Serovar Enteritidis Research Articles

Occurrence of ciprofloxacin resistance, plasmid-mediated quinolone resistance genes and virulence factors in Salmonella enterica serovar Enteritidis isolated from broilers in Thailand isolated from broilers in Thailand

Salmonella Enteritidis is one of the most common serovars associated with gastroenteritis in humans. Fluoroquinolone resistance in non-typhoidal Salmonella including S. Enteritidis has increased globally and is considered as a threat to public health. In this study, we aimed to investigate the occurrence of ciprofloxacin resistance and plasmid-mediated quinolone resistance (PMQR) genes, and examine virulence gene profiles of 69 S. Enteritidis isolates recovered from 46 boot swab and 23 intestinal samples collected from 69 commercial intensive broiler farms in Thailand. Ciprofloxacin susceptibility of these isolates was determined using microbroth dilution method. PCR was used to detect 5 common PMQR genes (qnrA, qnrB, qnrS, aac(6')-Ib-cr and qepA) and 12 important virulence genes (agfA, invA, spaN, prgH, sitC, ssaQ, mgtC, sopB, sifA, tolC, cdtB and spvC). All S. Enteritidis showed reduced susceptibility to ciprofloxacin, with the MIC values of 0.125-0.50 µg/mL. However, these isolates did not carry PMQR genes investigated. The same virulence profile was observed among 69 S. Enteritidis isolates in which 11 virulence genes, except cdtB, were detected. The presence of virulence genes identified in invasive salmonellosis in the S. Enteritidis isolates with reduced susceptibility to ciprofloxacin could be of public health concerns. Our findings underline the need for constant monitoring of ciprofloxacin-resistant S. Enteritidis in poultry production chain to reduce public health risk.

Mitigation of Salmonella on Food Contact Surfaces by Using Organic Acid Mixtures Containing 2-Hydroxy-4-(methylthio) Butanoic Acid (HMTBa)

Contaminated surfaces can transmit pathogens to food in industrial and domestic food-handling environments. Exposure to pathogens on food contact surfaces may take place via the cross-contamination of pathogens during postprocessing activities. Formaldehyde-based commercial sanitizers in recent years are less commonly being used within food manufacturing facilities due to consumer perception and labeling concerns. There is interest in investigating clean-label, food-safe components for use on food contact surfaces to mitigate contamination from pathogenic bacteria, including Salmonella. In this study, the antimicrobial effects of two types of organic acid mixtures containing 2-hydroxy-4-(methylthio) butanoic acid (HMTBa), Activate DA™ and Activate US WD-MAX™, against Salmonella when applied onto various food contact surfaces were evaluated. The efficacy of Activate DA (HMTBa + fumaric acid + benzoic acid) at 1% and 2% and Activate US WD-MAX (HMTBa + lactic acid + phosphoric acid) at 0.5% and 1% against Salmonella enterica (serovars Enteritidis, Heidelberg, and Typhimurium) were evaluated on six different material surfaces: plastic (bucket elevator and tote bag), rubber (bucket elevator belt and automobile tire), stainless steel, and concrete. There was a significant difference in the Salmonella log reduction on the material surfaces due to the organic acid treatments when compared to the untreated surfaces. The type of material surface also had an effect on the log reductions obtained. Stainless steel and plastic (tote) had the highest Salmonella log reductions (3-3.5 logs), while plastic (bucket elevator) and rubber (tire) had the lowest log reductions (1-1.7 logs) after treatment with Activate US WD-MAX. For Activate DA, the lowest log reductions (~1.6 logs) were observed for plastic (bucket elevator) and rubber (tire), and the highest reductions were observed for plastic (tote), stainless steel, and concrete (2.8-3.2 logs). Overall, the results suggested that Activate DA at 2% and Activate US WD-MAX at 1% are potentially effective at reducing Salmonella counts on food contact surfaces by 1.6-3.5 logs.

Salmonella Enteritidis RfbD enhances bacterial colonization and virulence through inhibiting autophagy

Autophagy is a crucial innate immune response that clears pathogens intracellularly. Salmonella enterica serovar Enteritidis (S.E) has emerged as one of the most important food-borne pathogens. Here, we reported that dTDP-4-dehydro-β-ւ-rhamnose reductase (RfbD) was able to enhance bacterial colonization in vivo and in vitro by regulating autophagy. We screened the transposon mutant library of Salmonella Enteritidis strain Z11 by High-Content Analysis System, found that rfbD gene has an effect on autophagy. The Z11ΔrfbD-infected group showed greater expression of LC3-II than the Z11-infected group in HeLa, RAW264.7, and J774A.1 cells. Overall, the survival of Z11ΔrfbD in RAW264.7 cells was reduced after 8 h of infection compared to that of the Z11 wild-type strain. In addition, we observed that inhibition of autophagic flux significantly increased the survival of Z11ΔrfbD in RAW264.7 cells. Mice infection experiments revealed that Z11ΔrfbD virulence was significantly reduced, and bacterial load was reduced in the liver and cecum in mice model, and LC3-II expression was significantly increased. These findings indicate an important role of Salmonella Enteritidis protein as a strategy to suppress autophagy and provides new ideas for manipulating autophagy as a novel strategy to treat infectious diseases.

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.

Prebiotic properties of fermented ceri Terengganu (Lepisanthes fruticosa) beverage by survival of lactic acid bacteria and its antibacterial activity

The present study was undertaken to investigate the survival of lactic acid bacteria (LAB) in the fermented ceri Terengganu beverage and its antibacterial activity. The preliminary study of the survival of five selected strains of LAB involved was Bifidobacterium bifidum UABb-10™, Lactobacillus acidophilus DDS®-1, Lactobacillus paracasei UALpc -04™, Lactobacillus plantarum UALp-05™, and Streptococcus thermophilus UASt-09™. The viability of each strain was tested in fermented ceri Terengganu beverage (pH 3.25) where all strains showed a survival rate of at least 92.5%. A total of five types of foodborne pathogens namely Escherichia coli O517:H7 UPMEC32, Listeria monocytogenes ATCC® 51772TM, Salmonella enterica serovar Enteritidis MDC15, Salmonella enterica serovar Typhimurium ATCC® 53648TM and Streptococcus gallolyticus ATCC® 9809TM were selected to determine the antibacterial activity and minimum bactericidal concentration (MBC>99) of fermented ceri Terengganu beverage. Antibacterial activity using the agar well diffusion method inhibited five tested food-borne pathogens at varying extents of inhibition zone ranging between 12.92 to 18.50 mm in diameter. Another antibacterial activity assay using the broth microdilution method also confirmed the 100% inhibition effect of fermented ceri Terengganu beverage against these selected pathogenic microorganisms even though the beverage has been diluted to 50%. The synergetic effect of a significant amount of multiple organic acids present in fermented ceri Terengganu beverages was the main factor contributing to its potent antibacterial properties. This finding indicated the potential of fermented ceri Terengganu beverage as a prebiotic beverage and might be able to reduce the risk of food poisoning incidence as it has shown a good antibacterial effect against selected foodborne pathogens.

Genotyping and antimicrobial resistance profiles of chicken originated Salmonella Enteritidis isolates.

Salmonellosis is a common foodborne zoonosis worldwide. The most common Salmonella serovar in humans is Salmonella enterica subsp. enterica serovar Enteritidis (50.3%) in the world. The main transmission route for S. Enteritidis is consumption of contaminated poultry products. Therefore, it is important to determine the diversity and spread of chicken-originated S. Enteritidis isolates in order to monitor and control salmonellosis. Pulsed-field gel electrophoresis (PFGE) and multiple locus variable number of tandem repeats analysis (MLVA) are frequently used for typing of S. Enteritidis isolates. This study aimed to determine the antimicrobial resistance (AMR) profiles and MLVA and PFGE genotypes of chicken-originated S. Enteritidis isolates. A total of 200 S. Enteritidis isolated from chicken broiler, layer, and breeder flocks from different locations in Turkey were investigated by Kirby-Bauer disk diffusion method, PFGE, and MLVA. The AMR test indicated that 57% of the S. Enteritidis isolates were susceptible to all antimicrobials, while 39% were resistant to at least one antimicrobial. The highest resistance (25%) was against ampicillin. Multi-drug resistance rate was low (21%) and mostly from broiler flocks (93%). All isolates were genotyped into 32 different PFGE genotypes (PT) and 34 different MLVA genotypes (MT). The dominant genotypes were PT6 (12.5%) and MT22 (50%). In specific sample groups, there was a correlation between genotypes, breeding type, geographic location, and isolation years of the isolates. There was no significant difference in the discrimination power of PFGE and MLVA. However, MLVA was more suitable for large sample groups and routine genotyping because it was easier, quicker, and less labor-intensive to use.

Genetic and phenotypic assessment of the antimicrobial activity of three potential probiotic lactobacilli against human enteropathogenic bacteria.

Lactobacilli are avid producers of antimicrobial compounds responsible for their adaptation and survival in microbe-rich matrices. The bactericidal or bacteriostatic ability of lactic acid bacteria (LAB) can be exploited for the identification of novel antimicrobial compounds to be incorporated in functional foodstuffs or pharmaceutical supplements. In this study, the antimicrobial and antibiofilm properties of Lactiplantibacillus pentosus L33, Lactiplantibacillus plantarum L125 and Lacticaseibacillus paracasei SP5, previously isolated form fermented products, were examined, against clinical isolates of Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Enteritidis and Escherichia coli. The ability of viable cells to inhibit pathogen colonization on HT-29 cell monolayers, as well as their co-aggregation capacity, were examined utilizing the competitive exclusion assay. The antimicrobial activity of cell-free culture supernatants (CFCS) was determined against planktonic cells and biofilms, using microbiological assays, confocal microscopy, and gene expression analysis of biofilm formation-related genes. Furthermore, in vitro analysis was supplemented with in silico prediction of bacteriocin clusters and of other loci involved in antimicrobial activity. The three lactobacilli were able to limit the viability of planktonic cells of S. aureus and E. coli in suspension. Greater inhibition of biofilm formation was recorded after co-incubation of S. enterica with the CFCS of Lc. paracasei SP5. Predictions based on sequence revealed the ability of strains to produce single or two-peptide Class II bacteriocins, presenting sequence and structural conservation with functional bacteriocins. The efficiency of the potentially probiotic bacteria to elicit antimicrobial effects presented a strain- and pathogen-specific pattern. Future studies, utilizing multi-omic approaches, will focus on the structural and functional characterization of molecules involved in the recorded phenotypes.

Addition of a protected complex of biofactors and antioxidants to breeder hen diets confers transgenerational protection against Salmonella enterica serovar Enteritidis in progeny chicks

Addition of vitamins and antioxidants has been long associated with increased immunity and are commonly used in the poultry industry; however, less is known regarding their use in broiler breeder hens. The objective of this study was to determine if feeding a complex of protected biofactors and antioxidants composed of vitamins and fermentation extracts to broiler breeder hens conferred resistance against Salmonella enterica serovar Enteritidis (S. Enteritidis) in the progeny chicks. Three-day-old chicks from control- and supplement-fed hens were challenged with S. Enteritidis and necropsied 4- and 11-days postchallenge (dpc) to determine if there were differences in invasion and colonization. Serum and jejunum were evaluated for various cytokine and chemokine production. Fewer (P=0.002) chicks from supplement-fed hens had detectable S. Enteritidis in the ceca (32.6%) compared to chicks from control-fed hens (64%). By 11 dpc, significantly (P < 0.001) fewer chicks from supplement-fed hens were positive for S. Enteritidis (liver [36%]; ceca [16%]) compared to chicks from the control hens (liver [76%]; ceca [76%]). The recoverable S. Enteritidis in the cecal content was also lower (P=0.01) at 11 dpc. In additional to the differences in invasion and colonization, cytokine and chemokine production were distinct between the 2 groups of chicks. Chicks from supplement-fed hens had increased production of IL-16, IL-6, MIP-3α, and RANTES in the jejunum while IL-16 and MIP-1β were higher in the serum of chicks from the control-fed hens. By 11 dpc, production of IFN-γ was decreased in the jejunum of chicks from supplement-fed hens. Collectively, these data demonstrate adding a protected complex of biofactors and antioxidants to the diet of broiler breeder hens offers a measure of transgenerational protection to the progeny against S. Enteritidis infection and reduces colonization that is mediated, in part, by a robust and distinct cytokine and chemokine response locally at the intestine and systemically in the blood.

Genomic and phylogenetic analysis of Salmonella enterica serovar Enteritidis strains linked to multiple outbreaks in Brazil.

Salmonella enterica subsp. enterica serovar Enteritidis (SE) has become the prevalent serovar isolated from gastroenteritis cases in Brazil since the 1990s. To better understand the genomic diversity and phylogenetic relationship amongst SE epidemic isolates from Brazil, 30 SE isolates from a variety of implicated foods and case patients of outbreaks between 1999 and 2006 were selected for genome comparison analyses. SE genomes were also compared against publicly available Brazilian SE isolates from pre- and postepidemic period. MLST analysis revealed that all isolates belong to sequence type (ST) 11. A total of seven Salmonella pathogenicity islands (SPIs) (SPI-1, SPI-3-5, SPI-13, SPI14, and C63PI) were identified in the evaluated genomes and all studied SE genomes carried similar prophage profiling. Resistome analysis revealed the presence of resistance genes to aminoglycosides [aac(6')laa, aph(3")-lb, aph(6)-ld], as well as point mutations in gyrA. Phylogenetic analysis demonstrated that certain isolates have circulated in Brazil for years and been involved in distinct outbreaks.

Antimicrobial Resistance and Mechanisms of Azithromycin Resistance in Nontyphoidal Salmonella Isolates in Taiwan, 2017 to 2018.

Antimicrobial resistance was investigated in 2,341 nontyphoidal Salmonella (NTS) isolates recovered from humans in Taiwan from 2017 to 2018 using antimicrobial susceptibility testing. Azithromycin resistance determinants were detected in 175 selected isolates using PCR and confirmed in 81 selected isolates using whole-genome sequencing. Multidrug resistance was found in 47.3% of total isolates and 96.2% of Salmonella enterica serovar Anatum and 81.7% of S. enterica serovar Typhimurium isolates. Resistance to the conventional first-line drugs (ampicillin, chloramphenicol, and cotrimoxazole), cefotaxime and ceftazidime, and ciprofloxacin was found in 32.5 to 49.0%, 20.3 to 20.4%, and 3.2% of isolates, respectively. A total of 76 (3.1%) isolates were resistant to azithromycin, which was associated with mph(A), erm(42), erm(B), and possibly the enhanced expression of efflux pump(s) due to ramAp or defective ramR. mph(A) was found in 53% of the 76 azithromycin-resistant isolates from 11 serovars and located in an IS26-mph(A)-mrx(A)-mphR(A)-IS6100 unit in various incompatibility plasmids and the chromosomes. erm(42) in S. enterica serovar Albany was carried by an integrative and conjugative element, ICE_erm42, and in S. enterica serovar Enteritidis and S. Typhimurium was located in IS26 composite transposons in the chromosomes. erm(B) was carried by IncI1-I(α) plasmids in S. Enteritidis and S. Typhimurium. ramAp was a plasmid-borne ramA, a regulatory activator of efflux pump(s), found in only S. enterica serovar Goldcoast. Since the azithromycin resistance determinants are primarily carried on mobile genetic elements, they could easily be disseminated among human bacterial pathogens. The ramAp-carrying S. Goldcoast isolates displayed azithromycin MICs of 16 to 32 mg/L. Thus, the epidemiological cutoff value of ≤16 mg/L of azithromycin proposed for wild-type NTS should be reconsidered. IMPORTANCE Antimicrobial resistance in NTS isolates is a major public health concern in Taiwan, and the mechanisms of azithromycin resistance are rarely investigated. Azithromycin and carbapenems are the last resort for the treatment of invasive salmonellosis caused by multidrug-resistant (MDR) and extensively drug-resistant Salmonella strains. Our study reports the epidemiological trend of resistance in NTS in Taiwan and the genetic determinants involved in azithromycin resistance. We point out that nearly half of NTS isolates from 2017 to 2018 are MDR, and 20% are resistant to third-generation cephalosporins. The azithromycin resistance rate (3.1%) for the NTS isolates from Taiwan is much higher than those for the NTS isolates from the United States and Europe. Our study also indicates that azithromycin resistance is primarily mediated by mph(A), erm(42), erm(B), and ramAp, which are frequently carried on mobile genetic elements. Thus, the azithromycin resistance determinants could be expected to be disseminated among diverse bacterial pathogens.

Membrane vesicles released by Lacticaseibacillus casei BL23 inhibit the biofilm formation of Salmonella Enteritidis

Biofilms represent a major concern in the food industry and healthcare. The use of probiotic bacteria and their derivatives as an alternative to conventional treatments to fight biofilm development is a promising option that has provided convincing results in the last decades. Recently, membrane vesicles (MVs) produced by probiotics have generated considerable interest due to the diversity of roles they have been associated with. However, the antimicrobial activity of probiotic MVs remains to be studied. In this work, we showed that membrane vesicles produced by Lacticaseibacillus casei BL23 (LC-MVs) exhibited strong antibiofilm activity against Salmonella enterica serovar Enteritidis (S. Enteritidis) without affecting bacterial growth. Furthermore, we found that LC-MVs affected the early stages of S. Enteritidis biofilm development and prevented attachment of bacteria to polystyrene surfaces. Importantly, LC-MVs did not impact the biomass of already established biofilms. We also demonstrated that the antibiofilm activity depended on the proteins associated with the LC-MV fraction. Finally, two peptidoglycan hydrolases (PGHs) were found to be associated with the antibiofilm activity of LC-MVs. Overall, this work allowed to identify the antibiofilm properties of LC-MVs and paved the way for the use of probiotic MVs against the development of negative biofilms.

Abstract 2647: Antibiotic resistance of Salmonella enterica serovar Enteritidis mediated by EnvZ/OmpR two-component system in response to β-lactams

Abstract 2647: Antibiotic resistance of Salmonella enterica serovar Enteritidis mediated by EnvZ/OmpR two-component system in response to β-lactams

Atividade antimicrobiana de produtos à base de monopersulfato de potássio em bactérias associadas a infecções aviárias

Abstract The objective of this work was to evaluate the antimicrobial activity of four new potassium monopersulfate-based products on bacterial biofilms associated with avian infections, in order to disinfect drinking fountains in poultry farms. Initially, tests were performed in planktonic bacterial cells, to verify the antimicrobial activity and the minimum inhibitory concentration of the products, named PA, PB, PC, and PD. These products were tested on mature biofilms of the avian pathogens Escherichia coli, Salmonella enterica subsp. enterica serovar Enteritidis, and Listeria monocytogenes, grown on specimens preserved in acrylic blocks, by counting colony-forming units, scanning electron microscopy, and confocal fluorescence microscopy. All products were effective against the evaluated bacterial species. The PA and PB products inhibited the bacterial growth at ≤ 0.13% concentrations, and PC and PD showed the same effect at ≤ 0.25% concentrations. Furthermore, the PA product was able to eliminate mature biofilms of S. enterica subsp. enterica serovar Enteritidis and L. monocytogenes. The evaluated monopersulfate-based products, notably PA, are effective against bacterial biofilms associated with avian infections and show potential as sanitizers and disinfectants for drinking fountains in poultry farms.

Genotyping of Salmonella isolates from chickens and ducks and sequence analysis of two chicken isolates of Salmonella Enterica serovar Enteritidis

Salmonella causes drastic losses in chicken and duck farms and is considered a major food-borne pathogen with public health concern. Out of 400 chicken and duck samples distributed as follows: (260 samples were collected from native species and 140 from imported species). Salmonella infection in local species represented 5.4% (14/260) and 3.6% (5/140) in imported poultry. A total seven Salmonella serotypes were obtained as follows: S. Enteritidis, S. Typhimurum, S. Kentuky, S. Virchow, S. Infantis, S. Remo, and S. Seremban by serotyping using the (Kauffmann–White scheme). In the same time, all the isolates were subjected to molecular identification by using five primers targeting (sefA, STM4495, ISR2-ISR3, ORF, and Hypothertical protein) which were specific for S. Enteritidis, S. Typhimurum, S. Infantis, S. Kentuky, and S. Virchow, respectively. Genotyping provided rapid, sensitive, and accurate method for Salmonella classification. Phylogenetic analysis was performed onto 2 strains of isolated Salmonella enteritidis (one isolated from imported chicks and the other one from local chicks based on sequencing of the PCR product of the sefA gene. The sequences showed close identity of local and imported isolates with other Salmonella reference strains in different countries. On conclusion, it is important to implement strict monitoring policy of Salmonella in imported and local poultry for prevention and control of Salmonella and formulation of effective vaccines to protect the poultry industry.

Hybrid Genomic Analysis of Salmonella enterica Serovar Enteritidis SE3 Isolated from Polluted Soil in Brazil.

In Brazil, Salmonella enterica serovar Enteritidis is a significant health threat. Salmonella enterica serovar Enteritidis SE3 was isolated from soil at the Subaé River in Santo Amaro, Brazil, a region contaminated with heavy metals and organic waste. Illumina HiSeq and Oxford Nanopore Technologies MinION sequencing were used for de novo hybrid assembly of the Salmonella SE3 genome. This approach yielded 10 contigs with 99.98% identity with S. enterica serovar Enteritidis OLF-SE2-98984-6. Twelve Salmonella pathogenic islands, multiple virulence genes, multiple antimicrobial gene resistance genes, seven phage defense systems, seven prophages and a heavy metal resistance gene were encoded in the genome. Pangenome analysis of the S. enterica clade, including Salmonella SE3, revealed an open pangenome, with a core genome of 2137 genes. Our study showed the effectiveness of a hybrid sequence assembly approach for environmental Salmonella genome analysis using HiSeq and MinION data. This approach enabled the identification of key resistance and virulence genes, and these data are important to inform the control of Salmonella and heavy metal pollution in the Santo Amaro region of Brazil.

The occurrence of antibiotic-resistant enteric bacteria in Selected Nigerian traditional dairy products.

Wara and nono are popular dairy products in Nigeria, rich in nutrients, highly exposed to microbial contaminants during processing and sale and support microbial growth. To investigate occurrence and antibiotic resistance pattern of enteric bacterial pathogens in dairy products. Dairy products were serially diluted and cultured on Eosin Methylene Blue agar, Salmonella-Shigella agar, McConkey agar and nutrient agar at 37°C for 24 h. Characterisation and identification of isolates with API 20E kit (Biomereux, France). Antibiotic susceptibility was with agar disc diffusion. Polyvalent O and H antisera for Salmonella serotyping. Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Typhi, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter aerogenes, Enterobacter cloacae and Serratia marcescens, were identified. Dominant enteric bacterium detected was E. coli followed by Salmonella spp. Serratia marcescens was the least occurring. The isolates were most resistant to trimethoprim-sulfamethoxazole (96.7%), amoxicillin (83.3%), augmentin (83.3%), chloramphenicol (66.7%), streptomycin (50%). They were resistant to ≥ 4 (multiple) antibiotics, E. coli 8, Salmonella spp. 7, Serratia marcescens 6 and Klebsiella spp. and Enterobacter spp. 4 each. The presence of enteric bacterial pathogens in wara and nono and their resistance to multiple antibiotics was reported in this study.

Phosphoproteomics Profile of Chicken Cecum in the Response to Salmonella enterica Serovar Enteritidis Inoculation

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a foodborne pathogen, which can cause great threats to human health through the consumption of contaminated poultry products. This research combines TMT labeling, HPLC and mass-spectrometry-based phosphoproteomics on cecum of the F1 cross of Guangxi Yao chicken and Jining Bairi chicken. The treated group was inoculated with 0.3 mL inoculum S. Enteritidis, and the control group was inoculated with 0.3 mL phosphate-buffered saline (PBS). A total of 338 differentially phosphorylated modification sites in 243 differentially phosphorylated proteins (DPPs) were chosen for downstream analyses. A total of 213 sites in 146 DPPs were up-regulated and 125 sites in 97 DPPs were down-regulated. Functional analysis was performed for DPPs based on gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and the protein domain. The DPPs were mainly enriched in immune- and metabolic-related GO-BP (biological process) and KEGG pathways. We predicted and classified the subcellular structure and COG/KOG of DPPs. Furthermore, protein-protein interaction network analyses were performed by using multiple algorithms. We identified 71 motifs of the phosphorylated modification sites and selected 18 sites randomly to detect the expression level through parallel reaction monitoring (PRM). S. Enteritidis inoculation caused phosphorylation alteration in immune- and metabolic-related proteins. The invasion of S. Enteritidis may be actualized by inducing cecum cell apoptosis through the endoplasmic reticulum pathway, and chickens could resist the invasion of S. Enteritidis by affecting the function of ECM receptors. The findings herein provide a crucial theoretical foundation to understand the molecular mechanism and epigenetic regulation in response to S. Enteritidis inoculation in chickens.

Regulation of mRNA and miRNA in the response to Salmonella enterica serovar Enteritidis infection in chicken cecum

BackgroundSalmonella enterica, serovar Enteritidis (SE) is a food-borne pathogen, which can cause great threat to human health through consumption of the contaminated poultry products. Chicken is the main host of SE. The mRNA and microRNA (miRNA) expression profiles were analyzed on cecum of Shouguang chicken via next-generation sequencing and bioinformatics approaches. The treated group was inoculated SE, and the control group was inoculated with phosphate buffer saline (PBS).ResultsThere were 1760 differentially expressed mRNAs in the SE-infected group, of which 1046 were up-regulated mRNA, and 714 were down-regulated mRNA. In addition, a total of 821 miRNAs were identified, and 174 miRNAs were differentially expressed, of which 100 were up-regulated and 74 were down-regulated. Functional enrichment of differentially expressed mRNAs was similar to miRNA target genes. The functional analysis results of differentially expressed mRNAs and miRNAs were performed. Immune-related processes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways were enriched by up-regulated mRNA. The down-regulated mRNAs were enriched in tissue development and metabolic-related KEGG pathways. The functional analysis of up-regulated miRNA target genes was similar to the down-regulated mRNAs. The down-regulated miRNA target genes were enriched in metabolic-related GO (Gene Ontology) -BP (Biological process) terms and KEGG pathways. The overlap of the up-regulated mRNA and the up-regulated miRNA target genes (class I) was 325, and the overlap of the down-regulated miRNA target genes (class II) was 169. The class I enriched in the immune-related GO-BP terms and KEGG pathways. The class II mainly enriched in metabolic-related GO-BP terms and KEGG pathways. Then we detected the expression of mRNA and miRNA through qRT-PCR. The results shown that the expression of HHIP, PGM1, HTR2B, ITGB5, RELN, SFRP1, TCF7L2, SCNN1A, NEK7, miR-20b-5p, miR-1662, miR-15a, miR-16-1-3p was significantly different between two groups. Dual-luciferase reporter assay was used to detect the relationship between miR-20b-5p and SCNN1A. The result indicated that miR-20b-5p regulate immune or metabolic responses after SE infection in Shouguang chickens by directly targeting SCNN1A.ConclusionsThe findings here contribute to the further analysis of the mechanism of mRNA and miRNA defense against SE infection, and provide a theoretical foundation for the molecular disease-resistant breeding of chickens.

Detection and Molecular Identification of Salmonella Pathogenic Islands and Virulence Plasmid Genes of Salmonella in Xuzhou Raw Meat Products

Virulence genes expressed in Salmonella are a primary contributing factor leading to the high morbidity and mortality of salmonellosis in humans. The pathogenicity of Salmonella is mainly determined by the specific virulence factors that it carries. These factors also confer greater virulence and play a role in infection of a host and transmission of disease, and most Salmonella enterica can cause cross-infections between humans and animals. In this study, 265 samples in total were collected from a farmer's market and two supermarkets in Xuzhou, Jiangsu province, China, including 205 pork samples and 60 chicken samples. The suspected Salmonella isolates were isolated and identified using microbiological and molecular methods, and the confirmed isolates were used for serovar analysis and antimicrobial susceptibility testing. The virulence genes of Salmonella pathogenic islands (SPIs) and Salmonella virulence plasmids (Spv) in Salmonella-positive isolates were subsequently detected. Salmonella was isolated from 29.0% of samples, and all isolates were confirmed by PCR targeting the stn gene. Among the Salmonella isolates, resistance was most frequently observed against ciprofloxacin (84.4%), followed by tetracycline (71.4%) and streptomycin (68.8%). Resistance to amoxicillin-clavulanic acid (6.3%) and aztreonam (5%) was less commonly detected. The presence of the following virulence genes was determined by specific PCRs: hilA (SPI-1), sifA (SPI-2), misL (SPI-3), siiE (SPI-4), sopB (SPI-5), and spvC. The detection rate for SPI-1 to SPI-5 was 93.5, 87.0, 97.4, 97.4, and 97.4%, respectively. In addition, the detection rate of the spvC gene was 96.1%. Except for sopB (94.7%), all isolates of the dominant serovar S. enterica subsp.. enterica serovar Enteritidis contained all virulence genes from SPI-1 to SPI-5. This study demonstrated the epidemiological status of Salmonella in raw meat products in Xuzhou, and the complex antibiotic resistance and high isolation rate of virulence genes observed reveal many potential risks of which the findings presented herein will provide orientation to improve public health safeguards.

Symbiotic bacteria stabilize the intestinal environment by producing phenylpropanoids.

Salmonella enterica serovar Enteritidis (S. Enteritidis) can colonize in the intestinal tract of chickens and transmit to humans. In order to decrypt the mechanism of avian resistance to S. Enteritidis, we utilized two China local chicken breeds to generate the reciprocal crosses (the Cross and the Reverse-cross). The two lines of hybrids were orally inoculated with S. Enteritidis at 2-day old and sampled at 3 days post-inoculation. Along the analysis direction of multi-omics, differential metabolites, functional pathways and correlated microbes, we found that 12 species of microbes thrived upon S. Enteritidis challenge and probably contributed to the intestinal stability in the Cross by enhancing the production of phenylpropanoids. Our findings can help to understand the symbiotic and resistant mechanisms derived from the intestinal microbiota.