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April 21, 2020  |  

The complete genome sequence and comparative genome analysis of the multi-drug resistant food-borne pathogen Bacillus cereus.

Bacillus cereus is an opportunistic human pathogen causing food-borne gastrointestinal infections and non-gastrointestinal infections worldwide. The strain B. cereus FORC_013 was isolated from fried eel. Its genome was completely sequenced by PacBio technology, analyzed and compared with other complete genome sequences of Bacillus to elucidate the distinct pathogenic features of the strain isolated in South Korea. Genomic analysis revealed pathogenesis and host immune evasion-associated genes encoding tissue-destructive exoenzymes, and pore-forming toxins. In particular, tissue-destructive (hemolysin BL, nonhaemolytic enterotoxins) and cytolytic proteins (cytolysin) were observed in the genome, which damage the plasma membrane of the epithelial cells of the small intestine causing diarrhea in humans. Capsule biosynthesis gene found in both chromosome and plasmid, which might be responsible for protecting the pathogen from the host cell immune defense system after host cell invasion. Additionally, multidrug resistance operon and efflux pumps were identified in the genome, which play a prominent role in multi-antibiotic resistance. Comparative phylogenetic tree analysis of the strain FORC_013 and other B. cereus strains revealed that the closest strains are ATCC 14579 and B4264. This genome data can be used to identify virulence factors that can be applied for the development of novel biomarkers for the rapid detection of this pathogen in foods.Copyright © 2018. Published by Elsevier Inc.

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April 21, 2020  |  

Tracking short-term changes in the genetic diversity and antimicrobial resistance of OXA-232-producing Klebsiella pneumoniae ST14 in clinical settings.

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.

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April 21, 2020  |  

Potent LpxC Inhibitors with In Vitro Activity Against Multi-Drug Resistant Pseudomonas aeruginosa.

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.

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April 21, 2020  |  

Evolution of a 72-kb cointegrant, conjugative multiresistance plasmid from early community-associated methicillin-resistant Staphylococcus aureus isolates.

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.

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April 21, 2020  |  

Resistome and a Novel blaNDM-1-Harboring Plasmid of an Acinetobacter haemolyticus Strain from a Children’s Hospital in Puebla, Mexico.

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.

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April 21, 2020  |  

An Outbreak of KPC-Producing Klebsiella pneumoniae Linked with an Index Case of Community-Acquired KPC-Producing Isolate: Epidemiological Investigation and Whole Genome Sequencing Analysis.

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.

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April 21, 2020  |  

A robust benchmark for germline structural variant detection

New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based, from short-, linked-, and long-read sequencing, as well as optical and electronic mapping. The final benchmark set contains 12745 isolated, sequence-resolved insertion and deletion calls =50 base pairs (bp) discovered by at least 2 technologies or 5 callsets, genotyped as heterozygous or homozygous variants by long reads. The Tier 1 benchmark regions, for which any extra calls are putative false positives, cover 2.66 Gbp and 9641 SVs supported by at least one diploid assembly. Support for SVs was assessed using svviz with short-, linked-, and long-read sequence data. In general, there was strong support from multiple technologies for the benchmark SVs, with 90 % of the Tier 1 SVs having support in reads from more than one technology. The Mendelian genotype error rate was 0.3 %, and genotype concordance with manual curation was >98.7 %. We demonstrate the utility of the benchmark set by showing it reliably identifies both false negatives and false positives in high-quality SV callsets from short-, linked-, and long-read sequencing and optical mapping.

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April 21, 2020  |  

Complete genome sequence of Paracoccus sp. Arc7-R13, a silver nanoparticles synthesizing bacterium isolated from Arctic Ocean sediments

Paracoccus sp. Arc7-R13, a silver nanoparticles (AgNPs) synthesizing bacterium, was isolated from Arctic Ocean sediment. Here we describe the complete genome of Paracoccus sp. Arc7-R13. The complete genome contains 4,040,012?bp with 66.66?mol%?G?+?C content, including one circular chromosome of 3,231,929?bp (67.45?mol%?G?+?C content), and eight plasmids with length ranging from 24,536?bp to 199,685?bp. The genome contains 3835 protein-coding genes (CDSs), 49 tRNA genes, as well as 3 rRNA operons as 16S-23S-5S rRNA. Based on the gene annotation and Swiss-Prot analysis, a total of 15 genes belonging to 11 kinds, including silver exporting P-type ATPase (SilP), alkaline phosphatase, nitroreductase, thioredoxin reductase, NADPH dehydrogenase and glutathione peroxidase, might be related to the synthesis of AgNPs. Meanwhile, many additional genes associated with synthesis of AgNPs such as protein-disulfide isomerase, c-type cytochrome, glutathione synthase and dehydrogenase reductase were also identified.

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April 21, 2020  |  

Whole genome sequence of first Candida auris strain, isolated in Russia.

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.

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April 21, 2020  |  

Comparative Genomic Analysis of Virulence, Antimicrobial Resistance, and Plasmid Profiles of Salmonella Dublin Isolated from Sick Cattle, Retail Beef, and Humans in the United States.

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. Fifteen Salmonella pathogenicity islands (SPIs) and a spv operon (spvRABCD), which encodes important virulence factors, were identified in all 69 (100%) isolates. The 15 SPIs were located on the chromosome of the 5 closed genomes, with each of these isolates also carrying 1 or 2 plasmids with sizes between 36 and 329?kb. Multiple antimicrobial resistance genes (ARGs), including blaCMY-2, blaTEM-1B, aadA12, aph(3′)-Ia, aph(3′)-Ic, strA, strB, floR, sul1, sul2, and tet(A), along with spv operons were identified on these plasmids. Comprehensive antimicrobial resistance genotypes were determined, including 17 genes encoding resistance to 5 different classes of antimicrobials, and mutations in the housekeeping gene (gyrA) associated with resistance or decreased susceptibility to fluoroquinolones. Together these data revealed that this panel of Salmonella Dublin commonly carried 15 SPIs, MDR/virulence plasmids, and ARGs against several classes of antimicrobials. Such genomic elements may make important contributions to the severity of disease and treatment failures in Salmonella Dublin infections in both humans and cattle.

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April 21, 2020  |  

Plasmid-encoded tet(X) genes that confer high-level tigecycline resistance in Escherichia coli.

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.

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April 21, 2020  |  

Antibiotic susceptibility of plant-derived lactic acid bacteria conferring health benefits to human.

Lactic acid bacteria (LAB) confer health benefits to human when administered orally. We have recently isolated several species of LAB strains from plant sources, such as fruits, vegetables, flowers, and medicinal plants. Since antibiotics used to treat bacterial infection diseases induce the emergence of drug-resistant bacteria in intestinal microflora, it is important to evaluate the susceptibility of LAB strains to antibiotics to ensure the safety and security of processed foods. The aim of the present study is to determine the minimum inhibitory concentration (MIC) of antibiotics against several plant-derived LAB strains. When aminoglycoside antibiotics, such as streptomycin (SM), kanamycin (KM), and gentamicin (GM), were evaluated using LAB susceptibility test medium (LSM), the MIC was higher than when using Mueller-Hinton (MH) medium. Etest, which is an antibiotic susceptibility assay method consisting of a predefined gradient of antibiotic concentrations on a plastic strip, is used to determine the MIC of antibiotics world-wide. In the present study, we demonstrated that Etest was particularly valuable while testing LAB strains. We also show that the low susceptibility of the plant-derived LAB strains against each antibiotic tested is due to intrinsic resistance and not acquired resistance. This finding is based on the whole-genome sequence information reflecting the horizontal spread of the drug-resistance genes in the LAB strains.

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April 21, 2020  |  

RNA sequencing: the teenage years.

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions such as when and where transcription occurs to the folding and intermolecular interactions that govern RNA function.

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April 21, 2020  |  

Emergence of plasmid-mediated high-level tigecycline resistance genes in animals and humans.

Tigecycline is a last-resort antibiotic that is used to treat severe infections caused by extensively drug-resistant bacteria. tet(X) has been shown to encode a flavin-dependent monooxygenase that modifies tigecycline1,2. Here, we report two unique mobile tigecycline-resistance genes, tet(X3) and tet(X4), in numerous Enterobacteriaceae and Acinetobacter that were isolated from animals, meat for consumption and humans. Tet(X3) and Tet(X4) inactivate all tetracyclines, including tigecycline and the newly FDA-approved eravacycline and omadacycline. Both tet(X3) and tet(X4) increase (by 64-128-fold) the tigecycline minimal inhibitory concentration values for Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii. In addition, both Tet(X3) (A. baumannii) and Tet(X4) (E. coli) significantly compromise tigecycline in in vivo infection models. Both tet(X3) and tet(X4) are adjacent to insertion sequence ISVsa3 on their respective conjugative plasmids and confer a mild fitness cost (relative fitness of >0.704). Database mining and retrospective screening analyses confirm that tet(X3) and tet(X4) are globally present in clinical bacteria-even in the same bacteria as blaNDM-1, resulting in resistance to both tigecycline and carbapenems. Our findings suggest that both the surveillance of tet(X) variants in clinical and animal sectors and the use of tetracyclines in food production require urgent global attention.

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April 21, 2020  |  

Whole-genome analysis of New Delhi Metallo-Beta-Lactamase-1-producing Acinetobacter haemolyticus from China.

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 was resistant to carbapenems and other tested agents except for amikacin, tigecycline and colistin. The production of MBLs was confirmed by CDT. Transfer of carbapenem resistance was not successful. WGS analysis showed the genome of sz1652 was comprised of chromosome and two plasmids, and sixteen genomic islands (GIs) were predicted. Genes associated with resistance were found in this strain including the beta-lactamase genes blaNDM-1, blaOXA-214 and blaLRA-18, the ?uoroquinolone resistant-related mutations [GyrA subunits (Ser81Ile) and ParC subunits (Ser84Tyr)], and efflux pump genes related to tetracycline and macrolide resistance. Analysis of the genetic environment showed that blaNDM-1was embedded in Tn125 transposon. The Tn125 structure was chromosomally located and shared more than 99% sequence identity with previously reported blaNDM-1 carrying region.The NDM-1-producing A.haemolyticus coexisted multiple durg-resistant determinants. The acquisition of the blaNDM-1 gene was probably facilitated by Tn125 in this strain. Non-A.baumannii species also contain GIs.Copyright © 2019. Published by Elsevier Ltd.

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