Learn how Single Molecule, Real-Time (SMRT) Sequencing and the Sequel IIe System and will accelerate your research by delivering highly accurate long reads to provide the most comprehensive view of genomes, transcriptomes and epigenomes.
The newer hierarchical genome assembly process (HGAP) performs de novo assembly using data from a single PacBio long insert library. To assess the benefits of this method, DNA from several Salmonella enterica serovars was isolated from a pure culture. Genome sequencing was performed using Pacific Biosciences RS sequencing technology. The HGAP process enabled us to close sixteen Salmonella subsp. enterica genomes and their associated mobile elements: The ten serotypes include: Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) S. Bareilly, S. Heidelberg, S. Cubana, S. Javiana and S. Typhimurium, S. Newport, S. Montevideo, S. Agona, and S. Tennessee. In addition,…
For comprehensive metabolic reconstructions and a resulting understanding of the pathways leading to natural products, it is desirable to obtain complete information about the genetic blueprint of the organisms used. Traditional Sanger and next-generation, short-read sequencing technologies have shortcomings with respect to read lengths and DNA-sequence context bias, leading to fragmented and incomplete genome information. The development of long-read, single molecule, real-time (SMRT) DNA sequencing from Pacific Biosciences, with >10,000 bp average read lengths and a lack of sequence context bias, now allows for the generation of complete genomes in a fully automated workflow. In addition to the genome sequence,…
In this PacBio User Group Meeting presentation, PacBio scientist Meredith Ashby shared several examples of analysis — from full-length 16S sequencing to shotgun sequencing — showing how SMRT Sequencing enables accurate representation for metagenomics and microbiome characterization, in some cases even without fully assembling genomes. New updates will provide users with a dedicated microbial assembly pipeline, optimized for all classes of bacteria, as well as increased multiplexing on the Sequel II System, now with 48 validated barcoded adapters. That throughput could reduce the cost of microbial analysis substantially.
Understanding interactions among plants and the complex communities of organisms living on, in and around them requires more than one experimental approach. A new method for de novo metagenome assembly, PacBio HiFi sequencing, has unique strengths for determining the functional capacity of metagenomes. With HiFi sequencing, the accuracy and median read length of unassembled data outperforms the quality metrics for many existing assemblies generated with other technologies, enabling cost-competitive recovery of full-length genes and operons even from rare species. When paired with the ability to close the genomes of even challenging isolates like Xanthomonas, the PacBio Sequel II System is…
To track stepwise changes in genetic diversity and antimicrobial resistance in rapidly evolving OXA-232-producing Klebsiella pneumoniae ST14, an emerging carbapenem-resistant high-risk clone, in clinical settings.Twenty-six K. pneumoniae ST14 isolates were collected by the Korean Nationwide Surveillance of Antimicrobial Resistance system over the course of 1 year. Isolates were subjected to whole-genome sequencing and MIC determinations using 33 antibiotics from 14 classes.Single-nucleotide polymorphism (SNP) typing identified 72 unique SNP sites spanning the chromosomes of the isolates, dividing them into three clusters (I, II and III). The initial isolate possessed two plasmids with 18 antibiotic-resistance genes, including blaOXA-232, and exhibited resistance to 11 antibiotic…
New drugs with novel mechanisms of resistance are desperately needed to address both community and nosocomial infections due to Gram-negative bacteria. One such potential target is LpxC, an essential enzyme that catalyzes the first committed step of Lipid A biosynthesis. Achaogen conducted an extensive research campaign to discover novel LpxC inhibitors with activity against Pseudomonas aeruginosa We report here the in vitro antibacterial activity and pharmacodynamics of ACHN-975, the only molecule from these efforts and the first ever LpxC inhibitor to be evaluated in Phase 1 clinical trials. In addition, we describe the profile of three additional LpxC inhibitors that…
Horizontal transfer of plasmids encoding antimicrobial-resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s the first CA-MRSA isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline and penicillin-resistance genes on plasmid pWBG753 (~30 kb). WA-5 and pWBG753 appeared only briefly in WA, however, fusidic-acid-resistance plasmids related to pWBG753 were also present in the first European CA-MRSA at the time. Here we characterized a 72-kb conjugative plasmid pWBG731 present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a…
Salmonella enterica serovar Dublin is a host-adapted serotype associated with typhoidal disease in cattle. While rare in humans, it usually causes severe illness, including bacteremia. In the United States, Salmonella Dublin has become one of the most multidrug-resistant (MDR) serotypes. To understand the genetic elements that are associated with virulence and resistance, we sequenced 61 isolates of Salmonella Dublin (49 from sick cattle and 12 from retail beef) using the Illumina MiSeq and closed 5 genomes using the PacBio sequencing platform. Genomic data of eight human isolates were also downloaded from NCBI (National Center for Biotechnology Information) for comparative analysis.…
Tigecycline is one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria1. Tigecycline resistance has sporadically occurred in recent years, primarily due to chromosome-encoding mechanisms, such as overexpression of efflux pumps and ribosome protection2,3. Here, we report the emergence of the plasmid-mediated mobile tigecycline resistance mechanism Tet(X4) in Escherichia coli isolates from China, which is capable of degrading all tetracyclines, including tigecycline and the US FDA newly approved eravacycline. The tet(X4)-harbouring IncQ1 plasmid is highly transferable, and can be successfully mobilized and stabilized in recipient clinical and laboratory strains of Enterobacteriaceae bacteria. It…
Infections caused by multi-drug resistant Acinetobacter spp. has aroused worldwide attention. With the increasing isolation of non-baumannii Acinetobacter, the nature of infection and resistance associated with them needs to be elaborated. This study aimed to analyze the characteristics of New Delhi Metallo-Beta-Lactamase-1 (NDM-1)-producing Acinetobacter haemolyticus (named sz1652) isolated from Shenzhen city, China.Antibiotic spectrum was analyzed after antimicrobial susceptibility test. Combined disk test (CDT) was used to detecting the metallo-beta-lactamases (MBLs). Transferability of carbapenem resistance was tested by filter mating experiments and plasmid transformation assays. Whole-genome sequencing (WGS) was performed using HiSeq 2000 and PacBio RS system.The A. haemolyticus strain sz1652…
This study describes the characterization of type 2 IncC plasmids pC-Ec20-KPC and pC-Ec2-KPC, carrying blaKPC-2 gene, from two multiresistant E. coli recovered in the University Hospital of Larissa, in 2018.Escherichia coli, Ec-2Lar and Ec-20Lar, were recovered from rectal swabs from two patients, during the monthly surveillance cultures. Transfer experiments by conjugation were carried out with E. coli recipients. blaKPC-carrying plasmids were characterized by S1 profiling. Isolates were typed by MLST. Whole bacterial genome was sequenced using the Sequel platform.Both E. coli isolates, belonging to ST648, transferred the blaKPC-2 to E. coli A15 laboratory strain by conjugation. Plasmid analysis revealed that…
Bacteremia can be caused by Acinetobacter baumannii with clinical manifestations ranging from transient bacteremia to septic shock. Extensively drug-resistant A. baumannii (XDRAB) strains producing the New Delhi metallo-ß-lactamase, which confers resistance to all ß-lactams including carbapenems, have emerged and infected patients suffer increased mortality, morbidity and length of hospitalization. The lack of new antimicrobials led to a renewed interest into phage therapy, the so-called forgotten cure. Accordingly, we tested new lytic bacteriophages in a Galleria mellonella and a mouse model of XDRAB-induced bacteremia.Galleria mellonella were challenged with 5.105 CFU of the XDRAB strain FER. Phages vB_AbaM_3054 and vB_AbaM_3090 were administrated…
The aim of this study was to detect the transferable oxazolidinone resistance determinants (cfr, optrA and poxtA) in E. faecalis and E. faecium of swine origin in Sichuan Province, China.A total of 158 enterococci strains (93 E. faecalis and 65 E. faecium) isolated from 25 large-scale swine farms were screened for the presence of cfr, optrA and poxtA by PCR. The genetic environments of cfr, optrA and poxtA were characterized by whole genome sequencing. Transfer of oxazolidinone resistance determinants was determined by conjugation or electrotransformation experiments.The transferable oxazolidinone resistance determinants, cfr, optrA and poxtA, were detected in zero, six, and…
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…