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Auto Tag: Klebsiella pneumoniae

September 22, 2019  |  

SMRT-Cappable-seq reveals complex operon variants in bacteria.

Current methods for genome-wide analysis of gene expression require fragmentation of original transcripts into small fragments for short-read sequencing. In bacteria, the resulting fragmented information hides operon complexity. Additionally, in vivo processing of transcripts confounds the accurate identification of the 5′ and 3′ ends of operons. Here we develop a methodology called SMRT-Cappable-seq that combines the isolation of un-fragmented primary transcripts with single-molecule long read sequencing. Applied to E. coli, this technology results in an accurate definition of the transcriptome with 34% of known operons from RegulonDB being extended by at least one gene. Furthermore, 40% of transcription termination sites have read-through that alters the gene content of the operons. As a result, most of the bacterial genes are present in multiple operon variants reminiscent of eukaryotic splicing. By providing such granularity in the operon structure, this study represents an important resource for the study of prokaryotic gene network and regulation.

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September 22, 2019  |  

Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation.

Shotgun metagenomics methods enable characterization of microbial communities in human microbiome and environmental samples. Assembly of metagenome sequences does not output whole genomes, so computational binning methods have been developed to cluster sequences into genome ‘bins’. These methods exploit sequence composition, species abundance, or chromosome organization but cannot fully distinguish closely related species and strains. We present a binning method that incorporates bacterial DNA methylation signatures, which are detected using single-molecule real-time sequencing. Our method takes advantage of these endogenous epigenetic barcodes to resolve individual reads and assembled contigs into species- and strain-level bins. We validate our method using synthetic and real microbiome sequences. In addition to genome binning, we show that our method links plasmids and other mobile genetic elements to their host species in a real microbiome sample. Incorporation of DNA methylation information into shotgun metagenomics analyses will complement existing methods to enable more accurate sequence binning.

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September 22, 2019  |  

Clinical PathoScope: rapid alignment and filtration for accurate pathogen identification in clinical samples using unassembled sequencing data.

The use of sequencing technologies to investigate the microbiome of a sample can positively impact patient healthcare by providing therapeutic targets for personalized disease treatment. However, these samples contain genomic sequences from various sources that complicate the identification of pathogens.Here we present Clinical PathoScope, a pipeline to rapidly and accurately remove host contamination, isolate microbial reads, and identify potential disease-causing pathogens. We have accomplished three essential tasks in the development of Clinical PathoScope. First, we developed an optimized framework for pathogen identification using a computational subtraction methodology in concordance with read trimming and ambiguous read reassignment. Second, we have demonstrated the ability of our approach to identify multiple pathogens in a single clinical sample, accurately identify pathogens at the subspecies level, and determine the nearest phylogenetic neighbor of novel or highly mutated pathogens using real clinical sequencing data. Finally, we have shown that Clinical PathoScope outperforms previously published pathogen identification methods with regard to computational speed, sensitivity, and specificity.Clinical PathoScope is the only pathogen identification method currently available that can identify multiple pathogens from mixed samples and distinguish between very closely related species and strains in samples with very few reads per pathogen. Furthermore, Clinical PathoScope does not rely on genome assembly and thus can more rapidly complete the analysis of a clinical sample when compared with current assembly-based methods. Clinical PathoScope is freely available at: http://sourceforge.net/projects/pathoscope/.

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September 22, 2019  |  

The methylome of the gut microbiome: disparate Dam methylation patterns in intestinal Bacteroides dorei

Despite the large interest in the human microbiome in recent years, there are no reports of bacterial DNA methylation in the microbiome. Here metagenomic sequencing using the Pacific Biosciences platform allowed for rapid identification of bacterial GATC methylation status of a bacterial species in human stool samples. For this work, two stool samples were chosen that were dominated by a single species, Bacteroides dorei. Based on 16S rRNA analysis, this species represented over 45% of the bacteria present in these two samples. The B. dorei genome sequence from these samples was determined and the GATC methylation sites mapped. The Bacteroides dorei genome from one subject lacked any GATC methylation and lacked the DNA adenine methyltransferase genes. In contrast, B. dorei from another subject contained 20,551 methylated GATC sites. Of the 4970 open reading frames identified in the GATC methylated B. dorei genome, 3184 genes were methylated as well as 1735 GATC methylations in intergenic regions. These results suggest that DNA methylation patterns are important to consider in multi-omic analyses of microbiome samples seeking to discover the diversity of bacterial functions and may differ between disease states.

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September 22, 2019  |  

Identification of Burkholderia fungorum in the urine of an individual with spinal cord injury and augmentation cystoplasty using 16S sequencing: copathogen or innocent bystander?

People with neuropathic bladder (NB) secondary to spinal cord injury (SCI) are at risk for multiple genitourinary complications, the most frequent of which is urinary tract infection (UTI). Despite the high frequency with which UTI occurs, our understanding of the role of urinary microbes in health and disease is limited. In this paper, we present the first prospective case study integrating symptom reporting, urinalysis, urine cultivation, and 16S ribosomal ribonucleic acid (rRNA) sequencing of the urine microbiome.A 55-year-old male with NB secondary to SCI contributed 12 urine samples over an 8-month period during asymptomatic, symptomatic, and postantibiotic periods. All bacteria identified on culture were present on 16S rRNA sequencing, however, 16S rRNA sequencing revealed the presence of bacteria not isolated on culture. In particular, Burkholderia fungorum was present in three samples during both asymptomatic and symptomatic periods. White blood cells of =5-10/high power field and leukocyte esterase =2 on urinalysis was associated with the presence of symptoms.In this patient, there was a predominance of pathogenic bacteria and a lack of putative probiotic bacteria during both symptomatic and asymptomatic states. Urinalysis-defined inflammatory markers were present to a greater extent during symptomatic periods compared to the asymptomatic state, which may underscore a role for urinalysis or other inflammatory markers in differentiating asymptomatic bacteriuria from UTI in patients with NB. The finding of potentially pathogenic bacteria identified by sequencing but not cultivation, suggests a need for greater understanding of the relationships amongst bacterial species in the bacteriuric neuropathic bladder.

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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  |  

Composition and pathogenic potential of a microbial bioremediation product used for crude oil degradation.

A microbial bioremediation product (MBP) used for large-scale oil degradation was investigated for microbial constituents and possible pathogenicity. Aerobic growth on various media yielded >108 colonies mL-1. Full-length 16S rDNA sequencing and fatty acid profiling from morphologically distinct colonies revealed =13 distinct genera. Full-length 16S rDNA library sequencing, by either Sanger or long-read PacBio technology, suggested that up to 21% of the MBP was composed of Arcobacter. Other high abundance microbial constituents (>6%) included the genera Proteus, Enterococcus, Dysgonomonas and several genera in the order Bacteroidales. The MBP was most susceptible to ciprofloxacin, doxycycline, gentamicin, and meropenam. MBP exposure of human HT29 and A549 cells caused significant cytotoxicity, and bacterial growth and adherence. An acellular MBP filtrate was also cytotoxic to HT29, but not A549. Both MBP and filtrate exposures elevated the neutrophil chemoattractant IL-8. In endotracheal murine exposures, bacterial pulmonary clearance was complete after one-week. Elevation of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-a, and chemokines KC and MCP-1 occurred between 2h and 48h post-exposure, followed by restoration to baseline levels at 96h. Cytokine/chemokine signalling was accompanied by elevated blood neutrophils and monocytes at 4h and 48h, respectively. Peripheral acute phase response markers were maximal at 24h. All indicators examined returned to baseline values by 168h. In contrast to HT29, but similar to A549 observations, MBP filtrate did not induce significant murine effects with the indicators examined. The results demonstrated the potentially complex nature of MBPs and transient immunological effects during exposure. Products containing microbes should be scrutinized for pathogenic components and subjected to characterisation and quality validation prior to commercial release.

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September 22, 2019  |  

Next-generation sequencing for pathogen detection and identification

Over the past decade, the field of genomics has seen such drastic improvements in sequencing chemistries that high-throughput sequencing, or next-generation sequencing (NGS), is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, clinical use of NGS has been mostly focused on the human genome, for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples whereby NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, ability to detect fastidious or non-culturable organisms, and ability to detect co-infections. One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. We discuss these advantages and challenges in the context of recently described research studies.

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September 22, 2019  |  

A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome.

The initiating nucleotide found at the 5′ end of primary transcripts has a distinctive triphosphorylated end that distinguishes these transcripts from all other RNA species. Recognizing this distinction is key to deconvoluting the primary transcriptome from the plethora of processed transcripts that confound analysis of the transcriptome. The currently available methods do not use targeted enrichment for the 5’end of primary transcripts, but rather attempt to deplete non-targeted RNA.We developed a method, Cappable-seq, for directly enriching for the 5′ end of primary transcripts and enabling determination of transcription start sites at single base resolution. This is achieved by enzymatically modifying the 5′ triphosphorylated end of RNA with a selectable tag. We first applied Cappable-seq to E. coli, achieving up to 50 fold enrichment of primary transcripts and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. We also applied Cappable-seq to a mouse cecum sample and identified TSS in a microbiome.Cappable-seq allows for the first time the capture of the 5′ end of primary transcripts. This enables a unique robust TSS determination in bacteria and microbiomes.  In addition to and beyond TSS determination, Cappable-seq depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per transcript enabling digital profiling of gene expression in any microbiome.

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September 22, 2019  |  

Metataxonomic and metagenomic approaches vs. culture-based techniques for clinical pathology.

Diagnoses that are both timely and accurate are critically important for patients with life-threatening or drug resistant infections. Technological improvements in High-Throughput Sequencing (HTS) have led to its use in pathogen detection and its application in clinical diagnoses of infectious diseases. The present study compares two HTS methods, 16S rRNA marker gene sequencing (metataxonomics) and whole metagenomic shotgun sequencing (metagenomics), in their respective abilities to match the same diagnosis as traditional culture methods (culture inference) for patients with ventilator associated pneumonia (VAP). The metagenomic analysis was able to produce the same diagnosis as culture methods at the species-level for five of the six samples, while the metataxonomic analysis was only able to produce results with the same species-level identification as culture for two of the six samples. These results indicate that metagenomic analyses have the accuracy needed for a clinical diagnostic tool, but full integration in diagnostic protocols is contingent on technological improvements to decrease turnaround time and lower costs.

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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  |  

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  |  

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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