To start Day 1 of the PacBio User Group Meeting, Jonas Korlach, PacBio CSO, provides an update on the latest releases and performance metrics for the Sequel II System. The longest reads generated on this system with the SMRT Cell 8M now go beyond 175,000 bases, while maintaining extremely high accuracy. HiFi mode, for example, uses circular consensus sequencing to achieve accuracy of Q40 or even Q50.
In this introductory talk to our PAG 2020 workshop, PacBio Chief Scientific Officer Jonas Korlach presents the evolution of Single Molecule, Real-Time (SMRT) Sequencing technology over the past decade and highlights recent developments, including the Sequel II System performance and reliability
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…
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…
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…
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…
This study aimed to investigate the spread of the blaKPC-2 gene among Klebsiella pneumoniae and to illustrate the mechanism of dissemination of KPC-producing K. pneumoniae (KPC-Kp) ST11 in China.A total of 354 K. pneumoniae isolates were collected from four hospitals in China and were characterized by Multilocus sequence typing (MLST). Mobile genetic elements (MGEs) and pulsed-field gel electrophoresis (PFGE) analysis were used to identify the subtypes of K. pneumoniae ST11. PCR-based amplification and sequencing were performed to analyze Tn1721 transposons and IncFII-like plasmids. Electroporation experiments and whole-genome sequencing (WGS) analysis were used to reveal the genetic environment of the blaKPC-2…
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…
Enteropathogenic Escherichia coli (EPEC) is a leading cause of moderate to severe diarrhea among young children in developing countries, and EPEC isolates can be subdivided into two groups. Typical EPEC (tEPEC) bacteria are characterized by the presence of both the locus of enterocyte effacement (LEE) and the plasmid-encoded bundle-forming pilus (BFP), which are involved in adherence and translocation of type III effectors into the host cells. Atypical EPEC (aEPEC) bacteria also contain the LEE but lack the BFP. In the current report, we describe the complete genome of outbreak-associated aEPEC isolate E110019, which carries four plasmids. Comparative genomic analysis demonstrated…
Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum ß-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed…
Four Enterobacteriaceae clinical isolates bearing mcr-1 gene-harboring plasmids were characterized. All isolates demonstrated the ability to transfer colistin resistance to Escherichia coli; plasmids were stable in conjugants after multiple passages on nonselective media. mcr-1 was located on an IncX4 (n?=?3) or IncN (n?=?1) plasmid. The IncN plasmid harbored 13 additional antimicrobial resistance genes. Results indicate that the mcr-1-bearing plasmids in this study were highly transferable in vitro and stable in the recipients.This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
In 2011, VIM-1-producing Salmonella enterica serovar Infantis and Escherichia coli were isolated for the first time in four German livestock farms. In 2015/2016, highly related isolates were identified in German pig production. This raised the issue of potential reservoirs for these isolates, the relation of their mobile genetic elements, and potential links between the different affected farms/facilities. In a piglet-producing farm suspicious for being linked to some blaVIM-1 findings in Germany, fecal and environmental samples were examined for the presence of carbapenemase-producing Enterobacteriaceae and Salmonella spp. Newly discovered isolates were subjected to Illumina whole-genome sequencing (WGS) and S1 pulsed-field gel…
The spread of carbapenemase-producing Enterobacteriaceae (CPE), contributing to widespread carbapenem resistance, has become a global concern. However, the specific dissemination patterns of carbapenemase genes have not been intensively investigated in developing countries, including Myanmar, where NDM-type carbapenemases are spreading in clinical settings. In the present study, we phenotypically and genetically characterized 91 CPE isolates obtained from clinical (n = 77) and environmental (n = 14) samples in Yangon, Myanmar. We determined the dissemination of plasmids harboring genes encoding NDM-1 and its variants using whole-genome sequencing and plasmid analysis. IncFII plasmids harboring blaNDM-5 and IncX3 plasmids harboring blaNDM-4 or blaNDM-7 were…
New Delhi metallo-beta-lactamases (NDMs) are an uncommon but emerging cause of carbapenem resistance in the United States. Genomic factors promoting their domestic spread remain poorly characterized. A prospective genomic surveillance program among Boston-area hospitals identified multiple new occurrences of NDM-carrying strains of Escherichia coli and Enterobacter cloacae complex in inpatient and outpatient settings, representing the first occurrences of NDM-mediated resistance since initiating genomic surveillance in 2011. Cases included domestic patients with no international exposures. PacBio sequencing of isolates identified strain characteristics, resistance genes, and the complement of mobile vectors mediating spread. Analyses revealed a common 3,114-bp region containing the blaNDM…