Dr. Wenger gives attendees an update on PacBio’s long-read sequencing and variant detection capabilities on the Sequel II System and shares recommendations on how to design your own study using…
In this ASHG 2020 PacBio Workshop Emily Farrow of Children’s Mercy Kansas City, shares how the incorporation of long-read sequencing into the Genomic Answers for Kids research study is increasing…
In this SMRT Leiden 2020 Online Virtual Event presentation Pedro Oliveira of Mount Sinai shares his research on Clostridioides – a leading cause of nosocomial-acquired diarrhea and colitis across the…
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 classes. Four other plasmids containing 12 different resistance genes, including blaCTX-M-15 and strA/B, were introduced over time, providing additional resistance to aztreonam and streptomycin. Moreover, chromosomal integration of insertion sequence Ecp1-blaCTX-M-15 mediated the inactivation of mgrB responsible for colistin resistance in four isolates from cluster III. To the best of our knowledge, this is the first description of K. pneumoniae ST14 resistant to both carbapenem and colistin in South Korea. Furthermore, although some acquired genes were lost over time, the retention of 12 resistance genes and inactivation of mgrB provided resistance to 13 classes of antibiotics.We describe stepwise changes in OXA-232-producing K. pneumoniae ST14 in vivo over time in terms of antimicrobial resistance. Our findings contribute to our understanding of the evolution of emerging high-risk K. pneumoniae clones and provide reference data for future outbreaks.Copyright © 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Pacific Biosciences’ SMRT sequencing method was used to extend the sequence of HLA-A*02:13. © 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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 were identified as potential lead molecules. These efforts did not produce an additional development candidate with a sufficiently large therapeutic window and the program was subsequently terminated.Copyright © 2019 American Society for Microbiology.
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 pWBG749-family conjugative plasmid. pWBG731 carried mupirocin, trimethoprim, cadmium and penicillin-resistance genes. The stepwise evolution of pWBG731 likely occurred through the combined actions of IS257, IS257-dependent miniature inverted-repeat transposable elements (MITEs) and the BinL resolution system of the ß-lactamase transposon Tn552 An evolutionary intermediate ~42-kb non-conjugative plasmid pWBG715, possessed the same resistance genes as pWBG731 but retained an integrated copy of the small tetracycline-resistance plasmid pT181. IS257 likely facilitated replacement of pT181 with conjugation genes on pWBG731, thus enabling autonomous transfer. Like conjugative plasmid pWBG749, pWBG731 also mobilized non-conjugative plasmids carrying oriT mimics. It seems likely that pWBG731 represents the product of multiple recombination events between the WA-5 pWBG753 plasmid and other mobile genetic elements present in indigenous CA-MSSA. The molecular evolution of pWBG731 saliently illustrates how diverse mobile genetic elements can together facilitate rapid accrual and horizontal dissemination of multiresistance in S. aureus CA-MRSA.Copyright © 2019 American Society for Microbiology.
Acinetobacter calcoaceticus-baumannii complex isolates have been frequently associated with hospital and community infections, with A. baumannii being the most common. Other Acinetobacter spp. not belonging to this complex also cause infections in hospital settings, and the incidence has increased over the past few years. Some species of the Acinetobacter genus possess a great diversity of antibiotic resistance mechanisms, such as efflux pumps, porins, and resistance genes that can be acquired and disseminated by mobilizable genetic elements. By means of whole-genome sequencing, we describe in the clinical Acinetobacter haemolyticus strain AN54 different mechanisms of resistance that involve blaOXA-265, blaNDM-1, aphA6, aac(6′)-Ig, and a resistance-nodulation-cell division-type efflux pump. This strain carries six plasmids, of which the plasmid pAhaeAN54e contains blaNDM-1 in a Tn125-like transposon that is truncated at the 3′ end. This strain also has an insertion sequence IS91 and seven genes encoding hypothetical proteins. The pAhaeAN54e plasmid is nontypable and different from other plasmids carrying blaNDM-1 that have been reported in Mexico and other countries. The presence of these kinds of plasmids in an opportunistic pathogen such as A. haemolyticus highlights the role that these plasmids play in the dissemination of antibiotic resistance genes, especially against carbapenems, in Mexican hospitals.
Aims: A hospital outbreak of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPN) linked with an index case of community-acquired infection occurred in an urban tertiary care hospital in Seoul, South Korea. Therefore, we performed an outbreak investigation and whole genome sequencing (WGS) analysis to trace the outbreak and investigate the molecular characteristics of the isolates. Results: From October 2014 to January 2015, we identified a cluster of three patients in the neurosurgery ward with sputum cultures positive for carbapenem-resistant KPN. An epidemiological investigation, including pulsed-field gel electrophoresis analysis was performed to trace the origins of this outbreak. The index patient’s infection was community acquired. Active surveillance cultures using perirectal swabbing from exposed patients, identified one additional patient with KPC-producing KPN colonization. WGS analyses using PacBio RSII instruments were performed for four linked isolates. WGS revealed a genetic linkage of the four isolates belonging to the same sequence type (ST307). All KPN isolates harbored conjugative resistance plasmids, which has blaKPC-2 carbapenemase genes contained within the Tn4401 “a” isoform and other resistance genes. However, WGS showed only three isolates among four KPC-producing KPN were originated from a common origin. Conclusions: This report demonstrates the challenge that KPC-2-producing KPN with the conjugative resistance plasmid may spread not only in hospitals but also in community, and WGS can help to accurately characterize the outbreak.
Candida auris is an emergent yeast pathogen, easily transmissible between patients and with high percent of multidrug resistant strains. Here we present a draft genome sequence of the first known Russian strain of C. auris, isolated from a case of candidemia. The strain clustered within South Asian C. auris clade and seemingly represented an independent event of dissemination from the original species range. Observed fluconazole resistance was probably due to F105L and K143R mutations in ERG11. © The Author(s) 2019. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
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 is noteworthy that tet(X4)-positive E.?coli strains, including isolates co-harbouring mcr-1, have been widely detected in pigs, chickens, soil and dust samples in China. In vivo murine models demonstrated that the presence of Tet(X4) led to tigecycline treatment failure. Consequently, the emergence of plasmid-mediated Tet(X4) challenges the clinical efficacy of the entire family of tetracycline antibiotics. Importantly, our study raises concern that the plasmid-mediated tigecycline resistance may further spread into various ecological niches and into clinical high-risk pathogens. Collective efforts are in urgent need to preserve the potency of these essential antibiotics.
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 transconjugants harbored blaKPC-positive plasmids of different sizes. Analysis of plasmid sequences showed that, in both isolates, blaKPC-2 gene was carried on type 2 IncC plasmids pC-Ec20-KPC and pC-Ec2-KPC. Both plasmids carried the ARI-B resistance island, which consisted of several resistance genes, intact and truncated copies of several mobile elements, and a 25,571-bp segment harboring coding sequences for an iron transporter. The blaKPC-2 gene was part of the transposon Tn4401a, which was bounded by direct repeats of 5 bp (TCCTT) suggesting its transposition into the IncC plasmids.To our knowledge, this is the first report on complete nucleotide sequences of type 2 IncC plasmids. These findings, which hypothesize the acquisition of KPC-2-encoding transposon Tn4401a by an IncC replicon, indicate the ongoing need for molecular surveillance studies of MDR pathogens. Additionally, they underline the increasing clinical importance of the IncC plasmid family.Copyright © 2019. Published by Elsevier Ltd.
Carbapenem-resistant Enterobacteriaceae (CRE) represent one of the most urgent threats to human health posed by antibiotic resistant bacteria. Enterobacter hormaechei and other members of the Enterobacter cloacae complex are the most commonly encountered Enterobacter spp. within clinical settings, responsible for numerous outbreaks and ultimately poorer patient outcomes. Here we applied three complementary whole genome sequencing (WGS) technologies to characterise a hospital cluster of blaIMP-4 carbapenemase-producing E. hormaechei.In response to a suspected CRE outbreak in 2015 within an Intensive Care Unit (ICU)/Burns Unit in a Brisbane tertiary referral hospital we used Illumina sequencing to determine that all outbreak isolates were sequence type (ST)90 and near-identical at the core genome level. Comparison to publicly available data unequivocally linked all 10 isolates to a 2013 isolate from the same ward, confirming the hospital environment as the most likely original source of infection in the 2015 cases. No clonal relationship was found to IMP-4-producing isolates identified from other local hospitals. However, using Pacific Biosciences long-read sequencing we were able to resolve the complete context of the blaIMP-4 gene, which was found to be on a large IncHI2 plasmid carried by all IMP-4-producing isolates. Continued surveillance of the hospital environment was carried out using Oxford Nanopore long-read sequencing, which was able to rapidly resolve the true relationship of subsequent isolates to the initial outbreak. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing.Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak, including the transmission dynamics of a carbapenemase-producing E. hormaechei cluster, identification of possible hospital reservoirs and the full context of blaIMP-4 on a multidrug resistant IncHI2 plasmid that appears to be widely distributed in Australia.
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 one enterococci strains, respectively. The poxtA in one E. faecalis strain was located on a 37,990 bp plasmid, which co-harbored fexB, cat, tet(L) and tet(M), and could be conjugated to E. faecalis JH2-2. One E. faecalis strain harbored two different OptrA variants, including one variant with a single substitution, Q219H, which has not been reported previously. Two optrA-carrying plasmids, pC25-1, with a size of 45,581 bp, and pC54, with a size of 64,500 bp, shared a 40,494 bp identical region that contained genetic context IS1216E-fexA-optrA-erm(A)-IS1216E, which could be electrotransformed into Staphylococcus aureus. Four different chromosomal optrA gene clusters were found in five strains, in which optrA was associated with Tn554 or Tn558 that were inserted into the radC gene.Our study highlights the fact that mobile genetic elements, such as plasmids, IS1216E, Tn554 and Tn558, may facilitate the horizontal transmission of optrA or poxtA.Copyright © 2019. Published by Elsevier Ltd.
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.
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