Bioinformatic and experimental characterization of SEN1998: a conserved gene carried by the Enterobacteriaceae-associated ROD21-like family of genomic islands.

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.

The combined effect of stressful factors (temperature and pH) on the expression of biofilm, stress, and virulence genes in Salmonella enterica ser. Enteritidis and Typhimurium.

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.

Prevalence, virulence factor and antimicrobial resistance analysis of Salmonella Enteritidis from poultry and egg samples in Iran.

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.

Sodium butyrate modulates chicken macrophage proteins essential for Salmonella Enteritidis invasion.

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.

Positive regulation of Type III secretion effectors and virulence by RyhB paralogs in Salmonella enterica serovar Enteritidis.

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.

A non-coding small RNA MicC contributes to virulence in outer membrane proteins in Salmonella Enteritidis.

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 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 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 enterica Serovars Dublin and Enteritidis Comparative Proteomics Reveals Differential Expression of Proteins Involved in Stress Resistance, Virulence, and Anaerobic Metabolism.

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.

Phylogenetic structure of Salmonella Enteritidis provides context for a foodborne outbreak in Peru.

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.

Evaluation of propanediol and cobalamin metabolism in the intestinal colonization and systemic invasion of Salmonella Enteritidis in laying hens / [Avaliação do metabolismo do propanodiol e da cobalamina na colonização intestinal e na infecção sistêmica de Salmonella Enteritidis em poedeiras]

Embora Salmonella Enteritidis (SE) seja capaz de metabolizar 1,2-propanodiol (1,2-Pd), utilizado como fonte de carbono e de energia ao longo de uma rota dependente de vitamina B12, a importância deste composto na infeção de Gallus gallus domesticus por SE permanece desconhecida. No presente estudo, foram construídos um mutante de SE sem os genes pduCDE, que codifica a propanodiol desidratase (Pdu), e outro contendo as deleções no pduCDE e também nos genes cobS e cbiA, responsáveis pela síntese de vitamina B12. Em seguida, avaliou-se a importância do metabolismo do 1,2-Pd em SE para colonização intestinal de infecção sistêmica de poedeiras comerciais. As estirpes mutantes de SE foram capazes de colonizar o intestino, de serem excretadas nas fezes e de invadir o baço e o fígado na mesma intensidade que a estirpe selvagem, o que sugere que os produtos dos genes pduC, pduD, pduE, cobS e cbiA não são essenciais durante infecção por Salmonella Enteritidis nessa espécie.(AU)

Research on the Effect of Pediococcus pentosaceus on Salmonella enteritidis-Infected Chicken.

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 × 109 CFU/g. All the birds were given with 0.5 ml of S. enteritidis bacterial suspension (109 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-ß, 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.

Role of OB-Fold Protein YdeI in Stress Response and Virulence of Salmonella enterica Serovar Enteritidis.

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 ΔydeI mutant showed reduced survival during exposure to AMPs or glucose and Mg2+ 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 ΔydeI 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 ΔydeI 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.IMPORTANCES 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.

Case Study of Emphysematous Osteomyelitis of Both Femoral Heads.

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.

Serotypes, antibiotic resistance, and virulence genes of Salmonella in children with diarrhea.

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.

AvrA Exerts Inhibition of NF-κB Pathway in Its Naïve Salmonella Serotype through Suppression of p-JNK and Beclin-1 Molecules.

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.

Systematic evaluation of potential pathogenicity of Salmonella Indiana.

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.

Genomic analyses of multidrug-resistant Salmonella Indiana, Typhimurium, and Enteritidis isolates using MinION and MiSeq sequencing technologies.

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.

The complete genome of lytic Salmonella phage vB_SenM-PA13076 and therapeutic potency in the treatment of lethal Salmonella Enteritidis infections in mice.

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_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 13076Amp (5 × 108 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>104 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.

RpoS-regulated SEN1538 gene promotes resistance to stress and influences Salmonella enterica serovar enteritidis virulence.

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.

A ROD9 island encoded gene in Salmonella Enteritidis plays an important role in acid tolerance response and helps in systemic infection in mice.

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 ΔSEN1008 failed to survive during ATR assay, indicating its role under acid stress. Together, our findings suggested ΔSEN1008 to be significantly attenuated and we propose this gene to be a potent factor responsible for S. Enteritidis pathogenesis.