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September 22, 2019

Complete genome sequence of multidrug-resistant Staphylococcus cohnii ssp. urealyticus strain SNUDS-2 isolated from farmed duck, Republic of Korea.

Staphylococcus cohnii has become increasingly recognized as a potential pathogen of clinically significant nosocomial and farm animal infections. This study was designed to determine the genome of a multidrug-resistant S. cohnii subsp. urealyticus strain SNUDS-2 isolated from a farmed duck in Korea.Genomic DNA was sequenced using the PacBio RS II system. The complete genome was annotated and the presence of antimicrobial resistance and virulence genes were identified.The annotated 2,625,703 bp genome contained various antimicrobial resistance genes conferring resistance to ß-lactam, aminoglycosides, fluoroquinolones, phenicols and trimethoprim. The virulence-associated three synergistic hemolysins have been identified in the strain.To the best of our knowledge, this is the first complete genome of S. cohnii, and will provide important insights into the biodiversity of CoNS and valuable information for the control of this emerging pathogen. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

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September 22, 2019

Evaluating the mobility potential of antibiotic resistance genes in environmental resistomes without metagenomics.

Antibiotic resistance genes are ubiquitous in the environment. However, only a fraction of them are mobile and able to spread to pathogenic bacteria. Until now, studying the mobility of antibiotic resistance genes in environmental resistomes has been challenging due to inadequate sensitivity and difficulties in contig assembly of metagenome based methods. We developed a new cost and labor efficient method based on Inverse PCR and long read sequencing for studying mobility potential of environmental resistance genes. We applied Inverse PCR on sediment samples and identified 79 different MGE clusters associated with the studied resistance genes, including novel mobile genetic elements, co-selected resistance genes and a new putative antibiotic resistance gene. The results show that the method can be used in antibiotic resistance early warning systems. In comparison to metagenomics, Inverse PCR was markedly more sensitive and provided more data on resistance gene mobility and co-selected resistances.

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September 22, 2019

Effects of metal and metalloid pollutants on the microbiota composition of feces obtained from twelve commercial pig farms across China.

Understanding the metal and metalloid contamination and microbiota composition of pig feces is an important step required to support the design and implementation of effective pollution control and prevention strategies. A survey was implemented in 12 locations across China to investigate the content of metals and metalloids, and the main composition of the microbial communities of commercially reared pigs during two growth periods, defined as the early (Q group) and the later fattening growth phases (H group). These data showed widespread Al, Mn, Cu, Zn, and Fe pollution in pig feces. The concentration of Zn in the Q group feces was nearly two times higher than the levels measured in the H group. The microbial composition of the Q group exhibited greater richness of operational taxonomic units (OTUs) and fewer bacteria associated with zoonotic diseases compared with the microbial composition of the H group. Spearman rank correlation analysis showed that Cu and northern latitudes had a significant positive effect on the richness of bacterial communities in pig feces. Zn and Cd exhibited the biggest impact on microbial community composition based on canonical correspondence analysis. Functional metagenomic prediction indicated that about 0.8% genes present in the pig feces bacteria community are related to human diseases, and significantly more predicted pathogenic genes were detected in the H group than in the Q group. These results support the need to monitor heavy metal contamination and to control for zoonotic pathogens disseminated from pig feces in Chinese pig farms. Copyright © 2018. Published by Elsevier B.V.

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September 22, 2019

Reduction in fecal microbiota diversity and short-chain fatty acid producers in Methicillin-resistant Staphylococcus aureus infected individuals as revealed by PacBio single molecule, real-time sequencing technology.

Methicillin-resistant Staphylococcus aureus (MRSA) may cause potentially lethal infections. Increasing evidence suggests that the gut microbiota is associated with human health. Yet, whether patients with MRSA infections carry specific signatures in their fecal microbiota composition has not been determined. Thus, this study aimed to compare the fecal microbiota profile of MRSA-positive patients (n=15) with individuals without MRSA infection (n=15) by using the PacBio single molecule, real-time (SMRT) DNA sequencing system and real-time quantitative polymerase chain reaction (qPCR). Mann-Whitney tests and unweighted UniFrac principal coordinate analysis (PCoA) showed that the profile of fecal microbiota was apparently different between the two populations. Both the community richness and diversity were reduced in the MRSA-positive group (p<0.050). The genera Acinetobacter and Enterococcus were highly enriched in the MRSA-positive group, whereas less short-chain fatty acid (SCFA)-producing bacteria, including Butyricimonas, Faecalibacterium, Roseburia, Ruminococcus, Megamonas and Phascolarctobacterium, were detected in the MRSA-positive group. At species level, the species Acinetobacter baumannii and Bacteroides thetaiotaomicron were prevalent in the MRSA-positive group, whereas opposite trends were observed in 17 other species, such as Faecalibacterium prausnitzii, Lactobacillus rogosae, Megamonas rupellensis and Phascolarctobacterium faecium. Positive correlations were observed between Acinetobacter baumannii and erythrocyte sedimentation rate (ESR) (R=0.554, p=0.001), as well as hypersensitive C reactive protein (hsCRP) (R=0.406, p=0.026). Faecalibacterium prausnitzii was negatively associated with ESR (R=-0.545, p=0.002), hsCRP (R=-0.401, p=0.028) and total bile acids (TBA) (R=-0.364, p=0.048). In conclusion, the fecal microbiota structure was different between MRSA-positive and -negative patients. The increase in potential pathogens with the reduction of beneficial populations, such as SCFA-producing bacteria, in MRSA-positive patients may affect prognosis.

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September 22, 2019

Long-read, Single Molecule, Real-Time (SMRT) DNA Sequencing for metagenomic applications

In this chapter, we describe applications of single molecule, real-time (SMRT) DNA sequencing toward metagenomic research. The long sequence reads, combined with a lack of bias with respect to DNA sequence context or GC content, facilitate a more comprehensive analysis of the genomic constitution of microbial communities. Full-length 16S RNA gene sequencing at high (>99%) accuracy allows for species-level characterization of community members concomitant with the determination of community structure. The application of SMRT sequencing to whole-community shotgun microbial metagenomics has also been discussed.

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September 22, 2019

Cataloguing over-expressed genes in Epstein Barr Virus immortalized lymphoblastoid cell lines through consensus analysis of PacBio transcriptomes corroborates hypomethylation of chromosome 1

The ability of Epstein Barr Virus (EBV) to transform resting cell B-cells into immortalized lymphoblastoid cell lines (LCL) provides a continuous source of peripheral blood lymphocytes that are used to model conditions in which these lymphocytes play a key role. Here, the PacBio generated transcriptome of three LCLs from a parent-daughter trio (SRAid:SRP036136) provided by a previous study [1] were analyzed using a kmer-based version of YeATS (KEATS). The set of over-expressed genes in these cell lines were determined based on a comparison with the PacBio transcriptome of twenty tissues pro- vided by another study (hOPTRS) [2]. MIR155 long non-coding RNA (MIR155HG), Fc fragment of IgE receptor II (FCER2), T-cell leukemia/lymphoma 1A (TCL1A), and germinal center associated signaling and motility (GCSAM) were genes having the highest expression counts in the three LCLs with no expression in hOPTRS. Other over-expressed genes, having low expression in hOPTRS, were membrane spanning 4-domains A1 (MS4A1) and ribosomal protein S2 pseudogene 55 (RPS2P55). While some of these genes are known to be over-expressed in LCLs, this study provides a comprehensive cataloguing of such genes. A recent work involving a patient with EBV-positive large B-cell lymphoma was “unusually lacking various B-cell markers”, but over-expressing CD30 [3] – a gene ranked 79 among uniquely expressed genes here. Hypomethylation of chromosome 1 observed in EBV immortalized LCLs [4, 5] is also corroborated here by mapping the genes to chromosomes. Extending previous work identifying un-annotated genes [6], 80 genes were identified which are expressed in the three LCLs, not in hOPTRS, and missing in the GENCODE, RefSeq and RefSeqGene databases. KEATS introduces a method of determining expression counts based on a partitioning of the known annotated genes, has runtimes of a few hours on a personal workstation and provides detailed reports enabling proper debugging.

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September 22, 2019

Shorter unreported sequences in a RACE-Seq study involving seven tissues confirms ~150 novel transcripts identified in MCF-7 cell line PacBio transcriptome, leaving ~100 non-redundant transcripts exclusive to the cancer cell line.

PacBio sequencing generates much longer reads compared to second-generation sequencing technologies, with a trade-off of lower throughput, higher error rate and more cost per base. The PacBio transcriptome of the breast cancer cell line MCF-7 was found to have ~300 transcripts un-annotated in the current GENCODE (v25) or RefSeq, and missing in the liver, heart and brain PacBio transcriptomes [1]. RACE-sequencing (RACE-seq [2]) extends a well-established method of characterizing cDNA molecules generated by rapid amplification of cDNA ends (RACE [3]) using high-throughput sequencing technologies, reducing costs compared to PacBio. Here, shorter fragments of ~150 transcripts were found to be present in seven tissues analyzed in a recent RACE-seq study (Accid:ERP012249) [4]. These transcripts were not among the ~2500 novel transcripts reported in that study, tested separately here using the genomic coordinates provided, although “all curated novel isoforms were incorporated into the human GENCODE set (v22)” in that study. Non-redundancy analysis of the exclusive transcripts identified one transcript mapping to Chr1 with seven different splice variants, and erroneously mapped to Chr15 (PAC clone 15q11-q13) from the Prader-Willi/Angelman Syndrome region (Accid:AC004137.1). Finally, there are ~100 non-redundant transcripts missing in the seven tissues, in addition to other three tissues analyzed previously. Their absence in GENCODE and RefSeq databases rule them out as commonly transcribed regions, further increasing their likelihood as biomarkers.

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September 22, 2019

Discovery of the fourth mobile sulfonamide resistance gene.

Over the past 75 years, human pathogens have acquired antibiotic resistance genes (ARGs), often from environmental bacteria. Integrons play a major role in the acquisition of antibiotic resistance genes. We therefore hypothesized that focused exploration of integron gene cassettes from microbial communities could be an efficient way to find novel mobile resistance genes. DNA from polluted Indian river sediments were amplified using three sets of primers targeting class 1 integrons and sequenced by long- and short-read technologies to maintain both accuracy and context.Up to 89% of identified open reading frames encode known resistance genes, or variations thereof (>?1000). We identified putative novel ARGs to aminoglycosides, beta-lactams, trimethoprim, rifampicin, and chloramphenicol, including several novel OXA variants, providing reduced susceptibility to carbapenems. One dihydropteroate synthase gene, with less than 34% amino acid identity to the three known mobile sulfonamide resistance genes (sul1-3), provided complete resistance when expressed in Escherichia coli. The mobilized gene, here named sul4, is the first mobile sulfonamide resistance gene discovered since 2003. Analyses of adjacent DNA suggest that sul4 has been decontextualized from a set of chromosomal genes involved in folate synthesis in its original host, likely within the phylum Chloroflexi. The presence of an insertion sequence common region element could provide mobility to the entire integron. Screening of 6489 metagenomic datasets revealed that sul4 is already widespread in seven countries across Asia and Europe.Our findings show that exploring integrons from environmental communities with a history of antibiotic exposure can provide an efficient way to find novel, mobile resistance genes. The mobilization of a fourth sulfonamide resistance gene is likely to provide expanded opportunities for sulfonamide resistance to spread, with potential impacts on both human and animal health.

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September 22, 2019

Lactobacillus fermentum FTDC 8312 combats hypercholesterolemia via alteration of gut microbiota.

In this study, hypercholesterolemic mice fed with Lactobacillus fermentum FTDC 8312 after a seven-week feeding trial showed a reduction in serum total cholesterol (TC) levels, accompanied by a decrease in serum low-density lipoprotein cholesterol (LDL-C) levels, an increase in serum high-density lipoprotein cholesterol (HDL-C) levels, and a decreased ratio of apoB100:apoA1 when compared to those fed with control or a type strain, L. fermentum JCM 1173. These have contributed to a decrease in atherogenic indices (TC/HDL-C) of mice on the FTDC 8312 diet. Serum triglyceride (TG) levels of mice fed with FTDC 8312 and JCM 1173 were comparable to those of the controls. A decreased ratio of cholesterol and phospholipids (C/P) was also observed for mice fed with FTDC 8312, leading to a decreased number of spur red blood cells (RBC) formation in mice. Additionally, there was an increase in fecal TC, TG, and total bile acid levels in mice on FTDC 8312 diet compared to those with JCM 1173 and controls. The administration of FTDC 8312 also altered the gut microbiota population such as an increase in the members of genera Akkermansia and Oscillospira, affecting lipid metabolism and fecal bile excretion in the mice. Overall, we demonstrated that FTDC 8312 exerted a cholesterol lowering effect that may be attributed to gut microbiota modulation. Copyright © 2017 Elsevier B.V. All rights reserved.

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September 22, 2019

Single-cell (meta-)genomics of a dimorphic Candidatus Thiomargarita nelsonii reveals genomic plasticity.

The genus Thiomargarita includes the world’s largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group I intron also carried a MITE sequence that, like the hupL MITE family, occurs broadly across the genome. The presence of a high degree of mobile elements in genes central to Thiomargarita’s core metabolism has not been previously reported in free-living bacteria and suggests a highly mutable genome.

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September 22, 2019

MCF-7 breast cancer cell line PacBio generated transcriptome has ~300 novel transcribed regions, un-annotated in both RefSeq and GENCODE, and absent in the liver, heart and brain transcriptomes

Illuminating the “dark” regions of the human genome remains an ongoing effort, a decade and a half after the human genome was sequenced – RefSeq and GENCODE being two of the major annotation databases. Pacific Biosciences (PacBio) has provided open access to the transcriptome of MCF-7, a breast cancer cell line that has provided significant therapeutic advancement in breast cancer research since the 1970s. PacBio sequencing generates much longer reads compared to second-generation sequencing technologies, with a trade-off of lower throughput, higher error rate and more cost per base. Here, this transcriptome was analyzed using the YeATS pipeline, with additionally introduced kmer based algorithms, reducing computational times to a few hours on a simple workstation. Out of ~300 transcripts that have no match in both RefSeq and GENCODE, ~250 are absent in the transcriptomes of the heart, liver and brain, also provided by PacBio. Also, ~200 transcripts are absent in a recent catalogue of un-annotated long non-coding RNAs from 6,503 samples (~43 Terabases of sequence data) [1], and only two present in common in an experimental workflow RACE-Seq that reported 2,556 novel transcripts [2]. ~100 transcripts have >100 amino acid open reading frames, and have the potential of being protein coding genes. ORF based annotation also identified few bacterial transcripts in the PacBio database mapped to the human genome, and one human transcript that has been annotated as bacterial in the NCBI database. The current work reiterates the under-utilization of transcriptomes for annotating genomes. It also provides new leads for investigating breast cancer by virtue of exclusively expressed transcripts not expressed in other tissues, which have the prospects of breast cancer biomarkers based on further investigations.

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September 22, 2019

Genomic insights into the acid adaptation of novel methanotrophs enriched from acidic forest soils.

Soil acidification is accelerated by anthropogenic and agricultural activities, which could significantly affect global methane cycles. However, detailed knowledge of the genomic properties of methanotrophs adapted to acidic soils remains scarce. Using metagenomic approaches, we analyzed methane-utilizing communities enriched from acidic forest soils with pH 3 and 4, and recovered near-complete genomes of proteobacterial methanotrophs. Novel methanotroph genomes designated KS32 and KS41, belonging to two representative clades of methanotrophs (Methylocystis of Alphaproteobacteria and Methylobacter of Gammaproteobacteria), were dominant. Comparative genomic analysis revealed diverse systems of membrane transporters for ensuring pH homeostasis and defense against toxic chemicals. Various potassium transporter systems, sodium/proton antiporters, and two copies of proton-translocating F1F0-type ATP synthase genes were identified, which might participate in the key pH homeostasis mechanisms in KS32. In addition, the V-type ATP synthase and urea assimilation genes might be used for pH homeostasis in KS41. Genes involved in the modification of membranes by incorporation of cyclopropane fatty acids and hopanoid lipids might be used for reducing proton influx into cells. The two methanotroph genomes possess genes for elaborate heavy metal efflux pumping systems, possibly owing to increased heavy metal toxicity in acidic conditions. Phylogenies of key genes involved in acid adaptation, methane oxidation, and antiviral defense in KS41 were incongruent with that of 16S rRNA. Thus, the detailed analysis of the genome sequences provides new insights into the ecology of methanotrophs responding to soil acidification.

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September 22, 2019

Automated broad range molecular detection of bacteria in clinical samples.

Molecular detection methods, such as quantitative PCR (qPCR), have found their way into clinical microbiology laboratories for the detection of an array of pathogens. Most routinely used methods, however, are directed at specific species. Thus, anything that is not explicitly searched for will be missed. This greatly limits the flexibility and universal application of these techniques. We investigated the application of a rapid universal bacterial molecular identification method, IS-pro, to routine patient samples received in a clinical microbiology laboratory. IS-pro is a eubacterial technique based on the detection and categorization of 16S-23S rRNA gene interspace regions with lengths that are specific for each microbial species. As this is an open technique, clinicians do not need to decide in advance what to look for. We compared routine culture to IS-pro using 66 samples sent in for routine bacterial diagnostic testing. The samples were obtained from patients with infections in normally sterile sites (without a resident microbiota). The results were identical in 20 (30%) samples, IS-pro detected more bacterial species than culture in 31 (47%) samples, and five of the 10 culture-negative samples were positive with IS-pro. The case histories of the five patients from whom these culture-negative/IS-pro-positive samples were obtained suggest that the IS-pro findings are highly clinically relevant. Our findings indicate that an open molecular approach, such as IS-pro, may have a high added value for clinical practice. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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September 22, 2019

Bacteroides dorei dominates gut microbiome prior to autoimmunity in Finnish children at high risk for type 1 diabetes.

The incidence of the autoimmune disease, type 1 diabetes (T1D), has increased dramatically over the last half century in many developed countries and is particularly high in Finland and other Nordic countries. Along with genetic predisposition, environmental factors are thought to play a critical role in this increase. As with other autoimmune diseases, the gut microbiome is thought to play a potential role in controlling progression to T1D in children with high genetic risk, but we know little about how the gut microbiome develops in children with high genetic risk for T1D. In this study, the early development of the gut microbiomes of 76 children at high genetic risk for T1D was determined using high-throughput 16S rRNA gene sequencing. Stool samples from children born in the same hospital in Turku, Finland were collected at monthly intervals beginning at 4-6 months after birth until 2.2 years of age. Of those 76 children, 29 seroconverted to T1D-related autoimmunity (cases) including 22 who later developed T1D, the remaining 47 subjects remained healthy (controls). While several significant compositional differences in low abundant species prior to seroconversion were found, one highly abundant group composed of two closely related species, Bacteroides dorei and Bacteroides vulgatus, was significantly higher in cases compared to controls prior to seroconversion. Metagenomic sequencing of samples high in the abundance of the B. dorei/vulgatus group before seroconversion, as well as longer 16S rRNA sequencing identified this group as Bacteroides dorei. The abundance of B. dorei peaked at 7.6 months in cases, over 8 months prior to the appearance of the first islet autoantibody, suggesting that early changes in the microbiome may be useful for predicting T1D autoimmunity in genetically susceptible infants. The cause of increased B. dorei abundance in cases is not known but its timing appears to coincide with the introduction of solid food.

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September 22, 2019

The human microbiome and understanding the 16S rRNA gene in translational nursing science.

As more is understood regarding the human microbiome, it is increasingly important for nurse scientists and healthcare practitioners to analyze these microbial communities and their role in health and disease. 16S rRNA sequencing is a key methodology in identifying these bacterial populations that has recently transitioned from use primarily in research to having increased utility in clinical settings.The objectives of this review are to (a) describe 16S rRNA sequencing and its role in answering research questions important to nursing science; (b) provide an overview of the oral, lung, and gut microbiomes and relevant research; and (c) identify future implications for microbiome research and 16S sequencing in translational nursing science.Sequencing using the 16S rRNA gene has revolutionized research and allowed scientists to easily and reliably characterize complex bacterial communities. This type of research has recently entered the clinical setting, one of the best examples involving the use of 16S sequencing to identify resistant pathogens, thereby improving the accuracy of bacterial identification in infection control. Clinical microbiota research and related requisite methods are of particular relevance to nurse scientists-individuals uniquely positioned to utilize these techniques in future studies in clinical settings.

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