Hepatocyte-specific CCAAT/enhancer binding protein α restricts liver fibrosis progression.

Metabolic dysfunction-associated steatohepatitis (MASH) - previously described as nonalcoholic steatohepatitis (NASH) - is a major driver of liver fibrosis in humans, while liver fibrosis is a key determinant of all-cause mortality in liver disease independent of MASH occurrence. CCAAT/enhancer binding protein α (CEBPA), as a versatile ligand-independent transcriptional factor, has an important function in myeloid cells, and is under clinical evaluation for cancer therapy. CEBPA is also expressed in hepatocytes and regulates glucolipid homeostasis; however, the role of hepatocyte-specific CEBPA in modulating liver fibrosis progression is largely unknown. Here, hepatic CEBPA expression was found to be decreased during MASH progression both in humans and mice, and hepatic CEBPA mRNA was negatively correlated with MASH fibrosis in the human liver. CebpaΔHep mice had markedly enhanced liver fibrosis induced by a high-fat, high-cholesterol, high-fructose diet or carbon tetrachloride. Temporal and spatial hepatocyte-specific CEBPA loss at the progressive stage of MASH in CebpaΔHep,ERT2 mice functionally promoted liver fibrosis. Mechanistically, hepatocyte CEBPA directly repressed Spp1 transactivation to reduce the secretion of osteopontin, a fibrogenesis inducer of hepatic stellate cells. Forced hepatocyte-specific CEBPA expression reduced MASH-associated liver fibrosis. These results demonstrate an important role for hepatocyte-specific CEBPA in liver fibrosis progression, and may help guide the therapeutic discoveries targeting hepatocyte CEBPA for the treatment of liver fibrosis.

Identification of Bacteria and Viruses Associated with Patients with Acute Febrile Illness in Khon Kaen Province, Thailand.

The majority of cases of undifferentiated acute febrile illness (AFI) in the tropics have an undefined etiology. In Thailand, AFI accounts for two-thirds of illnesses reported to the Ministry of Public Health. To characterize the bacterial and viral causes of these AFIs, we conducted molecular pathogen screening and serological analyses in patients who sought treatment in Chum Phae Hospital, Khon Kaen province, during the period from 2015 to 2016. Through integrated approaches, we successfully identified the etiology in 25.5% of cases, with dengue virus infection being the most common cause, noted in 17% of the study population, followed by scrub typhus in 3.8% and rickettsioses in 6.8%. Further investigations targeting viruses in patients revealed the presence of Guadeloupe mosquito virus (GMV) in four patients without other pathogen co-infections. The characterization of four complete genome sequences of GMV amplified from AFI patients showed a 93-97% nucleotide sequence identity with GMV previously reported in mosquitoes. Nucleotide substitutions resulted in amino acid differences between GMV amplified from AFI patients and mosquitoes, observed in 37 positions. However, these changes had undergone purifying selection pressure and potentially had a minimal impact on protein function. Our study suggests that the GMV strains identified in the AFI patients are relatively similar to those previously reported in mosquitoes, highlighting their potential role associated with febrile illness.

Full-length 16S rDNA sequencing based on Oxford Nanopore Technologies revealed the association between gut-pharyngeal microbiota and tuberculosis in cynomolgus macaques.

Tuberculosis (TB) is an infectious disease caused by the Mycobacterium tuberculosis complex (Mtbc), which develops from asymptomatic latent TB to active stages. The microbiome was purposed as a potential factor affecting TB pathogenesis, but the study was limited. The present study explored the association between gut-pharyngeal microbiome and TB stages in cynomolgus macaques using the full-length 16S rDNA amplicon sequencing based on Oxford Nanopore Technologies. The total of 71 macaques was divided into TB (-) control, TB (+) latent and TB (+) active groups. The differential abundance analysis showed that Haemophilus hemolyticus was decreased, while Prevotella species were increased in the pharyngeal microbiome of TB (+) macaques. In addition, Eubacterium coprostanoligenes in the gut was enriched in TB (+) macaques. Alteration of these bacteria might affect immune regulation and TB severity, but details of mechanisms should be further explored and validated. In summary, microbiota may be associated with host immune regulation and affect TB progression. The findings suggested the potential mechanisms of host-microbes interaction, which may improve the understanding of the role of microbiota and help develop therapeutics for TB in the future.

"Nano COVID-19": Nanopore sequencing of spike gene to identify SARS-CoV-2 variants of concern.

Coronavirus disease 2019 (COVID-19) is a worldwide pandemic infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). World Health Organization (WHO) has defined the viral variants of concern (VOC) which cause more severe disease, higher transmissibility, and reduced vaccine efficacy. In this study, the "Nano COVID-19" workflow based on Oxford nanopore sequencing of the full-length spike gene combined with flexible data analysis options was developed to identify SARS-CoV-2 VOCs. The primers were designed to cover the full-length spike gene and can amplify all VOC strains. The results of VOC identification based on phylogenetic analysis of the full-length spike gene were comparable to the whole genome sequencing (WGS). Compared to the standard VOC identification pipeline, the fast analysis based on Read Assignment, Mapping, and Phylogenetic Analysis in Real Time (RAMPART) and the user-friendly method based on EPI2ME yielded 89.3% and 97.3% accuracy, respectively. The EPI2ME pipeline is recommended for researchers without bioinformatic skills, whereas RAMPART is more suitable for bioinformaticians. This workflow provides a cost-effective, simplified pipeline with a rapid turnaround time. Furthermore, it is portable to point-of-care SARS-CoV-2 VOC identification and compatible with large-scale analysis. Therefore, "Nano COVID-19" is an alternative viral epidemic screening and transmission tracking workflow.

Comparative analysis of gut microbiota between common (Macaca fascicularis fascicularis) and Burmese (M. f. aurea) long-tailed macaques in different habitats.

The environment has an important effect on the gut microbiota-an essential part of the host's health-and is strongly influenced by the dietary pattern of the host as these together shape the composition and functionality of the gut microbiota in humans and other animals. This study compared the gut microbiota of Macaca fascicularis fascicularis and M. f. aurea in mangrove and island populations using 16S rRNA gene sequencing on a nanopore platform to investigate the effect of the environment and/or diet. The results revealed that the M. f. fascicularis populations that received anthropogenic food exhibited a higher richness and evenness of gut microbiota than the M. f. aurea populations in different habitats. Firmicutes and Bacteroidetes were the two most abundant bacterial phyla in the gut microbiota of both these subspecies; however, the relative abundance of these phyla was significantly higher in M. f. aurea than in M. f. fascicularis. This variation in the gut microbiota between the two subspecies in different habitats mostly resulted from the differences in their diets. Moreover, the specific adaptation of M. f. aurea to different environments with a different food availability had a significant effect on their microbial composition.

Investigation of the Molecular Epidemiology and Evolution of Circulating Severe Acute Respiratory Syndrome Coronavirus 2 in Thailand from 2020 to 2022 via Next-Generation Sequencing.

Coronavirus disease 2019 (COVID-19) is an infectious condition caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which surfaced in Thailand in early 2020. The current study investigated the SARS-CoV-2 lineages circulating in Thailand and their evolutionary history. Complete genome sequencing of 210 SARS-CoV-2 samples collected from collaborating hospitals and the Institute of Urban Disease Control and Prevention over two years, from December 2020 to July 2022, was performed using next-generation sequencing technology. Multiple lineage introductions were observed before the emergence of the B.1.1.529 omicron variant, including B.1.36.16, B.1.351, B.1.1, B.1.1.7, B.1.524, AY.30, and B.1.617.2. The B.1.1.529 omicron variant was subsequently detected between January 2022 and June 2022. The evolutionary rate for the spike gene of SARS-CoV-2 was estimated to be between 0.87 and 1.71 × 10-3 substitutions per site per year. There was a substantial prevalence of the predominant mutations C25672T (L94F), C25961T (T190I), and G26167T (V259L) in the ORF3a gene during the Thailand outbreaks. Complete genome sequencing can enhance the prediction of future variant changes in viral genomes, which is crucial to ensuring that vaccine strains are protective against worldwide outbreaks.

Development of a simplified and cost-effective sample preparation method for genotyping of human papillomavirus by next-generation sequencing.

High-risk human papillomavirus (HPV) infection is the most common cause of cervical cancer, but low-risk HPV strains can sometimes also be involved. Although HPV genotyping techniques used in clinical diagnosis cannot detect low-risk HPV, next-generation sequencing (NGS) can detect both types. However, DNA library preparation is complicated and expensive. The aim of this study was to develop a simplified, cost-effective sample preparation procedure for HPV genotyping based on next-generation sequencing (NGS). After DNA extraction, a first round of PCR was performed using modified MY09/11 primers specific for the L1 region of the HPV genome, followed by a second round of PCR to add the indexes and adaptors. Then, the DNA libraries were purified and quantified, and high-throughput sequencing was performed using an Illumina MiSeq platform. The sequencing reads were compared with reference sequences for HPV genotyping. The limit of detection for HPV amplification was 100 copies/µl. Analysis of the correlation of pathological cytology with the HPV genotype in individual clinical samples showed that HPV66 was the most common genotype found in the normal stage, whereas HPV16 was the main genotype found in low-grade squamous intraepithelial lesions, high-grade squamous intraepithelial lesions, and cervical cancer. This NGS method can detect and identify several HPV genotypes with 92% accuracy and 100% reproducibility, and it shows potential as a simplified and cost-effective technique for large-scale HPV genotyping in clinical samples.

Intrahost Genetic Diversity of Dengue Virus in Human Hosts and Mosquito Vectors under Natural Conditions Which Impact Replicative Fitness In Vitro.

Dengue virus (DENV) is an arbovirus whose transmission cycle involves disparate hosts: humans and mosquitoes. The error-prone nature of viral RNA replication drives the high mutation rates, and the consequently high genetic diversity affects viral fitness over this transmission cycle. A few studies have been performed to investigate the intrahost genetic diversity between hosts, although their mosquito infections were performed artificially in the laboratory setting. Here, we performed whole-genome deep sequencing of DENV-1 (n = 11) and DENV-4 (n = 13) derived from clinical samples and field-caught mosquitoes from the houses of naturally infected patients, in order to analyze the intrahost genetic diversity of DENV between host types. Prominent differences in DENV intrahost diversity were observed in the viral population structure between DENV-1 and DENV-4, which appear to be associated with differing selection pressures. Interestingly, three single amino acid substitutions in the NS2A (K81R), NS3 (K107R), and NS5 (I563V) proteins in DENV-4 appear to be specifically acquired during infection in Ae. aegypti mosquitoes. Our in vitro study shows that the NS2A (K81R) mutant replicates similarly to the wild-type infectious clone-derived virus, while the NS3 (K107R), and NS5 (I563V) mutants have prolonged replication kinetics in the early phase in both Vero and C6/36 cells. These findings suggest that DENV is subjected to selection pressure in both mosquito and human hosts. The NS3 and NS5 genes may be specific targets of diversifying selection that play essential roles in early processing, RNA replication, and infectious particle production, and they are potentially adaptive at the population level during host switching.

Alteration of gut microbiota in wild-borne long-tailed macaques after 1-year being housed in hygienic captivity.

The wild-born long-tailed macaques (Macaca fascicularis) were recently recruited and used as breeders for the National Primate Research Center of Thailand, Chulalongkorn University (NPRCT-CU), and changes in their in-depth gut microbiota profiles were investigated. The Oxford Nanopore Technology (ONT) was used to explore full-length 16S rDNA sequences of gut microbiota in animals once captured in their natural habitat and 1-year following translocation and housing in a hygienic environment at NPRCT-CU. Our findings show that the gut microbiota of macaques after 1 year of hygienic housing and programmed diets feeding was altered and reshaped. The prevalent gut bacteria such as Prevotella copri and Faecalibacterium prausnitzii were enriched after translocation, causing the lower alpha diversity. The correlation analysis revealed that Prevotella copri, Phascolarctobacterium succinatutens, and Prevotella stercorea, showed a positive correlation with each other. Significantly enriched pathways in the macaques after translocation included biosynthesis of essential amino acids, fatty acids, polyamine and butanoate. The effects of microbiota change could help macaques to harvest the energy from programmed diets and adapt their gut metabolism. The novel probiotics and microbiota engineering approach could be further developed based on the current findings and should be helpful for captive animal health care management.

Effect of a Multispecies Synbiotic Supplementation on Body Composition, Antioxidant Status, and Gut Microbiomes in Overweight and Obese Subjects: A Randomized, Double-Blind, Placebo-Controlled Study.

Studies investigating the effect of multispecies synbiotic supplementation in obesity management are limited. The current study was performed to evaluate the effects of multispecies probiotics mixed with fructooligosaccharides on body composition, antioxidant status, and gut microbiome composition in overweight and obese individuals. We employed a randomized, double-blind, placebo-controlled trial design, in which 63 individuals aged 18-45 years were assigned to receive either a synbiotic supplement or placebo for 12 weeks. The synbiotic group consumed a daily dose of 37 × 109 colony-forming units (CFU) of a unique blend of seven different probiotics, along with 2 g of fructooligosaccharides, while the placebo group consumed 2 g of maltodextrin daily. Assessments were performed at baseline, week 6, and the end of the study. The results of the study indicated that synbiotic supplementation resulted in a significant reduction in waist circumference and body fat percentage compared to the baseline measurements, as observed at 12 weeks. At the end of the study, there were no significant differences observed in body weight, BMI, waist circumference, or percentage of body fat between the synbiotic group and the placebo group. An analysis of plasma antioxidant capacity revealed that synbiotic supplementation caused a significant increase in Trolox equivalent antioxidant capacity (TEAC) and a concomitant decrease in malondialdehyde (MDA) in the test group when compared to the placebo. For the gut microbiota analysis, synbiotic supplementation significantly decreased Firmicutes abundance and the Firmicutes/Bacteroidetes (F/B) ratio at week 12 as compared to the placebo group. Nevertheless, the synbiotic group did not exhibit any substantial alterations in other biochemical blood parameters compared to the placebo group. These findings suggest that multispecies synbiotic supplementation could be a beneficial strategy to improve body composition, antioxidant status, and gut microbiome composition in overweight and obese subjects.

Comparison of circulating bacterial profiles between mild and severe COVID-19 patients.

Recent reports revealed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients can develop bacteremia; however, the circulating bacterial profile is not well studied. Therefore, this study has aimed to investigate circulating bacterial profile in mild (n = 15) and severe (n = 13) SARS-CoV-2-infected patients as well as healthy controls (n = 10), using 16S rDNA (V4) sequencing approach. The alpha diversity indexes and Bray-Curtis dissimilarity matrix revealed that the bacterial profiles between the two conditions are significantly different. Correspondingly, the relative abundance indicates that the predominant bacterial phylum in both conditions was Proteobacteria. At genus level, the dominant bacterial genera in the mild patients belonged to Sphingomonas, Stenotrophomonas, and Achromobacter, while bacterial genera belonging to Enhydrobacter, Comamonas, and Acinetobacter were dominant in the severe patients. Furthermore, Linear discriminant analysis (LDA) Effect Size (LEfSe). revealed that Stenotrophomonas, Delftia, Achromobacter, and Neisseria were enriched in the mild condition, while Agrobacterium, Comamonas, Pseudomonas, Corynebacterium, Alkaliphilus, and Kocuria were enriched in the severe patients. These results revealed a distinct circulating bacterial profile in the mild and severe SARS-CoV-2-infected patients, which may provide an insight for further therapeutic strategy.

Description of potential vectors of zoonotic filarial nematodes, Brugia pahangi, Setaria digitata, and Setaria labiatopapillosa in Thai mosquitoes.

Filariasis is classified as a vector-borne zoonotic disease caused by several filarial nematodes. The disease is widely distributed in tropical and subtropical regions. Understanding the relationship between mosquito vectors, filarial parasites, and vertebrate hosts is therefore essential for determining the probability of disease transmission and, correspondingly, developing effective strategies for prevention and control of diseases. In this study, we aimed to investigate the infection of zoonotic filarial nematodes in field-caught mosquitoes, observe the potential vectors of filaria parasites in Thailand using a molecular-based survey, conduct a study of host-parasite relationship, and propose possible coevolution of the parasites and their hosts. Mosquitoes were collected around cattle farms in Bangkok, Nakhon Si Thammarat, Ratchaburi, and Lampang provinces from May to December 2021 using a CDC Backpack aspirator for 20-30 minutes in each area (intra-, peri-, and wild environment). All mosquitoes were identified and morphologically dissected to demonstrate the live larvae of the filarial nematode. Furthermore, all samples were tested for filarial infections using PCR and sequencing. A total of 1,273 adult female mosquitoes consisted of five species: 37.78% Culex quinquefasciatus, 22.47% Armigeres subalbatus, 4.71% Cx. tritaeniorhynchus, 19.72% Anopheles peditaeniatus, and 15.32% An. dirus. Larvae of Brugia pahangi and Setaria labiatopapillosa were found in Ar. subalbatus and An. dirus mosquitoes, respectively. All mosquito samples were processed by PCR of ITS1 and COXI genes for filaria nematode species identification. Both genes showed that B. pahangi was found in four mosquitoes of Ar. subalbatus from Nakhon Si Thammarat, S. digitata was detected in three samples of An. peditaeniatus from Lampang, and S. labiatopapillosa was detected in one of An. dirus from Ratchaburi. However, filarial nematodes were not found in all Culex species. This study infers that this is the first data regarding the circulation of Setaria parasites in Anopheles spp. from Thailand. The phylogenetic trees of the hosts and parasites are congruent. Moreover, the data could be used to develop more effective prevention and control strategies for zoonotic filarial nematodes before they spread in Thailand.

Gut bacteriome and metabolome of Ascaris lumbricoides in patients.

The most frequent intestinal helminth infections in humans are attributed to Ascaris lumbricoides, and there are concerns over the anthelminthic resistance of this species. The gut microbiota has essential roles in host physiology. Therefore, discovering host-parasite-microbiota interactions could help develop alternative helminthiasis treatments. Additionally, these interactions are modulated by functional metabolites that can reveal the mechanisms of infection and disease progression. Thus, we aimed to investigate bacteriomes in the gut of helminths and fecal samples of patients via next-generation sequencing. Our results showed that infection intensity was associated with the bacterial composition of helminth guts but not with the intestinal bacteriome of human hosts. Moreover, the metabolomes of A. lumbricoides in the heavy and light ascariasis cases were characterized using ultra-high performance liquid chromatography/time-of-flight mass spectrometry. Increased levels of essential biomolecules, such as amino acids, lipids, and nucleotide precursors, were found in the guts of helminths isolated from heavily infected patients, implying that these metabolites are related to egg production and ascariasis pathogenicity. These findings are the first step towards a more complete understanding of the mechanisms by which the bacteriome of helminth guts affect their colonization and may reveal novel and more effective approaches to parasitic disease therapy.

Classification of salivary bacteriome in asymptomatic COVID-19 cases based on long-read nanopore sequencing.

The coronavirus (COVID-19) global pandemic has impacted the health of almost everyone, including changes in their salivary microbiota. Since 2019, there has been an increase in the number of new COVID-19 cases in Thailand. Therefore, COVID-19 active case finding is important for early detection and epidemic control. Moreover, the dynamic changes of salivary bacteriome in asymptomatic COVID-19 cases are largely unknown. This research aimed to investigate and compare the salivary bacteriome and the co-infectious bacterial pathogens in the asymptomatic COVID-19 positive group to the negative group, based on novel nanopore sequencing. This cohort was a cross-sectional study including saliva samples collected from 82 asymptomatic participants (39 COVID-19 positive and 43 COVID-19 negative cases). All samples were sequenced for the full-length bacterial 16S rDNA. The alpha and beta diversity analyses were not significantly different between groups. The three major species in salivary bacteriome including Veillonella parvula, Streptococcus mitis, and Prevotella melaninogenica were observed in both groups. Interestingly, Lautropia mirabilis was a significantly enriched species in the saliva of the asymptomatic COVID-19-positive cases based on linear discriminant analysis effect size (LEfSe) analysis. The results suggested that L. mirabilis was a co-infectious agent in the asymptomatic COVID-19 group. However, the potential role of L. mirabilis should be validated in further experimental studies.

Skin Microbiota Profiles from Tape Stripping and Skin Biopsy Samples of Patients with Psoriasis Treated with Narrowband Ultraviolet B.

Purpose: Although the pathogenesis of psoriasis involves the dermis, most previous studies collected samples using the swab technique. A recent study examining the microbiomes obtained via both skin biopsies and swabs revealed a significant difference in normal skin. We hypothesized that the microbiome profile of patients with psoriasis from tape stripping and skin biopsy might be different. This study sought to contribute to microbiome research on psoriasis by investigating the changes in the microbiome during narrowband ultraviolet B (NBUVB) therapy by comparing the results from the different sampling techniques of tape stripping and skin biopsy. Patients and Methods: Twenty-three participants, including 14 patients with chronic plaque psoriasis and nine healthy controls, were recruited, and nine patients with psoriasis completed 20-sessions of NBUVB treatment. Skin microbiota from both techniques was analyzed using the 16S rRNA gene at baseline and after treatment. Results: A clear difference was observed between the results from the two sampling techniques. Alpha diversity of the microbiota obtained from tape stripping was higher than that of the microbiota from skin biopsy, whereas beta diversity was clustered into two groups by sampling technique. The microbiome was altered during NBUVB treatment using both sampling techniques. Conclusion: Different sampling techniques resulted in different microbiome profiles in patients with psoriasis. Tape stripping and swabs are feasible procedures and are mostly used in psoriasis and other skin microbiome studies; however, skin biopsy may also expand our understanding of psoriasis and other skin diseases that pathophysiology involves deeper to the dermis or subcutaneous tissue.

Critical roles of sepsis-reshaped fecal virota in attenuating sepsis severity.

Because studies on all fecal organisms (bacteria, fungi, and viruses) in sepsis are rare and bacteriophages during sepsis might have adapted against gut bacteria with possible pathogenicity, cecal ligation and puncture (CLP; a sepsis mouse model) was evaluated. In fecal bacteriome, sepsis increased Bacteroides and Proteobacteria but decreased Firmicutes, while fecal virome demonstrated increased Podoviridae when compared with sham feces. There was no difference in the fungal microbiome (predominant Ascomycota in both sham and CLP mice) and the abundance of all organisms between sepsis and control groups. Interestingly, the transfers of feces from CLP mice worsened sepsis severity when compared with sham fecal transplantation, as evaluated by mortality, renal injury (serum creatinine and histology), liver damage (liver enzyme and histology), spleen apoptosis, serum cytokines, endotoxemia, and bacteremia. In contrast, the transfers of fecal viral particles from sepsis mice, but not from sham mice, attenuated inflammation in CLP sepsis possibly through the decrease in several fecal pathogenic bacteria (such as Proteobacteria, Gammaproteobacteria, and Prevotellaceae) as evaluated by fecal microbiome analysis. Perhaps the isolation of favorable bacteriophages in sepsis feces and increased abundance ex vivo before oral treatment in a high concentration are beneficial.

Regulation of mi-RNAs Target Cancer Genes Between Exercise and Non-exercise in Rat Rheumatoid Arthritis Induction: Pilot Study.

Introduction: Rheumatoid arthritis is associated with various cancers. Many studies have investigated physical exercise interventions as health improvements to ameliorate the risk of cancer during rheumatoid arthritis diagnosis. Recently, microRNAs were used as biomarkers for health assessment and cancer prediction in rheumatoid arthritis patients. Methods: The effects of exercise interventions on serum microRNAs were investigated in pristane-induced arthritis (PIA) rat models. Twelve Sprague-Dawley male rats were divided into 4 groups including non-exercise without PIA (N-EX), non-exercise with PIA (N-EX + PIA), exercise without PIA (EX) and exercise with PIA (EX + PIA). Blood samples were collected at the end of the study period to analyze miRNA biomarkers and target cancer gene predictions. Results: Four significant Rattus norvegicus (rno-microRNAs) may purpose as tumor suppressors were identified as potential target cancer gene candidate expressions within the 4 comparative interventional exercise groups. One rno-microRNA and target cancer gene candidate was up-regulated and 3 rno-microRNAs and their target cancer genes were down-regulated. Conclusions: Exercise interventions affected rno-miRNAs regulated target cancer gene candidates ITPR3, SOCS6, ITGA6, and NKX2-1 as biomarkers for cancer prognosis in rheumatoid arthritis diagnosis.

Genome characterization and mutation analysis of human influenza A virus in Thailand.

The influenza A viruses have high mutation rates and cause a serious health problem worldwide. Therefore, this study focused on genome characterization of the viruses isolated from Thai patients based on the next-generation sequencing technology. The nasal swabs were collected from patients with influenza-like illness in Thailand during 2017-2018. Then, the influenza A viruses were detected by reverse transcription-quantitative polymerase chain reaction and isolated by MDCK cells. The viral genomes were amplified and sequenced by Illumina MiSeq platform. Whole genome sequences were used for characterization, phylogenetic construction, mutation analysis and nucleotide diversity of the viruses. The result revealed that 90 samples were positive for the viruses including 44 of A/ H1N1 and 46 of A/H3N2. Among these, 43 samples were successfully isolated and then the viral genomes of 25 samples were completely amplified. Finally, 17 whole genomes of the viruses (A/H1N1, n=12 and A/H3N2, n=5) were successfully sequenced with an average of 232,578 mapped reads and 1,720 genome coverage per sample. Phylogenetic analysis demonstrated that the A/H1N1 viruses were distinguishable from the recommended vaccine strains. However, the A/H3N2 viruses from this study were closely related to the recommended vaccine strains. The nonsynonymous mutations were found in all genes of both viruses, especially in hemagglutinin (HA) and neuraminidase (NA) genes. The nucleotide diversity analysis revealed negative selection in the PB1, PA, HA, and NA genes of the A/H1N1 viruses. High-throughput data in this study allow for genetic characterization of circulating influenza viruses which would be crucial for preparation against pandemic and epidemic outbreaks in the future.

Comparison of Malassezia spp. colonization between human skin exposed to high- and low-ambient air pollution.

The skin microbiota is essential for human health; altered skin microbiome colonization and homeostasis may be associated with several inflammatory skin conditions and other inflammatory diseases. Malassezia spp. are commensal fungi commonly found on the human skin, and they also play a pathogenic role in various skin diseases. It is hypothesized that the exposure of human skin to air pollution might be associated with Malassezia spp. colonization. The aim of this study was to compare Malassezia spp. colonization on healthy human skin between people living in two major cities in Thailand with different air qualities: one city with highly polluted ambient air and the other with less polluted air. Skin microbiome samples from 66 participants were collected using swabbing and scraping techniques. The skin fungal composition was analysed using high-throughput sequencing based on internal transcribed spacer 2 (ITS2) rDNA. A significant difference was found in alpha and beta diversities and the relative abundance of fungal profiles between the groups. The relative abundance of Malassezia spp. was found to be significantly higher in the highly polluted area than in the less polluted area. This study demonstrates that high-ambient air pollution may alter Malassezia spp. colonization on healthy human skin, which could lead to dysbiosis of the cutaneous ecosystem and eventually result in some skin disorders.

Metagenomic analysis of the gut microbiota in piglets either challenged or not with enterotoxigenic Escherichia coli reveals beneficial effects of probiotics on microbiome composition, resistome, digestive function and oxidative stress responses.

This study used metagenomic analysis to investigate the gut microbiota and resistome in piglets that were or were not challenged with enterotoxigenic Escherichia coli (ETEC) and had or had not received dietary supplementation with microencapsulated probiotics. The 72 piglets belonged to six groups that were either non-ETEC challenged (groups 1-3) or ETEC challenged (receiving 5ml of 109 CFU/ml pathogenic ETEC strain L3.2 one week following weaning at three weeks of age: groups 4-6). On five occasions at 2, 5, 8, 11, and 14 days of piglet age, groups 2 and 5 were supplemented with 109 CFU/ml of multi-strain probiotics (Lactiplantibacillus plantarum strains 22F and 25F, and Pediococcus acidilactici 72N) while group 4 received 109 CFU/ml of P. acidilactici 72N. Group 3 received 300mg/kg chlortetracycline in the weaner diet to mimic commercial conditions. Rectal faecal samples were obtained for metagenomic and resistome analysis at 2 days of age, and at 12 hours and 14 days after the timing of post-weaning challenge with ETEC. The piglets were all euthanized at 42 days of age. The piglets in groups 2 and 5 were enriched with several desirable microbial families, including Lactobacillaceae, Lachnospiraceae and Ruminococcaceae, while piglets in group 3 had increases in members of the Bacteroidaceae family and exhibited an increase in tetW and tetQ genes. Group 5 had less copper and multi-biocide resistance. Mobile genetic elements IncQ1 and IncX4 were the most prevalent replicons in antibiotic-fed piglets. Only groups 6 and 3 had the integrase gene (intl) class 2 and 3 detected, respectively. The insertion sequence (IS) 1380 was prevalent in group 3. IS3 and IS30, which are connected to dietary intake, were overrepresented in group 5. Furthermore, only group 5 showed genes associated with detoxification, with enrichment of genes associated with oxidative stress, glucose metabolism, and amino acid metabolism compared to the other groups. Overall, metagenomic analysis showed that employing a multi-strain probiotic could transform the gut microbiota, reduce the resistome, and boost genes associated with food metabolism.