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Catalytic DNA circuits, serving as signal amplification strategies, can enable simple and accurate detection of pathogenic bacteria in complex matrices but suffer from low reaction rates and depths. Herein, we design an enzyme-accelerated catalytic hairpin assembly (EACHA) in which duplex DNA products are converted into hairpin reactants to continue participating in the next circuit reaction with the assistance of RNase H. Profiting from the high recyclability of the reactants, EACHA exhibits an approximately 37.6-fold enhancement in the rate constant and a two-order-of-magnitude improvement in sensitivity compared to conventional catalytic hairpin assembly (CHA). By integrating an allosteric probe with EACHA, a one-pot method is developed for rapid and direct detection of S. enterica Enteritidis (S. Enteritidis). This method is capable of detecting 15 CFU mL -1 of S. Enteritidis within 20 min, which is superior to that of real-time PCR. By testing 60 milk samples, we demonstrate this method's high accuracy in discriminating contaminated samples, with an area under the curve (AUC) of 0.997. Moreover, this method can be employed to accurately diagnose early-stage infected mice, with an AUC of 1.00 for feces samples and 0.986 for serum samples. Therefore, this study offers a simple and feasible method for identifying pathogens in complex matrices., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Invasive non-typhoidal Salmonella (iNTS) disease is an under-recognized high-burden disease causing major health and socioeconomic issues in sub-Saharan Africa (sSA), predominantly among immune-naïve infants and young children, including those with recognized comorbidities such as HIV infection. iNTS disease is primarily caused by Salmonella enterica serovar Typhimurium sequence type (ST) 313 and 'African-restricted clades' of Salmonella Enteritidis ST11 that have emerged across the African continent as a series of epidemics associated with acquisition of new antimicrobial resistance. Due to genotypes with a high prevalence of antimicrobial resistance and scarcity of therapeutic options, these NTS serovars are designated by the World Health Organization as a priority pathogen for research and development of interventions, including vaccines, to address and reduce NTS associated bacteremia and meningitis in sSA. Novel and traditional vaccine technologies are being applied to develop vaccines against iNTS disease, and the results of the first clinical trials in the infant target population should become available in the near future. The "Vaccine Value Profile" (VVP) addresses information related predominantly to invasive disease caused by Salmonella Enteritidis and Salmonella Typhimurium prevalent in sSA. Information is included on stand-alone iNTS disease candidate vaccines and candidate vaccines targeting iNTS disease combined with another invasive serotype, Salmonella Typhi, that is also common across sSA. Out of scope for the first version of this VVP is a wider discussion on either diarrheagenic NTS disease (dNTS) also associated with Salmonella Enteritidis and Salmonella Typhimurium or the development of a multivalent Salmonella vaccines targeting key serovars for use globally. This VVP for vaccines to prevent iNTS disease is intended to provide a high-level, holistic assessment of the information and data that are currently available to inform the potential public health, economic, and societal value of pipeline vaccines and vaccine-like products. Future versions of this VVP will be updated to reflect ongoing activities such as vaccine development strategies and a "Full Vaccine Value Assessment" that will inform the value proposition of an iNTS disease vaccine. This VVP was developed by a working group of subject matter experts from academia, non-profit organizations, public private partnerships, and multi-lateral organizations, and in collaboration with stakeholders from the World Health Organization African Region. All contributors have extensive expertise on various elements of the iNTS disease VVP and collectively aimed to identify current research and knowledge gaps. The VVP was developed using only existing and publicly available information., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: LBM and MJS report financial support and administrative support by the World Health Organization. LBM was an independent consultant during VVP development; she is currently employed by the US Pharmacopeial Convention. CSM and JAC received support through the received funding from the EU Horizon 2020 research and innovation programme under the project Vacc-iNTS (grant agreement number 815439).VM was employed by the International Vaccines Institute during VVP development; he is currently employed by the World Health Organization. All other authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

The injectisome encoded by Salmonella pathogenicity island 2 (SPI-2) had been thought to translocate 28 effectors. Here, we used a proteomic approach to characterize the secretome of a clinical strain of invasive non-typhoidal Salmonella enterica serovar Enteritidis that had been mutated to cause hyper-secretion of the SPI-2 injectisome effectors. Along with many known effectors, we discovered the novel SseM protein. sseM is widely distributed among the five subspecies of Salmonella enterica, is found in many clinically relevant serovars, and is co-transcribed with pipB2 , a SPI-2 effector gene. The translocation of SseM required a functional SPI-2 injectisome. Following expression in human cells, SseM interacted with five components of the dystrophin-associated protein complex (DAPC), namely, β-2-syntrophin, utrophin/dystrophin, α-catulin, α-dystrobrevin, and β-dystrobrevin. The interaction between SseM and β-2-syntrophin and α-dystrobrevin was verified in Salmonella Typhimurium-infected cells and relied on the postsynaptic density-95/discs large/zonula occludens-1 (PDZ) domain of β-2-syntrophin and a sequence corresponding to a PDZ-binding motif (PBM) in SseM. A Δ sseM mutant strain had a small competitive advantage over the wild-type strain in the S . Typhimurium/mouse model of systemic disease. This phenotype was complemented by a plasmid expressing wild-type SseM from S . Typhimurium or S . Enteritidis and was dependent on the PBM of SseM. Therefore, a PBM within a Salmonella effector mediates interactions with the DAPC and modulates the systemic growth of bacteria in mice. Furthermore, the Δ sseM mutant strain displayed enhanced replication in bone marrow-derived macrophages, demonstrating that SseM restrains intracellular bacterial growth to modulate Salmonella virulence., Importance: In Salmonella enterica , the injectisome machinery encoded by Salmonella pathogenicity island 2 (SPI-2) is conserved among the five subspecies and delivers proteins (effectors) into host cells, which are required for Salmonella virulence. The identification and functional characterization of SPI-2 injectisome effectors advance our understanding of the interplay between Salmonella and its host(s). Using an optimized method for preparing secreted proteins and a clinical isolate of the invasive non-typhoidal Salmonella enterica serovar Enteritidis strain D24359, we identified 22 known SPI-2 injectisome effectors and one new effector-SseM. SseM modulates bacterial growth during murine infection and has a sequence corresponding to a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif that is essential for interaction with the PDZ-containing host protein β-2-syntrophin and other components of the dystrophin-associated protein complex (DAPC). To our knowledge, SseM is unique among Salmonella effectors in containing a functional PDZ-binding motif and is the first bacterial protein to target the DAPC., Competing Interests: The authors declare no conflict of interest.

Bacterial resistance to serum is a key virulence factor for the development of systemic infections. The amount of lipopolysaccharide (LPS) and the O-antigen chain length distribution on the outer membrane, predispose Salmonella to escape complement-mediated killing. In Salmonella enterica serovar Enteritidis (S. Enteritidis) a modal distribution of the LPS O-antigen length can be observed. It is characterized by the presence of distinct fractions: low molecular weight LPS, long LPS and very long LPS. In the present work, we investigated the effect of the O-antigen modal length composition of LPS molecules on the surface of S. Enteritidis cells on its ability to evade host complement responses. Therefore, we examined systematically, by using specific deletion mutants, roles of different O-antigen fractions in complement evasion. We developed a method to analyze the average LPS lengths and investigated the interaction of the bacteria and isolated LPS molecules with complement components. Additionally, we assessed the aspect of LPS O-antigen chain length distribution in S. Enteritidis virulence in vivo in the Galleria mellonella infection model. The obtained results of the measurements of the average LPS length confirmed that the method is suitable for measuring the average LPS length in bacterial cells as well as isolated LPS molecules and allows the comparison between strains. In contrast to earlier studies we have used much more precise methodology to assess the LPS molecules average length and modal distribution, also conducted more subtle analysis of complement system activation by lipopolysaccharides of various molecular mass. Data obtained in the complement activation assays clearly demonstrated that S. Enteritidis bacteria require LPS with long O-antigen to resist the complement system and to survive in the G. mellonella infection model., (© 2024. The Author(s).)

Genomic islands (GIs) are horizontally transferred elements that shape bacterial genomes and contributes to the adaptation to different environments. Some GIs encode an integrase and a recombination directionality factor (RDF), which are the molecular GI-encoded machinery that promotes the island excision from the chromosome, the first step for the spread of GIs by horizontal transfer. Although less studied, this process can also play a role in the virulence of bacterial pathogens. While the excision of GIs is thought to be similar to that observed in bacteriophages, this mechanism has been only studied in a few families of islands. Here, we aimed to gain a better understanding of the factors involved in the excision of ROD21 a pathogenicity island of the food-borne pathogen Salmonella enterica serovar Enteritidis and the most studied member of the recently described Enterobacteriaceae-associated ROD21-like family of GIs. Using bioinformatic and experimental approaches, we characterized the conserved gene SEN1998, showing that it encodes a protein with the features of an RDF that binds to the regulatory regions involved in the excision of ROD21. While deletion or overexpression of SEN1998 did not alter the expression of the integrase-encoding gene SEN1970, a slight but significant trend was observed in the excision of the island. Surprisingly, we found that the expression of both genes, SEN1998 and SEN1970, were negatively correlated to the excision of ROD21 which showed a growth phase-dependent pattern. Our findings contribute to the growing body of knowledge regarding the excision of GIs, providing insights about ROD21 and the recently described EARL family of genomic islands., (© 2022. The Author(s).)

Salmonella enterica is a major food borne pathogen that creates biofilm. Salmonella biofilm formation under different environmental conditions is a public health problem. The present study was aimed to evaluate the combined effects of stressful factors (temperature and pH) on the expression of biofilm, stress, and virulence genes in Salmonella Enteritidis and Salmonella Typhimurium. In this study, the effect of temperature (2, 8, 22.5, 37, 43 °C) and pH (2.4, 3, 4.5, 6, 6.6) on the expression of biofilm production genes (adr A, bap A), virulence genes (hil A, inv A) and the stress gene (RpoS) of S. Enteritidis and S. Typhimurium was evaluated. The response surface methodology (RSM) approach was used to evaluate the combined effect of the above factors. The highest expression of adr A, bap A, hil A, and RpoS gene for S. Typhimurium was at 22 °C-pH 4.5 (6.39-fold increase), 37 °C-pH 6 (3.92-fold increase), 37 °C-pH 6 (183-fold increase), and 37 °C-pH 3 (43.8-fold increase), respectively. The inv A gene of S. Typhimurium was decreased in all conditions. The adr A, bap A, hil A, inv A, and RpoS gene of S. Enteritidis had the highest expression level at 8 °C-pH 3 (4.09-fold increase), 22 °C-pH 6 (2.71-fold increase), 8 °C pH 3 (190-fold increase), 22 °C-pH 4.5 (9.21-fold increase), and 8 °C-pH 3 (16.6-fold), respectively. Response surface methodology (RSM) indicated that the temperature and pH had no significant effect on the expression level of adr A, bap A, hil A, Inv A, and RpoS gene in S. Enteritidis and S. Typhimurium. The expression of biofilm production genes (adr A, bap A), virulence genes (hil A, inv A) and the stress gene (RpoS) of S. Enteritidis and S. Typhimurium is not directly and exclusively associated with temperature and pH conditions., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the most common serovars, associated with human salmonellosis. The food-borne outbreak of this bacterium is mainly related to the consumption of contaminated poultry meat and poultry products, including eggs. Therefore, rapid and accurate detection, besides investigation of virulence characteristics and antimicrobial resistance profiles of S. Enteritidis in poultry and poultry egg samples is essential. A total of 3125 samples (2250 poultry and 875 poultry egg samples), sent to the administrative centers of veterinary microbiology laboratories in six provinces of Iran, were examined for Salmonella contamination, according to the ISO 6579 guideline. Next, duplex PCR was conducted on 250 presumptive Salmonella isolates to detect invA gene for identification of the genus Salmonella and sdf gene for identification of S. Enteritidis. Subsequently, the S. Enteritidis isolates were examined for detection of important virulence genes (pagC, cdtB, msgA, spaN, tolC, lpfC, and spvC) and determination of antibiotic resistance patterns against nalidixic acid, trimethoprim-sulfamethoxazole, cephalothin, ceftazidime, colistin sulfate, and kanamycin by the disk diffusion method., Results: Overall, 8.7 and 2.3% of poultry samples and 6.3 and 1.3% of eggs were contaminated with Salmonella species and S. Enteritidis, respectively. The invA and msgA genes (100%) and cdtB gene (6.3%) had the highest and the lowest prevalence rates in S. Enteritidis isolates. The spvC gene, which is mainly located on the Salmonella virulence plasmid, was detected in 50.8% of S. Enteritidis isolates. The S. Enteritidis isolates showed the highest and the lowest resistance to nalidixic acid (87.3%) and ceftazidime (11.1%), respectively. Unfortunately, 27.0% of S. Enteritidis isolates were multidrug-resistant (MDR)., Conclusion: The rate of contamination with Salmonella in the poultry and egg samples, besides the presence of antimicrobial resistant and MDR Salmonella isolates harboring the virulence genes in these samples, could significantly affect food safety and subsequently, human health. Therefore, continuous monitoring of animal-source foods, enhancement of poultry farm control measures, and limiting the use of antibiotics for prophylactic purposes in food producing animals, are essential for reducing the zoonotic risk of this foodborne pathogen for consumers and also choosing effective antibiotics for the treatment of salmonellosis.

Salmonella Enteritidis is an intracellular foodborne pathogen that has developed multiple mechanisms to alter poultry intestinal physiology and infect the gut. Short chain fatty acid butyrate is derived from microbiota metabolic activities, and it maintains gut homeostasis. There is limited understanding on the interaction between S. Enteritidis infection, butyrate, and host intestinal response. To fill this knowledge gap, chicken macrophages (also known as HTC cells) were infected with S. Enteritidis, treated with sodium butyrate, and proteomic analysis was performed. A growth curve assay was conducted to determine sub-inhibitory concentration (SIC, concentration that do not affect bacterial growth compared to control) of sodium butyrate against S. Enteritidis. HTC cells were infected with S. Enteritidis in the presence and absence of SIC of sodium butyrate. The proteins were extracted and analyzed by tandem mass spectrometry. Our results showed that the SIC was 45 mM. Notably, S. Enteritidis-infected HTC cells upregulated macrophage proteins involved in ATP synthesis through oxidative phosphorylation such as ATP synthase subunit alpha (ATP5A1), ATP synthase subunit d, mitochondrial (ATP5PD) and cellular apoptosis such as Cytochrome-c (CYC). Furthermore, sodium butyrate influenced S. Enteritidis-infected HTC cells by reducing the expression of macrophage proteins mediating actin cytoskeletal rearrangements such as WD repeat-containing protein-1 (WDR1), Alpha actinin-1 (ACTN1), Vinculin (VCL) and Protein disulfide isomerase (P4HB) and intracellular S. Enteritidis growth and replication such as V-type proton ATPase catalytic subunit A (ATPV1A). Interestingly, sodium butyrate increased the expression of infected HTC cell protein involving in bacterial killing such as Vimentin (VIM). In conclusion, sodium butyrate modulates the expression of HTC cell proteins essential for S. Enteritidis invasion., Competing Interests: The authors have declared that no competing interests exist.

Small non-coding RNA RyhB is a key regulator of iron homeostasis in bacteria by sensing iron availability in the environment. Although RyhB is known to influence bacterial virulence by interacting with iron metabolism related regulators, its interaction with virulence genes, especially the Type III secretion system (T3SS), has not been reported. Here, we demonstrate that two RyhB paralogs of Salmonella enterica serovar Enteritidis upregulate Type III secretion system (T3SS) effectors, and consequently affect Salmonella invasion into intestinal epithelial cells. Specifically, we found that RyhB-1 modulate Salmonella response to stress condition of iron deficiency and hypoxia, and stress in simulated intestinal environment (SIE). Under SIE culture conditions, both RyhB-1 and RyhB-2 are drastically induced and directly upregulate the expression of T3SS effector gene sipA by interacting with its 5' untranslated region (5' UTR) via an incomplete base-pairing mechanism. In addition, the RyhB paralogs upregulate the expression of T3SS effector gene sopE. By regulating the invasion-related genes, RyhBs in turn affect the ability of S. Enteritidis to adhere to and invade into intestinal epithelial cells. Our findings provide evidence that RyhBs function as critical virulence factors by directly regulating virulence-related gene expression. Thus, inhibition of RyhBs may be a potential strategy to attenuate Salmonella.

The Enteritidis and Dublin serovars of Salmonella enterica are phylogenetically closely related yet differ significantly in host range and virulence. S Enteritidis is a broad-host-range serovar that commonly causes self-limited gastroenteritis in humans, whereas S Dublin is a cattle-adapted serovar that can infect humans, often resulting in invasive extraintestinal disease. The mechanism underlying the higher invasiveness of S Dublin remains undetermined. In this work, we quantitatively compared the proteomes of clinical isolates of each serovar grown under gut-mimicking conditions. Compared to S Enteritidis, the S Dublin proteome was enriched in proteins linked to response to several stress conditions, such as those encountered during host infection, as well as to virulence. The S Enteritidis proteome contained several proteins related to central anaerobic metabolism pathways that were undetected in S Dublin. In contrast to what has been observed in other extraintestinal serovars, most of the coding genes for these pathways are not degraded in S Dublin. Thus, we provide evidence that S Dublin metabolic functions may be much more affected than previously reported based on genomic studies. Single and double null mutants in stress response proteins Dps, YciF, and YgaU demonstrate their relevance to S Dublin invasiveness in a murine model of invasive salmonellosis. All in all, this work provides a basis for understanding interserovar differences in invasiveness and niche adaptation, underscoring the relevance of using proteomic approaches to complement genomic studies., (Copyright © 2021 American Society for Microbiology.)

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336ΔmicC by the λ Red-based recombination system and the complemented mutant 50336ΔmicC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336ΔmicC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336ΔmicC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336ΔmicC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336ΔmicC and 50336ΔmicC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 10 9 CFU, 1.55 x 10 8 CFU, and 2.54 x 10 8 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

Salmonella Enteritidis, an important foodborne zoonosis, has a dramatically increased number of cases around the world. To explore the phylogenetic structure of Peruvian Salmonella Enteritidis strains and their relationship with an outbreak occurred in 2018, we analyzed a comprehensive strains of S. Enteritidis received by the National Institute of Health during the period 2000-2018. A total of 180 strains were characterized by microbiological procedures, serotyping and whole genome sequencing. Based on genome sequences annotated, virulence factors and accessory genes were identified. Phylogenetic and population structure analysis were also analyzed based on SNPs. The phylogenetic analysis grouped the genomes into two well-supported clades that were consistent with population structure analysis. The clinical and food strains corresponding to the outbreak were included in the same cluster, which presented the sdhA gene, related to the increase of the virulence of this pathogen. The phylogenetic relationship of Peruvian S. Enteritidis suggests the presence of four S. enteritidis population with high epidemiological importance.

An essential feature of the pathogenesis of the Salmonella enterica serovar Enteritidis wild type (WT) is its ability to survive under diverse microenvironmental stress conditions, such as encountering antimicrobial peptides (AMPs) or glucose and micronutrient starvation. These stress factors trigger virulence genes carried on Salmonella pathogenicity islands (SPIs) and determine the efficiency of enteric infection. Although the oligosaccharide/oligonucleotide binding-fold (OB-fold) family of proteins has been identified as an important stress response and virulence determinant, functional information on members of this family is currently limited. In this study, we decipher the role of YdeI, which belongs to OB-fold family of proteins, in stress response and virulence of S Enteritidis. When ydeI was deleted, the Δ ydeI mutant showed reduced survival during exposure to AMPs or glucose and Mg 2+ starvation stress compared to the WT. Green fluorescent protein (GFP) reporter and quantitative real-time PCR (qRT-PCR) assays showed ydeI was transcriptionally regulated by PhoP, which is a major regulator of stress and virulence. Furthermore, the Δ ydeI mutant displayed ∼89% reduced invasion into HCT116 cells, ∼15-fold-reduced intramacrophage survival, and downregulation of several SPI-1 and SPI-2 genes encoding the type 3 secretion system apparatus and effector proteins. The mutant showed attenuated virulence compared to the WT, confirmed by its reduced bacterial counts in feces, mesenteric lymph node (mLN), spleen, and liver of C57BL/6 mice. qRT-PCR analyses of the Δ ydeI mutant displayed differential expression of 45 PhoP-regulated genes, which were majorly involved in metabolism, transport, membrane remodeling, and drug resistance under different stress conditions. YdeI is, therefore, an important protein that modulates S Enteritidis virulence and adaptation to stress during infection. IMPORTANCE S Enteritidis during its life cycle encounters diverse stress factors inside the host. These intracellular conditions allow activation of specialized secretion systems to cause infection. We report a conserved membrane protein, YdeI, and elucidate its role in protection against various intracellular stress conditions. A key aspect of the study of a pathogen's stress response mechanism is its clinical relevance during host-pathogen interaction. Bacterial adaptation to stress plays a vital role in evolution of a pathogen's resistance to therapeutic agents. Therefore, investigation of the role of YdeI is vital for understanding the molecular basis of regulation of Salmonella pathogenesis. In conclusion, our findings may contribute to finding potential targets to develop new intervention strategies for treatment and prevention of enteric diseases., (Copyright © 2020 American Society for Microbiology.)

Background: Salmonella is an important foodborne pathogen that causes acute diarrhea in humans worldwide. This study analyzed the relationships of serotypes and antibiotic resistance with virulence genes of Salmonella isolated from children with salmonellosis., Methods: Serological typing was performed using the slide-agglutination method. The Kirby-Bauer disk diffusion method was used to test antibiotic susceptibility. Twenty virulence genes were detected by PCR., Results: Salmonella Typhimurium (21 isolates, 34.43%) and S Enteritidis (12 isolates, 19.67%) were the predominant species among the 61 isolates. Ampicillin resistance was most common (63.93%), and among the cephalosporins, resistance was most often found to cefotaxime, a third-generation cephalosporin (19.67%). Among the 20 virulence genes, prgH, ssrB, and pagC were detected in all Salmonella isolates. In S Typhimurium, the detection rates of hilA, sipB, marT, mgtC, sopB, pagN, nlpI, bapA, oafA, and tolC were high. In S Enteritidis, the detection rates of icmF, spvB, spvR, and pefA were high. Nitrofurantoin resistance was negatively correlated with the virulence gene bapA (P = .005) and was positively correlated with icmF, spvB, spvR, and pefA (P = .012, .008, .002, and .005, respectively), The P values between all other virulence genes and antibiotic resistance were >.05., Conclusion: Salmonella Typhimurium and S Enteritidis were the main serotypes in children with diarrhea in Hangzhou, China. Salmonella exhibited a high level of resistance to common antibiotics, and a high rate of bacteria carrying virulence genes was observed. However, no significant correlation was found between virulence genes and resistance to common antibiotics., (© 2020 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Salmonella , like other pathogenic bacteria has undergone multiple genomic alterations to adapt itself into specific host environments executing varied degrees of virulence through evolution. Such variations in genome content have been assumed to lead the closely related non-typhoidal serovars, S . Enteritidis, and S . Typhimurium to exhibit Type Three Secretion System -2 (T3SS-2) based diverse colonization and inflammation kinetics. Mutually exclusive genes present in either of the serovars are recently being studied and in our currentwork, we focused on a particular island ROD9, present in S . Enteritidis but not in S . Typhimurium. Earlier reports have identified a few genes from this island to be responsible for virulence in vitro as well as in vivo . In this study, we have identified another gene, SEN1008 from the same island encoding a hypothetical protein to be a potential virulence determinant showing systemic attenuation upon mutation in C57BL/6 mice infection model. The isogenic mutant strain displayed reduced adhesion to epithelial cells in vitro as well as was highly immotile. It was also deficient in intracellular replication in vitro , with a highly suppressed SPI-2and failed to cause acute colitis at 72-h p.i. in vivo . Moreover, on acid exposure, SEN1008 showed 17 folds and 2 fold up-regulations during adaptation and challenge phases,respectively and Δ SEN1008 failed to survive during ATR assay, indicating its role under acid stress. Together, our findings suggested Δ SEN1008 to be significantly attenuated and we propose this gene to be a potent factor responsible for S . Enteritidis pathogenesis.

Salmonella enterica serovar Enteritidis ( S . Enteritidis; wild type (WT)) is a major cause of foodborne illness globally. The ability of this pathogen to survive stress inside and outside the host, such as encountering antimicrobial peptides and heat stress, determines the efficiency of enteric infection. These stressors concertedly trigger virulence factors encoded on Salmonella pathogenicity islands (SPIs). Although RpoS is a well-known central transcriptional stress and virulence regulator, functional information regarding the genes of the regulon is currently limited. Here, we identified SEN1538 as a conserved RpoS-regulated gene belonging to the KGG protein superfamily. We further assessed its role in pathogenic stress responses and virulence. When SEN1538 was deleted (Δ1538), the pathogen showed reduced survival during antimicrobial peptide introduction and heat stress at 55°C compared to WT. The mutant displayed 70% reduced invasion in the HCT116 colon epithelial cell line, 5-fold attenuated phagocytic survival in RAW264.7 cells, and downregulation of several SPI-1 and SPI-2 genes encoding the three secretion system apparatus and effector proteins. Δ1538 also showed decreased virulence compared to WT, demonstrated by its reduced bacterial counts in the feces, mLN, spleen, and cecum of C57BL/6 mice. Comparative transcriptomic analysis of Δ1538 against WT revealed 111 differentially regulated genes, 103 of which were downregulated (fold change ≤ -1.5, P < 0.05). The majority of these genes were in clusters for metabolism, transporters, and pathogenesis, driving pathogenic stress responses and virulence. SEN1538 is, therefore, an important virulence determinant contributing to the resilience of S . Enteritidis to stress factors during infection.

Salmonella enteritidis can cause significant morbidity and mortality in humans and economic loss in the animal industry. Improving the innate immunity is an effective method to prevent S. enteritidis infection. Pediococcus pentosaceus is a Gram-positive coccus which had probiotics properties. Numerous previously published studies reported that probiotics were beneficial to gut microbiota by changing the intestinal flora structure and inhibiting the harmful microbial growth to enhance the innate immunity. We investigated the immunological effects of P. pentosaceus on Salmonella-infected chickens by the following experiment. A total of 120 broilers from AA line were fed and divided into 2 groups (treated and control groups) for the experiment from day 1. The control group was fed with the basic diet, while the treated group was fed with the basic diet adding P. pentosaceus microcapsule with the bacterial concentration of 1 g/kg in the feed and bacterial counts 2.5 × 10 9  CFU/g. All the birds were given with 0.5 ml of S. enteritidis bacterial suspension (10 9  CFU/ml) through oral cavity at day 9. The number of dead birds was recorded and used in the analysis. The bacterial culture method and quantitative real-time PCR analysis were used to evaluate the effects of P. pentosaceus on chickens infected with S. enteritidis and to ascertain the mechanism of the effect. The results showed that the P. pentosaceus could restrain the pathogenicity of S. enteritidis and reduce the death rate from 44.4% to 23.3%. The flora in the caecum exhibited "rising-declining" trends, and the gene ( TLR4 , MyD88 , TRAF6 NF-κB , IFN-β , TNF-a , IL6 , and IL8 ) expression pattern was different between the experimental and control group. P. pentosaceus as a probiotic may competitively inhibit the growth of S. enteritidis and control the inflammatory response through regulating the gene expression which involved in the toll-like receptor pathway and inflammation pathway., Competing Interests: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the manuscript., (Copyright © 2020 Dan Lan et al.)

Early references of emphysematous osteomyelitis (EO) in the literature trace back to 1981, when the presence of intraosseous gas was mentioned in a paper of Ram et al.. Subsequently, 48 cases of EO were described. A significant underlying comorbidity was reported in most EO cases, especially diabetes and malignancy. This report presents a 37-year-old male with undiagnosed diabetes mellitus which was accompanied by emphysematous osteomyelitis of both femoral heads.

Avian salmonellosis caused by Salmonella enterica serovar Enteritidis ( S . Enteritidis) and Pullorum ( S. Pullorum) remains a big threat to the poultry industry and public hygiene. AvrA is an effector involved in inhibiting inflammation. Compared to AvrA from S . Enteritidis (SE-AvrA), the AvrA from S. Pullorum (SP-AvrA) lacks ten amino acids at the C-terminal. In this study, we compared the anti-inflammatory response induced by SP-AvrA to that of SE-AvrA. Transient expression of SP-AvrA in epithelial cells resulted in significantly weaker inhibition of NF-κB pathway activation when treated with TNF-α compared to the inhibition by SE-AvrA. SP-AvrA expression in the S . Enteritidis resulted in weaker suppression of NF-κB pathway in infected HeLa cells compared to SE-AvrA expression in the cells, while SP-AvrA expressed in S. Pullorum C79-13 suppressed NF-κB activation in infected HeLa and Caco 2 BBE cells to a greater extent than did SE-AvrA because of the higher expression of SP-AvrA than SE-AvrA in S. Pullorum. Further analysis demonstrated that the inhibition of NF-κB pathway in Salmonella -infected cells corresponded to the downregulation of the p-JNK and Beclin-1 protein molecules. Our study reveals that AvrA modifies the anti-inflammatory response in a manner dependent on the Salmonella serotype through inhibition of NF-κB pathway.

S. Enteritidis continues to be the most common pathogen of farm animals and a major public health burden worldwide. Using bacteriophages is a potential alternative to antibiotics against S. Enteritidis infection. In this study, the genome analysis of the lytic phage vB&#95;SenM-PA13076 (PA13076) infecting S. Enteritidis revealed a linear, double-stranded DNA genome, which comprised of 52,474 bp and contained 69 ORFs. It belongs to the order Caudovirales; family Myoviridae, genus unclassified. The genes coded for DNA packaging, phage structural proteins, lysis components, DNA recombination, regulation, modification, and replication. No bacterial virulence or drug-resistance genes were detected. The phage PA13076 protected mice from a lethal dose of S. Enteritidis 13076 Amp (5 × 10 8 CFU) by reducing the concentration of bacterial cells in blood, intestine, liver, spleen, and kidney. The phage PA13076 achieved at least 2.5 log reductions of S. Enteritidis cells in infected mice within 24 h (P < 0.05) when compared to the organs of control mice. The data also indicated that phage PA13076 could rapidly enter the blood and four organs of infected mice, remaining therein at concentrations of>10 4 PFU/g for at least 72 h. These results show that phage PA13076 has definite potential as an antibacterial therapeutic agent for attenuating S. Enteritidis infections., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Salmonella Indiana has emerged in recent years as an important zoonotic pathogen, but its pathogenicity has not been fully elucidated. In this study, using in vivo and in vitro animal and cellular experimental model systems, we evaluated the pathogenicity of Salmonella Indiana (S. Indiana) compared with three other serotypes of Salmonella, S. Enteritidis, S. Typhimurium and S. Thompson. The animal experiments included observations of clinical symptoms, pathological changes and determination of median lethal dose in mice. The adhesion and invasiveness and intracellular proliferative capacity of Salmonella in vitro were measured with the murine macrophage-like cell line RAW264.7 cells and the human colon adenocarcinoma cell line Caco-2 cells. The results of animal experiments showed that S. Indiana, S. Enteritidis, S. Typhimurium and S. Thompson caused histopathological changes in most organs to varying degrees, primarily in the liver and intestine of mice. The gross lesions included white necrotic foci on the liver surface with different levels. The histopathological changes of monocyte/macrophage infiltration and coagulative necrosis were observed in the liver. Intestinal villi became short and were sloughed off, and lymphocyte infiltration was found in the submucosa. Compared with the other serotypes, the pathological changes caused by S. Indiana were slighter and had a relatively high median lethal dose in mice. The results of adhesion and invasion tests showed that the intracellular growth trend of most Salmonella strains was positively correlated with the number of pathogens adhering to and invading cells. Compared with the strains of the other three serotypes, most S. Indiana strains exhibited significantly lower adhesion and invasiveness to RAW264.7 and Caco-2 cells within 30 min. Most S. Indiana strains displayed twice to four times lower intracellular proliferation within 24 h in RAW264.7 cells. In conclusion, S. Indiana was pathogenic, but its pathogenicity was lower than that of S. Typhimurium and S. Enteritidis, and was similar to that of S. Thompson., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

We sequenced 25 isolates of phenotypically multidrug-resistant Salmonella Indiana (n = 11), Typhimurium (n = 8), and Enteritidis (n = 6) using both MinION long-read [SQK-LSK109 and flow cell (R9.4.1)] and MiSeq short-read (Nextera XT and MiSeq Reagent Kit v2) sequencing technologies to determine the advantages of each approach in terms of the characteristics of genome structure, antimicrobial resistance (AMR), virulence potential, whole-genome phylogeny, and pan-genome. The MinION reads were base-called in real-time using MinKnow 3.4.8 integrated with Guppy 3.0.7. The long-read-only assembly, Illumina-only assembly, and hybrid assembly pipelines of Unicycler 0.4.8 were used to generate the MinION, MiSeq, and hybrid assemblies, respectively. The MinION assemblies were highly contiguous compared to the MiSeq assemblies but lacked accuracy, a deficiency that was mitigated by adding the MiSeq short reads through the Unicycler hybrid assembly which corrected erroneous single nucleotide polymorphisms (SNPs). The MinION assemblies provided similar predictions of AMR and virulence potential compared to the MiSeq and hybrid assemblies, although they produced more total false negatives of AMR genotypes, primarily due to failure in identifying tetracycline resistance genes in 11 of the 19 MinION assemblies of tetracycline-resistant isolates. The MinION assemblies displayed a large genetic distance from their corresponding MiSeq and hybrid assemblies on the whole-genome phylogenetic tree, indicating that the lower read accuracy of MinION sequencing caused incorrect clustering. The pan-genome of the MinION assemblies contained significantly more accessory genes and less core genes compared to the MiSeq and hybrid assemblies, suggesting that although these assemblies were more contiguous, their sequencing errors reduced accurate genome annotations. Our research demonstrates that MinION sequencing by itself provides an efficient assessment of the genome structure, antimicrobial resistance, and virulence potential of Salmonella; however, it is not sufficient for whole-genome phylogenetic and pan-genome analyses. MinION in combination with MiSeq facilitated the most accurate genomic analyses., Competing Interests: The authors have declared that no competing interests exist.

Bloodstream infections and invasive nontyphoidal Salmonellosis in particular remain a major health and economic burden worldwide. The complexity of blood matrixes along with extremely low concentration of pathogens in blood poses a great challenge for rapid and ultrasensitive detection. Sample preparation has been the critical step that should provide blood-matrix-free sample with the targeted pathogen in the highest possible concentration. In this work, we addressed this challenge by combining magnetic-bead-based pathogen concentration and solid-phase PCR (SP-PCR). The SP-PCR performed on a supercritical angle fluorescence (SAF) microlens array embedded in a microchip enabled quick and accurate detection of low levels of Salmonella enterica serovar typhimurium and enteritidis in blood samples without culture enrichment. Protein AG-magnetic beads immobilized with antisalmonella antibody could efficiently concentrate both Salmonella serovars with a capturing efficiency >95%. Higher tolerance of Phusion hot start DNA polymerase to PCR inhibitors and its compatibility with protein AG-magnetic beads allowed the integration of SP-PCR. Analysis of Salmonella -spiked blood samples with the SP-PCR resulted in a limit of detection (LoD) as low as 86 CFU/mL and 94 CFU/mL for S . typhimurium and S . enteritidis , respectively, that could be attributed to the high fluorescence collection efficiency of the SAF microlens array. These combinations reduced the duration of analysis to less than 3 h including sample preparation. This platform has the potential for wide application as a high-throughput biosensor to analyze pathogens in clinical, food, and environmental samples.

The effect of dietary aflatoxin B 1 (AFB 1 ) and Salmonella Enteritidis infection on intestinal permeability was investigated. Two hundred 1-day-old male Ross 308 broiler chickens were randomly divided into 4 treatments of 5 replicates each (10 birds per replicate), which were fed ad libitum for 3 weeks with the following treatments: control, chickens fed an AFB 1 -free diet; AF, chickens fed an AFB 1 -contaminated diet at 470 ng/g; SE, chickens fed an AFB 1 -free diet and challenged with 10 8  cfu of S. Enteritidis per bird at 18 days old; AF + SE, chickens fed an AFB 1 -contaminated diet and challenged with 10 8  cfu of S. Enteritidis per bird at 18 days old. At day 21 of age, chicks received an oral gavage dose of fluorescein isothiocyanate dextran (FITC-dextran) to evaluate gastrointestinal leakage. Blood and intestinal samples were collected to evaluate serum biochemistry and total intestinal IgA secretion, respectively. Liver tissues were aseptically collected to assess bacterial invasiveness and for histomorphological studies. The results showed that chickens receiving AFB 1 presented a significant increment (up to 2.4-fold) in serum FITC-dextran concentration (p < 0.05). Nevertheless, S. Enteritidis infection had no additional effect on gastrointestinal leakage. Furthermore, the ingestion of AFB 1 had no impact on the invasive potential of S. Enteritidis. These results suggest that moderate-dose AFB 1 adversely affects intestinal barrier function resulting in increased gut permeability in broiler chickens.

Salmonella Enteritidis is an important foodborne pathogen that can infect a wide range of animal species including human beings, resulting in great losses to commercial husbandry and human health. CirA is an outer membrane receptor involved in iron uptake and colicin1A/B-mediated competitive killing. Although iron uptake is crucial to bacterial virulence, limited literature is available about the role of CirA in infection. In the present work, we aimed to evaluate the role of CirA during S. Enteritidis infection. For this purpose, we generated a CirA-deficient mutant of the S. Enteritidis strain C50336 and examined its biological characteristics. The results showed that cirA gene inactivation caused sharply decreased biofilm formation and apparently impaired antibiotic resistance. Furthermore, the cirA gene deletion mutant showed markedly reduced adhesion and invasion to human epithelial cell line Caco-2 cells and decreased proliferation in mouse macrophage cell line RAW264.7 cells. Moreover, attenuated virulence was determined by a mouse model, with an LD 50 increase of approximately 1,000-fold. These data indicated that CirA plays critical roles in the S. Enteritidis infection process., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Although disinfection procedures are widely implemented in food environments, bacteria can survive and present increased virulence/resistance. Since little is known about these phenomena regarding biofilms, this study aimed to investigate the effect of chemical disinfection on biofilm-derived cells of Salmonella Enteritidis. Using a reference strain (NCTC 13349) and a food isolate (350), biofilm susceptibility to benzalkonium chloride (BAC), sodium hypochlorite (SH) and hydrogen peroxide (HP) was evaluated and biofilms were exposed to sub-lethal concentrations of each disinfectant. Biofilm-derived cells were characterized for their biofilm forming ability, antibiotic resistance and expression of virulence-associated genes. Except for a few instances, disinfectant exposure did not alter antibiotic susceptibility. However, SH and HP exposure enhanced the biofilm forming ability of Salmonella Enteritidis NCTC 13349. After BAC and HP exposure, biofilm-derived cells presented a down-regulation of rpoS . Exposure to BAC also revealed an up-regulation of invA, avrA and csgD on Salmonella Enteritidis NCTC 13349. The results obtained suggest that biofilm-derived cells that survive disinfection may represent an increased health risk.

Pathogenicity island excision is a phenomenon that occurs in several Salmonella enterica serovars and other members of the family Enterobacteriaceae. ROD21 is an excisable pathogenicity island found in the chromosome of S. Enteritidis, S. Dublin and S. Typhi among others, which contain several genes encoding virulence-associated proteins. Excision of ROD21 may play a role in the ability of S. Enteritidis to cause a systemic infection in mice. Our previous studies have shown that Salmonella strains unable to excise ROD21 display a reduced ability to colonize the liver and spleen. In this work, we determined the kinetics of ROD21 excision in vivo in C57BL/6 mice and its effect on virulence. We quantified bacterial burden and excision frequency in different portions of the digestive tract and internal organs throughout the infection. We observed that the frequency of ROD21 excision was significantly increased in the bacterial population colonizing mesenteric lymph nodes at early stages of the infective cycle, before 48 hours post-infection. In contrast, excision frequency remained very low in the liver and spleen at these stages. Interestingly, excision increased drastically after 48 h post infection, when intestinal re-infection and mortality begun. Moreover, we observed that the inability to excise ROD21 had a negative effect on S. Enteritidis capacity to translocate from the intestine to deeper organs, which correlates with an abnormal transcription of invA in the S. Enteritidis strain unable to excise ROD21. These results suggest that excision of ROD21 is a genetic mechanism required by S. Enteritidis to produce a successful invasion of the intestinal epithelium, a step required to generate systemic infection in mice., Competing Interests: The authors have declared that no competing interests exist.

We studied the interaction of Salmonella enteritidis and Salmonella typhimurium in the microbial association formed by these microorganisms in vitro. It was found that S. typhimurium in microbial association in LB-broth inhibited reproduction of S. enteritidis, which attested to its higher competitiveness. The concentration of S. typhimurium in the medium in 6 h reached 92-93% of the total number of bacteria. Parallel growth of the populations of S. enteritidis and S. typhimurium was observed only during the first hour, but later, S. typhimurium always exceeded S. enteritidis by the growth dynamics. The competitive advantage of S. typhimurium was observed both in the absence and presence of 60-MDa virulence plasmid.

Salmonella enterica serovar Enteritidis strain SE86 has been associated with several foodborne diseases occurring in Southern Brazil, becoming and important causative agent of human salmonellosis. In this work, the complete genome of the bacterium Salmonella Enteritidis SE86 was sequenced using the Illumina MiSeq platform. An in silico analysis of the SE86 genome was performed in order to compare it with different Salmonella strains as well as to investigate the presence of stress-resistance and virulence genes. This strain showed a variety of genes that can be involved in antimicrobial and biocide resistance, acid and thermal resistance as well as virulence and adhesion. These genetic features could explain its increased resistance and the prevalence of this strain in foodborne outbreaks in Southern Brazil., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Small non-coding RNAs (sRNAs) regulate bacterial gene expression at the post-transcriptional level. STnc640 is a type of sRNA that was identified in Salmonella Typhimurium., Results: In this study, STnc640 in Salmonella Enteritidis was confirmed to be an Hfq-dependent sRNA. TargetRNA software analysis showed that fimbrial genes fimA and bcfA were likely to be the target genes of STnc640. To investigate the target mRNAs and function of STnc640 in pathogenicity, we constructed the deletion mutant strain 50336△stnc640 and the complemented strain 50336△stnc640/pstnc640 in Salmonella Enteritidis 50336. The RT-qPCR results showed that the mRNA level of fimA was decreased, while bcfA was unchanged in 50336△stnc640 compared with that in the wild type (WT) strain. The adhesion ability of 50336△stnc640 to Caco-2 cells was increased compared to the 50336 WT strain. The virulence of 50336△stnc640 was enhanced in a one-day-old chicken model of S. Enteritidis disease as determined by quantifying the 50% lethal dose (LD 50 ) of the bacterial strains., Conclusions: The results demonstrate that STnc640 contributes to the virulence of Salmonella Enteritidis.

Antimicrobial resistance (AMR) is now a major global problem largely resulting from the overuse of antibiotics in humans and livestock. In some AMR bacteria, resistance is encoded by conjugative plasmids expressing sex-pili that can readily spread resistance through bacterial populations. The aim of this study was to use sex pilus-specific (SPS) phage to reduce the carriage of AMR plasmids. Here, we demonstrate that SPS phage can kill AMR Escherichia coli and select for AMR plasmid loss in vitro. For the first time, we also demonstrate that SPS phage can both prevent the spread of AMR Salmonella Enteritidis infection in chickens and shift the bacterial population towards antibiotic sensitivity.

Toll-like receptors (TLRs) activate innate immunity via interactions between their Toll/interleukin-1 (IL-1) receptor (TIR) domain and downstream adaptor proteins. Here we report that Salmonella Enteritidis produces a secreted protein (TcpS) that contains both a TIR domain and a coiled-coil domain. TcpS blocks MyD88- and TRIF-mediated TLR signaling, inhibits inflammatory responses, and promotes bacterial survival. Early-stage immune evasion by TcpS results in severe tissue damage in the late stage of infection and contributes to Salmonella virulence. TcpS-derived peptides inhibit nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) activation and reduce lipopolysaccharide (LPS)-elicited systemic inflammation. Therapeutic peptide administration alleviates weight loss of mice infected with H1N1 influenza. Importantly, maximal TcpS-mediated TLR inhibition requires the critical TIR-TcpS residues Y191 and I284, as well as TcpS homodimerization via its N-terminal coiled-coil domain. Our study unveils a mechanism in which TcpS suppresses innate immunity via both its homodimerization and interaction with MyD88. TcpS is also a potential therapeutic agent for inflammation-associated diseases., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Non-typhoidal Salmonella enterica serovars continue to be an important food safety issue worldwide. Cranberry (Vaccinium macrocarpon Ait) fruits possess antimicrobial properties due to their various acids and phenolic compounds; however, the underlying mechanism of actions is poorly understood. We evaluated the effects of cranberry extracts on the growth rate of Salmonella enterica serovars Typhimurium, Enteritidis and Heidelberg and on the transcriptomic profile of Salmonella Enteritidis to gain insight into phenotypic and transcriptional changes induced by cranberry extracts on this pathogen. An ethanolic extract from cranberry pomaces (KCOH) and two of its sub-fractions, anthocyanins (CRFa20) and non-anthocyanin polyphenols (CRFp85), were used. The minimum inhibitory (MICs) and bactericidal (MBCs) concentrations of these fractions against tested pathogens were obtained using the broth micro-dilution method according to the Clinical Laboratory Standard Institute's guidelines. Transcriptional profiles of S. Enteritidis grown in cation-adjusted Mueller-Hinton broth supplemented with or without 2 or 4 mg/ml of KCOH were compared by RNASeq to reveal gene modulations serving as markers for biological activity. The MIC and MBC values of KCOH were 8 and 16 mg/mL, respectively, against all tested S. enterica isolates. The MIC value was 4 mg/mL for both CRFa20 and CRFp85 sub-fractions, and a reduced MBC value was obtained for CRFp85 (4 mg/ml). Treatment of S. Enteritidis with KCOH revealed a concentration-dependent transcriptional signature. Compared to the control, 2 mg/ml of KCOH exposure resulted in 89 differentially expressed genes (DEGs), of which 53 and 36 were downregulated and upregulated, respectively. The upregulated genes included those involved in citrate metabolism, enterobactin synthesis and transport, and virulence. Exposure to 4 mg/ml KCOH led to the modulated expression of 376 genes, of which 233 were downregulated and 143 upregulated, which is 4.2 times more DEGs than from exposure to 2 mg/ml KCOH. The downregulated genes were related to flagellar motility, Salmonella Pathogenicity Island-1 (SPI-1), cell wall/membrane biogenesis, and transcription. Moreover, genes involved in energy production and conversion, carbohydrate transport and metabolism, and coenzyme transport and metabolism were upregulated during exposure to 4 mg/ml KCOH. Overall, 57 genes were differentially expressed (48 downregulated and 9 upregulated) in response to both concentrations. Both concentrations of KCOH downregulated expression of hilA, which is a major SPI-1 transcriptional regulator. This study provides information on the response of Salmonella exposed to cranberry extracts, which could be used in the control of this important foodborne pathogen., Competing Interests: The authors have declared that no competing interests exist.

The first synthetic carbohydrate based potential anti-Salmonella Enteritidis vaccine has been developed by conjugating a synthetic tetrasaccharide antigen with bacteriophage Qβ. High levels of specific and long lasting anti-glycan IgG antibodies were induced by the conjugate, which completely protected mice from lethal bacterial challenges in a passive transfer model.

Salmonella is a common foodborne and zoonotic pathogen. Only a few serovars carry a virulence plasmid (pSV), which enhances the pathogenicity of the host. Here, we investigated the pathogenicity roles of the pSVs among wild-type, plasmid-less, and complemented S. Typhimurium, S. Enteritidis S. Choleraesuis in invasion, phagocytosis, and intracellular bacterial survival in human THP-1 cells and cell death patterns by flow cytometry and difference in cell death patterns between pig and human S. Choleraesuis isolates with large pSCVs. Virulence plasmid (pSTV) led to slightly increasing cellular apoptosis for S. Typhimurium; virulence plasmid (pSEV) enhanced apoptosis and necrosis significantly for S. Enteritidis; and pSCV reduced apoptosis significantly for S. Choleraesuis. After complementation, pSTV increased the intracellular survival of pSCV-less Choleraesuis and the cytotoxicity against human THP-1 cells. Using the Cytochalasin D to differentiate the invasion of S. Choleraaesuis and phagocytosis of THP-1 cells determined that pSCV were responsible for invasion and phagocytosis at 0 h and inhibited intracellular replication in THP-1 cells, and pSTV were responsible for invasion and increased intracellular survival for S. Choleraesuis in THP-1 cells. The human isolates with large pSCV induced more cellular apoptosis and necrosis than the pig isolates. In conclusion, human S. Choleraesuis isolates carrying large pSCVs were more adapted to human THP-1 cells for more cell death than pig isolates with large pSCV. The role of pSVs in invasion, phagocytosis, intracellular survival and apoptosis differed among hosted serovars., (Copyright © 2018. Published by Elsevier Ltd.)

The survival mechanism of Salmonella enterica serovar Enteritidis in antibacterial egg white is not fully understood. In our lab, an egg white-resistant strain, S. Enteritidis SJTUF 10978, was identified. Cell envelope damage and osmotic stress response (separation of cell wall and inner membrane as well as cytoplasmic shrinkage) of this strain surviving in egg white were identified through microscopic observation. RNA-Seq analysis of the transcriptome of Salmonella survival in egg white showed that a considerable number of genes involved in DNA damage repair, alkaline pH adaptation, osmotic stress adaptation, envelope damage repair, Salmonella pathogenicity island 2 (SPI-2), iron absorption, and biotin synthesis were significantly upregulated (fold change ≥ 2) in egg white, indicating that these pathways or genes might be critical for bacterial survival. RNA-Seq results were confirmed by qRT-PCR, and the survival analysis of six gene deletion mutants confirmed their importance in the survival of bacteria in egg white. The importance of alkaline pH adaptation and envelope damage repair for Salmonella to survive in egg white were further confirmed by analysis of nhaA , cpxR , waaH , and eco deletion mutants. According to the RNA-Seq results, we propose that alkaline pH adaptation might be the cause of bacterial osmotic stress phenotype and that the synergistic effect between alkaline pH and other inhibitory factors can enhance the bacteriostatic effect of egg white. Moreover, cpxR and sigE were recognized as the central regulators that coordinate bacterial metabolism to adapt to envelope damage and alkaline pH. IMPORTANCE Salmonella enterica serovar Enteritidis is a major foodborne pathogen that causes salmonellosis mainly through contaminated chicken eggs or egg products and has been a worldwide public health threat since 1980. Frequent outbreaks of this serotype through eggs correlate significantly with its exceptional survival ability in the antibacterial egg white. Research on the survival mechanism of S. Enteritidis in egg white will help to further understand the complex and highly effective antibacterial mechanisms of egg white and lay the foundation for the development of safe and effective vaccines to prevent egg contamination by this Salmonella serotype. Key pathways and genes that were previously overlooked under bactericidal conditions were characterized as being induced in egg white, and synergistic effects between different antimicrobial factors appear to exist according to the gene expression changes. Our work provides new insights into the survival mechanism of S. Enteritidis in egg white., (Copyright © 2019 Huang et al.)

Neonates are highly susceptible to infection with enteric pathogens, but the underlying mechanisms are not resolved. We show that neonatal chick colonization with Salmonella enterica serovar Enteritidis requires a virulence-factor-dependent increase in epithelial oxygenation, which drives pathogen expansion by aerobic respiration. Co-infection experiments with an Escherichia coli strain carrying an oxygen-sensitive reporter suggest that S. Enteritidis competes with commensal Enterobacteriaceae for oxygen. A combination of Enterobacteriaceae and spore-forming bacteria, but not colonization with either community alone, confers colonization resistance against S. Enteritidis in neonatal chicks, phenocopying germ-free mice associated with adult chicken microbiota. Combining spore-forming bacteria with a probiotic E. coli isolate protects germ-free mice from pathogen colonization, but the protection is lost when the ability to respire oxygen under micro-aerophilic conditions is genetically ablated in E. coli. These results suggest that commensal Enterobacteriaceae contribute to colonization resistance by competing with S. Enteritidis for oxygen, a resource critical for pathogen expansion., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Nontyphoidal salmonellae (NTS) are a major cause of invasive (iNTS) disease in sub-Saharan Africa, manifesting as bacteremia and meningitis. Available epidemiological data indicate that iNTS disease is endemic in much of the region. Antimicrobial resistance is common and case fatality rates are high. There are well-characterized clinical associations with iNTS disease, including young age, HIV infection, malaria, malnutrition, anemia, and sickle cell disease. However, the clinical presentation of iNTS disease is often with fever alone, so clinical diagnosis is impossible without blood culture confirmation. No vaccine is currently available, making this a priority area for global health research. Over the past ten years, it has emerged that iNTS disease in Africa is caused by distinct pathovars of Salmonella Typhimurium, belonging to sequence type ST313, and Salmonella Enteritidis. These are characterized by genome degradation and appear to be adapting to an invasive lifestyle. Investigation of rare patients with primary immunodeficiencies has suggested a key role for interferon gamma-mediated immunity in host defense against NTS. This concept has been supported by recent population-based host genetic studies in African children. In contrast, immunoepidemiological studies from Africa indicate an important role for antibody for protective immunity, supporting the development of antibody-inducing vaccines against iNTS disease. With candidate O-antigen-based vaccines due to enter clinical trials in the near future, research efforts should focus on understanding the relative contributions of antibody and cell-mediated immunity to protection against iNTS disease in humans.

The increasing demand for high quality and safe food led to important technological innovations in food processing. Cold plasma appears as an emerging technology that has demonstrated efficiency in the removal of microbial contamination from fresh and minimally processed food. In this study, the proteomic profile of Salmonella Enteritidis SE86 subjected to cold plasma treatment was investigated. The number of viable S. Enteritidis SE86 cells was analyzed at different time intervals upon exposure to cold plasma and approximately 100 μg of S. Enteritidis SE86 protein extracts were analyzed by Multidimensional Protein Identification Technology (MudPIT). The results demonstrated that no significant changes in cell counts were detected for up to 20 min exposure to cold plasma, and 2 log reduction was achieved after 60 min. Overall, 1096 proteins were identified, with 249 detected only in plasma-treated samples, and 9 exclusive in non-treated control samples. The proteins uniquely detected in cold plasma-treated cells that showed higher abundance were glyoxalase I, ABC transporter substrate-binding protein and transcriptional activator OsmE, followed by some oxidoreductases. Proteins related with carbohydrate and nucleotide metabolism were mostly overexpressed in cold plasma treated cells, suggesting energy metabolism was increased., (Copyright © 2018. Published by Elsevier Ltd.)

Salmonella pathogenicity island 13 (SPI-13) contributes to the virulence of Salmonella. The majority of the SPI-13 genes encode proteins putatively involved in bacterial metabolism, however, their functions largely remain uncharacterized. It is currently unknown if SPI-13 contributes to metabolic fitness of Salmonella and, if so, what are the metabolic substrates for the protein encoded by genes within SPI-13. We employed Phenotype Microarray (Biolog, USA) to compare the metabolic properties of SPI-13 deficient mutant (ΔSPI-13) and the WT parent strain of non-typhoidal Salmonella enterica sub sp. enterica serovar Enteritidis (S. Enteritidis). The results of Phenotype Microarray revealed that SPI-13 is required for efficient utilization of two micronutrients, namely, d-glucuronic acid (DGA) and tyramine (TYR), as sole sources of carbon and/or nitrogen. By systematic deletion of the individual gene(s), we identified specific genes within SPI-13 that are required for efficient utilization of DGA (SEN2977-80) and TYR (SEN2967 and SEN2971-72) as sole nutrient sources. The results show that SPI-13 mediated DGA and TYR metabolic pathways afford nutritional fitness to S. Enteritidis. Comparative genomics analysis of the SPI-13 locus from 247 Salmonella strains belonging to 57 different serovars revealed that SPI-13 genes specifically involved in the metabolism of DGA and TYR are highly conserved in Salmonella enterica. Because DGA and TYR are naturally present as metabolic byproducts in the gastrointestinal tract and other host tissues, we propose a metabolic model that shows that the role of SPI-13 mediated DGA and TYR metabolism in the nutritional fitness of Salmonella is likely linked to nutritional virulence of this pathogen., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

We previously reported that inactivation of a universally conserved dimethyl adenosine transferase (KsgA) attenuates virulence and increases sensitivity to oxidative and osmotic stress in Salmonella Enteritidis. Here, we show a role of KsgA in cell-envelope fitness as a potential mechanism underlying these phenotypes in Salmonella. We assessed structural integrity of the cell-envelope by transmission electron microscopy, permeability barrier function by determining intracellular accumulation of ethidium bromide and electrophysical properties by dielectrophoresis, an electrokinetic tool, in wild-type and ksgA knock-out mutants of S. Enteritidis. Deletion of ksgA resulted in disruption of the structural integrity, permeability barrier and distorted electrophysical properties of the cell-envelope. The cell-envelope fitness defects were alleviated by expression of wild-type KsgA (WT-ksgA) but not by its catalytically inactive form (ksgAE66A), suggesting that the dimethyl transferase activity of KsgA is important for cell-envelope fitness in S. Enteritidis. Upon expression of WT-ksgA and ksgAE66A in inherently permeable E. coli cells, the former strengthened and the latter weakened the permeability barrier, suggesting that KsgA also contributes to the cell-envelope fitness in E. coli. Lastly, expression of ksgAE66A exacerbated the cell-envelope fitness defects, resulting in impaired S. Enteritidis interactions with human intestinal epithelial cells, and human and avian phagocytes. This study shows that KsgA contributes to cell-envelope fitness and opens new avenues to modulate cell-envelopes via use of KsgA-antagonists., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Emerging evidence suggests that microbial pathogens may induce oxidative stress in infected hosts. The aim of the present study was to investigate the relationship between changes in oxidative stress and intestinal infection with and without antibiotic treatment in animal models. Sprague-Dawley (SD) rats were divided into three groups: rats infected with Salmonella enterica serovar Enteritidis (S. enteritidis), rats infected with S. enteritidis followed by norfloxacin treatment, and the control group. To evaluate oxidative stress changes, levels of 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn), which represented oxidative damage to RNA and DNA, respectively, were analysed in urine and tissue samples. In urine, the level of 8-oxo-Gsn increased significantly after oral exposure to S. enteritidis (p ≤ 0.001) and returned to baseline after recovery. Notably, norfloxacin treatment decreased the level of 8-oxo-Gsn in urine significantly (p = 0.001). Changes of 8-oxo-Gsn measured in tissues from the small intestine, colon, liver and spleen were consistent with 8-oxo-Gsn measured in urine. Our study suggested that 8-oxo-Gsn in urine may serve as a highly sensitive biomarker for evaluating the severity of S. enteritidis infection and the effectiveness of antibiotic treatment against infection.

In this study, an amino-modified aptasensor using multi-walled carbon nanotubes (MWCNTs)-deposited ITO electrode was prepared and evaluated for the detection of pathogenic Salmonella bacteria. An amino-modified aptamer (ssDNA) which binds selectively to whole-cell Salmonella was immobilised on the COOH-rich MWCNTs to produce the ssDNA/MWCNT/ITO electrode. The morphology of the MWCNT before and after interaction with the aptamers were observed using scanning electron microscopy (SEM). Cyclic voltammetry and electrochemical impedance spectroscopy techniques were used to investigate the electrochemical properties and conductivity of the aptasensor. The results showed that the impedance measured at the ssDNA/MWCNT/ITO electrode surface increased after exposure to Salmonella cells, which indicated successful binding of Salmonella on the aptamer-functionalised surface. The developed ssDNA/MWCNT/ITO aptasensor was stable and maintained linearity when the scan rate was increased from 10 mV s -1 to 90 mV s -1 . The detection limit of the ssDNA/MWCNT/ITO aptasensor, determined from the sensitivity analysis, was found to be 5.5 × 10 1  cfu mL -1 and 6.7 × 10 1  cfu mL -1 for S. Enteritidis and S. Typhimurium, respectively. The specificity test demonstrated that Salmonella bound specifically to the ssDNA/MWCNT/ITO aptasensor surface, when compared with non-Salmonella spp. The prepared aptasensor was successfully applied for the detection of Salmonella in food samples., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) is a major cause of foodborne diseases for humans. The completeness of the O-chain antigen of Lipopolysaccharide (LPS) determines whether a S. Enteritidis strain is smooth or rough. However, genes that are involved in the synthesis of LPS and rough-smooth variation are not completely understood. In this study, we used monoclonal antibody against O-antigens (O 9 mAb) to identify novel factors that are involved in LPS synthesis and rough variation in S. Enteritidis by using signature-tagged mutagenesis (STM) technique. Our results showed that transposon insertion in the gene rfbH led to different LPS phenotype, auto-aggregation characteristic, motility and resistance to environmental stress compared with SE wild-type strain C50041. In addition, sera tests showed that rfbH mutant does not elicit specific antibodies against O-antigens in vaccinated animals. Taken together, the S. Enteritidis rfbH gene is implicated in LPS biosynthesis, rough variation, sera distinguishable reaction, motility and stress resistance. The rfbH mutant strain could be potentially used as a distinguishable vaccine or a live vector to deliver drugs and antibodies in vivo., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

This study assessed the antagonistic activity of fruit-derived lactic acid bacteria (LAB) strains against food-related bacteria and the effects of the highest organic acids LAB producers on the survival of Listeria monocytogenes and Salmonella Enteritidis PT4 in cheese and chicken meat, respectively. The production of organic acids by the Lactobacillus strains in the tested food matrices was also monitored. All tested LAB strains showed antagonistic activity in vitro on the growth of pathogenic or spoiling food-related bacteria, particularly on L. monocytogenes and/or S. Enteritidis PT4, through the action of non-proteinaceous substances. The highest amounts of acetic and lactic acid were detected in cell free culture supernatants of L. paracasei 108 and L. plantarum 201. In "Minas Frescal" cheese, L. plantarum 49 and L. paracasei 108 decreased the counts of L. monocytogenes, and L. plantarum 201 showed bacteriostatic effects on this pathogen over time. L. paracasei 108 decreased the counts of S. Enteritidis PT4 in ground chicken breast; L. plantarum 49 and L. plantarum 201 failed to decrease the counts of this pathogen. Decreases in counts of L. monocytogenes or S. Enteritidis in "Minas Frescal" cheese and ground chicken breast, respectively, were related with increases in lactic and acetic acid contents and decreases in pH values. L. plantarum 49 and L. paracasei 108 could be used as biopreservation tools in cheese and chicken breast meat, respectively., (Copyright © 2018. Published by Elsevier Ltd.)

Salmonella enterica serovar Enteritidis is the leading global cause of salmonellosis. A total of 146 Salmonella Enteritidis isolates obtained from retail chicken products in Shanghai, China were characterized for their antimicrobial susceptibilities, virulence and antibiotic resistance gene profiles, and molecular subtypes using pulsed-field gel electrophoresis (PFGE). Approximately 42% (61/146) of the isolates were susceptible to all 13 antimicrobials tested. More than half of the isolates (50.70%) were resistant to ampicillin, 49.32% to sulfisoxazole, 17.12% to tetracycline, and 15.75% to doxycycline. Thirty (20.55%) isolates were resistant to three or more antimicrobials. The avrA, mgtC, and sopE virulence genes were identified in all isolates, while 97.2% and 92.4% were positive for bcfC and spvC genes, respectively. Genes associated with resistance to streptomycin (aadA), β-lactams (blaTEM, blaCMY, blaSHV, and blaCTX), tetracycline (tetA and tetB), and sulfonamides (sulI, sulII, and sulIII) were detected among corresponding resistant isolates. A total of 41 PFGE patterns were identified from 77 antimicrobial resistance (AMR) isolates and were primarily grouped into seven clusters (A-G), each with 90% similarity. The majority of Salmonella Enteritidis isolates (63.63%, 49/77) shared the same PFGE cluster, indicating potential cross contamination during processing and cutting or working during retailing and marketing. A significantly (p < 0.05) lower percentage (<25%) of isolates belonging to clusters D and E were resistant to sulfisoxazole compared with those belonging to clusters A, B, C, F, and G (>80%), indicating that sulfisoxazole resistance might be associated with genetic content (PFGE profiles) of Salmonella Enteritidis. This study provides important and updated information about the baseline antimicrobial-resistant data for food safety risk assessment of Salmonella Enteritidis from retailed chicken in Shanghai, which is the first step for the development and implementation of China's AMR National Action Plan, and can be helpful for future surveillance activities to ensure the safety of the chicken supply.

Salmonella Enteritidis and Salmonella Typhimurium are among the most prevalent serotypes isolated from salmonellosis outbreaks and poultry. Salmonella spp. have the capacity to form biofilms on several surfaces, which can favour survival in hostile environments, such as slaughterhouses. Salmonella strains present differences in pathogenicity. However, there is little information regarding the pathogenicity of S. Enteritidis and S. Typhimurium isolated from avian sources and their relationship to biofilm production. The aim of this study was to use a novel pathogenicity index and a biofilm production assay to evaluate their relationships within these serotypes. In addition, we detected the presence of the spiA and agfA genes in these strains. Biofilm formation was investigated at two temperatures (37 °C and 28 °C) using microtiter plate assay, and the results were compared with the individual pathogenicity index of each strain. PCR was used to detect spiA and agfA, virulence genes associated with biofilm production. S. Enteritidis and S. Typhimurium strains were capable of producing biofilm at 37 °C and 28 °C. Sixty-two percent and 59.5% of S. Enteritidis and 73.8% and 46.2% of S. Typhimurium produced biofilm at 37 °C and 28 °C, respectively. Biofilm production at 37 °C was significantly higher in both serotypes. Only S. Enteritidis was capable of adhering strongly at both temperatures. Biofilm production was related to pathogenicity index only at 28 °C for S. Enteritidis. spiA and agfA were found in almost all strains and were not statistically associated with biofilm production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: To determine the prevalence and antimicrobial susceptibility of Salmonella isolated from broiler production process in 4 provinces of China. Methods: Using convenience sampling method, 238 sample sites from broiler whole production process were chosen in Henan, Jiangsu, Heilongjiang and Shandong provinces in 2012. A total of 11 592 samples were collected and detected to analyze prevalence baseline, including 2 090 samples from breeding chicken farms and hatcheries, 1 421 samples from broiler farms, 5 610 samples from slaughterhouses and 2 471 samples from distribution and retail stores. All Salmonella strains were isolated through selective enrichment, and were serotyped according to Kauffmann-White scheme. The antimicrobial susceptibilities of selected Salmonella strains were determined by the broth microdilution method and fourteen antimicrobial agents were examined. Results: During incubation course, the average prevalence of Salmonella was 5.5% in feces of breeding hens, feces of chicks, and hatching eggs, 123 Salmonella strains were isolated. During cultivation course, the prevalence of Salmonella was 8.0% in feces from broiler farms, soil, feed, and workers, 114 Salmonella strains were isolated. During slaughter course, the prevalence of Salmonella was 24.9% in swabs pre-slaughter, dressed broiler carcasses, pre-cooled broiler carcasses, water from precooling pool, cutter and chipping boards, frozen chicken portions, and workers, 1 438 Salmonella strains were isolated. During distribution and sale course, the prevalence of Salmonella was 20.9% in transport carts, frozen chicken portions, retail chicken portions and workers, 551 Salmonella strains were isolated. The dominant Salmonella serotypes were Salmonella Enteritidis ( n= 1 229) and Salmonella Indiana ( n= 621). Among 1 231 examined strains, 97.2% Salmonella isolates were resistant to at least one antimicrobial, 69.9% Salmonella strains were multi-drug resistant isolates. Conclusion: Our findings indicated that Salmonella contamination was common and serious in commercial broiler whole production process in China, especially in the course of defeathering, precooling and selling. The environment of broiler farm is the important source of Salmonella contamination. Additionally, antibiotic resistance of Salmonella was serious for common antimicrobials and multi-drug resistant strains existed widespread, which can pose potential risk on public health and clinical therapy.

Salmonella enteritidis is a common food-borne pathogen associated with consumption of contaminated poultry meat and eggs, which frequently causes gastroenteritis in humans. Salmonella secreted effector K1 (SseK1), as a translocated and secreted protein has been identified to be essential for the virulence of Salmonella typhimurium in host cells. However, the role of the sseK1 gene in the pathogenicity of S. enteritidis remain unclear. In this study, a sseK1 deletion mutant of S. enteritidis was constructed and its biological characteristics were examined. It was found that the sseK1 deletion mutant did not affect the growth, adherence and invasion of Salmonella enteritidis when compared to the wild-type S. enteritidis. However, the mutant showed decreased formation of biofilm and significantly reduced intracellular survival of bacteria in activated mouse peritoneal macrophages, as well as showed reduced pathogenicity to a murine model by increasing the lethal dose 50% (LD 50 ) value and decreasing the proliferation ratio of bacteria in vivo. Taken together, this study determined an important role for SseK1 in the pathogenicity of S. enteritidis in vitro and in vivo., (Copyright © 2018 Elsevier Ltd. All rights reserved.)