Guodong Zhang

Background: As a leading cause of foodborne illness worldwide, detection of Salmonella enterica subsp. enterica serovar Typhimurium is essential for food safety and public health. Objective: This study aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella ser. Typhimurium in egg products. Methods: The primer set targeting the open reading frame STM3845 of Salmonella ser. Typhimurium was designed using PrimerExplorer v.4. The LAMP assay was optimized by adjusting reagent concentrations, reaction temperature, and incubation time, achieving the highest amplification/fluorescence in a 25.0 µL reaction at 65°C for 30 min. Results: Results indicated that the newly designed assay could successfully detect and differentiate Salmonella ser. Typhimurium from other Salmonella serotypes and non-Salmonella bacterial pathogens except for Salmonella serotypes Montevideo, Michigan, and Senftenberg after testing 73 Salmonella ser. Typhimurium, 100 non-Typhimurium Salmonella, and 35 non-Salmonella bacterial pathogens of pure cultures. The LAMP assay was further compared with a commercial real-time PCR and FDA BAM culture method by testing pure culture and 200 inoculated (1–5 CFU/25g) egg and egg product samples, and proved to be comparable to the FDA BAM culture method; it also demonstrated 100 times more sensitivity than the real-time PCR assay in pure culture testing, with a detection limit of 0.56 log CFU/mL. Conclusions: The newly developed LAMP assay offers a rapid, specific, and sensitive method for detecting Salmonella ser. Typhimurium in egg products. Its simplicity, speed, and sensitivity position it as a powerful tool for routine monitoring, outbreak investigation, and on-site testing in the food industry. Highlights: Developed a LAMP assay for specific detection Salmonella ser. Typhimurium in egg products. The newly developed LAMP assay was 100 times more sensitive than the real-time PCR assay. Our new LAMP assay was validated with hundreds of pure isolates and food samples.

For pathogen detection in food, there are occasions where samples cannot be processed immediately after selective enrichment or need to be reexamined days or weeks later for confirmation or retest. This study aimed to investigate the effect of different prolonged period of storage of selective enrichments of food at 4 ± 2°C and room temperature (20–22°C) on the detection and isolation of Salmonella. This study included two experiments involving 34 types of foods to compare the effect of 4 ± 2°C and room temperature storage on the detection of Salmonella in 204 selective enrichments (Rappaport-Vassiliadis [RV] and Tetrathionate [TT] broths) during a 42-day storage (Experiment I); and to monitor the survival of Salmonella in 300 selective enrichments (RV and TT) with different pre-enrichment broths (Lactose broth [LB] or Buffered peptone water broth [BPW]), stored at 4°C for 60 days (Experiment II). All the samples were subjected to Salmonella analysis following the FDA BAM method. During multiple samplings, the positive detection rate for Salmonella remained consistent through Day 42 after selective enrichment, irrespective of Salmonella serotype, storage temperature, pre-enrichment broth, or selective enrichment broth in both Experiment I and II. However, on Day 60 sampling in Experiment II, seven previously positive results turned to negatives. These data indicated that storage of RV and TT enrichments at 4 ± 2°C or room temperature for up to 42 days after selective enrichment did not compromise the detection of Salmonella in the tested food categories, regardless of Salmonella serotypes and the broths used for pre-enrichment and selective enrichment. At least for the food types studied in this experiment, the recovery of Salmonella from selective enrichments could be postponed for a limited period of time (e.g., Salmonella from selective TT and RV enrichments stored at 4 ± 2°C or room temperature could be deferred for a period (up to 42 days) without any negative effect on the test results, if necessary.

Introduction: The objective of this study was to analyze antimicrobial resistance (AMR), stress resistance, and virulence factors through whole genome sequencing (WGS) of 192 isolates comprising 164 Salmonella isolates, 8 non-O157 Shiga toxin-producing Escherichia coli (STEC) isolates, and 20 Listeria monocytogenes isolates.Methods: These isolates were sourced from a national survey conducted between 2010 and 2017, involving 31,322 samples of produce (31 isolates), nuts (43 isolates), and spices (118 isolates).Results: The findings yielded several key insights: (1) Within all Salmonella isolates studied, the most prevalent Salmonella serotypes included Give, Kentucky, Senftenberg, Mbandaka, Anatum, Newport, and Weltevreden. (2) All eight non-O157 STEC isolates were found to carry the genes blaEC, acrF, and mdtM, while all 20 L. monocytogenes isolates possessed fosX and lin genes. The Salmonella isolates displayed diverse AMR gene profiles, with 3.65% exhibiting multi-drug resistance. (3) Both Salmonella and non-O157 STEC isolates were discovered to carry stress genes associated with acid resistance, but none of the L. monocytogenes isolates carried an acid resistance gene. Salmonella isolates were found to carry multiple metal-resistance genes. The non-O157 STEC isolates universally exhibited acid resistance genes, and 4 out of the 20 L. monocytogenes isolates were equipped with resistance genes against biocides. (4) All of the STEC isolates (100%) carried stx1 and stx2 genes, while none of them carried eae and wyz genes. Most L. monocytogenes isolates were found to contain 29 virulence genes and 1 pathogenicity island. All Salmonella isolates carried SPI-9, but lacked SPI-7, SPI-10, SPI-11, SPI-12, mig-5, prefA, tviA, and viaB genes.Conclusion: These findings on AMR, stress resistance, and virulence factors among the investigated isolates highlight the potential risks they pose to public health and provide the scientific foundation for the development of preventative and control strategies and guidance pertaining to these major foodborne pathogens.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are recognized as an important group of bacterial enteropathogens. Here, we report the draft genome sequence of nine strains of non-O157 STEC isolated from ready-to-eat foods in Argentina. The whole-genome sequence data provide a better understanding of these isolates and will aid epidemiological investigation during outbreaks.

Bacillus species have been widely selected and used as probiotics for humans and animals. In this article, we reported draft whole-genome sequences of four Bacillus strains isolated from sourdough and chicken cecum and previously selected as potential probiotics for poultry. These genome sequences will provide a foundation for further characterization and understanding of their probiotic attributes.

Competitive exclusion (CE) bacteria have been used to control the colonization of chickens by major foodborne pathogens. In this article, we report draft whole-genome sequences of three Ligilactobacillus salivarius strains isolated from chicken gastrointestinal tracts and previously selected as CE for poultry. These genome sequences will provide a foundation for further characterization and understanding of their CE attributes.

Background: Shiga toxin-producing Escherichia coli (STEC) is a significant cause of foodborne illness causing various gastrointestinal diseases including hemolytic uremic syndrome (HUS), the most severe form, which can lead to kidney failure or even death. Objective: Here, we report the development of recombinase aided amplification (RAA)-exo-probe assays targeting the stx1 and stx2 genes for the rapid detection of STEC in food samples. Methods: Primers and exo-probes were designed and optimized for the detection of stx1 and stx2 using RAA technology. The optimal STEC RAA-exo-probe assays were then tested for specificity and sensitivity, and validated in both spiked and real food samples. Results: These assays were found to be 100% specific to STEC strains and were also highly sensitive with a detection limit of 1.6 × 103 CFU/mL or 32 copies/reaction. Importantly, the assays were able to successfully detect STEC in spiked and real food samples (beef, mutton, and pork), with a detection limit as low as 0.35 CFU/25g in beef samples after an overnight enrichment step. Conclusions: Overall, the RAA assay reactions completed within ∼20 min and were less dependent on expensive equipment, suggesting they can be easily adopted for in-field testing requiring only a fluorescent reader. Highlights: As such, we have developed two rapid, sensitive, and specific assays that can be used for the routine monitoring of STEC contamination in food samples, particularly in the field or in poorly equipped labs.

Rapid and sensitive detection of Salmonella is a critical step in routine food quality control, outbreak investigation, and food recalls. Although various genes have been the targets in the design of rapid molecular detection methods for Salmonella, there is limited information on the diversity of these target genes at the level of DNA sequence and the encoded protein structures. In this study, we investigated the diversity of ten target genes (invA, fimA, phoP, spvC, and agfA; ttrRSBCA operon including 5 genes) commonly used in the detection and identification of Salmonella. To this end, we performed whole genome sequencing of 143 isolates of Salmonella serotypes (Enteritidis, Typhimurium, and Heidelberg) obtained from poultry (eggs and chicken). Phylogenetic analysis showed that Salmonella ser. Typhimurium was more diverse than either Enteritidis or Heidelberg. Forty-five non-synonymous mutations were identified in the target genes from the 143 isolates, with the two most common mutations as T ↔ C (15 times) and A ↔ G (13 times). The gene spvC was primarily present in Salmonella ser. Enteritidis isolates and absent from Heidelberg isolates, whereas ttrR was more conserved (0 non-synonymous mutations) than ttrS, ttrB, ttrC, and ttrA (7, 2, 2, and 7 non-synonymous mutations, respectively). Notably, we found one non-synonymous mutation (fimA-Mut.6) across all Salmonella ser. Enteritidis and Salmonella ser. Heidelberg, C → T (496 nt postion), resulting in the change at AA 166 position, Glutamine (Q) → Stop condon (TAG), suggesting that the fimA gene has questionable sites as a target for detection. Using Phyre² and SWISS-MODEL software, we predicted the structures of the proteins encoded by some of the target genes, illustrating the positions of these non-synonymous mutations that mainly located on the α-helix and β-sheet which are key elements for maintaining the conformation of proteins. These results will facilitate the development of sensitive molecular detection methods for Salmonella.

The objective of this survey was to estimate the prevalence, contamination level, and genetic diversity of Salmonella in selected raw, shelled tree nuts (Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, and walnuts) at retail markets in the United States. A total of 3,374 samples of eight tree nuts were collected from different types of retail stores and markets nationwide between September 2015 and March 2017. These samples (375 g) were analyzed using a modified FDA's BAM Salmonella culture method. Of the 3,374 samples, 15 (0.44%) (95% confidence interval [CI] [0.25, 0.73]) were culturally confirmed as containing Salmonella ; 17 isolates were obtained. Among these isolates, there were 11 serotypes. Salmonella was not detected in Brazil nuts (296), hazelnuts (487), pecans (510), pine nuts (500), and walnuts (498). Salmonella prevalence estimates in cashews (510), macadamia (278), and pistachios (295) were 0.20% (95% CI [Salmonella isolation from major/big‐chain supermarkets (1381), small‐chain supermarkets (328), discount/variety/drug stores (1329), and online (336) were 0.29% (95% CI [0.08, 0.74]), 0.30% (95% CI [0.01, 1.69]), 0.45% (95% CI [0.17, 0.98]), and 1.19% (95% CI [0.33, 3.02]), respectively. Salmonella prevalence in organic (530) and conventional (2,844) nuts was not different statistically ( P = 0.0601). Of the enumerated samples (15), 80% had Salmonella levels ≤0.0092 most probable number (MPN)/g. The highest contamination level observed was 0.75 MPN/g. The prevalence and contamination levels of Salmonella in the tree nuts analyzed were generally comparable to previous reports. Pulsed‐field gel electrophoresis, serotype, and sequencing data all demonstrated that Salmonella population in nuts is very diverse genetically. Practical Application: The prevalence, contamination level, and genetic diversity of Salmonella in eight types of tree nuts (3,374 samples collected nationwide) revealed in this survey could help the development of mitigation strategies to reduce public health risks associated with consumption of these nuts.