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Asset Tag: Infectious disease

September 22, 2019

The global distribution and spread of the mobilized colistin resistance gene mcr-1.

Colistin represents one of the few available drugs for treating infections caused by carbapenem-resistant Enterobacteriaceae. As such, the recent plasmid-mediated spread of the colistin resistance gene mcr-1 poses a significant public health threat, requiring global monitoring and surveillance. Here, we characterize the global distribution of mcr-1 using a data set of 457 mcr-1-positive sequenced isolates. We find mcr-1 in various plasmid types but identify an immediate background common to all mcr-1 sequences. Our analyses establish that all mcr-1 elements in circulation descend from the same initial mobilization of mcr-1 by an ISApl1 transposon in the mid 2000s (2002-2008; 95% highest posterior density), followed by a marked demographic expansion, which led to its current global distribution. Our results provide the first systematic phylogenetic analysis of the origin and spread of mcr-1, and emphasize the importance of understanding the movement of antibiotic resistance genes across multiple levels of genomic organization.

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September 22, 2019

CliqueSNV: Scalable reconstruction of intra-host viral populations from NGS reads

Highly mutable RNA viruses such as influenza A virus, human immunodeficiency virus and hepatitis C virus exist in infected hosts as highly heterogeneous populations of closely related genomic variants. The presence of low-frequency variants with few mutations with respect to major strains may result in an immune escape, emergence of drug resistance, and an increase of virulence and infectivity. Next-generation sequencing technologies permit detection of sample intra-host viral population at extremely great depth, thus providing an opportunity to access low-frequency variants. Long read lengths offered by single-molecule sequencing technologies allow all viral variants to be sequenced in a single pass. However, high sequencing error rates limit the ability to study heterogeneous viral populations composed of rare, closely related variants. In this article, we present CliqueSNV, a novel reference-based method for reconstruction of viral variants from NGS data. It efficiently constructs an allele graph based on linkage between single nucleotide variations and identifies true viral variants by merging cliques of that graph using combinatorial optimization techniques. The new method outperforms existing methods in both accuracy and running time on experimental and simulated NGS data for titrated levels of known viral variants. For PacBio reads, it accurately reconstructs variants with frequency as low as 0.1%. For Illumina reads, it fully reconstructs main variants. The open source implementation of CliqueSNV is freely available for download at https://github.com/vyacheslav-tsivina/CliqueSNV

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September 22, 2019

New Delhi metallo-beta-lactamase-producing Enterobacteriaceae in South Korea between 2010 and 2015.

This study was carried out to investigate the epidemiological time-course of New Delhi metallo-beta-lactamase- (NDM-) mediated carbapenem resistance in Enterobacteriaceae in South Korea. A total of 146 non-duplicate NDM-producing Enterobacteriaceae recovered between 2010 and 2015 were voluntarily collected from 33 general hospitals and confirmed by PCR. The species were identified by sequences of the 16S rDNA. Antimicrobial susceptibility was determined either by the disk diffusion method or by broth microdilution, and the carbapenem MICs were determined by agar dilution. Then, multilocus sequence typing and PCR-based replicon typing was carried out. Co-carried genes for drug resistance were identified by PCR and sequencing. The entire genomes of eight random selected NDM producers were sequenced. A total of 69 Klebsiella pneumoniae of 12 sequence types (STs), 34 Escherichia coli of 15 STs, 28 Enterobacter spp. (including one Enterobacter aerogenes), nine Citrobacter freundii, four Raoultella spp., and two Klebsiella oxytoca isolates produced either NDM-1 (n = 126), NDM-5 (n = 18), or NDM-7 (n = 2). The isolates co-produced CTX-M-type ESBL (52.1%), AmpCs (27.4%), additional carbapenemases (7.1%), and/or 16S rRNA methyltransferases (4.8%), resulting in multidrug-resistance (47.9%) or extensively drug-resistance (52.1%). Among plasmids harboring blaNDM, IncX3 was predominant (77.4%), followed by the IncFII type (5.8%). Genome analysis revealed inter-species and inter-strain horizontal gene transfer of the plasmid. Both clonal dissemination and plasmid transfer contributed to the wide dissemination of NDM producers in South Korea.

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September 22, 2019

Challenges of Francisella classification exemplified by an atypical clinical isolate.

The accumulation of sequenced Francisella strains has made it increasingly apparent that the 16S rRNA gene alone is not enough to stratify the Francisella genus into precise and clinically useful classifications. Continued whole-genome sequencing of isolates will provide a larger base of knowledge for targeted approaches with broad applicability. Additionally, examination of genomic information on a case-by-case basis will help resolve outstanding questions regarding strain stratification. We report the complete genome sequence of a clinical isolate, designated here as F. novicida-like strain TCH2015, acquired from the lymph node of a 6-year-old male. Two features were atypical for F. novicida: exhibition of functional oxidase activity and additional gene content, including proposed virulence determinants. These differences, which could potentially impact virulence and clinical diagnosis, emphasize the need for more comprehensive methods to profile Francisella isolates. This study highlights the value of whole-genome sequencing, which will lead to a more robust database of environmental and clinical genomes and inform strategies to improve detection and classification of Francisella strains. Copyright © 2017 Elsevier Inc. All rights reserved.

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September 22, 2019

Genetic characterization of a VanG-type vancomycin-resistant Enterococcus faecium clinical isolate.

To characterize, phenotypically and genotypically, the first Enterococcus faecium clinical isolate harbouring a vanG operon.The antibiotic resistance profile of E. faecium 16-346 was determined and its whole genome sequenced using PacBio technology. Attempts to transfer vancomycin resistance by filter mating were performed and the inducibility of expression of the vanG operon was studied by reverse-transcription quantitative PCR (RT-qPCR) in the presence or absence of subinhibitory concentrations of vancomycin.E. faecium 16-346 was resistant to rifampicin (MIC >4?mg/L), erythromycin (MIC >4?mg/L), tetracycline (MIC >16?mg/L) and vancomycin (MIC 8?mg/L), but susceptible to teicoplanin (MIC 0.5?mg/L). The strain harboured the vanG operon in its chromosome, integrated in a 45.5?kb putative mobile genetic element, similar to that of Enterococcus faecalis BM4518. We were unable to transfer vancomycin resistance from E. faecium 16-346 to E. faecium BM4107 and E. faecalis JH2-2. Lastly, transcription of the vanG gene was inducible by vancomycin.This is, to the best of our knowledge, the first report of a VanG-type vancomycin-resistant strain of E. faecium. Despite the alarm pulled because of the therapeutic problems caused by VRE, our work shows that new resistant loci can still be found in E. faecium.

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September 22, 2019

Comparative genome analysis reveals a complex population structure of Legionella pneumophila subspecies.

The majority of Legionnaires’ disease (LD) cases are caused by Legionella pneumophila, a genetically heterogeneous species composed of at least 17 serogroups. Previously, it was demonstrated that L. pneumophila consists of three subspecies: pneumophila, fraseri and pascullei. During an LD outbreak investigation in 2012, we detected that representatives of both subspecies fraseri and pascullei colonized the same water system and that the outbreak-causing strain was a new member of the least represented subspecies pascullei. We used partial sequence based typing consensus patterns to mine an international database for additional representatives of fraseri and pascullei subspecies. As a result, we identified 46 sequence types (STs) belonging to subspecies fraseri and two STs belonging to subspecies pascullei. Moreover, a recent retrospective whole genome sequencing analysis of isolates from New York State LD clusters revealed the presence of a fourth L. pneumophila subspecies that we have termed raphaeli. This subspecies consists of 15 STs. Comparative analysis was conducted using the genomes of multiple members of all four L. pneumophila subspecies. Whereas each subspecies forms a distinct phylogenetic clade within the L. pneumophila species, they share more average nucleotide identity with each other than with other Legionella species. Unique genes for each subspecies were identified and could be used for rapid subspecies detection. Improved taxonomic classification of L. pneumophila strains may help identify environmental niches and virulence attributes associated with these genetically distinct subspecies. Published by Elsevier B.V.

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September 22, 2019

Genetic basis of chromosomally-encoded mcr-1 gene.

Compared with plasmid-borne mcr-1, the occurrence of chromosomally-encoded mcr-1 is rare although it has been reported in several cases. This study aimed to investigate the genetic features of chromosomally-encoded mcr-1 among Escherichia coli strains as well as the potential genetic basis governing mobilisation of mcr-1 in bacterial chromosomes. The genome sequences of 16 E. coli strains containing a chromosomal mcr-1 gene were obtained and analysed. Phylogenetic and whole-genome sequencing (WGS) analysis demonstrated that mcr-1 was associated with four major types of genetic arrangements, namely ISApl1-mcr1-orf, Tn6330, complex Tn6330 and ?Tn6330 in chromosomes of genetically unrelated E. coli strains. The mcr-1-carrying mobile elements were shown to insert into the AT-rich region, which was also the case for ISApl1. Analysis of complete E. coli genome sequences showed that there were multiple copies of ISApl1 present in E. coli chromosomes that also carried mcr-1, whilst all mcr-1-negative chromosomes were absent of any copy of ISApl1, suggesting the strong association of ISApl1 and mcr-1. Insertion of ISApl1 into E. coli chromosomes may be a prerequisite for the insertion of mcr-1-carrying mobile elements. Insertion of mcr-1 into E. coli chromosomes would enable it to become intrinsically resistant, which is expected to become more prevalent. Policy on the prudent use of colistin both in veterinary and clinical settings should be imposed globally to further prevent dissemination of mcr-1 in E. coli and other bacterial pathogens. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

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September 22, 2019

Autologous cell therapy approach for Duchenne muscular dystrophy using PiggyBac transposons and mesoangioblasts.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease currently without cure. We investigated the use of the PiggyBac transposon for full-length dystrophin expression in murine mesoangioblast (MABs) progenitor cells. DMD murine MABs were transfected with transposable expression vectors for full-length dystrophin and transplanted intramuscularly or intra-arterially into mdx/SCID mice. Intra-arterial delivery indicated that the MABs could migrate to regenerating muscles to mediate dystrophin expression. Intramuscular transplantation yielded dystrophin expression in 11%-44% of myofibers in murine muscles, which remained stable for the assessed period of 5 months. The satellite cells isolated from transplanted muscles comprised a fraction of MAB-derived cells, indicating that the transfected MABs may colonize the satellite stem cell niche. Transposon integration site mapping by whole-genome sequencing indicated that 70% of the integrations were intergenic, while none was observed in an exon. Muscle resistance assessment by atomic force microscopy indicated that 80% of fibers showed elasticity properties restored to those of wild-type muscles. As measured in vivo, transplanted muscles became more resistant to fatigue. This study thus provides a proof-of-principle that PiggyBac transposon vectors may mediate full-length dystrophin expression as well as functional amelioration of the dystrophic muscles within a potential autologous cell-based therapeutic approach of DMD. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Horizontal antimicrobial resistance transfer drives epidemics of multiple Shigella species.

Horizontal gene transfer has played a role in developing the global public health crisis of antimicrobial resistance (AMR). However, the dynamics of AMR transfer through bacterial populations and its direct impact on human disease is poorly elucidated. Here, we study parallel epidemic emergences of multiple Shigella species, a priority AMR organism, in men who have sex with men to gain insight into AMR emergence and spread. Using genomic epidemiology, we show that repeated horizontal transfer of a single AMR plasmid among Shigella enhanced existing and facilitated new epidemics. These epidemic patterns contrasted with slighter, slower increases in disease caused by organisms with vertically inherited (chromosomally encoded) AMR. This demonstrates that horizontal transfer of AMR directly affects epidemiological outcomes of globally important AMR pathogens and highlights the need for integration of genomic analyses into all areas of AMR research, surveillance and management.

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September 22, 2019

Extensive gene amplification as a mechanism for piperacillin-tazobactam resistance in Escherichia coli.

Although the TEM-1 ß-lactamase (BlaTEM-1) hydrolyzes penicillins and narrow-spectrum cephalosporins, organisms expressing this enzyme are typically susceptible to ß-lactam/ß-lactamase inhibitor combinations such as piperacillin-tazobactam (TZP). However, our previous work led to the discovery of 28 clinical isolates of Escherichia coli resistant to TZP that contained only blaTEM-1 One of these isolates, E. coli 907355, was investigated further in this study. E. coli 907355 exhibited significantly higher ß-lactamase activity and BlaTEM-1 protein levels when grown in the presence of subinhibitory concentrations of TZP. A corresponding TZP-dependent increase in blaTEM-1 copy number was also observed, with as many as 113 copies of the gene detected per cell. These results suggest that TZP treatment promotes an increase in blaTEM-1 gene dosage, allowing BlaTEM-1 to reach high enough levels to overcome inactivation by the available tazobactam in the culture. To better understand the nature of the blaTEM-1 copy number proliferation, whole-genome sequence (WGS) analysis was performed on E. coli 907355 in the absence and presence of TZP. The WGS data revealed that the blaTEM-1 gene is located in a 10-kb genomic resistance module (GRM) that contains multiple resistance genes and mobile genetic elements. The GRM was found to be tandemly repeated at least 5 times within a p1ESCUM/p1ECUMN-like plasmid when bacteria were grown in the presence of TZP.IMPORTANCE Understanding how bacteria acquire resistance to antibiotics is essential for treating infected patients effectively, as well as preventing the spread of resistant organisms. In this study, a clinical isolate of E. coli was identified that dedicated more than 15% of its genome toward tandem amplification of a ~10-kb resistance module, allowing it to escape antibiotic-mediated killing. Our research is significant in that it provides one possible explanation for clinical isolates that exhibit discordant behavior when tested for antibiotic resistance by different phenotypic methods. Our research also shows that GRM amplification is difficult to detect by short-read WGS technologies. Analysis of raw long-read sequence data was required to confirm GRM amplification as a mechanism of antibiotic resistance. Copyright © 2018 Schechter et al.

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September 22, 2019

A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions.

Many bacteria utilize simple DNA sequence repeats as a mechanism to randomly switch genes on and off. This process is called phase variation. Several phase-variable N6-adenine DNA-methyltransferases from Type III restriction-modification systems have been reported in bacterial pathogens. Random switching of DNA methyltransferases changes the global DNA methylation pattern, leading to changes in gene expression. These epigenetic regulatory systems are called phasevarions – phase-variable regulons. The extent of these phase-variable genes in the bacterial kingdom is unknown. Here, we interrogated a database of restriction-modification systems, REBASE, by searching for all simple DNA sequence repeats in mod genes that encode Type III N6-adenine DNA-methyltransferases. We report that 17.4% of Type III mod genes (662/3805) contain simple sequence repeats. Of these, only one-fifth have been previously identified. The newly discovered examples are widely distributed and include many examples in opportunistic pathogens as well as in environmental species. In many cases, multiple phasevarions exist in one genome, with examples of up to 4 independent phasevarions in some species. We found several new types of phase-variable mod genes, including the first example of a phase-variable methyltransferase in pathogenic Escherichia coli. Phasevarions are a common epigenetic regulation contingency strategy used by both pathogenic and non-pathogenic bacteria.

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September 22, 2019

Identification and pathogenomic analysis of an Escherichia coli strain producing a novel Shiga toxin 2 subtype.

Shiga toxin (Stx) is the key virulent factor in Shiga toxin-producing Escherichia coli (STEC). To date, three Stx1 subtypes and seven Stx2 subtypes have been described in E. coli, which differed in receptor preference and toxin potency. Here, we identified a novel Stx2 subtype designated Stx2h in E. coli strains isolated from wild marmots in the Qinghai-Tibetan plateau, China. Stx2h shares 91.9% nucleic acid sequence identity and 92.9% amino acid identity to the nearest Stx2 subtype. The expression of Stx2h in type strain STEC299 was inducible by mitomycin C, and culture supernatant from STEC299 was cytotoxic to Vero cells. The Stx2h converting prophage was unique in terms of insertion site and genetic composition. Whole genome-based phylo- and patho-genomic analysis revealed STEC299 was closer to other pathotypes of E. coli than STEC, and possesses virulence factors from other pathotypes. Our finding enlarges the pool of Stx2 subtypes and highlights the extraordinary genomic plasticity of E. coli strains. As the emergence of new Shiga toxin genotypes and new Stx-producing pathotypes pose a great threat to the public health, Stx2h should be further included in E. coli molecular typing, and in epidemiological surveillance of E. coli infections.

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September 22, 2019

Epigenetic landscape influences the liver cancer genome architecture.

The accumulations of different types of genetic alterations such as nucleotide substitutions, structural rearrangements and viral genome integrations and epigenetic alterations contribute to carcinogenesis. Here, we report correlation between the occurrence of epigenetic features and genetic aberrations by whole-genome bisulfite, whole-genome shotgun, long-read, and virus capture sequencing of 373 liver cancers. Somatic substitutions and rearrangement breakpoints are enriched in tumor-specific hypo-methylated regions with inactive chromatin marks and actively transcribed highly methylated regions in the cancer genome. Individual mutation signatures depend on chromatin status, especially, signatures with a higher transcriptional strand bias occur within active chromatic areas. Hepatitis B virus (HBV) integration sites are frequently detected within inactive chromatin regions in cancer cells, as a consequence of negative selection for integrations in active chromatin regions. Ultra-high structural instability and preserved unmethylation of integrated HBV genomes are observed. We conclude that both precancerous and somatic epigenetic features contribute to the cancer genome architecture.

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September 22, 2019

RTS,S/AS01 malaria vaccine mismatch observed among Plasmodium falciparum isolates from southern and central Africa and globally.

The RTS,S/AS01 malaria vaccine encompasses the central repeats and C-terminal of Plasmodium falciparum circumsporozoite protein (PfCSP). Although no Phase II clinical trial studies observed evidence of strain-specific immunity, recent studies show a decrease in vaccine efficacy against non-vaccine strain parasites. In light of goals to reduce malaria morbidity, anticipating the effectiveness of RTS,S/AS01 is critical to planning widespread vaccine introduction. We deep sequenced C-terminal Pfcsp from 77 individuals living along the international border in Luapula Province, Zambia and Haut-Katanga Province, the Democratic Republic of the Congo (DRC) and compared translated amino acid haplotypes to the 3D7 vaccine strain. Only 5.2% of the 193 PfCSP sequences from the Zambia-DRC border region matched 3D7 at all 84 amino acids. To further contextualize the genetic diversity sampled in this study with global PfCSP diversity, we analyzed an additional 3,809 Pfcsp sequences from the Pf3k database and constructed a haplotype network representing 15 countries from Africa and Asia. The diversity observed in our samples was similar to the diversity observed in the global haplotype network. These observations underscore the need for additional research assessing genetic diversity in P. falciparum and the impact of PfCSP diversity on RTS,S/AS01 efficacy.

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September 22, 2019

Genomic analysis of oral Campylobacter concisus strains identified a potential bacterial molecular marker associated with active Crohn’s disease.

Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD) including Crohn’s disease (CD) and ulcerative colitis (UC). C. concisus consists of two genomospecies (GS) and diverse strains. This study aimed to identify molecular markers to differentiate commensal and IBD-associated C. concisus strains. The genomes of 63 oral C. concisus strains isolated from patients with IBD and healthy controls were examined, of which 38 genomes were sequenced in this study. We identified a novel secreted enterotoxin B homologue, Csep1. The csep1 gene was found in 56% of GS2 C. concisus strains, presented in the plasmid pICON or the chromosome. A six-nucleotide insertion at the position 654-659?bp in csep1 (csep1-6bpi) was found. The presence of csep1-6bpi in oral C. concisus strains isolated from patients with active CD (47%, 7/15) was significantly higher than that in strains from healthy controls (0/29, P?=?0.0002), and the prevalence of csep1-6bpi positive C. concisus strains was significantly higher in patients with active CD (67%, 4/6) as compared to healthy controls (0/23, P?=?0.0006). Proteomics analysis detected the Csep1 protein. A csep1 gene hot spot in the chromosome of different C. concisus strains was found. The pICON plasmid was only found in GS2 strains isolated from the two relapsed CD patients with small bowel complications. This study reports a C. concisus molecular marker (csep1-6bpi) that is associated with active CD.

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