Evaluation of crAssphages as a potential marker of human viral contamination in environmental water and fresh leafy greens.

CrAssphages are human gut bacteriophages with potential use as an indicator of human fecal contamination in water and other environmental systems. We determined the prevalence and abundance of crAssphages in water, food, and fecal samples and compared these estimates with the prevalence of norovirus. Samples were tested using two crAssphage-specific qPCR assays (CPQ056 and TN201-203) and for norovirus using TaqMan realtime RT-PCR. CrAssphage was detected in 40% of human fecal specimens, 61% of irrigation water samples, 58.5% of stream water samples, and 68.5% of fresh leafy greens samples. Interestingly, across all sample categories, crAssphage concentrations were 2-3 log10 higher than norovirus concentrations. The correlation of detection of crAssphage and norovirus was significant for the irrigation water samples (r = 0.74, p = 7.4e-06). Sequences obtained from crAssphage positive samples from human fecal and stream water samples phylogenetically clustered with genotype I crAssphages, whereas sequences derived from irrigation water samples clustered differently from other genotypes. Our data show that crAssphages were prevalent in norovirus-positive water samples and in fresh leafy green samples, there was a strong correlation between the presence of crAssphage and norovirus. CrAssphage genomic copies were consistently higher than norovirus copies in all sample types. Overall, our findings suggest that crAssphages could be used as reliable indicators to monitor fecal-borne virus contamination within the food safety chain.

Comparison of DNA Extraction Methods for the Quantification of Listeria monocytogenes in Dairy Products by Real-Time Quantitative PCR>.

ABSTRACT: Listeria monocytogenes is a common foodborne pathogen affecting public health. Thus, detecting L. monocytogenes, even at low levels, in food matrices is essential. However, the current culture methods used for its detection and quantification are time consuming and difficult owing to background flora and interference by food matrices. DNA-based assays depend on DNA extraction and purification techniques. No optimal DNA extraction kit has been developed for analyzing L. monocytogenes in dairy products by real-time quantitative PCR (RT-qPCR). Therefore, in this study, we aimed to determine the efficiency of three DNA extraction kits for detecting L. monocytogenes in dairy products by RT-qPCR. We tested the efficiency of three commercial kits for DNA extraction from L. monocytogenes artificially inoculated in milk and dairy products. For the PrepSEQ rapid spin sample preparation kit and Exgene Cell SV mini, the limit of detection of was 100, 100, and 101 CFU/mL L. monocytogenes in milk, processed cheese, and infant formula, respectively, whereas that of the QIAamp DNA mini kit was 101, 103, and 102 CFU/mL, respectively. In addition, the Exgene Cell SV mini was better than the PrepSEQ rapid spin sample preparation kit for obtaining a standard curve for RT-qPCR of L. monocytogenes DNA in milk and dairy products, with a high correlation coefficient and amplification efficiency. The results of this study may be valuable for diagnostic laboratories and for developing an effective extraction method for processing food samples, such as dairy products, to subsequently detect and quantify L. monocytogenes by RT-qPCR.

Comparison of enrichment methods for isolating Enterohemorrhagic Escherichia coli in kimchi.

This study was conducted to compare the efficiency of four enrichment methods of Enterohemorrhagic Escherichia coli by using the 16S rRNA amplicon sequencing and a predictive model. Four different methods (US FDA, ISO, Japan Food Hygiene Association and Korea Ministry of Food and Drug Safety) were used to enrich EHEC in kimchi inoculated with cocktails of EHEC strains (NCCP 13720, NCCP 13721, and NCCP 14134). The maximum growth rate (µmax) and lag phase duration (LPD) were compared using the Baranyi model, and 16S rRNA targeted sequencing was performed with samples at the end of the exponential phase. As a result, the µmax and LPD values of Baranyi model developed for the four enriched media ranged from 0.82 to 0.92 and from 2.35 to 2.68, respectively, suggesting that the growth of EHEC was similar in all four enrichment media. As for the relative abundance of the bacterial composition at the family level, Enterobacteriaceae was identified as the major component (>50%) in all four enriched media. The relative abundance of Enterobacteriaceae was highest (>90%) in the two enriched media with 20 mg/L novobiocin, demonstrating that significant growth of non-targeted bacteria takes place in enrichment broths utilizing <20 mg/L novobiocin or different antibiotics. In conclusion, this study suggests that all four enrichment broth are suitable for growing EHEC in kimchi and the use and concentration of antibiotics such as novobiocin in enrichment media may have a critical role in species diversity.

Gut Microbiome Changes Occurring with Norovirus Infection and Recovery in Infants Enrolled in a Longitudinal Birth Cohort in Leon, Nicaragua.

Noroviruses are associated with one fifth of diarrheal illnesses globally and are not yet preventable with vaccines. Little is known about the effects of norovirus infection on infant gut microbiome health, which has a demonstrated role in protecting hosts from pathogens and a possible role in oral vaccine performance. In this study, we characterized infant gut microbiome changes occurring with norovirus-associated acute gastroenteritis (AGE) and the extent of recovery. Metagenomic sequencing was performed on the stools of five infants participating in a longitudinal birth cohort study conducted in León, Nicaragua. Taxonomic and functional diversities of gut microbiomes were profiled at time points before, during, and after norovirus infection. Initially, the gut microbiomes resembled those of breastfeeding infants, rich in probiotic species. When disturbed by AGE, Gammaproteobacteria dominated, particularly Pseudomonas species. Alpha diversity increased but the genes involved in carbohydrate metabolism and glycan biosynthesis decreased. After the symptoms subsided, the gut microbiomes rebounded with their taxonomic and functional communities resembling those of the pre-infection microbiomes. In this study, during disruptive norovirus-associated AGE, the gut microbiome was temporarily altered, returning to a pre-infection composition a median of 58 days later. Our study provides new insights for developing probiotic treatments and furthering our understanding of the role that episodes of AGE have in shaping the infant gut microbiome, their long-term outcomes, and implications for oral vaccine effectiveness.

Evaluation of Hygiene Indicators and Sampling Plan for Detecting Microbial Contamination in Health Functional Foods.

ABSTRACT: This study aimed to monitor microbial contamination levels in a variety of health functional foods and to establish new microbial criteria. Indicator organisms (i.e., aerobic bacteria, coliform bacteria, and Escherichia coli) were monitored in 10 health functional food categories (743 items, 3,715 samples). The mean total aerobic counts of ginseng and Korean red ginseng were -0.35 and -0.74 log CFU/g; and the mean total coliform counts were -1.4 and -1.39 log CFU/g, respectively. In addition, the mean total coliform counts of fiber and protein products were -1.34 and -1.22 log CFU/g, respectively. However, no aerobic or coliform cells were detected in any other health functional food products (vitamins, minerals, probiotics, milk thistle extract, propolis, eicosapentaenoic acid, docosahexaenoic acid, or lutein products), and no E. coli was detected in any of the categories. These results can potentially be used to update the microbial criteria of the Health Functional Food Code.

Development and validation of a predictive model for pathogenic Escherichia coli in fresh-cut produce.

This study was performed to develop and validate a predictive growth model of pathogenic Escherichia coli to ensure the safety of fresh-cut produce. Samples were inoculated with a cocktail of seven E. coli strains of five pathotypes (EHEC, Enterohemorrhagic E. coli; ETEC, Enterotoxigenic E. coli; EPEC, Enteropathogenic E. coli; EIEC, Enteroinvasive E. coli, and EAEC, Enteroaggregative E. coli) and stored at 4, 10, 12, 15, 25, 30, and 37°C. Growth of pathogenic E. coli was observed above 12°C. The primary growth model for pathogenic E. coli in fresh-cut produce was developed based on the Baranyi model. The secondary model was developed as a function of temperature for lag phase duration (LPD) and maximum specific growth rate (µmax) based on the polynomial second-order model. The primary and secondary models for pathogenic E. coli were fitted with a high degree of goodness of fit (R2 ≥ 0.99). The bias factor (Bf), accuracy factor (Af), and root mean square error (RMSE) were 0.995, 1.011, and 0.084, respectively. The growth model we developed can provide useful data for assessing the quantitative microbial risk of pathogenic E. coli in fresh-cut produce intended for human consumption. In addition, it is thought to be widely available in industries that produce, process, distribute, and sell fresh-cut produce.

Quantitative microbial risk assessment of pathogenic Escherichia coli in commercial kimchi in South Korea.

Owing to convenience, ease of preparation, and price, the consumption of commercial kimchi is gradually rising in South Korea. Here, we estimated the risk level posed by pathogenic Escherichia coli in commercial kimchi products using the quantitative microbial risk assessment (QMRA) approach to develop measures for preventing potential foodborne outbreaks from kimchi consumption. We collected 610 samples of commercial kimchi products produced in Korea, 267 kimchi samples from foreign countries imported to Korea, and 187 raw materials used in kimchi preparation, and analyzed them for contamination with pathogenic E. coli. A Predictive model was developed to observe the survival characteristics of pathogenic E. coli. A dose-response model was selected, and the risk level was estimated using @RISK software. Although a prior epidemiological study indicated the frequent occurrence of foodborne outbreaks arising from contaminated kimchi products consumed in food service facilities, we found a low probability of foodborne illness caused by pathogenic E. coli in commercial kimchi products.

Serratia nevei sp. nov. and Serratia bockelmannii sp. nov., isolated from fresh produce in Germany and reclassification of Serratia marcescens subsp. sakuensis Ajithkumar et al. 2003 as a later heterotypic synonym of Serratia marcescens subsp. marcescens.

Fifteen enterobacterial strains were isolated from fresh produce. The 16S rRNA gene sequences indicated that these belong to Serratia, with twelve strains showing 99.57%-99.93% and three strains showing 99.86-100% 16S rRNA gene sequence similarity with Serratia marcescens and Serratia nematodiphila as nearest neighbors, respectively. Further comparative multi locus sequence analyses, as well as phylogenomic comparisons, revealed that 6 of the 15 strains were well-separated from their nearest neighbors and formed two clearly distinct taxa. Strains S2, S9, S10 and S15T were urease-positive, while strains S3T and S13 were urease-negative. Average nucleotide identity and digital DNA-DNA hybridization comparisons of representative strains S3T and S15T with type strains of S. marcescens, S. nematodiphila and S. ureilytica indicated that these shared less than 96% and 70% homology, respectively. Major fatty acids of strains S3T and S15T included C16:0, C16:1 ω7c/C16:1 ω6c, C17:0 Cyclo and C18:1 ω6c /C18:1 ω7c. The mol% G+C of genomic DNA of strain S15T was 59.49% and of strain S3T was 59.04. These results support the description of two novel species, Serratia nevei and Serratia bockelmannii, with strains S15T (=LMG 31536T =DSM 110085T) and S3T (=LMG 31535T =DSM 110152T) as type strains, respectively. Although Serratia marcescens subsp. sakuensis was previously described to form spores, spores could not be determined in this study. As spore formation was the only differential characteristic of this subspecies, S. marcescens subsp. sakuensis is a later heterotypic synonym of Serratia marcescens.

Predictive model of Staphylococcus aureus growth on egg products.

Egg products are widely consumed in Korea and continue to be associated with risks of Staphylococcus aureus-induced food poisoning. This prompted the development of predictive mathematical models to understand growth kinetics of S. aureus in egg products in order to improve the production of domestic food items. Egg products were inoculated with S. aureus and observe S. aureus growth. The growth kinetics of S. aureus was used to calculate lag-phase duration (LPD) and maximum specific growth rate (µmax) using Baranyi model as the primary growth model. The secondary models provided predicted values for the temperature changes and were created using the polynomial equation for LPD and a square root model for µmax. In addition, root mean square errors (RMSE) were analyzed to evaluate the suitability of the mathematical models. The developed models demonstrated 0.16-0.27 RMSE, suggesting that models properly represented the actual growth of S. aureus in egg products.

Use of DNA aptamer for sandwich type detection of Listeria monocytogenes.

A single stranded (ss) DNA aptamer, specific to members of Listeria genus, was used to develop a two-site binding sandwich assay for capture and detection of L. monocytogenes. Antibody-immobilized immunomagnetic beads were used to capture L. monocytogenes, followed by their exposure to the aptamer detector. Detection was achieved by amplification of cell-bound aptamers by qPCR. The lower limit of detection for the combined assay was 2.5 CFU L. monocytogenes in 500 µl buffer. This is juxtaposed to a detection limit of 2.4 log10 CFU in 500 µl buffer for immunomagnetic separation coupled with qPCR detection of L. monocytogenes targeting the hly gene. When applied to turkey deli meat, subjected to 24 h of non-selective enrichment, the two-site binding sandwich assay showed positive results at initial inoculum levels of 1-2 log10 CFU per 25 g sample. Because of its lower limit of detection, the assay reported here could be useful for detection of L. monocytogenes in foods and environmental samples.

Perilla frutescens Extract Ameliorates Acetylcholinesterase and Trimethyltin Chloride-Induced Neurotoxicity.

One of the critical features of Alzheimer's disease is cognitive dysfunction, which is, in part, due to decreases in acetylcholine (ACh). The ethanol extract of Perilla frutescens was selected for isolating the acetylcholinesterase (AChE) inhibitor based on preliminary screening. In vivo behavioral tests were performed to examine the effects of the P. frutescens extract on trimethyltin chloride-induced impairment of learning and memory in mice. A diet containing P. frutescens extract effectively reversed learning and memory impairment on the Y-maze and passive avoidance tests. To isolate the active compound from the P. frutescens extract, solvent partitioning, silica gel open column chromatography, thin-layer chromatography, and high-performance liquid chromatography were used. The AChE inhibitor was identified as rosmarinic acid.

Characterization of Nutritional Composition, Antioxidative Capacity, and Sensory Attributes of Seomae Mugwort, a Native Korean Variety of Artemisia argyi H. Lév. & Vaniot.

Few studies have investigated Seomae mugwort (a Korean native mugwort variety of Artemisia argyi H. Lév. & Vaniot), exclusively cultivated in the southern Korean peninsula, and the possibility of its use as a food resource. In the present study, we compared the nutritional and chemical properties as well as sensory attributes of Seomae mugwort and the commonly consumed species Artemisia princeps Pamp. In comparison with A. princeps, Seomae mugwort had higher contents of polyunsaturated fatty acids, total phenolic compounds, vitamin C, and essential amino acids. In addition, Seomae mugwort had better radical scavenging activity and more diverse volatile compounds than A. princeps as well as favorable sensory attributes when consumed as tea. Given that scant information is available regarding the Seomae mugwort and its biological, chemical, and sensory characteristics, the results herein may provide important characterization data for further industrial and research applications of this mugwort variety.

Generation and characterization of nucleic acid aptamers targeting the capsid P domain of a human norovirus GII.4 strain.

Human noroviruses (NoV) are the leading cause of acute viral gastroenteritis worldwide. Significant antigenic diversity of NoV strains has limited the availability of broadly reactive ligands for design of detection assays. The purpose of this work was to produce and characterize single stranded (ss)DNA aptamers with binding specificity to human NoV using an easily produced NoV target-the P domain protein. Aptamer selection was done using SELEX (Systematic Evolution of Ligands by EXponential enrichment) directed against an Escherichia coli-expressed and purified epidemic NoV GII.4 strain P domain. Two of six unique aptamers (designated M1 and M6-2) were chosen for characterization. Inclusivity testing using an enzyme-linked aptamer sorbent assay (ELASA) against a panel of 14 virus-like particles (VLPs) showed these aptamers had broad reactivity and exhibited strong binding to GI.7, GII.2, two GII.4 strains, and GII.7 VLPs. Aptamer M6-2 exhibited at least low to moderate binding to all VLPs tested. Aptamers significantly (p<0.05) bound virus in partially purified GII.4 New Orleans outbreak stool specimens as demonstrated by ELASA and aptamer magnetic capture (AMC) followed by RT-qPCR. This is the first demonstration of human NoV P domain protein as a functional target for the selection of nucleic acid aptamers that specifically bind and broadly recognize diverse human NoV strains.

Selection, characterization and application of nucleic acid aptamers for the capture and detection of human norovirus strains.

Human noroviruses (HuNoV) are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ss)DNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV). Four aptamer candidates (designated 19, 21, 25 and 26) were identified and screened for binding affinity to 14 different virus-like particles (VLPs) corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA). Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV) and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC) method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5-36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.

Ligularia fischeri extract protects against oxidative-stress-induced neurotoxicity in mice and PC12 cells.

Alzheimer's disease (AD) is pathologically characterized by the presence of amyloid plaques in brain and the overproduction of amyloid beta (Aß), leading to learning and memory impairment and intense oxidative stress. In this study, the protective effect of Ligularia fischeri extract was investigated using PC12 cells. L. fischeri extract attenuated hydrogen-peroxide-induced DNA fragmentation in cells. In vivo behavioral tests were performed to examine the effects of the extract on amyloid-ß peptide1-42-induced impairment of learning and memory in mice. A diet containing the extract increased alternation behaviors in the Y-maze test and step-through latency of passive avoidance task. Moreover, we found that consumption of the extract decreased lipid peroxidation in a biochemical study of brain tissue in mice. High-performance liquid chromatography was used to identify the active compounds in the extract. These results suggest that L. fischeri extract could be protective against Aß-induced neurotoxicity, possibly due to the antioxidative capacity of its constituent, 3-O-caffeoylquinic acid.

Selection and characterization of DNA aptamers specific for Listeria species.

Single-stranded (ss) DNA aptamers with binding affinity to Listeria spp. were selected using a whole-cell SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. Listeria monocytogenes cells were grown at 37°C and harvested at mid-log phase or early stationary phase to serve as the targets in SELEX. A total of 10 unique aptamer sequences were identified, six associated with log phase cells and four with stationary phase cells. Binding affinity of the aptamers was determined using flow cytometry and ranged from 10% to 44%. Four candidates having high binding affinity were further studied and found to show genus-specific binding affinity when screened against five different species within the Listeria genus. Using sequential binding assays combined with flow cytometry, it was determined that three of the aptamers (LM6-2, LM12-6, and LM12-13) bound to one apparent cell surface moiety, while a fourth aptamer (LM6-116) appeared to bind to a different cell surface region. This is the first study in which SELEX targeted bacterial cells at different growth phases. When used together, aptamers that bind to different cell surface moieties could increase the analytical sensitivity of future capture and detection assays.

Nucleic acid aptamers for capture and detection of Listeria spp.

The purpose of this study was to identify biotinylated single-stranded (ss) DNA aptamers with binding specificity to Listeria and use these for capture and subsequent qPCR detection of the organism. For aptamer selection, SELEX (systematic evolution of ligands by exponential enrichment) was applied to a biotin-labeled ssDNA combinatorial library. After multiple rounds of selection and counter-selection, aptamers separated, sequenced, and characterized by flow cytometry showed binding affinities to L. monocytogenes of 18-23%. Although selected for using L. monocytogenes, these aptamers showed similar binding affinity for other members of the Listeria genus and low binding affinity for non-Listeria species. One aptamer, Lbi-17, was chosen for development of a prototype capture and detection assay. When Lbi-17 was conjugated to magnetic beads and used in a combined aptamer magnetic capture (AMC)-qPCR assay, the pathogen could be detected at concentrations <60 CFU/500 µl buffer in the presence of a heterogeneous cocktail of non-Listeria bacterial cells, with a capture efficiency of 26-77%. Parallel experiments using immunomagnetic separation (IMS)-qPCR produced the same detection limit but lower capture efficiency (16-21%). Increasing assay volume to 10 and 50 ml resulted in reduced capture efficiency and higher limits of detection, at 2.7 and 4.8 log10 CFU L. monocytogenes per sample, respectively, for the AMC-qPCR assay. Biotinylated ssDNA aptamers are promising ligands for food-borne pathogen concentration prior to detection using molecular methods.

Punica granatum protects against oxidative stress in PC12 cells and oxidative stress-induced Alzheimer's symptoms in mice.

Alzheimer's disease (AD) is a progressive degenerative brain disorder that is characterized by neuronal loss, neurofibrillary tangles, and the abnormal deposition of senile plaque and amyloid ß peptide (Aß). The brains of AD patients are under intense oxidative stress. The overproduction of Aß leads to Aß-associated free radical oxidative stress. In this study, the antioxidative and neuronal protective effects of Punica granatum extract were investigated against oxidative stress-induced cytotoxicity in PC12 cells. The ethanol extracts of P. granatum protected PC12 cells from hydrogen peroxide (H2O2-induced oxidative stress. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assays revealed a significant increase in cell viability when oxidatively stressed PC12 cells were treated with the P. granatum extract. To examine the effects of P. granatum on Aß1₋42-induced learning and memory impairment in mice, in vivo behavioral tests were performed. Treatment with the extract of P. granatum increased step-through latency in mice injected with Aß1₋42. The results of this study suggest that the ethanol extract of P. granatum mitigated H2O2-induced oxidative stress in PC12 cells. In addition, the extract inhibited neuronal cell death caused by Aß-induced oxidative stress and Aß-induced learning and memory deficiency.

Optimization of production of monacolin K from gamma-irradiated Monascus mutant by use of response surface methodology.

Monascus isolate number 711, which is capable of producing monacolin K as an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the key enzyme of cholesterol synthesis, was isolated from Ang-kak, the red yeast rice koji. To increase the monacolin K-producing activity of the strain, spore suspensions of the strain were subjected to gamma-irradiation. One thousand mutants were generated via gamma-irradiation and screened using bioassay and high performance liquid chromatography analysis. Several mutants with higher productivities of monacolin K than that of the parent strain were primarily selected. Mutant KU609 was finally selected because of its characteristics of high monacolin K production and non-citrinin-producing activity under our test conditions. Response surface methodology was used to analyze the effect of culture medium on the production of monacolin K in mixed solid-state cultures. The optimal values of nutritional ingredients for the maximal production were soytone, glucose, MgSO4, and barley at concentrations of 0.5 g, 0.48 g, 0.053 g, and 9 g, respectively. The final monacolin K production of Monascus KU609 was increased almost 100-fold compared to that of the parent strain.