Structure and rational engineering of the PglX methyltransferase and specificity factor for BREX phage defence.

Bacteria have evolved a broad range of systems that provide defence against their viral predators, bacteriophages. Bacteriophage Exclusion (BREX) systems recognise and methylate 6 bp non-palindromic motifs within the host genome, and prevent replication of non-methylated phage DNA that encodes these same motifs. How BREX recognises cognate motifs has not been fully understood. In this study we characterise BREX from pathogenic Salmonella and present X-ray crystallographic structures of the conserved BREX protein, PglX. The PglX N-terminal domain encodes the methyltransferase, whereas the C-terminal domain is for motif recognition. We also present the structure of PglX bound to the phage-derived DNA mimic, Ocr, an inhibitor of BREX activity. Our analyses propose modes for DNA-binding by PglX and indicate that both methyltransferase activity and defence require larger BREX complexes. Through rational engineering of PglX we broaden both the range of phages targeted, and the host motif sequences that are methylated by BREX. Our data demonstrate that PglX is used to recognise specific DNA sequences for BREX activity, contributing to motif recognition for both phage defence and host methylation.

Prevalence and antimicrobial susceptibility profile of Salmonella isolated from vegetable farms fertilized with animal manure in Addis Ababa Ethiopia.

The resistance of foodborne pathogens to antimicrobial agents is a potential danger to human health. Hence, establishing the status of good agricultural practices (GAPs) and the antimicrobial susceptibility of major foodborne pathogens has a significant programmatic implication in planning interventions. The objective of this study was to assess the gap in attaining GAP and estimate the prevalence and antimicrobial susceptibility profile of Salmonella in vegetable farms fertilized with animal manure in Addis Ababa, Ethiopia. A total of 81 vegetable farms from four sub-cities in Addis Ababa were visited, and 1119 samples were collected: soil (n = 271), manure (n = 375), vegetables (n = 398), and dairy cattle feces (n = 75). Additional data were collected using a structured questionnaire. Isolation of Salmonella was done using standard microbiology techniques and antimicrobial susceptibility testing was conducted using disk diffusion assays. Carriage for antimicrobial resistance genes was tested using polymerase chain reaction (PCR). Among the 81 vegetable farms visited, 24.7% used animal manure without any treatment, 27.2% used properly stored animal manure and 80.2% were easily accessible to animals. The prevalence of Salmonella was 2.3% at the sample level, 17.3% at the vegetable farm level, and 2.5% in vegetables. The highest rate of resistance was recorded for streptomycin, 80.7% (21 of 26), followed by kanamycin, 65.4% (17 of 26), and gentamicin, 61.5% (16 of 26). Multidrug resistance was detected in 61.5% of the Salmonella isolates. Vegetable farms have a gap in attaining GAPs, which could contribute to increased contamination and the transfer of antimicrobial resistance to the vegetables. The application of GAPs, including proper preparation of compost and the appropriate use of antimicrobials in veterinary practices, are recommended to reduce the emergence and spread of antimicrobial resistance.

Structure, biosynthesis and regulation of the T1 antigen, a phase-variable surface polysaccharide conserved in many Salmonella serovars.

The bacterial genus Salmonella includes diverse isolates with multiple variations in the structure of the main polysaccharide component (O antigen) of membrane lipopolysaccharides. In addition, some isolates produce a transient (T) antigen, such as the T1 polysaccharide identified in the 1960s in an isolate of Salmonella enterica Paratyphi B. The structure and biosynthesis of the T1 antigen have remained enigmatic. Here, we use biophysical, biochemical and genetic methods to show that the T1 antigen is a complex linear glycan containing tandem homopolymeric domains of galactofuranose and ribofuranose, linked to lipid A-core, like a typical O antigen. T1 is a phase-variable antigen, regulated by recombinational inversion of the promoter upstream of the T1 genetic locus through a mechanism not observed for other bacterial O antigens. The T1 locus is conserved across many Salmonella isolates, but is mutated or absent in most typhoidal serovars and in serovar Enteritidis.

Comprehensive blueprint of Salmonella genomic plasticity identifies hotspots for pathogenicity genes.

Understanding the dynamic evolution of Salmonella is vital for effective bacterial infection management. This study explores the role of the flexible genome, organised in regions of genomic plasticity (RGP), in shaping the pathogenicity of Salmonella lineages. Through comprehensive genomic analysis of 12,244 Salmonella spp. genomes covering 2 species, 6 subspecies, and 46 serovars, we uncover distinct integration patterns of pathogenicity-related gene clusters into RGP, challenging traditional views of gene distribution. These RGP exhibit distinct preferences for specific genomic spots, and the presence or absence of such spots across Salmonella lineages profoundly shapes strain pathogenicity. RGP preferences are guided by conserved flanking genes surrounding integration spots, implicating their involvement in regulatory networks and functional synergies with integrated gene clusters. Additionally, we emphasise the multifaceted contributions of plasmids and prophages to the pathogenicity of diverse Salmonella lineages. Overall, this study provides a comprehensive blueprint of the pathogenicity potential of Salmonella. This unique insight identifies genomic spots in nonpathogenic lineages that hold the potential for harbouring pathogenicity genes, providing a foundation for predicting future adaptations and developing targeted strategies against emerging human pathogenic strains.

A Newly Incompatibility F Replicon Allele (FIB81) in Extensively Drug-Resistant Escherichia coli Isolated from Diseased Broilers.

Multiple drug resistance (MDR) has gained pronounced attention among Enterobacterales. The transfer of multiple antimicrobial resistance genes, frequently carried on conjugative incompatibility F (IncF) plasmids and facilitating interspecies resistance transmission, has been linked to Salmonella spp. and E. coli in broilers. In Egypt, the growing resistance is exacerbated by the limited clinical efficacy of many antimicrobials. In this study, IncF groups were screened and characterized in drug-resistant Salmonella spp. and E. coli isolated from broilers. The antimicrobial resistance profile, PCR-based replicon typing of bacterial isolates pre- and post-plasmid curing, and IncF replicon allele sequence typing were investigated. Five isolates of E. coli (5/31; 16.13%) and Salmonella spp. (5/36; 13.89%) were pan-susceptible to the examined antimicrobial agents, and 85.07% of tested isolates were MDR and extensively drug-resistant (XDR). Twelve MDR and XDR E. coli and Salmonella spp. isolates were examined for the existence of IncF replicons (FII, FIA, and FIB). They shared resistance to ampicillin, ampicillin/sulbactam, amoxicillin/clavulanate, doxycycline, cefotaxime, and colistin. All isolates carried from one to two IncF replicons. The FII-FIA-FIB+ and FII-FIA+FIB- were the predominant replicon patterns. FIB was the most frequently detected replicon after plasmid curing. Three XDR E. coli isolates that were resistant to 12-14 antimicrobials carried a newly FIB replicon allele with four nucleotide substitutions: C99→A, G112→T, C113→T, and G114→A. These findings suggest that broilers are a significant reservoir of IncF replicons with highly divergent IncF-FIB plasmid incompatibility groups circulating among XDR Enterobacterales. Supporting these data with additional comprehensive epidemiological studies involving replicons other than the IncF can provide insights for implementing efficient policies to prevent the spreading of new replicons to humans.

Phenotypic characterization and genomic analysis of a Salmonella phage L223 for biocontrol of Salmonella spp. in poultry.

The escalating incidence of foodborne salmonellosis poses a significant global threat to food safety and public health. As antibiotic resistance in Salmonella continues to rise, there is growing interest in bacteriophages as potential alternatives. In this study, we isolated, characterized, and evaluated the biocontrol efficacy of lytic phage L223 in chicken meat. Phage L223 demonstrated robust stability across a broad range of temperatures (20-70 °C) and pH levels (2-11) and exhibited a restricted host range targeting Salmonella spp., notably Salmonella Typhimurium and Salmonella Enteritidis. Characterization of L223 revealed a short latent period of 30 min and a substantial burst size of 515 PFU/cell. Genomic analysis classified L223 within the Caudoviricetes class, Guernseyvirinae subfamily and Jerseyvirus genus, with a dsDNA genome size of 44,321 bp and 47.9% GC content, featuring 72 coding sequences devoid of antimicrobial resistance, virulence factors, toxins, and tRNA genes. Application of L223 significantly (p < 0.005) reduced Salmonella Typhimurium ATCC 14,028 counts by 1.24, 2.17, and 1.55 log CFU/piece after 2, 4, and 6 h of incubation, respectively, in experimentally contaminated chicken breast samples. These findings highlight the potential of Salmonella phage L223 as a promising biocontrol agent for mitigating Salmonella contamination in food products, emphasizing its relevance for enhancing food safety protocols.

An integrated comparative genomics, subtractive proteomics and immunoinformatics framework for the rational design of a Pan-Salmonella multi-epitope vaccine.

Salmonella infections pose a significant global public health concern due to the substantial expenses associated with monitoring, preventing, and treating the infection. In this study, we explored the core proteome of Salmonella to design a multi-epitope vaccine through Subtractive Proteomics and immunoinformatics approaches. A total of 2395 core proteins were curated from 30 different isolates of Salmonella (strain NZ CP014051 was taken as reference). Utilizing the subtractive proteomics approach on the Salmonella core proteome, Curlin major subunit A (CsgA) was selected as the vaccine candidate. csgA is a conserved gene that is related to biofilm formation. Immunodominant B and T cell epitopes from CsgA were predicted using numerous immunoinformatics tools. T lymphocyte epitopes had adequate population coverage and their corresponding MHC alleles showed significant binding scores after peptide-protein based molecular docking. Afterward, a multi-epitope vaccine was constructed with peptide linkers and Human Beta Defensin-2 (as an adjuvant). The vaccine could be highly antigenic, non-toxic, non-allergic, and have suitable physicochemical properties. Additionally, Molecular Dynamics Simulation and Immune Simulation demonstrated that the vaccine can bind with Toll Like Receptor 4 and elicit a robust immune response. Using in vitro, in vivo, and clinical trials, our findings could yield a Pan-Salmonella vaccine that might provide protection against various Salmonella species.

Virulence factors of Salmonella spp. isolated from free-living grass snakes Natrix natrix.

Salmonellosis associated with reptiles is a well-researched topic, particularly in China and the United States, but it occurs less frequently in Europe. The growth of the human population and changes in the environment could potentially increase the interaction between humans and free-living reptiles, which are an unidentified source of Salmonella species. In this study, we sought to explore this issue by comparing the microbiota of free-living European grass snakes, scientifically known as Natrix natrix, with that of captive banded water snakes, or Nerodia fasciata. We were able to isolate 27 strains of Salmonella species from cloacal swabs of 59 N. natrix and 3 strains from 10 N. fasciata. Our findings revealed that free-living snakes can carry strains of Salmonella species that are resistant to normal human serum (NHS). In contrast, all the Salmonella species strains isolated from N. fasciata were sensitive to the action of the NHS, further supporting our findings. We identified two serovars from N. natrix: Salmonella enterica subspecies diarizonae and S. enterica subspecies houtenae. Additionally, we identified three different virulotypes (VT) with invA, sipB, prgH, orgA, tolC, iroN, sitC, sifA, sopB, spiA, cdtB and msgA genes, and ß-galactosidase synthesised by 23 serovars. The identification of Salmonella species in terms of their VT is a relatively unknown aspect of their pathology. This can be specific to the serovar and pathovar and could be a result of adaptation to a new host or environment.

Retrospective study on the occurrence of Salmonella serotypes in veterinary specimens of Atlantic Canada (2012-2021).

AIM: This study aimed to summarize the frequency and the antimicrobial susceptibility profiles of the Salmonella serotypes identified from the specimens of companion animals, livestock, avian, wildlife and exotic species within Atlantic Canada. MATERIALS AND METHODS: The retrospective electronic laboratory data of microbiological analyses of a selected subset of samples from 03 January 2012 to 29 December 2021 submitted from various animal species were retrieved. The frequency of Salmonella serotypes identified, and their antimicrobial susceptibility results obtained using the disk diffusion or broth method were analysed. The test results were interpreted according to the Clinical and Laboratory Standards Institute standard. The Salmonella serotypes were identified by slide agglutination (Kauffman-White-Le-Minor Scheme) and/or the Whole Genome Sequencing for the Salmonella in silico Serovar Typing Resource-based identification. RESULTS: Of the cases included in this study, 4.6% (n = 154) had at least one Salmonella isolate, corresponding to 55 different serovars. Salmonella isolation was highest from exotic animal species (n = 40, 1.20%), followed by porcine (n = 26, 0.78%), and canine (n = 23, 0.69%). Salmonella subsp. enterica serovar Typhimurium was predominant among exotic mammals, porcine and caprine samples, whereas S. Enteritidis was mostly identified in bovine and canine samples. S. Typhimurium of porcine origin was frequently resistant (>70.0%) to ampicillin. In contrast, S. Typhimurium isolates from porcine and caprine samples were susceptible (>70.0%) to florfenicol. S. Oranienburg from equine samples was susceptible to chloramphenicol, but frequently resistant (>90.0%) to azithromycin. In avian samples, S. Copenhagen was susceptible (>90.0%) to florfenicol, whereas Muenchen was frequently resistant (>90.0%) to florfenicol. S. subsp. diarizonae serovar IIIb:61:k:1,5 of ovine origin was resistant (50.0% isolates) to sulfadimethoxine. No significant changes were observed in the antibiotic resistance profiles across the study years. CONCLUSIONS: This report provides data for surveillance studies, distribution of Salmonella serotypes and their antimicrobial resistance among veterinary specimens of Atlantic Canada.

Characterization of the prevalence of Salmonella in different retail chicken supply modes using genome-wide and machine-learning analyses.

Salmonella is a foodborne pathogen that causes salmonellosis, of which retail chicken meat is a major source. However, the prevalence of Salmonella in different retail chicken supply modes and the threat posed to consumers remains unclear. The prevalence, serotype distribution, antibiotic resistance, and genomic characteristics of Salmonella in three supply modes of retail chicken (live poultry, frozen, and chilled) were investigated using whole-genome sequencing (WGS) and machine learning (ML). In this study, 480 retail chicken samples from live poultry, frozen, and chilled supply modes in Guangzhou from 2020 to 2021, as well as 253 Salmonella isolates (total isolation rate = 53.1 %), were collected. The prevalence of isolates in the live poultry mode (67.5 %, 81/120) was statistically higher than in the frozen (50.0 %, 120/240) and chilled (43.3 %, 52/120) (P < 0.05) modes. Serotype identification showed significant differences in the serotype distribution of Salmonella in different supply modes. S. Enteritis (46.7 %) and S. Indiana (14.2 %) were predominant in the frozen mode. S. Agona (23.5 %) and S. Saintpaul (13.6 %) were predominant in live poultry, while S. Enteritis (40.4 %) and S. Kentucky (17.3 %) were predominant in chilled mode. Antibiotic testing showed that frozen mode isolates were more resistant; the multidrug-resistant (MDR) rate of isolates in the frozen mode reached 91.8 %, significantly higher than in the chilled (86.5 %) and live (74.1 %) (P < 0.05) modes. WGS was performed on 155 top serotypes (S. Enteritidis, S. Kentucky, S. Indiana, and S. Agona). The antibiotic resistance gene analysis showed that the abundance and carrying rate of antibiotic resistance genes of Salmonella in the frozen mode (54 types, 16.1 %) were significantly higher than in other modes (live poultry: 36 types, 9.4 %, P < 0.05; chilled: 31 types, 11.6 %). The blaNDM-1 and blaNDM-9 genes encoding carbapenem resistance were found in frozen mode isolates on a complex transposon consisting of TnAS3-IS26. Virulence factors and plasmid replicons were abundant in the studied frozen mode isolates. In addition, single nucleotide polymorphism (SNP) phylogenetic tree results showed that in the frozen supply mode, the S. Enteritidis clonal clade continued to contaminate retail chicken meat and was homologous to S. Enteritidis strains found in farm chicken embryos, slaughterhouse chicken carcasses, and patients from hospitals in China (SNP 0 = 10). Notably, the pan-genome-based ML model showed that characteristic genes in frozen and live poultry isolates differed. The narZ gene was a key characteristic gene in frozen isolates, encoding nitrate reductase, relating to anaerobic bacterial growth. The ydgJ gene is a key characteristic gene in the live mode and encodes an oxidoreductase related to oxidative function in bacteria. The high prevalence of live poultry mode Salmonella and the transmission of frozen mode MDR Salmonella in this study pose serious risks to food safety and public health, emphasizing the importance of improving disinfection and cold storage measures to reduce Salmonella contamination and transmission. In conclusion, the continued surveillance of Salmonella across different supply models and the development of an epidemiological surveillance system based on WGS is necessary.

Microbiota in lymph nodes of cattle harvested in a Canadian meat processing plant.

Lymph nodes (LN) harboring bacteria, when being incorporated into ground beef, may impact the microbial safety and quality of such products. We tested two main foodborne pathogens Salmonella and Shiga toxin-producing Escherichia coli (STEC) and profiled the microbiota in LNs (n = 160) of cattle harvested at a Canadian abattoir, by conventional plating methods, PCR, and high throughput sequencing. LNs at two anatomical locations, subiliac and popliteal from 80 cattle were included. All cattle had bacteria detected in popliteal and/or subiliac LNs with the maximum bacterial load of 5.4 and 2.8 log10CFU/g in popliteal and subiliac LNs, respectively. Neither Salmonella nor STEC was found in LNs although STEC was detected in a significant percentage of samples from beef hides (50.6 %) by plating and/or PCR. Both 16S rRNA gene amplicon and metagenome sequencing found the predominance of Escherichia (13-34.6 % among bacterial community), Clostridium (12.6-20.6 %) and Streptococcus (9.7-10 %) in popliteal LNs. Metagenomic sequencing was able to identify the predominant taxa at species level with E. coli (13 %), Clostridium perfringens (11.1 %) and Streptococcus uberis (6 %) predominant in LNs. Low prevalence/abundance of Salmonella was found by metagenomic sequencing. In conclusion, the relatively high bacterial load and diversity in LNs may affect the shelf life of ground beef and high relative abundance of E. coli would warrant further monitoring.

Prevalence, serotype, antimicrobial susceptibility, contamination factors, and control methods of Salmonella spp. in retail fresh fruits and vegetables: A systematic review and meta-analysis.

This research presents a comprehensive review of Salmonella presence in retail fresh fruits and vegetables from 2010 to 2023, utilizing data from recognized sources such as PubMed, Scopus, and Web of Science. The study incorporates a meta-analysis of prevalence, serovar distribution, antimicrobial susceptibility, and antimicrobial resistance genes (ARGs). Additionally, it scrutinizes the heterogeneous sources across various food categories and geographical regions The findings show a pooled prevalence of 2.90% (95% CI: 0.0180-0.0430), with an increase from 4.63% in 2010 to 5.32% in 2022. Dominant serovars include S. Typhimurium (29.14%, 95% CI: 0.0202-0.6571) and S. Enteritidis (21.06%, 95% CI: 0.0181-0.4872). High resistance rates were noted for antimicrobials like erythromycin (60.70%, 95% CI: 0.0000-1.0000) and amoxicillin (39.92%, 95% CI: 0.0589-0.8020). The most prevalent ARGs were blaTEM (80.23%, 95% CI: 0.5736-0.9692) and parC mutation (66.67%, 95% CI: 0.3213-0.9429). Factors such as pH, water activity, and nutrient content, along with external factors like the quality of irrigation water and prevailing climatic conditions, have significant implications on Salmonella contamination. Nonthermal sterilization technologies, encompassing chlorine dioxide, ozone, and ultraviolet light, are emphasized as efficacious measures to control Salmonella. This review stresses the imperative need to bolster prevention strategies and control measures against Salmonella in retail fresh fruits and vegetables to alleviate related food safety risks.

Development of a Colorimetric and SERS Dual-Signal Platform via dCas9-Mediated Chain Assembly of Bifunctional Au@Pt Nanozymes for Ultrasensitive and Robust Salmonella Assay.

Timely screening for harmful pathogens is a great challenge in emergencies where traditional culture methods suffer from long assay time and alternative methods are limited by poor accuracy and low robustness. Herein, we present a dCas9-mediated colorimetric and surface-enhanced Raman scattering (SERS) dual-signal platform (dCas9-CSD) to address this challenge. Strategically, the platform used dCas9 to accurately recognize the repetitive sequences in amplicons produced by loop-mediated isothermal amplification (LAMP), forming nucleic acid frameworks that assemble numerous bifunctional gold-platinum (Au@Pt) nanozymes into chains on the surface of streptavidin-magnetic beads (SA-MB). The collected Au@Pt converted colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB (oxTMB) via its Pt shell and then enhanced the Raman signal of oxTMB by its Au core. Therefore, the presence of Salmonella could be dexterously converted into cross-validated colorimetric and SERS signals, providing more reliable conclusions. Notably, dCas9-mediated secondary recognition of amplicons reduced background signal caused by nontarget amplification, and two-round signal amplification consisting of LAMP reaction and Au@Pt catalysis greatly improved the sensitivity. With this design, Salmonella as low as 1 CFU/mL could be detected within 50 min by colorimetric and SERS modes. The robustness of dCas9-CSD was further confirmed by various real samples such as lake water, cabbage, milk, orange juice, beer, and eggs. This work provides a promising point-of-need tool for pathogen detection.

Sensitive and rapid detection of three foodborne pathogens in meat by recombinase polymerase amplification with lateral flow dipstick (RPA-LFD).

Foodborne illnesses, caused by harmful microorganisms in food, are a significant global health issue. Current methods for identifying these pathogens are both labor-intensive and time-consuming. In this research, we devised a swift and precise detection technique using recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) for three foodborne pathogens found in meat. By employing a dedicated detection device, RPA-LFD allows for the rapid analysis of DNA from Escherichia coli O157 (E. coli O157), Salmonella, and Shigella-pathogens that are prohibited in food. The detection thresholds for E. coli O157, Salmonella, and Shigella are 0.168 fg/µl (1.04 CFU/ml), 0.72 fg/µl (27.49 CFU/ml), and 1.25 fg/µl (48.84 CFU/ml), respectively. This method provides a short detection window, operates at low temperatures, follows simple procedures, and exhibits high sensitivity. Our study establishes the RPA-LFD method for simultaneously identifying the nucleic acid of three foodborne pathogens, offering an efficient solution for quickly identifying multiple contaminants.

Prospective modeling and estimating the epidemiologically informative match rate within large foodborne pathogen genomic databases.

OBJECTIVES: Much has been written about the utility of genomic databases to public health. Within food safety these databases contain data from two types of isolates-those from patients (i.e., clinical) and those from non-clinical sources (e.g., a food manufacturing environment). A genetic match between isolates from these sources represents a signal of interest. We investigate the match rate within three large genomic databases (Listeria monocytogenes, Escherichia coli, and Salmonella) and the smaller Cronobacter database; the databases are part of the Pathogen Detection project at NCBI (National Center for Biotechnology Information). RESULTS: Currently, the match rate of clinical isolates to non-clinical isolates is 33% for L. monocytogenes, 46% for Salmonella, and 7% for E. coli. These match rates are associated with several database features including the diversity of the organism, the database size, and the proportion of non-clinical BioSamples. Modeling match rate via logistic regression showed relatively good performance. Our prediction model illustrates the importance of populating databases with non-clinical isolates to better identify a match for clinical samples. Such information should help public health officials prioritize surveillance strategies and show the critical need to populate fledgling databases (e.g., Cronobacter sakazakii).

Enhancing Nanobody Immunoassays through Ferritin Fusion: Construction of a Salmonella-Specific Fenobody for Improved Avidity and Sensitivity.

Nanobodies (Nbs) serve as powerful tools in immunoassays. However, their small size and monovalent properties pose challenges for practical application. Multimerization emerges as a significant strategy to address these limitations, enhancing the utilization of nanobodies in immunoassays. Herein, we report the construction of a Salmonella-specific fenobody (Fb) through the fusion of a nanobody to ferritin, resulting in a self-assembled 24-valent nanocage-like structure. The fenobody exhibits a 35-fold increase in avidity compared to the conventional nanobody while retaining good thermostability and specificity. Leveraging this advancement, three ELISA modes were designed using Fb as the capture antibody, along with unmodified Nb422 (FbNb-ELISA), biotinylated Nb422 (FbBio-ELISA), and phage-displayed Nb422 (FbP-ELISA) as the detection antibody, respectively. Notably, the FbNb-ELISA demonstrates a detection limit (LOD) of 3.56 × 104 CFU/mL, which is 16-fold lower than that of FbBio-ELISA and similar to FbP-ELISA. Moreover, a fenobody and nanobody sandwich chemiluminescent enzyme immunoassay (FbNb-CLISA) was developed by replacing the TMB chromogenic substrate with luminal, resulting in a 12-fold reduction in the LOD. Overall, the ferritin-displayed technology represents a promising methodology for enhancing the detection performance of nanobody-based sandwich ELISAs, thereby expanding the applicability of Nbs in food detection and other fields requiring multivalent modification.

DODGE: automated point source bacterial outbreak detection using cumulative long term genomic surveillance.

SUMMARY: The reliable and timely recognition of outbreaks is a key component of public health surveillance for foodborne diseases. Whole genome sequencing (WGS) offers high resolution typing of foodborne bacterial pathogens and facilitates the accurate detection of outbreaks. This detection relies on grouping WGS data into clusters at an appropriate genetic threshold. However, methods and tools for selecting and adjusting such thresholds according to the required resolution of surveillance and epidemiological context are lacking. Here we present DODGE (Dynamic Outbreak Detection for Genomic Epidemiology), an algorithm to dynamically select and compare these genetic thresholds. DODGE can analyse expanding datasets over time and clusters that are predicted to correspond to outbreaks (or "investigation clusters") can be named with established genomic nomenclature systems to facilitate integrated analysis across jurisdictions. DODGE was tested in two real-world Salmonella genomic surveillance datasets of different duration, 2 months from Australia and 9 years from the United Kingdom. In both cases only a minority of isolates were identified as investigation clusters. Two known outbreaks in the United Kingdom dataset were detected by DODGE and were recognized at an earlier timepoint than the outbreaks were reported. These findings demonstrated the potential of the DODGE approach to improve the effectiveness and timeliness of genomic surveillance for foodborne diseases and the effectiveness of the algorithm developed. AVAILABILITY AND IMPLEMENTATION: DODGE is freely available at https://github.com/LanLab/dodge and can easily be installed using Conda.

Isolation, whole genome sequencing and application of a broad-spectrum Salmonella phage.

Salmonella is considered as one of the most common zoonotic /foodborne pathogens in the world. The application of bacteriophages as novel antibacterial agents in food substrates has become an emerging strategy. Bacteriophages have the potential to control Salmonella contamination.We have isolated and characterized a broad-spectrum Salmonella phage, SP154, which can lyse 9 serotypes, including S. Enteritidis, S. Typhimurium, S. Pullorum, S. Arizonae, S. Dublin, S. Cholerasuis, S. Chester, S. 1, 4, [5], 12: i: -, and S. Derby, accounting for 81.9% of 144 isolates. SP154 showed a short latent period (40 min) and a high burst size (with the first rapid burst size at 107 PFUs/cell and the second rapid burst size at approximately 40 PFUs/cell). Furthermore, SP154 activity has higher survival rates across various environmental conditions, including pH 4.0-12.0 and temperatures ranging from 4 to 50 °C for 60 min, making it suitable for diverse food processing and storage applications. Significant reductions in live Salmonella were observed in different foods matrices such as milk and chicken meat, with a decrease of up to 1.9 log10 CFU/mL in milk contamination and a 1 log10 CFU/mL reduction in chicken meat. Whole genome sequencing analysis revealed that SP154 belongs to the genus Ithacavirus, subfamily Humphriesvirinae, within the family Schitoviridae. Phylogenetic analysis based on the terminase large subunit supported this classification, although an alternate tree using the tail spike protein gene suggested affiliation with the genus Kuttervirus, underscoring the limitations of relying on a single gene for phylogenetic inference. Importantly, no virulence or antibiotic resistance genes were detected in SP154. Our research highlights the potential of using SP154 for biocontrol of Salmonella in the food industry.

[Effect of Quorum Sensing Systems on Biofilm Formation and Virulence in Salmonella]. / Salmonella'da Çogunlugu Algilama Sistemlerinin Biyofilm Olusumu ve Virülans Üzerine Etkisi.

In recent years, as the paradigm of communication between cells has been clarified, the ability of bacteria to change their gene expression patterns in response to various extracellular signals has attracted great interest. In particular, intracellular and intercellular communication between bacterial populations, called quorum sensing (QS), is essential for coordinating physiological and genetic activities. QS studies are critical, particularly in elucidating the regulatory mechanisms of infectious processes in food-borne pathogens. Elucidating the QS mechanisms in Salmonella is effective in silencing the virulence factors in the fight against this bacterium. The aims of this study were; to create luxS gene mutants that play a vital role in the QS activity of Salmonella and to determine the effect of this mutation on the expression of virulence genes in the bacteria and to determine the impact of synthetic N-hexanoyl-homoserine lactone (C6HSL) on biofilm formation and AI-2 signaling pathway of Salmonella wild strain and luxS gene mutants. luxS gene mutants were constructed by recombining the gene region with the chloramphenicol gene cassette based on homologous region recombination. In the luxS mutants obtained in this way, the expression of eight different virulence genes (hilA, invA, inv, glgC, fimF, fliF, lpfA, gyrA), which have essential roles in Salmonella pathogenicity, was determined by quantitative real-time reverse transcriptase polymerase chain reaction (rRT-qPCR) method and compared with natural strains. As a result of these studies, it was determined that the expression of each gene examined was significantly reduced in luxS mutant strains. The relative AI-2 activities of Salmonella strains were analyzed depending on time. It was determined that the highest activity occurred at the fourth hour and the AI-2 activities of luxS mutants were reduced compared to the wild strain. Finally, it was determined that C6HSL increased the biofilm activity of Salmonella Typhimurium DMC4, SL1344 wild strains, and mutants, mainly at the 72nd hour. In conclusion, our results proved that C6HSL stimulated QS communication in all strains and increased biofilm of Salmonella formation and autoinducer activity. This situation determines that Salmonella responds to external signals by using QS systems. In addition, this research contributed to provide additional information on interspecies communication mechanisms to develop strategies to prevent biofilm formation of this pathogen.

The microbiological quality of flour products in the UK with respect to Salmonella and Shiga-toxin-producing Escherichia coli.

AIM: To investigate the possible contamination of raw flour and raw flour-based products, such as pancake/batter mixes, with Salmonella, generic Escherichia coli, and Shiga-toxin-producing E. coli (STEC). Samples included flours available for sale in the UK over a period of four months (January to April 2020). The Bread and Flour regulations, 1998 state the permitted ingredients in flour and bread but it does not specify the regular monitoring of the microbiological quality of flour and flour-based products. METHODS AND RESULTS: Samples of raw flour were collected by local authority sampling officers in accordance with current guidance on microbiological food sampling then transported to the laboratory for examination. Microbiological testing was performed to detect Salmonella spp., generic E. coli, and STEC characterized for the presence of STEC virulence genes: stx1, stx2, and subtypes, eae, ipah, aggR, lt, sth, and stp, using molecular methods Polymerase Chain Reaction (PCR). Of the 882 flours sampled, the incidence of Salmonella was 0.1% (a single positive sample that contained multiple ingredients such as flour, dried egg, and dried milk, milled in the UK), and 68 samples (7.7%) contained generic E. coli at a level of >20 CFU/g. Molecular characterization of flour samples revealed the presence of the Shiga-toxin (stx) gene in 10 samples (5 imported and 5 from the UK) (1.1%), from which STEC was isolated from 7 samples (0.8%). Salmonella and STEC isolates were sequenced to provide further characterization of genotypes and to compare to sequences of human clinical isolates held in the UKHSA archive. Using our interpretive criteria based on genetic similarity, none of the STEC flour isolates correlated with previously observed human cases, while the singular Salmonella serotype Newport isolate from the mixed ingredient product was similar to a human case in 2019, from the UK, of S. Newport. Although there have been no reported human cases of STEC matching the isolates from these flour samples, some of the same serotypes and stx subtypes detected are known to have caused illness in other contexts. CONCLUSION: Results indicate that while the incidence was low, there is a potential for the presence of Salmonella and STEC in flour, and a genetic link was demonstrated between a Salmonella isolate from a flour-based product and a human case of salmonellosis.