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Asset Tag: Resistance gene

July 19, 2019  |  

Insertion sequence IS26 reorganizes plasmids in clinically isolated multidrug-resistant bacteria by replicative transposition.

Carbapenemase-producing Enterobacteriaceae (CPE), which are resistant to most or all known antibiotics, constitute a global threat to public health. Transposable elements are often associated with antibiotic resistance determinants, suggesting a role in the emergence of resistance. One insertion sequence, IS26, is frequently associated with resistance determinants, but its role remains unclear. We have analyzed the genomic contexts of 70 IS26 copies in several clinical and surveillance CPE isolates from the National Institutes of Health Clinical Center. We used target site duplications and their patterns as guides and found that a large fraction of plasmid reorganizations result from IS26 replicative transpositions, including replicon fusions, DNA inversions, and deletions. Replicative transposition could also be inferred for transposon Tn4401, which harbors the carbapenemase blaKPC gene. Thus, replicative transposition is important in the ongoing reorganization of plasmids carrying multidrug-resistant determinants, an observation that carries substantial clinical and epidemiological implications for understanding how such extreme drug resistance phenotypes evolve.Although IS26 is frequently reported to reside in resistance plasmids of clinical isolates, the characteristic hallmark of transposition, target site duplication (TSD), is generally not observed, raising questions about the mode of transposition for IS26. The previous observation of cointegrate formation during transposition implies that IS26 transposes via a replicative mechanism. The other possible outcome of replicative transposition is DNA inversion or deletion, when transposition occurs intramolecularly, and this would also generate a specific TSD pattern that might also serve as supporting evidence for the transposition mechanism. The numerous examples we present here demonstrate that replicative transposition, used by many mobile elements (including IS26 and Tn4401), is prevalent in the plasmids of clinical isolates and results in significant plasmid reorganization. This study also provides a method to trace the evolution of resistance plasmids based on TSD patterns. Copyright © 2015 He et al.

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July 19, 2019  |  

Parallel epidemics of community-associated methicillin-resistant Staphylococcus aureus USA300 infection in North and South America.

The community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) epidemic in the United States is attributed to the spread of the USA300 clone. An epidemic of CA-MRSA closely related to USA300 has occurred in northern South America (USA300 Latin-American variant, USA300-LV). Using phylogenomic analysis, we aimed to understand the relationships between these 2 epidemics.We sequenced the genomes of 51 MRSA clinical isolates collected between 1999 and 2012 from the United States, Colombia, Venezuela, and Ecuador. Phylogenetic analysis was used to infer the relationships and times since the divergence of the major clades.Phylogenetic analyses revealed 2 dominant clades that segregated by geographical region, had a putative common ancestor in 1975, and originated in 1989, in North America, and in 1985, in South America. Emergence of these parallel epidemics coincides with the independent acquisition of the arginine catabolic mobile element (ACME) in North American isolates and a novel copper and mercury resistance (COMER) mobile element in South American isolates.Our results reveal the existence of 2 parallel USA300 epidemics that shared a recent common ancestor. The simultaneous rapid dissemination of these 2 epidemic clades suggests the presence of shared, potentially convergent adaptations that enhance fitness and ability to spread.© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 19, 2019  |  

TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonas oryzae pv. oryzicola while strict conservation suggests universal importance of five TAL effectors.

Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL effectors.

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July 19, 2019  |  

Mind the gap; seven reasons to close fragmented genome assemblies.

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions. Copyright © 2015 Elsevier Inc. All rights reserved.

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July 19, 2019  |  

Recurrent methicillin-resistant Staphylococcus aureus cutaneous abscesses and selection of reduced chlorhexidine susceptibility during chlorhexidine use.

We describe the selection of reduced chlorhexidine susceptibility during chlorhexidine use in a patient with two episodes of cutaneous USA300 methicillin-resistant Staphylococcus aureus abscess. The second clinical isolate harbors a novel plasmid that encodes the QacA efflux pump. Greater use of chlorhexidine for disease prevention warrants surveillance for resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 19, 2019  |  

Stepwise evolution of pandrug-resistance in Klebsiella pneumoniae.

Carbapenem resistant Enterobacteriaceae (CRE) pose an urgent risk to global human health. CRE that are non-susceptible to all commercially available antibiotics threaten to return us to the pre-antibiotic era. Using Single Molecule Real Time (SMRT) sequencing we determined the complete genome of a pandrug-resistant Klebsiella pneumoniae isolate, representing the first complete genome sequence of CRE resistant to all commercially available antibiotics. The precise location of acquired antibiotic resistance elements, including mobile elements carrying genes for the OXA-181 carbapenemase, were defined. Intriguingly, we identified three chromosomal copies of an ISEcp1-blaOXA-181 mobile element, one of which has disrupted the mgrB regulatory gene, accounting for resistance to colistin. Our findings provide the first description of pandrug-resistant CRE at the genomic level, and reveal the critical role of mobile resistance elements in accelerating the emergence of resistance to other last resort antibiotics.

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July 19, 2019  |  

Pangenome analysis of Bifidobacterium longum and site-directed mutagenesis through by-pass of restriction-modification systems.

Bifidobacterial genome analysis has provided insights as to how these gut commensals adapt to and persist in the human GIT, while also revealing genetic diversity among members of a given bifidobacterial (sub)species. Bifidobacteria are notoriously recalcitrant to genetic modification, which prevents exploration of their genomic functions, including those that convey (human) health benefits.PacBio SMRT sequencing was used to determine the whole genome seqeunces of two B. longum subsp. longum strains. The B. longum pan-genome was computed using PGAP v1.2 and the core B. longum phylogenetic tree was constructed using a maximum-likelihood based approach in PhyML v3.0. M.blmNCII was cloned in E. coli and an internal fragment if arfBarfB was cloned into pORI19 for insertion mutagenesis.In this study we present the complete genome sequences of two Bifidobacterium longum subsp. longum strains. Comparative analysis with thirty one publicly available B. longum genomes allowed the definition of the B. longum core and dispensable genomes. This analysis also highlighted differences in particular metabolic abilities between members of the B. longum subspecies infantis, longum and suis. Furthermore, phylogenetic analysis of the B. longum core genome indicated the existence of a novel subspecies. Methylome data, coupled to the analysis of restriction-modification systems, allowed us to substantially increase the genetic accessibility of B. longum subsp. longum NCIMB 8809 to a level that was shown to permit site-directed mutagenesis.Comparative genomic analysis of thirty three B. longum representatives revealed a closed pan-genome for this bifidobacterial species. Phylogenetic analysis of the B. longum core genome also provides evidence for a novel fifth B. longum subspecies. Finally, we improved genetic accessibility for the strain B. longum subsp. longum NCIMB 8809, which allowed the generation of a mutant of this strain.

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July 19, 2019  |  

Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships.

Pathogen-injected, direct transcriptional activators of host genes, TAL (transcription activator-like) effectors play determinative roles in plant diseases caused by Xanthomonas spp. A large domain of nearly identical, 33-35 aa repeats in each protein mediates DNA recognition. This modularity makes TAL effectors customizable and thus important also in biotechnology. However, the repeats render TAL effector (tal) genes nearly impossible to assemble using next-generation, short reads. Here, we demonstrate that long-read, single molecule real-time (SMRT) sequencing solves this problem. Taking an ensemble approach to first generate local, tal gene contigs, we correctly assembled de novo the genomes of two strains of the rice pathogen X. oryzae completed previously using the Sanger method and even identified errors in those references. Sequencing two more strains revealed a dynamic genome structure and a striking plasticity in tal gene content. Our results pave the way for population-level studies to inform resistance breeding, improve biotechnology and probe TAL effector evolution.

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July 19, 2019  |  

AnnoTALE: bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences.

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present ‘AnnoTALE’, a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities.

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July 19, 2019  |  

Large genomic differences between Moraxella bovoculi isolates acquired from the eyes of cattle with infectious bovine keratoconjunctivitis versus the deep nasopharynx of asymptomatic cattle.

Moraxella bovoculi is a recently described bacterium that is associated with infectious bovine keratoconjunctivitis (IBK) or “pinkeye” in cattle. In this study, closed circularized genomes were generated for seven M. bovoculi isolates: three that originated from the eyes of clinical IBK bovine cases and four from the deep nasopharynx of asymptomatic cattle. Isolates that originated from the eyes of IBK cases profoundly differed from those that originated from the nasopharynx of asymptomatic cattle in genome structure, gene content and polymorphism diversity and consequently placed into two distinct phylogenetic groups. These results suggest that there are genetically distinct strains of M. bovoculi that may not associate with IBK.

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July 19, 2019  |  

Highly efficient CRISPR/Cas9-mediated cloning and functional characterization of gastric cancer-derived Epstein-Barr virus strains.

The Epstein-Barr virus (EBV) is etiologically linked to approximately 10% of gastric cancers, in which viral genomes are maintained as multicopy episomes. EBV-positive gastric cancer cells are incompetent for progeny virus production, making viral DNA cloning extremely difficult. Here we describe a highly efficient strategy for obtaining bacterial artificial chromosome (BAC) clones of EBV episomes by utilizing a CRISPR/Cas9-mediated strand break of the viral genome and subsequent homology-directed repair. EBV strains maintained in two gastric cancer cell lines (SNU719 and YCCEL1) were cloned, and their complete viral genome sequences were determined. Infectious viruses of gastric cancer cell-derived EBVs were reconstituted, and the viruses established stable latent infections in immortalized keratinocytes. While Ras oncoprotein overexpression caused massive vacuolar degeneration and cell death in control keratinocytes, EBV-infected keratinocytes survived in the presence of Ras expression. These results implicate EBV infection in predisposing epithelial cells to malignant transformation by inducing resistance to oncogene-induced cell death.Recent progress in DNA-sequencing technology has accelerated EBV whole-genome sequencing, and the repertoire of sequenced EBV genomes is increasing progressively. Accordingly, the presence of EBV variant strains that may be relevant to EBV-associated diseases has begun to attract interest. Clearly, the determination of additional disease-associated viral genome sequences will facilitate the identification of any disease-specific EBV variants. We found that CRISPR/Cas9-mediated cleavage of EBV episomal DNA enabled the cloning of disease-associated viral strains with unprecedented efficiency. As a proof of concept, two gastric cancer cell-derived EBV strains were cloned, and the infection of epithelial cells with reconstituted viruses provided important clues about the mechanism of EBV-mediated epithelial carcinogenesis. This experimental system should contribute to establishing the relationship between viral genome variation and EBV-associated diseases. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 19, 2019  |  

Nested Russian doll-like genetic mobility drives rapid dissemination of the Carbapenem resistance gene blaKPC

The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort against this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms, including conjugation and transposition; however, the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is unknown. Using a combination of short- and long-read whole-genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolates from a single hospital over 5 years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested genetic levels, with transmission of blaKPC strains between individuals, frequent transfer of blaKPC plasmids between strains/species, and frequent transposition of blaKPC transposon Tn4401 between plasmids. We also identify a common insertion site for Tn4401 within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this by the use of reference-based methods. We also demonstrate that, as a consequence of the genetic mobility observed in this study, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that nonclonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance. Copyright © 2016 Sheppard et al.

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July 19, 2019  |  

Initial assessment of the molecular epidemiology of blaNDM-1 in Colombia.

We report complete genome sequences of fourblaNDM-1-harboring Gram-negative multidrug resistant (MDR) isolates from Colombia. TheblaNDM-1genes were located 193Kb-Inc FIA, 178Kb-Inc A/C2 and 47Kb (unknown Inc type) plasmids. MLST revealed that isolates belong to ST10 (Escherichia coli), ST392 (Klebsiella pneumoniae), and ST322 and ST464 (Acinetobacter baumanniiandA. nosocomialis, respectively). Our analysis identified that the Inc A/C2 plasmid inE. colicontained a novel complex transposon (Tn125and Tn5393with 3 copies ofblaNDM-1) and a recombination “hotspot” for the acquisition of new resistance determinants. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 19, 2019  |  

Targeted capture and sequencing of gene-sized DNA molecules.

Targeted capture provides an efficient and sensitive means for sequencing specific genomic regions in a high-throughput manner. To date, this method has mostly been used to capture exons from the genome (the exome) using short insert libraries and short-read sequencing technology, enabling the identification of genetic variants or new members of large gene families. Sequencing larger molecules results in the capture of whole genes, including intronic and intergenic sequences that are typically more polymorphic and allow the resolution of the gene structure of homologous genes, which are often clustered together on the chromosome. Here, we describe an improved method for the capture and single-molecule sequencing of DNA molecules as large as 7 kb by means of size selection and optimized PCR conditions. Our approach can be used to capture, sequence, and distinguish between similar members of the NB-LRR gene family-key genes in plant immune systems.

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July 19, 2019  |  

Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli.

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common infections. bla KPC, encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10?kb). Here we characterize the genetic features of bla KPC emergence in global E. coli, 2008-2013, using both long- and short-read whole-genome sequencing. Amongst 43/45 successfully sequenced bla KPC-E. coli strains, we identified substantial strain diversity (n?=?21 sequence types, 18% of annotated genes in the core genome); substantial plasmid diversity (=9 replicon types); and substantial bla KPC-associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of inter-species, regional and international plasmid spread. In several cases bla KPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures. E. coli is a common human pathogen, but also a commensal in multiple environmental and animal reservoirs, and easily transmissible. The association of bla KPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. bla TEM, bla CTX-M) suggests that it may become similarly prevalent.

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