Metagenomic characterization of bacterial community and antibiotic resistance genes found in the mass transit system in Seoul, South Korea.

Mass transit systems, including subways and buses, are useful environments for studying the urban microbiome, as the vast majority of populations in urban areas use public transportation. Microbial communities in urban environments include both human- and environment-associated bacteria that play roles in health and pathogen transmission. In this study, we used shotgun metagenomic sequencing to profile microbial communities sampled from various surfaces found in subway stations and bus stops within the Seoul mass transit system. The metagenomic approach and network analysis were used to investigate broad-spectrum antibiotic resistance genes (ARGs) and their co-occurrence patterns. We uncovered 598 bacterial species in 76 samples collected from various surfaces within the Seoul mass transit system. All samples were dominated by the potential human pathogen Salmonella enterica (40 %) and the human skin bacterium Cutibacterium acnes (19 %). Significantly abundant biomarkers detected in subway station samples were associated with bacteria typically found in the human oral cavity and respiratory tract, whereas biomarkers detected in bus stop samples were associated with bacteria commonly found in soil, water, and plants. Temperature and location had significant effects on microbial community structure and diversity. In total, 41 unique ARG subtypes were identified, associated with single-drug or multidrug resistance to clinically important and extensively used antibiotics, including aminoglycosides, carbapenem, glycopeptide, and sulfonamides. We revealed that Seoul subway stations and bus stops possess unique microbiomes containing potential human pathogens and ARGs. These findings provide insights for refining location-specific responses to reduce exposure to potentially causative agents of infectious diseases, improving public health.

Gut microbial shifts by synbiotic combination of Pediococcus acidilactici and lactulose in weaned piglets challenged with Shiga toxin-producing Escherichia coli.

Development of alternatives to in-feed antibiotics in the swine industry have been the focused of many pig gut microbiota studies to improve animal health. In this study, we evaluated the effects of probiotic Pediococcus acidilactici (PRO), prebiotic lactulose (PRE), and their synbiotic combination (SYN) on gut microbiota using 16S rRNA gene sequencing in weaned piglets challenged with Shiga-toxin producing Escherichia coli (STEC). Our data showed that prebiotics, probiotics and synbiotics improved the intestinal health in weaned piglets. No significant differences were observed in species richness and species diversity in weaned piglets fed prebiotics, probiotics and their synbiotic combination. However, beta diversity analysis revealed distinct clustering of the microbiota of according to dietary treatment and by oral challenge of STEC. At the phylum level, Firmicutes to Bacteroidetes ratio was lower in the dietary treatment groups than the control group. Oral supplementation of prebiotics, probiotics and synbiotics enriched the abundance of Prevotella and Roseburia. Succinivibrio was elevated in PRO group; however, Phascolarctobacterium was depleted with STEC challenge regardless of dietary treatment. Overall, our data showed that administration of synbiotics in piglets improved intestinal health through gut microbiota modulation. Our data indicated that prebiotics, probiotics and their synbiotic combination could promote intestinal health through gut microbiota modulation in weaned piglets.

Fecal microbiome shifts by different forms of copper supplementations in growing pigs.

Copper is an essential mineral for pigs, thus it is used as a feed additive in the forms of copper sulfate. Therefore, this study aimed at characterizing the fecal microbiota shifts in pigs as fed by different forms of copper supplementation. 40 growing pigs aged 73 ± 1 days with an average weight of 30.22 ± 1.92kg were randomly divided into 5 groups. The control group (CON) fed with basal diet, while treatment groups were fed a basal diet supplemented with 100 ppm/kg of copper sulfate (CuSO4), Cu-glycine complex (CuGly), Cu-amino acid complex (CuAA), and Cu-hydroxy(4methylthio)butanoate chelate complex (CuHMB) for 28 days of trial, respectively. The data presented the comparison between inorganic and organic copper supplementation through gut microbiota in growing pigs. Alpha and Beta diversity anaylsis resulted in copper supplementation did shifted gut microbioal community structure. At the phylum level, Firmicutes and Bacteroidetes were the most abundant phyla at all times regardless of treatment. At the genus level, the relative abundances of Prevotella, Lactobacillus, Megasphaera, and SMB53 of the CuGly and CuHMB groups were significantly higher than those of copper sulfate and basal diet groups. Overall, this study may provide the potential role of organic copper replacing inorganic copper, resulting in increased beneficial bacteria in the pig gut.

Single-cell transcriptome profiling and the use of AID deficient mice reveal that B cell activation combined with antibody class switch recombination and somatic hypermutation do not benefit the control of experimental trypanosomosis.

Salivarian trypanosomes are extracellular protozoan parasites causing infections in a wide range of mammalian hosts, with Trypanosoma evansi having the widest geographic distribution, reaching territories far outside Africa and occasionally even Europe. Besides causing the animal diseases, T. evansi can cause atypical Human Trypanosomosis. The success of this parasite is attributed to its capacity to evade and disable the mammalian defense response. To unravel the latter, we applied here for the first time a scRNA-seq analysis on splenocytes from trypanosome infected mice, at two time points during infection, i.e. just after control of the first parasitemia peak (day 14) and a late chronic time point during infection (day 42). This analysis was combined with flow cytometry and ELISA, revealing that T. evansi induces prompt activation of splenic IgM+CD1d+ Marginal Zone and IgMIntIgD+ Follicular B cells, coinciding with an increase in plasma IgG2c Ab levels. Despite the absence of follicles, a rapid accumulation of Aicda+ GC-like B cells followed first parasitemia peak clearance, accompanied by the occurrence of Xbp1+ expressing CD138+ plasma B cells and Tbx21+ atypical CD11c+ memory B cells. Ablation of immature CD93+ bone marrow and Vpreb3+Ly6d+Ighm+ expressing transitional spleen B cells prevented mature peripheral B cell replenishment. Interestingly, AID-/- mice that lack the capacity to mount anti-parasite IgG responses, exhibited a superior defense level against T. evansi infections. Here, elevated natural IgMs were able to exert in vivo and in vitro trypanocidal activity. Hence, we conclude that in immune competent mice, trypanosomosis associated B cell activation and switched IgG production is rapidly induced by T. evansi, facilitating an escape from the detrimental natural IgM killing activity, and resulting in increased host susceptibility. This unique role of IgM and its anti-trypanosome activity are discussed in the context of the dilemma this causes for the future development of anti-trypanosome vaccines.

Oral Vaccination against Lawsoniaintracellularis Changes the Intestinal Microbiome in Weaned Piglets.

Lawsoniaintracellularis, which causes porcine proliferative enteropathy (PPE), is a common swine intestinal pathogen that is prevalent in pig production sites worldwide. In this study, the alteration in the microbiome composition of weaned pigs was investigated in response to vaccination against L. intracellularis, using 16S rRNA gene sequencing. A total of 64 crossbred (Duroc × [Landrace × Yorkshire]) healthy weanling pigs weaned at 4 weeks of age were randomly assigned to four treatment groups (four pigs/pen; four pens/treatment), using a randomized complete block design for the 42-day trial. Pigs in the treatment groups were orally administered with three different doses (1 dose = 2 mL) of vaccine against L. intracellularis (Enterisol® Ileitis, Boehringer Ingelheim Vetmedica GmbH), namely the following: LAW1 (0.5 dose), LAW2 (1 dose), LAW3 (2 dose). A non-vaccinated group served as a negative control (CONT). Alpha diversity analysis revealed that vaccination led to significant changes in species evenness but not species richness of the gut microbiota. Beta diversity analysis revealed that vaccination against L. intracellularis caused a significant shift in the microbial community structure. At the genus level, there was a significant increase in Streptococcus and a significant decrease in Clostridium in the fecal microbiota of vaccinated pigs, regardless of dose.

Complete genome sequence of Lactococcus lactis strain K_LL005, a xylose-utilizing bacterium isolated from grasshopper (Oxya chinensis sinuosa).

Lactococcus lactis is a fermentative lactic acid bacterium that is used extensively in food fermentations. The L. lactis strain K_LL005 was isolated from the grasshopper (Oxya chinensis sinuosa) gut in Korea. In this study, we reported the complete genome sequence of Lactococcus lactis K_LL005. The final complete genome assembly consist of one circular chromosome (2,375,093 bp) with an overall guanine + cytosine (G + C) content of 35.0%. Annotation results revealed 2,281 protein-coding sequences (CDSs), 19 rRNAs, and 68 tRNA genes. Lactococcus lactis K_LL005 has a gene encoding xylose metabolism such as xylR, xylA, and xylB (xylRAB).

Comprehensive genomic analysis reveals virulence factors and antibiotic resistance genes in Pantoea agglomerans KM1, a potential opportunistic pathogen.

Pantoea agglomerans is a Gram-negative facultative anaerobic bacillus causing a wide range of opportunistic infections in humans including septicemia, pneumonia, septic arthritis, wound infections and meningitis. To date, the determinants of virulence, antibiotic resistance, metabolic features conferring survival and host-associated pathogenic potential of this bacterium remain largely underexplored. In this study, we sequenced and assembled the whole-genome of P. agglomerans KM1 isolated from kimchi in South Korea. The genome contained one circular chromosome of 4,039,945 bp, 3 mega plasmids, and 2 prophages. The phage-derived genes encoded integrase, lysozyme and terminase. Six CRISPR loci were identified within the bacterial chromosome. Further in-depth analysis showed that the genome contained 13 antibiotic resistance genes conferring resistance to clinically important antibiotics such as penicillin G, bacitracin, rifampicin, vancomycin, and fosfomycin. Genes involved in adaptations to environmental stress were also identified which included factors providing resistance to osmotic lysis, oxidative stress, as well as heat and cold shock. The genomic analysis of virulence factors led to identification of a type VI secretion system, hemolysin, filamentous hemagglutinin, and genes involved in iron uptake and sequestration. Finally, the data provided here show that, the KM1 isolate exerted strong immunostimulatory properties on RAW 264.7 macrophages in vitro. Stimulated cells produced Nitric Oxide (NO) and pro-inflammatory cytokines TNF-α, IL-6 and the anti-inflammatory cytokine IL-10. The upstream signaling for production of TNF-α, IL-6, IL-10, and NO depended on TLR4 and TLR1/2. While production of TNF-α, IL-6 and NO involved solely activation of the NF-κB, IL-10 secretion was largely dependent on NF-κB and to a lesser extent on MAPK Kinases. Taken together, the analysis of the whole-genome and immunostimulatory properties provided in-depth characterization of the P. agglomerans KM1 isolate shedding a new light on determinants of virulence that drive its interactions with the environment, other microorganisms and eukaryotic hosts.

Intestinal Microbial Dysbiosis in Beagles Naturally Infected with Canine Parvovirus.

Canine parvoviral enteritis (PVE) is an important intestinal disease of the puppies; however, the potential impact of the canine parvovirus (CPV) on the gut microbiota has not been investigated. Therefore, the aim of this study was to evaluate the gut microbial shifts in puppies naturally infected with CPV. Fecal samples were collected from healthy dogs and those diagnosed with PVE at 4, 6, 8, and 12 weeks of age. The distal gut microbiota of dogs was characterized using Illumina MiSeq sequencing of the bacterial 16S rRNA genes. The sequence data were analyzed using QIIME with an Operational Taxonomic Unit definition at a similarity cutoff of 97%. Our results showed that the CPV was associated with significant microbial dysbiosis of the intestinal microbiota. Alpha diversity and species richness and evenness in dogs with PVE decreased compared to those of healthy dogs. At the phylum level, the proportion of Proteobacteria was significantly enriched in dogs with PVE while Bacteroidetes was significantly more abundant in healthy dogs (p < 0.05). In dogs with PVE, Enterobacteriaceae was the most abundant bacterial family accounting for 36.44% of the total bacterial population compared to only 0.21% in healthy puppies. The two most abundant genera in healthy dogs were Prevotella and Lactobacillus and their abundance was significantly higher compared to that of dogs with PVE (p < 0.05). These observations suggest that disturbances of gut microbial communities were associated with PVE in young dogs. Evaluation of the roles of these bacterial groups in the pathophysiology of PVE warrants further studies.

Role of Probiotics in Human Gut Microbiome-Associated Diseases.

Probiotics, including bacteria and yeast, are live microorganisms that have demonstrated beneficial effects on human health. Recently, probiotic bacteria are constantly being studied and their applications are also being considered in promising adjuvant treatments for various intestinal diseases. Clinical trials and in vivo experiments have extended our current understanding of the important roles that probiotics play in human gut microbiomeassociated diseases. It has been documented through many clinical trials that probiotics could shape the intestinal microbiota leading to potential control of multiple bowel diseases and promotion of overall wellness. In this review, we focused on the relationship between probiotics and the human gut microbiota and its roles in gut microbiome-associated diseases. Here, we also discuss future directions and research areas that need further elucidation in order to better understand the roles of probiotics in the treatment of intestinal diseases.

Evaluation of synbiotics as gut health improvement agents against Shiga toxin-producing Escherichia coli isolated from the pig.

Colibacillosis is one of the major health problems in young piglets resulting in poor health and death caused by Escherichia coli producing F18 pili and Shiga toxin 2e. It is pivotal to reduce colibacillosis in weaned piglets to enhance production performance. In this study, we evaluated synbiotics as the gut health improvement agents in the mouse model challenged with Shiga toxin-producing E. coli (STEC) isolated from piglets. Prebiotic lactulose was formulated with each 5.0 × 106 CFU/mL of Pediococcus acidilactici GB-U15, Lactobacillus plantarum GB-U17, and Lactobacillus plantarum GB 1-3 to produce 3 combinations of synbiotics. A total of 40 three weeks old BALB/c mice were randomly assigned to 4 groups (n = 10): a control group and 3 synbiotics treated groups. Each treatment groups were daily administrated with 5.0 × 106 CFU/mL of one synbiotics for the first week, and every 3 days during the second week. All the mice were challenged with 8.0 × 108 CFU/mL of STEC 5 days after animals began to receive synbiotics. Mice treated with synbiotics based on Pediococcus acidilactici GB-U15 and Lactobacillus plantarum GB-U17 significantly improved daily weight gain compared to mice in other groups. While mice treated with GB-U15 showed better fecal index, no significant differences were observed among groups. Gross lesion and histopathological evaluations showed that mice treated with GB-U15 moderately improved recovery from STEC infection. In conclusion, our results suggest that the synbiotics formulated with lactulose and Pediococcus acidilactici GB-U15 have potential benefits to prevent and improve colibacillosis in weaned piglets.

Piglet gut microbial shifts early in life: causes and effects.

The gut microbiome has long been known to play fundamentally important roles in the animal health and the well-being of its host. As such, the establishment and maintenance of a beneficial gut microbiota early in life is crucial in pigs, since early gut colonizers are pivotal in the establishment of permanent microbial community structures affecting the health and growth performance of pigs later in life. Emphasizing this importance of early gut colonizers, it is critical to understand the factors impacting the establishment of the piglet gut microbiome at weaning. Factors include, among others, diet, in-feed antibiotics, probiotics and prebiotic administration. The impact of these factors on establishment of the gut microbiome of piglets at weaning includes effects on piglet gut microbial diversity, structure, and succession. In this review, we thoroughly reviewed the most recent findings on the piglet gut microbiome shifts as influenced by weaning, and how these microbiome changes brought about by various factors that have been shown to affect the development of microbiota in piglets. This review will provide a general overview of recent studies that can help to facilitate the design of new strategies to modulate the gut microbiome in order to enhance gastrointestinal health, growth performance and well-being of piglets.

The dynamics of the piglet gut microbiome during the weaning transition in association with health and nutrition.

BACKGROUND: Understanding the composition of the microbial community and its functional capacity during weaning is important for pig production as bacteria play important roles in the pig's health and growth performance. However, limited information is available regarding the composition and function of the gut microbiome of piglets in early-life. Therefore, we performed 16S rRNA gene and whole metagenome shotgun sequencing of DNA from fecal samples from healthy piglets during weaning to measure microbiome shifts, and to identify the potential contribution of the early-life microbiota in shaping piglet health with a focus on microbial stress responses, carbohydrate and amino acid metabolism. RESULTS: The analysis of 16S rRNA genes and whole metagenome shotgun sequencing revealed significant compositional and functional differences between the fecal microbiome in nursing and weaned piglets. The fecal microbiome of the nursing piglets showed higher relative abundance of bacteria in the genus Bacteroides with abundant gene families related to the utilization of lactose and galactose. Prevotella and Lactobacillus were enriched in weaned piglets with an enrichment for the gene families associated with carbohydrate and amino acid metabolism. In addition, an analysis of the functional capacity of the fecal microbiome showed higher abundances of genes associated with heat shock and oxidative stress in the metagenome of weaned piglets compared to nursing piglets. CONCLUSIONS: Overall, our data show that microbial shifts and changes in functional capacities of the piglet fecal microbiome resulted in potential reductions in the effects of stress, including dietary changes that occur during weaning. These results provide us with new insights into the piglet gut microbiome that contributes to the growth of the animal.

Analysis of Microbiota in Bellflower Root, Platycodon grandiflorum, Obtained from South Korea.

Bellflower root (Platycodon grandiflorum), which belongs to the Campanulaceae family, is a perennial grass that grows naturally in Korea, northeastern China, and Japan. Bellflower is widely consumed as both food and medicine owing to its high nutritional value and potential therapeutic effects. Since foodborne disease outbreaks often come from vegetables, understanding the public health risk of microorganisms on fresh vegetables is pivotal to predict and prevent foodborne disease outbreaks. We investigated the microbial communities on the bellflower root (n = 10). 16S rRNA gene amplicon sequencing targeting the V6-V9 regions of 16S rRNA genes was conducted via the 454-Titanium platform. The sequence quality was checked and phylogenetic assessments were performed using the RDP classifier implemented in QIIME with a bootstrap cutoff of 80%. Principal coordinate analysis was performed using the weighted Fast UniFrac distance. The average number of sequence reads generated per sample was 67,192 sequences. At the phylum level, bacterial communities from the bellflower root were composed primarily of Proteobacteria, Firmicutes, and Actinobacteria in March and September samples. Genera Serratia, Pseudomonas, and Pantoea comprised more than 54% of the total bellflower root bacteria. Principal coordinate analysis plots demonstrated that the microbial community of bellflower root in March samples was different from those in September samples. Potential pathogenic genera, such as Pantoea, were detected in bellflower root samples. Even though further studies will be required to determine if these species are associated with foodborne illness, our results indicate that the 16S rRNA gene-based sequencing approach can be used to detect pathogenic bacteria on fresh vegetables.

Laminarin favorably modulates gut microbiota in mice fed a high-fat diet.

We investigated the anti-obesity effects of the potential prebiotic, laminarin, on mice fed a high-fat diet. A metagenomics approach was applied to characterize the ecological and functional differences of gut microbiota among mice fed a normal diet (CTL), a high-fat diet (HFD), and a laminarin-supplemented high-fat diet (HFL). The HFL mice showed a slower weight gain than the HFD mice during the laminarin-feeding period, but the rate of weight gain increased after the termination of laminarin supplementation. Gut microbial community analysis showed clear differences between the CTL and HFD mice, whereas the HFL mice were between the two. A higher abundance of carbohydrate active enzymes was observed in the HFL mice compared to the HFD mice, with especially notable increases in glycoside hydrolase and polysaccharide lyases. A significant decrease in Firmicutes and an increase in the Bacteroidetes phylum, especially the genus Bacteroides, were observed during laminarin ingestion. Laminarin ingestion altered the gut microbiota at the species level, which was re-shifted after termination of laminarin ingestion. Therefore, supplementing laminarin could reduce the adverse effects of a high-fat diet by shifting the gut microbiota towards a higher energy metabolism. Thus, laminarin could be used to develop anti-obesity functional foods. Our results also suggest that laminarin would need to be consumed regularly in order to prevent or manage obesity.

Influence of seawater intrusion on microbial communities in groundwater.

Groundwater is the sole source of potable water on Jeju Island in the Republic of (South) Korea. Groundwater is also used for irrigation and industrial purposes, and it is severely impacted by seawater intrusion in coastal areas. Consequently, monitoring the intrusion of seawater into groundwater on Jeju is very important for health and environmental reasons. A number of studies have used hydrological models to predict the deterioration of groundwater quality caused by seawater intrusion. However, there is conflicting evidence of intrusion due to complicated environmental influences on groundwater quality. Here we investigated the use of next generation sequencing (NGS)-based microbial community analysis as a way to monitor groundwater quality and detect seawater intrusion. Pristine groundwater, groundwater from three coastal areas, and seawater were compared. Analysis of the distribution of bacterial species clearly indicated that the high and low salinity groundwater differed significantly with respect to microbial composition. While members of the family Parvularculaceae were only identified in high salinity water samples, a greater percentage of the phylum Actinobacteria was predominantly observed in pristine groundwater. In addition, we identified 48 shared operational taxonomic units (OTUs) with seawater, among which the high salinity groundwater sample shared a greater number of bacterial species with seawater (6.7%). In contrast, other groundwater samples shared less than 0.5%. Our results suggest that NGS-based microbial community analysis of groundwater may be a useful tool for monitoring groundwater quality and detect seawater intrusion. This technology may also provide additional insights in understanding hydrological dynamics.

Analysis of swine fecal microbiota at various growth stages.

Recent obesity studies in humans and rodents have suggested that host weight gain is significantly associated with energy harvesting efficiency which is regulated by gut microbiota. Antibiotic growth promoters have been banned as feed additives in many countries. In this study, we aimed to provide knowledge of swine fecal microbiota by analyzing bacterial 16S rRNA gene sequences. Our results showed that swine fecal bacterial composition varied at each growth stage. Bacteroidetes decreased as the swine gained weight and unclassified genera significantly increased at later growth stages. Operational taxonomic unit (OTU) distribution analysis showed that the bacterial community difference was most significant between growers and finishers, while analysis of shared OTUs indicated a greater proportion of common species between growers and finishers. The differential abundance test between growers and finishers detected that nearly half of the species were shared OTUs, suggesting that differential abundance of each bacterial species predominantly controls bacterial community differences. Although functions of these bacteria are yet to be identified, understanding differences in fecal microbiota between each growth stage will provide additional insights for further studies related to swine gut microbiota.

Effects of antibiotic growth promoter and characterization of ecological succession in Swine gut microbiota.

Ever since the ban on antibiotic growth promoters (AGPs), the livestock death rate has increased owing to pathogenic bacterial infections. There is a need of developing AGP alternatives; however, the mechanisms by which AGP enhances livestock growth performance are not clearly understood. In this study, we fed 3-week-old swine for 9 weeks with and without AGPs containing chlortetracycline, sulfathiazole, and penicillin to investigate the effects of AGPs on swine gut microbiota. Microbial community analysis was done based on bacterial 16S rRNA genes using MiSeq. The use of AGP showed no growth promoting effect, but inhibited the growth of potential pathogens during the early growth stage. Our results showed the significant increase in species richness after the stabilization of gut microbiota during the post-weaning period (4-week-old). Moreover, the swine gut microbiota was divided into four clusters based on the distribution of operational taxonomic units, which was significantly correlated to the swine weight regardless of AGP treatments. Taxonomic abundance analysis indicated a negative correlation between host weight and the abundance of the family Prevotellaceae species, but showed positive correlation to the abundance of the family Spirochaetaceae, Clostridiaceae_1, and Peptostreptococcaeae species. Although no growth performance enhancement was observed, the use of AGP inhibited the potential pathogens in the early growth stage of swine. In addition, our results indicated the ecological succession of swine gut microbiota according to swine weight. Here, we present a characterization of swine gut microbiota with respect to the effects of AGPs on growth performance.