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July 19, 2019

Single-molecule sequencing (PacBio) of the Staphylococcus capitis NRCS-A clone reveals the basis of multidrug resistance and adaptation to the Neonatal Intensive Care Unit environment.

The multi-resistant Staphylococcus capitis clone NRCS-A has recently been described as a major pathogen causing nosocomial, late-onset sepsis (LOS) in preterm neonates worldwide. NRCS-A representatives exhibit an atypical antibiotic resistance profile. Here, the complete closed genome (chromosomal and plasmid sequences) of NRCS-A prototype strain CR01 and the draft genomes of three other clinical NRCS-A strains from Australia, Belgium and the United Kingdom are annotated and compared to available non-NRCS-A S. capitis genomes. Our goal was to delineate the uniqueness of the NRCS-A clone with respect to antibiotic resistance, virulence factors and mobile genetic elements. We identified 6 antimicrobial resistance genes, all carried by mobile genetic elements. Previously described virulence genes present in the NRCS-A genomes are shared with the six non-NRCS-A S. capitis genomes. Overall, 63 genes are specific to the NRCS-A lineage, including 28 genes located in the methicillin-resistance cassette SCCmec. Among the 35 remaining genes, 25 are of unknown function, and 9 correspond to an additional type I restriction modification system (n = 3), a cytosine methylation operon (n = 2), and a cluster of genes related to the biosynthesis of teichoic acids (n = 4). Interestingly, a tenth gene corresponds to a resistance determinant for nisin (nsr gene), a bacteriocin secreted by potential NRCS-A strain niche competitors in the gut microbiota. The genomic characteristics presented here emphasize the contribution of mobile genetic elements to the emergence of multidrug resistance in the S. capitis NRCS-A clone. No NRCS-A-specific known virulence determinant was detected, which does not support a role for virulence as a driving force of NRCS-A emergence in NICUs worldwide. However, the presence of a nisin resistance determinant on the NRCS-A chromosome, but not in other S. capitis strains and most coagulase-negative representatives, might confer a competitive advantage to NRCS-A strains during the early steps of gut colonization in neonates. This suggests that the striking adaptation of NRCS-A to the NICU environment might be related to its specific antimicrobial resistance and also to a possible enhanced ability to challenge competing bacteria in its ecological niche.

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July 19, 2019

Gorilla MHC class I gene and sequence variation in a comparative context.

Comparisons of MHC gene content and diversity among closely related species can provide insights into the evolutionary mechanisms shaping immune system variation. After chimpanzees and bonobos, gorillas are humans’ closest living relatives; but in contrast, relatively little is known about the structure and variation of gorilla MHC class I genes (Gogo). Here, we combined long-range amplifications and long-read sequencing technology to analyze full-length MHC class I genes in 35 gorillas. We obtained 50 full-length genomic sequences corresponding to 15 Gogo-A alleles, 4 Gogo-Oko alleles, 21 Gogo-B alleles, and 10 Gogo-C alleles including 19 novel coding region sequences. We identified two previously undetected MHC class I genes related to Gogo-A and Gogo-B, respectively, thereby illustrating the potential of this approach for efficient and highly accurate MHC genotyping. Consistent with their phylogenetic position within the hominid family, individual gorilla MHC haplotypes share characteristics with humans and chimpanzees as well as orangutans suggesting a complex history of the MHC class I genes in humans and the great apes. However, the overall MHC class I diversity appears to be low further supporting the hypothesis that gorillas might have experienced a reduction of their MHC repertoire.

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July 19, 2019

Comprehensive bioinformatics analysis of Mycoplasma pneumoniae genomes to investigate underlying population structure and type-specific determinants.

Mycoplasma pneumoniae is a significant cause of respiratory illness worldwide. Despite a minimal and highly conserved genome, genetic diversity within the species may impact disease. We performed whole genome sequencing (WGS) analysis of 107 M. pneumoniae isolates, including 67 newly sequenced using the Pacific BioSciences RS II and/or Illumina MiSeq sequencing platforms. Comparative genomic analysis of 107 genomes revealed >3,000 single nucleotide polymorphisms (SNPs) in total, including 520 type-specific SNPs. Population structure analysis supported the existence of six distinct subgroups, three within each type. We developed a predictive model to classify an isolate based on whole genome SNPs called against the reference genome into the identified subtypes, obviating the need for genome assembly. This study is the most comprehensive WGS analysis for M. pneumoniae to date, underscoring the power of combining complementary sequencing technologies to overcome difficult-to-sequence regions and highlighting potential differential genomic signatures in M. pneumoniae.

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July 19, 2019

Complete genome sequences of isolates of Enterococcus faecium sequence type 117, a globally disseminated multidrug-resistant clone.

The emergence of nosocomial infections by multidrug-resistant sequence type 117 (ST117) Enterococcus faecium has been reported in several European countries. ST117 has been detected in Spanish hospitals as one of the main causes of bloodstream infections. We analyzed genome variations of ST117 strains isolated in Madrid and describe the first ST117 closed genome sequences. Copyright © 2017 Tedim et al.

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July 19, 2019

SMRT genome assembly corrects reference errors, resolving the genetic basis of virulence in Mycobacterium tuberculosis.

The genetic basis of virulence in Mycobacterium tuberculosis has been investigated through genome comparisons of virulent (H37Rv) and attenuated (H37Ra) sister strains. Such analysis, however, relies heavily on the accuracy of the sequences. While the H37Rv reference genome has had several corrections to date, that of H37Ra is unmodified since its original publication.Here, we report the assembly and finishing of the H37Ra genome from single-molecule, real-time (SMRT) sequencing. Our assembly reveals that the number of H37Ra-specific variants is less than half of what the Sanger-based H37Ra reference sequence indicates, undermining and, in some cases, invalidating the conclusions of several studies. PE_PPE family genes, which are intractable to commonly-used sequencing platforms because of their repetitive and GC-rich nature, are overrepresented in the set of genes in which all reported H37Ra-specific variants are contradicted. Further, one of the sequencing errors in H37Ra masks a true variant in common with the clinical strain CDC1551 which, when considered in the context of previous work, corresponds to a sequencing error in the H37Rv reference genome.Our results constrain the set of genomic differences possibly affecting virulence by more than half, which focuses laboratory investigation on pertinent targets and demonstrates the power of SMRT sequencing for producing high-quality reference genomes.

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July 19, 2019

An integrated strategy combining DNA walking and NGS to detect GMOs.

Recently, we developed a DNA walking system for the detection and characterization of a broad spectrum of GMOs in routine analysis of food/feed matrices. Here, we present a new version with improved throughput and sensitivity by coupling the DNA walking system to Pacific Bioscience® Next-generation sequencing technology. The performance of the new strategy was thoroughly assessed through several assays. First, we tested its detection and identification capability on grains with high or low GMO content. Second, the potential impacts of food processing were investigated using rice noodle samples. Finally, GMO mixtures and a real-life sample were analyzed to illustrate the applicability of the proposed strategy in routine GMO analysis. In all tested samples, the presence of multiple GMOs was unambiguously proven by the characterization of transgene flanking regions and the combinations of elements that are typical for transgene constructs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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July 19, 2019

Quasispecies composition and evolution of a typical Zika virus clinical isolate from Suriname.

The arthropod-borne Zika virus (ZIKV) is currently causing a major international public health threat in the Americas. This study describes the isolation of ZIKV from the plasma of a 29-year-old female traveler that developed typical symptoms, like rash, fever and headache upon return from Suriname. The complete genome sequence including the 5′ and 3′ untranslated regions was determined and phylogenetic analysis showed the isolate clustering within the Asian lineage, close to other viruses that have recently been isolated in the Americas. In addition, the viral quasispecies composition was analyzed by single molecule real time sequencing, which suggested a mutation frequency of 1.4?×?10(-4) for this ZIKV isolate. Continued passaging of the virus in cell culture led to the selection of variants with mutations in NS1 and the E protein. The latter might influence virus binding to cell surface heparan sulfate.

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July 19, 2019

Comparative genomics of two sequential Candida glabrata clinical isolates.

Candida glabrata is an important fungal pathogen which develops rapid antifungal resistance in treated patients. It is known that azole treatments lead to antifungal resistance in this fungal species and that multidrug efflux transporters are involved in this process. Specific mutations in the transcriptional regulator PDR1 result in upregulation of the transporters. In addition, we showed that the PDR1 mutations can contribute to enhance virulence in animal models. In this study, we were interested to compare genomes of two specific C. glabrata-related isolates, one of which was azole susceptible (DSY562) while the other was azole resistant (DSY565). DSY565 contained a PDR1 mutation (L280F) and was isolated after a time-lapse of 50 d of azole therapy. We expected that genome comparisons between both isolates could reveal additional mutations reflecting host adaptation or even additional resistance mechanisms. The PacBio technology used here yielded 14 major contigs (sizes 0.18-1.6 Mb) and mitochondrial genomes from both DSY562 and DSY565 isolates that were highly similar to each other. Comparisons of the clinical genomes with the published CBS138 genome indicated important genome rearrangements, but not between the clinical strains. Among the unique features, several retrotransposons were identified in the genomes of the investigated clinical isolates. DSY562 and DSY565 each contained a large set of adhesin-like genes (101 and 107, respectively), which exceed by far the number of reported adhesins (63) in the CBS138 genome. Comparison between DSY562 and DSY565 yielded 17 nonsynonymous SNPs (among which the was the expected PDR1 mutation) as well as small size indels in coding regions (11) but mainly in adhesin-like genes. The genomes contained a DNA mismatch repair allele of MSH2 known to be involved in the so-called hyper-mutator phenotype of this yeast species and the number of accumulated mutations between both clinical isolates is consistent with the presence of a MSH2 defect. In conclusion, this study is the first to compare genomes of C. glabrata sequential clinical isolates using the PacBio technology as an approach. The genomes of these isolates taken in the same patient at two different time points exhibited limited variations, even if submitted to the host pressure. Copyright © 2017 Vale-Silva et al.

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July 19, 2019

Quality control of the traditional patent medicine Yimu Wan based on SMRT Sequencing and DNA barcoding.

Substandard traditional patent medicines may lead to global safety-related issues. Protecting consumers from the health risks associated with the integrity and authenticity of herbal preparations is of great concern. Of particular concern is quality control for traditional patent medicines. Here, we establish an effective approach for verifying the biological composition of traditional patent medicines based on single-molecule real-time (SMRT) sequencing and DNA barcoding. Yimu Wan (YMW), a classical herbal prescription recorded in the Chinese Pharmacopoeia, was chosen to test the method. Two reference YMW samples were used to establish a standard method for analysis, which was then applied to three different batches of commercial YMW samples. A total of 3703 and 4810 circular-consensus sequencing (CCS) reads from two reference and three commercial YMW samples were mapped to the ITS2 and psbA-trnH regions, respectively. Moreover, comparison of intraspecific genetic distances based on SMRT sequencing data with reference data from Sanger sequencing revealed an ITS2 and psbA-trnH intergenic spacer that exhibited high intraspecific divergence, with the sites of variation showing significant differences within species. Using the CCS strategy for SMRT sequencing analysis was adequate to guarantee the accuracy of identification. This study demonstrates the application of SMRT sequencing to detect the biological ingredients of herbal preparations. SMRT sequencing provides an affordable way to monitor the legality and safety of traditional patent medicines.

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July 19, 2019

Comparative analysis of extended-spectrum-ß-lactamase CTX-M-65-producing Salmonella enterica serovar Infantis isolates from humans, food animals, and retail chickens in the United States.

We sequenced the genomes of ten Salmonella enterica serovar Infantis containing blaCTX-M-65 isolated from chicken, cattle, and human sources collected between 2012 and 2015 in the United States through routine NARMS surveillance and product sampling programs. We also completely assembled the plasmids from four of the isolates. All isolates had a D87Y mutation in the gyrA gene and harbored between 7 and 10 resistance genes (aph (4)-Ia, aac (3)-IVa, aph(3′ )-Ic, blaCTX-M-65, fosA3, floR, dfrA14, sul1, tetA, aadA1) located in two distinct sites of a megaplasmid (~316-323kb) similar to that described in a blaCTX-M-65-positive S. Infantis isolated from a patient in Italy. High-quality single nucleotide polymorphism (hqSNP) analysis revealed that all U.S. isolates were closely related, separated by only 1 to 38 pairwise high quality SNPs, indicating a high likelihood that strains from humans, chicken, and cattle recently evolved from a common ancestor. The U.S. isolates were genetically similar to the blaCTX-M-65-positive S. Infantis isolate from Italy, with a separation of 34 to 47 SNPs. This is the first report of the blaCTX-M-65 gene and the pESI-like megaplasmid from S. Infantis in the United States, and illustrates the importance of applying a global One Health, human and animal perspective to combat antimicrobial resistance. Copyright © 2017 American Society for Microbiology.

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July 19, 2019

Characterization of a large antibiotic resistance plasmid found in enteropathogenic Escherichia coli strain B171 and its relatedness to plasmids of diverse E. coli and Shigella.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe infantile diarrhea in developing countries. Previous research has focused on the diversity of the EPEC virulence plasmid, whereas less is known regarding the genetic content and distribution of antibiotic resistance plasmids carried by EPEC. A previous study demonstrated that in addition to the virulence plasmid, reference EPEC strain B171 harbors a second, larger plasmid that confers antibiotic resistance. To further understand the genetic diversity and dissemination of antibiotic resistance plasmids among EPEC strains, we describe the complete sequence of an antibiotic resistance plasmid from EPEC strain B171. The resistance plasmid, pB171_90, has a completed sequence length of 90,229 bp, a GC content of 54.55%, and carries protein-encoding genes involved in conjugative transfer, resistance to tetracycline (tetA), sulfonamides (sulI), and mercury, as well as several virulence-associated genes, including the transcriptional regulator hha and the putative calcium sequestration inhibitor (csi). In silico detection of the pB171_90 genes among 4,798 publicly available E. coli genome assemblies indicates that the unique genes of pB171_90 (csi and traI) are primarily restricted to genomes identified as EPEC or enterotoxigenic E. coli However, conserved regions of the pB171_90 plasmid containing genes involved in replication, stability, and antibiotic resistance were identified among diverse E. coli pathotypes. Interestingly, pB171_90 also exhibited significant similarity with a sequenced plasmid from Shigella dysenteriae type I. Our findings demonstrate the mosaic nature of EPEC antibiotic resistance plasmids and highlight the need for additional sequence-based characterization of antibiotic resistance plasmids harbored by pathogenic E. coli. Copyright © 2017 American Society for Microbiology.

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July 19, 2019

Genomic epidemiology of global Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli.

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common infections. bla KPC, encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10?kb). Here we characterize the genetic features of bla KPC emergence in global E. coli, 2008-2013, using both long- and short-read whole-genome sequencing. Amongst 43/45 successfully sequenced bla KPC-E. coli strains, we identified substantial strain diversity (n?=?21 sequence types, 18% of annotated genes in the core genome); substantial plasmid diversity (=9 replicon types); and substantial bla KPC-associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of inter-species, regional and international plasmid spread. In several cases bla KPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures. E. coli is a common human pathogen, but also a commensal in multiple environmental and animal reservoirs, and easily transmissible. The association of bla KPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. bla TEM, bla CTX-M) suggests that it may become similarly prevalent.

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July 19, 2019

An improved Plasmodium cynomolgi genome assembly reveals an unexpected methyltransferase gene expansion.

Plasmodium cynomolgi, a non-human primate malaria parasite species, has been an important model parasite since its discovery in 1907. Similarities in the biology of P. cynomolgi to the closely related, but less tractable, human malaria parasite P. vivax make it the model parasite of choice for liver biology and vaccine studies pertinent to P. vivax malaria. Molecular and genome-scale studies of P. cynomolgi have relied on the current reference genome sequence, which remains highly fragmented with 1,649 unassigned scaffolds and little representation of the subtelomeres.  Methods: Using long-read sequence data (Pacific Biosciences SMRT technology), we assembled and annotated a new reference genome sequence, PcyM, sourced from an Indian rhesus monkey. We compare the newly assembled genome sequence with those of several other Plasmodium species, including a re-annotated P. coatneyi assembly.The new PcyM genome assembly is of significantly higher quality than the existing reference, comprising only 56 pieces, no gaps and an improved average gene length. Detailed manual curation has ensured a comprehensive annotation of the genome with 6,632 genes, nearly 1,000 more than previously attributed to P. cynomolgi. The new assembly also has an improved representation of the subtelomeric regions, which account for nearly 40% of the sequence. Within the subtelomeres, we identified more than 1300 Plasmodium interspersed repeat ( pir) genes, as well as a striking expansion of 36 methyltransferase pseudogenes that originated from a single copy on chromosome 9.The manually curated PcyM reference genome sequence is an important new resource for the malaria research community. The high quality and contiguity of the data have enabled the discovery of a novel expansion of methyltransferase in the subtelomeres, and illustrates the new comparative genomics capabilities that are being unlocked by complete reference genomes.

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July 19, 2019

A case study into microbial genome assembly gap sequences and finishing strategies.

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.

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July 19, 2019

Increased risk of low birth weight in women with placental malaria associated with P. falciparum VAR2CSA clade.

Pregnancy associated malaria (PAM) causes adverse pregnancy and birth outcomes owing to Plasmodium falciparum accumulation in the placenta. Placental accumulation is mediated by P. falciparum protein VAR2CSA, a leading PAM-specific vaccine target. The extent of its antigen diversity and impact on clinical outcomes remain poorly understood. Through amplicon deep-sequencing placental malaria samples from women in Malawi and Benin, we assessed sequence diversity of VAR2CSA’s ID1-DBL2x region, containing putative vaccine targets and estimated associations of specific clades with adverse birth outcomes. Overall, var2csa diversity was high and haplotypes subdivided into five clades, the largest two defined by homology to parasites strains, 3D7 or FCR3. Across both cohorts, compared to women infected with only FCR3-like variants, women infected with only 3D7-like variants delivered infants with lower birthweight (difference: -267.99?g; 95% Confidence Interval [CI]: -466.43?g,-69.55?g) and higher odds of low birthweight (<2500?g) (Odds Ratio [OR] 5.41; 95% CI:0.99,29.52) and small-for-gestational-age (OR: 3.65; 95% CI: 1.01,13.38). In two distinct malaria-endemic African settings, parasites harboring 3D7-like variants of VAR2CSA were associated with worse birth outcomes, supporting differential effects of infection with specific parasite strains. The immense diversity coupled with differential clinical effects of this diversity suggest that an effective VAR2CSA-based vaccine may require multivalent activity.

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