June 1, 2021  |  

Getting the most out of your PacBio libraries with size selection.

PacBio RS II sequencing chemistries provide read lengths beyond 20 kb with high consensus accuracy. The long read lengths of P4-C2 chemistry and demonstrated consensus accuracy of 99.999% are ideal for applications such as de novo assembly, targeted sequencing and isoform sequencing. The recently launched P5-C3 chemistry generates even longer reads with N50 often >10,000 bp, making it the best choice for scaffolding and spanning structural rearrangements. With these chemistry advances, PacBio’s read length performance is now primarily determined by the SMRTbell library itself. Size selection of a high-quality, sheared 20 kb library using the BluePippin™ System has been demonstrated to increase the N50 read length by as much as 5 kb with C3 chemistry. BluePippin size selection or a more stringent AMPure® PB selection cutoff can be used to recover long fragments from degraded genomic material. The selection of chemistries, P4-C2 versus P5-C3, is highly dependent on the final size distribution of the SMRTbell library and experimental goals. PacBio’s long read lengths also allow for the sequencing of full-length cDNA libraries at single-molecule resolution. However, longer transcripts are difficult to detect due to lower abundance, amplification bias, and preferential loading of smaller SMRTbell constructs. Without size selection, most sequenced transcripts are 1-1.5 kb. Size selection dramatically increases the number of transcripts >1.5 kb, and is essential for >3 kb transcripts.


April 21, 2020  |  

The use of Online Tools for Antimicrobial Resistance Prediction by Whole Genome Sequencing in MRSA and VRE.

The antimicrobial resistance (AMR) crisis represents a serious threat to public health and has resulted in concentrated efforts to accelerate development of rapid molecular diagnostics for AMR. In combination with publicly-available web-based AMR databases, whole genome sequencing (WGS) offers the capacity for rapid detection of antibiotic resistance genes. Here we studied the concordance between WGS-based resistance prediction and phenotypic susceptibility testing results for methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE) clinical isolates using publicly-available tools and databases.Clinical isolates prospectively collected at the University of Pittsburgh Medical Center between December 2016 and December 2017 underwent WGS. Antibiotic resistance gene content was assessed from assembled genomes by BLASTn search of online databases. Concordance between WGS-predicted resistance profile and phenotypic susceptibility as well as sensitivity, specificity, positive and negative predictive values (NPV, PPV) were calculated for each antibiotic/organism combination, using the phenotypic results as the gold standard.Phenotypic susceptibility testing and WGS results were available for 1242 isolate/antibiotic combinations. Overall concordance was 99.3% with a sensitivity, specificity, PPV, NPV of 98.7% (95% CI, 97.2-99.5%), 99.6% (95 % CI, 98.8-99.9%), 99.3% (95% CI, 98.0-99.8%), 99.2% (95% CI, 98.3-99.7%), respectively. Additional identification of point mutations in housekeeping genes increased the concordance to 99.4% and the sensitivity to 99.3% (95% CI, 98.2-99.8%) and NPV to 99.4% (95% CI, 98.4-99.8%).WGS can be used as a reliable predicator of phenotypic resistance for both MRSA and VRE using readily-available online tools.Copyright © 2019. Published by Elsevier Ltd.


April 21, 2020  |  

Mce3R Stress-Resistance Pathway Is Vulnerable to Small-Molecule Targeting That Improves Tuberculosis Drug Activities.

One-third of the world’s population carries Mycobacterium tuberculosis ( Mtb), the infectious agent that causes tuberculosis (TB), and every 17 s someone dies of TB. After infection, Mtb can live dormant for decades in a granuloma structure arising from the host immune response, and cholesterol is important for this persistence of Mtb. Current treatments require long-duration drug regimens with many associated toxicities, which are compounded by the high doses required. We phenotypically screened 35 6-azasteroid analogues against Mtb and found that, at low micromolar concentrations, a subset of the analogues sensitized Mtb to multiple TB drugs. Two analogues were selected for further study to characterize the bactericidal activity of bedaquiline and isoniazid under normoxic and low-oxygen conditions. These two 6-azasteroids showed strong synergy with bedaquiline (fractional inhibitory concentration index = 0.21, bedaquiline minimal inhibitory concentration = 16 nM at 1 µM 6-azasteroid). The rate at which spontaneous resistance to one of the 6-azasteroids arose in the presence of bedaquiline was approximately 10-9, and the 6-azasteroid-resistant mutants retained their isoniazid and bedaquiline sensitivity. Genes in the cholesterol-regulated Mce3R regulon were required for 6-azasteroid activity, whereas genes in the cholesterol catabolism pathway were not. Expression of a subset of Mce3R genes was down-regulated upon 6-azasteroid treatment. The Mce3R regulon is implicated in stress resistance and is absent in saprophytic mycobacteria. This regulon encodes a cholesterol-regulated stress-resistance pathway that we conclude is important for pathogenesis and contributes to drug tolerance, and this pathway is vulnerable to small-molecule targeting in live mycobacteria.


April 21, 2020  |  

Evolution and global transmission of a multidrug-resistant, community-associated MRSA lineage from the Indian subcontinent

The evolution and global transmission of antimicrobial resistance has been well documented in Gram-negative bacteria and healthcare-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. Here, we trace the recent origins and global spread of a multidrug resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data shows that the clone emerged on the Indian subcontinent in the early 1970s and disseminated rapidly in the 1990s. Short-term outbreaks in community and healthcare settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the divergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional healthcare-associated clones with the epidemiological transmission of community-associated MRSA. Our study demonstrates the importance of whole genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere.Importance The Bengal Bay clone (ST772) is a community-acquired and multidrug-resistant Staphylococcus aureus lineage first isolated from Bangladesh and India in 2004. In this study, we show that the Bengal Bay clone emerged from a virulent progenitor circulating on the Indian subcontinent. Its subsequent global transmission was associated with travel or family contact in the region. ST772 progressively acquired specific resistance elements at limited cost to its fitness and continues to be exported globally resulting in small-scale community and healthcare outbreaks. The Bengal Bay clone therefore combines the virulence potential and epidemiology of community-associated clones with the multidrug-resistance of healthcare-associated S. aureus lineages. This study demonstrates the importance of whole genome sequencing for the surveillance of highly antibiotic resistant pathogens, which may emerge in the community setting of regions with poor antibiotic stewardship and rapidly spread into hospitals and communities across the world.


April 21, 2020  |  

The replication-competent HIV-1 latent reservoir is primarily established near the time of therapy initiation.

Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4+ T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting peripheral CD4+ T cells from nine HIV-positive women on therapy to viral sequences circulating in blood collected longitudinally before therapy. We found that, on average, 71% of the unique viruses induced from the post-therapy latent reservoir were most genetically similar to viruses replicating just before ART initiation. This proportion is far greater than would be expected if the reservoir formed continuously and was always long lived. We conclude that ART alters the host environment in a way that allows the formation or stabilization of most of the long-lived latent HIV-1 reservoir, which points to new strategies targeted at limiting the formation of the reservoir around the time of therapy initiation.Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.


April 21, 2020  |  

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens.

Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases.IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

The ADEP Biosynthetic Gene Cluster in Streptomyces hawaiiensis NRRL 15010 Reveals an Accessory clpP Gene as a Novel Antibiotic Resistance Factor.

The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpPADEP) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpPADEP in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant.IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpPADEP) as an ADEP resistance gene. ClpPADEP constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Mitochondrial DNA Variants in Patients with Liver Injury Due to Anti-Tuberculosis Drugs.

Hepatotoxicity is the most severe adverse effect of anti-tuberculosis therapy. Isoniazid’s metabolite hydrazine is a mitochondrial complex II inhibitor. We hypothesized that mitochondrial DNA variants are risk factors for drug-induced liver injury (DILI) due to isoniazid, rifampicin or pyrazinamide.We obtained peripheral blood from tuberculosis (TB) patients before anti-TB therapy. A total of 38 patients developed DILI due to anti-TB drugs. We selected 38 patients with TB but without DILI as controls. Next-generation sequencing detected point mutations in the mitochondrial DNA genome. DILI was defined as ALT =5 times the upper limit of normal (ULN), or ALT =3 times the ULN with total bilirubin =2 times the ULN.In 38 patients with DILI, the causative drug was isoniazid in eight, rifampicin in 14 and pyrazinamide in 16. Patients with isoniazid-induced liver injury had more variants in complex I’s NADH subunit 5 and 1 genes, more nonsynonymous mutations in NADH subunit 5, and a higher ratio of nonsynonymous to total substitutions. Patients with rifampicin- or pyrazinamide-induced liver injury had no association with mitochondrial DNA variants.Variants in complex I’s subunit 1 and 5 genes might affect respiratory chain function and predispose isoniazid-induced liver injury when exposed to hydrazine, a metabolite of isoniazid and a complex II inhibitor.


April 21, 2020  |  

Whole-genome sequence of the oriental lung fluke Paragonimus westermani.

Foodborne infections caused by lung flukes of the genus Paragonimus are a significant and widespread public health problem in tropical areas. Approximately 50 Paragonimus species have been reported to infect animals and humans, but Paragonimus westermani is responsible for the bulk of human disease. Despite their medical and economic importance, no genome sequence for any Paragonimus species is available.We sequenced and assembled the genome of P. westermani, which is among the largest of the known pathogen genomes with an estimated size of 1.1 Gb. A 922.8 Mb genome assembly was generated from Illumina and Pacific Biosciences (PacBio) sequence data, covering 84% of the estimated genome size. The genome has a high proportion (45%) of repeat-derived DNA, particularly of the long interspersed element and long terminal repeat subtypes, and the expansion of these elements may explain some of the large size. We predicted 12,852 protein coding genes, showing a high level of conservation with related trematode species. The majority of proteins (80%) had homologs in the human liver fluke Opisthorchis viverrini, with an average sequence identity of 64.1%. Assembly of the P. westermani mitochondrial genome from long PacBio reads resulted in a single high-quality circularized 20.6 kb contig. The contig harbored a 6.9 kb region of non-coding repetitive DNA comprised of three distinct repeat units. Our results suggest that the region is highly polymorphic in P. westermani, possibly even within single worm isolates.The generated assembly represents the first Paragonimus genome sequence and will facilitate future molecular studies of this important, but neglected, parasite group.


April 21, 2020  |  

Genome-Wide Screening for Enteric Colonization Factors in Carbapenem-Resistant ST258 Klebsiella pneumoniae.

A diverse, antibiotic-naive microbiota prevents highly antibiotic-resistant microbes, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp), from achieving dense colonization of the intestinal lumen. Antibiotic-mediated destruction of the microbiota leads to expansion of CR-Kp in the gut, markedly increasing the risk of bacteremia in vulnerable patients. While preventing dense colonization represents a rational approach to reduce intra- and interpatient dissemination of CR-Kp, little is known about pathogen-associated factors that enable dense growth and persistence in the intestinal lumen. To identify genetic factors essential for dense colonization of the gut by CR-Kp, we constructed a highly saturated transposon mutant library with >150,000 unique mutations in an ST258 strain of CR-Kp and screened for in vitro growth and in vivo intestinal colonization in antibiotic-treated mice. Stochastic and partially reversible fluctuations in the representation of different mutations during dense colonization revealed the dynamic nature of intestinal microbial populations. We identified genes that are crucial for early and late stages of dense gut colonization and confirmed their role by testing isogenic mutants in in vivo competition assays with wild-type CR-Kp Screening of the transposon library also identified mutations that enhanced in vivo CR-Kp growth. These newly identified colonization factors may provide novel therapeutic opportunities to reduce intestinal colonization by CR-KpIMPORTANCEKlebsiella pneumoniae is a common cause of bloodstream infections in immunocompromised and hospitalized patients, and over the last 2 decades, some strains have acquired resistance to nearly all available antibiotics, including broad-spectrum carbapenems. The U.S. Centers for Disease Control and Prevention has listed carbapenem-resistant K. pneumoniae (CR-Kp) as an urgent public health threat. Dense colonization of the intestine by CR-Kp and other antibiotic-resistant bacteria is associated with an increased risk of bacteremia. Reducing the density of gut colonization by CR-Kp is likely to reduce their transmission from patient to patient in health care facilities as well as systemic infections. How CR-Kp expands and persists in the gut lumen, however, is poorly understood. Herein, we generated a highly saturated mutant library in a multidrug-resistant K. pneumoniae strain and identified genetic factors that are associated with dense gut colonization by K. pneumoniae This study sheds light on host colonization by K. pneumoniae and identifies potential colonization factors that contribute to high-density persistence of K. pneumoniae in the intestine. Copyright © 2019 Jung et al.


April 21, 2020  |  

Genomic characterization of Nocardia seriolae strains isolated from diseased fish.

Members of the genus Nocardia are widespread in diverse environments; a wide range of Nocardia species are known to cause nocardiosis in several animals, including cat, dog, fish, and humans. Of the pathogenic Nocardia species, N. seriolae is known to cause disease in cultured fish, resulting in major economic loss. We isolated two N. seriolae strains, CK-14008 and EM15050, from diseased fish and sequenced their genomes using the PacBio sequencing platform. To identify their genomic features, we compared their genomes with those of other Nocardia species. Phylogenetic analysis showed that N. seriolae shares a common ancestor with a putative human pathogenic Nocardia species. Moreover, N. seriolae strains were phylogenetically divided into four clusters according to host fish families. Through genome comparison, we observed that the putative pathogenic Nocardia strains had additional genes for iron acquisition. Dozens of antibiotic resistance genes were detected in the genomes of N. seriolae strains; most of the antibiotics were involved in the inhibition of the biosynthesis of proteins or cell walls. Our results demonstrated the virulence features and antibiotic resistance of fish pathogenic N. seriolae strains at the genomic level. These results may be useful to develop strategies for the prevention of fish nocardiosis. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


April 21, 2020  |  

Genome mining identifies cepacin as a plant-protective metabolite of the biopesticidal bacterium Burkholderia ambifaria.

Beneficial microorganisms are widely used in agriculture for control of plant pathogens, but a lack of efficacy and safety information has limited the exploitation of multiple promising biopesticides. We applied phylogeny-led genome mining, metabolite analyses and biological control assays to define the efficacy of Burkholderia ambifaria, a naturally beneficial bacterium with proven biocontrol properties but potential pathogenic risk. A panel of 64 B.?ambifaria strains demonstrated significant antimicrobial activity against priority plant pathogens. Genome sequencing, specialized metabolite biosynthetic gene cluster mining and metabolite analysis revealed an armoury of known and unknown pathways within B.?ambifaria. The biosynthetic gene cluster responsible for the production of the metabolite cepacin was identified and directly shown to mediate protection of germinating crops against Pythium damping-off disease. B.?ambifaria maintained biopesticidal protection and overall fitness in the soil after deletion of its third replicon, a non-essential plasmid associated with virulence in Burkholderia?cepacia complex bacteria. Removal of the third replicon reduced B.?ambifaria persistence in a murine respiratory infection model. Here, we show that by using interdisciplinary phylogenomic, metabolomic and functional approaches, the mode of action of natural biological control agents related to pathogens can be systematically established to facilitate their future exploitation.


April 21, 2020  |  

Human contamination in bacterial genomes has created thousands of spurious proteins.

Contaminant sequences that appear in published genomes can cause numerous problems for downstream analyses, particularly for evolutionary studies and metagenomics projects. Our large-scale scan of complete and draft bacterial and archaeal genomes in the NCBI RefSeq database reveals that 2250 genomes are contaminated by human sequence. The contaminant sequences derive primarily from high-copy human repeat regions, which themselves are not adequately represented in the current human reference genome, GRCh38. The absence of the sequences from the human assembly offers a likely explanation for their presence in bacterial assemblies. In some cases, the contaminating contigs have been erroneously annotated as containing protein-coding sequences, which over time have propagated to create spurious protein “families” across multiple prokaryotic and eukaryotic genomes. As a result, 3437 spurious protein entries are currently present in the widely used nr and TrEMBL protein databases. We report here an extensive list of contaminant sequences in bacterial genome assemblies and the proteins associated with them. We found that nearly all contaminants occurred in small contigs in draft genomes, which suggests that filtering out small contigs from draft genome assemblies may mitigate the issue of contamination while still keeping nearly all of the genuine genomic sequences. © 2019 Breitwieser et al.; Published by Cold Spring Harbor Laboratory Press.


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