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July 19, 2019

Deep genome annotation of the opportunistic human pathogen Streptococcus pneumoniae D39.

A precise understanding of the genomic organization into transcriptional units and their regulation is essential for our comprehension of opportunistic human pathogens and how they cause disease. Using single-molecule real-time (PacBio) sequencing we unambiguously determined the genome sequence of Streptococcus pneumoniae strain D39 and revealed several inversions previously undetected by short-read sequencing. Significantly, a chromosomal inversion results in antigenic variation of PhtD, an important surface-exposed virulence factor. We generated a new genome annotation using automated tools, followed by manual curation, reflecting the current knowledge in the field. By combining sequence-driven terminator prediction, deep paired-end transcriptome sequencing and enrichment of primary transcripts by Cappable-Seq, we mapped 1015 transcriptional start sites and 748 termination sites. We show that the pneumococcal transcriptional landscape is complex and includes many secondary, antisense and internal promoters. Using this new genomic map, we identified several new small RNAs (sRNAs), RNA switches (including sixteen previously misidentified as sRNAs), and antisense RNAs. In total, we annotated 89 new protein-encoding genes, 34 sRNAs and 165 pseudogenes, bringing the S. pneumoniae D39 repertoire to 2146 genetic elements. We report operon structures and observed that 9% of operons are leaderless. The genome data are accessible in an online resource called PneumoBrowse (https://veeninglab.com/pneumobrowse) providing one of the most complete inventories of a bacterial genome to date. PneumoBrowse will accelerate pneumococcal research and the development of new prevention and treatment strategies.

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July 7, 2019

A novel Tn3-like composite transposon harboring blaVIM-1 in Klebsiella pneumoniae spp. pneumoniae isolated from river water.

We present a new plasmid (pOW16C2) with a novel Tn3-like transposon harboring blaVIM-1 from a Klebsiella pneumoniae strain isolated from river water in Switzerland.Complete nucleotide sequence of pOW16C2 was obtained using a Pacific Biosciences SMRT sequencing approach and coding sequences were predicted.The 59,228?bp sequence included a typical IncN-like backbone and a mosaic structure with blaVIM-1, aacA4, aphA15, aadA1, catB2, qnrS1, sul1, and dfrA14 conferring resistance to carbapenems and other ß-lactam antibiotics, aminoglycosides, chloramphenicol, quinolones, sulfonamides, and trimethoprim, respectively. Most of these resistance genes were inserted in a class 1 integron that was embedded in a novel Tn3-like composite transposon.IncN plasmids carrying carbapenemases are frequently isolated from K. pneumoniae strains in clinical settings. The dissemination of K. pneumoniae harboring blaVIM-1 in surface water is a cause for increased concern to public health.

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July 7, 2019

Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles.

Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity. © 2015 Nandi et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae at a single institution: insights into endemicity from whole-genome sequencing.

The global emergence of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) multilocus sequence type ST258 is widely recognized. Less is known about the molecular and epidemiological details of non-ST258 K. pneumoniae in the setting of an outbreak mediated by an endemic plasmid. We describe the interplay of blaKPC plasmids and K. pneumoniae strains and their relationship to the location of acquisition in a U.S. health care institution. Whole-genome sequencing (WGS) analysis was applied to KPC-Kp clinical isolates collected from a single institution over 5 years following the introduction of blaKPC in August 2007, as well as two plasmid transformants. KPC-Kp from 37 patients yielded 16 distinct sequence types (STs). Two novel conjugative blaKPC plasmids (pKPC_UVA01 and pKPC_UVA02), carried by the hospital index case, accounted for the presence of blaKPC in 21/37 (57%) subsequent cases. Thirteen (35%) isolates represented an emergent lineage, ST941, which contained pKPC_UVA01 in 5/13 (38%) and pKPC_UVA02 in 6/13 (46%) cases. Seven (19%) isolates were the epidemic KPC-Kp strain, ST258, mostly imported from elsewhere and not carrying pKPC_UVA01 or pKPC_UVA02. Using WGS-based analysis of clinical isolates and plasmid transformants, we demonstrate the unexpected dispersal of blaKPC to many non-ST258 lineages in a hospital through spread of at least two novel blaKPC plasmids. In contrast, ST258 KPC-Kp was imported into the institution on numerous occasions, with other blaKPC plasmid vectors and without sustained transmission. Instead, a newly recognized KPC-Kp strain, ST941, became associated with both novel blaKPC plasmids and spread locally, making it a future candidate for clinical persistence and dissemination. Copyright © 2015, Mathers et al.

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July 7, 2019

Characterization of the effect of the histidine kinase CovS on response regulator phosphorylation in group A Streptococcus.

Two-component gene regulatory systems (TCSs) are a major mechanism by which bacteria respond to environmental stimuli and thus are critical to infectivity. For example, the control of virulence regulator/sensor kinase (CovRS) TCS is central to the virulence of the major human pathogen group A Streptococcus (GAS). Here, we used a combination of quantitative in vivo phosphorylation assays, isoallelic strains that varied by only a single amino acid in CovS, and transcriptome analyses to characterize the impact of CovS on CovR phosphorylation and GAS global gene expression. We discovered that CovS primarily serves to phosphorylate CovR, thereby resulting in the repression of virulence factor-encoding genes. However, a GAS strain selectively deficient in CovS phosphatase activity had a distinct transcriptome relative to that of its parental strain, indicating that both CovS kinase and phosphatase activities influence the CovR phosphorylation status. Surprisingly, compared to a serotype M3 strain, serotype M1 GAS strains had high levels of phosphorylated CovR, low transcript levels of CovR-repressed genes, and strikingly different responses to environmental cues. Moreover, the inactivation of CovS in the serotype M1 background resulted in a greater decrease in phosphorylated CovR levels and a greater increase in the transcript levels of CovR-repressed genes than did CovS inactivation in a serotype M3 strain. These data clarify the influence of CovS on the CovR phosphorylation status and provide insight into why serotype M1 GAS strains have high rates of spontaneous mutations in covS during invasive GAS infection, thus providing a link between TCS molecular function and the epidemiology of deadly bacterial infections. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Complete sequences of six IncA/C plasmids of multidrug-resistant Salmonella enterica subsp. enterica serotype Newport.

Multidrug-resistant (MDR) Salmonella enterica subsp. enterica serotype Newport has been a long-standing public health concern in the United States. We present the complete sequences of six IncA/C plasmids from animal-derived MDR S. Newport ranging from 80.1 to 158.5 kb. They shared a genetic backbone with S. Newport IncA/C plasmids pSN254 and pAM04528. Copyright © 2015 Cao et al.

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July 7, 2019

Ceftriaxone-resistant Salmonella enterica serotype Typhimurium sequence type 313 from Kenyan patients is associated with the blaCTX-M-15 gene on a novel IncHI2 plasmid.

Multidrug-resistant bacteria pose a major challenge to the clinical management of infections in resource-poor settings. Although nontyphoidal Salmonella (NTS) bacteria cause predominantly enteric self-limiting illness in developed countries, NTS is responsible for a huge burden of life-threatening bloodstream infections in sub-Saharan Africa. Here, we characterized nine S. Typhimurium isolates from an outbreak involving patients who initially failed to respond to ceftriaxone treatment at a referral hospital in Kenya. These Salmonella enterica serotype Typhimurium isolates were resistant to ampicillin, chloramphenicol, cefuroxime, ceftriaxone, aztreonam, cefepime, sulfamethoxazole-trimethoprim, and cefpodoxime. Resistance to ß-lactams, including to ceftriaxone, was associated with carriage of a combination of blaCTX-M-15, blaOXA-1, and blaTEM-1 genes. The genes encoding resistance to heavy-metal ions were borne on the novel IncHI2 plasmid pKST313, which also carried a pair of class 1 integrons. All nine isolates formed a single clade within S. Typhimurium ST313, the major clone of an ongoing invasive NTS epidemic in the region. This emerging ceftriaxone-resistant clone may pose a major challenge in the management of invasive NTS in sub-Saharan Africa. Copyright © 2015, Kariuki et al.

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July 7, 2019

Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40.

Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type.We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro).Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

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July 7, 2019

Insights on the emergence of Mycobacterium tuberculosis from the analysis of Mycobacterium kansasii.

By phylogenetic analysis, Mycobacterium kansasii is closely related to Mycobacterium tuberculosis. Yet, although both organisms cause pulmonary disease, M. tuberculosis is a global health menace, whereas M. kansasii is an opportunistic pathogen. To illuminate the differences between these organisms, we have sequenced the genome of M. kansasii ATCC 12478 and its plasmid (pMK12478) and conducted side-by-side in vitro and in vivo investigations of these two organisms. The M. kansasii genome is 6,432,277 bp, more than 2 Mb longer than that of M. tuberculosis H37Rv, and the plasmid contains 144,951 bp. Pairwise comparisons reveal conserved and discordant genes and genomic regions. A notable example of genomic conservation is the virulence locus ESX-1, which is intact and functional in the low-virulence M. kansasii, potentially mediating phagosomal disruption. Differences between these organisms include a decreased predicted metabolic capacity, an increased proportion of toxin-antitoxin genes, and the acquisition of M. tuberculosis-specific genes in the pathogen since their common ancestor. Consistent with their distinct epidemiologic profiles, following infection of C57BL/6 mice, M. kansasii counts increased by less than 10-fold over 6 weeks, whereas M. tuberculosis counts increased by over 10,000-fold in just 3 weeks. Together, these data suggest that M. kansasii can serve as an image of the environmental ancestor of M. tuberculosis before its emergence as a professional pathogen, and can be used as a model organism to study the switch from an environmental opportunistic pathogen to a professional host-restricted pathogen. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Core genome and plasmidome of the quorum-quenching bacterium Rhodococcus erythropolis.

Rhodococcus erythropolis is a worldwide-distributed actinobacterium that exhibits a remarkable metabolic versatility illustrated by its ability to degrade complex compounds, such as quorum-sensing signals N-acylhomoserine lactones (NAHLs), phenols, sterols and fuel derivatives. Because of its catabolic properties, R. erythropolis strains are proposed as anti-biofouling agents against NAHL-dependent biofilms, biocontrol agents against NAHL-emitting plant pathogens, and bioremediation agents in contaminated waters and soils. Here, we used the PacBio technology to resolve the complete genome sequence of the biocontrol strain R. erythropolis R138. Its genome consisted in a circular chromosome (6,236,862 bp), a linear plasmid pLRE138 (477,915 bp) and a circular plasmid pCRE138 (91,729 bp). In addition, draft genomes of five R. erythropolis strains were determined by Illumina technology and compared with the other five R. erythropolis genomes that are available in public databases: 5,825 common CDSs were present in all of the eleven analyzed genomes and represented up to 87 % of those identified in R. erythropolis R138. This study highlighted the high proportion of core-genome genes in R. erythropolis, but a high variability of the plasmid content. Key-metabolic pathways which are involved in the degradation of complex molecules, such as NAHLs and phenol, catechol and sterol derivatives are coded by the R. erythropolis core-genome.

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July 7, 2019

Phylogeographical analysis of the dominant multidrug-resistant H58 clade of Salmonella Typhi identifies inter- and intracontinental transmission events.

The emergence of multidrug-resistant (MDR) typhoid is a major global health threat affecting many countries where the disease is endemic. Here whole-genome sequence analysis of 1,832 Salmonella enterica serovar Typhi (S. Typhi) identifies a single dominant MDR lineage, H58, that has emerged and spread throughout Asia and Africa over the last 30 years. Our analysis identifies numerous transmissions of H58, including multiple transfers from Asia to Africa and an ongoing, unrecognized MDR epidemic within Africa itself. Notably, our analysis indicates that H58 lineages are displacing antibiotic-sensitive isolates, transforming the global population structure of this pathogen. H58 isolates can harbor a complex MDR element residing either on transmissible IncHI1 plasmids or within multiple chromosomal integration sites. We also identify new mutations that define the H58 lineage. This phylogeographical analysis provides a framework to facilitate global management of MDR typhoid and is applicable to similar MDR lineages emerging in other bacterial species.

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July 7, 2019

Genomes of ‘Candidatus Liberibacter solanacearum’ Haplotype A from New Zealand and the United States Suggest Significant Genome Plasticity in the Species.

‘Candidatus Liberibacter solanacearum’ contains two solanaceous crop-infecting haplotypes, A and B. Two haplotype A draft genomes were assembled and compared with ZC1 (haplotype B), revealing inversion and relocation genomic rearrangements, numerous single-nucleotide polymorphisms, and differences in phage-related regions. Differences in prophage location and sequence were seen both within and between haplotype comparisons. OrthoMCL and BLAST analyses identified 46 putative coding sequences present in haplotype A that were not present in haplotype B. Thirty-eight of these loci were not found in sequences from other Liberibacter spp. Quantitative polymerase chain reaction (qPCR) assays designed to amplify sequences from 15 of these loci were screened against a panel of ‘Ca. L. solanacearum’-positive samples to investigate genetic diversity. Seven of the assays demonstrated within-haplotype diversity; five failed to amplify loci in at least one haplotype A sample while three assays produced amplicons from some haplotype B samples. Eight of the loci assays showed consistent A-B differentiation. Differences in genome arrangements, prophage, and qPCR results suggesting locus diversity within the haplotypes provide more evidence for genetic complexity in this emerging bacterial species.

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July 7, 2019

Complete genome sequence of ER2796, a DNA methyltransferase-deficient strain of Escherichia coli K-12.

We report the complete sequence of ER2796, a laboratory strain of Escherichia coli K-12 that is completely defective in DNA methylation. Because of its lack of any native methylation, it is extremely useful as a host into which heterologous DNA methyltransferase genes can be cloned and the recognition sequences of their products deduced by Pacific Biosciences Single-Molecule Real Time (SMRT) sequencing. The genome was itself sequenced from a long-insert library using the SMRT platform, resulting in a single closed contig devoid of methylated bases. Comparison with K-12 MG1655, the first E. coli K-12 strain to be sequenced, shows an essentially co-linear relationship with no major rearrangements despite many generations of laboratory manipulation. The comparison revealed a total of 41 insertions and deletions, and 228 single base pair substitutions. In addition, the long-read approach facilitated the surprising discovery of four gene conversion events, three involving rRNA operons and one between two cryptic prophages. Such events thus contribute both to genomic homogenization and to bacteriophage diversification. As one of relatively few laboratory strains of E. coli to be sequenced, the genome also reveals the sequence changes underlying a number of classical mutant alleles including those affecting the various native DNA methylation systems.

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July 7, 2019

Complete genome sequencing of a multidrug-resistant and human-invasive Salmonella enterica serovar Typhimurium strain of the emerging sequence type 213 genotype.

Salmonella enterica subsp. enterica serovar Typhimurium strain YU39 was isolated in 2005 in the state of Yucatán, Mexico, from a human systemic infection. The YU39 strain is representative of the multidrug-resistant emergent sequence type 213 (ST213) genotype. The YU39 complete genome is composed of a chromosome and seven plasmids. Copyright © 2015 Calva et al.

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July 7, 2019

Complete genome sequence of Salmonella enterica subsp. enterica serovar Agona 460004 2-1, associated with a multistate outbreak in the United States.

Within the last several years, Salmonella enterica subsp. enterica serovar Agona has been among the 20 most frequently isolated serovars in clinical cases of salmonellosis. In this report, the complete genome sequence of S. Agona strain 460004 2-1 isolated from unsweetened puffed-rice cereal during a multistate outbreak in 2008 was sequenced using single-molecule real-time DNA sequencing. Copyright © 2015 Hoffmann et al.

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