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September 22, 2019

Single-molecule long-read 16S sequencing to characterize the lung microbiome from mechanically ventilated patients with suspected pneumonia.

In critically ill patients, the development of pneumonia results in significant morbidity and mortality and additional health care costs. The accurate and rapid identification of the microbial pathogens in patients with pulmonary infections might lead to targeted antimicrobial therapy with potentially fewer adverse effects and lower costs. Major advances in next-generation sequencing (NGS) allow culture-independent identification of pathogens. The present study used NGS of essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial aspirates of intubated patients with suspected pneumonia. The results from 61 patients demonstrated that sufficient DNA was obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27 sequenced samples, only 20 had bacterial culture growth, while the microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples. Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The numbers of NGS-identifiable bacterial genera were consistently higher than identified by standard microbiological methods. As technical advances reduce the processing and sequencing times, NGS-based methods will ultimately be able to provide clinicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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September 22, 2019

Long-term changes of bacterial and viral compositions in the intestine of a recovered Clostridium difficile patient after fecal microbiota transplantation

Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infections (RCDIs). However, long-term effects on the patients’ gut microbiota and the role of viruses remain to be elucidated. Here, we characterized bacterial and viral microbiota in the feces of a cured RCDI patient at various time points until 4.5 yr post-FMT compared with the stool donor. Feces were subjected to DNA sequencing to characterize bacteria and double-stranded DNA (dsDNA) viruses including phages. The patient’s microbial communities varied over time and showed little overall similarity to the donor until 7 mo post-FMT, indicating ongoing gut microbiota adaption in this time period. After 4.5 yr, the patient’s bacteria attained donor-like compositions at phylum, class, and order levels with similar bacterial diversity. Differences in the bacterial communities between donor and patient after 4.5 yr were seen at lower taxonomic levels. C. difficile remained undetectable throughout the entire timespan. This demonstrated sustainable donor feces engraftment and verified long-term therapeutic success of FMT on the molecular level. Full engraftment apparently required longer than previously acknowledged, suggesting the implementation of year-long patient follow-up periods into clinical practice. The identified dsDNA viruses were mainly Caudovirales phages. Unexpectedly, sequences related to giant algae–infecting Chlorella viruses were also detected. Our findings indicate that intestinal viruses may be implicated in the establishment of gut microbiota. Therefore, virome analyses should be included in gut microbiota studies to determine the roles of phages and other viruses—such as Chlorella viruses—in human health and disease, particularly during RCDI.

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September 22, 2019

Metabolism of toxic sugars by strains of the bee gut symbiont Gilliamella apicola.

Social bees collect carbohydrate-rich food to support their colonies, and yet, certain carbohydrates present in their diet or produced through the breakdown of pollen are toxic to bees. The gut microbiota of social bees is dominated by a few core bacterial species, including the Gram-negative species Gilliamella apicola We isolated 42 strains of G. apicola from guts of honey bees and bumble bees and sequenced their genomes. All of the G. apicola strains share high 16S rRNA gene similarity, but they vary extensively in gene repertoires related to carbohydrate metabolism. Predicted abilities to utilize different sugars were verified experimentally. Some strains can utilize mannose, arabinose, xylose, or rhamnose (monosaccharides that can cause toxicity in bees) as their sole carbon and energy source. All of the G. apicola strains possess a manO-associated mannose family phosphotransferase system; phylogenetic analyses suggest that this was acquired from Firmicutes through horizontal gene transfer. The metabolism of mannose is specifically dependent on the presence of mannose-6-phosphate isomerase (MPI). Neither growth rates nor the utilization of glucose and fructose are affected in the presence of mannose when the gene encoding MPI is absent from the genome, suggesting that mannose is not taken up by G. apicola strains which harbor the phosphotransferase system but do not encode the MPI. Given their ability to simultaneously utilize glucose, fructose, and mannose, as well as the ability of many strains to break down other potentially toxic carbohydrates, G. apicola bacteria may have key roles in improving dietary tolerances and maintaining the health of their bee hosts.Bees are important pollinators of agricultural plants. Our study documents the ability of Gilliamella apicola, a dominant gut bacterium in honey bees and bumble bees, to utilize several sugars that are harmful to bee hosts. Using genome sequencing and growth assays, we found that the ability to metabolize certain toxic carbohydrates is directly correlated with the presence of their respective degradation pathways, indicating that metabolic potential can be accurately predicted from genomic data in these gut symbionts. Strains vary considerably in their range of utilizable carbohydrates, which likely reflects historical horizontal gene transfer and gene deletion events. Unlike their bee hosts, G. apicola bacteria are not detrimentally affected by growth on mannose-containing medium, even in strains that cannot metabolize this sugar. These results suggest that G. apicola may be an important player in modulating nutrition in the bee gut, with ultimate effects on host health. Copyright © 2016 Zheng et al.

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September 22, 2019

Resistance to ceftazidime-avibactam in Klebsiella pneumoniae due to porin mutations and the increased expression of KPC-3.

We reported the first clinical case of a ceftazidime-avibactam resistant KPC-3-producing Klebsiella pneumoniae (1), from a patient with no history of ceftazidime-avibactam therapy. We now present data documenting mechanisms of ceftazidime-avibactam resistance in this isolate. Whole-genome sequencing (WGS) was performed on two isolates: KP1245 (ceftazidime-avibactam MIC, 4 µg/ml; from blood on hospital day 1; referred to as isolate 1 in our previous report [1]) and KP1244 (ceftazidime-avibactam MIC, 32 µg/ml; from blood on hospital day 2; referred to as isolate 2 in our previous report [2]), using MiSeq (Illumina, San Diego, CA) and PacBio RSII (Menlo Park, CA) systems (2). The in silico multilocus sequence type (ST) was ST258. Single nucleotide polymorphism (SNP) analysis revealed 17 SNPs between KP1245 and KP1244, indicating that the isolates were related but that significant diversity existed in this patient (2). Nonsynonymous mutations are shown in Table 1; the most striking of these is in the OmpK36 porin gene. KP1244 contained a missense mutation predicted to encode a T333N mutation. Both isolates also harbored a mutation predicted to encode R191L in OmpK36 and had a nonfunctional OmpK35, due to a frameshift mutation that truncated the protein at amino acid 42, common to K. pneumoniae ST258 (3). Association between mutations in ompK36 and elevated ceftazidime-avibactam MICs has been shown previously (4). However, T333N, found in one of the ß-sheet domains of the OmpK36 subunit, has not been described in K. pneumoniae; as such, further validation is required to confirm the role of the OmpK36 mutation in this isolate’s ceftazidime-avibactam resistance phenotype.

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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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September 22, 2019

Interaction between the microbiome and TP53 in human lung cancer.

Lung cancer is the leading cancer diagnosis worldwide and the number one cause of cancer deaths. Exposure to cigarette smoke, the primary risk factor in lung cancer, reduces epithelial barrier integrity and increases susceptibility to infections. Herein, we hypothesize that somatic mutations together with cigarette smoke generate a dysbiotic microbiota that is associated with lung carcinogenesis. Using lung tissue from 33 controls and 143 cancer cases, we conduct 16S ribosomal RNA (rRNA) bacterial gene sequencing, with RNA-sequencing data from lung cancer cases in The Cancer Genome Atlas serving as the validation cohort.Overall, we demonstrate a lower alpha diversity in normal lung as compared to non-tumor adjacent or tumor tissue. In squamous cell carcinoma specifically, a separate group of taxa are identified, in which Acidovorax is enriched in smokers. Acidovorax temporans is identified within tumor sections by fluorescent in situ hybridization and confirmed by two separate 16S rRNA strategies. Further, these taxa, including Acidovorax, exhibit higher abundance among the subset of squamous cell carcinoma cases with TP53 mutations, an association not seen in adenocarcinomas.The results of this comprehensive study show both microbiome-gene and microbiome-exposure interactions in squamous cell carcinoma lung cancer tissue. Specifically, tumors harboring TP53 mutations, which can impair epithelial function, have a unique bacterial consortium that is higher in relative abundance in smoking-associated tumors of this type. Given the significant need for clinical diagnostic tools in lung cancer, this study may provide novel biomarkers for early detection.

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September 22, 2019

Role of clinicogenomics in infectious disease diagnostics and public health microbiology.

Clinicogenomics is the exploitation of genome sequence data for diagnostic, therapeutic, and public health purposes. Central to this field is the high-throughput DNA sequencing of genomes and metagenomes. The role of clinicogenomics in infectious disease diagnostics and public health microbiology was the topic of discussion during a recent symposium (session 161) presented at the 115th general meeting of the American Society for Microbiology that was held in New Orleans, LA. What follows is a collection of the most salient and promising aspects from each presentation at the symposium. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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September 22, 2019

Recent developments in using advanced sequencing technologies for the genomic studies of lignin and cellulose degrading microorganisms.

Lignin is a complex polyphenyl aromatic compound which exists in tight associations with cellulose and hemicellulose to form plant primary and secondary cell wall. Lignocellulose is an abundant renewable biomaterial present on the earth. It has gained much attention in the scientific community in recent years because of its potential applications in bio-based industries. Microbial degradation of lignocellulose polymers was well studied in wood decaying fungi. Based on the plant materials they degrade these fungi were classified as white rot, brown rot and soft rot. However, some groups of bacteria belonging to the actinomycetes, a-proteobacteria and ß-proteobacteria were also found to be efficient in degrading lignocellulosic biomass but not well understood unlike the fungi. In this review we focus on recent advancements deployed for finding and understanding the lignocellulose degradation by microorganisms. Conventional molecular methods like sequencing 16s rRNA and Inter Transcribed Spacer (ITS) regions were used for identification and classification of microbes. Recent progression in genomics mainly next generation sequencing technologies made the whole genome sequencing of microbes possible in a great ease. The whole genome sequence studies reveals high quality information about genes and canonical pathways involved in the lignin and other cell wall components degradation.

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September 22, 2019

Discovery of enzymes for toluene synthesis from anoxic microbial communities.

Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, but the enzyme catalyzing this biochemically challenging reaction has never been identified. Here we report the toluene-producing enzyme PhdB, a glycyl radical enzyme of bacterial origin that catalyzes phenylacetate decarboxylation, and its cognate activating enzyme PhdA, a radical S-adenosylmethionine enzyme, discovered in two distinct anoxic microbial communities that produce toluene. The unconventional process of enzyme discovery from a complex microbial community (>300,000 genes), rather than from a microbial isolate, involved metagenomics- and metaproteomics-enabled biochemistry, as well as in vitro confirmation of activity with recombinant enzymes. This work expands the known catalytic range of glycyl radical enzymes (only seven reaction types had been characterized previously) and aromatic-hydrocarbon-producing enzymes, and will enable first-time biochemical synthesis of an aromatic fuel hydrocarbon from renewable resources, such as lignocellulosic biomass, rather than from petroleum.

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September 22, 2019

Characteristics of ARG-carrying plasmidome in the cultivable microbial community from wastewater treatment system under high oxytetracycline concentration.

Studies on antibiotic production wastewater have shown that even a single antibiotic can select for multidrug resistant bacteria in aquatic environments. It is speculated that plasmids are an important mechanism of multidrug resistance (MDR) under high concentrations of antibiotics. Herein, two metagenomic libraries were constructed with plasmid DNA extracted from cultivable microbial communities in a biological wastewater treatment reactor supplemented with 0 (CONTROL) or 25 mg/L of oxytetracycline (OTC-25). The OTC-25 plasmidome reads were assigned to 72 antibiotic resistance genes (ARGs) conferring resistance to 13 types of antibiotics. Dominant ARGs, encoding resistance to tetracycline, aminoglycoside, sulfonamide, and multidrug resistance genes, were enriched in the plasmidome under 25 mg/L of oxytetracycline. Furthermore, 17 contiguous multiple-ARG carrying contigs (carrying =?2 ARGs) were discovered in the OTC-25 plasmidome, whereas only nine were found in the CONTROL. Mapping of the OTC-25 plasmidome reads to completely sequenced plasmids revealed that the conjugative IncU resistance plasmid pFBAOT6 of Aeromonas caviae, carrying multidrug resistance transporter (pecM), tetracycline resistance genes (tetA, tetR), and transposase genes, might be a potential prevalent resistant plasmid in the OTC-25 plasmidome. Additionally, two novel resistant plasmids (containing contig C301682 carrying multidrug resistant operon mexCD-oprJ and contig C301632 carrying the tet36 and transposases genes) might also be potential prevalent resistant plasmids in the OTC-25 plasmidome. This study will be helpful to better understand the role of plasmids in the development of MDR in water environments under high antibiotic concentrations.

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September 22, 2019

Precise fecal microbiome of the herbivorous Tibetan antelope inhabiting high-altitude alpine plateau

The metataxonomic approach combining 16S rRNA gene amplicon sequencing using the PacBio technology with the application of the operational phylogenetic unit (OPU) approach, has been used to analyze the fecal microbial composition of the high-altitude and herbivorous Tibetan antelopes. The fecal samples of the antelope were collected in Hoh Xil National Nature Reserve, at an altitude over 4500 m, the largest depopulated zone in Qinghai-Tibetan Plateau, China, where non-native animals or humans may experience life-threatening acute mountain sickness. In total, 104 antelope fecal samples were enrolled in this study, and were clustered into 61,258 operational taxonomic units (OTUs) at an identity of 98.7% and affiliated with 757 OPUs, including 144 known species, 256 potentially new species, 103 potentially higher taxa within known lineages. In addition, 254 comprised sequences not affiliating with any known family, and the closest relatives were unclassified lineages of existing orders or classes. A total of 42 out of 757 OPUs conformed to the core fecal microbiome, of which four major lineages, namely, un-cultured Ruminococcaceae, Lachnospiraceae, Akkermansia and Christensenellaceae were associated with human health or longevity. The current study reveals that the fecal core microbiome of antelope is mainly composited of uncultured bacteria. The most abundant core taxa, namely, uncultured Ruminococcaceae, uncultured Akkermansia, uncultured Bacteroides, uncultured Christensenellaceae, uncultured Mollicutes, and uncultured Lachnospiraceae, may represent new bacterial candidates at high taxa levels, and several may have beneficial roles in health promotion or anti-intestinal dysbiosis. These organisms should be further isolated and evaluated for potential effect on human health and longevity.

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September 22, 2019

Diverse antibiotic resistance genes in dairy cow manure.

Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings. IMPORTANCE The increasing prevalence of antibiotic resistance among bacteria is one of the most intractable challenges in 21st-century public health. The origins of resistance are complex, and a better understanding of the impacts of antibiotics used on farms would produce a more robust platform for public policy. Microbiomes of farm animals are reservoirs of antibiotic resistance genes, which may affect distribution of antibiotic resistance genes in human pathogens. Previous studies have focused on antibiotic resistance genes in manures of animals subjected to intensive antibiotic use, such as pigs and chickens. Cow manure has received less attention, although it is commonly used in crop production. Here, we report the discovery of novel and diverse antibiotic resistance genes in the cow microbiome, demonstrating that it is a significant reservoir of antibiotic resistance genes. The genomic resource presented here lays the groundwork for understanding the dispersal of antibiotic resistance from the agroecosystem to other settings.

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September 22, 2019

Towards long-read metagenomics: complete assembly of three novel genomes from bacteria dependent on a diazotrophic cyanobacterium in a freshwater lake co-culture.

Here we report three complete bacterial genome assemblies from a PacBio shotgun metagenome of a co-culture from Upper Klamath Lake, OR. Genome annotations and culture conditions indicate these bacteria are dependent on carbon and nitrogen fixation from the cyanobacterium Aphanizomenon flos-aquae, whose genome was assembled to draft-quality. Due to their taxonomic novelty relative to previously sequenced bacteria, we have temporarily designated these bacteria as incertae sedis Hyphomonadaceae strain UKL13-1 (3,501,508 bp and 56.12% GC), incertae sedis Betaproteobacterium strain UKL13-2 (3,387,087 bp and 54.98% GC), and incertae sedis Bacteroidetes strain UKL13-3 (3,236,529 bp and 37.33% GC). Each genome consists of a single circular chromosome with no identified plasmids. When compared with binned Illumina assemblies of the same three genomes, there was ~7% discrepancy in total genome length. Gaps where Illumina assemblies broke were often due to repetitive elements. Within these missing sequences were essential genes and genes associated with a variety of functional categories. Annotated gene content reveals that both Proteobacteria are aerobic anoxygenic phototrophs, with Betaproteobacterium UKL13-2 potentially capable of phototrophic oxidation of sulfur compounds. Both proteobacterial genomes contain transporters suggesting they are scavenging fixed nitrogen from A. flos-aquae in the form of ammonium. Bacteroidetes UKL13-3 has few completely annotated biosynthetic pathways, and has a comparatively higher proportion of unannotated genes. The genomes were detected in only a few other freshwater metagenomes, suggesting that these bacteria are not ubiquitous in freshwater systems. Our results indicate that long-read sequencing is a viable method for sequencing dominant members from low-diversity microbial communities, and should be considered for environmental metagenomics when conditions meet these requirements.

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September 22, 2019

Diversified microbiota of meconium is affected by maternal diabetes status.

This study was aimed to assess the diversity of the meconium microbiome and determine if the bacterial community is affected by maternal diabetes status.The first intestinal discharge (meconium) was collected from 23 newborns stratified by maternal diabetes status: 4 mothers had pre-gestational type 2 diabetes mellitus (DM) including one mother with dizygotic twins, 5 developed gestational diabetes mellitus (GDM) and 13 had no diabetes. The meconium microbiome was profiled using multi-barcode 16S rRNA sequencing followed by taxonomic assignment and diversity analysis.All meconium samples were not sterile and contained diversified microbiota. Compared with adult feces, the meconium showed a lower species diversity, higher sample-to-sample variation, and enrichment of Proteobacteria and reduction of Bacteroidetes. Among the meconium samples, the taxonomy analyses suggested that the overall bacterial content significantly differed by maternal diabetes status, with the microbiome of the DM group showing higher alpha-diversity than that of no-diabetes or GDM groups. No global difference was found between babies delivered vaginally versus via Cesarean-section. Regression analysis showed that the most robust predictor for the meconium microbiota composition was the maternal diabetes status that preceded pregnancy. Specifically, Bacteroidetes (phyla) and Parabacteriodes (genus) were enriched in the meconium in the DM group compared to the no-diabetes group.Our study provides evidence that meconium contains diversified microbiota and is not affected by the mode of delivery. It also suggests that the meconium microbiome of infants born to mothers with DM is enriched for the same bacterial taxa as those reported in the fecal microbiome of adult DM patients.

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September 22, 2019

Genomic microdiversity of Bifidobacterium pseudocatenulatum underlying differential strain-level responses to dietary carbohydrate intervention.

The genomic basis of the response to dietary intervention of human gut beneficial bacteria remains elusive, which hinders precise manipulation of the microbiota for human health. After receiving a dietary intervention enriched with nondigestible carbohydrates for 105 days, a genetically obese child with Prader-Willi syndrome lost 18.4% of his body weight and showed significant improvement in his bioclinical parameters. We obtained five isolates (C1, C15, C55, C62, and C95) of one of the most abundantly promoted beneficial species, Bifidobacterium pseudocatenulatum, from a postintervention fecal sample. Intriguingly, these five B. pseudocatenulatum strains showed differential responses during the dietary intervention. Two strains were largely unaffected, while the other three were promoted to different extents by the changes in dietary carbohydrate resources. The differential responses of these strains were consistent with their functional clustering based on the COGs (Clusters of Orthologous Groups), including those involved with the ABC-type sugar transport systems, suggesting that the strain-specific genomic variations may have contributed to the niche adaption. Particularly, B. pseudocatenulatum C15, which had the most diverse types and highest gene copy numbers of carbohydrate-active enzymes targeting plant polysaccharides, had the highest abundance after the dietary intervention. These studies show the importance of understanding genomic diversity of specific members of the gut microbiota if precise nutrition approaches are to be realized.IMPORTANCE The manipulation of the gut microbiota via dietary approaches is a promising option for improving human health. Our findings showed differential responses of multiple B. pseudocatenulatum strains isolated from the same habitat to the dietary intervention, as well as strain-specific correlations with bioclinical parameters of the host. The comparative genomics revealed a genome-level microdiversity of related functional genes, which may have contributed to these differences. These results highlight the necessity of understanding strain-level differences if precise manipulation of gut microbiota through dietary approaches is to be realized. Copyright © 2017 Wu et al.

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