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July 7, 2019  |  

From isolate to answer: how whole genome sequencing is helping us rapidly characterise nosocomial bacterial outbreaks

The occurrence of highly resistant bacterial pathogens has risen in recent years, causing immense strain on the healthcare industry. Hospital-acquired infections are arguably of most concern, as bacterial outbreaks in clinical settings provide an ideal environment for proliferation among vulnerable populations. Understanding these outbreaks beyond what can be determined with traditional clinical diagnostics and implementing these new techniques routinely in the hospital environment has now become a major focus. This brief review will discuss the three main whole genome sequence techniques available today, and how they are being used to further discriminate bacterial outbreaks in nosocomial settings.


July 7, 2019  |  

Bow-tie signaling in c-di-GMP: Machine learning in a simple biochemical network.

Bacteria of many species rely on a simple molecule, the intracellular secondary messenger c-di-GMP (Bis-(3′-5′)-cyclic dimeric guanosine monophosphate), to make a vital choice: whether to stay in one place and form a biofilm, or to leave it in search of better conditions. The c-di-GMP network has a bow-tie shaped architecture that integrates many signals from the outside world-the input stimuli-into intracellular c-di-GMP levels that then regulate genes for biofilm formation or for swarming motility-the output phenotypes. How does the ‘uninformed’ process of evolution produce a network with the right input/output association and enable bacteria to make the right choice? Inspired by new data from 28 clinical isolates of Pseudomonas aeruginosa and strains evolved in laboratory experiments we propose a mathematical model where the c-di-GMP network is analogous to a machine learning classifier. The analogy immediately suggests a mechanism for learning through evolution: adaptation though incremental changes in c-di-GMP network proteins acquires knowledge from past experiences and enables bacteria to use it to direct future behaviors. Our model clarifies the elusive function of the ubiquitous c-di-GMP network, a key regulator of bacterial social traits associated with virulence. More broadly, the link between evolution and machine learning can help explain how natural selection across fluctuating environments produces networks that enable living organisms to make sophisticated decisions.


July 7, 2019  |  

Resistance to ceftazidime-avibactam is due to tranposition of KPC in a porin-deficient strain of Klebsiella pneumoniae with increased efflux activity.

Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 µg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype. Copyright © 2017 American Society for Microbiology.


July 7, 2019  |  

Parallel evolution of two clades of a major Atlantic endemic Vibrio parahaemolyticus pathogen lineage by independent acquisition of related pathogenicity islands.

Shellfish-transmitted Vibrio parahaemolyticus infections have recently increased from locations with historically low disease incidence, such as the Northeast United States (US). This change coincided with a bacterial population shift towards human pathogenic variants occurring in part through the introduction of several Pacific native lineages (ST36, ST43 and ST636) to near-shore areas off the Atlantic coast of the Northeast US. Concomitantly, ST631 emerged as a major endemic pathogen. Phylogenetic trees of clinical and environmental isolates indicated that two clades diverged from a common ST631 ancestor, and in each of these clades, a human pathogenic variant evolved independently through acquisition of distinct Vibrio pathogenicity islands (VPaI). These VPaI differ from each other and bear little resemblance to hemolysin-containing VPaI from isolates of the pandemic clonal complex. Clade I ST631 isolates either harbored no hemolysins, or contained a chromosome I-inserted island we call VPaIß that encodes a type three secretion system (T3SS2ß) typical of Trh hemolysin-producers. The more clinically prevalent and clonal ST631 clade II had an island we call VPaI? that encodes both tdh and trh and that was inserted in chromosome II. VPaI? was derived from VPaIß but with some additional acquired elements in common with VPaI carried by pandemic isolates, exemplifying the mosaic nature of pathogenicity islands. Genomics comparisons and amplicon assays identified VPaI?-type islands containing tdh inserted adjacent to the ure cluster in the three introduced Pacific and most other emergent lineages. that collectively cause 67% of Northeast US infections as of 2016.IMPORTANCE The availability of three different hemolysin genotypes in the ST631 lineage provided a unique opportunity to employ genome comparisons to further our understanding of the processes underlying pathogen evolution. The fact that two different pathogenic clades arose in parallel from the same potentially benign lineage by independent VPaI acquisition is surprising considering the historically low prevalence of community members harboring VPaI in waters along the Northeast US coast that could serve as the source of this material. This illustrates a possible predisposition of some lineages to not only acquire foreign DNA but also to become human pathogens. Whereas the underlying cause for the expansion of V. parahaemolyticus lineages harboring VPaI? along the US Atlantic coast and spread of this element to multiple lineages that underlies disease emergence is not known, this work underscores the need to define the environment factors that favor bacteria harboring VPaI in locations of emergent disease. Copyright © 2017 American Society for Microbiology.


July 7, 2019  |  

First complete genome sequences of Xanthomonas citri pv. vignicola strains CFBP7111, CFBP7112, and CFBP7113 obtained using long-read technology

Xanthomonas citri pv. vignicola strains cause bacterial blight of the legume crop cowpea. We report whole-genome sequences of three X. citri pv. vignicola strains obtained using PacBio single-molecule real-time sequencing. Such genomic data provide new information on pathogenicity factors, such as transcription activator-like effectors. Copyright © 2017 Ruh et al.


July 7, 2019  |  

Genome architecture and evolution of a unichromosomal asexual nematode.

Asexual reproduction in animals, though rare, is the main or exclusive mode of reproduction in some long-lived lineages. The longevity of asexual clades may be correlated with the maintenance of heterozygosity by mechanisms that rearrange genomes and reduce recombination. Asexual species thus provide an opportunity to gain insight into the relationship between molecular changes, genome architecture, and cellular processes. Here we report the genome sequence of the parthenogenetic nematode Diploscapter pachys with only one chromosome pair. We show that this unichromosomal architecture is shared by a long-lived clade of asexual nematodes closely related to the genetic model organism Caenorhabditis elegans. Analysis of the genome assembly reveals that the unitary chromosome arose through fusion of six ancestral chromosomes, with extensive rearrangement among neighboring regions. Typical nematode telomeres and telomeric protection-encoding genes are lacking. Most regions show significant heterozygosity; homozygosity is largely concentrated to one region and attributed to gene conversion. Cell-biological and molecular evidence is consistent with the absence of key features of meiosis I, including synapsis and recombination. We propose that D. pachys preserves heterozygosity and produces diploid embryos without fertilization through a truncated meiosis. As a prelude to functional studies, we demonstrate that D. pachys is amenable to experimental manipulation by RNA interference. Copyright © 2017 Elsevier Ltd. All rights reserved.


July 7, 2019  |  

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOS(YA), replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.


July 7, 2019  |  

Evaluation of oritavancin dosing strategies against vancomycin-resistant Enterococcus faecium isolates with or without reduced susceptibility to daptomycin in an in vitro pharmacokinetic/pharmacodynamic model.

Clinical development of nonsusceptibility to the lipopeptide antibiotic daptomycin remains a serious concern during therapy for infections caused by vancomycin-resistant Enterococcus faecium (VREfm). The long-acting lipoglycopeptide oritavancin exhibits potent in vitro activity against VREfm although its safety and efficacy in treating clinical VREfm infections have not been established. In this study, novel dosing regimens of daptomycin and oritavancin were assessed against both VREfm and daptomycin-nonsusceptible VREfm isolates in an in vitro pharmacokinetic/pharmacodynamic model. Copyright © 2017 American Society for Microbiology.


July 7, 2019  |  

Copy number variation probes inform diverse applications

A major contributor to inter-individual genomic variability is copy number variation (CNV). CNVs change the diploid status of the DNA, involve one or multiple genes, and may disrupt coding regions, affect regulatory elements, or change gene dosage. While some of these changes may have no phenotypic consequences, others underlie disease, explain evolutionary processes, or impact the response to medication.


July 7, 2019  |  

A 3-way hybrid approach to generate a new high-quality chimpanzee reference genome (Pan_tro_3.0).

The chimpanzee is arguably the most important species for the study of human origins. A key resource for these studies is a high-quality reference genome assembly; however, as with most mammalian genomes, the current iteration of the chimpanzee reference genome assembly is highly fragmented. In the current iteration of the chimpanzee reference genome assembly (Pan_tro_2.1.4), the sequence is scattered across more then 183 000 contigs, incorporating more than 159 000 gaps, with a genome-wide contig N50 of 51 Kbp. In this work, we produce an extensive and diverse array of sequencing datasets to rapidly assemble a new chimpanzee reference that surpasses previous iterations in bases represented and organized in large scaffolds. To this end, we show substantial improvements over the current release of the chimpanzee genome (Pan_tro_2.1.4) by several metrics, such as increased contiguity by >750% and 300% on contigs and scaffolds, respectively, and closure of 77% of gaps in the Pan_tro_2.1.4 assembly gaps spanning >850 Kbp of the novel coding sequence based on RNASeq data. We further report more than 2700 genes that had putatively erroneous frame-shift predictions to human in Pan_tro_2.1.4 and show a substantial increase in the annotation of repetitive elements. We apply a simple 3-way hybrid approach to considerably improve the reference genome assembly for the chimpanzee, providing a valuable resource for the study of human origins. Furthermore, we produce extensive sequencing datasets that are all derived from the same cell line, generating a broad non-human benchmark dataset.© The Author 2017. Published by Oxford University Press.


July 7, 2019  |  

Institutional profile: translational pharmacogenomics at the Icahn School of Medicine at Mount Sinai.

For almost 50 years, the Icahn School of Medicine at Mount Sinai has continually invested in genetics and genomics, facilitating a healthy ecosystem that provides widespread support for the ongoing programs in translational pharmacogenomics. These programs can be broadly cataloged into discovery, education, clinical implementation and testing, which are collaboratively accomplished by multiple departments, institutes, laboratories, companies and colleagues. Focus areas have included drug response association studies and allele discovery, multiethnic pharmacogenomics, personalized genotyping and survey-based education programs, pre-emptive clinical testing implementation and novel assay development. This overview summarizes the current state of translational pharmacogenomics at Mount Sinai, including a future outlook on the forthcoming expansions in overall support, research and clinical programs, genomic technology infrastructure and the participating faculty.


July 7, 2019  |  

High incidence of invasive group A Streptococcus disease caused by strains of uncommon emm types in Thunder Bay, Ontario, Canada.

An outbreak of type emm59 invasive group A Streptococcus (iGAS) disease was declared in 2008 in Thunder Bay District, Northwestern Ontario, two years after a country-wide emm59 epidemic was recognized in Canada. Despite a declining number of emm59 infections since 2010, numerous cases of iGAS disease continue to be reported in the area. We collected clinical information on all iGAS cases recorded in Thunder Bay District from 2008-2013. We also emm typed and sequenced the genomes of all available strains isolated in 2011-2013 from iGAS infections, and from severe cases of soft tissue infections. We used whole-genome data to investigate the population structure of GAS strains of the most frequently isolated emm types. We report increased incidence of iGAS in Thunder Bay compared to the metropolitan area of Toronto/Peel and the province of Ontario. Illicit drug use, alcohol abuse, homelessness and hepatitis C infection were underlying diseases or conditions that might have predisposed patients to iGAS disease. Most cases were caused by clonal strains of “skin” or “generalist” emm types (i.e. emm82, emm87, emm101, emm4, emm83, and emm114), uncommonly seen in other areas of the province. We observed rapid waxing and waning of emm types causing disease and their replacement by other emm types associated with the same tissue tropisms. Thus, iGAS disease in Thunder Bay District predominantly affects a select population of disadvantaged persons and is caused by clonally related strains of a few “skin” and “generalist” emm types less commonly associated with iGAS in other areas of Ontario. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


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