Biology subjects

Robert Sebra

Publications and source records attributed to Robert Sebra.

5 recordsLinked to original sources

bioRxiv2016

Genome-wide biases in the rate and molecular spectrum of spontaneous mutations in Vibrio cholerae and Vibrio fischeri

The vast diversity in nucleotide composition and architecture among bacterial genomes may be partly explained by inherent biases in the rates and spectra of spontaneous mutations. Bacterial genomes with multiple chromosomes are relatively unusual but some are relevant to human health, none more so than the causative agent of cholera, Vibrio cholerae. Here, we present the genome-wide mutation spectra in wild-type and mismatch repair (MMR) defective backgrounds of two Vibrio species, the low-GC% squid symbiont V. fischeri and the pathogen V. cholerae, collected under conditions that greatly minimize the efficiency of natural selection. In apparent contrast to their high diversity in nature, both wild-type V. fischeri and V. cholerae have among the lowest rates for base-substitution mutations (bpsms) and insertion-deletion mutations (indels) that have been measured, below 10-3/genome/generation. V. fischeri and V. cholerae have distinct mutation spectra, but both are AT-biased and produce a surprising number of multi-nucleotide indels. Furthermore, the loss of a functional MMR system caused the mutation spectra of these species to converge, implying that the MMR system itself contributes to species-specific mutation patterns. Bpsm and indel rates varied among genome regions, but do not explain the more rapid evolutionary rates of genes on chromosome 2, which likely result from weaker purifying selection. More generally, the very low mutation rates of Vibrio species correlate inversely with their immense population sizes and suggest that selection may not only have maximized replication fidelity but also optimized other polygenic traits relative to the constraints of genetic drift.

Genetics↗

bioRxiv2015

Nested Russian Doll-like Genetic Mobility Drives Rapid Dissemination of the Carbapenem Resistance Gene blaKPC

The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort in this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms including conjugation and transposition, however the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is currently unknown. Using a combination of short- and long-read whole genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolated from a single hospital over five years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested genetic levels, with transmission of blaKPC strains between individuals, frequent transfer of blaKPC plasmids between strains/species, and frequent transposition of the blaKPC transposon Tn4401 between plasmids. We also identify a common insertion site for Tn4401 within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this with the use of reference-based methods. We also demonstrate that as a consequence of the genetic mobility observed herein, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that non-clonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance.\n\nImportanceIncreasing antibiotic resistance is a major threat to human health, as highlighted by the recent emergence of multi-drug resistant \"superbugs\". Here, we tracked how one important multi-drug resistance gene spread in a single hospital over five years. This revealed high levels of resistance gene mobility to multiple bacterial species, which was facilitated by various different genetic mechanisms. The mobility occurred at multiple nested genetic levels, analogous to a Russian doll set where smaller dolls may be carried along inside larger dolls. Our results challenge traditional views that drug-resistance outbreaks are due to transmission of a single pathogenic strain. Instead, outbreaks can be \"gene-based\", and we must therefore focus on tracking specific resistance genes and their context rather than only specific bacteria.

Microbiology↗

bioRxiv2015

Evolutionary history of the global emergence of the Escherichia coli epidemic clone ST131

BackgroundEscherichia coli sequence type 131 (ST131) has emerged globally as the most predominant lineage within this clinically important species, and its association with fluoroquinolone and extended-spectrum cephalosporin resistance impacts significantly on treatment. The evolutionary histories of this lineage, and of important antimicrobial resistance elements within it, remain unclearly defined.\n\nResultsThis study of the largest worldwide collection (n = 215) of sequenced ST131 E. coli isolates to date demonstrates that clonal expansion of two previously recognized antimicrobial-resistant clades, C1/H30R and C2/H30Rx, started around 25 years ago, consistent with the widespread introduction of fluoroquinolones and extended-spectrum cephalosporins in clinical medicine. These two clades appear to have emerged in the United States, with the expansion of the C2/H30Rx clade driven by the acquisition of a blaCTX-M-15-containing IncFII-like plasmid that has subsequently undergone extensive rearrangement. Several other evolutionary processes influencing the trajectory of this drug-resistant lineage are described, including sporadic acquisitions of CTX-M resistance plasmids, and chromosomal integration of blaCTX-M within sub-clusters followed by vertical evolution. These processes are also occurring for another family of CTX-M gene variants more recently observed amongst ST131, the blaCTX-M-14/14-like group.\n\nConclusionsThe complexity of the evolutionary history of ST131 has important implications for antimicrobial resistance surveillance, epidemiological analysis, and control of emerging clinical lineages of E. coli. These data also highlight the global imperative to reduce specific antibiotic selection pressures, and demonstrate the important and varied roles played by plasmids and other mobile genetic elements in the perpetuation of antimicrobial resistance within lineages.

Genomics↗

bioRxiv2015

Extensive sequencing of seven human genomes to characterize benchmark reference materials

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.

Genomics↗

bioRxiv2014

Single molecule-level detection and long read-based phasing of epigenetic variations in bacterial methylomes

Comprehensive genome-wide analyses of bacterial DNA methylation have not been possible until the recent advent of single molecule, real-time (SMRT) sequencing. This technology enables the direct detection of N6-methyladenine (6mA) and 4-methylcytosine (4mC) at single nucleotide resolution on a genome-wide scale. The distributions of these two major types of DNA methylation, along with 5-methylcytosine (5mC), comprise the bacterial methylome, some rare exceptions notwithstanding. SMRT sequencing has already revealed marked diversity in bacterial methylomes as well as the existence of heterogeneity of methylation in cells in single bacterial colonies, where such epigenetic variation can enable bacterial populations to rapidly adapt to changing conditions. However, current methods for studying bacterial methylomes using SMRT sequencing mainly rely on population-level summaries that do not provide the single-cell resolution necessary for dissecting the epigenetic heterogeneity in bacterial populations. Here, we present a novel SMRT sequencing-based framework, consisting of two complementary methods, for single molecule-level detection of DNA methylation and assessment of methyltransferase activity through single molecule-level long read-based epigenetic phasing. Using seven bacterial strains and integrating data from SMRT and Illumina sequencing, we show that our method yields significantly improved resolution compared to existing population-level methods, and reveals several distinct types of epigenetic heterogeneity. Our approach enables new investigations of the complex architecture and dynamics of bacterial methylomes and provides a powerful new tool for the study of bacterial epigenetic control.

Microbiology↗