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July 19, 2019  |  

Chromosomal integration of the Klebsiella pneumoniae carbapenemase gene, blaKPC, in Klebsiella species is elusive but not rare.

Carbapenemase genes in Enterobacteriaceae are mostly described as being plasmid associated. However, the genetic context of carbapenemase genes is not always confirmed in epidemiological surveys, and the frequency of their chromosomal integration therefore is unknown. A previously sequenced collection of blaKPC-positive Enterobacteriaceae from a single U.S. institution (2007 to 2012; n = 281 isolates from 182 patients) was analyzed to identify chromosomal insertions of Tn4401, the transposon most frequently harboring blaKPC Using a combination of short- and long-read sequencing, we confirmed five independent chromosomal integration events from 6/182 (3%) patients, corresponding to 15/281 (5%) isolates. Three patients had isolates identified by perirectal screening, and three had infections which were all successfully treated. When a single copy of blaKPC was in the chromosome, one or both of the phenotypic carbapenemase tests were negative. All chromosomally integrated blaKPC genes were from Klebsiella spp., predominantly K. pneumoniae clonal group 258 (CG258), even though these represented only a small proportion of the isolates. Integration occurred via IS15-?I-mediated transposition of a larger, composite region encompassing Tn4401 at one locus of chromosomal integration, seen in the same strain (K. pneumoniae ST340) in two patients. In summary, we identified five independent chromosomal integrations of blaKPC in a large outbreak, demonstrating that this is not a rare event. blaKPC was more frequently integrated into the chromosome of epidemic CG258 K. pneumoniae lineages (ST11, ST258, and ST340) and was more difficult to detect by routine phenotypic methods in this context. The presence of chromosomally integrated blaKPC within successful, globally disseminated K. pneumoniae strains therefore is likely underestimated. Copyright © 2017 Mathers et al.


July 19, 2019  |  

Genomic confirmation of vancomycin-resistant Enterococcus transmission from deceased donor to liver transplant recipient.

In a liver transplant recipient with vancomycin-resistant Enterococcus (VRE) surgical site and bloodstream infection, a combination of pulsed-field gel electrophoresis, multilocus sequence typing, and whole genome sequencing identified that donor and recipient VRE isolates were highly similar when compared to time-matched hospital isolates. Comparison of de novo assembled isolate genomes was highly suggestive of transplant transmission rather than hospital-acquired transmission and also identified subtle internal rearrangements between donor and recipient missed by other genomic approaches. Given the improved resolution, whole-genome assembly of pathogen genomes is likely to become an essential tool for investigation of potential organ transplant transmissions.


July 19, 2019  |  

TAL effector driven induction of a SWEET gene confers susceptibility to bacterial blight of cotton.

Transcription activator-like (TAL) effectors from Xanthomonas citri subsp. malvacearum (Xcm) are essential for bacterial blight of cotton (BBC). Here, by combining transcriptome profiling with TAL effector-binding element (EBE) prediction, we show that GhSWEET10, encoding a functional sucrose transporter, is induced by Avrb6, a TAL effector determining Xcm pathogenicity. Activation of GhSWEET10 by designer TAL effectors (dTALEs) restores virulence of Xcm avrb6 deletion strains, whereas silencing of GhSWEET10 compromises cotton susceptibility to infections. A BBC-resistant line carrying an unknown recessive b6 gene bears the same EBE as the susceptible line, but Avrb6-mediated induction of GhSWEET10 is reduced, suggesting a unique mechanism underlying b6-mediated resistance. We show via an extensive survey of GhSWEET transcriptional responsiveness to different Xcm field isolates that additional GhSWEETs may also be involved in BBC. These findings advance our understanding of the disease and resistance in cotton and may facilitate the development cotton with improved resistance to BBC.


July 19, 2019  |  

Amplification-free, CRISPR-Cas9 targeted enrichment and SMRT Sequencing of repeat-expansion disease causative genomic regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods require amplification. Some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, many human genetic disorders are caused by repeat expansions, including difficult to sequence tandem repeats. We have developed a novel, amplification-free enrichment technique that employs the CRISPR-Cas9 system for specific targeting multiple genomic loci. This method, in conjunction with long reads generated through Single Molecule, Real-Time (SMRT) sequencing and unbiased coverage, enables enrichment and sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples, we demonstrate successful targeting of causative loci for Huntingtontextquoterights disease (HTT; CAG repeat), Fragile X syndrome (FMR1; CGG repeat), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72; GGGGCC repeat), and spinocerebellar ataxia type 10 (SCA10) (ATXN10; variable ATTCT repeat). The method, amenable to multiplexing across multiple genomic loci, uses an amplification-free approach that facilitates the isolation of hundreds of individual on-target molecules in a single SMRT Cell and accurate sequencing through long repeat stretches, regardless of extreme GC percent or sequence complexity content. Our novel targeted sequencing method opens new doors to genomic analyses independent of PCR amplification that will facilitate the study of repeat expansion disorders.


July 19, 2019  |  

Coupling of single molecule, long read sequencing with IMGT/HighV-QUEST analysis expedites identification of SIV gp140-specific antibodies from scFv phage display libraries.

The simian immunodeficiency virus (SIV)/macaque model of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome pathogenesis is critical for furthering our understanding of the role of antibody responses in the prevention of HIV infection, and will only increase in importance as macaque immunoglobulin (IG) gene databases are expanded. We have previously reported the construction of a phage display library from a SIV-infected rhesus macaque (Macaca mulatta) using oligonucleotide primers based on human IG gene sequences. Our previous screening relied on Sanger sequencing, which was inefficient and generated only a few dozen sequences. Here, we re-analyzed this library using single molecule, real-time (SMRT) sequencing on the Pacific Biosciences (PacBio) platform to generate thousands of highly accurate circular consensus sequencing (CCS) reads corresponding to full length single chain fragment variable. CCS data were then analyzed through the international ImMunoGeneTics information system®(IMGT®)/HighV-QUEST (www.imgt.org) to identify variable genes and perform statistical analyses. Overall the library was very diverse, with 2,569 different IMGT clonotypes called for the 5,238 IGHV sequences assigned to an IMGT clonotype. Within the library, SIV-specific antibodies represented a relatively limited number of clones, with only 135 different IMGT clonotypes called from 4,594 IGHV-assigned sequences. Our data did confirm that the IGHV4 and IGHV3 gene usage was the most abundant within the rhesus antibodies screened, and that these genes were even more enriched among SIV gp140-specific antibodies. Although a broad range of VH CDR3 amino acid (AA) lengths was observed in the unpanned library, the vast majority of SIV gp140-specific antibodies demonstrated a more uniform VH CDR3 length (20 AA). This uniformity was far less apparent when VH CDR3 were classified according to their clonotype (range: 9-25 AA), which we believe is more relevant for specific antibody identification. Only 174 IGKV and 588 IGLV clonotypes were identified within the VL sequences associated with SIV gp140-specific VH. Together, these data strongly suggest that the combination of SMRT sequencing with the IMGT/HighV-QUEST querying tool will facilitate and expedite our understanding of polyclonal antibody responses during SIV infection and may serve to rapidly expand the known scope of macaque V genes utilized during these responses.


July 19, 2019  |  

Full-Length Envelope Analyzer (FLEA): A tool for longitudinal analysis of viral amplicons.

Next generation sequencing of viral populations has advanced our understanding of viral population dynamics, the development of drug resistance, and escape from host immune responses. Many applications require complete gene sequences, which can be impossible to reconstruct from short reads. HIV env, the protein of interest for HIV vaccine studies, is exceptionally challenging for long-read sequencing and analysis due to its length, high substitution rate, and extensive indel variation. While long-read sequencing is attractive in this setting, the analysis of such data is not well handled by existing methods. To address this, we introduce FLEA (Full-Length Envelope Analyzer), which performs end-to-end analysis and visualization of long-read sequencing data. FLEA consists of both a pipeline (optionally run on a high-performance cluster), and a client-side web application that provides interactive results. The pipeline transforms FASTQ reads into high-quality consensus sequences (HQCSs) and uses them to build a codon-aware multiple sequence alignment. The resulting alignment is then used to infer phylogenies, selection pressure, and evolutionary dynamics. The web application provides publication-quality plots and interactive visualizations, including an annotated viral alignment browser, time series plots of evolutionary dynamics, visualizations of gene-wide selective pressures (such as dN/dS) across time and across protein structure, and a phylogenetic tree browser. We demonstrate how FLEA may be used to process Pacific Biosciences HIV env data and describe recent examples of its use. Simulations show how FLEA dramatically reduces the error rate of this sequencing platform, providing an accurate portrait of complex and variable HIV env populations. A public instance of FLEA is hosted at http://flea.datamonkey.org. The Python source code for the FLEA pipeline can be found at https://github.com/veg/flea-pipeline. The client-side application is available at https://github.com/veg/flea-web-app. A live demo of the P018 results can be found at http://flea.murrell.group/view/P018.


July 19, 2019  |  

HIV envelope glycoform heterogeneity and localized diversity govern the initiation and maturation of a V2 apex broadly neutralizing antibody lineage.

Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.


July 7, 2019  |  

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae at a single institution: insights into endemicity from whole-genome sequencing.

The global emergence of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) multilocus sequence type ST258 is widely recognized. Less is known about the molecular and epidemiological details of non-ST258 K. pneumoniae in the setting of an outbreak mediated by an endemic plasmid. We describe the interplay of blaKPC plasmids and K. pneumoniae strains and their relationship to the location of acquisition in a U.S. health care institution. Whole-genome sequencing (WGS) analysis was applied to KPC-Kp clinical isolates collected from a single institution over 5 years following the introduction of blaKPC in August 2007, as well as two plasmid transformants. KPC-Kp from 37 patients yielded 16 distinct sequence types (STs). Two novel conjugative blaKPC plasmids (pKPC_UVA01 and pKPC_UVA02), carried by the hospital index case, accounted for the presence of blaKPC in 21/37 (57%) subsequent cases. Thirteen (35%) isolates represented an emergent lineage, ST941, which contained pKPC_UVA01 in 5/13 (38%) and pKPC_UVA02 in 6/13 (46%) cases. Seven (19%) isolates were the epidemic KPC-Kp strain, ST258, mostly imported from elsewhere and not carrying pKPC_UVA01 or pKPC_UVA02. Using WGS-based analysis of clinical isolates and plasmid transformants, we demonstrate the unexpected dispersal of blaKPC to many non-ST258 lineages in a hospital through spread of at least two novel blaKPC plasmids. In contrast, ST258 KPC-Kp was imported into the institution on numerous occasions, with other blaKPC plasmid vectors and without sustained transmission. Instead, a newly recognized KPC-Kp strain, ST941, became associated with both novel blaKPC plasmids and spread locally, making it a future candidate for clinical persistence and dissemination. Copyright © 2015, Mathers et al.


July 7, 2019  |  

Whole-genome sequencing identifies emergence of a quinolone resistance mutation in a case of Stenotrophomonas maltophilia bacteremia.

Whole-genome sequences for Stenotrophomonas maltophilia serial isolates from a bacteremic patient before and after development of levofloxacin resistance were assembled de novo and differed by one single-nucleotide variant in smeT, a repressor for multidrug efflux operon smeDEF. Along with sequenced isolates from five contemporaneous cases, they displayed considerable diversity compared against all published complete genomes. Whole-genome sequencing and complete assembly can conclusively identify resistance mechanisms emerging in S. maltophilia strains during clinical therapy. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


July 7, 2019  |  

The complete genome sequence of the emerging pathogen Mycobacterium haemophilum explains its unique culture requirements.

Mycobacterium haemophilum is an emerging pathogen associated with a variety of clinical syndromes, most commonly skin infections in immunocompromised individuals. M. haemophilum exhibits a unique requirement for iron supplementation to support its growth in culture, but the basis for this property and how it may shape pathogenesis is unclear. Using a combination of Illumina, PacBio, and Sanger sequencing, the complete genome sequence of M. haemophilum was determined. Guided by this sequence, experiments were performed to define the basis for the unique growth requirements of M. haemophilum. We found that M. haemophilum, unlike many other mycobacteria, is unable to synthesize iron-binding siderophores known as mycobactins or to utilize ferri-mycobactins to support growth. These differences correlate with the absence of genes associated with mycobactin synthesis, secretion, and uptake. In agreement with the ability of heme to promote growth, we identified genes encoding heme uptake machinery. Consistent with its propensity to infect the skin, we show at the whole-genome level the genetic closeness of M. haemophilum with Mycobacterium leprae, an organism which cannot be cultivated in vitro, and we identify genes uniquely shared by these organisms. Finally, we identify means to express foreign genes in M. haemophilum. These data explain the unique culture requirements for this important pathogen, provide a foundation upon which the genome sequence can be exploited to improve diagnostics and therapeutics, and suggest use of M. haemophilum as a tool to elucidate functions of genes shared with M. leprae.Mycobacterium haemophilum is an emerging pathogen with an unknown natural reservoir that exhibits unique requirements for iron supplementation to grow in vitro. Understanding the basis for this iron requirement is important because it is fundamental to isolation of the organism from clinical samples and environmental sources. Defining the molecular basis for M. haemophilium’s growth requirements will also shed new light on mycobacterial strategies to acquire iron and can be exploited to define how differences in such strategies influence pathogenesis. Here, through a combination of sequencing and experimental approaches, we explain the basis for the iron requirement. We further demonstrate the genetic closeness of M. haemophilum and Mycobacterium leprae, the causative agent of leprosy which cannot be cultured in vitro, and we demonstrate methods to genetically manipulate M. haemophilum. These findings pave the way for the use of M. haemophilum as a model to elucidate functions of genes shared with M. leprae. Copyright © 2015 Tufariello et al.


July 7, 2019  |  

Whole-genome sequences of Burkholderia pseudomallei isolates exhibiting decreased meropenem susceptibility.

We report here paired isogenic Burkholderia pseudomallei genomes obtained from three patients receiving intravenous meropenem for melioidosis treatment, with post-meropenem isolates developing decreased susceptibility. Two genomes were finished, and four were drafted to improved high-quality standard. These genomes will be used to identify meropenem resistance mechanisms in B. pseudomallei. Copyright © 2017 Price et al.


July 7, 2019  |  

Emergence and evolution of multidrug-resistant Klebsiella pneumoniae with both blaKPC and blaCTX-M integrated in the chromosome.

The extended-spectrum-ß-lactamase (ESBL)- and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae represent serious and urgent threats to public health. In a retrospective study of multidrug-resistant K. pneumoniae, we identified three clinical isolates, CN1, CR14, and NY9, carrying both blaCTX-M and blaKPC genes. The complete genomes of these three K. pneumoniae isolates were de novo assembled by using both short- and long-read whole-genome sequencing. In CR14 and NY9, blaCTX-M and blaKPC were carried on two different plasmids. In contrast, CN1 had one copy of blaKPC-2 and three copies of blaCTX-M-15 integrated in the chromosome, for which the blaCTX-M-15 genes were linked to an insertion sequence, ISEcp1, whereas the blaKPC-2 gene was in the context of a Tn4401a transposition unit conjugated with a PsP3-like prophage. Intriguingly, downstream of the Tn4401a-blaKPC-2-prophage genomic island, CN1 also carried a clustered regularly interspaced short palindromic repeat (CRISPR)-cas array with four spacers targeting a variety of K. pneumoniae plasmids harboring antimicrobial resistance genes. Comparative genomic analysis revealed that there were two subtypes of type I-E CRISPR-cas in K. pneumoniae strains and suggested that the evolving CRISPR-cas, with its acquired novel spacer, induced the mobilization of antimicrobial resistance genes from plasmids into the chromosome. The integration and dissemination of multiple copies of blaCTX-M and blaKPC from plasmids to chromosome depicts the complex pandemic scenario of multidrug-resistant K. pneumoniae Additionally, the implications from this study also raise concerns for the application of a CRISPR-cas strategy against antimicrobial resistance. Copyright © 2017 American Society for Microbiology.


July 7, 2019  |  

Comparative genomics of Burkholderia multivorans, a ubiquitous pathogen with a highly conserved genomic structure.

The natural environment serves as a reservoir of opportunistic pathogens. A well-established method for studying the epidemiology of such opportunists is multilocus sequence typing, which in many cases has defined strains predisposed to causing infection. Burkholderia multivorans is an important pathogen in people with cystic fibrosis (CF) and its epidemiology suggests that strains are acquired from non-human sources such as the natural environment. This raises the central question of whether the isolation source (CF or environment) or the multilocus sequence type (ST) of B. multivorans better predicts their genomic content and functionality. We identified four pairs of B. multivorans isolates, representing distinct STs and consisting of one CF and one environmental isolate each. All genomes were sequenced using the PacBio SMRT sequencing technology, which resulted in eight high-quality B. multivorans genome assemblies. The present study demonstrated that the genomic structure of the examined B. multivorans STs is highly conserved and that the B. multivorans genomic lineages are defined by their ST. Orthologous protein families were not uniformly distributed among chromosomes, with core orthologs being enriched on the primary chromosome and ST-specific orthologs being enriched on the second and third chromosome. The ST-specific orthologs were enriched in genes involved in defense mechanisms and secondary metabolism, corroborating the strain-specificity of these virulence characteristics. Finally, the same B. multivorans genomic lineages occur in both CF and environmental samples and on different continents, demonstrating their ubiquity and evolutionary persistence.


July 7, 2019  |  

Generation of a collection of mutant tomato lines using pooled CRISPR libraries.

The high efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mutagenesis in plants enables the development of high-throughput mutagenesis strategies. By transforming pooled CRISPR libraries into tomato (Solanum lycopersicum), collections of mutant lines were generated with minimal transformation attempts and in a relatively short period of time. Identification of the targeted gene(s) was easily determined by sequencing the incorporated guide RNA(s) in the primary transgenic events. From a single transformation with a CRISPR library targeting the immunity-associated leucine-rich repeat subfamily XII genes, heritable mutations were recovered in 15 of the 54 genes targeted. To increase throughput, a second CRISPR library was made containing three guide RNAs per construct to target 18 putative transporter genes. This resulted in stable mutations in 15 of the 18 targeted genes, with some primary transgenic plants having as many as five mutated genes. Furthermore, the redundancy in this collection of plants allowed for the association of aberrant T0 phenotypes with the underlying targeted genes. Plants with mutations in a homolog of an Arabidopsis (Arabidopsis thaliana) boron efflux transporter displayed boron deficiency phenotypes. The strategy described here provides a technically simple yet high-throughput approach for generating a collection of lines with targeted mutations and should be applicable to any plant transformation system.© 2017 American Society of Plant Biologists. All Rights Reserved.


July 7, 2019  |  

Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors.

Common bacterial blight is a devastating bacterial disease of common bean (Phaseolus vulgaris) caused by Xanthomonas citri pv. fuscans and Xanthomonas phaseoli pv. phaseoli. These phylogenetically distant strains are able to cause similar symptoms on common bean, suggesting that they have acquired common genetic determinants of adaptation to common bean. Transcription Activator-Like (TAL) effectors are bacterial type III effectors that are able to induce the expression of host genes to promote infection or resistance. Their capacity to bind to a specific host DNA sequence suggests that they are potential candidates for host adaption.To study the diversity of tal genes from Xanthomonas strains responsible for common bacterial blight of bean, whole genome sequences of 17 strains representing the diversity of X. citri pv. fuscans and X. phaseoli pv. phaseoli were obtained by single molecule real time sequencing. Analysis of these genomes revealed the existence of four tal genes named tal23A, tal20F, tal18G and tal18H, respectively. While tal20F and tal18G were chromosomic, tal23A and tal18H were carried on plasmids and shared between phylogenetically distant strains, therefore suggesting recent horizontal transfers of these genes between X. citri pv. fuscans and X. phaseoli pv. phaseoli strains. Strikingly, tal23A was present in all strains studied, suggesting that it played an important role in adaptation to common bean. In silico predictions of TAL effectors targets in the common bean genome suggested that TAL effectors shared by X. citri pv. fuscans and X. phaseoli pv. phaseoli strains target the promoters of genes of similar functions. This could be a trace of convergent evolution among TAL effectors from different phylogenetic groups, and comforts the hypothesis that TAL effectors have been implied in the adaptation to common bean.Altogether, our results favour a model where plasmidic TAL effectors are able to contribute to host adaptation by being horizontally transferred between distant lineages.


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