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Asset Tag: Human Biomedical Research,

July 7, 2019

Complete genome sequence of Acinetobacter baumannii A1296 (ST1469) with a small plasmid harbouring the tet(39) tetracycline resistance gene.

Acinetobacter baumannii is considered an important nosocomial pathogen worldwide owing to its increasing antibiotic resistance. This study aimed to determine the complete genome sequence of A. baumannii strain A1296 and to perform a comparative analysis among A. baumannii.The complete genome sequence of A. baumannii A1296 was sequenced on two SMRT cells using P6C4 chemistry on a PacBio Single Molecule, Real-Time (SMRT) RS II instrument. The A1296 genome sequence was annotated using Prokaryotic Genome Automatic Annotation Pipeline (PGAAP), and the sequence type and resistance genes of the strain were analysed.Here we present the complete genome sequence of A. baumannii strain A1296, belonging to a novel sequence type (ST1469) and isolated from patient in China, that was sensitive to multiple antibiotics. The genome of A. baumannii A1296 was 3810701bp in length, including one circular chromosome and two plasmids. The tet(39) resistance gene was located on the small plasmid in this A. baumannii strain.The genome sequence of A. baumannii strain A1296 can be used as a reference sequence for comparative analysis aimed at elucidating the acquisition, dissemination and mobilisation of resistance genes among A. baumannii. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

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July 7, 2019

Complete genome sequence of Salmonella enterica subsp. enterica serovar Minnesota strain

Mango has been implicated as food vehicle in several Salmonella-causing foodborne outbreaks. Here, Salmonella enterica subsp. enterica serovar Minnesota was isolated from fresh mango fruit imported from Mexico in 2014. The complete genome sequence of S. Minnesota CFSAN017963 was sequenced using single-molecule real-time DNA sequencing. Distinct prophage regions, Salmonella pathogenicity islands, and fimbrial gene clusters were observed in comparative genomic analysis on S. Minnesota CFSAN017963 with other phylogenetically closely related Salmonella serovars. Core genome multilocus sequencing typing analysis of all the S. Minnesota isolates in the Genbank and Enterobase also revealed a high genomic diversity among the genomes analyzed.

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July 7, 2019

Detection of complex structural variation from paired-end sequencing data

Detecting structural variants (SVs) from sequencing data is a key problem in genome analysis, but the full diversity of SVs is not captured by most methods. We introduce the Automated Reconstruction of Complex Structural Variants (ARC-SV) method, which detects a broad class of structural variants from paired-end whole genome sequencing (WGS) data. Analysis of samples from NA12878 and HuRef suggests that complex SVs are often misclassified by traditional methods. We validated our results both experimentally and by comparison to whole genome assembly and PacBio data; ARC-SV compares favorably to existing algorithms in general and gives state-of-the-art results on complex SV detection. By expanding the range of detectable SVs compared to commonly-used algorithms, ARC-SV allows additional information to be extracted from existing WGS data.

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July 7, 2019

Dissemination and characteristics of a novel plasmid-encoded carbapenem-hydrolyzing class D beta-lactamase, OXA-436 from four patients involving six different hospitals in Denmark.

The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D carbapenemase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates including Enterobacter asburiae (n=3), Citrobacter freundii (n=2) and Klebsiella pneumoniae (n=1) were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4-92.8% identity to other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to OXA-48 and OXA-181 with activity against penicillins including temocillin, limited or no activity against extended-spectrum cephalosporins and activity against carbapenems. The blaOXA-436 gene was located on a conjugative ~314 kb IncHI2/IncHI2A plasmid belonging to pMLST ST1, in a region surrounded by chromosomal genes previously identified adjacent to blaOXA-genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with similar functional properties as OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and plasmid location of blaOXA-436 illustrates its potential for further dissemination. Copyright © 2017 American Society for Microbiology.

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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.

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July 7, 2019

Global phylogenetic analysis of Escherichia coli and plasmids carrying the mcr-1 gene indicates bacterial diversity but plasmid restriction.

To understand the dynamics behind the worldwide spread of the mcr-1 gene, we determined the population structure of Escherichia coli and of mobile genetic elements (MGEs) carrying the mcr-1 gene. After a systematic review of the literature we included 65 E. coli whole genome sequences (WGS), adding 6 recently sequenced travel related isolates, and 312 MLST profiles. We included 219 MGEs described in 7 Enterobacteriaceae species isolated from human, animal and environmental samples. Despite a high overall diversity, 2 lineages were observed in the E. coli population that may function as reservoirs of the mcr-1 gene, the largest of which was linked to ST10, a sequence type known for its ubiquity in human faecal samples and in food samples. No genotypic clustering by geographical origin or isolation source was observed. Amongst a total of 13 plasmid incompatibility types, the IncI2, IncX4 and IncHI2 plasmids accounted for more than 90% of MGEs carrying the mcr-1 gene. We observed significant geographical clustering with regional spread of IncHI2 plasmids in Europe and IncI2 in Asia. These findings point towards promiscuous spread of the mcr-1 gene by efficient horizontal gene transfer dominated by a limited number of plasmid incompatibility types.

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July 7, 2019

DNA methylation profiling using long-read Single Molecule Real-Time bisulfite sequencing (SMRT-BS).

For the past two decades, bisulfite sequencing has been a widely used method for quantitative CpG methylation detection of genomic DNA. Coupled with PCR amplicon cloning, bisulfite Sanger sequencing allows for allele-specific CpG methylation assessment; however, its time-consuming protocol and inability to multiplex has recently been overcome by next-generation bisulfite sequencing techniques. Although high-throughput sequencing platforms have enabled greater accuracy in CpG methylation quantitation as a result of increased bisulfite sequencing depth, most common sequencing platforms generate reads that are similar in length to the typical bisulfite PCR size range (~300-500 bp). Using the Pacific Biosciences (PacBio) sequencing platform, we developed single molecule real-time bisulfite sequencing (SMRT-BS), which is an accurate targeted CpG methylation analysis method capable of a high degree of multiplexing and long read lengths. SMRT-BS is reproducible and was found to be concordant with other lower throughput quantitative CpG methylation methods. Moreover, the ability to sequence up to ~1.5-2.0 kb amplicons, when coupled with an optimized bisulfite-conversion protocol, allows for more thorough assessment of CpG islands and increases the capacity for studying the relationship between single nucleotide variants and allele-specific CpG methylation.

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July 7, 2019

pSY153-MDR, a p12969-DIM-related mega plasmid carrying blaIMP-45 and armA, from clinical Pseudomonas putida.

This work characterized mega plasmid pSY153-MDR, carrying blaIMP-45 and armA, from a multidrug-resistant (MDR) Pseudomonas putida isolate from the urine of a cerebral infarction patient in China. The backbone of pSY153-MDR was closely related to Pseudomonas plasmids p12969-DIM, pOZ176, pBM413, pTTS12, and pRBL16, and could not be assigned to any of the known incompatibility groups. The accessory modules of pSY153-MDR were composed of 10 individual insertion sequence elements and two different MDR regions, and differed dramatically from the above plasmids. Fifteen non-redundant resistance markers were identified to be involved in resistance to at least eight distinct classes of antibiotics. All of these resistance genes were associated with mobile elements, and were embedded within the two MDR regions. blaIMP-45 and armA coexisted in a Tn1403-Tn1548 region, which was generated from homologous recombination of Tn1403- and Tn1548-like transposons. The second copy of armA was a component of the ISCR28-armA-?ISCR28 structure, representing a novel armA vehicle. This vehicle was located within In48, which was related to In363 and In1058. Data presented here provide a deeper insight into the evolutionary history of SY153, especially in regard to how it became extensively drug-resistant.

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July 7, 2019

Complete genome sequence of a colistin-resistant Escherichia coli strain harboring mcr-1 on an IncHI2 plasmid in the United States.

We report here the incidental detection and complete genome sequence of a urinary Escherichia coli strain harboring mcr-1 and resistant to colistin in a New York patient returning from Portugal in 2016. This strain, with sequence type 1485 (ST1485), was a non-extended-spectrum beta-lactamase (ESBL) and non-carbapenemase producer and carried the mcr-1 gene on an IncHI2 plasmid. Copyright © 2017 Gilrane et al.

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July 7, 2019

Remarkable diversity of Escherichia coli carrying mcr-1 from hospital sewage with the identification of two new mcr-1 variants.

The plasmid-borne colistin-resistant gene mcr-1 has rapidly become a worldwide public health concern. This study aims to determine the host bacterial strains, plasmids, and genetic contexts of mcr-1 in hospital sewage. A 1-ml hospital sewage sample was cultured. Colistin-resistant bacterial colonies were selected on agar plates and were subjected to whole genome sequencing and subsequent analysis. The transfer of mcr-1 between bacterial strains was tested using conjugation. New variants of mcr-1 were cloned to test the impact of variations on the function of mcr-1. Plasmids carrying mcr-1 were retrieved from GenBank for comparison based on concatenated backbone genes. In the sewage sample, we observed that mcr-1 was located in various genetic contexts on the chromosome, or plasmids of four different replicon types (IncHI2, IncI2, IncP, and IncX4), in Klebsiella pneumoniae, Kluyvera spp. and seven Escherichia coli strains of six different sequence types (ST10, ST34, ST48, ST1196, ST7086, and ST7087). We also identified two new variants of mcr-1, mcr-1.4 and mcr-1.7, both of which encode an amino acid variation from mcr-1. mcr-1-carrying IncX4 plasmids, which have a global distribution across the Enterobacteriaceae, are the result of global dissemination of a single common plasmid, while IncI2 mcr-1 plasmids appear to acquire mcr-1 in multiple events. In conclusion, the unprecedented remarkable diversity of species, strains, plasmids, and genetic contexts carrying mcr-1 present in a single sewage sample from a single healthcare site highlights the continued evolution and dynamic transmission of mcr-1 in healthcare-associated environments.

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July 7, 2019

Characterization of oqxAB in Escherichia coli isolates from animals, retail meat, and human patients in Guangzhou, China.

The purpose of this study was to investigate the prevalence and genetic elements of oqxAB among Escherichia coli isolates from animals, retail meat, and humans (patients with infection or colonization) in Guangzhou, China. A total of 1,354 E. coli isolates were screened for oqxAB by PCR. Fifty oqxAB-positive isolates were further characterized by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), S1-PFGE, genetic environment analysis, plasmid replicon typing, and plasmid sequencing. oqxAB was detected in 172 (33.79%), 60 (17.34%), and 90 (18.07%) E. coli isolates from animal, food, and human, respectively. High clonal diversity was observed among oqxAB-positive isolates. In 21 oqxAB-containing transformants, oqxAB was flanked by two IS26 elements in the same orientation, formed a composite transposon Tn6010 in 19 transformants, and was located on plasmids (33.3~500 kb) belonging to IncN1-F33:A-:B- (n = 3), IncHI2/ST3 (n = 3), F-:A18:B- (n = 2), F-:A-:B54 (n = 2), or others. Additionally, oqxAB was co-located with multiple resistance genes on the same plasmid, such as aac(6′)-Ib-cr and/or qnrS, which were identified in two F-:A18:B- plasmids from pigs, and blaCTX-M-55, rmtB, fosA3, and floR, which were detected in two N1-F33:A-:B- plasmids from patients. The two IncHI2/ST3 oqxAB-bearing plasmids, pHNLDF400 and pHNYJC8, which were isolated from human patient and chicken meat, respectively, contained a typical IncHI2-type backbone, and were similar to each other with 2-bp difference, and also showed 99% identity to the Salmonella Typhimurium oqxAB-carrying plasmids pHXY0908 (chicken) and pHK0653 (human patient). Horizontal transfer mediated by mobile elements may be the primary mechanism underlying oqxAB spread in E. coli isolates obtained from various sources in Guangzhou, China. The transmission of identical oqxAB-carrying IncHI2 plasmids between food products and humans might pose a serious threat to public health.

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July 7, 2019

ICESag37, a novel integrative and conjugative element carrying antimicrobial resistance genes and potential virulence factors in Streptococcus agalactiae.

ICESag37, a novel integrative and conjugative element carrying multidrug resistance and potential virulence factors, was characterized in a clinical isolate of Streptococcus agalactiae. Two clinical strains of S. agalactiae, Sag37 and Sag158, were isolated from blood samples of new-borns with bacteremia. Sag37 was highly resistant to erythromycin and tetracycline, and susceptible to levofloxacin and penicillin, while Sag158 was resistant to tetracycline and levofloxacin, and susceptible to erythromycin. Transfer experiments were performed and selection was carried out with suitable antibiotic concentrations. Through mating experiments, the erythromycin resistance gene was found to be transferable from Sag37 to Sag158. SmaI-PFGE revealed a new SmaI fragment, confirming the transfer of the fragment containing the erythromycin resistance gene. Whole genome sequencing and sequence analysis revealed a mobile element, ICESag37, which was characterized using several molecular methods and in silico analyses. ICESag37 was excised to generate a covalent circular intermediate, which was transferable to S. agalactiae. Inverse PCR was performed to detect the circular form. A serine family integrase mediated its chromosomal integration into rumA, which is a known hotspot for the integration of streptococcal ICEs. The integration site was confirmed using PCR. ICESag37 carried genes for resistance to multiple antibiotics, including erythromycin [erm(B)], tetracycline [tet(O)], and aminoglycosides [aadE, aphA, and ant(6)]. Potential virulence factors, including a two-component signal transduction system (nisK/nisR), were also observed in ICESag37. S1-PFGE analysis ruled out the existence of plasmids. ICESag37 is the first ICESa2603 family-like element identified in S. agalactiae carrying both resistance and potential virulence determinants. It might act as a vehicle for the dissemination of multidrug resistance and pathogenicity among S. agalactiae.

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July 7, 2019

Genomic patterns of de novo mutation in simplex autism.

To further our understanding of the genetic etiology of autism, we generated and analyzed genome sequence data from 516 idiopathic autism families (2,064 individuals). This resource includes >59 million single-nucleotide variants (SNVs) and 9,212 private copy number variants (CNVs), of which 133,992 and 88 are de novo mutations (DNMs), respectively. We estimate a mutation rate of ~1.5 × 10(-8) SNVs per site per generation with a significantly higher mutation rate in repetitive DNA. Comparing probands and unaffected siblings, we observe several DNM trends. Probands carry more gene-disruptive CNVs and SNVs, resulting in severe missense mutations and mapping to predicted fetal brain promoters and embryonic stem cell enhancers. These differences become more pronounced for autism genes (p = 1.8 × 10(-3), OR = 2.2). Patients are more likely to carry multiple coding and noncoding DNMs in different genes, which are enriched for expression in striatal neurons (p = 3 × 10(-3)), suggesting a path forward for genetically characterizing more complex cases of autism. Copyright © 2017 Elsevier Inc. All rights reserved.

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July 7, 2019

Methylomic and phenotypic analysis of the ModH5 phasevarion of Helicobacter pylori.

The Helicobacter pylori phase variable gene modH, typified by gene HP1522 in strain 26695, encodes a N6-adenosine type III DNA methyltransferase. Our previous studies identified multiple strain-specific modH variants (modH1 – modH19) and showed that phase variation of modH5 in H. pylori P12 influenced expression of motility-associated genes and outer membrane protein gene hopG. However, the ModH5 DNA recognition motif and the mechanism by which ModH5 controls gene expression were unknown. Here, using comparative single molecule real-time sequencing, we identify the DNA site methylated by ModH5 as 5′-Gm6ACC-3′. This motif is vastly underrepresented in H. pylori genomes, but overrepresented in a number of virulence genes, including motility-associated genes, and outer membrane protein genes. Motility and the number of flagella of H. pylori P12 wild-type were significantly higher than that of isogenic modH5 OFF or ?modH5 mutants, indicating that phase variable switching of modH5 expression plays a role in regulating H. pylori motility phenotypes. Using the flagellin A (flaA) gene as a model, we show that ModH5 modulates flaA promoter activity in a GACC methylation-dependent manner. These findings provide novel insights into the role of ModH5 in gene regulation and how it mediates epigenetic regulation of H. pylori motility.

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