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.

Genomic portraits of methicillin-resistant staphylococci (MRS) from food fish unveiled the genes associated with staphylococcal food poisoning (SFP), virulence and antimicrobial resistance.

BACKGROUND: Characteristics of non-clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) especially from fishery environment are poorly understood. This research, in addition to comprehensive characterisation, sought to delineate the genetic relatedness between the MRSA strains originating from clinical as well as non-clinical settings. Out of 39 methicillin-resistant staphylococcal isolates from 197 fish samples, 6 (Three each of methicillin-resistant S. haemolyticus (MRSH) and MRSA) with distinct resistance profiles were selected for whole-genome sequencing. Using respective bioinformatics tools, MRSA genomes were comprehensively characterized for resistome, virulomes, molecular epidemiology and phylogenetic analysis. Simultaneously, MRSH genomes were specifically examined to characterize antimicrobial resistance genes (ARGs), owing to the fact that MRSH is often recognized as a reservoir for resistance determinants. RESULTS: Three MRSA clones identified in this study include ST672-IVd/t13599 (sequence type-SCCmec type/spa type), ST88-V/t2526, and ST672-IVa/t1309. Though, the isolates were phenotypically vancomycin-sensitive, five of the six genomes carried vancomycin resistance genes including the VanT (VanG cluster) or VanY (VanM cluster). Among the three MRSA, only one harbored the gene encoding Panton-Valentine Leukocidin (PVL) toxin, while staphylococcal enterotoxin (SEs) genes such as sea and seb, associated with staphylococcal food poisoning were identified in two other MRSA. Genomes of MRSH carried a composite of type V staphylococcal cassette chromosome mec (SCCmec) elements (5C2 & 5). This finding may be explained by the inversion and recombination events that may facilitate the integration of type V elements to the SCC elements of S. aureus with a methicillin-susceptible phenotype. Phylogenetically, MRSA from a non-clinical setting displayed a considerable relatedness to that from clinical settings. CONCLUSION: This study highlights the genetic diversity and resistance profiles of MRSA and MRSH, with non-clinical MRSA showing notable relatedness to clinical strains. Future research should explore resistance gene transfer mechanisms and environmental reservoirs to better manage MRSA spread.

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.

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.

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.

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.

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.

[Development of a Genus Identification Method for Poisonous Plants Using Real-Time PCR].

We have developed a rapid genus identification method for poisonous plants. The real-time PCR using the TaqMan® probe method was employed for detection, with the amplified targets being the "trnL (UAA)-intron" or "trnL-trnF intergenic spacer" regions of chloroplast DNA. The targeted plants were selected six genera (Aconitum, Colchicum, Veratrum, Brugmansia, Scopolia and Narcissus), which have been implicated in many instances of food poisoning in Japan. A tissue lysis solution was used for DNA extraction, which can be completed within approximate 30 min. A master mix corresponding to the tissue lysis solution was used for real-time PCR reagents. As a result, we were able to complete the entire process from DNA extraction to genus identification in 4 to 5 hr. The detection sensitivity was estimated at approximately 1 pg of DNA for all six plant genera. Remarkably, an amplification plot was discerned even with the crude cell lysates of all samples. It was also possible to obtain amplification curves for three plant samples that had been subjected to simulated cooking (boiling). This study suggests that the developed method can rapidly identify six genera of poisonous plants.

[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.

An Outbreak of Bacillus cereus Emetic Toxin Mediated Food Poisoning After Consumption of Fried Rice in Assam.

Bacillus cereus is an emerging food-borne pathogen responsible for two types of food poisoning: emetic and diarrhoeal type. Here we report an emetic type of food-borne illness attributable to Bacillus cereus. On 2nd February, 2021, 202 people suffered from pain in abdomen and vomiting after consuming the rice provided during a public gathering in Diphu, Assam. Culture of leftover fried rice showed growth of Bacillus cereus group of organisms. Molecular detection of enterotoxin and emetic toxin genes was done in the isolated strains by polymerase chain reaction. Multi locus sequence typing (MLST) and phylogenetic analysis was done to characterise the isolated strains. A total of five strains of Bacillus cereus were isolated. Ces gene was found in isolates GMC22 & GMC24 and other enterotoxins producing genes were found in isolates GMC23 and GMC24. MLST identified four sequence types (STs) (ST1051, ST1616, ST998 and ST1000). Phylogenetic analysis clustered ST-1051 assigned to the GMC22 strain into the previously defined clade I and was in close relation with ST-144, representing a new cereulide-producing emetic cluster. As Bacillus cereus is a common contaminant of foods, it is essential to evaluate the pathogenic potential of the bacteria for a definite link between causative agents and the illness. MLST can be used to characterize the Bacillus cereus strains isolated from outbreak samples in order to link the probable pathogens with the illness. In this outbreak, we suggest that ST-1051 is the strain responsible for the food-borne illness, which was predominantly of emetic type.

Emergence of Novel Type C Botulism Strain in Household Outbreak, Japan.

In 2021, an outbreak of food poisoning caused by Clostridium botulinum type C occurred in Kumamoto, Japan. Analysis of the isolated strain revealed that it possessed the bont/C gene and was slightly different from the reference bont/C gene. The risk for human infection with this new toxin type may be low.

Types A and F Clostridium perfringens in healthcare wastewater from Ghana.

IMPORTANCE: Clostridium perfringens causes gas gangrene and food poisoning in humans, and monitoring this bacterium is important for public health. Although whole-genome sequencing is useful to comprehensively understand the virulence, resistome, and global genetic relatedness of bacteria, limited genomic data from environmental sources and developing countries hamper our understanding of the richness of the intrinsic genomic diversity of this pathogen. Here, we successfully accumulated the genetic data on C. perfringens strains isolated from hospital effluent and provided the first evidence that predicted pathogenic C. perfringens may be disseminated in the clinical environment in Ghana. Our findings suggest the importance of risk assessment in the environment as well as the clinical setting to mitigate the potential outbreak of C. perfringens food poisoning in Ghana.

Atypical diarrhoeagenic Escherichia coli in milk related to a large foodborne outbreak.

A foodborne outbreak related to milk cartons served in school lunches occurred in June 2021, which involved more than 1,800 cases from 25 schools. The major symptoms were abdominal pain, diarrhoea, vomiting, and fever. Although major foodborne toxins and pathogens were not detected, a specific Escherichia coli strain, serotype OUT (OgGp9):H18, was predominantly isolated from milk samples related to the outbreak and most patients tested. The strains from milk and patient stool samples were identified as the same clone by core genome multilocus sequence typing and single-nucleotide polymorphism analysis. The strain was detected in milk samples served for two days related to the foodborne outbreak at a rate of 69.6% and levels of less than ten most probable number/100 mL but not on days unrelated to the outbreak. The acid tolerance of the strain for survival in the stomach was similar to that of enterohaemorrhagic E. coli O157:H7, and the same inserts in the chu gene cluster in the acid fitness island were genetically revealed. The pathogenicity of the strain was not clear; however, it was indicated that the causative pathogen was atypical diarrhoeagenic E. coli OUT (OgGp9):H18.

Genomic analysis and biological characterization of a novel Schitoviridae phage infecting Vibrio alginolyticus.

Vibrio alginolyticus is a Gram-negative bacterium commonly associated with mackerel poisoning. A bacteriophage that specifically targets and lyses this bacterium could be employed as a biocontrol agent for treating the bacterial infection or improving the shelf-life of mackerel products. However, only a few well-characterized V. alginolyticus phages have been reported in the literature. In this study, a novel lytic phage, named ΦImVa-1, specifically infecting V. alginolyticus strain ATCC 17749, was isolated from Indian mackerel. The phage has a short latent period of 15 min and a burst size of approximately 66 particles per infected bacterium. ΦImVa-1 remained stable for 2 h at a wide temperature (27-75 °C) and within a pH range of 5 to 10. Transmission electron microscopy revealed that ΦImVa-1 has an icosahedral head of approximately 60 nm in diameter with a short tail, resembling those in the Schitoviridae family. High throughput sequencing and bioinformatics analysis elucidated that ΦImVa-1 has a linear dsDNA genome of 77,479 base pairs (bp), with a G + C content of ~ 38.72% and 110 predicted gene coding regions (106 open reading frames and four tRNAs). The genome contains an extremely large virion-associated RNA polymerase gene and two smaller non-virion-associated RNA polymerase genes, which are hallmarks of schitoviruses. No antibiotic genes were found in the ΦImVa-1 genome. This is the first paper describing the biological properties, morphology, and the complete genome of a V. alginolyticus-infecting schitovirus. When raw mackerel fish flesh slices were treated with ΦImVa-1, the pathogen loads reduced significantly, demonstrating the potential of the phage as a biocontrol agent for V. alginolyticus strain ATCC 17749 in the food. KEY POINTS: • A novel schitovirus infecting Vibrio alginolyticus ATCC 17749 was isolated from Indian mackerel. • The complete genome of the phage was determined, analyzed, and compared with other phages. • The phage is heat stable making it a potential biocontrol agent in extreme environments.

The affinity of histamine to serum albumin: Capillary electrophoresis-frontal analysis and in-silico molecular docking approaches.

Histamine is a biogenic amine found in various body tissues and responsible for many critical vital activities. It is also responsible for allergic reactions in the body. Ingestion of foods containing high amounts of histamine can cause fatal allergic reactions. Albumin in plasma controls drugs and free concentrations of bioactive constituents taken to the body with food. Hence, this study aimed to characterise the interactions of histamine with bovine serum albumin. Capillary electrophoresis in the frontal analysis mode was employed in this study as a practical approach for assessing histamine-bovine serum albumin affinity. The plateau-shaped free histamine peak was well separated from the bovine serum albumin (BSA)-histamine complex peak. The free histamine concentration was obtained by following the height of the free histamine peak. Whereas the bound histamine concentrations were obtained by calculating the difference between the height of total and free histamine peaks. Histamine bound to BSA at one independent site with a Kb value of 2.50 × 103 L/mol. Moreover, an in-silico molecular docking method was performed, and it was revealed that the binding site of histamine was located closer to Lysine-131 in subdomain IIA of bovine serum albumin.

The Epidemiological and Clinical Characteristics of the Largest Outbreak of Enterohemorrhagic Escherichia coli in Korea.

BACKGROUND: The largest outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 occurred at a preschool in South Korea from June 12 to 29, 2020. This study aimed to analyze the epidemiological and clinical characteristics of EHEC infection in this outbreak. METHODS: Epidemiological investigation was performed on all 184 children and 19 workers at the preschool using a standard questionnaire to assess symptoms, food intake, attendance, and special activity history. Pulsed-field gel electrophoresis analysis of confirmed cases was performed to determine genetic relevance. RESULTS: During this outbreak, 103 children were affected, whereas only one infection was identified in adults. Of the 103 pediatric patients, 85 had symptoms (82.5%), including diarrhea, abdominal pain, bloody stool, fever, and vomiting. Thirty-two patients (31.1%) were hospitalized, 15 (14.6%) were diagnosed with hemolytic uremic syndrome, and 4 (3.9%) received dialysis treatment. Pulsed-field gel electrophoresis analysis identified 4 genotypes with high genetic relevance (92.3%). Epidemiological investigation revealed that this outbreak might have occurred from ingesting foods stored in a refrigerator with a constant temperature above 10°C, which was conducive to bacterial growth. Despite several measures after outbreak recognition, new infections continued to appear. Therefore, the preschool was forced to close on June 19 to prevent further person-to-person transmission. CONCLUSION: Our findings from the response to the largest outbreak will help prepare countermeasures against future EHEC outbreak.

Molecular Mechanisms of Scombroid Food Poisoning.

Scombroid food poisoning (SFP) is a foodborne disease that develops after consumption of fresh fish and, rarely, seafood that has fine organoleptic characteristics but contains a large amount of exogenous histamine. SFP, like other food pseudo-allergic reactions (FPA), is a disorder that is clinically identical to allergic reactions type I, but there are many differences in their pathogenesis. To date, SFP has been widespread throughout the world and is an urgent problem, although exact epidemiological data on incidence varies greatly. The need to distinguish SFP from true IgE-associated allergy to fish and seafood is one of the most difficult examples of the differential diagnosis of allergic conditions. The most important difference is the absence of an IgE response in SFP. The pathogenesis of SFP includes a complex system of interactions between the body and chemical triggers such as exogenous histamine, other biogenic amines, cis-urocanic acid, salicylates, and other histamine liberators. Because of the wide range of molecular pathways involved in this process, it is critical to understand their differences. This may help predict and prevent poor outcomes in patients and contribute to the development of adequate hygienic rules and regulations for seafood product safety. Despite the vast and lengthy history of research on SFP mechanisms, there are still many blank spots in our understanding of this condition. The goals of this review are to differentiate various molecular mechanisms of SFP and describe methods of hygienic regulation of some biogenic amines that influence the concentration of histamine in the human body and play an important role in the mechanism of SFP.

[Development of a Simple Identification Method for Omphalotus guepiniformis by Color Reaction].

A simple method of identification using a color reaction was developed for Omphalotus guepiniformis. Only Omphalotus guepiniformis turned turquoise green. Other edible mushrooms resembling the mushroom did not change color when the beam reagent (5 w/v% potassium hydroxide ethanolic solution) was dripped onto the mushroom pileus. Furthermore, ethanol extract and mock cooking products of this mushroom exhibited the same color reaction. These results demonstrate this method as useful for identifying Omphalotus guepiniformis during mushroom hunting or during investigations of food poisoning.

[Color Reaction Identification of Omphalotus guepiniformis Causing Food Poisoning].

Simple identification using a color reaction was applied to investigate poisoning, putatively caused by Omphalotus guepiniformis. Some leftover uncooked mushrooms had turned turquoise green when a beam reagent (5 w/v% potassium hydroxide ethanolic solution) was dripped onto the mushroom pileus. Furthermore, ethanol extract of the mushrooms exhibited the same color reaction. Then, illudin S, a toxic compound contained in O. guepiniformis, was detected in uncooked leftover mushrooms using LC-MS/MS analysis. Therefore, this case was inferred as caused by O. guepiniformis. These results indicate the identification method described above as useful for screening tests for investigating food poisoning caused by O. guepiniformis.