion animals, and humans. [ABSTRACT FROM AUTHOR]

Simple Summary: Salmonella infection is an urgent problem in animal husbandry, which causes salmonellosis in livestock and poses threats to human health through contaminated livestock products. As an alternative to antibiotics, probiotics play an important role in maintaining animal health. However, some probiotics, such as lactic acid bacteria, have limitations in storage and hostile environments. In this context, exploring the bacterial components or metabolites of probiotics has gradually drawn more research interest, and these are termed "postbiotics". This study investigated the antibacterial effect of Lactiplantibacillus plantarum (LP) postbiotics and compared the effects to the live bacteria on intestinal health and autoimmunity in mice challenged with Salmonella. The results showed LP culture supernatant directly inhibited Salmonella growth and pathogenicity. LP postbiotics (the heat-killed bacteria and metabolites) showed similar or even superior effects to the active probiotic against Salmonella in mice. Furthermore, we found that LP postbiotics alleviated Salmonella infection via modulating bacterial pathogenicity, autophagy and inflammatory response. These results provide a theoretical basis for the protection of LP postbiotics against Salmonella and help to further explore its role in animal husbandry. Our study aimed to explore the effects of postbiotics on protecting against Salmonella infection in mice and clarify the underlying mechanisms. Eighty 5-week-old C57BL/6 mice were gavaged daily with Lactiplantibacillus plantarum (LP)-derived postbiotics (heat-killed bacteria, LPBinactive; culture supernatant, LPC) or the active bacteria (LPBactive), and gavaged with Salmonella enterica Typhimurium (ST). The Turbidimetry test and agar diffusion assay indicated that LPC directly inhibited Salmonella growth. Real-time PCR and biofilm inhibition assay showed that LPC had a strong ability in suppressing Salmonella pathogenicity by reducing virulence genes (SopE, SopB, InvA, InvF, SipB, HilA, SipA and SopD2), pili genes (FilF, SefA, LpfA, FimF), flagellum genes (FlhD, FliC, FliD) and biofilm formation. LP postbiotics were more effective than LP on attenuating ST-induced intestinal damage in mice, as indicated by increasing villus/crypt ratio and increasing the expression levels of tight junction proteins (Occludin and Claudin-1). Elisa assay showed that LP postbiotics significantly reduced ST-induced inflammation by regulating the levels of inflammatory cytokines (the increased IL-4 and IL-10 and the decreased TNF-α) in serum and ileum (p < 0.05). Furthermore, LP postbiotics inhibited the activation of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) inflammasome by decreasing the protein expression of NLRP3 and Caspase-1, and the gene expression of Caspase-1, IL-1β and IL-18. Meanwhile, both LPC and LPB observably activated autophagy under ST infection, as indicated by the up-regulated expression of LC3 and Beclin1 and the downregulated p62 level (p < 0.05). Finally, we found that LP postbiotics could trigger an AMP-activated protein kinase (AMPK) signaling pathway to induce autophagy. In summary, Lactiplantibacillus plantarum-derived postbiotics alleviated Salmonella infection via modulating bacterial pathogenicity, autophagy and NLRP3 inflammasome in mice. Our results confirmed the effectiveness of postbiotics agents in the control of Salmonella infection. [ABSTRACT FROM AUTHOR]

On account of the widespread development and propagation of antimicrobial-resistant (AMR) bacteria, essential oils (EOs) have emerged as potential alternatives to antibiotics. However, as already observed for antibiotics, recent studies have raised concerns regarding the potential emergence of resistant variants (RVs) to EOs. In this study, we assessed the emergence of RVs in Escherichia coli and Salmonella enterica Typhimurium after evolution assays under extended exposure to subinhibitory doses of two commercial EOs (AEN and COLIFIT) as well as to two antibiotics (amoxicillin and colistin). Phenotypic characterization of RVs from evolution assays with commercial EOs yielded no relevant increases in the minimum inhibitory concentration (MIC) of E. coli and did not even modify MIC values in S. Typhimurium. Conversely, RVs of E. coli and S. Typhimurium isolated from evolution assays with antibiotics showed increased resistance. Genotypic analysis demonstrated that resistance to commercial EOs was associated with enhanced protection against oxidative stress and redirection of cell energy toward efflux activity, while resistance to antibiotics was primarily linked to modifications in the cell binding sites of antibiotics. These findings suggest that AEN and COLIFIT could serve as safe alternatives to antibiotics in combating the emergence and dissemination of antimicrobial resistance within the agrifood system. [ABSTRACT FROM AUTHOR]

Simple Summary: Salmonella species and Escherichia coli infections in pigs can result in substantial economic losses and frequently necessitate antibiotic treatment. The misuse of antibiotics has led to the emergence of antibiotic resistance, and residues in the human food chain may appear. It is pivotal for the swine industry to seek feed additives that can contribute to the health of the gastrointestinal tract. Probiotics are promising candidates for this purpose. Probiotic bacteria offer various advantageous effects, including the prevention of pathogen adhesion, the promotion of heat shock proteins, the modulation of cytokine production, antioxidative qualities, and the reinforcement of barrier function. The beneficial effect of probiotics is species/strain specific; the potential benefits need to be individually assessed for each probiotic strain or species. The objective of this study was to investigate the impact of Lactobacillus rhamnosus in in vitro porcine gastrointestinal infection models. Two economically significant swine pathogens, namely Escherichia and Salmonella enterica serovar Typhimurium, were applied to model gastrointestinal infections. L. rhamnosus demonstrated numerous advantageous effects, such as its antioxidative properties, ability to inhibit pathogens, anti-inflammatory characteristics, and capacity to enhance barrier function. This study makes a substantial contribution to the understanding of the specific advantageous effects associated with L. rhamnosus (which has relevance when choosing a probiotic in practice) and serves as a basis for further in vivo research. Antimicrobial resistance is one of the biggest health challenges nowadays. Probiotics are promising candidates as feed additives contributing to the health of the gastrointestinal tract. The beneficial effect of probiotics is species/strain specific; the potential benefits need to be individually assessed for each probiotic strain or species. We established a co-culture model, in which gastrointestinal infection was modeled using Escherichia coli (E. coli) and Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium). Using intestinal porcine epithelial cells (IPEC-J2), the effects of pre-, co-, and post-treatment with Lactobacillus (L.) rhamnosus on the barrier function, intracellular (IC) reactive oxygen species (ROS) production, proinflammatory cytokine (IL-6 and IL-8) response, and adhesion inhibition were tested. E. coli- and S. Typhimurium-induced barrier impairment and increased ROS production could be counteracted using L. rhamnosus (p < 0.01). S. Typhimurium-induced IL-6 production was reduced via pre-treatment (p < 0.05) and post-treatment (p < 0.01); increased IL-8 secretion was decreased via pre-, co-, and post-treatment (p < 0.01) with L. rhamnosus. L. rhamnosus demonstrated significant inhibition of adhesion for both S. Typhimurium (p < 0.001) and E. coli (p < 0.001 in both pre-treatment and post-treatment; p < 0.05 in co-treatment). This study makes a substantial contribution to the understanding of the specific benefits of L. rhamnosus. Our findings can serve as a basis for further in vivo studies carried out in pigs and humans. [ABSTRACT FROM AUTHOR]

Salmonella enterica serotype 1,4,[5],12:i:- (Typhimurium monophasic variant) of sequence type (ST) 34 has emerged as the predominant pandemic genotype in recent decades. Despite increasing reports of resistance to antimicrobials in Southeast Asia, Salmonella ST34 population structure and evolution remained understudied in the region. Here we performed detailed genomic investigations on 454 ST34 genomes collected from Vietnam and diverse geographical sources to elucidate the pathogen's epidemiology, evolution and antimicrobial resistance. We showed that ST34 has been introduced into Vietnam in at least nine occasions since 2000, forming five co-circulating major clones responsible for paediatric diarrhoea and bloodstream infection. Most expansion events were associated with acquisitions of large multidrug resistance plasmids of IncHI2 or IncA/C2. Particularly, the self-conjugative IncA/C2 pST34VN2 (co-transferring blaCTX-M-55, mcr-3.1, and qnrS1) underlies local expansion and intercontinental spread in two separate ST34 clones. At the global scale, Southeast Asia was identified as a potential hub for the emergence and dissemination of multidrug resistant Salmonella ST34, and mutation analysis suggests of selection in antimicrobial responses and key virulence factors. A genomic epidemiology approach describes the population structure of Salmonella monophasic Typhimurium ST34 from Vietnam, and identifies Southeast Asia as a potential hub for the emergence and international spread of multidrug resistant Salm. ST34. [ABSTRACT FROM AUTHOR]

The transcriptional regulator SsrB acts as a switch between virulent and biofilm lifestyles of non-typhoidal Salmonella enterica serovar Typhimurium. During infection, phosphorylated SsrB activates genes on Salmonella Pathogenicity Island-2 (SPI-2) essential for survival and replication within the macrophage. Low pH inside the vacuole is a key inducer of expression and SsrB activation. Previous studies demonstrated an increase in SsrB protein levels and DNA-binding affinity at low pH; the molecular basis was unknown (Liew et al., 2019). This study elucidates its underlying mechanism and in vivo significance. Employing single-molecule and transcriptional assays, we report that the SsrB DNA-binding domain alone (SsrBc) is insufficient to induce acid pH-sensitivity. Instead, His12, a conserved residue in the receiver domain confers pH sensitivity to SsrB allosterically. Aciddependent DNA binding was highly cooperative, suggesting a new configuration of SsrB oligomers at SPI-2-dependent promoters. His12 also plays a role in SsrB phosphorylation; substituting His12 reduced phosphorylation at neutral pH and abolished pH-dependent differences. Failure to flip the switch in SsrB renders Salmonella avirulent and represents a potential means of controlling virulence. [ABSTRACT FROM AUTHOR]

In many Gram-negative bacteria, the stress sigma factor of RNA polymerase, σS/RpoS, remodels global gene expression to reshape the physiology of stationary phase cells and ensure their survival under non-optimal growth conditions. In the foodborne pathogen Salmonella enterica serovar Typhimurium, σS is also required for biofilm formation and virulence. We have recently shown that a ΔrpoS mutation decreases the magnesium content and expression level of the housekeeping Mg2+-transporter CorA in stationary phase Salmonella. The other two Mg2+-transporters of Salmonella are encoded by the PhoP-activated mgtA and mgtB genes and are expressed under magnesium starvation. The σS control of corA prompted us to evaluate the impact of CorA in stationary phase Salmonella cells, by using global and analytical proteomic analyses and physiological assays. The ΔcorA mutation conferred a competitive disadvantage to exit from stationary phase, and slightly impaired motility, but had no effect on total and free cellular magnesium contents. In contrast to the wild-type strain, the ΔcorA mutant produced MgtA, but not MgtB, in the presence of high extracellular magnesium concentration. Under these conditions, MgtA production in the ΔcorA mutant did not require PhoP. Consistently, a ΔmgtA, but not a ΔphoP, mutation slightly reduced the magnesium content of the ΔcorA mutant. Synthetic phenotypes were observed when the ΔphoP and ΔcorA mutations were combined, including a strong reduction in growth and motility, independently of the extracellular magnesium concentration. The abundance of several proteins involved in flagella formation, chemotaxis and secretion was lowered by the ΔcorA and ΔphoP mutations in combination, but not alone. These findings unravel the importance of PhoP-dependent functions in the absence of CorA when magnesium is sufficient. Altogether, our data pinpoint a regulatory network, where the absence of CorA is sensed by the cell and compensated by MgtA and PhoP- dependent mechanisms. [ABSTRACT FROM AUTHOR]

A series of novel 5‐amido‐2‐carboxypyrazine derivatives were designed, synthesized and evaluated for the inhibitory activities against the T3SS of Salmonella enterica serovar Typhimurium. Preliminary results displayed that the compounds 2f, 2g, 2h and 2i showed potent inhibitory activities against T3SS. Compound 2h was identified as the most potent T3SS inhibitor and the SPI‐1 effector secretion was strongly inhibited by 2h in a dose‐dependent manner. The effects of compound 2h on the SPI‐1 genes transcription might be via impacting the SicA/InvF regulatory pathway. [ABSTRACT FROM AUTHOR]

Background: Bacteria in nature live together in communities called biofilms, where they produce a matrix that protects them from hostile environments. The components of this matrix vary among species, with Salmonella enterica serovar Typhimurium (STm- WT) primarily producing curli and cellulose, which are regulated by the master regulator csgD. Interactions between bacteria can be competitive or cooperative, with cooperation more commonly observed among the kin population. This study refers to STm- WT as the generalist which produces all the matrix components and knockout strains that are defective in either curli or cellulose as the specialists, which produces one of the matrix components but not both. We have asked whether two different specialists will cooperate and share matrix components during biofilm formation to match the ability of the generalist which produces both components. Results: In this study, the response of the specialists and generalist to physical, chemical, and biological stress during biofilm formation is also studied to assess their abilities to cooperate and produce biofilms like the generalist. STm WT colony biofilm which produces both the major biofilm matrix component were protected from stress whereas the non-matrix producer (∆csgD), the cellulose, and curli alone producers ∆csgA, ∆bcsA respectively were affected. During the exposure to various stresses, the majority of killing occurred in ∆csgD. Whereas the co-culture (∆csgA: ∆bcsA) was able to resist stress like that of the STm WT. Phenotypic and morphological characteristics of the colonies were typed using congo red assay and the Influence of matrix on the architecture of biofilms was confirmed by scanning electron microscopy. Conclusion: Our results show that matrix aids in survival during antibiotic, chlorine, and predatory stress. And possible sharing of the matrix is occurring in co-culture, with one counterbalancing the inability of the other when confronted with stress. [ABSTRACT FROM AUTHOR]

Background: Bacterial surface proteins play key roles in pathogenicity and often contribute to microbial adhesion and invasion. Pasteurella lipoprotein E (PlpE), a Pasteurella multocida (P. multocida) surface protein, has recently been identified as a potential vaccine candidate. Live attenuated Salmonella strains have a number of potential advantages as vaccine vectors, including immunization with live vector can mimic natural infections by organisms, lead to the induction of mucosal, humoral, and cellular immune responses. In this study, a previously constructed recombinant attenuated Salmonella Choleraesuis (S. Choleraesuis) vector rSC0016 was used to synthesize and secrete the surface protein PlpE of P. multocida to form the vaccine candidate rSC0016(pS-PlpE). Subsequently, the immunogenicity of S. Choleraesuis rSC0016(pS-PlpE) as an oral vaccine to induce protective immunity against P. multocida in mice was evaluated. Results: After immunization, the recombinant attenuated S. Choleraesuis vector can efficiently delivered P. multocida PlpE protein in vivo and induced a specific immune response against this heterologous antigen in mice. In addition, compared with the inactivated vaccine, empty vector (rSC0016(pYA3493)) and PBS immunized groups, the rSC0016(pS-PlpE) vaccine candidate group induced higher antigen-specific mucosal, humoral and mixed Th1/Th2 cellular immune responses. After intraperitoneal challenge, the rSC0016(pS-PlpE) immunized group had a markedly enhanced survival rate (80%), a better protection efficiency than 60% of the inactivated vaccine group, and significantly reduced tissue damage. Conclusions: In conclusion, our study found that the rSC0016(pS-PlpE) vaccine candidate provided good protection against challenge with wild-type P. multocida serotype A in a mouse infection model, and may potentially be considered for use as a universal vaccine against multiple serotypes of P. multocida in livestock, including pigs. [ABSTRACT FROM AUTHOR]

Gut microbial communities protect the host against a variety of major human gastrointestinal pathogens. Bacteriophages (phages) are ubiquitous in nature and frequently ingested via food and drinking water. Moreover, they are an attractive tool for microbiome engineering due to the lack of known serious adverse effects on the host. However, the functional role of phages within the gastrointestinal microbiome remain poorly understood. Here, we investigated the effects of microbiota-directed phages on infection with the human enteric pathogen Salmonella enterica serovar Typhimurium (S. Tm), using a gnotobiotic mouse model (OMM14) for colonization resistance (CR). We show, that phage cocktails targeting Escherichia coli and Enterococcus faecalis acted in a strain-specific manner. They transiently reduced the population density of their respective target before establishing coexistence for up to 9 days. Infection susceptibility to S. Tm was markedly increased at an early time point after challenge with both phage cocktails. Surprisingly, OMM14 mice were also susceptible 7 days after a single phage inoculation, when the targeted bacterial populations were back to pre-phage administration density. Concluding, our work shows that phages that dynamically modulate the density of protective members of the gut microbiota can provide opportunities for invasion of bacterial pathogens, in particular at early time points after phage application. This suggests, that phages targeting protective members of the microbiota may increase the risk for Salmonella infection. Author summary: Metagenomic studies revealed that bacteria and their specific viruses, bacteriophages (phages), are the most abundant and diverse community of microbes in the human intestines. However, functional studies for identifying the role of phages in shaping intestinal bacterial communities are still lacking. A fundamental function of the intestinal microbiota is colonization resistance (CR) to the invasion of intestinal pathogens such as Salmonella. Here, we use a simplified gnotobiotic mouse model and two phage cocktails to investigate the impact of phages in a bacterial community, on their target strains and on CR. We found, that oral application of phages leads to a decrease in CR against Salmonella. Thus, ingestion of phages might be a risk factor predisposing to bacterial infections and could explain interpersonal variability of infection susceptibility. We report a previously unanticipated impact of phages related to the impairment of CR against gastrointestinal infections. [ABSTRACT FROM AUTHOR]

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. Author summary: As causative agents of infectious diseases, intracellular bacterial pathogens have evolved diverse immune escape and survival strategies while thriving inside their hosts. Whereas the residence of the pathogen inside the host can impact the functioning of the latter drastically, the bacterial macromolecular content is typically vastly outstripped by that of the host. This sizeable difference in macromolecular content has thus far hindered proteome profiling of bacteria and its infected host in an integrated and unbiased manner. Here, the power of dual proteome profiling–a universal mass spectrometry-based proteomic strategy for unravelling the intricate interplay between pathogen and host—is presented for the exploration of host as well as pathogen proteomic responses over the time course of the infection, thereby advancing our understanding of infectious diseases. [ABSTRACT FROM AUTHOR]