Detection of Salmonella Pathogenicity Islands and Antimicrobial-Resistant Genes in Salmonella enterica Serovars Enteritidis and Typhimurium Isolated from Broiler Chickens.

Rapid growth in commercial poultry production is one of the major sources of Salmonella infections that leads to human salmonellosis. The two main Salmonella enterica serovars associated with human salmonellosis are enteritidis and typhimurium. The aim of this study was to determine the prevalence of S. enterica serovars Enteritidis and S. Typhimurium as well as their Salmonella pathogenicity islands (SPI) and antibiotic resistance profiles in broiler chicken feces from slaughterhouses. A total of 480 fecal samples from broiler chickens that were grouped into 96 pooled samples were identified to have Salmonella spp. using the invA gene, whilst the Spy and sdfI genes were used to screen for the presence of S. Enteritidis and S. Typhimurium serovars, respectively, by polymerase chain reaction (PCR) assays. The isolates were also screened for the presence of Salmonella pathogenicity islands (SPIs) using PCR. The disc diffusion assay was performed to determine the antibiotic resistance profiles of the isolates. A total of 36 isolates were confirmed as Salmonella spp. through amplification of the invA gene. Out of 36 confirmed Salmonella spp. a total of 22 isolates were classified as S. Enteritidis (n = 8) and were S. Typhimurium (n = 14) serovars. All (n = 22) S. Enteritidis and S. Typhimurium isolates possessed the hilA (SPI-1), ssrB (SPI-2) and pagC (SPI-11) pathogenicity islands genes. Amongst these serovars, 50% of the isolates (n = 11/22) were resistant to tetracycline and nalidixic acid. Only 22% of the isolates, S. Typhimurium (13.6%) and S. Enteritidis (9.1%) demonstrated resistance against three or more antibiotic classes. The most detected antibiotic resistance genes were tet(K), mcr-1, sulI and strA with 13 (59.1%), 9 (40.9%), 9 (40.9%) and 7 (31.8%), respectively. The findings of this study revealed that S. Typhimurium is the most prevalent serotype detected in chicken feces. To reduce the risk to human health posed by salmonellosis, a stringent public health and food safety policy is required.

Impact of packaging atmosphere, oregano essential oil, and storage temperature on cold-adapted Salmonella Enteritidis and Salmonella Typhimurium on ready-to-eat smoked turkey.

The hazard of diseases created by S. Enteritidis and S. Typhimurium is relatively high in turkey meat products. Combinations of preservation methods are utilized in many strategies, such as mild heat with decreased water activity, a changed atmosphere, refrigerated storage, and decreased heat treatment with some acidification. Within the domain of ready-to-eat food technology, a range of preservation methods are typically utilized to enhance shelf life, such as applying mild heat in tandem with reduced water activity, employing modified atmosphere packaging, utilizing refrigerated storage, and utilizing reduced heat treatment combined with acidification. This investigation aimed to determine how S. Enteritidis and S. Typhimurium grew when sliced ready-to-eat smoked turkey (RTE-SM) was stored at 0, 5, 10, and 15°C for various periods. The study also examined the effects of modified atmosphere packaging (MAP) (40% CO2 and 60% N2) and VP on these growth patterns. Total viable count (TVC), lactic acid bacteria (LAB), pH, and redox potential levels were determined. The control experiment on RTE-SM showed no Salmonella growth within 30 d of storage at any temperature. This indicated that the RTE-SM in use did not initially contain S. Typhimurium and S. Enteritidis. Results indicated that the storage of RTE-SM using a combination of VP, MAP, and MAPEO with storage at 0 and 5°C did not allow for the pathogen to grow throughout storage. In comparison, at 10 and 15°C after one day, which allowed for minor growth (0.17-0.5 log CFU/g)? In contrast, at 0 and 5°C, Salmonella survives until the end of storage (173 d). However, the combination of MAPEO with the same storage temperatures achieved the elimination of the pathogen in the meat after 80 d. The combination of both packaging systems with high temperatures (10 or 15°C) allowed for the multiplication and growth of the bacterium through the product's shelf life of more than 1 log CFU/g. Thus, a combination of MAP or MAPEO with low storage temperatures (0 or 5°C) inhibited the growth of the pathogen.

Comparative efficacy of difloxacin and nano-emulsion difloxacin as antibacterial agents against Salmonella enterica Serovar enteritidis in chickenss.

Background: Avian salmonellosis is a group of diseases caused by bacteria from the genus Salmonella with a negative impact on poultry, particularly chickens. In addition, salmonellosis is a global food-borne infection. Aim: The aim of this study was to evaluate the effect of nano-emulsion difloxacin (NED) and commercial difloxacin (CD) water supplement on broiler's growth, feed intake, and body weight, weight gain, growth rate, feed conversion ratio (FCR), and mortality rate (MR). The antibiotic sensitivity was determined both in-vivo and in-vitro for NED against Salmonella enterica Serovar enteritidis in chickens. Methods: 1500 one-day of age chicks were grouped into five groups as follows: group 1 (G1) control negative group, G2 control positive group (infected and not treated), G3 (infected and treated with CD, and G4 and G5 (infected and treated with NED at different doses). Samples, including the intestine, liver, and spleen were collected. Agar well diffusion test and minimum inhibitory concentrations were adopted. Histopathological lesions on different tissues were studied. During 35 days of the experiment, the feed intake, growth rate, growth gain, FCR, and MR were recorded daily. In addition, a variety of analytical techniques including transmission electron microscopic analysis, dynamic light scattering, UV-visible spectroscopy, and zeta-potential analysis were applied to characterize NED. Results: The agar well diffusion test indicated that NED was in-vitro effective against S. enteritidis isolates than CD. The minimum inhibitory concentration was recorded as NED inhibited bacterial growth till well 8 at a concentration of 0.78 µg/ml; on the other hand, the CD inhibited bacterial growth till well 6 at a concentration of 0.62 µg/ml. Growth performance and MRs in the groups treated with NED are significantly reduced. Conclusion: Treatment of broiler's drinking water with NED at doses of 0.5 and 1 ml instead of pure CD was able to enforce a new perspective, antibacterial efficacy, enhancing the productive performance, and reducing the MRs of broilers.

Identification of metabolite biomarkers in Salmonella enteritidis-contaminated chickens using UHPLC-QTRAP-MS-based targeted metabolomics.

This study aimed to characterize the metabolic profile of Salmonella enteritidis (S. enteritidis) in chicken matrix and to identify metabolic biomarkers of S. enteritidis in chicken. The UHPLC-QTRAP-MS high-throughput targeted metabolomics approach was employed to analyze the metabolic profiles of contaminated and control group chickens. A total of 348 metabolites were quantified, and the application of deep learning least absolute shrinkage and selection operator (LASSO) modelling analysis obtained eight potential metabolite biomarkers for S. enteritidis. Metabolic abundance change analysis revealed significantly enriched abundances of anthranilic acid, l-pyroglutamic acid, 5-hydroxylysine, n,n-dimethylarginine, 4-hydroxybenzoic acid, and menatetrenone in contaminated chicken samples. The receiver operating characteristic (ROC) curve analysis demonstrated the strong ability of these six metabolites as biomarkers to distinguish S. enteritidis contaminated and fresh chicken samples. The findings presented in this study offer a theoretical foundation for developing an innovative approach to identify and detect foodborne contamination caused by S. enteritidis.

Evaluation of the Antimicrobial Activity of a Formulation Containing Ascorbic Acid and Eudragit FS 30D Microparticles for the Controlled Release of a Curcumin-Boric Acid Solid Dispersion in Turkey Poults Infected with Salmonella enteritidis: A Therapeutic Model.

The selection of components within a formulation or for treatment must stop being arbitrary and must be focused on scientific evidence that supports the inclusion of each one. Therefore, the objective of the present study was to obtain a formulation based on ascorbic acid (AA) and Eudragit FS 30D microparticles containing curcumin-boric acid (CUR-BA) considering interaction studies between the active components carried out via Fourier transform infrared spectrometry (FTIR) and differential scanning calorimetry (DSC) to minimize antagonistic effects, and comprehensively and effectively treat turkey poults infected with Salmonella enteritidis (S. enteritidis). The DSC and FTIR studies clearly demonstrated the interactions between AA, BA, and CUR. Consequently, the combination of AA with CUR and/or BA should be avoided, but not CUR and BA. Furthermore, the Eudragit FS 30D microparticles containing CUR-BA (SD CUR-BA MP) showed a limited release of CUR-BA in an acidic medium, but they were released at a pH 6.8-7.0, which reduced the interactions between CUR-BA and AA. Finally, in the S. enteritidis infection model, turkey poults treated with the combination of AA and SD CUR-BA MP presented lower counts of S. enteritidis in cecal tonsils after 10 days of treatment. These results pointed out that the use of an adequate combination of AA and CUR-BA as an integral treatment of S. enteritidis infections could be a viable option to replace the indiscriminate use of antibiotics.

Resistance profiles, virulence and antimicrobial resistance genes of XDR S. Enteritidis and S. Typhimurium.

Avian salmonellosis is concomitant with high financial crises in the poultry industry as well as food-borne illness in man. The present study is designed to investigate the emergence of Salmonella Enteritidis and Salmonella Typhimurium in diseased broilers, resistance profiles, and monitoring virulence and antibiotic resistance genes. Consequently, 450 samples (cloacal swabs, liver, and spleen) were collected from 150 diseased birds from different farms in Giza Governorate, Egypt. Subsequently, the bacteriological examination was done. Afterward, the obtained Salmonella isolates were tested for serogrouping, antibiogram, PCR monitoring of virulence (invA, stn, hilA, and pefA), and antimicrobial resistance genes (blaTEM, blaCTX-M, blaNDM, ermA, sul1, tetA, and aadA1). The total prevalence of Salmonella in the examined diseased broilers was 9.3%, and the highest prevalence was noticed in cloacal swabs. Among the recovered Salmonella isolates (n = 35), 20 serovars were recognized as S. Enteritidis and 15 serovars were identified as S. Typhimurium. Almost 60% of the retrieved S. Enteritidis serovars were extensively drug-resistant (XDR) to seven antimicrobial classes and inherited sul1, blaTEM, tetA, blaCTX-M, ereA, and aadA1 genes. Likewise, 25% of the recovered S. Enteritidis serovars were multidrug-resistant (MDR) to six classes and have sul1, blaTEM, tetA, blaCTX-M, and ereA resistance genes. Also, 66.7% of the retrieved S. Typhimurium serovars were XDR to seven classes and have sul1, blaTEM, tetA, blaCTX-M, ereA, and aadA1 genes. Succinctly, this report underlined the reemergence of XDR S. Typhimurium and S. Enteritidis in broiler chickens. Meropenem and norfloxacin exposed a hopeful antimicrobial activity toward the re-emerging XDR S. Typhimurium and S. Enteritidis in broilers. Moreover, the recurrence of these XDR Salmonella strains poses a potential public health threat.

Flaxseed Supplementation in Chicken Feed Accelerates Salmonella enterica subsp. enterica Serovar Enteritidis Clearance, Modulates Cecum Microbiota, and Influences Ovarian Gene Expression in Laying Hens.

Salmonella is a foodborne pathogen that poses a serious threat to both human and animal health and food safety. Flaxseed is rich in unsaturated fatty acids; has anti-metabolic syndrome, anti-inflammatory, and neuroprotective properties; and may be a potential source of feed additives. To investigate the impact of flaxseed on Salmonella-infected laying hens, we administered Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) after adding flaxseed to the feed of laying hens (15% [750 mg/kg]). S. Enteritidis colonization was reduced and its clearance was accelerated from the laying hens. Furthermore, flaxseed supplementation mitigated the damage to the ileum caused by S. Enteritidis. We analyzed alterations in intestinal flora through 16S rRNA amplicon sequencing. S. Enteritidis infection increased the abundance of Akkermansia and triggered the host inflammatory response. Conversely, the addition of flaxseed to the feed increased the abundance of beneficial intestinal bacteria, such as Lactobacilli and Bacteroides. Ovarian health is important for egg production performance in laying hens and our findings indicate that S. Enteritidis can persist in the ovaries for an extended period. Therefore, we further performed transcriptome sequencing analysis of ovarian tissues on day seven after S. Enteritidis infection. S. Enteritidis infection leads to altered ovarian gene expression, including the downregulation of lipid metabolism and growth and development genes and the upregulation of host immune response genes in laying hens. The upregulation of genes associated with growth and development may have stimulated ovarian growth and development.

Probiotic Escherichia coli Nissle 1917 protect chicks from damage caused by Salmonella enterica serovar Enteritidis colonization.

As a foodborne pathogen of global importance, Salmonella enterica serovar Enteritidis (S. Enteritidis) is a threat to public health that is mainly spread by poultry products. Intestinal Enterobacteriaceae can inhibit the colonization of S. Enteritidis and are regarded as a potential antibiotic substitute. We investigated, in chicks, the anti-S. Enteritidis effects of Escherichia coli (E. coli) Nissle 1917, the most well-known probiotic member of Enterobacteriaceae. Eighty 1-d-old healthy female AA broilers were randomly divided into 4 groups, with 20 in each group, namely the negative control (group P), the E. coli Nissle 1917-treated group (group N), the S. Enteritidis-infected group (group S) and the E. coli Nissle 1917-treated and S. Enteritidis-infected group (group NS). From d 5 to 7, chicks in groups N and NS were orally gavaged once a day with E. coli Nissle 1917 and in groups P and S were administered the same volume of sterile PBS. At d 8, the chicks in groups S and NS were orally gavaged with S. Enteritidis and in groups P and N were administered the same volume of sterile PBS. Sampling was conducted 24 h after challenge. Results showed that gavage of E. coli Nissle 1917 reduced the spleen index, Salmonella loads, and inflammation (P < 0.05). It improved intestinal morphology and intestinal barrier function (P < 0.05). S. Enteritidis infection significantly reduced mRNA expression of angiotensin-converting enzyme 2 (ACE2) and solute carrier family 6-member 19 (SLC6A19) in the cecum and the content of Gly, Ser, Gln, and Trp in the serum (P < 0.05). Pretreatment with E. coli Nissle 1917 yielded mRNA expression of ACE2 and SLC6A19 in the cecum and levels of Gly, Ser, Gln, and Trp in the serum similar to that of uninfected chicks (P < 0.05). Additionally, E. coli Nissle 1917 altered cecum microbiota composition and enriched the abundance of E. coli, Lactobacillales, and Lachnospiraceae. These findings reveal that the probiotic E. coli Nissle 1917 reduced S. Enteritidis infection and shows enormous potential as an alternative to antibiotics.

Application of a novel lytic Jerseyvirus phage LPSent1 for the biological control of the multidrug-resistant Salmonella Enteritidis in foods.

Non-typhoidal Salmonella is the tremendously predominant source of acquired foodborne infection in humans, causing salmonellosis which is a global threat to the healthcare system. This threat is even worse when it is combined with the incidence of multidrug-resistant Salmonella strains. Bacteriophage therapy has been proposed as a promising potential candidate to control a diversity of foodborne infective bacteria. The objective of this study designed to isolate and characterize lytic phages infecting zoonotic multi-drug resistant and strong biofilm producer Salmonella enterica serovar Enteritidis EG.SmE1 and then apply the isolated phage/s as a biocontrol agent against infections in ready-to-eat food articles including milk, water, apple juice, and chicken breasts. One lytic phage (LPSent1) was selected based on its robust and stable lytic activity. Phage LPSent1 belonged to the genus Jerseyvirus within the Jerseyvirinae subfamily. The lysis time of phage LPSent1 was 60 min with a latent period of 30 min and each infected cell burst about 112 plaque-forming units. Phage LPSent1 showed a narrow host range. Furthermore, the LPSent1 genome did not encode any virulence or lysogenic genes. In addition, phage LPSent1 had wide pH tolerance, prolonged thermal stability, and was stable in food articles lacking its susceptible host for 48 h. In vitro applications of phage LPSent1 inhibited free planktonic cells and biofilms of Salmonella Enteritidis EG.SmE1 with a lower occurrence to form phage-resistant bacterial mutants which suggests promising applications on food articles. Application of phage LPSent1 at multiplicities of infections of 100 or 1000 showed significant inhibition in the bacterial count of Salmonella Enteritidis EG.SmE1 by 5 log10/sample in milk, water, apple juice, and chicken breasts at either 4°C or 25°C. Accordingly, taken together these findings establish phage LPSent1 as an effective, promising candidate for the biocontrol of MDR Salmonella Enteritidis in ready-to-eat food.

Antioxidative and Cytoprotective Efficacy of Ethanolic Extracted Cranberry Pomace against Salmonella Enteritidis Infection in Chicken Liver Cells.

Salmonella enterica serovar Enteritidis is a globally significant zoonotic foodborne pathogen. Chicken liver is a vital organ that has been recently implicated in several reported human salmonellosis outbreaks in the U.S. One promising strategy for reducing Salmonella in chickens could be through supplementation with natural antimicrobial additives. Ethanolic extracted cranberry pomace (CPOH) is an excellent source of bioactive polyphenolic compounds with antioxidant and antimicrobial activities. However, the protective effect of CPOH against S. Enteritidis-induced chicken hepatic cell damage remains unclear. In this study, we used a chicken hepatoma cell (LMH) infection model to investigate the protective effects and potential mechanisms of CPOH. CPOH increased the viability of S. Enteritidis-infected LMH cells. Furthermore, CPOH reduced the adhesion and invasion of S. Enteritidis to LMH cells. CPOH downregulated the expression of Rho GTPase genes that are essential for Salmonella's entry into LMH cells. Additionally, the expression of antioxidant regulatory genes, such as Nrf2, HO-1, Txn, and Gclc, was increased. Our data show that CPOH effectively protected LMH cells from cell damage through the inhibition of S. Enteritidis adhesion and invasion, as well as the induction of the expression of master antioxidant genes. These findings offer opportunities to develop sustainable, safe, and economic strategies to reduce the colonization and pathogenesis of Salmonella.

The vertical transmission of Salmonella Enteritidis in a One-Health context.

Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) is a foodborne zoonotic pathogen, causing economic losses in animal husbandry and large numbers of human deaths and critically threatening economic development and public health. Human infection with SE has complex transmission routes, involving the environment, animal reservoirs, and water in a One-Health context. Food-producing animals, particularly poultry and livestock, are regarded as the most common sources of SE infection in humans. However, there is little known about the vertical transmission of SE in a One-Health context. In this review, we analyze the ecological significance of SE in a One-Health context. Importantly, we focus on the difference in vertical transmission of SE in poultry, livestock, and humans. We introduce the transmission pathway, describe the immune mechanisms, and discuss the models that could be used for studying the vertical transmission of SE and the strategy that prevention and control for vertical transmission of SE into the future from a One-Health perspective. Together, considering the vertical transmission of SE, it is helpful to provide important insights into the control and decontamination pathways of SE in animal husbandry and enhance knowledge about the prevention of fetal infection in human pregnancy.

Isolation and Characterization of Chi-like Salmonella Bacteriophages Infecting Two Salmonella enterica Serovars, Typhimurium and Enteritidis

Salmonella enterica Serovar Typhimurium and Salmonella enterica Serovar Enteritidis are well-known pathogens that cause foodborne diseases in humans. The emergence of antibiotic-resistant Salmonella serovars has caused serious public health problems worldwide. In this study, two lysogenic phages, STP11 and SEP13, were isolated from a wastewater treatment plant in Jeddah, KSA. Transmission electron microscopic images revealed that both phages are new members of the genus "Chivirus" within the family Siphoviridae. Both STP11 and SEP13 had a lysis time of 90 min with burst sizes of 176 and 170 PFU/cell, respectively. The two phages were thermostable (0 °C ≤ temperature < 70 °C) and pH tolerant at 3 ≤ pH < 11. STP11 showed lytic activity for approximately 42.8% (n = 6), while SEP13 showed against 35.7% (n = 5) of the tested bacterial strains. STP11 and STP13 have linear dsDNA genomes consisting of 58,890 bp and 58,893 bp nucleotide sequences with G + C contents of 57% and 56.5%, respectively. Bioinformatics analysis revealed that the genomes of phages STP11 and SEP13 contained 70 and 71 ORFs, respectively. No gene encoding tRNA was detected in their genome. Of the 70 putative ORFs of phage STP11, 27 (38.6%) were assigned to functional genes and 43 (61.4%) were annotated as hypothetical proteins. Similarly, 29 (40.8%) of the 71 putative ORFs of phage SEP13 were annotated as functional genes, whereas the remaining 42 (59.2%) were assigned as nonfunctional proteins. Phylogenetic analysis of the whole genome sequence demonstrated that the isolated phages are closely related to Chi-like Salmonella viruses.

Antimicrobial resistance patterns and virulence gene profiles of Salmonella enteritidis and Salmonella typhimurium recovered from patients with gastroenteritis in three cities of Iran.

This study evaluated distribution of virulence factors and antibiotic resistance in clinical isolates of Salmonella enteritidis and Salmonella typhimurium in three cities of Iran. Altogether 48 S. enteritidis and S. typhimurium isolates were collected from patients at certain Iranian hospitals between May 2018 and September 2021. Antimicrobial susceptibility testing was performed by disk diffusion and broth microdilution methods. The presence of antibiotic-resistance genes (blaTEM,blaSHV,blaCTX-M,blaNDM,strA, strB, aadA1, tetA, tetB, floR, sul1, sul2, dfrA), integrons (classe 1 and 2), and virulence-associated genes (invA, stn, sopB, spvC, rck, phoPQ) was investigated by PCR and sequencing. Antimicrobial agents like trimethoprim-sulfamethoxazole and imipenem represent highly efficient agents with 97% susceptibility. S. enteritidis and S. typhimurium exhibited high resistance to ciprofloxacin (n = 20, 71.43%) and ceftazidime (n = 9, 45%), respectively. Overall, 3 (6.25%), 13 (27.08%), and 6 (12.5%) isolates were divided into strong, moderate, and weak biofilm producers, respectively. Moreover, blaCTX-M,blaTEM, blaSHV, sul1, sul2, tetA, tetB, floR, strA, and strB resistant genes were detected in 10 (20.8%), 5 (10.4%), 1 (2.08%), 7 (14.58%), 1 (2.08%), 3 (6.25%), 2 (4.1%), 1 (2.08%), 2 (4.1%), 2 (4.1%), respectively. Furthermore, 7 (14.58%) strains had classe 1 integron. All tested S. enteritidis strains had invA and sopB, and all S. typhimurium strains had invA and phoPQ. However, spvC remained undetected in all isolates. Extensive surveillance and efficient control measures against infection help to stop the upsurge of various antibiotic-resistant isolates.

Preclinical evaluation of OMVs as potential vaccine candidates against Salmonella enterica serovar Enteritidis infection.

Salmonella enterica serovar Enteritidis is the most prevalent serotype that causes human infections worldwide. Consumption of S. Enteritidis-contaminated animal foods is a major source of human infections; however, eradicating bacteria from animals remains difficult. Therefore, it is necessary to develop new measures to prevent and control salmonellosis. Here, we used the outer-membrane vesicles (OMVs) of S. Enteritidis and assessed their protective efficacy and immune response in mice. Deletion of tolR in S. Enteritidis increased the production and size of OMVs compared to those in the wild type (WT) and ΔrfaQ strains. Intramuscular immunization with OMVs conferred greater protection than intraperitoneal and intranasal immunization. Moreover, OMVs extracted from both WT and ΔtolR strains provided an 83.3% protective rate in mice challenged with S. Enteritidis, which was higher than that provided by OMVs extracted from the ΔrfaQ strain. However, compared with OMVs from the ΔtolR strain, OMVs from WT and ΔrfaQ strains rapidly eradicated S. Enteritidis colonizing the liver, spleen, ileum, and cecum of BALB/c mice after immunization. Immunization with OMVs from each of the three strains induced humoral immune responses and showed no side effects on the growth of mice. Our study revealed that OMVs from various S. Enteritidis strains could be developed for use as subunit vaccine candidates against nontyphoidal Salmonella infections in mammals.

Evaluation of the use of CRISPR loci for discrimination of Salmonella enterica subsp. enterica serovar Enteritidis strains recovered in Canada and comparison with other subtyping methods.

Salmonella enterica subsp. enterica serovar Enteritidis remains one of the most important foodborne pathogens worldwide. To minimise its public health impact when outbreaks of the disease occur, timely investigation to identify and recall the contaminated food source is necessary. Central to this approach is the need for rapid and accurate identification of the bacterial subtype epidemiologically linked to the outbreak. While traditional methods of S. Enteritidis subtyping, such as pulsed field gel electrophoresis (PFGE) and phage typing (PT), have played an important role, the clonal nature of this organism has spurred efforts to improve subtyping resolution and timeliness through molecular based approaches. This study uses a cohort of 92 samples, recovered from a variety of sources, to compare these two traditional methods for S. Enteritidis subtyping with recently developed molecular techniques. These latter methods include the characterisation of two clustered regularly interspaced short palindromic repeats (CRISPR) loci, either in isolation or together with sequence analysis of virulence genes such as fimH. For comparison, another molecular technique developed in this laboratory involved the scoring of 60 informative single nucleotide polymorphisms (SNPs) distributed throughout the genome. Based on both the number of subtypes identified and Simpson's index of diversity, the CRISPR method was the least discriminatory and not significantly improved with the inclusion of fimH gene sequencing. While PT analysis identified the most subtypes, the SNP-PCR process generated the greatest index of diversity value. Combining methods consistently improved the number of subtypes identified, with the SNP/CRISPR typing scheme generating a level of diversity comparable with that of PT/PFGE. While these molecular methods, when combined, may have significant utility in real-world situations, this study suggests that CRISPR analysis alone lacks the discriminatory capability required to support investigations of foodborne disease outbreaks.

Effect of Lactobacillus reuteri S5 Intervention on Intestinal Microbiota Composition of Chickens Challenged with Salmonella enteritidis.

To understand the mechanism of lactic acid bacteria against Salmonella enteritidis infection; we examined how lactic acid bacteria regulated the intestinal microbiota to resist infection by pathogenic bacteria. The probiotic strain Lactobacillus reuteri S5 was used to construct an animal model of S. enteritidis infected broilers. A high-throughput sequencing technology was used to analyze the regulatory effects of L. reuteri S5 on the structure of the intestinal microbiota of broilers infected with S. enteritidis; and to examine the possible defense mechanism they used. Our results showed that the administration of L. reuteri S5 reduced colonization of S. enteritidis (p < 0.05), decreased intestinal permeability (p < 0.05), and reduced the bacterial displacement likely due by S. enteritidis colonization (p < 0.05), suggesting some enhancement of the intestinal barrier function. Furthermore, L. reuteri S5 increased the number of operational taxonomic units (OTUs) in the chicken cecal microflora and the relative abundance of Lactobacillaceae and decreased the relative abundance of Enterobacteriaceae. These results suggest that the lactic acid bacterium L. reuteri S5 protected the intestinal microbiota of chickens against S. enteritidis infection.

Characterization of the Role of Two-Component Systems in Antibiotic Resistance Formation in Salmonella enterica Serovar Enteritidis.

The two-component system (TCS) is one of the primary pathways by which bacteria adapt to environmental stresses such as antibiotics. This study aimed to systematically explore the role of TCSs in the development of multidrug resistance (MDR) in Salmonella enterica serovar Enteritidis. Twenty-six in-frame deletion mutants of TCSs were generated from S. Enteritidis SJTUF12367 (the wild type [WT]). Antimicrobial susceptibility tests with these mutants revealed that 10 TCSs were involved in the development of antibiotic resistance in S. Enteritidis. In these 10 pairs of TCSs, functional defects in CpxAR, PhoPQ, and GlnGL in various S. Enteritidis isolates led to a frequent decrease in MIC values against at least three classes of clinically important antibiotics, including cephalosporins and quinolones, which indicated the importance of these TCSs to the formation of MDR. Interaction network analysis via STRING revealed that the genes cpxA, cpxR, phoP, and phoQ played important roles in the direct interaction with global regulatory genes and the relevant genes of efflux pumps and outer membrane porins. Quantitative reverse transcription-PCR analysis further demonstrated that the increased susceptibility to cephalosporins and quinolones in ΔphoP and ΔcpxR mutant cells was accompanied by increased expression of membrane porin genes (ompC, ompD, and ompF) and reduced expression of efflux pump genes (acrA, macB, and mdtK), as well as an adverse transcription of the global regulatory genes (ramA and crp). These results indicated that CpxAR and PhoPQ played an important role in the development of MDR in S. Enteritidis through regulation of cell membrane permeability and efflux pump activity. IMPORTANCE S. Enteritidis is a predominant Salmonella serotype that causes human salmonellosis and frequently exhibits high-level resistance to commonly used antibiotics, including cephalosporins and quinolones. Although TCSs are known as regulators for bacterial adaptation to stressful conditions, which modulates ß-lactam resistance in Vibrio parahaemolyticus and colistin resistance in Salmonella enterica serovar Typhimurium, there is little knowledge of their functional mechanisms underlying the development of antibiotic resistance in S. Enteritidis. Here, we systematically identified the TCS elements in S. Enteritidis SJTUF12367, revealed that the three TCSs CpxAR, PhoPQ, and GlnGL were crucial for the MDR formation in S. Enteritidis, and preliminarily illustrated the regulatory functions of CpxAR and PhoPQ for antimicrobial resistance genes. Our work provides the basis to understand the important TCSs that regulate formation of antibiotic resistance in S. Enteritidis.

Antimicrobial resistance genes, virulence markers and prophage sequences in Salmonella enterica serovar Enteritidis isolated in Tunisia using whole genome sequencing.

Salmonella Enteritidis causes a major public health problem in the world. Whole genome sequencing can give us a lot of information not only about the phylogenetic relatedness of these bacteria but also in antimicrobial resistance and virulence gene predictions. In this study, we analyzed the whole genome data of 45 S. Enteritidis isolates recovered in Tunisia from different origins, human, animal, and foodborne samples. Two major lineages (A and B) were detected based on 802 SNPs differences. Among these SNPs, 493 missense SNPs were identified. A total of 349 orthologue genes mutated by one or two missense SNPs were classified in 22 functional groups with the prevalence of carbohydrate transport and metabolism group. A good correlation between genotypic antibiotic resistance profiles and phenotypic analysis were observed. Only resistant isolates carried the respective molecular resistant determinants. The investigation of virulence markers showed the distribution of 11 Salmonella pathogenicity islands (SPI) out of 23 previously described. The SPI-1 and SPI-2 genes encoding type III secretion systems were highly conserved in all isolates except one. In addition, the virulence plasmid genes were present in all isolates except two. We showed the presence of two fimbrial operons sef and ste previously considered to be specific for typhoidal Salmonella. Our collection of S. Enteritidis reveal a diversity among prophage profiles. SNPs analysis showed that missense mutations identified in fimbriae and in SPI-1 and SPI-2 genes were mostly detected in lineage B. In conclusion, WGS is a powerful application to study functional genomic determinants of S. Enteritidis such as antimicrobial resistance genes, virulence markers and prophage sequences. Further studies are needed to predict the impact of the missenses SNPs that can affect the protein functions associated with pathogenicity.

Molecular detection of Salmonella isolated from commercial chicken.

Background: Salmonella in chicken, specially, the motile salmonellae, causes the food chain unsafe from farm to table and is considered a significant global threat to public health. Aims: The present study was carried out for molecular detection of Salmonellae in commercial poultry using PCR. Methods: The study was conducted for eight months, from July 2019 to February 2020, and a total of 26 poultry farms, including 15 broiler and 11-layer farms, were visited individually. Pooled faecal samples were obtained from the sheds. A total of 189 necropsy cases were examined for gastrointestinal lesions. Isolation and identification of the organism were done using microbe culture method, and the molecular characterization was performed via PCR targeting invA and ent genes. Results: The prevalence of salmonellosis in the broiler and layer farms was recorded at 20.0% and 45.4%, respectively, through the traditional gold standard culture method. From 189 necropsy birds, salmonellosis was recorded at 1.58% dead cases. Molecular detection of Salmonella isolates by PCR targeting invA gene was confirmed in 13.33% of the broiler farms and 36.3% of the layer farms. Further detection of Salmonella enteritidis was performed by PCR targeting ent gene by which 11.11% positivity was determined. Conclusion: This study, focused on the Salmonella prevalence, highlighted the zoonotic importance of the bacterium in the commercial poultry farms, which can subsequently be dispersed into the human food chain causing harmful health effects.

Salmonella Enteritidis GalE Protein Inhibits LPS-Induced NLRP3 Inflammasome Activation.

Microbial infection can trigger the assembly of inflammasomes and promote secretion of cytokines, such as IL-1ß and IL-18. It is well-known that Salmonella modulates the activation of NLRC4 (NLR family CARD domain-containing protein 4) and NLRP3 (NLR family pyrin domain-containing 3) inflammasomes, however the mechanisms whereby Salmonella avoids or delays inflammasome activation remain largely unknown. Therefore, we used Salmonella Enteritidis C50336ΔfliC transposon library to screen for genes involved in modulating inflammasomes activation. The screen revealed the galactose metabolism-related gene galE to be essential for inflammasome activation. Here, we found that inflammasome activation was significantly increased in J774A.1 cells or wild-type bone marrow-derived macrophages (BMDMs) during infection by ΔfliCΔgalE compared to cells infected with ΔfliC. Importantly, we found that secretion of IL-1ß was Caspase-1-dependent, consistent with canonical NLRP3 inflammasome activation. Furthermore, the virulence of ΔfliCΔgalE was significantly decreased compared to ΔfliC in a mouse model. Finally, RNA-seq analysis showed that multiple signaling pathways related to the inflammasome were subject to regulation by GalE. Taken together, our results suggest that GalE plays an important role in the regulatory network of Salmonella evasion of inflammasome activation.