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Salmonella Thompson is a prevalent foodborne pathogen and a major threat to food safety and public health. This study aims to reveal the dissemination mechanism of S. Thompson with co-resistance to ceftriaxone and ciprofloxacin. In this study, 181 S. Thompson isolates were obtained from a retrospective screening on 2118 serotyped Salmonella isolates from foods and patients, which were disseminated in 12 of 16 districts in Shanghai, China. A total of 10 (5.5 %) S. Thompson isolates exhibited resistance to ceftriaxone (MIC ranging from 8 to 32 μg/mL) and ciprofloxacin (MIC ranging from 2 to 8 μg/mL). The AmpC β-lactamase gene bla CMY-2 and plasmid-mediated quinolone resistance (PMQR) genes of qnrS and qepA were identified in the 9 isolates. Conjugation results showed that the co-transfer of bla CMY-2 , qnrS, and qepA occurred on the IncC plasmids with sizes of ∼150 (n = 8) or ∼138 (n = 1) kbp. Three typical modules of ISEcp1-bla CMY-2 -blc-sugE, IS26-IS15DIV-qnrS-ISKpn19, and ISCR3-qepA-intl1 were identified in an ST3 IncC plasmid pSH11G0791. Phylogenetic analysis indicated that IncC plasmids evolved into Lineages 1, 2, and 3. IncC plasmids from China including pSH11G0791 in this study fell into Lineage 1 with those from the USA, suggesting their close genotype relationship. In conclusion, to our knowledge, it is the first report of the co-existence of bla CMY-2 , qnrS, and qepA in IncC plasmids, and the conjugational transfer contributed to their dissemination in S. Thompson. These findings underline further challenges for the prevention and treatment of Enterobacteriaceae infections posed by IncC plasmids bearing bla CMY-2 , qnrS, and qepA., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Indiana ( S. Indiana) is an extremely expanded foodborne pathogen in China in recent years. This study aimed to elucidate the national prevalence and phylogenomic characterization of this pathogen in China. Among 5, 287 serotyped Salmonella isolates collected during 2002 to 2018, 466 S. Indiana isolates were found in 15 provinces, and 407 were identified to be ST17, and the rest were ST2040. Among 407 ST17 isolates, 372 (91.4%) were multidrug resistant, and 366 (89.9%) were resistant to ciprofloxacin, 235 (57.7%) were further resistant to ceftriaxone. Phylogenomic analysis revealed that ST17 isolates were classified into four clades (I, II, III and IV), which appeared in international clonal dissemination. ST17 isolates from China fell into Clade IV with part of isolates from the United Kingdom, the United States, South Korea, and Thailand, suggesting their close genetic relationship. Mutations in quinolone resistance-determining regions (QRDR) of GyrA and ParC, and plasmid-mediated quinolone resistance (PMQR) genes aac (6')-Ib-cr, oqx AB, and qnr S as well as extended spectrum β-lactamases (ESBL) genes bla CTX-M , bla OXA , and bla TEM in isolates from Clade IV were much higher than those from other three clades. Various bla CTX-M subtypes ( bla CTX-M-65 , bla CTX-M-55 , bla CTX-M-27 , bla CTX-M-14 , and bla CTX-M-123 ) with IS Ecp1 , IS 903B , IS Vsa5 , and IS 1R were found in ST17 isolates, especially Tn 1721 containing ΔIS Ecp1 - bla CTX-M-27 -IS 903B in P1-like bacteriophage plasmids. These findings on the prevalent and genomic characterization for the S . Indiana multidrug-resistant ST17 clone in China, which have not been reported yet, provide valuable insights into the potential risk of this high-resistant clone. IMPORTANCE Fluoroquinolones and cephalosporins are the primary choices for severe salmonellosis treatment. S. Indiana has become one of the most prevalent serovars in breeding poultry and poultry meats in China in recent years. ST17 was recognized as the leading epidemiological importance in S. Indiana because of its high-level resistance to the most of common antibiotics, including ciprofloxacin and ceftriaxone. However, the prevalence and phylogenomic characterization of ST17 isolates are unclear. Here, we did a retrospective screening on a large scale for S. Indiana in China, and performed its phylogenomic analysis. It was found that ST17 isolates had extensive spread in 15 provinces of China and became a multidrug-resistant clone. The international spread of the ST17 isolates was observed among several countries, especially China, the United Kingdom, and the United States. Our study emphasized the importance of surveillance of a high-resistant S. Indiana ST17 clone to combat its threat to public health.

This study characterized the prevalence and antimicrobial resistance characteristics of foodborne Salmonella isolates from March 2016 to February 2017 in Shanghai, China. A total of 147 (14.2%) nonduplicate foodborne Salmonella isolates were obtained from 1035 food samples. The Salmonella isolates were most frequently identified in fresh meat samples (28.0%), followed by ready-to-eat foods (9.0%), frozen convenience foods (7.1%), and fresh produce (4.5%). The top 3 serovars were Salmonella Enteritidis (46.3%; 68/147), Salmonella Typhimurium (32.7%; 48/147), and Salmonella Derby (6.8%; 10/147). The majority of isolates were resistant to sulfisoxazole (93.9%; 138/147) and trimethoprim/sulfamethoxazole (61.2%; 90/147). Interestingly, frozen convenience food isolates exhibited an extremely high multidrug resistance rate (86.7%; resistant to ≥3 classes of antimicrobials). Among 81 quinolone-resistant isolates, aac(6')-Ib-cr (100%), oqx AB (84.0%), qnr S1 (23.5%), D87Y (49.4%), and D87N (33.3%) mutations in GyrA, and T57S in ParC (12.3%) were observed. The β-lactamase genes bla TEM-1 (100%) were present in 63 ampicillin-resistant isolates. Polymerase chain reaction-based plasmid replicon typing revealed that 147 isolates represented 6 plasmid incompatibility groups (IncFIIs, IncHI2, IncI1, IncP, IncFIC, and IncA/C), among which, IncFIIs (59.2%) and IncHI2 (26.5%) were predominant. The genetic relationship of isolates was elucidated using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). MLST results indicated that ST34 and ST11 were predominate types in Salmonella Typhimurium (56.3%; 27/48) and Salmonella Enteritidis (95.6%; 65/68), respectively. Importantly, 96.3% (26/27) of ST34 Salmonella Typhimurium isolates possessed the ACSSuT resistance pattern (ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline). PFGE analysis of ST34 isolates showed clonal dissemination across all four types of retail foods. Our findings highlight the high prevalence of antimicrobial-resistant Salmonella isolates in retail foods in Shanghai, especially the clonal expansion of ST34 isolates with MDR-ACSSuT resistance, which might pose a public health threat.

Salmonella enterica is the most common foodborne pathogen worldwide, with 2,500 recognized serovars. Detection of S. enterica and its classification into serovars are essential for food safety surveillance and clinical diagnosis. The PCR method is useful for these applications because of its rapidity and high accuracy. We obtained 412 candidate detection targets for S. enterica using a comparative genomics mining approach. Gene ontology (GO) functional enrichment analysis of these candidate targets revealed that the GO term with the largest number of unigenes with known function (38 of 177, 21.5%) was significantly involved in pathogenesis (P < 10(-24)). All the candidate targets were then evaluated by PCR assays. Fifteen targets showed high specificity for the detection of S. enterica by verification with 151 S. enterica strains and 34 non-Salmonella strains. The phylogenetic trees of verified targets were highly comparable with those of housekeeping genes, especially for differentiating S. enterica strains into serovars. The serovar prediction ability was validated by sequencing one target (S9) for 39 S. enterica strains belonging to six serovars. Identical mutation sites existed in the same serovar, and different mutation sites were found in diverse serovars. Our findings revealed that 15 verified targets can be potentially used for molecular detection, and some of them can be used for serotyping of S. enterica strains.

As the development of molecular serotyping approaches is critical for Salmonella spp., which include >2,600 serovars, we performed an initial evaluation of the ability to identify Salmonella serovars using (i) different molecular subtyping methods and (ii) a newly implemented combined PCR- and sequencing-based approach that directly targets O- and H-antigen-encoding genes. Initial testing was performed using 46 isolates that represent the top 40 Salmonella serovars isolated from human and nonhuman sources, as reported by the U.S. Centers for Disease Control and Prevention and the World Health Organization. Multilocus sequence typing (MLST) was able to accurately predict the serovars for 42/46 isolates and showed the best ability to predict serovars among the subtyping methods tested. Pulsed-field gel electrophoresis (PFGE), ribotyping, and repetitive extragenic palindromic sequence-based PCR (rep-PCR) were able to accurately predict the serovars for 35/46, 34/46, and 30/46 isolates, respectively. Among the methods, S. enterica subsp. enterica serovars 4,5,12:i:-, Typhimurium, and Typhimurium var. 5- were frequently not classified correctly, which is consistent with their close phylogenetic relationship. To develop a PCR- and sequence-based serotyping approach, we integrated available data sources to implement a combination PCR-based O-antigen screening and sequencing of internal fliC and fljB fragments. This approach correctly identified the serovars for 42/46 isolates in the initial set representing the most common Salmonella serovars, as well as for 54/63 isolates representing less common Salmonella serovars. Our study not only indicates that different molecular approaches show the potential to allow for rapid serovar classification of Salmonella isolates, but it also provides data that can help with the selection of molecular serotyping methods to be used by different laboratories.

This report presents SMM-system, a software package that implements various personalized pre- and post-BLASTN tasks for mining specific markers of microbial pathogens. The main functionalities of SMM-system are summarized as follows: (i) converting multi-FASTA file, (ii) cutting interesting genomic sequence, (iii) automatic high-throughput BLASTN searches, and (iv) screening target sequences. The utility of SMM-system was demonstrated by using it to identify 214 Salmonella enterica-specific protein-coding sequences (CDSs). Eighteen primer pairs were designed based on eighteen S. enterica-specific CDSs, respectively. Seven of these primer pairs were validated with PCR assay, which showed 100% inclusivity for the 101 S. enterica genomes and 100% exclusivity of 30 non-S. enterica genomes. Three specific primer pairs were chosen to develop a multiplex PCR assay, which generated specific amplicons with a size of 180bp (SC1286), 238bp (SC1598) and 405bp (SC4361), respectively. This study demonstrates that SMM-system is a high-throughput specific marker generation tool that can be used to identify genus-, species-, serogroup- and even serovar-specific DNA sequences of microbial pathogens, which has a potential to be applied in food industries, diagnostics and taxonomic studies. SMM-system is freely available and can be downloaded from http://foodsafety.sjtu.edu.cn/SMM-system.html., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Comparative genomic approaches provide abundant information to reveal the diversity among Salmonella serogroups. In a local genomic sequence database, twenty-five Salmonella whole genomic sequences were divided into 6 (A, B, C1, C2, D and others) serogroups for mining the DNA fragments specific for serogroups A through D. For each serogroup, a reference sequence was selected and split into 1000-bp fragments in silico to align against all the other genomic sequences to obtain one or more serogroup-specific fragments. As a result, 2, 6, 7, 10, and 7 specific fragments were found for A, B, C1, C2 and D serogroups, respectively. Specific primer sets targeting these DNA fragments were designed for multiplex PCR assays identifying 21 Salmonella standard strains and 86 additional food isolates. The PCR results demonstrated good agreement with those from Salmonella serotyping. This means that the PCR assay may be able to identify 5 (A, B, C1, C2 and D) Salmonella serogroups and elucidate differences among them. Based on the gene annotations, the 32 serogroup-specific fragments were divided into 3 categories (membrane protein genes, rfb gene clusters, and fimbrial genes). Each gene from these three groups was conserved within the serogroup and was closely correlated with phenotypic characterization. This finding implies that these genes, which are associated with sugar synthesis and metabolism or glycosyl and O-actetyl transfer, impart the differences among Salmonella serogroups., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

A 5'-nuclease real-time PCR assay using a minor groove binding probe was developed for the detection of Salmonella enterica from artificially contaminated foods. S. enterica-specific sequences were identified by a comparative genomic approach. Several species-specific target sequences were evaluated for specificity. A real-time PCR assay was developed targeting a nucleotide sequence within the putative type III secretion ATP synthase gene (ssaN). An internal amplification control (IAC) probe was designed by randomly shuffling the target probe sequence and a single-stranded oligonucleotide was synthesized to serve as an IAC. The assay demonstrated 100% inclusivity for the 40 Salmonella strains tested and 100% exclusivity for 24 non-Salmonella strains. The detection limit of the real-time PCR assay was 41.2 fg/PCR with Salmonella Typhimurium genomic DNA and 18.6 fg/PCR using Salmonella Enteritidis genomic DNA; 8 and 4 genome equivalents, respectively. In the presence of a natural background flora derived from chicken meat enrichment cultures, the sample preparation and PCR method were capable of detecting as few as 130 Salmonella cfu/mL. Using the developed real-time PCR method to detect Salmonella in artificially contaminated chicken, liquid egg and peanut butter samples, as few as 1 cfu/10 g of sample was detectable after a brief (6h) non-selective culture enrichment., ((c) 2009. Published by Elsevier B.V. All rights reserved.)

Colistin resistance in bacteria has become a significant threat to food safety and public health, and its development was mainly attributed to the plasmid-mediated mcr genes. This study aimed to determine the global prevalence and molecular characteristics of mcr-producing Salmonella enterica isolates. A total of 2279 mcr-producing Salmonella genomes were obtained from the public database, which were disseminated in 37 countries from five continents worldwide, including Asia, Europe, America, Australia, and Africa. Human samples (39.5%; 900/2279) were the predominant sources of mcr-producing Salmonella isolates, followed by foods (32.6%), animals (13.7%), and environment (4.4%). Furthermore, 80 Salmonella serotypes were identified, and Typhimurium and 1,4,[5],12:i:- were the predominant serotypes, accounting for 18.3% and 18.7%, respectively. Twenty mcr variants were identified, and the most common ones were mcr-9.1 (65.2%) and mcr-1.1 (24.4%). Carbapenems-resistance gene blaNDM-1 and tigecycline-resistance gene tet(X4) were identified in one isolate, respectively. Phylogenetic results indicated that mcr-producing Salmonella fell into nine lineages (Lineages I-IX), and Salmonella Typhimurium, 1,4,[5],12:i:- and 4,[5],12:i:- isolates from different countries were mixed in Lineages I, II and III, suggesting that international spread occurred. These findings underline further challenges for the spread of Salmonella-bearing mcr genes. [ABSTRACT FROM AUTHOR]

Salmonella enterica, Staphylococcus aureus, Escherichia coli O157: H7, and Listeria monocytogenes may contaminate similar types of food and cause foodborne disease. The objective of this study was to develop a selective enrichment broth for simultaneous enrichment of Salmonella enterica, Staphylococcus aureus, Escherichia coli O157: H7, and Listeria monocytogenes (SSEL) using nalidixic acid, acriflavine, lithium chloride, and sodium cholate as selective agents. Developed SSEL broth not only enriched the target pathogens to 5 log10 CFU/ml after 18 hr incubation at 37°C with 10–100 CFU/mL of inoculation concentration, but also could successfully support the simultaneous enrichment of target pathogens with similar growth rates and inhibit the growth of most nontarget bacteria effectively. The enrichment effect of SSEL was confirmed by artificial contamination test coupled with multiplex PCR. In summary, SSEL has been shown to be a promising multiplex selective enrichment broth for the detection of the four pathogens on a single‐assay platform. [ABSTRACT FROM AUTHOR]

Abstract Salmonella enterica serovar Enteritidis (S. Enteritidis) is a food-borne bacterial pathogen that can cause human salmonellosis predominately by contamination of eggs and egg products. However, its survival mechanisms in egg white are not fully understood, especially from a proteomic point of view. In this study, the proteomic profiles of S. Enteritidis in Luria-Bertani (LB) broth containing 50% and 80% egg white, and in whole egg white were compared with the profile in LB broth using iTRAQ technology to identify key proteins that were involved in S. Enteritidis survival in egg white. It was found that there were 303, 284 and 273 differentially expressed proteins in S. Enteritidis after 6 h exposure to whole, 80% and 50% egg white, respectively. Most of up-regulated proteins were primarily associated with iron acquisition, cofactor and amino acid biosynthesis, transporter, regulation and stress responses, whereas down-regulated proteins were mainly involved in energy metabolism, virulence as well as motility and chemotaxis. Three stress response-related proteins (YbgC, TolQ, TolA) of the tol - pal system responsible for maintaining cell membrane stability of Gram-negative bacteria were up-regulated in S. Enteritidis in response to whole egg white. Interestingly, deletion of ybgC resulted in a decreased resistance of S. Enteritidis to egg white. Compared with the wild type and complementary strains, a 3-log population reduction was observed in △ ybgC mutant strain after incubation in whole egg white for 24 h. Cellular morphology of △ ybgC mutant strain was altered from rods to spheres along with cell lysis in whole egg white. Furthermore, deletion of ybgC decreased the expression of tol - pal system-related genes (tolR , tolA). Collectively, these proteomic and mutagenic analysis reveal that YbgC is essential for S. Enteritidis survival in egg white. Highlights • Iron acquisition system-related proteins were induced by egg white. • Amino acid biosynthesis and transporter-related proteins were induced. • Stress responses-related proteins (i. e. DNA repair, cell membrane) were induced. • Energy metabolism, virulence and motility-related proteins were repressed. • YbgC was essential for S. Enteritidis survival in egg white. [ABSTRACT FROM AUTHOR]

Abstract: Using a comparative genomic method, 38 and 8 fragments were verified as specific identification targets for Salmonella Typhimurium and Enteritidis, respectively. Primer sets were designed based on these sequences and evaluated by PCR assays. Two primer sets targeting the STM4495 and SEN1392 genes with high specificity were selected for S. Typhimurium and Enteritidis, respectively, and a multiplex PCR method was developed for the identification of these bacteria based on these two primer sets and another primer set targeting the srfC gene specific for Salmonella enterica. The multiplex PCR also included an internal amplification control (IAC), which was constructed by psy gene from Chlorella protothecoides for process control to monitor potential PCR inhibitors. The sensitivity of this multiplex PCR was 89 fg and 138 fg of DNA per PCR for S. Typhimurium and S. Enteritidis, respectively. The detection limits were as low as 22–23 CFU per PCR for pure cultures of both serovars. Moreover, positive results were observed from milk samples that were initially contaminated with 2–3 CFU Salmonella after 12 h of enrichment at 37 °C. These results demonstrate that the comparative genomic method is a valuable tool for identifying new specific targets, a necessary step for developing rapid and accurate detection methods for foodborne pathogens. [Copyright &y& Elsevier]

Salmonella enterica serovar Indiana has become a widely prevalent non-typhoidal serovar in China in recent years. To improve the understanding of antimicrobial resistance and genetic bias in this emerging pathogen, a total of 214 S. Indiana isolates from foods, patients, and the environments in China from 2007 to 2016 were characterized for antimicrobial susceptibility, antimicrobial resistance-determining factors, and molecular typing. It was found that 208 (97.2%) exhibited resistance to ciprofloxacin, and 102 (47.7%) isolates to ceftriaxone. Ciprofloxacin resistance increased from 82.4% in 2007–2008 to 100.0% in 2015–2016. Ceftriaxone resistance increased from 29.4% in 2007–2008 to 47.1% in 2015–2016. In particular, the proportion of multidrug-resistant (MDR) S. Indiana isolates increased from 64.7% in 2007–2008 to 91.4% in 2015–2016. Mutations in the quinolone resistance determining region (QRDR) (gyr A and par C), plasmid-mediated quinolone resistance (PMQR) genes [ aac(6')-Ib-cr , oqx AB, qnr A, qnr S, qnr B and qep A], extended-spectrum beta-lactamase (ESBL) genes (bla OXA , bla CTX-M , bla TEM , bla CMY and bla VEB), and class I integrons were identified in these S. Indiana isolates, along with transposon units of ΔIS Ecp1 - bla CTX-M-65 -IS 903B , IS Ecp1 -IS Vsa5 - bla CTX-M-14 -IS 903B , and IS Ecp1 - bla CTX-M-55/15 -Δ orf 477. To our knowledge, the ESBL gene bla VEB was identified in Salmonella for the first time. Diverse plasmid types (IncHI2, IncN, IncA/C, IncFIB, IncI1 and IncFII) were identified in these isolates, among which IncHI2-type plasmid (49.1%) was the most common type in S. Indiana. Multilocus sequence typing (MLST) showed a single ST17 in S. Indiana. Pulsed-field gel electrophoresis (PFGE) results showed that there was a genotypic diversity, and some clones might have existed in S. Indiana between patients and foods for several years. These findings suggested that the frequent emergence of S. Indiana isolates with resistance to ciprofloxacin and ceftriaxone would pose a huge threat to food safety and public health. ∙ Ciprofloxacin and ceftriaxone co-resistant isolates were found. ∙ESBLs genes, PMQR genes with mutations in QRDR and class I integrons were observed. ∙ bla VEB was identified in Salmonella for the first time. ∙IS Ecp1 , IS Vsa5 and IS 903B was adjacent to bla CTX-M. ∙ST17 and genotypic diversity were identified in S. Indiana. [ABSTRACT FROM AUTHOR]

Salmonellosis is an important global foodborne disease, and Salmonella isolates that are resistant to a wide variety of antibiotics have become a major public health concern. The aim of this study was to investigate the distribution and transmission of mph A gene in azithromycin-resistant Salmonella isolates and characterize the plasmid bearing mph A. In this study, the azithromycin resistance was shown in 15 Salmonella enterica serovar Indiana (S. Indiana) isolates from chicken, duck and humans with concurrent resistance to ciprofloxacin and ceftriaxone, two of which were resistant to all antibiotics tested except colistin and imipenem. MLST and PFGE results suggested that these 15 isolates were closely related in phylogeny. The azithromycin resistance gene (mph A), extended-spectrum beta-lactamase (ESBL)-encoding genes (bla CTX-M-65 , bla CTX-M-14 and bla CTX-M-15) and plasmid-mediated quinolone resistance (PMQR) genes [ oqx AB, qep A, qnr A and aac(6′)-Ib-cr ] with mutations in the quinolone resistance determining region (QRDR) (gyr A and par C) were identified in these S. Indiana isolates. Plasmid conjugation and transformation were performed in these 15 isolates. The co-existence of the mph A gene with oqx AB- bla CTX-M-65 was observed on the IncHI2 plasmid with a size of~240 kbp in 7 transformants, and these 7 transformants exhibited the resistance to azithromycin (MIC from 32 μg/mL to 64 μg/mL) and ceftriaxone (MIC≥ 256 μg/mL). The complete sequence of an IncHI2 type plasmid, p13520, was determined. An approximately 80-kbp of multidrug resistance region (MRR) comprising 29 antimicrobial resistance genes (ARGs) including mph A, oqx AB, bla CTX-M-65 , fos A3 and rmt B interspersed with different insertion sequences (ISs) (IS 26 , IS Aba1 , IS 91 , IS 1006 and IS 903B) was located on the plasmid. The typical IS26- mph A- mrx - mph R-IS 6100 transposable structure was identified in the IncHI2 plasmid of nine transformants via genome sequencing and PCR mapping, which suggested that azithromycin resistance in S. Indiana was probably due to the dissemination of the IncHI2 plasmid carrying the mph A- mrx - mph R cluster. This study highlighted the emergence of S. Indiana isolates with concurrent resistance to azithromycin, ciprofloxacin and ceftriaxone in humans and retail foods in China. This is likely due to the co-existence of mph A, oqx AB and bla CTX-M-65 carried by the IncHI2 plasmid. These findings emphasized the importance of ongoing surveillance for the prevalence and transmission of the p13520-like plasmid in S. Indiana, so as to better understand the potential threat to public health. • Azithromycin, ciprofloxacin and ceftriaxone co-resistant isolates were found. • mph A, PMQR genes with mutations in QRDR and ESBLs genes were detected. • mph A co-existence with oqx AB- bla CTX-M-65 was found on ～240 kbp IncHI2 plasmid. • IS26- mph A- mrx - mph R-IS 6100 transponson was identified in the IncHI2 plasmid. [ABSTRACT FROM AUTHOR]