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To disclose the antimicrobial susceptibility and wide adaptability of commonly occurring genotypes of Salmonella enterica serovar Typhimurium, the antimicrobial resistance and multilocus sequence t...

Salmonellosis occurs frequently worldwide, causing serious threats to public health safety. The abuse of antibiotics is increasing the antibiotic resistance in bacteria, thereby making the prevention and control of Salmonella more difficult. A phage can help control the spread of bacteria. In this study, S55, a lytic phage, was isolated from faecal samples obtained from poultry farms using Salmonella Pullorum ( S . Pullorum) as the host bacterium. This phage belongs to Siphoviridae and has a polyhedral head and a retraction-free tail. S55 showed a strong ability to lyse Salmonella serovars, such as S . Pullorum (58/60, 96.67%) and S . Enteritidis (97/104, 93.27%). One-step growth kinetics showed that the latent period was 10 min, burst period was 80 min and burst size was 40 pfu/cell. The optimal multiplicity of infection was 0.01, and the phage was able to survive at a pH of 4-11 and temperature of 40°C-60°C for 60 min. Complete genome sequence analysis revealed that the S55 genome length is 42,781 bp (GC content, 50.28%) and it contains 58 open reading frames (ORF), including 25 ORFs with known or assumed functions, without tRNA genes. Moreover, S55 does not carry genes that encode virulence or resistance factors. At different temperatures (4°C or 25°C), S55 was found to lower the populations of S . Pullorum and S . Enteritidis on chicken skin surface. Its bacteriostatic effect at 4°C was higher than that at 25°C. In conclusion, S55 can be considered a promising biological agent for the prevention and control of Salmonella .

Salmonella spp. is a major foodborne pathogen that is distributed among most pork production chains worldwide. This study aimed to investigate the dynamic changes in Salmonella spp. along the pig breeding process monthly from April 2018 to March 2019 in a pig farm in Shanghai, China, and identify the potential critical control points during the production. In total, 239 Salmonella spp. isolates were obtained from 1389 samples, in which Salmonella were detected from 26.3% (222/843) of fecal samples, 7.1% (17/240) of feed samples, and 0.0% (0/306) of both water and insect samples. Seven different serotypes were identified, with the predominant serotype being Salmonella Derby (21.8%), followed by Salmonella Typhimurium (18.8%), Salmonella Rissen (16.3%), Salmonella Mbandaka (12.6%), and Salmonella 1,4,[5],12:i:- (11.8%). Most probable number (MPN) analysis revealed that the load of Salmonella spp. gradually increased along the pig production chain, while the highest number of Salmonella spp. isolates was at the fattening stage (MPN value, 11-15 MPN/g). The pulsed-field gel electrophoresis showed that both Salmonella Typhimurium and Salmonella Derby isolates were grouped to six clusters. The antimicrobial resistance analyzed demonstrated that 80.0% of the isolates were of multidrug resistance and resistant to sulfamethoxazole (84.5%), lincomycin (89.4%), ampicillin (96.9%), oxytetracycline (93.8%), and tetracycline (95.1%). We further evaluated the Salmonella spp. Resistance to quaternary ammonium compounds (QACs) showed an increasing trend along with the testing period indicating that the use of QACs could induce the resistance of Salmonella spp. to QACs. Our study confirmed the dynamic changes in Salmonella spp. over time and space in this pig farm and identified feed and the fattening house as the key points for the prevention and control of Salmonella spp. contamination.

The type VI secretion system (T6SS) encoded by Salmonella Pathogenicity Island 19 (SPI-19) has been confirmed to be involved in bacterial infection or colonization in hosts and in the inhibition of the host T-cell immune response. However, deletion of the core genes (clpV, vgrG, and hcp2) encoding the T6SS apparatus does not affect the phenotypes caused by SPI-19-encoded T6SS. As Salmonella infection in host cells and survival in chickens are closely associated with the type III secretion system (T3SS), RNA-Seq was performed, and the results revealed that most T3SS genes were downregulated in the C79-13ΔSPI-19 mutant. To identify the SPI-19 genes involved in regulating T3SS genes expression, we constructed mutants of genes encoding potential regulators (RS09140 and RS09275) or proteins with Sel1-like motifs (RS09150 and RS09155) and analyzed their associated phenotypes. Deletion of RS09150 and RS09155 caused the decreased bacterial infection in avian cells and bacterial colonization in chicken organs. In addition, qRT-PCR results revealed that both mutants showed decreased expression levels of regulatory genes of T3SS. The present findings demonstrate that the two Sel1-like proteins RS09150 and RS09155 in S. Pullorum SPI-19 contribute to bacterial infection in chickens by mediating the expression of T3SS genes, indicating a potential crosstalk between SPI-19 and T3SS in Salmonella.

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Salmonella enterica serovar Pullorum (S. Pullorum) causes pullorum disease (PD), which is an acute systemic disease, in chickens, and leads to serious economic losses in many developing countries because of its high morbidity and mortality rate in young chicks. The live-attenuated vaccine is considered to be an effective measure to control the Salmonella infection. In addition, the DIVA (differentiation of infected and vaccinated animals) feature without the interference of serological monitoring of Salmonella infection is an important consideration in the development of the Salmonella vaccine. In this study, we evaluated the immunogenicity and protective efficacy of a S. Pullorum rough mutant S06004ΔspiCΔrfaH as a live attenuated DIVA vaccine candidate in chickens. The S06004ΔspiCΔrfaH exhibited a significant rough lipopolysaccharides (LPS) phenotype which was agglutinated with the acriflavine, not with the O9 mono antibody. Compared to the wild-type, 50% lethal dose (LD50) of the rough mutant increased 100-fold confirmed its attenuation. The mutant strain also showed a decreased bacterial colonization in the spleen and liver. The immunization with the mutant strain had no effect on the body weight and no tissue lesions were observed in the liver and spleen. The high level of the S. Pullorum-specific IgG titers in the serum indicated that significant humoral immune responses were induced in the immunization group. The cellular immune responses were also elicited from the analysis of lymphocyte proliferation and expression of cytokines in the spleen. In addition, the S06004ΔspiCΔrfaH immunized group exhibited a negative response for the serological test, while the wild-type S06004 infection group was strongly positive for the serological test showing a DIVA capability. The survival rates in the vaccinated chickens were 87% after intramuscular challenge with wild-type S. Pullorum, while the survival rates were 20% in the control groups. Overall, these results have demonstrated that the rough mutant S06004ΔspiCΔrfaH strain can be developed as an efficient live attenuated DIVA vaccine candidate to control the systemic S. Pullorum infection without the interference of salmonellosis monitoring program in poultry.

Background Polymyxins are currently regarded as a possible last-resort therapy to eradicate multidrug-resistant (MDR) gram-negative bacteria. Meanwhile, the old antimicrobial agent fosfomycin has recently been reintroduced into clinical use for the treatment of extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant Enterobacteriaceae. This study investigated a multidrug-resistant Salmonella 4,[5],12:i:- strain from a food catering handler, which had the potential to act as a vehicle for transmitting MDR foodborne pathogens. Methods A Salmonella 4,[5],12:i:- YZU1189 strain was isolated from the fecal sample of a food catering worker according to the standard protocol of the Salmonella detection method from World Health Organization in 2003. Serotyping of YZU1189 was performed according to the Kauffmann-White scheme. The antimicrobial resistance phenotype of the strain was determined by the agar dilution method according to the instruction from Clinical and Laboratory Standards Institute (CLSI). Plasmid conjugation was performed between the donor strain Salmonella 4,[5],12:i:- YZU1189 and the recipient strain Escherichia coli C600. The genetic locations of mcr-1, bla CTX-M-14 and fosA3 genes were determined by the whole genome sequence analysis. Results Salmonella 4,[5],12:i:- YZU1189 was an ESBL-producing stain isolated from a healthy catering worker. The strain displayed resistance to aminoglycosides, beta-lactams, polymyxins, fosfomycins, phenicols, trimethoprims, sulfonamides, tetracyclines and fluoroquinolones. Whole genome sequence analysis and plasmid conjugation revealed that the strain had a transferable IncHI2 plasmid carrying the mcr-1, bla CTX-M-14 and fosA3 genes. Sequence homology analysis showed that this plasmid possessed high sequence similarity to previously reported mcr-1, bla CTX-M-14 and fosA3 positive plasmids in China. Conclusion This study reported a the multidrug-resistant Salmonella 4,[5],12:i:- isolate harboring mcr-1, bla CTX-M-14 and fosA3 from human for the first time in China. The occurrence of mcr-1 and fosA3 genes in the transferable IncHI2 plasmid pYZU1189 from the ESBL-producing Salmonella 4,[5],12:i:- isolate showed a potential threat to public health. Great concern should be taken for the spread of multidrug-resistant ESBL-producing Salmonella isolates from food catering workers to consumers.

Salmonella spp. are important zoonotic pathogens that are responsible for severe diseases in both animals and humans. Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) and biovar Pullorum (S. Pullorum) are typical infectious pathogens detected in the chicken industry that have caused great economic losses. To facilitate their detection and prevent contamination, we developed a rapid multiple PCR method, which can simultaneously detect Salmonella spp. and further identify the biovars S. Pullorum/Gallinarum. This PCR detection method is based on the cigR gene, which is conserved among Salmonella spp. but has a 42-bp deletion in S. Pullorum/Gallinarum. The specificity and sensitivity of the PCR assay was evaluated with 41 different strains: 34 Salmonella strains, including 5 S. Pullorum/Gallinarum strains, and 7 non-Salmonella strains. The lower limit of detection was 8.15 pg of S. Pullorum (S06004) genomic DNA and 20 cfu in PCR, which shows a great sensitivity. In addition, this method was applied to detect or identify Salmonella from processing chicken liver and egg samples, and the results corresponded to those obtained from serotype analysis using the conventional slide agglutination test. Overall, the new cigR-based PCR assay is efficient and practical for Salmonella detection and S. Pullorum/Gallinarum identification and will greatly reduce the workload of epidemiologic investigation.

In this study, a carbapenem-resistant Salmonella enterica serovar Typhimurium strain W131 (sequence type 34) was isolated from retail pork in Jiangsu province, China. An IncX3 plasmid carrying blaNDM-5 element was identified in this strain by whole-genome sequencing analysis. The conjugation experiment demonstrated that the plasmid can be transferred to Escherichia coli recipient J53 and conferred carbapenem resistance to the recipient strain. This study first reported a Salmonella Typhimurium strain with blaNDM-5 from retail pork, which revealed that retail meat as a potential transmission factor of carbapenem-resistant blaNDM-5 to be a threat human health.

TNF receptor-associated factor 6 (TRAF6) is a signal transducer, which plays a pivotal role in triggering a variety of signalling cascades. Here, we cloned and identified the TRAF6 gene from the King pigeon. The open reading frame sequence of pigeon TRAF6 (piTRAF6) is 1638 bp long and encodes a 545 aa protein, including a low-complexity domain, RING finger, Zinc finger, coiled coil domain, and meprin and TRAF homology domain. The aa sequence of piTRAF6 shared a strong identity with that of other birds. PiTRAF6 transcripts were broadly expressed in all the tested tissues; piTRAF6 levels were the highest and lowest in the heart and stomach, respectively. Overexpression of piTRAF6 activated NF-κB in a dose-dependent manner and induced IFN-β expression. Upon piTRAF6 knockdown by small interfering RNAs, NF-κB activation was markedly inhibited in HEK293T cells. The expression of piTRAF6, as well as pro-inflammatory cytokines and antiviral molecules, were obviously increased after TLR ligand stimulation and Newcastle disease virus or Salmonella Pullorum inoculation. These results suggest that piTRAF6 may play a key immunoregulatory role in the innate immune response against viral and bacterial infections.

The applications of carvacrol are limited due to its poor stability and water solubility, and high volatility; however, ovalbumin can be used to encapsulate hydrophobic molecules, improve their aqueous solubility, and reduce their volatility. In this study, we fabricated ovalbumin-carvacrol nanoparticles (OCGns) under different pH (2, 5, 7, and 9) conditions using a gel embedding method and investigated their physicochemical and antibacterial properties. Rheological experiments revealed that the G' of ovalbumin gels (OGs) prepared under different pH conditions were OG-2 > OG-7 > OG-9 > OG-5. Carvacrol addition reduced the tight structure of ovalbumin and carvacrol under pH 5 and 7 conditions, with hardness first decreasing and then increasing, but increasing under pH 2 and 9 conditions. Fluorescence and infrared spectroscopy indicated complex formation, with carvacrol increasing the average diameter of nanoparticles prepared at pH 2, 5, 7, and 9. Encapsulation reached 89.34 and 91.86% at pH 2 and 9, respectively; however, inhibition experiments revealed that the minimum inhibitory concentration of OCGn-2 against Gram-positive Bacillus cereus and Salmonella (0.08 and 0.16 mg mL-1, respectively) was lower than that of OCGn-9 (both 0.28 mg mL-1). Moreover, OCGn-2 possessed a better dense gel structure and a higher stability, encapsulation rate, and antibacterial activity, suggesting that pH affects gel microstructure and thus the encapsulation efficiency and bacteriostatic properties of the prepared nanoparticles. These results contribute to our knowledge of the design and fabrication of polymeric nanoparticle delivery systems for bioactive compounds with beneficial properties.

CRISPR-based typing was performed to subtype isolates of S. Typhimurium and its monophasic variant Salmonella 4,[5],12:i:- from humans and animals between 2009 and 2017 in China. CRISPR typing classified all isolates into two lineages and four sub-lineages. All isolates from Lineage II and Lineage IB-1 were Salmonella Typhimurium. All of Salmonella 4,[5],12:i: – isolates were distributed in Lineage IA and Lineage IB-2, which all belonged to ST34 by MLST typing. Only Lineage IB-2 contained ST34 isolates from both Salmonella Typhimurium and Salmonella 4,[5],12:i:-. Among the isolates of ST34, TST4 was identified as the most common CRISPR type representing 86.5% of Salmonella 4,[5],12:i:- and 14.5 % of Salmonella Typhimurium mainly from pigs and humans. This study demonstrated that TST4-ST34 isolates were predominant in Salmonella 4,[5],12:i:-, and pig was the main reservoir for Salmonella 4,[5],12:i:- in China, which might have the potential to transmit to humans by pig production.

Made available in DSpace on 2022-04-29T08:36:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-10-01 One characteristic of the few Salmonella enterica serovars that produce typhoid-like infections is that disease-free persistent infection can occur for months or years in a small number of individuals post-convalescence. The bacteria continue to be shed intermittently which is a key component of the epidemiology of these infections. Persistent chronic infection occurs despite high levels of circulating specific IgG. We have reviewed the information on the basis for persistence in S. Typhi, S. Dublin, S. Gallinarum, S. Pullorum, S. Abortusovis and also S. Typhimurium in mice as a model of persistence. Persistence appears to occur in macrophages in the spleen and liver with shedding either from the gall bladder and gut or the reproductive tract. The involvement of host genetic background in defining persistence is clear from studies with the mouse but less so with human and poultry infections. There is increasing evidence that the organisms (i) modulate the host response away from the typical Th1-type response normally associated with immune clearance of an acute infection to Th2-type or an anti-inflammatory response, and that (ii) the bacteria modulate transformation of macrophage from M1 to M2 type. The bacterial factors involved in this are not yet fully understood. There are early indications that it might be possible to remodulate the response back towards a Th1 response by using cytokine therapy. SRUC Aberdeen Campus, Craibstone Estate, Ferguson Building Institute of Molecular Physiology Shenzhen Bay Laboratory Departamento de Patologia Veterinária Faculdade de Ciências Agrárias e Veterinárias Univ Estadual Paulista, Via de Acesso Paulo Donato Castellane, s/n Jiangsu Key Laboratory of Zoonosis Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses Yangzhou University School of Veterinary Medicine University of Surrey, Daphne Jackson Road Departamento de Patologia Veterinária Faculdade de Ciências Agrárias e Veterinárias Univ Estadual Paulista, Via de Acesso Paulo Donato Castellane, s/n

One characteristic of the few Salmonella enterica serovars that produce typhoid-like infections is that disease-free persistent infection can occur for months or years in a small number of individuals post-convalescence. The bacteria continue to be shed intermittently which is a key component of the epidemiology of these infections. Persistent chronic infection occurs despite high levels of circulating specific IgG. We have reviewed the information on the basis for persistence in S. Typhi, S. Dublin, S. Gallinarum, S. Pullorum, S. Abortusovis and also S. Typhimurium in mice as a model of persistence. Persistence appears to occur in macrophages in the spleen and liver with shedding either from the gall bladder and gut or the reproductive tract. The involvement of host genetic background in defining persistence is clear from studies with the mouse but less so with human and poultry infections. There is increasing evidence that the organisms (i) modulate the host response away from the typical Th1-type response normally associated with immune clearance of an acute infection to Th2-type or an anti-inflammatory response, and that (ii) the bacteria modulate transformation of macrophage from M1 to M2 type. The bacterial factors involved in this are not yet fully understood. There are early indications that it might be possible to remodulate the response back towards a Th1 response by using cytokine therapy.

Egg safety and quality is a long-standing concern for producers and consumers. Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) is the prevalent egg-product-related foodborne pathogen. The treatment with organic acids, including lactic acid, for the elimination and control of foodborne pathogens is generally recognized as safe, and is widely applied in many foods. In this study, we found that spraying with 2% lactic acid on the eggshell surface effectively reduced Salmonella Enteritidis counts. Agar-filled eggs were used to study the bacterial penetration of the eggshell (eggshell and membranes), and the contamination of the whole egg contents was investigated. No significant differences were observed between the Salmonella Enteritidis penetration in lactic acid sprayed and unsprayed eggs stored at 4 °C. The Haugh unit, albumin pH, and eggshell strength values were similar in the sprayed and washed eggs. Average Haugh unit scores for sprayed eggs were still Grade A within 3 weeks. However, the lactic acid treatment decreased the eggshell strength and cuticle layer thickness. SEM observations confirmed that spraying of lactic acid damaged the cuticle integrity. The results from our research will indicate that lactic acid spray is highly effective in reducing Salmonella contamination on the eggshell surface, but trans-shell penetration was demonstrated in the eggs stored at room temperature. Consequently, low-temperature storage is important for the prevention of recontamination of the egg after spraying.

Salmonella is a worldwide foodborne pathogen causing human disease. Food handlers, who are potential carriers of Salmonella, may transmit the pathogen to consumers through food. To determine the prevalence of Salmonella enterica serovars among food handlers working in the catering industry in Nantong, China, a total of 214,542 food handlers' fecal samples were tested for Salmonella in the Nantong CDC (Centers for Disease Control) from 2012 to 2017. Among those tested, 193 (0.09%) were identified to be positive for Salmonella, and the highest detection rate was 0.16% during the period of July to September. Serotyping analysis showed that Salmonella enterica serovar Typhimurium was the predominant serotype (16.1%), followed by Salmonella Derby (13.5%), Salmonella Enteritidis (11.4%), and Salmonella London (11.4%). The high detection rate of Salmonella Derby was probably closely related to its high prevalence of the serotype in pork, which is the primary meat consumed by the Chinese. Antibiotic susceptibility analysis demonstrated that 73.4% of the isolates were multidrug-resistant (MDR) strains with predominant resistance to ampicillin (AMP, 64.6%), followed by resistance to sulfisoxazole (SUL, 58.1%), nalidixic acid (55.8%), and tetracycline (TET, 44.5%). Therefore, MDR Salmonella strain carriage among food handlers working in the catering industry might be a potential source of human salmonellosis, especially for the predominant MDR genotype isolates (32.3%) with resistance to AMP, SUL, and TET.

In this study, Salmonella prevalence and antimicrobial resistance were evaluated at various production stages in 2 geographically separated breeder farms (referred to as G and F). Day-old chicks for the breeder flock at farm F were purchased from farm G. A total of 219 Salmonella isolates, all identified as Salmonella enterica subsp. enterica serovar Enteritidis, were recovered from 1,430 samples (sick chicken carcasses and/or dead embryos). The isolation rates at breeder farms G and F were 10.53% (56/532) and 18.15% (163/898), respectively. Resistance to 4–6 antimicrobial agents was the most frequent phenotype during the laying stage at both farms, suggesting that chicks are exposed to higher risk of antimicrobial-resistant Salmonella infection during this stage of the breeding process. Using clustered regularly interspaced short palindromic repeat (CRISPR) typing, 5 CRISPR patterns were identified, out of which one pattern was shared by the 2 farms. In addition, pulsed-field gel electrophoresis (PFGE) typing result indicated that 2 clusters (PF-1 and PF-2) were shared among the 2 breeder farms, suggesting that strains were transmitted from breeder farm G to farm F via the trade of day-old chicks. Our findings suggested that the trade of day-old breeder chicks could be one of the potential Salmonella transmission routes, and antibiotics should be administered with caution during the laying stage.

Salmonella enterica is a major foodborne pathogen causing symptomatic diseases or asymptomatic infections in humans. To reveal the genetic difference of Salmonella strains from patients to that from asymptomatic people, we used CRISPR typing to analyze the phylogenetic relationship of 180 clinical strains during 2017-2018 in Jiangsu, China. The CRISPR typing divided these isolates into 76 CRISPR types with a discriminatory power of 97.6%. S. Typhimurium and its monophasic variants of 6 CRISPR types are the significant serotypes causing both human diseases and asymptomatic infection, while S. Enteritidis mainly resulted in diseases and shared one CRISPR type. The spacer HadB20 displayed as a new molecular marker to differentiate ST34-S. Typhimurium monophasic variant from ST19-S. Typhimurium. S. Derby, S. London, and S. Senftenberg frequently caused asymptomatic infection with diverse CRISPR types, while S. Mbandaka and S. Meleagridis, occasionally isolated from patients, had conserved CRISPR types. Additionally, 30 of 516 newly identified spacers showed homology to sequences in both plasmids and bacteriophages. Interestingly, some spacers from one serotype showed homology to the correspondent prophage or plasmid sequences in another serotype; and more than two spacers identified in one strain showed homology to the sequences located in the identical plasmids or phages, revealing the constant evolution of Salmonella CRISPR arrays during the interactions between bacteria and phages or plasmids.

The monophasic variant of Salmonella Typhimurium has emerged and increased rapidly worldwide during the past two decades. The loss of genes encoding the second-phase flagella and the acquirement of the multi-drug resistance cassette are the main genomic characteristics of the S. Typhimurium monophasic variant. In this study, two Salmonella strains were isolated from the knee effusion and feces of a 4-year-old girl who presented with a case of septic arthritis and fever, respectively. Primary serovar identification did not detect the second-phase flagellar antigens of the strains using the classical slide agglutination test. Whole-genome sequencing analysis was performed to reveal that the replacement of the fljAB operon by a 4.8-kb cassette from E. coli caused the non-expression of phase-2 flagellar antigens of the strains, which were confirmed to be a novel S. Virchow monophasic variant (Salmonella 6,7,14:r:-) by core-genome multi-locus sequence typing (cgMLST). Compared to the 16 published S. Virchow genomes, the two strains shared a unique CRISPR type of VCT12, and showed a close genetic relationship to S. Virchow BCW_2814 and BCW_2815 strains, isolated from Denmark and China, respectively, based on cgMLST and CRISPR typing. Additionally, the acquisition of Salmonella genomic island 2 (SGI2) with an antimicrobial resistance gene cassette enabled the strains to be multidrug-resistant to chloramphenicol, tetracycline, trimethoprim, and sulfamethoxazole. The emergence of the multidrug-resistant S. Virchow monophasic variant revealed that whole-genome sequencing and CRISPR typing could be applied to identify the serovaraints of Salmonella enterica strains in the national Salmonella surveillance system.

The study of the interaction mechanism between bacteriophage and host is helpful in promoting development of bacteriophage applications. The mechanism of the interaction with the phage was studied by constructing the rfbN gene deletion and complemented with strains of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium, S. Typhimurium) D6. The rfbN gene deletion strain could not be lysed by phage S55 and led to a disorder of lipopolysaccharide (LPS) biosynthesis, which changed from the smooth type to rough type. Also, the RfbN protein lacking any of the three-segment amino acid (aa) sequences (90-120 aa, 121-158 aa, and 159-194 aa) produces the same result. Transmission electron microscopy and confocal microscopy assays demonstrated that phage S55 dramatically reduced adsorption to the rfbN deletion strain as compared to the wild strain D6. After co-incubation of the S55 with the purified smooth LPS, D6 could not be lysed, indicating that the smooth LPS binds to the S55 in vitro and then inhibits the cleavage activity of the S55. To sum up, the rfbN gene affects phage adsorption by regulating LPS synthesis. Furthermore, the functioning of the RfbN protein requires the involvement of multiple structures. To the best of our knowledge, this study is the first report of the involvement of the bacterial rfbN gene involved in the phage-adsorption process.

Salmonella Enteritidis (SE) are important zoonotic pathogens, and can be easily transferred to humans by contaminated animal products. Epidemic surveys of SE are necessary in current modern large-scale chicken farms. In this study, Salmonella strains were isolated from possibly infected samples collected at 3 independent farms, and their serotype, drug resistances, virulence genes, and genetic similarity were analyzed by molecular genetic analysis technologies including multilocus sequence typing (MLST), clustered regularly interspaced short palindromic repeats (CRISPR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). A total of 346 Salmonella strains were isolated from 3,598 samples (9.61%); 329 isolates were identified as SE (95.09%) and 308 isolates were multidrug resistant (93.62%). Virulotyping based on 6 virulence genes showed high similarity in SE isolates of each farm, with the exception of 2 isolates. All SE isolates were found to be the same ST11 type by MLST, and 22 strains of 150 SE isolates selected at random were found to belong to 1 cluster by PFGE and the same SET1 type by CRISPR. WGS results further revealed that these isolates belonged to the same clonal cluster, with high genetic similarity of 99.80 to 100.00%. All these results indicated that these SE isolates were overwhelmingly dominant and demonstrated high genetic similarity, which revealed that the same SE clone might be transmitted in these farms.

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-&kappa, B pathway activation when treated with TNF-&alpha, compared to the inhibition by SE-AvrA. SP-AvrA expression in the S. Enteritidis resulted in weaker suppression of NF-&kappa, B pathway in infected HeLa cells compared to SE-AvrA expression in the cells, while SP-AvrA expressed in S. Pullorum C79-13 suppressed NF-&kappa, B 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-&kappa, B 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-&kappa, B pathway.

Salmonella spp. can survive and replicate in macrophage cells to cause persistent infection, SpiC is a necessary T3SS effector, but its pathogenic mechanism is still not known completely. In our study, Salmonella Enteritidis spiC mutant (SEΔspiC) was found to have stronger swarming motility and intramacrophage hyperproliferation which was closely related to glucose metabolism. SEΔspiC wbaP::Tn5 mutant was screened out by transposon mutagenesis, which had weaker swarming motility and intramacrophage replication ability than SEΔspiC in the presence of glucose. Bioinformatics displayed that undecaprenyl-phosphate galactose phosphotransferase (Wbap), encoded by wbaP gene, was a key enzyme for glucose metabolism and Lipopolysaccharide(LPS) synthesis, which confirmed our outcome that Wbap was involved in intramacrophage replication ability by glucose use in addition to swarming motility based on SEΔspiC. This discovery will further promote the understanding of the interaction between wbaP gene and spiC gene and the intracellular Salmonella replication mechanism.

L ive a ttenuated S almonella v accine (LASV) is considered to be an effective contributory measure during the control of Salmonella infection. A Salmonella Pullorum spiC mutant was evaluated comprehensively as a LASV candidate (LASV-p) for broilers in terms of safety and immunogenicity. LASV-p was adminstered to 3-day broilers by intramuscular injection. The LD50 increased 126 fold, and no tissue lesions were observed in the liver, spleen and cecum, in comparison with the control group inoculated with PBS and a passive group by wild-type Salmonella. Growth rates of all broilers were normal and not affected. LASV-p persisted in vivo until 21 days in liver, 28 days in spleen and 35 days in feces, and induced high levels of humoral IgG and mucosal IgA. Cellular immunity was also stimulated in the form of antigen-specific lymphocyte proliferation and higher counts of CD3+CD8+ T cells and increased expression of mRNA of Th1 cytokines, IFN-γ and IL-2, in the early stage, and Th2 cytokines, IL-4 and IL-10, in the later stages. LASV-p provided at least 90% immuneprotection against a wild-type Salmonella Pullorum and cross-protection in different degree against other Salmonella searovars. Oral vaccine could also offer high immune protection of 87.5%. These results indicated that LASV-p vaccine candidate had a high level of safety and immune protection and it might be developed as a novel easy-to-use oral vaccine to improve poultry health in the future.

S. Pullorum and S. Enteritidis are closely related in genetic terms, but they show very different pathogenicity and host range. S. Enteritidis infects many different hosts, usually causing acute gastroenteritis, while S. Pullorum is restricted to avian, where it causes systemic disease in young animals. The reason why they differ in host range and pathogenicity is unknown. The core-genome denotes those genes that are present in all strains within a clade, and in the present work, an automated bioinformatics workflow was developed and applied to identify core-genome differences between these two serovars with the aim to identify genome features associated with host specificity of S. Pullorum. Results showed that S. Pullorum unique coding sequences (CDS) were mainly concentrated in three regions not present in S. Enteritidis, suggesting that such CDS were taken up probably during the separation of the two types from their common ancestor. One of the unique regions encoded Pathogenicity Islands 19 (SPI-19), which encodes a type VI secretion system (T6SS). Single-nucleotide polymorphism (SNP) analysis identified 1791 conserved SNPs in coding sequences between the two serovars, including several SNPs located in a type IV secretion system (T4SS). Analyzing of 100 bp regions upstream of coding sequences identified 443 conserved SNPs between the two serovars, including SNP variations in type III secretion system effector (T3SE). In conclusion, this analysis has identified genetic features encoding putative factors controlling host-specificity in S. Pullorum. The novel bioinformatic workflow and associated scripts can directly be applied to other bacteria to uncover the genome difference between clades.

Salmonella enterica serovar Pullorum is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. In contrast to the strong inflammatory response induced by Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis, S. Pullorum causes systemic infection with little inflammation. The effector proteins secreted by Salmonella often play a crucial role in modulating host signal transduction and cellular processes to the pathogen’s advantage. In the present study, the invasion plasmid antigen J (IpaJ) protein specifically identified in S. Pullorum was found to significantly inhibit activation of the key proinflammatory transcription factor, NF-κB, which was induced by tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and lipopolysaccharide (LPS). IpaJ inhibited the NF-κB pathway in cells infected with S. Pullorum through the stabilization of IκBα. Deletion of ipaJ in S. Pullorum caused a significantly increased level of ubiquitinated IκBα that was subsequently degraded by the proteasome in HeLa cells. Moreover, IpaJ was efficient in the prevention of NF-κB translocation to the nucleus and ultimately interfered with the secretion of the proinflammatory cytokines IL-1β, IL-6, and IL-8 in infected HeLa cells. Additionally, the transformation of ipaJ into S. Enteritidis decreased the secretion of proinflammatory cytokines in HeLa cells through suppression of the NF-κB pathway. The infection of chicken peripheral blood monocyte-derived macrophages (chMDM) confirmed that ipaJ-deleted S. Pullorum induced a stronger expression of proinflammatory cytokines than the wild-type and complementary strains. In summary, the present study revealed that IpaJ functions as an important anti-inflammatory protein involved in S. Pullorum infection through inhibition of the NF-κB pathway and the subsequent inflammatory response.

Autophagy is a cellular process to clear pathogens. Salmonella enterica serovar Enteritidis (S.E) has emerged as one of the most important food-borne pathogens. However, major studies still focus on Salmonella enterica serovar Typhimurium. Here, we reported that AvrA, a S. Enteritidis effector, inhibited autophagy to promote bacterial survival in the host. We found that AvrA regulates the conversion of LC3 I into LC3 II and the enrichment of lysosomes. Beclin-1, a key molecular regulator of autophagy, was decreased after AvrA expressed strain colonization. In S.E-AvrA--infected cells, we found the increases of protein levels of p-JNK and p-c-Jun and the transcription level of AP-1. AvrA-reduction of Beclin-1 protein expression is through the JNK pathway. The JNK inhibitor abolished the AvrA-reduced Beclin-1 protein expression. Moreover, we identified that the AvrA mutation C186A abolished its regulation of Beclin-1 expression. In addition, AvrA protein interacted with Beclin-1. In organoids and infected mice, we explored the physiologically related effects and mechanism of AvrA in reducing Beclin-1 through the JNK pathway, thus attenuating autophagic responses.ImportanceSalmonella Enteritidis is an important pathogen with a public health concern and farm production risk, yet the host-pathogen interactions that govern the survival of S. Enteritidis infections are incompletely understood. Anti-bacterial autophagy provides potent cell-autonomous immunity against bacterial colonization. Here, we report that a new role for effector AvrA of S. Enteritidis in the reduction of Beclin-1 protein expression through the JNK pathway and the attenuation of the autophagic response in intestinal epithelial cells. This finding not only indicates an important role of S. Enteritidis effector in reducing host protein as a strategy to suppress autophagy, but also suggests manipulating autophagy as a new strategy to treat infectious diseases.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum) and biovar Pullorum (S. Pullorum) are gram-negative bacteria, members of the most important infectious pathogens, and have caused common problems in the poultry industry, especially in the developing countries. O- and H-antigen specific anti-sera are commonly for slide and tube agglutination tests to identify Salmonella serovars. However, it is both labor intensive and time consuming, so there is an urgent need for a new technique for the rapid detection of the major Salmonella serovars. In this study, we developed a 1-step PCR assay to identify the serovar Gallinarum. This PCR-based assay was based on the SPUL_2693 gene, which was located in SPI-19 and found by comparing the genomes of the S. Pullorum and S. Gallinarum in the whole data of NCBI. The specificity of this gene was evaluated by bioinformatics analysis, and the results showed that the SPUL_2693 gene exists in all serovar Gallinarum. The specificity and sensitivity of this PCR assay were evaluated in our study. The developed PCR assay was able to distinguish the serovar Gallinarum from 27 different Salmonella serovars and 5 different non-Salmonella pathogens. The minimum limit of genomic DNA of S. Pullorum for PCR detection was 2.143 pg/μL, and the minimum limit number of cells was 6 CFU. This PCR assay was also applied to analyze Salmonella strains isolated from a chicken farm in this study. The PCR assay properly identified the serovar Gallinarum from other Salmonella serovars, and the results were in agreement with the results of a traditional serotyping assay. In general, the newly developed PCR-based assay can be used to accurately judge the presence of the serovar Gallinarum and can be combined with traditional serotyping assays, especially in the case of large quantities of samples.

In Salmonella, plasmids participate in many pathways involved in virulence, metabolism, and antibiotic resistance. To investigate the function of the ipaJ gene in a multi-copy plasmid pSPI12 prevalent in Salmonella enterica serovar Pullorum (S. Pullorum), we established a method to eliminate the plasmid and constructed the plasmid-cured bacteria C79-13-ΔpSPI12 by using the suicide vector pDM4. Briefly, a 500 bp fragment ipaJU from pSPI12 was cloned into pDM4 and transformed into S. Pullorum C79-13 by conjugative transfer. After homologous recombination, the suicide vector was inserted into pSPI12 to produce pSPI12-pDM4-ipaJU. Induction of the expression of the sacB gene in the suicide vector killed the bacteria harbouring plasmid, while the progeny losing the plasmid survived in the plate with sucrose. The plasmid-cured strain showed extremely decreased ability to infect chicken macrophage HD11 cells and LMH hepatic epithelial cells compared to wild type strain and complementary strain carrying ipaJ. Additionally, IFN-γ mRNA levels were up-regulated in HD11 cells or chicken spleens infected by plasmid-cured strain, but no difference was detected in IL-4 among the three strains. Transforming ipaJ into S. Enteritidis also decreased expression of proinflammatory cytokines in infected macrophages or chicken spleens compared to wild type strain. These results suggest that the ipaJ gene in pSPI12 is involved in S. Pullorum infection and that IpaJ protein modulates immune response.

Salmonella enterica serovar Enteritidis (SE) is a communicable zoonotic bacterium. Macrophages are essential for Salmonella survival, transmission, and infection. In this study, selective capture of transcribed sequences (SCOTS) was used to screen genes preferentially expressed by SE during contact with macrophages from different hosts. We found 57 predicted genes and 52 genes expressed by SE during interaction with avian HD-11 and murine RAW264.7 cells, respectively. These expressed genes were involved in virulence, metabolism, stress response, transport, regulation, and other functions. Although genes related to survival or metabolic pathways were needed during SE infection, different gene expression profiles of SE occurred in the two macrophage cell lines. qRT-PCR results confirmed that most screened genes were upregulated during infection in contrast to the observation during in vitro cultivation, with different expression levels in infected avian macrophages at 2-h and 7-h post-infection. In addition, in vitro and in vivo competition assays confirmed that SEN3610 (a putative deoR family regulator) and rfaQ (related to LPS synthesis) were closely related to SE virulence in both mice and chickens. Three putative transcriptional regulators, SEN2967, SEN4299, and rtcR, were related to SE colonization in mice, while the ycaM mutation caused decreased infection and survival of SE in HD-11 cells without influencing virulence in mice or chicken. Genes showing differential expression between SE-infected avian and murine macrophages indicate specific pathogen adaptation to enable infection of various hosts.

We investigated Salmonella contamination in a slaughterhouse in Jiangsu province (China) by analysing prevalences, loads, and serotypes of Salmonella isolates. In total, 480 samples were collected, with total prevalence of 25.4%. High Salmonella prevalence and load were observed at exsanguination and splitting stages (40.6% and 75.0%; 2.50 ± 0.94 and 2.24 ± 0.72 logMPN/m2, respectively), with low prevalence and load at dehairing, flaming, and chilling stages (2.5%, 5.0%, and 15.0%; 1.39 ± 0.42, 1.36 ± 0.31, and 1.38 ± 0.30 logMPN/m2, respectively). The Salmonella prevalence and load increased substantially from flaming to splitting stage. Six serovars were represented among 122 Salmonella isolates; S. Derby and S. Typhimurium were predominant and were subtyped using PFGE and CRISPR typing approaches. Fourteen PFGE clusters were identified, with discrimination indices (DI) of 0.929 and 0.976 for S. Derby and S. Typhimurium, respectively. Clusters A1, A2, B, C1, C3, C4, D1, F, I, J, L1, M2, and N2 indicated the isolates from same sampling visit were distributed in same cluster. Cluster D1 and K showed that the strains isolated from splitter and carcass swab-samples were highly resemble. Salmonella serovars with the same CRISPR type were mostly isolated after splitting (represented by Dercr 1, Dercr 2, Dercr 3, Tycr 1, Tycr 2, Tycr 3, Tycr 4, and Tycr 6). Our findings revealed that introduced Salmonella was the major source of swine carcass contamination; three slaughtering steps (polishing, rectal drilling, and evisceration) between flaming and splitting were important risk points for Salmonella release, post-splitting slaughtering processes were major contamination risk points, and the splitter was a contamination factor. Our data suggest routes for controlling Salmonella contamination in a swine slaughterhouse.

This study was undertaken to acquire data on Salmonella contamination of whole raw chickens, eggs, and vegetables available to consumers in Yangzhou city, China, between April 2011 and March 2012. In total, 240 chicken carcasses were tested, and the overall contamination rate for Salmonella was 33.8%. While the prevalence of Salmonella in 100 eggs and 155 vegetable samples was 7.0% and 3.2%, respectively. The 84 isolated strains were identified in 19 different serotypes with Salmonella enterica serovar Indiana ( S. Indiana) (25.0%), S. Typhimurium (21.4%) and S. Enteritidis (17.9%) as the predominant serovars. Moreover, the median load of the contaminated chicken samples reached 6.4 MPN/100 g with 3.6 MPN/100 g as the 25th percentile and 15.0 MPN/100 g as the 75th percentile. Chicken carcasses collected in October had not only the highest prevalence of Salmonella (70%), but also the highest median load (33 MPN/100 g) and 75th percentile load (460 MPN/100 g), while the lowest prevalence (10%) was in April. The clustered regularly interspaced short palindromic repeats (CRISPR) subtyping method was then used to identify serotypes of Salmonella and distinguish strains from the same Salmonella serotypes. We found that 85.7% of strains were distributed in 11 serotypes speculated by CRISPR typing, which corresponded to the identified serotypes by O and H antiserum. The speculated serotypes of 7.1% of the strains by CRISPR typing are very close to the identified ones, as they belong to the same O group with a small difference in the O or H antigen. All of the above findings implied that CRISPR typing could be applied to serotyping of Salmonella . In addition, CRISPR typing method could be used to subtype different strains from the same serotype, specifically S. Hadar.

A quantitative microbiological risk assessment model was used to quantify the risk of salmonellosis caused by bacterial growth and cross-contamination of chicken meals prepared in households of China. Chinese data on initial loads of Salmonella in chicken carcasses sold at retail, storage time and handling of raw chicken meat in household kitchens and confirmatory transfer rates of Salmonella among different kitchen objects were collected. Only one third of Chinese families in our sample separated the cutting board between raw and ready-to-eat foods. The cross-contamination of ready-to-eat foods from chicken meals via the cutting board, the knife and cooks’ hands increased the frequency of pathogen ingestion and the risk of salmonellosis. A significant decrease in the risk of salmonellosis could be achieved by reducing the cross-contamination when handling raw chicken meat and ready-to-eat foods. Decreasing the prevalence of Salmonella contamination to 8.8% or removing chicken carcasses with contamination densities higher than 100 MPN/100 g at retail was less effective. Using transfer rates of Salmonella from raw chicken meat to the wooden cutting board instead of that from references, a statistically higher risk of salmonellosis per serve due to the cross-contamination in households was observed. The present study validated values of hygiene practices in China to reduce the risk of salmonellosis from contaminated raw chicken meat at retail. Deliberate surveys for cooking behaviors and transfer rates of Salmonella from and to different objects including wooden cutting boards were needed.

In order to ascertain the contamination status of Salmonella from carcass, equipment, environment and the intestinal contents in a pig slaughterhouse in Huaian, China, we investigated the prevalence and characteristics of Salmonella isolates in one slaughterhouse. A total of 636 samples were collected; Salmonella was laboratory-confirmed from 154/441 (34.9%) carcass swab-samples, 13/57 (22.8%) pieces of equipment, 30/86 (34.9%) environmental samples and the intestinal contents of 19/52 (36.5%) animals. Salmonella was found to be at its highest and lowest frequency during splitting (55.9%) and flaming (17.2%) processes, respectively. All Salmonella isolates were characterized by serotyping, multilocus sequence typing (MLST) and 69 S. Derby isolates were analyzed using pulsed-field gel electrophoresis (PFGE). Four serovars (S. Derby, S. Rissen, S. Typhimurium, and S. London) were shared from the four sources and predominant serovars were S. Derby and S. Rissen. MLST results indicated that ten different STs were distinguished, of which ST40 was identified as the most prominent ST. Moreover, ST40, ST469, ST34 and ST155 comprised Salmonella isolates obtained from carcass, equipment, the slaughterhouse environment and intestinal contents. PFGE results showed that 33 pulsotypes (PF1-PF33) were identified. PF21, PF25 and PF29 suggested that many isolates from carcass-swab samples at different slaughterhouse process points were of the same pulsotype which also partly comprised isolates from equipment, the environment and intestinal contents. The present study provides an insight into Salmonella distribution in a pig slaughterhouse and revealed that cross-contamination occurs frequently at different sites, which provides data support for proposing some interventions to control Salmonella.

Salmonella is considered one of the most important foodborne pathogens, and is commonly associated with the consumption of poultry meat and eggs. Multidrug-resistant (MDR) Salmonella strains are highly adaptive and have been responsible for foodborne disease outbreaks with high mortality worldwide. Therefore, we investigated Salmonella prevalence and antimicrobial resistance at different production stages in a chicken breeder farm in Jiangsu Province, China. A total of 115 Salmonella isolates were recovered from 638 samples. Interestingly, serotyping revealed all of these to be Salmonella enterica subsp. enterica serovar Enteritidis ( S . Enteritidis). Prevalence was highest at the laying stage, with 29.17% of samples containing Salmonella , followed by the hatching phase (21.56%). Tests of susceptibility to 16 antimicrobial agents using a disk diffusion assay showed that all isolates were resistant to at least one compound, and 31.30% exhibited MDR phenotypes, covering all five production stages. Of the Salmonella isolates recovered during the rearing and laying periods, a large proportion (>50%) were MDR (100 and 71.43%, respectively). Our results imply that the laying period constitutes a high-risk stage in breeder farms. Clustered regularly interspaced short palindromic repeat (CRISPR) sequence typing identified three CRISPR patterns among the isolates. Two of these were detected in samples from four of the five production stages, suggesting that certain genetically similar strains may have spread across the farm. Our study provides data concerning Salmonella epidemiology and antimicrobial resistance in breeder farms, which may aid the optimization of hazard analysis and critical control point strategies for such sites.

Salmonella is an important zoonotic agent and a vehicle for antibiotic resistance genes. Here, 294 isolates from humans and food-producing animals were subjected to serotyping, multilocus sequence typing, and assessment of phenotypic (15 antibiotics) and genotypic (32 resistance genes) antimicrobial resistance. Twenty-two serotypes and 35 sequence types (STs) were identified, the most common STs being ST11, including S. Enteritidis from chickens and humans; ST17, including S. Indiana from chickens; and ST40, including S. Derby from pigs and humans. Antimicrobial resistance phenotypes and genotypes exhibited ST- and serovar-specific features. ST11, clonal complex (CC) 19, ST40, and ST155 were moderately multidrug-resistant (MDR) clones, most of the isolates of which were resistant to between 3 and 6 antibiotics. Isolates of a super-MDR clone, ST17, demonstrated resistance against 9 to 14 antimicrobials, in particular, ampicillin, amoxicillin-clavulanic acid, cefotaxime, cefepime, cefoperazone, ceftriaxone, and ciprofloxacin. Consistent with this, ST17 ( S. Indiana) was associated with a gene cluster comprising bla CTX-M (and/or bla OXA-1 -like together with bla TEM-1 -like), sul1 , aacC4 , aac(6)-1b , floR , and dfrA17 , while the moderately-MDR clones (ST11, S. Enteritidis) were more closely linked to the bla TEM-1 -like gene. The similar genetic clones isolated from animals and humans indicate a common ancestor, and implicate animals as a major salmonellae source. Antibiotic abuse in animal production appears to be the origin of MDR and super-MDR isolates, the latter being closely associated with the β -lactamase genes bla CTX-M , bla OXA-1 -like, and bla TEM-1 -like. Carried by chickens, ST17 ( S. Indiana) is an emerging super-MDR clone whose associated resistance genes are expanding to other ST clones and serotypes being transmitted to animals and humans.