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July 7, 2019

Lightning-fast genome variant detection with GROM.

Current human whole genome sequencing projects produce massive amounts of data, often creating significant computational challenges. Different approaches have been developed for each type of genome variant and method of its detection, necessitating users to run multiple algorithms to find variants.We present GROM (Genome Rearrangement OmniMapper), a novel comprehensive variant detection algorithm accepting aligned read files as input and finding SNVs, indels, structural variants (SVs), and copy number variants (CNVs). We show that GROM outperforms state-of-the-art methods on seven validated benchmarks using two whole genome sequencing (WGS) datasets. Additionally, GROM boasts lightning fast run times, analyzing a 50x WGS human dataset (NA12878) on commonly available computer hardware in 11 minutes, more than an order of magnitude (up to 72 times) faster than tools detecting a similar range of variants.Addressing the needs of big data analysis, GROM combines in one algorithm SNV, indel, SV, and CNV detection providing superior speed, sensitivity, and precision. GROM is also able to detect CNVs, SNVs and indels in non-paired read WGS libraries, as well as SNVs and indels in whole exome or RNA sequencing datasets.


July 7, 2019

Exception to the rule: Genomic characterization of naturally occurring unusual Vibrio cholerae strains with a single chromosome.

The genetic make-up of most bacteria is encoded in a single chromosome while about 10% have more than one chromosome. Among these, Vibrio cholerae, with two chromosomes, has served as a model system to study various aspects of chromosome maintenance, mainly replication, and faithful partitioning of multipartite genomes. Here, we describe the genomic characterization of strains that are an exception to the two chromosome rules: naturally occurring single-chromosome V. cholerae. Whole genome sequence analyses of NSCV1 and NSCV2 (natural single-chromosome vibrio) revealed that the Chr1 and Chr2 fusion junctions contain prophages, IS elements, and direct repeats, in addition to large-scale chromosomal rearrangements such as inversions, insertions, and long tandem repeats elsewhere in the chromosome compared to prototypical two chromosome V. cholerae genomes. Many of the known cholera virulence factors are absent. The two origins of replication and associated genes are generally intact with synonymous mutations in some genes, as are recA and mismatch repair (MMR) genes dam, mutH, and mutL; MutS function is probably impaired in NSCV2. These strains are ideal tools for studying mechanistic aspects of maintenance of chromosomes with multiple origins and other rearrangements and the biological, functional, and evolutionary significance of multipartite genome architecture in general.


July 7, 2019

Complete genome sequence of the pathogenic Vibrio vulnificus type strain ATCC 27562.

Vibrio vulnificus has the highest death rate and economic burden per case of any foodborne pathogen in the United States. A complete genome sequence of the type strain promotes comparative analyses with other clinical and environmental isolates, improving our understanding of this important human pathogen and successful environmental organism. Copyright © 2017 Rusch and Rowe-Magnus.


July 7, 2019

Novel multiresistance cfr plasmids in linezolid-resistant methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus faecium (VRE) from a hospital outbreak: co-location of cfr and optrA in VRE.

Linezolid is often the drug of last resort to treat infections caused by Gram-positive cocci. Linezolid resistance can be mutational (23S rRNA or L-protein) or, less commonly, acquired [predominantly cfr, conferring resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A compounds (PhLOPSA) or optrA, encoding oxazolidinone and phenicol resistance].To investigate the clonality and genetic basis of linezolid resistance in 13 linezolid-resistant (LZDR) methicillin-resistant Staphylococcus epidermidis (MRSE) isolates recovered during a 2013/14 outbreak in an ICU in an Irish hospital and an LZDR vancomycin-resistant Enterococcus faecium (VRE) isolate from an LZDR-MRSE-positive patient.All isolates underwent PhLOPSA susceptibility testing, 23S rRNA sequencing, DNA microarray profiling and WGS.All isolates exhibited the PhLOPSA phenotype. The VRE harboured cfr and optrA on a novel 73?kb plasmid (pEF12-0805) also encoding erm(A), erm(B), lnu(B), lnu(E), aphA3 and aadE. One MRSE (M13/0451, from the same patient as the VRE) harboured cfr on a novel 8.5?kb plasmid (pSEM13-0451). The remaining 12 MRSE lacked cfr but exhibited linezolid resistance-associated mutations and were closely related to (1-52 SNPs) but distinct from M13/0451 (202-223 SNPs).Using WGS, novel and distinct cfr and cfr/optrA plasmids were identified in an MRSE and VRE isolate, respectively, as well as a cfr-negative LZDR-MRSE ICU outbreak and a distinct cfr-positive LZDR-MRSE from the same ICU. To our knowledge, this is the first report of cfr and optrA on a single VRE plasmid. Ongoing surveillance of linezolid resistance is essential to maintain its therapeutic efficacy.© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.


July 7, 2019

Analysis of resistance genes in pan-resistant Myroides odoratimimus clinical strain PR63039 using whole genome sequencing.

To clarify the antibiotic resistance mechanisms of Myroides odoratimimus, pan-resistant M. odoratimimus strain PR63039 was isolated and its genome sequenced and analyzed. Antimicrobial susceptibility testing was conducted using the Kirby-Bauer disk diffusion method, and the Phoenix-100 Automated Microbiology System with a NMIC/ID-4 panel including aminoglycosides, ß-lactams, polypeptides, quinolones, sulfonamides, chloramphenicols, and tetracyclines. Single-molecule real-time whole genome sequencing was conducted using the PacBio RSII system, and genome annotation was performed using RAST and IMG ER. To characterize the genome features, a number of databases and software programs, including GC-Profile, CG viewer, the VFDB database, ISfinder, RADB, CARD, ResFinder, and PHAST, were used. M. odoratimimus isolate PR63039 was resistant to almost all antibiotics tested, suggesting pan-drug resistance. The genome consisted of a 4,366,950-bp chromosome and a 90,798-bp plasmid (p63039), which contained a large number of resistance genes and virulence factors. The distribution of the resistance genes was distinctive, and a resistance region, designated MY63039-RR, was identified. RAST analysis indicated that 108 of the annotated genes were potentially involved in virulence, disease, and defense, all of which could be associated with resistance and pathogenicity. Prophage analysis also identified two incomplete prophages in the genome of M. odoratimimus PR63039. Multiple antibiotic-resistance genes were identified, including those associated with resistance to tetracycline (tetX), macrolides (ereB, cfrA, lasE), sulfonamides (sul2, sul3), ß-lactams (blaMUS-1, blaTUS-1, blaSFB-1, blaSLB-1, blaOXA-209, blaOXA-347), and chloramphenicol (cat). Further, the presence of 18 antibiotic efflux pump-encoding resistance genes, including acrB, acrD, acrF, adeB, adeG, adeJ, amrB, ceoB, cmeB, mdsB, mexB, mexD, mexF, mtrD, smeE, mdtF, macB, likely accounts for the observed quinolone resistance of strain PR63039. To the best of our knowledge, this is the first report of the presence of the blaSFB-1, blaSLB-1, blaOXA-209, blaOXA-347, and tetX resistance genes in M. odoratimimus. Copyright © 2017 Elsevier Ltd. All rights reserved.


July 7, 2019

Whole-genome sequencing identification of a multidrug-resistant Salmonella enterica serovar Typhimurium strain carrying blaNDM-5 from Guangdong, China.

A carbapenem-resistant Salmonella enterica serovar Typhimurium (sequence type 34 [ST34]) strain was isolated from a fecal specimen from a child with acute diarrhea. Whole-genome sequencing revealed that the 84.5-kb IncFII plasmid pST41-NDM carrying the NDM-5 carbapenemase gene possesses a structure identical to that of the IncFII-type plasmid backbone. However, the blaNDM-5 flanking sequence found in this plasmid is identical to the blaNDM-5-positive IncX3 plasmids carried by 10 strains of Enterobacteriaceae identified in the same hospital. Copyright © 2017 Elsevier B.V. All rights reserved.


July 7, 2019

Identification of low allele frequency mosaic mutations in Alzheimer disease

Germline mutations ofAPP,PSEN1, andPSEN2 genes cause autosomal dominant Alzheimer disease (AD). Somatic variants of the same genes may underlie pathogenesis in sporadic AD, which is the most prevalent form of the disease. Importantly, such somatic variants may be present at very low allelic frequency, confined to the brain, and are thus very difficult or impossible to detect in blood-derived DNA. Ever-refined methodologies to identify mutations present in a fraction of the DNA of the original tissue are rapidly transforming our understanding of DNA mutation and their role in complex pathologies such as tumors. These methods stand poised to test to what extend somatic variants may play a role in AD and other neurodegenerative diseases.


July 7, 2019

Complete genetic analysis of a Salmonella enterica serovar Indiana isolate accompanying four plasmids carrying mcr-1, ESBL and other resistance genes in China

One mcr-1-carrying Salmonella enterica serovar Indiana strain D90, was identified from 1320 Salmonella enterica isolates from poultry slaughterhouse in 2012 in China. The objective of this study was to verify the transferability of the mcr-1 gene and also completely characterize the sequence of the strain at the whole-genome level. Broth matting assays were carried out to detect the transferability and whole-genome sequencing (WGS) of S. enterica serovar Indiana D90 was performed using the PacBio RS II system. Open reading frames were assigned using Rapid Annotation using Subsystem Technology (RAST) and analysed by BLASTn and BLASTp. Salmonella Pathogenisity Islands (SPIs) were annotated by SPIFinder platform. The complete genome sequence of S. enterica serovar Indiana D90 contained a circular 4,779,514-bp chromosome and four plasmids. Genome analysis and sequencing revealed that 24 multi-drug resistance (MDR) genes were located on plasmids. The largest plasmid pD90-1, was found to be of an IncHI2/HI2A/Q1/N type that encoded a blaCTX-M-65 gene along with 20 additional antimicrobial resistance genes. A 60.5-kbp IncI2 plasmid pD90-2 contained a nikA-nikB-mcr-1 genetic structure, that can be successfully transferred to E. coli and S. enterica serovar Typhimurium at low transfer rates. Interestingly, comparative sequence analysis revealed the plasmids pD90-1 and pD90-2 showed considerable nucleotide similarity to pHNSHP45-2 and pHNSHP45, respectively. Moreover, the genome and the plasmid pD90-2 also showed high similarity to one carbapenem resistant S. enterica serovar Indiana strain, C629 and its plasmid pC629, respectively. This is the first report of the complete nucleotide sequence of one mcr-1-carrying MDR S. enterica serovar Indiana strain.


July 7, 2019

Methylation-dependent DNA discrimination in natural transformation of Campylobacter jejuni.

Campylobacter jejuni, a leading cause of bacterial gastroenteritis, is naturally competent. Like many competent organisms, C. jejuni restricts the DNA that can be used for transformation to minimize undesirable changes in the chromosome. Although C. jejuni can be transformed by C. jejuni-derived DNA, it is poorly transformed by the same DNA propagated in Escherichia coli or produced with PCR. Our work indicates that methylation plays an important role in marking DNA for transformation. We have identified a highly conserved DNA methyltransferase, which we term Campylobacter transformation system methyltransferase (ctsM), which methylates an overrepresented 6-bp sequence in the chromosome. DNA derived from a ctsM mutant transforms C. jejuni significantly less well than DNA derived from ctsM(+) (parental) cells. The ctsM mutation itself does not affect transformation efficiency when parental DNA is used, suggesting that CtsM is important for marking transforming DNA, but not for transformation itself. The mutant has no growth defect, arguing against ongoing restriction of its own DNA. We further show that E. coli plasmid and PCR-derived DNA can efficiently transform C. jejuni when only a subset of the CtsM sites are methylated in vitro. A single methylation event 1 kb upstream of the DNA involved in homologous recombination is sufficient to transform C. jejuni, whereas otherwise identical unmethylated DNA is not. Methylation influences DNA uptake, with a slight effect also seen on DNA binding. This mechanism of DNA discrimination in C. jejuni is distinct from the DNA discrimination described in other competent bacteria.


July 7, 2019

Genetic characterization of blaNDM-harboring plasmids in carbapenem-resistant Escherichia coli from Myanmar.

The bacterial enzyme New Delhi metallo-ß-lactamase hydrolyzes almost all ß-lactam antibiotics, including carbapenems, which are drugs of last resort for severe bacterial infections. The spread of carbapenem-resistant Enterobacteriaceae that carry the New Delhi metallo-ß-lactamase gene, blaNDM, poses a serious threat to public health. In this study, we genetically characterized eight carbapenem-resistant Escherichia coli isolates from a tertiary care hospital in Yangon, Myanmar. The eight isolates belonged to five multilocus-sequence types and harbored multiple antimicrobial-resistance genes, resulting in resistance against nearly all of the antimicrobial agents tested, except colistin and fosfomycin. Nine plasmids harboring blaNDM genes were identified from these isolates. Multiple blaNDM genes were found in the distinct Inc-replicon types of the following plasmids: an IncA/C2 plasmid harboring blaNDM-1 (n = 1), IncX3 plasmids harboring blaNDM-4 (n = 2) or blaNDM-7 (n = 1), IncFII plasmids harboring blaNDM-4 (n = 1) or blaNDM-5 (n = 3), and a multireplicon F plasmid harboring blaNDM-5 (n = 1). Comparative analysis highlighted the diversity of the blaNDM-harboring plasmids and their distinct characteristics, which depended on plasmid replicon types. The results indicate circulation of phylogenetically distinct strains of carbapenem-resistant E. coli with various plasmids harboring blaNDM genes in the hospital.


July 7, 2019

Shared features of cryptic plasmids from environmental and pathogenic Francisella species.

The Francisella genus includes several recognized species, additional potential species, and other representatives that inhabit a range of incredibly diverse ecological niches, but are not closely related to the named species. Francisella species have been obtained from a wide variety of clinical and environmental sources; documented species include highly virulent human and animal pathogens, fish pathogens, opportunistic human pathogens, tick endosymbionts, and free-living isolates inhabiting brackish water. While more than 120 Francisella genomes have been sequenced to date, only a few contain plasmids, and most of these appear to be cryptic, with unknown benefit to the host cell. We have identified several putative cryptic plasmids in the sequenced genomes of three Francisella novicida and F. novicida-like strains (TX07-6608, AZ06-7470, DPG_3A-IS) and two new Francisella species (F. frigiditurris CA97-1460 and F. opportunistica MA06-7296). These plasmids were compared to each other and to previously identified plasmids from other Francisella species. Some of the plasmids encoded functions potentially involved in replication, conjugal transfer and partitioning, environmental survival (transcriptional regulation, signaling, metabolism), and hypothetical proteins with no assignable functions. Genomic and phylogenetic comparisons of these new plasmids to the other known Francisella plasmids revealed some similarities that add to our understanding of the evolutionary relationships among the diverse Francisella species.


July 7, 2019

Avoidance of APOBEC3B-induced mutation by error-free lesion bypass.

APOBEC cytidine deaminases mutate cancer genomes by converting cytidines into uridines within ssDNA during replication. Although uracil DNA glycosylases limit APOBEC-induced mutation, it is unknown if subsequent base excision repair (BER) steps function on replication-associated ssDNA. Hence, we measured APOBEC3B-induced CAN1 mutation frequencies in yeast deficient in BER endonucleases or DNA damage tolerance proteins. Strains lacking Apn1, Apn2, Ntg1, Ntg2 or Rev3 displayed wild-type frequencies of APOBEC3B-induced canavanine resistance (CanR). However, strains without error-free lesion bypass proteins Ubc13, Mms2 and Mph1 displayed respective 4.9-, 2.8- and 7.8-fold higher frequency of APOBEC3B-induced CanR. These results indicate that mutations resulting from APOBEC activity are avoided by deoxyuridine conversion to abasic sites ahead of nascent lagging strand DNA synthesis and subsequent bypass by error-free template switching. We found this mechanism also functions during telomere re-synthesis, but with a diminished requirement for Ubc13. Interestingly, reduction of G to C substitutions in Ubc13-deficient strains uncovered a previously unknown role of Ubc13 in controlling the activity of the translesion synthesis polymerase, Rev1. Our results highlight a novel mechanism for error-free bypass of deoxyuridines generated within ssDNA and suggest that the APOBEC mutation signature observed in cancer genomes may under-represent the genomic damage these enzymes induce.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.


July 7, 2019

MCR-1 and OXA-48 in vivo acquisition in KPC-producing Escherichia coli after colistin treatment.

The spread of mcr-1-encoding plasmids into carbapenem-resistant Enterobacteriaceae raises concerns about the emergence of untreatable bacteria. We report the acquisition of mcr-1 in a carbapenem-resistant Escherichia coli strain after a 3-week course of colistin in a patient repatriated to France from Portugal. Whole-genome sequencing revealed that the Klebsiella pneumoniae carbapenemase-producing E. coli strain acquired two plasmids, an IncL OXA-48-encoding plasmid and an IncX4 mcr-1-encoding plasmid. This is the first report of mcr-1 in carbapenemase-encoding bacteria in France. Copyright © 2017 American Society for Microbiology.


July 7, 2019

Complete genome sequence of super biofilm-elaborating Staphylococcus aureus isolated in Japan.

Staphylococcus aureus JP080, previously named TF2758, is a clinical isolate from an atheroma and a super biofilm-elaborating strain whose biofilm elaboration is dependent solely on polysaccharide poly-N-acetylglucosamine/polysaccharide intercellular adhesin (PNAG/PIA). Here, we report the complete genome sequence of strain JP080, which consists of one chromosome and one circular plasmid. Copyright © 2017 Yu et al.


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