Microbiome and mycobiome interaction in house dust mites and impact on airway cells.

BACKGROUND: Major allergen sources Dermatophagoides farinae, Dermatophagoides pteronyssinus and Tyrophagus putrescentiae have been reported to have distinct microbiomes. The purpose of this study was to investigate the effect of each mite on airway epithelial cells as a model of airway allergic disease. METHODS: Transcriptomic analysis (RNA-seq) of an airway epithelial cell line (BEAS-2B) was performed to compare gene expression patterns after treatment with extracts of three mite species (D. farinae, D. pteronyssinus and T. putrescentiae). In addition, mycobiome deep sequencing of mites was employed to identify fungal species that interact with the microbiomes of the mites. RESULTS: Immune responses to bacteria were enriched only in the D. farinae-treated group as this species harboured larger numbers of bacteria than the other mites, and the high level of LPS in D. farinae caused proinflammatory cytokine production in airway epithelial cells. In addition, antibiotic metabolism pathways were enriched in D. pteronyssinus-treated cells but not in D. farinae -treated cells. Subsequent experiments revealed that D. pteronyssinus had a high fungal load that inhibited bacterial survival in this mite species. CONCLUSION: The large amount of bacteria in D. farinae causes airway epithelial cells to produce more allergy-related cytokines than D. pteronyssinus, since fungi present in D. pteronyssinus suppress the abundance of mite-associated bacteria.

Comparative Microbiome Analysis of Three Species of Laboratory-Reared Periplaneta Cockroaches.

Cockroaches inhabit various habitats, which will influence their microbiome. Although the microbiome can be influenced by the diet and environmental factors, it can also differ between species. Therefore, we conducted 16S rDNAtargeted high-throughput sequencing to evaluate the overall bacterial composition of the microbiomes of 3 cockroach species, Periplaneta americana, P. japonica, and P. fuliginosa, raised in laboratory for several generations under the same conditions. The experiments were conducted using male adult cockroaches. The number of operational taxonomic units (OTUs) was not significantly different among the 3 species. With regard to the Shannon and Pielou indexes, higher microbiome values were noted in P. americana than in P. japonica and P. fuliginosa. Microbiome composition was also evaluated, with endosymbionts accounting for over half of all OTUs in P. japonica and P. fuliginosa. Beta diversity analysis further showed that P. japonica and P. fuliginosa had similar microbiome composition, which differed from that of P. americana. However, we also identified that P. japonica and P. fuliginosa host distinct OTUs. Thus, although microbiome compositions may vary based on multiple conditions, it is possible to identify distinct microbiome compositions among different Periplaneta cockroach species, even when the individuals are reared under the same conditions.

Chinese liver fluke Clonorchis sinensis infection changes the gut microbiome and increases probiotic Lactobacillus in mice.

Chinese liver fluke Clonorchis sinensis changes the host's immune system. Recently, it has been reported that helminths including C. sinensis can ameliorate immune-related diseases such as allergy. In addition, recent studies showed that helminth infection can alleviate immune-mediated disorders by altering the gut microbiome. However, changes in the gut microbiome due to C. sinensis have not been reported yet. In this study, changes in the gut microbiome of C57BL/6 mice infected with C. sinensis metacercariae were evaluated over time. Stool was analyzed by 16S rRNA amplicon analysis using high-throughput sequencing technology. There was no apparent difference in species richness and diversity between the infected and control groups. However, the composition of the microbiome was different between the infected and control groups at 20 days and 30 days post-infection, and the difference disappeared at 50 days post-infection. In particular, this microbiome alteration was associated with a change in the relative abundance of genus Lactobacillus and the probiotic Lactobacillus species that are known to have an immune-modulation role in immune-mediated diseases.

Enzymatic hydrolysis increases ginsenoside content in Korean red ginseng (Panax ginseng CA Meyer) and its biotransformation under hydrostatic pressure.

BACKGROUND: Enzymatic hydrolysis and high hydrostatic pressure (HHP) are common processing techniques in the extraction of active compounds from food materials. The aim of this study was to investigate the effects of enzymatic hydrolysis combined with HHP treatments on ginsenoside metabolites in red ginseng. RESULTS: The yield and changes in the levels of polyphenol and ginsenoside were measured in red ginseng treated with commercial enzymes such as Ultraflo L, Viscozyme, Cytolase PCL5, Rapidase and Econase E at atmospheric pressure (0.1 MPa), 50 MPa, and 100 MPa. ß-Glucosidase activity of Cytolase was the highest at 4258.2 mg-1 , whereas Viscozyme showed the lowest activity at 10.6 mg-1 . Pressure of 100 MPa did not affect the stability or the activity of the ß-glucosidase. Treatment of red ginseng with Cytolase and Econase at 100 MPa significantly increased the dry weight and polyphenol content of red ginseng, compared with treatments at 0.1 MPa and 50 MPa (P < 0.05). The amounts of ginsenoside and ginsenoside metabolites derived from red ginseng processed using Cytolase were higher than those derived from red ginseng treated with the other enzymes. Treatment with Cytolase also significantly increased the skin and intestinal permeability of red ginseng-derived polyphenols. CONCLUSION: Cytolase could be useful as an enzymatic treatment to enhance the yield of bioactive compounds from ginseng under HHP. In addition, ginsenoside metabolites obtained by Cytolase hydrolysis combined with HHP are functional substances with increased intestinal and skin permeability. © 2019 Society of Chemical Industry.

Intestinal fluke Metagonimus yokogawai infection increases probiotic Lactobacillus in mouse cecum.

Helminth infection can alleviate immune-mediated disorders such as allergies and autoimmune diseases, by altering the gut microbiome. However, changes in gut microbiome due to intestinal trematodes remain unelucidated. Here, we evaluated the changes in the gut microbiome of ICR mice infected with Metagonimus yokogawai, a hypo-virulent intestinal trematode. Four weeks after infection, mouse cecal content was analyzed by 16S rRNA amplicon analysis. Although there was no apparent difference in species richness and diversity, the microbiome composition was different in the infected and control groups. Furthermore, several Lactobacillus species with known immunomodulatory role in immune-mediated diseases were increased in the infected group.

Production of Dermatophagoides farinae Having Low Bacterial Content Using Ampicillin.

Background: Symbiotic bacteria in house dust mites pose a risk of immunological side effects in the clinical use of immunotherapeutic agents. In this study, we investigated the duration for which the bacterial concentration in Dermatophagoides farinae could be kept low with antibiotic treatment, and whether the allergenic properties of the mite changed under ampicillin treatment. Methods: D. farinae was cultivated in the presence of ampicillin powder in an autoclaved medium for 6 weeks. After subsequent subcultures without ampicillin, the mites were harvested, and the extract was prepared. The amounts of bacteria, lipopolysaccharides (LPS), and two major allergens (Der f 1 and Der f 2) were measured. Human bronchial epithelial cells and mice were treated with the D. farinae extract to assess the allergic airway inflammation. Results: The number of bacteria and level of LPS were reduced by 150-fold and 33-fold, respectively, at least 18 weeks after ampicillin treatment. The concentration of Der f 1 and Der f 2 remained unchanged by ampicillin treatment. The secretion of interleukin (IL)-6 and IL-8 from the human airway epithelial cells decreased when treated with the extract of ampicillin-treated D. farinae compared with that of ampicillin-untreated D. farinae. A mouse asthma model was developed using ampicillin-treated D. farinae. We observed that the level of lung function, airway inflammation, and serum-specific immunoglobulin were not different for the mouse asthma model developed using ampicillin-treated D. farinae than the model developed using ampicillin-untreated D. farinae. Conclusions: We showed that bacterial content in D. farinae was reduced by ampicillin treatment, which was sufficient to induce allergic sensitization and an immune response. This method will be used to develop more controlled allergy immunotherapeutic agents.

Ampicillin treated German cockroach extract leads to reduced inflammation in human lung cells and a mouse model of Asthma.

Cockroaches can cause allergic sensitization in humans via contact with their feces or frass. Antibiotics can affect concentration of major allergen and total bacteria production in German cockroaches (Blattella germanica). This study examined the ability of antibiotic-treated German cockroaches to induce allergic airway inflammation and the effect of antibiotics on their lipopolysaccharide and Bla g1, 2, and 5 expression levels. Specifically, we measured the ability of German cockroach extract (with or without prior antibiotic exposure) to induce allergic inflammation in human bronchial epithelial cells and a mouse model of asthma. Bacterial 16S rRNA and lipopolysaccharide levels were lower in ampicillin-treated cockroaches than in the control group. The Bla g1, Bla g2, and Bla g5 expression in ampicillin-treated cockroaches decreased at both the protein and RNA levels. In human bronchial epithelial cell lines BEAS-2B exposed to the ampicillin-treated extract, expression levels of interleukin-6 and interleukin-8 were lower than that in the control group. The total cell count and eosinophil count in bronchoalveolar lavage fluid was also lower in mice exposed to the ampicillin-treated extract than in those exposed to normal cockroach extract. Mouse lung histopathology showed reduced immune cell infiltration and mucus production in the ampicillin group. Our results showed that ampicillin treatment reduced the symbiont bacterial population and major allergen levels in German cockroaches, leading to reduced airway inflammation in mice. These results can facilitate the preparation of protein extracts for immunotherapy or diagnostics applications.

The microbiota in feces of domestic pigeons in Seoul, Korea.

In Korea, feral pigeons pose significant public health risks because they carry various zoonotic pathogens. Human population density is a significant factor in zoonotic disease events. Seoul is one of the largest cities by population density among developed countries and where most of the homeless population in Korea exists. We designed this study to compare the microbiota of pigeon feces by regional characteristics and the presence of homeless individuals. Therefore, this study used 16S rRNA amplicon sequencing to detect possible pathogenic microbes and assess the current risk of zoonosis in Seoul, South Korea. Pigeon fecal samples (n = 144) obtained from 19 public sites (86 and 58 fecal samples from regions in and outside Seoul, respectively) were examined. Potentially pathogenic bacteria were also detected in the fecal samples; Campylobacter spp. was found in 19 samples from 13 regions, Listeriaceae was found in seven samples, and Chlamydia spp. was found in three samples from two regions. Principal coordinates analysis and permutational multivariate analysis of variance revealed a significant difference in bacterial composition between the regions in Seoul (n = 86) and outside Seoul (n = 58) and between the regions with (n = 81) and without (n = 63) homeless individuals. Overall, this study identified various potentially pathogenic microorganisms in pigeon feces at public sites in South Korea. Moreover, this study demonstrates that the microbial composition was influenced by regional characteristics and homelessness. Taken together, this study provides important information for public health strategic planning and disease control.

Metabarcoding of bacteria and parasites in the gut of Apodemus agrarius.

BACKGROUND: The striped field mouse Apodemus agrarius is a wild rodent commonly found in fields in Korea. It is a known carrier of various pathogens. Amplicon-based next-generation sequencing (NGS) targeting the 16S ribosomal RNA (rRNA) gene is the most common technique used to analyze the bacterial microbiome. Although many bacterial microbiome analyses have been attempted using feces of wild animals, only a few studies have used NGS to screen for parasites. This study aimed to rapidly detect bacterial, fungal and parasitic pathogens in the guts of A. agrarius using NGS-based metabarcoding analysis. METHODS: We conducted 18S/16S rDNA-targeted high-throughput sequencing on cecal samples collected from A. agrarius (n = 48) trapped in May and October 2017. Taxa of protozoa, fungi, helminths and bacteria in the cecal content were then identified. RESULTS: Among the protozoa identified, the most prevalent was Tritrichomonas sp., found in all of the cecal samples, followed by Monocercomonas sp. (95.8% prevalence; in 46/48 samples) and Giardia sp. (75% prevalence; in 36/48 samples). For helminths, Heligmosomoides sp. was the most common, found in 85.4% (41/48) of samples, followed by Hymenolepis sp. (10.4%; 5/48) and Syphacia sp. (25%; 12/48). The 16S rRNA gene analysis showed that the microbial composition of the cecal samples changed by season (P = 0.005), with the linear discriminant analysis effect size showing that in the spring Escherichia coli and Lactobacillus murinus were more abundant and Helicobacter rodentium was less abundant. Helicobacter japonicus was more abundant and Prevotella_uc was less abundant in males. The microbial composition changed based on the Heligmosomoides sp. infection status (P = 0.019); specifically, Lactobacillus gasseri and Lactobacillus intestinalis were more abundant in the Heligmosomoides sp.-positive group than in the Heligmosomoides sp.-negative group. CONCLUSIONS: This study demonstrated that bacterial abundance changed based on the season and specific parasitic infection status of the trapped mice. These results highlight the advantages of NGS technology in monitoring zoonotic disease reservoirs.

Measuring the absolute abundance of the microbiome by adding yeast containing 16S rRNA gene from a hyperthermophile.

High-throughput sequencing (HTS) of 16S rRNA gene amplicons provides compositional information regarding the microbial community, but not the absolute abundance of the bacteria. We aimed to develop a standardized method for quantifying the absolute abundance of bacteria in microbiome studies. To demonstrate the utility of our approach, we quantified the number of bacteria from the compositional data of the fecal and cecal microbiomes. The 16S rRNA gene of a hyperthermophile, Thermus aquaticus, was cloned into Pichia pastoris (yeast) genome, and an equivalent amount of the yeast was added to the stool and cecal samples of mice before DNA extraction. 16S rRNA gene library construction and HTS were performed after DNA extraction. The absolute abundances of bacteria were calculated using T. aquaticus reads. The average relative abundances of T. aquaticus in the five stool and five cecal samples were 0.95% and 0.33%, respectively, indicating that the number of bacteria in a cecum sample is 2.9 times higher than that in a stool sample. The method proposed for quantifying the absolute abundance of the bacterial population in this study is expected to overcome the limitation of showing only compositional data in most microbiome studies.

Reduced production of the major allergens Bla g 1 and Bla g 2 in Blattella germanica after antibiotic treatment.

PURPOSE: Allergens present in the feces or frass of cockroaches can cause allergic sensitization in humans. The use of fecal and frass extracts for immunotherapy has been previously investigated but has not yet been fully standardized. Here, we treated cockroaches with ampicillin to produce extracts with reduced amounts of total bacteria. METHODS: We performed targeted high-throughput sequencing of 16S rDNA to compare the microbiomes of ampicillin-treated and untreated (control) cockroaches. RNA-seq was performed to identify differentially expressed genes (DEGs) in ampicillin-treated cockroaches. RESULTS: Analysis of the microbiome revealed that alpha diversity was lower in the ampicillin-treated group than in the control group. Beta diversity analysis indicated that ampicillin treatment altered bacterial composition in the microbiome of cockroaches. Quantitative polymerase chain reaction revealed that almost all bacteria were removed from ampicillin-treated cockroaches. RNA-seq analysis revealed 1,236 DEGs in ampicillin-treated cockroaches (compared to untreated cockroaches). Unlike bacterial composition, the DEGs varied between the two groups. Among major allergens, the expression of Bla g 2 decreased significantly in ampicillin-treated cockroaches (compared to untreated group). CONCLUSIONS: In this study, the reduced level of allergens observed in cockroaches may be related to lower amounts of total bacteria caused by treatment with antibiotics. It is possible to make a protein extract with few bacteria for use in immunotherapy.

Comparative microbiomes of ticks collected from a black rhino and its surrounding environment.

'Eliska,' an endangered black rhino (Diceros bicornis), died suddenly in Mkomazi National Park in Tanzania in 2016. Three Amblyomma gemma ticks were collected from Eliska's body, and four ticks were collected from the surrounding field. We conducted 16S rRNA targeted high-throughput sequencing to evaluate the overall composition of bacteria in the ticks' microbiomes and investigate whether the ticks could be the cause of Eliska's death. The ticks collected from Eliska's body and the field were found to differ in their bacterial composition. Bacillus chungangensis and B. pumilus were the most commonly found bacteria in the ticks collected from the field, and B. cereus and Lysinibacillus sphaericus were the most commonly found in the ticks collected from Eliska's body. The abundance was higher in the ticks collected from the field. In contrast, the equity was higher in the ticks collected from Eliska's body. No known pathogenic bacteria that could explain Eliska's sudden death were found in any of the ticks. The differences between the microbiome of ticks collected from Eliska's body and from the field indicate that the microbiome of ticks' changes through the consumption of blood.

Effect of isomaltulose used for osmotic extraction of Prunus mume fruit juice substituting sucrose.

This study evaluated the applicability of isomaltulose as a sucrose substitute in the osmotic extraction of Prunus mume fruit juice. Isomaltulose (20 mM) significantly reduced the uptake of a fluorescent tracer-2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl) amino]-2-deoxyglucose. Juice extracted by isomaltulose had similar pH and titratable acidity values to those of the other sugars. Citric and malic acids were the main organic acids in the extracted juices. The radical-scavenging ability of the plum juice extracted by isomaltulose was significantly higher than in juices extracted by other sugars (p < 0.05) and polyphenols content of the juice was also significantly higher than those of other sugars. The blood glucose level of P. mume juice extracted by fructose or isomaltulose was increased slowly compared to the juice extracted by sucrose. Therefore, the use of isomaltulose or an isomaltulose mixture in the manufacture of P. mume juice will help maintain health by reducing sugar intake.

Portable Agrichemical Detection System for Enhancing the Safety of Agricultural Products Using Aggregation of Gold Nanoparticles.

Organophosphorus (OP) and triazole chemicals have been commonly used as insecticides and fungicides to protect agricultural foods from harmful insects and fungi. However, these agrichemicals sometimes remain after distribution and can cause serious health and environmental issues. Therefore, it is essential to detect OPs and triazole chemicals in agricultural products. Nowadays, many detection techniques for OPs and triazole chemicals are expensive and time-consuming and require highly trained technicians. Thus, particularly rapid, simple, and sensitive detection methods are in demand for on-site screening of agrichemicals. Gold nanoparticles (AuNPs) have been utilized for applications in analytical assays and real-time monitoring in the biosensor field because of their biocompatibility and outstanding size-dependent optical properties. In this study, we used AuNPs as a detection probe, which have a size of 17 nm in diameter, a red color, and the absorbance peak at 520 nm. When imidazole was added to AuNPs mixed with the agrichemicals, the AuNPs aggregated and their colors changed to purple, causing the appearance of a new peak at 660-670 nm, which could be measured within approximately 20 s. Moreover, we developed a novel device for multiple agrichemical detections using an AuNP-aggregation-based spectrometric detection system. This portable device is light, simple, fast, and highly sensitive as well as selective. With this system, agrichemical residues can be easily detected on the spot at a low cost and in a short reaction time.

Selective increase in pinolenic acid (all-cis-5,9,12-18:3) in Korean pine nut oil by crystallization and its effect on LDL-receptor activity.

The aims of this study were to obtain concentrated pinolenic acid (5,9,12-18:3) from dietary Korean pine (Pinus koraiensis) nut oil by urea complexation and to investigate its cholesterol-lowering effect on the LDL-receptor activity of human hepatoma HepG2 cells. Pine nut oil was hydrolyzed to provide a low-pinolenic acid-containing FA extract (LPAFAE), followed by crystallization with different ratios of urea in ethanol (EtOH) or methanol (MeOH) as a solvent to produce a high-pinolenic acid-containing FA extract (HPAFAE). The profiles of HPAFAE obtained by urea complexation showed different FA compositions compared with LPAFAE. The long-chain saturated FA palmitic acid (16:0) and stearic acid (18:0) were decreased with urea/FA ratios (UFR) of 1:1 (UFR1), 2:1 (UFR2), and 3:1 (UFR3). Linoleic acid (9,12-18:2) was increased 1.3 times with UFR2 in EtOH, and linolenic acid (9,12,15-18:3) was increased 1.5 times with UFR3 in MeOH after crystallization. The crystallization with UFR3 in EtOH provided the highest concentration of pinolenic acid, which was elevated by 3.2-fold from 14.1 to 45.1%, whereas that of linoleic acid (9,12-18:2) was not changed, and that of oleic acid (9-18:1) was decreased 7.2-fold. Treatment of HepG2 cells with HPAFE resulted in significantly higher internalization of 3,3'-dioctadecylindocarbocyanine-LDL (47.0 +/- 0.15) as compared with treatment with LPAFAE (25.6 +/- 0.36) (P< 0.05). Thus, we demonstrate a method for the concentration of pinolenic acid and suggest that this concentrate may have LDL-lowering properties by enhancing hepatic LDL uptake.

Influence of steeping solution and storage temperature on the color change of garlic.

UNLABELLED: The objective of this study was to investigate the browning of garlic under different steeping conditions and storage temperatures. The brown indices of steeped garlics showed lowest values (7.3 and 7) in 25% and 50% EtOH at 7 d of storage. The degree of browning of steeped garlics was lowest (10.2 in 25% EtOH and 10.4 in 50% EtOH) in the samples soaked for 8 h at 13 d of storage. As the storage temperature was increased from 10 to 40 degrees C, the brown indices of garlics revealed an increasing trend relative to storage time regardless of steeping treatment. Overall, the kinetic parameters showed relatively low R(2) and irregular reaction constants, but the k(o) values showed an increasing trend with temperature under a zero-order model. The highest polyphenol content within the garlic bulbs was seen in controls (without steeping treatment, 588.9 microg/g), than 0% EtOH (water, 392.5 microg/g), than 25% EtOH (211.3 microg/g), and finally 50% EtOH (155.6 microg/g). The polyphenol oxidase activity of garlic showed a similar trend to that of polyphenol content. However, the texture properties of garlics steeped with 25% and 50% did not change. PRACTICAL APPLICATION: The garlic color preferred by consumers is a creamy-white, but this is susceptible to enzymatic browning when pre-peeled and chopped. When garlic was steeped in the 25% and 50% alcohol, the browning of garlic was prevented during storage.

Effects of moisture content and drying temperature on the physicochemical properties of ostrich jerky.

The objective of this study was to investigate the influence of temperature (50, 60, and 70 degrees C) and moisture content (18, 21, and 24%) on the quality ofjerky products from ostrich meat. The jerky products were evaluated by physicochemical and sensory methods. The tensile strength of the samples prepared to 24% w.b. moisture content at 50 degrees C was lowest (p < 0.05; 124 N) while those prepared under other conditions were in the range of 208-268 N (p > 0.05). The water activity of the jerky products was 0.51-0.72, and significant influence of drying temperature on protein solubility was also observed, but the samples dried at the same temperature had similar levels of soluble protein (p > 0.05) regardless of moisture content of samples. The pH of the samples dried at 50 degrees C and 60 degrees C showed little change in different moisture content, but samples dried at 70 degrees C tended to affect the pH by moisture content. Hunter a-values showed a predominantly increasing trend in all moisture levels as the heating temperature increased. The jerky product prepared to 24% w.b. moisture content at 60 degrees C showed the best sensory overall acceptability.