Peripheral blood transcriptomic clusters uncovered immune phenotypes of asthma.

BACKGROUND: Transcriptomic analysis has been used to elucidate the complex pathogenesis of heterogeneous disease and may also contribute to identify potential therapeutic targets by delineating the hub genes. This study aimed to investigate whether blood transcriptomic clustering can distinguish clinical and immune phenotypes of asthmatics, and microbiome in asthmatics. METHODS: Transcriptomic expression of peripheral blood mononuclear cells (PBMCs) from 47 asthmatics and 21 non-asthmatics was measured using RNA sequencing. A hierarchical clustering algorithm was used to classify asthmatics. Differentially expressed genes, clinical phenotypes, immune phenotypes, and microbiome of each transcriptomic cluster were assessed. RESULTS: In asthmatics, three distinct transcriptomic clusters with numerously different transcriptomic expressions were identified. The proportion of severe asthmatics was highest in cluster 3 as 73.3%, followed by cluster 2 (45.5%) and cluster 1 (28.6%). While cluster 1 represented clinically non-severe T2 asthma, cluster 3 tended to include severe non-T2 asthma. Cluster 2 had features of both T2 and non-T2 asthmatics characterized by the highest serum IgE level and neutrophil-dominant sputum cell population. Compared to non-asthmatics, cluster 1 showed higher CCL23 and IL1RL1 expression while the expression of TREML4 was suppressed in cluster 3. CTSD and ALDH2 showed a significant positive linear relationship across three clusters in the order of cluster 1 to 3. No significant differences in the diversities of lung and gut microbiomes were observed among transcriptomic clusters of asthmatics and non-asthmatics. However, our study has limitations in that small sample size data were analyzed with unmeasured confounding factors and causal relationships or function pathways were not verified. CONCLUSIONS: Genetic clustering based on the blood transcriptome may provide novel immunological insight, which can be biomarkers of asthma immune phenotypes. Trial registration Retrospectively registered.

Human Alphacoronavirus Universal Primers for Genome Amplification and Sequencing.

Rapid and accurate sequencing covering the entire genome is essential to identify genetic variations of viral pathogens. However, due to the low viral titers in clinical samples, certain amplification steps are required for viral genome sequencing. At present, there are no universal primers available for alphacoronaviruses and that, since these viruses have diverse strains, new primers specific to the target strain must be continuously developed for sequencing. Thus, in this study, we aimed to develop a universal primer set valid for all human alphacoronaviruses and applicable to samples containing trace amounts of the virus. To this aim, we designed overlapping primer pairs capable of amplifying the entire genome of all known human alphacoronaviruses. The selected primers, named the AC primer set, were composed of 10 primer pairs stretching over the entire genome of alphacoronaviruses, and produced PCR products of the expected size (3-5 kb) from both the HCoV-229E and HCoV-NL63 strains. After genome amplification, an evaluation using various sequencing platforms was carried out. The amplicon library sequencing data were assembled into complete genome sequences in all sequencing strategies examined in this study. The sequencing accuracy varied depending on the sequencing technology, but all sequencing methods showed a sequencing error of less than 0.01%. In the mock clinical specimen, the detection limit was 10-3 PFU/ml (102 copies/ml). The AC primer set and experimental procedure optimized in this study may enable the fast diagnosis of mutant alphacoronaviruses in future epidemics.

Distribution of bacteriocin genes in the lineages of Lactiplantibacillus plantarum.

Lactiplantibacillus plantarum, previously named "Lactobacillus plantarum," is found in a wide variety of environments exhibiting a high level of intraspecies genetic diversity. To investigate the strain diversity, we performed comparative genomic analyses of the 54 complete genome sequences. The results revealed that L. plantarum subsp. plantarum was split into three lineages, A, B and C. Of the genes beneficial for probiotic activity, only those associated with the biosynthesis of plantaricin (Pln), an L. plantarum-specific bacteriocin, were found to be significantly different among the lineages. The genes related to the biosynthesis of plnE/F were conserved throughout the three lineages, whereas the outgroups did not possess any Pln-producing genes. In lineage C, the deepest and ancestral type branch, plnE/F genes, were well conserved. In lineage B, loss of gene function was observed due to mobile elements in the pln loci. In lineage A, most strains were predicted to produce more than one type of Pln by possessing diverse Pln-encoding genes. These results showed the presence of functional diversity arising from the trifurcating evolution in L. plantarum subsp. plantarum and demonstrated that Pln is an indicator for differentiating the three lineages.

Interactions between NCR+ILC3s and the Microbiome in the Airways Shape Asthma Severity.

Asthma is a heterogeneous disease whose development is shaped by a variety of environmental and genetic factors. While several recent studies suggest that microbial dysbiosis in the gut may promote asthma, little is known about the relationship between the recently discovered lung microbiome and asthma. Innate lymphoid cells (ILCs) have also been shown recently to participate in asthma. To investigate the relationship between the lung microbiome, ILCs, and asthma, we recruited 23 healthy controls (HC), 42 patients with non-severe asthma, and 32 patients with severe asthma. Flow cytometry analysis showed severe asthma associated with fewer natural cytotoxicity receptor (NCR)+ILC3s in the lung. Similar changes in other ILC subsets, macrophages, and monocytes were not observed. The asthma patients did not differ from the HC in terms of the alpha and beta-diversity of the lung and gut microbiomes. However, lung function correlated positively with both NCR+ILC3 frequencies and microbial diversity in the lung. Sputum NCR+ILC3 frequencies correlated positively with lung microbiome diversity in the HC, but this relationship was inversed in severe asthma. Together, these data suggest that airway NCR+ILC3s may contribute to a healthy commensal diversity and normal lung function.

Microbiome profiling of uncinate tissue and nasal polyps in patients with chronic rhinosinusitis using swab and tissue biopsy.

Chronic rhinosinusitis (CRS) is characterized according to the presence or absence of nasal polyps (NPs) and displays nasal microbiota dysbiosis. However, optimal sampling methods of the nasal microbiome in CRS have not been identified. We aimed to assess the microbial composition in patients with CRS, comparing different sampling methods (swab and tissue biopsy), tissue types (uncinate tissue and NP), and disease subtypes. Samples were obtained by swabbing the middle meatus and taking a biopsy of uncinate tissue (UT) in patients with CRS with (CRSwNP, N = 8) or without NP (CRSsNP, N = 6) and controls (N = 8). NPs were also harvested in CRSwNP. DNAs were extracted from fifty-two samples and analyzed by 16S rRNA gene amplicon sequencing. As a result, a great interpersonal variance was observed in nasal swabs, while UT samples presented distinct microbiome with low inter-personal differences. Moreover, the UT microbiomes were further differentiated into three clusters which are associated with disease status (control, CRSsNP, and CRSwNP). Compared to UT, NP revealed a unique microbiome profile with significantly less bacterial diversity. Prevotella was the genus whose abundance was negatively correlated with disease severity in NP. In conclusion, tissue samples are better specimens than nasal swabs for assessing the microbiomes of CRS patients. Several bacteria in UT and NP tissues revealed an association with clinical severity of CRSwNP.

Lung virome: New potential biomarkers for asthma severity and exacerbation.

BACKGROUND: Although some respiratory virus infections are known to contribute to the development and exacerbation of asthma, commensal viromes in airway have not been extensively studied due to technical challenges. OBJECTIVES: This study investigated the characteristics of the virome in asthmatic airways. METHODS: Both the bacteriome and virome profiles in sputum from 12 healthy individuals, 15 patients with nonsevere asthma, and 15 patients with severe asthma were analyzed and assessed for the association with clinical characteristics such as severity, exacerbation, Asthma Control Test (ACT), and lung function. RESULTS: While analysis of the 16S ribosomal RNA bacteriome in the airway showed no differences, clear contrasts in the diversity and composition of airway viromes were observed between healthy controls and patients with asthma. Herpesviruses were the most abundant type of virus in the asthma group (44.6 ± 4.6%), mainly with cytomegalovirus (CMV) and EBV accounting for 24.5 ± 3.3% and 16.9 ± 3.5%, respectively, in contrast to those in the healthy controls (5.4 ± 2.5% and 7.1 ± 3.0%, respectively). CMV and EBV were more abundant in patients with asthma who experienced exacerbation, and their abundance showed correlation with more severe asthma, lower ACT score, and lower lung function. On the contrary, bacteriophage that is abundant in healthy controls was severely reduced in patients with asthma in the order of nonsevere and severe asthma and presented significant positive correlation with ACT and FEV1/forced vital capacity. CONCLUSIONS: Lung viromes, especially, CMV, EBV, and bacteriophage may be potential biomarkers of asthma severity and exacerbation.

A Half-Day Genome Sequencing Protocol for Middle East Respiratory Syndrome Coronavirus.

Recent coronavirus (CoV) outbreaks, including that of Middle East respiratory syndrome (MERS), have presented a threat to public health worldwide. A primary concern in these outbreaks is the extent of mutations in the CoV, and the content of viral variation that can be determined only by whole genome sequencing (WGS). We aimed to develop a time efficient WGS protocol, using universal primers spanning the entire MERS-CoV genome. MERS and synthetic Neoromicia capensis bat CoV genomes were successfully amplified using our developed PCR primer set and sequenced with MinION. All experimental and analytical processes took 6 h to complete and were also applied to synthetic animal serum samples, wherein the MERS-CoV genome sequence was completely recovered. Results showed that the complete genome of MERS-CoV and related variants could be directly obtained from clinical samples within half a day. Consequently, this method will contribute to rapid MERS diagnosis, particularly in future CoV epidemics.

Alteration of Lung and Gut Microbiota in IL-13-Transgenic Mice Simulating Chronic Asthma.

Increasing evidence suggests a potential role of microbial colonization in the inception of chronic airway diseases. However, it is not clear whether the lung and gut microbiome dysbiosis is coincidental or a result of mutual interaction. In this study, we investigated the airway microbiome in interleukin 13 (IL-13)-rich lung environment and related alterations of the gut microbiome. IL-13- overexpressing transgenic (TG) mice presented enhanced eosinophilic inflammatory responses and mucus production, together with airway hyperresponsiveness and subepithelial fibrosis. While bronchoalveolar lavage fluid and cecum samples obtained from 10-week-old IL-13 TG mice and their C57BL/6 wild-type (WT) littermates showed no significant differences in alpha diversity of lung and gut microbiome, they presented altered beta diversity in both lung and gut microbiota in the IL-13 TG mice compared to the WT mice. Lung-specific IL-13 overexpression also altered the composition of the gut as well as the lung microbiome. In particular, IL-13 TG mice showed an increased proportion of Proteobacteria and Cyanobacteria and a decreased amount of Bacteroidetes in the lungs, and depletion of Firmicutes and Proteobacteria in the gut. The patterns of polymicrobial interaction within the lung microbiota were different between WT and IL-13 TG mice. For instance, in IL-13 TG mice, lung Mesorhizobium significantly affected the alpha diversity of both lung and gut microbiomes. In summary, chronic asthma-like pathologic changes can alter the lung microbiota and affect the gut microbiome. These findings suggest that the lung-gut microbial axis might actually work in asthma.

Difference in the Gut Microbiome between Ovariectomy-Induced Obesity and Diet-Induced Obesity.

During menopausal transition, the imbalance of estrogen causes body weight gain. Although gut microbiome dysbiosis has been reported in postmenopausal obesity, it is not clear whether there is any difference in the microbiome profile between dietary-induced obesity and postmenopausal obesity. Therefore, in this study, we analyzed intestinal samples from ovariectomized mice and compared them with those of mice with high-fat diet-induced obesity. To further evaluate the presence of menopause-specific bacteria-gene interactions, we also analyzed the liver transcriptome. Investigation of the 16S rRNA V3-V4 region amplicon sequence profile revealed that menopausal obesity and dietary obesity resulted in similar gut microbiome structures. However, Bifidobacterium animalis was exclusively observed in the ovariectomized mice, which indicated that menopausal obesity resulted in a different intestinal microbiome than dietary obesity. Additionally, several bacterial taxa (Dorea species, Akkermansia muciniphila, and Desulfovibrio species) were found when the ovariectomized mice were treated with a high-fat diet. A significant correlation between the above-mentioned menopause-specific bacteria and the genes for female hormone metabolism was also observed, suggesting the possibility of bacteria-gene interactions in menopausal obesity. Our findings revealed the characteristics of the intestinal microbiome in menopausal obesity in the mouse model, which is very similar to the dietary obesity microbiome but having its own diagnostic bacteria.

Hydrogenophaga crassostreae sp. nov., isolated from a Pacific oyster.

A Gram-negative, motile, rod-shaped, and aerobic bacterial strain, designated LPB0072T, was isolated from a Pacific oyster (Crassostrea gigas). Autotrophic growth with hydrogen gas was not observed. Cells oxidized thiosulfate to sulfate and reduced nitrate to nitrite. The complete genome sequence of strain LPB0072T (CP017476) is 4.94 Mb in length and contains 4459 protein-coding genes, with a G+C content of 61.3 mol%. Analysis of the 16S rRNA gene sequence indicated that strain LPB0072T belongs to the genus Hydrogenophaga, with greatest sequence similarity to the type strain of Hydrogenophaga taeniospiralis (97.5 %). The isoprenoid quinone (Q-8) and the major cellular fatty acids (C16 : 1ω7c and/or C16 : 1ω6c, C16 : 0 and C17 : 1ω6c) identified were concordant with the chemotaxonomic properties of the genus Hydrogenophaga. The average nucleotide identities with closely related species were below the suggested boundary for species delineation, indicating that the isolate is a novel species. Numerous physiological and biochemical features also distinguished the isolate from other known Hydrogenophaga species. Based on the polyphasic data presented in this study, strain LPB0072T should be classified as a novel species in the genus Hydrogenophaga, and the name Hydrogenophaga crassostreae sp. nov. is proposed. The type strain is LPB0072T (=KACC 18705T=JCM 31188T).

Thalassotalea crassostreae sp. nov., isolated from Pacific oyster.

A Gram-staining-negative, aerobic, rod-shaped bacterial strain, designated LPB0090T, was isolated from the Pacific oyster, Crassostreagigas, collected from the Yeongheung Island, Korea (37° 15' 16.1″ N; 126° 29' 46.5″ E). The complete genome sequence of LPB0090T (accession number CP017689) was 3 861 670 bp long with a DNA G+C content of 38.8 mol%. The genome included 3245 protein-coding genes and six copies of rRNA operons. On the basis of the results of 16S rRNA gene sequence analysis, LPB0090T was found to form an independent phyletic line within the genus Thalassotalea, with 94.7-96.0 % sequence similarities to the previously known species of the genus. The isoprenoid quinone (Q-8) and major fatty acids (C16 : 0, C17 : 1 ω8c, and C16 : 1 ω7c and/or C16 : 1 ω6c) of the isolate were similar to those of the other members of the genus Thalassotalea. A number of phenotypic features, however, distinguished LPB0090T from its closest neighbour Thalassotalea ponticola as well as other species of the genus Thalassotalea. On the basis of the phylogenetic, genomic and phenotypic data presented in this study, the strain was classified as representing a novel species of the genus Thalassotalea. Therefore, the name Thalassotalea crassostreae sp. nov. is proposed for the isolate. The type strain is LPB0090T (=KACC 18695T=JCM 31189T).

Antimicrobial activity of the chloroform fraction of Drynaria fortunei against oral pathogens.

Drynaria fortunei (D. fortunei), widely used in traditional Korean medicine, is reportedly effective in treating inflammation, hyperlipidemia, bone fractures, oxidative damage, arteriosclerosis, rheumatism, and gynecological diseases. The objective of this study was to evaluate the antibacterial effects of the chloroform fraction of D. fortunei (DFCF) and assess the synergistic effects of DFCF with antibiotics against bacterial pathogens. This was carried out by calculating the minimal inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) and performing checkerboard dilution test and time-kill assays. The MICs/MBCs for DFCF, ampicillin, and gentamicin against all oral strains were >39-2,500/5,000 µg/mL, 0.25-64/0.25-64 µg/mL, and 0.5-256/1-512 µg/mL, respectively. DFCF exhibited the highest activity against the periodontic pathogens Prevotella intermedia and Porphylomonas gingivalis. DFCF in combination with ampicillin showed a strong synergistic effect against oral bacteria (fractional inhibitory concentration (FIC) index ≤0.5), whereas on combining with gentamicin, it reduced the on half-eighth times than used alone (FICI ≤ 0.5). DFCF combined with ampicillin or gentamicin killed 100% of most tested bacteria within 3-4 h. The results of this study demonstrate the antimicrobial and synergistic activity of DFCF and antibiotics against oral pathogens.

Antibacterial and synergistic effects of the n-BuOH fraction of Sophora flavescens root against oral bacteria.

The antibacterial activity of an extract and several fractions of Sophora flavescens (S. flavescens) root alone and in combination with antibiotics against oral bacteria was investigated by checkerboard assay and time-kill assay. The minimum inhibitory concentration/minimum bactericidal concentration (MIC/MBC) values for all examined bacteria were 0.313-2.5/0.625-2.5 µg/mL for the n-BuOH fraction, 0.625-5/1.25-10 µg/mL for the EtOAc fraction, 0.25-8/0.25-16 µg/mL for ampicillin, 0.5-256/1-512 µg/mL for gentamicin, 0.008-32/0.016-64 µg/mL for erythromycin, and 0.25-64/0.5-128 µg/mL for vancomycin. The n-butanol (n-BuOH) and ethyl acetate (EtOAc) fractions exhibited stronger antibacterial activity against oral bacteria than other fractions and extracts. The MICs and MBCs were reduced to between one half and one quarter when the n-BuOH and EtOAc fractions were combined with antibiotics. After 24 h of incubation, combination of 1/2 MIC of the n-BuOH fraction with antibiotics increased the degree of bactericidal activity. The present results suggest that n-BuOH and EtOAc extracts of S. flavescens root might be applicable as new natural antimicrobial agents against oral pathogens.

Flavobacterium crassostreae sp. nov., isolated from Pacific oyster.

A yellow, rod-shaped, Gram-reaction-negative bacterial strain, designated LPB0076T, was isolated from a Pacific oyster. Results of 16S rRNA gene sequence analyses indicated that the strain represented a member of the genus Flavobacterium. It had the highest sequence similarity to the type strains of Flavobacterium frigidarium (97.6 %) and Flavobacterium omnivorum (97.0 %), and its similarities with all other species of the genus Flavobacterium were below 97.0 %. Its genome size (3.02 Mb), DNA G+C content (36.0 mol%), predominant cellular fatty acids (anteiso-C15 : 0, iso-C15 : 0 and C16 : 1ω7c and/or C17 : 1ω6c), and major polar lipid (phosphatidylethanolamine) were similar to those described previously for members of the genus Flavobacterium. In contrast, a number of phenotypic characteristics, including the inability to grow microaerophilically, absence of flexirubin-type pigments and gliding motility and differences in enzymatic reactions, clearly distinguished LPB0076T from other species of the genus Flavobacterium. The polyphasic data presented in this study indicate that this isolate should be classified as representing a novel species of the genus Flavobacterium. The name Flavobacterium crassostreae sp. nov. is therefore proposed for the isolate, with the type strain being LPB0076T (=KACC 18706T=JCM 31219T).

Polaribacter vadi sp. nov., isolated from a marine gastropod.

A Gram-reaction-negative, non-motile, yellow-pigmented, NaCl-requiring bacterial strain, designated LPB0003T, was isolated from a marine gastropod, the granulated dogwhelk (Thais luteostoma). The complete genome sequence of strain LPB0003T (GenBank accession number CP017477), sized 3.81 Mb, had 3296 protein-coding genes. 16S rRNA gene sequence analysis indicated that strain LPB0003T belongs to the genus Polaribacter within the family Flavobacteriaceae with sequence similarities of 95.3-97.8 % to other species of the genus Polaribacter. The average nucleotide identities with closely related species were not higher than 80 %. The isoprenoid quinone (MK-6) and DNA G+C content (29.6 mol%) of strain LPB0003T fell within the expected ranges for the genus Polaribacter. The major polar lipids of the isolate were phosphatidylethanolamine, two unidentified aminolipids, an unidentified phospholipid, an unidentified glycolipid, and two unidentified lipids. The predominant cellular fatty acids were iso-C15 : 0, iso-C15 : 0 3-OH, C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3), and C15 : 1ω6c. Based on phylogenetic, genomic and phenotypic data presented in this study, strain LPB0003T should be classified as a novel species of the genus Polaribacter, for which the name Polaribacter vadi sp. nov. is proposed. The type strain is LPB0003T (=KACC 18704T=JCM 31217T).

Cochleicola gelatinilyticus gen. nov., sp. nov., Isolated from a Marine Gastropod, Reichia luteostoma.

A yellow, rod-shaped, non-motile, gram-negative, and strictly aerobic bacterial strain, designated LPB0005(T), was isolated from a marine gastropod, Reichia luteostoma. Here the genome sequence was determined, which comprised 3,395,737 bp with 2,962 protein-coding genes. The DNA G+C content was 36.3 mol%. The 16S rRNA gene sequence analysis indicated that the isolate represents a novel genus and species in the family Flavobacteriaceae, with relatively low sequence similarities to other closely related genera. The isolate showed chemotaxonomic properties within the range reported for the family Flavobacteriaceae, but possesses many physiological and biochemical characteristics that distinguished it from species in the closely related genera Ulvibacter, Jejudonia, and Aureitalea. Based on phylogenetic, phenotypic, and genomic analyses, strain LPB0005(T) represents a novel genus and species, for which the name Cochleicola gelatinilyticus gen. nov., sp. nov. is proposed. The type strain is LPB0005(T) (= KACC 18693(T) = JCM 31218(T)).

Wolbachia Sequence Typing in Butterflies Using Pyrosequencing.

Wolbachia is an obligate symbiotic bacteria that is ubiquitous in arthropods, with 25-70% of insect species estimated to be infected. Wolbachia species can interact with their insect hosts in a mutualistic or parasitic manner. Sequence types (ST) of Wolbachia are determined by multilocus sequence typing (MLST) of housekeeping genes. However, there are some limitations to MLST with respect to the generation of clone libraries and the Sanger sequencing method when a host is infected with multiple STs of Wolbachia. To assess the feasibility of massive parallel sequencing, also known as next-generation sequencing, we used pyrosequencing for sequence typing of Wolbachia in butterflies. We collected three species of butterflies (Eurema hecabe, Eurema laeta, and Tongeia fischeri) common to Korea and screened them for Wolbachia STs. We found that T. fischeri was infected with a single ST of Wolbachia, ST41. In contrast, E. hecabe and E. laeta were each infected with two STs of Wolbachia, ST41 and ST40. Our results clearly demonstrate that pyrosequencing-based MLST has a higher sensitivity than cloning and Sanger sequencing methods for the detection of minor alleles. Considering the high prevalence of infection with multiple Wolbachia STs, next-generation sequencing with improved analysis would assist with scaling up approaches to Wolbachia MLST.

Combination effects of baicalein with antibiotics against oral pathogens.

BACKGROUND: Baicalein is one of the major flavonoids in Scutellaria baicalensis Georgi, which has long been used in Asia as herbal medicine. Several biological effects of baicalein, such as antiviral, anti-inflammatiom, anti-hepatotoxicity, and anti-tumour properties, have been reported. OBJECTIVE AND DESIGN: In this study, the antibacterial activities of baicalein were investigated in combination with ampicillin and/or gentamicin against oral bacteria. RESULTS: Baicalein was determined with MIC and MBC values ranging from 80 to 320 and 160 to 640 µg/mL against oral bacteria. The range of MIC50 and MIC90 were 20-160 µg/mL and 80-320 µg/mL, respectively. The combination effects of baicalein with antibiotics were synergistic (FIC index <0.375-0.5 and FBCI <0.5) against oral bacteria. Furthermore, a time-kill study showed that the growth of the tested bacteria was completely attenuated after 1-6 h of treatment with the MIC50 of baicalein, regardless of whether it was administered alone or with ampicillin or gentamicin. CONCLUSION: These results suggest that baicalein combined with other antibiotics may be microbiologically beneficial and not antagonistic.