Benchmarking short and long read polishing tools for nanopore assemblies: achieving near-perfect genomes for outbreak isolates.

BACKGROUND: Oxford Nanopore provides high throughput sequencing platforms able to reconstruct complete bacterial genomes with 99.95% accuracy. However, even small levels of error can obscure the phylogenetic relationships between closely related isolates. Polishing tools have been developed to correct these errors, but it is uncertain if they obtain the accuracy needed for the high-resolution source tracking of foodborne illness outbreaks. RESULTS: We tested 132 combinations of assembly and short- and long-read polishing tools to assess their accuracy for reconstructing the genome sequences of 15 highly similar Salmonella enterica serovar Newport isolates from a 2020 onion outbreak. While long-read polishing alone improved accuracy, near perfect accuracy (99.9999% accuracy or ~ 5 nucleotide errors across the 4.8 Mbp genome, excluding low confidence regions) was only obtained by pipelines that combined both long- and short-read polishing tools. Notably, medaka was a more accurate and efficient long-read polisher than Racon. Among short-read polishers, NextPolish showed the highest accuracy, but Pilon, Polypolish, and POLCA performed similarly. Among the 5 best performing pipelines, polishing with medaka followed by NextPolish was the most common combination. Importantly, the order of polishing tools mattered i.e., using less accurate tools after more accurate ones introduced errors. Indels in homopolymers and repetitive regions, where the short reads could not be uniquely mapped, remained the most challenging errors to correct. CONCLUSIONS: Short reads are still needed to correct errors in nanopore sequenced assemblies to obtain the accuracy required for source tracking investigations. Our granular assessment of the performance of the polishing pipelines allowed us to suggest best practices for tool users and areas for improvement for tool developers.

A Case of Food Poisoning Caused by Campylobacter jejuni due to Ingestion of Undercooked Chicken Meal with Subsequent Development of Guillain-Barré Syndrome.

Campylobacter jejuni is one of the major bacteria that causes diarrhea in humans. It has been associated with many cases of food poisoning in Japan caused by eating raw or undercooked chicken meat, chicken liver, and grilled chicken (Yakitori). Campylobacter jejuni is also known as the preceding infection pathogen of Guillain-Barré syndrome (GBS), which causes considerable health impact on humans. In January 2022, in a case of C. jejuni food poisoning that occurred at a restaurant in Tokyo, one of four patients with diarrhea developed GBS. The poisoning is presumed to have been caused by undercooked chicken dishes. Recently, it was one of the common cases in Japan. Moreover, C. jejuni isolates from three patients, including the patient with GBS, had the same genotype (ST22, HS19, and LOS A). This genotype was frequently detected from patients with GBS in our past surveys. Our findings confirmed that the patient developed GBS via food poisoning after consuming undercooked chicken dish.

The First Reported Foodborne Botulism Outbreak in Riyadh, Saudi Arabia: Lessons Learned.

BACKGROUND: Botulism has not been previously reported in the Kingdom of Saudi Arabia. This rare and sometimes fatal foodborne illness is caused by neurotoxins and primarily results from consuming home-canned fruits, vegetables, dairy, and seafood products & it can lead to paralysis. OBJECTIVE: The purpose of this study was to evaluate the clinical features of patients who developed botulism in Riyadh in 2024 after consuming mayonnaise from a well-known local chain of restaurants in Riyadh, Saudi Arabia. METHODS: We conducted a retrospective analysis of medical records and interviewed patients or their attendants for all hospitalized cases of foodborne botulism at Riyadh First Health Cluster. For each patient, a standard case report form was completed, containing information on demographics, clinical aspects, botulinum test results, and type of exposure. Descriptive statistics were applied to assess the data. During the outbreak, nineteen patients with foodborne diseases were admitted to Riyadh First Health Cluster Hospitals. Following thorough physical examinations, botulism was suspected in each case. RESULTS: Eight of the 19 suspected foodborne illness patients fully satisfied the botulism case definition requirements set forth by the Saudi Arabian Public Health Authority (Weqaya). Among these eight patients, 2 (25%) were male and 6 (75%) were female, with a mean age of 23.25 ± 9.29 years (range: 12-38 years). The incubation period for our patients was 36.25 ± 26.26 h. Notable symptoms included dysphagia in all eight patients (100%), dysarthria, generalized weakness, nausea and vomiting in seven patients (88%), diplopia in four patients (50%), and stomach discomfort in three patients (38%). Of the eight cases, six required intubation, one mimicked brain death, and two were stable. The presence of Clostridium botulinum spores as the cause of the outbreak was confirmed by detecting botulinum spores in contaminated food. CONCLUSION: Diplopia and dysarthria were the most common early sign of botulism. Early manifestations may include respiratory symptoms without any musculoskeletal symptoms. or nausea, vomiting and disorientation.

A Food Poisoning Caused by Salmonella Enterica (S. Enteritidis) ST11 Carrying Multi-Antimicrobial Resistance Genes in 2019, China.

Purpose: This study was to identify and analyze the pathogen responsible for food poisoning in a tourist group traveling from Macao to Zhuhai. Patients and Methods: Samples were obtained from 27 patients of 96 cases, as well as samples of contaminated food in Macau. The collected samples were subjected to serological identification, drug sensitivity analysis, drug resistance gene identification, virulence factor analysis, and tracing. Results: Twenty-six isolates and the salad isolate were S. enteritidis ST11. Isolates from patients were exhibited significant resistance to Penicillin AMP (Ampicillin) and quinolones NAL (Nalidixic acid). Among these isolates, 21 strains were resistant to two or more antibiotics, indicating the multi-drug resistance (MDR). Genomic characteristics and phylogenetic analysis were performed on 9 of the isolates using whole genome sequencing (WGS). The analysis revealed that the resistance to AMP and NAL was primarily caused by a gryA mutation D87Y (9/9, 100%), and the presence of beta-lactam resistance genes blaOXA-1 (1/9, 11.11%), blaTEM-141 (1/9, 11.11%), and blaTEM-1B (8/9, 88.89%). It was also found a strains isolated from patients had two resistance genes to quinolones or beta-lactam drugs (1/8, 12.5%), respectively. The strains were found to possess 165 virulence genes, one adherence class virulence factor, one invasion class virulence factor and various pathogenicity islands, including SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, SPI-10, SPI-13, SPI-14, SPI-15, SGI 1, CS54_island, and C63PI-1. Additionally, the virulence plasmids were detected, including IncFIB(s)-IncFII(s)-IncX1 (55.56%), IncFIB(s)-IncFII(s) (33.33%), and IncFIB(s)-IncFII(s)-IncHI2-IncHI2A (11.11%). PFGE (Pulsed Field Gel Electrophoresis) and phylogenetic tree analysis revealed a high degree of similarity between Salmonella isolates from patients and food samples from Macao. Conclusion: This study identified Salmonella enterica ST11 as the cause of the food poisoning outbreak. The findings highlight the importance of phenotypic characterization and next-generation sequencing (NGS) tools in epidemiological studies and emphasize the potential risk of a new emerging multi-antibiotic ST11 clone for S. enteritidis.

Prevalence and Antibiotic Resistance Profile of Clostridium perfringens Isolated from Pork and Chicken Meat in Vietnam.

Clostridium perfringens is one of the most important zoonotic pathogens as it can cause food poisoning in humans and necrotic enteritis in both animals and humans. Meat, especially pork and chicken meat, is considered the main vehicle for the transmission of C. perfringens from animals to humans. The purpose of this study was to determine the prevalence, toxinotype, and antimicrobial resistance profile of C. perfringens isolated from pork and chicken meat sold in Vietnam. The isolation results showed that 15/50 (30%) of pork samples and 8/50 (16%) of chicken meat samples were contaminated with C. perfringens. The isolates exhibited their highest resistance rate to tetracycline (21/23; 91.30%) and clindamycin (10/23; 43.48%). On the contrary, their lowest resistance rates were observed in response to imipenem (2/23; 8.70%) and cefoxitin (1/23; 4.35%). In particular, 34.78% (8/23) of C. perfringens isolates were identified to be multidrug-resistant strains. The results of toxin genotyping indicated that all isolates were positive for the cpa gene and belonged to type A.

Staphylococcal food poisoning outbreaks occurred in Sicily (Italy) from 2009 to 2016.

Notification of foodborne outbreaks has been mandatory in Europe since 2005, and surveillance is carried out along the entire food chain. Here we report the results obtained from laboratory investigations about four cases of foodborne outbreaks that occurred in Sicily between 2009 and 2016, deemed to be related to staphylococcal enterotoxins (SEs) and coagulase-positive Staphylococci (CPS) by the Local Public Health Authority. Primosale cheese samples were processed by culture methods for enumeration of CPS and immunoenzymatic assays for detection and differentiation of the SEs possibly contained in food samples. In all cases, the mistrusted foods were found to be contaminated by CPS at bacterial loads between 5 and 8 log CFU/g and contained SE type C (SEC). The reported data confirm the risk of staphylococcal food poisoning associated with the consumption of raw milk cheese. SEC is the most commonly occurring SE in goat milk and dairy products and the most represented enterotoxin in Sicilian dairy products. Our results highlighted the need for improving the current monitoring efficiency and implementing the available laboratory methods to collect more faithful epidemiological data on the current prevalence of staphylococcal toxins in the food chain, including SEs currently not detectable by validated analytical methods.

A poisonous cocktail: interplay of cereulide toxin and its structural isomers in emetic Bacillus cereus.

Food intoxications evoked by emetic Bacillus cereus strains constitute a serious threat to public health, leading to emesis and severe organ failure. The emetic peptide toxin cereulide, assembled by the non-ribosomal peptide synthetase CesNRPS, cannot be eradicated from contaminated food by usual hygienic measures due to its molecular size and structural stability. Next to cereulide, diverse chemical variants have been described recently that are produced concurrently with cereulide by CesNRPS. However, the contribution of these isocereulides to the actual toxicity of emetic B. cereus, which produces a cocktail of these toxins in a certain ratio, is still elusive. Since cereulide isoforms have already been detected in food remnants from foodborne outbreaks, we aimed to gain insights into the composition of isocereulides and their impact on the overall toxicity of emetic B. cereus. The amounts and ratios of cereulide and isocereulides were determined in B. cereus grown under standard laboratory conditions and in a contaminated sample of fried rice balls responsible for one of the most severe food outbreaks caused by emetic B. cereus in recent years. The ratios of variants were determined as robust, produced either under laboratory or natural, food-poisoning conditions. Examination of their actual toxicity in human epithelial HEp2-cells revealed that isocereulides A-N, although accounting for only 10% of the total cereulide toxins, were responsible for about 40% of the total cytotoxicity. An this despite the fact that some of the isocereulides were less cytotoxic than cereulide when tested individually for cytotoxicity. To estimate the additive, synergistic or antagonistic effects of the single variants, each cereulide variant was mixed with cereulide in a 1:9 and 1:1 binary blend, respectively, and tested on human cells. The results showed additive and synergistic impacts of single variants, highlighting the importance of including not only cereulide but also the isocereulides in routine food and clinical diagnostics to achieve a realistic toxicity evaluation of emetic B. cereus in contaminated food as well as in patient samples linked to foodborne outbreaks. Since the individual isoforms confer different cell toxicity both alone and in association with cereulide, further investigations are needed to fully understand their cocktail effect.

Usefulness and Limitations of PFGE Diagnosis and Nucleotide Sequencing Method in the Analysis of Food Poisoning Pathogens Found in Cooking Employees.

In the case of a food poisoning outbreak, it is essential to understand the relationship between cooking workers and food poisoning. Many biological diagnostic methods have recently been developed to detect food poisoning pathogens. Among these diagnostic tools, this study presents PCR-based pulsed-field gel electrophoresis and nucleotide sequencing diagnostic analysis results for diagnosing food poisoning outbreaks associated with cooking employees in Chungcheongnam-do, Republic of Korea. Pulsed-field gel electrophoresis was useful in identifying the food poisoning outbreaks caused by Staphylococcus aureus and Enteropathogenic Escherichia coli. In the case of Norovirus, nucleotide sequencing was used to identify the relationship between cooking workers and the food poisoning outbreak. However, it is difficult to determine whether cooking employees directly caused the food poisoning outbreaks based on these molecular biological diagnostic results alone. A system is needed to integrate epidemiological and diagnostic information to identify a direct correlation between the food poisoning outbreak and cooking employees.

Identification of toxic Gelsemium elegans in processed food and honey based on real-time PCR analysis.

Gelsemium elegans (GE) is a widely distributed hypertoxic plant that has caused many food poisoning incidents. Its pollen can also be collected by bees to produce toxic honey, posing a great threat to the health and safety of consumers. However, for the complex matrices such as cooked food and honey, it is challenging to perform composition analysis. It is necessary to establish more effective strategies for investigating GE contamination. In this study, the real-time PCR (qPCR) analysis combined with DNA barcode matK was proposed for the identification and detection of GE. Fifteen honey samples along with twenty-eight individuals of GE and the common confusable objects Lonicera japonica, Ficus hirta, Stellera chamaejasme and Chelidonium majus were gathered. Additionally, the food mixtures treated with 20-min boiling and 30-min digestion were prepared. Specific primers were designed, and the detection capability and sensitivity of qPCR in honey and boiled and digested food matrices were tested. The results demonstrated that the matK sequence with sufficient mutation sites was an effective molecular marker for species differentiation. GE and the confusable species could be clearly classified by the fluorescence signal of qPCR assay with a high sensitivity of 0.001 ng/µl. In addition, this method was successfully employed for the detection of deeply processed food materials and honey containing GE plants which even accounted for only 0.1 %. The sequencing-free qPCR approach undoubtedly can serve as a robust support for the quality supervision of honey industry and the prevention and diagnosis of food poisoning.

[Establishment of Rapid Simultaneous Analysis Method for Plant Toxins by LC-TOF-MS].

Assuming food poisoning caused by toxic plants, an LC-TOF-MS-based method for the rapid and simultaneous analysis of 16 plant toxins was established. After adding water-methanol (1 : 9) and n-hexane, the samples were homogenized and extracted, and then subjected to centrifugal separation. Without any purification procedures, LC-TOF-MS measurements were performed, and qualitative and quantitative analyses using monoisotopic ion [M+H]+ (m/z) were conducted. The addition-recovery test using curry showed that qualitative analysis was possible under a setting with a retention time of ±0.2 minutes or less and mass accuracy of 5 ppm or lower and that quantitative analysis was possible with a recovery rate of 68-142% and a repeatability of 1.4-10.1%. Furthermore, measurements of the amount of plant toxins in the boiled plants and broths of cooked toxic plants demonstrated the transfer of plant toxins to broths. These suggest that in the event of food poisoning, broths may be used as an analysis sample, even when plants are not available.

[Determination of tetrodotoxin in urine by liquid chromatography-tandem mass spectrometry with internal standard calibration].

OBJECTIVE: An analytical method was developed for tetrodotoxin(TTX) in urine by liquid chromatography-tandem mass spectrometry(LC-MS/MS) with internal standard calibration. METHODS: TTX in the sample was extracted with the mixture of acetic acid/methanol/acetonitrile(0.005 mL/0.8 mL/1.8 mL), cleaned by solid phase extraction(SPE) with cation exchange cartridge, eluted with 50% acetonitrile/water containing 0.3% hydrochloric acid, and neutralized with ammonia. The extract was separated by a Waters XBridge~(TM) BEH Amide column(150 mm×3.0mm, 1.7 µm) and measured by MS/MS. By optimizing sample extraction and SPE cleanup conditions, the problems of low recovery and strong suppression effects of MS signal for TTX in urine were resolved when cleaned with cation exchange cartridge. RESULTS: Quantitatively calibrated by the internal standard of Kasugamycin, good linear relationship was found for TTX in urine at the range of 0.2-200 µg/L with the correlation coefficient(r~2) of 0.997. The limits of detection and quantitation for TTX in sample matrix were 0.1 and 0.2µg/L, respectively. The average recoveries at three spiking levels(0.2, 10.0 and 200 µg/L) were 89.3%-95.3% with relative standard deviation(n=6) less than 5.1%. The concentrations of TTX in urine from 11 poisoning patients were 0.4-138 µg/L. The detection rate was 100% in urine collected within 3 days after poisoning. CONCLUSION: The established method was simple, accurate and sensitive. It can provide reliable technical support for the rapid treatment of TTX poisoning events and the study of toxin metabolism in vivo.

Molecular characterization and toxigenic profiles of Bacillus cereus isolates from foodstuff and food poisoning outbreaks in Brazil.

Bacillus cereus sensu stricto (s.s.) is a well-known foodborne pathogen that produces a range of enterotoxins and is able to cause two different types of foodborne illnesses-the emetic and the diarrheal syndromes. In this study, 54 B. cereus s.s. strains isolated from foodstuff and foods involved in food poisoning outbreaks were characterized according to the presence of toxin-encoding genes, virulence-encoding genes, and panC typing. Most isolates were assigned to panC groups IV (61.1%) and III (25.9%), but members of groups II and V could also be found. Investigation of specific alleles revealed high numbers of isolates carrying toxin and other virulence genes including nheA (100%), nheB (100%), hblA (79.6%), hblC (79.6%), hblD (74.1%), cytK-2 (61.1%), clo (100%), pc-plc (75.9%), sph (68.5%), pi-plc (66.6%), hlyIII (62.9%), and hlyII (24.1%). All isolates were negative for ces and cytK-1. In summary, we detected various enterotoxin and other virulence factor genes associated with diarrheal syndrome in strains analyzed, implicated or not with food poisoning. Furthermore, the most isolates analyzed belong to high-risk phylogenetic groups' panC types III and IV. Our study provides a convenient molecular scheme for characterization of B. cereus s.s. strains responsible for food poisoning outbreaks in order to improve the monitoring and investigation and assess emerging clusters and diversity of strains.

Coordination of prophage and global regulator leads to high enterotoxin production in staphylococcal food poisoning-associated lineage.

Staphylococcus species in food produce Staphylococcal enterotoxins (SEs) that cause Staphylococcal food poisoning (SFP). More than 20 SE types have been reported, among which Staphylococcal enterotoxin A (SEA) has been recognized as one of the most important SEs associated with SFP. However, the regulatory mechanisms underlying its production remain unclear. Previously, we identified a major SFP clone in Japan, CC81 subtype-1, which exhibits high SEA production. In this study, we attempted to identify the factors contributing to this phenomenon. Thus, we demonstrated that the attenuation of the activity of endogenous regulator, Staphylococcal accessory regulator S (SarS), and the lysogenization of a high SEA-producing phage contributed to this phenomenon in CC81 subtype-1. Furthermore, our results indicated that SarS could directly bind to the promoter upstream of the sea gene and suppress SEA expression; this low SarS repression activity was identified as one of the reasons for the high SEA production observed. Therefore, we revealed that both exogenous and endogenous factors may probably contribute to the high SEA production. Our results confirmed that SE production is a fundamental and critical factor in SFP and clarified the associated production mechanism while enhancing our understanding as to why a specific clone frequently causes SFP. IMPORTANCE: The importance of this study lies in its unveiling of a molecular regulatory mechanism associated with the most important food poisoning toxin and the evolution of Staphylococcal food poisoning (SFP)-associated clone. SFP is primarily caused by Staphylococcus aureus, with Staphylococcal enterotoxin A (SEA) being commonly involved in many cases. Thus, SEA has been recognized as a major toxin type. However, despite almost a century since its discovery, the complete mechanism of SEA production is as yet unknown. In this study, we analyzed an SEA-producing SFP clone isolated in East Asia and discovered that this strain, besides acquiring the high SEA-producing phage, exhibits remarkably high SEA production due to the low activity of SarS, an intrinsic regulatory factor. This is the first report documenting the evolution of the SFP clone through the coordinated action of exogenous mobile genetic factors and endogenous regulators on this notorious toxin.

Clinical and Laboratory Effects of Foodborne Illness in Children.

The World Health Organization estimates that 31 foodborne pathogen account for 600 million cases of illness annually. This study, conducted in a pediatric emergency department in Turkey, addresses the limited research on pediatric foodborne diseases (FD) in the country, exposing a significant knowledge gap. Analyzing 17,091 pediatric cases, 106 FD cases were identified, predominantly affecting boys (94.3%) with an average age of 7.65 ± 6.51 years. Remarkably, no patients required pediatric intensive care admission, and no mortalities were recorded. Hyponatremia emerged as a prevalent electrolyte disorder in pediatric FD, while hyperkalemia was notably observed in children under 5. The study emphasizes the severity of FD in children under 5, reflected in longer hospital stays, underscoring the urgent need for targeted interventions and improved detection methods in pediatric FD.

Lysozyme Peptides as a Novel Nutra-Preservative to Control Some Food Poisoning and Food Spoilage Microorganisms.

Foodborne illnesses and microbial food contamination are crucial concerns and still issues of great worldwide concern. Additionally, the serious health hazards associated with the use of chemical preservatives in food technology. Lysozyme (Lz) is an active protein against Gram-positive bacterial cell wall through its muramidase lytic activity; however, several authors could identify some antimicrobial peptides derived from Lz that have an exaggerated and broad-spectrum antibacterial activity. Therefore, a lysozyme peptides preparation (LzP) is developed to broaden the Lz spectrum. In this work, we investigated the potential efficacy of LzP as a novel Nutra-preservative (food origin) agent against some pathogenic and spoilage bacteria. Our results showed that LzP demonstrated only 11% of the lysozyme lytic activity. However, LzP exhibited strong antibacterial activity against Escherichia coli, Salmonella enteritidis, and Pseudomonas species, while Salmonella typhi and Aeromonas hydrophila exhibited slight resistance. Despite the lowest LzP concentration (0.1%) employed, it performs stronger antibacterial activity than weak organic acids (0.3%). Interestingly, the synergistic multi-component formulation (LzP, glycine, and citric acid) could inhibit 6 log10 cfu/ml of E. coli survival growth. The effect of heat treatment on LzP showed a decrease in its antibacterial activity at 5 and 67% by boiling at 100 °C/30 min, and autoclaving at 121 °C/15 min; respectively. On the other hand, LzP acquired stable antibacterial activity at different pH values (4-7). In conclusion, LzP would be an innovative, natural, and food origin preservative to control the growth of food poisoning and spoilage bacteria in food instead chemical one.

Exploring the potential human pathogenic bacteria in selected ready-to-eat leafy greens sold in Dhaka City, Bangladesh: Estimation of bacterial load and incidence.

This study was designed to investigate the presence of potential human pathogenic bacteria, bacterial load, and their incidence in ready-to-eat leafy greens viz., coriander, lettuce, and mint leaves sold at diverse marketplaces in Dhaka City. Multiple identification methods including cultural, morphological, biochemical, and molecular analysis were employed in the Plant Pathology Laboratory of Sher-e-Bangla Agricultural University to identify the human pathogenic bacteria. In molecular analysis, the DNA samples were put through PCR using bacterial primer 27F: AGAGTTTGATCMTGGCTGAG and universal primer 1942R: CGGTTACCTTGTTACGACTT. Initially, nine different bacterial genera viz. Bacillus, Escherichia, Pseudomonas, Neisseria, Klebsiella, Enterobacter, Shigella, Vibrio, and Staphylococcus were detected, and their incidence was 93%, 67%, 44%, 30%, 26%, 26%, 11%, 7%, and 7% respectively. A total of twelve bacteria have been identified from these genera out of which 7 bacteria viz. Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterobacter aerogenes, Staphylococcus aureus, and Shigella spp., were reported as human pathogenic bacteria in several pieces of literature. The highest colony-forming units per gram were shown in mint (4.27 ± 2.35 × 109) followed by lettuce (2.87 ± 0.76 × 109) and coriander (2.43 ± 1.32 × 109). Considering marketplaces, the highest colony-forming units per gram were observed in the samples of street markets (5.0 ± 1.72 × 109) and the lowest was in supermarkets (1.87 ± 0.46 × 109) followed by local markets (2.7 ± 0.91 × 109). All the leafy green samples crossed the acceptable level of bacterial load (106 CFU/g). The findings of the study highlight the urgency for improved food safety protocols in their production and distribution in Dhaka city.

Molecular Characteristics of Staphylococcus aureus Isolates form Food-Poisoning Outbreaks (2011-2022) in Sichuan, China.

Staphylococcal food poisoning (SFP) is one of the most common foodborne diseases in the world. This study aimed to investigate the molecular epidemiological characteristics of Staphylococcus aureus isolated from SFP. A total of 103 S. aureus isolates were obtained during 2011-2022 in Sichuan, southwest China. All isolates were tested for the genomic characteristics and phylogenetic analysis by performing whole-genome sequencing. Multilocus sequence typing analysis showed 17 multilocus sequence types (STs), ST7 (23.30%), ST5 (22.33%), and ST6 (16.50%) being the most common. A total of 45 virulence genes were detected, 22 of which were staphylococcal enterotoxin (SE) genes. Among the identified SE genes, selX exhibited the highest prevalence (86.4%). All isolates carried at least one SE gene. The results of the antimicrobial resistance (AMR) gene detection revealed 41 AMR genes of 12 classes. ß-lactam resistance genes (blal, blaR1, blaZ) and tetracycline resistance gene (tet(38)) exhibited a higher prevalence rate. Core genome single nucleotide polymorphism showed phylogenetic clustering of the isolates with the same region, year, and ST. The results indicated that the SFP isolates in southwest of China harbored multiple toxin and resistance genes, with a high prevalence of new SEs. Therefore, it is important to monitor the antimicrobial susceptibility and SE of S. aureus to reduce the potential risks to public health.

Foodborne Outbreak by Salmonella enterica Serovar Weltevreden in West Bengal, India.

Foodborne gastroenteritis outbreaks owing to Salmonella enterica serovar Weltevreden (Salmonella Weltevreden) represent a significant global public health problem. In the past two decades, Salmonella Weltevreden has emerged as a dominant foodborne pathogen, especially in South-East Asian countries. This report describes a community foodborne outbreak of gastroenteritis caused by Salmonella Weltevreden in August 2022 following consumption of panipuri from a street vendor in the Polba block in Hooghly district, West Bengal, India. This food item was consumed by 185 people, of whom 129 had acute watery diarrhea with other clinical symptoms and 65 of them were admitted to different District hospitals for treatment. Stool specimens collected from hospitalized cases were positive for S. enterica, and further serotyped as Salmonella Weltevreden. All the Salmonella Weltevreden strains possessed the Salmonella pathogenicity islands associated genes (invA/E, orgA, ttrc, ssaQ, mgtC, misL, spi4D), the enterotoxin (stn), and hyperinvasive locus gene (hilA). Except erythromycin, all the strains were susceptible for commonly used antimicrobials in the treatment of diarrhea. The XbaI-based pulsed-field gel electrophoresis analysis indicated that all the isolates responsible for the recent outbreak were similar, but diverged from other Salmonella Weltevreden that were previously reported in West Bengal. This report indicates that foodborne infection is a major public health concern in India and demands to strengthen capacity-building measures at the local health care levels for linking causative agents of outbreaks.

Detection of Toxoplasma gondii and Sarcocystis sp. in the meat of common minke whale (Balaenoptera acutorostrata): A case of suspected food poisoning in Japan.

A case of suspected food poisoning related to the consumption of raw meat from a common minke whale (Balaenoptera acutorostrata) was reported in Tokyo, Japan, in June 2020. Microscopic analysis revealed tissue cysts of Toxoplasma gondii and sarcocysts of Sarcocystis sp. in whale meat. The SAG2 and ITS1 region sequences of T. gondii were detected in the DNA extracted from the meat. Genotyping of the multilocus nested PCR-RFLP using the genetic markers SAG1, SAG2 (5'- SAG2, 3'-SAG2, and alt. SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed that the genotype of T. gondii was type II, with a type I pattern for the L358 locus. In the phylogenetic analyses of the six loci (GRA6, GRA7, SAG1, HP2, UPRT1, and UPRT7), these sequences clustered into haplogroup 2. Moreover, the sequences of the virulence-related genes ROP5 and ROP18 of T. gondii isolated from whale meat were similar to those of the type II ME49 reference strain. Sequence analyses of the mtDNA cox1 gene, 18S rRNA gene, and ITS1 region indicated the highest similarity of sarcocyst isolated from whale meat to Sarcocystis species that infect birds or carnivores as intermediate hosts; however, the species could not be identified. To our knowledge, this is the first report of T. gondii and Sarcocystis spp. being detected in same whale meat ingested by patients involved in a suspected food poisoning case in Japan.

Experimental Evidence of the Diarrheal Activity of Sarcocystis sp. in Sika Deer (Cervus nippon).

Recently, the wild deer population has been increasing in Japan, causing serious feeding-related damage to the agricultural and forestry industries. In conjunction with the government's promotion of hunting for population control, the effective utilization of resources and promotion of the game meat industry as a sixth sector of industrialization are desired by local governments. However, several cases in which patients showed intestinal symptoms such as diarrhea due to the consumption of sika deer meat infected with protozoan Sarcocystis spp. have been reported, and the pathogenic microorganisms found in wild deer should be investigated. In this study, Sarcocystis sp. parasitized Kyushu sika deer (Cervus nippon nippon) in Nagasaki Prefecture, Japan, was examined for its enterotoxicity. A phylogenetic analysis based on the sequence of the 18S rRNA gene and cox1 showed that the species was highly homologous to Sarcocystis japonica and/or Sarcocystis sp. HM050622. We attempted to confirm the diarrhea-evoking toxicity of Sarcocystis sp. in sika deer meat, which has been previously reported in human case reports. A mouse ileal loop assay showed that Sarcocystis sp. in sika deer meat induced significant fluid accumulation in the loop at doses of ∼5 × 106 bradyzoites. Western blotting showed that these Sarcocystis parasites possess actin-depolymerizing factor, a diarrhea-evoking factor, similar to Sarcocystis fayeri, which exists in horsemeat. However, the pathogenic conditions of the ileal loop were different from those of similar experiments with S. fayeri. This study suggests that S. japonica parasitizing C. n. nippon may cause diarrhea via a different mechanism from that of S. fayeri.