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Volume 6,
Issue 6,
2020
Volume 6, Issue 6, 2020
- Research Article
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- Microbial Evolution and Epidemiology
- Population Genomics
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Comparative genomics of global optrA-carrying Enterococcus faecalis uncovers a common chromosomal hotspot for optrA acquisition within a diversity of core and accessory genomes
More LessLinezolid-resistant Enterococcus faecalis (LREfs) carrying optrA are increasingly reported globally from multiple sources, but we lack a comprehensive analysis of human and animal optrA-LREfs strains. To assess if optrA is dispersed in isolates with varied genetic backgrounds or with common genetic features, we investigated the phylogenetic structure, genetic content [antimicrobial resistance (AMR), virulence, prophages, plasmidome] and optrA-containing platforms of 27 publicly available optrA-positive E. faecalis genomes from different hosts in seven countries. At the genome-level analysis, an in-house database with 64 virulence genes was tested for the first time. Our analysis showed a diversity of clones and adaptive gene sequences related to a wide range of genera from Firmicutes . Phylogenies of core and accessory genomes were not congruent, and at least PAI-associated and prophage genes contribute to such differences. Epidemiologically unrelated clones (ST21, ST476-like and ST489) obtained from human clinical and animal hosts in different continents over eight years (2010–2017) could be phylogenetically related (3–126 SNPs difference). optrA was located on the chromosome within a Tn6674-like element (n=10) or on medium-size plasmids (30–60 kb; n=14) belonging to main plasmid families (RepA_N/Inc18/Rep_3). In most cases, the immediate gene vicinity of optrA was generally identical in chromosomal (Tn6674) or plasmid (impB-fexA-optrA) backbones. Tn6674 was always inserted into the same ∆radC integration site and embedded in a 32 kb chromosomal platform common to strains from different origins (patients, healthy humans, and animals) in Europe, Africa, and Asia during 2012–2017. This platform is conserved among hundreds of E. faecalis genomes and proposed as a chromosomal hotspot for optrA integration. The finding of optrA in strains sharing common adaptive features and genetic backgrounds across different hosts and countries suggests the occurrence of common and independent genetic events occurring in distant regions and might explain the easy de novo generation of optrA-positive strains. It also anticipates a dramatic increase of optrA carriage and spread with a serious impact on the efficacy of linezolid for the treatment of Gram-positive infections.
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Assessing the genomic relatedness and evolutionary rates of persistent verotoxigenic Escherichia coli serotypes within a closed beef herd in Canada
Verotoxigenic Escherichia coli (VTEC) are food- and water-borne pathogens associated with both sporadic illness and outbreaks of enteric disease. While it is known that cattle are reservoirs of VTEC, little is known about the genomic variation of VTEC in cattle, and whether the variation in genomes reported for human outbreak strains is consistent with individual animal or group/herd sources of infection. A previous study of VTEC prevalence identified serotypes carried persistently by three consecutive cohorts of heifers within a closed herd of cattle. This present study aimed to: (i) determine whether the genomic relatedness of bovine isolates is similar to that reported for human strains associated with single source outbreaks, (ii) estimate the rates of genome change among dominant serotypes over time within a cattle herd, and (iii) identify genomic features of serotypes associated with persistence in cattle. Illumina MiSeq genome sequencing and genotyping based on allelic and single nucleotide variations were completed, while genome change over time was measured using Bayesian evolutionary analysis sampling trees. The accessory genome, including the non-protein-encoding intergenic regions (IGRs), virulence factors, antimicrobial-resistance genes and plasmid gene content of representative persistent and sporadic cattle strains were compared using Fisher’s exact test corrected for multiple comparisons. Herd strains from serotypes O6:H34 (n=22), O22:H8 (n=30), O108:H8 (n=39), O139:H19 (n=44) and O157:H7 (n=106) were readily distinguishable from epidemiologically unrelated strains of the same serotype using a similarity threshold of 10 or fewer allele differences between adjacent nodes. Temporal-cohort clustering within each serotype was supported by date randomization analysis. Substitutions per site per year were consistent with previously reported values for E. coli ; however, there was low branch support for these values. Acquisition of the phage-encoded Shiga toxin 2 gene in serotype O22:H8 was observed. Pan-genome analyses identified accessory regions that were more prevalent in persistent serotypes (P≤0.05) than in sporadic serotypes. These results suggest that VTEC serotypes from a specific cattle population are highly clonal with a similar level of relatedness as human single-source outbreak-associated strains, but changes in the genome occur gradually over time. Additionally, elements in the accessory genomes may provide a selective advantage for persistence of VTEC within cattle herds.
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- Mechanisms of Evolution
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Microevolution in the major outer membrane protein OmpA of Acinetobacter baumannii
More LessAcinetobacter baumannii is nowadays a relevant nosocomial pathogen characterized by multidrug resistance (MDR) and concomitant difficulties to treat infections. OmpA is the most abundant A. baumannii outer membrane (OM) protein, and is involved in virulence, host-cell recognition, biofilm formation, regulation of OM stability, permeability and antibiotic resistance. OmpA members are two‐domain proteins with an N‐terminal eight‐stranded β‐barrel domain with four external loops (ELs) interacting with the environment, and a C‐terminal periplasmic domain binding non‐covalently to the peptidoglycan. Here, we combined data from genome sequencing, phylogenetic and multilocus sequence analyses from 975 strains/isolates of the Acinetobacter calcoaceticus / Acinetobacter baumannii complex (ACB), 946 from A. baumannii , to explore ompA microevolutionary divergence. Five major ompA variant groups were identified (V1 to V5) in A. baumannii , encompassing 52 different alleles coding for 23 different proteins. Polymorphisms were concentrated in five regions corresponding to the four ELs and the C‐terminal end, and provided evidence for intra‐genic recombination. ompA variants were not randomly distributed across the A . baumannii phylogeny, with the most frequent V1(lct)a1 allele found in most clonal complex 2 (CC2) strains and the second most frequent V2(lct)a1 allele in the majority of CC1 strains. Evidence was found for assortative exchanges of ompA alleles not only between separate A . baumannii lineages, but also different ACB species. The overall results have implications for A. baumannii evolution, epidemiology, virulence and vaccine design.
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- Communicable Disease Genomics
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Duplication and diversification of a unique chromosomal virulence island hosting the subtilase cytotoxin in Escherichia coli ST58
More LessThe AB5 cytotoxins are important virulence factors in Escherichia coli . The most notable members of the AB5 toxin families include Shiga toxin families 1 (Stx1) and 2 (Stx2), which are associated with enterohaemorrhagic E. coli infections causing haemolytic uraemic syndrome and haemorrhagic colitis. The subAB toxins are the newest and least well understood members of the AB5 toxin gene family. The subtilase toxin genes are divided into a plasmid-based variant, subAB1, originally described in enterohaemorrhagic E. coli O113:H21, and distinct chromosomal variants, subAB2, that reside in pathogenicity islands encoding additional virulence effectors. Previously we identified a chromosomal subAB2 operon within an E. coli ST58 strain IBS28 (ONT:H25) taken from a wild ibis nest at an inland wetland in New South Wales, Australia. Here we show the subAB2 toxin operon comprised part of a 140 kb tRNA–Phe chromosomal island that co-hosted tia, encoding an outer-membrane protein that confers an adherence and invasion phenotype and additional virulence and accessory genetic content that potentially originated from known virulence island SE-PAI. This island shared a common evolutionary history with a secondary 90 kb tRNA–Phe pathogenicity island that was presumably generated via a duplication event. IBS28 is closely related [200 single-nucleotide polymorphisms (SNPs)] to four North American ST58 strains. The close relationship between North American isolates of ST58 and IBS28 was further supported by the identification of the only copy of a unique variant of IS26 within the O-antigen gene cluster. Strain ISB28 may be a historically important E. coli ST58 genome sequence hosting a progenitor pathogenicity island encoding subAB.
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- Microbial Communities
- Human
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Preterm infants harbour diverse Klebsiella populations, including atypical species that encode and produce an array of antimicrobial resistance- and virulence-associated factors
Klebsiella spp. are frequently enriched in the gut microbiota of preterm neonates, and overgrowth is associated with necrotizing enterocolitis (NEC), nosocomial infections and late-onset sepsis. Little is known about the genomic and phenotypic characteristics of preterm-associated Klebsiella , as previous studies have focused on the recovery of antimicrobial-resistant isolates or culture-independent molecular analyses. The aim of this study was to better characterize preterm-associated Klebsiella populations using phenotypic and genotypic approaches. Faecal samples from a UK cohort of healthy and sick preterm neonates (n=109) were screened on MacConkey agar to isolate lactose-positive Enterobacteriaceae . Whole-genome sequences were generated for Klebsiella spp., and virulence and antimicrobial resistance genes identified. Antibiotic susceptibility profiling and in vitro macrophage and iron assays were undertaken for the Klebsiella strains. Metapangenome analyses with a manually curated genome dataset were undertaken to examine the diversity of Klebsiella oxytoca and related bacteria in a publicly available shotgun metagenome dataset. Approximately one-tenth of faecal samples harboured Klebsiella spp. ( Klebsiella pneumoniae , 7.3 %; Klebsiella quasipneumoniae , 0.9 %; Klebsiella grimontii , 2.8 %; Klebsiella michiganensis , 1.8 %). Isolates recovered from NEC- and sepsis-affected infants and those showing no signs of clinical infection (i.e. ‘healthy’) encoded multiple β-lactamases. No difference was observed between isolates recovered from healthy and sick infants with respect to in vitro siderophore production (all encoded enterobactin in their genomes). All K. pneumoniae , K. quasipneumoniae , K. grimontii and K. michiganensis faecal isolates tested were able to reside and persist in macrophages, indicating their immune evasion abilities. Metapangenome analyses of published metagenomic data confirmed our findings regarding the presence of K. michiganensis in the preterm gut. There is little difference in the phenotypic and genomic characteristics of Klebsiella isolates recovered from healthy and sick infants. Identification of β-lactamases in all isolates may prove problematic when defining treatment regimens for NEC or sepsis, and suggests that healthy preterm infants contribute to the resistome. Refined analyses with curated sequence databases are required when studying closely related species present in metagenomic data.
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- Other - Animals, Insects, Plants
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Whole-genome sequence analysis of environmental Escherichia coli from the faeces of straw-necked ibis (Threskiornis spinicollis) nesting on inland wetlands
Wildlife, and birds in particular, play an increasingly recognized role in the evolution and transmission of Escherichia coli that pose a threat to humans. To characterize these lineages and their potential threat from an evolutionary perspective, we isolated and performed whole-genome sequencing on 11 sequence types (STs) of E. coli recovered from the desiccated faeces of straw-necked ibis (Threskiornis spinicollis) nesting on inland wetlands located in geographically different regions of New South Wales, Australia. Carriage of virulence-associated genes was limited, and no antimicrobial resistance genes were detected, but novel variants of an insertion element that plays an important role in capturing and mobilizing antibiotic resistance genes, IS26, were identified and characterized. The isolates belonged to phylogroups B1 and D, including types known to cause disease in humans and animals. Specifically, we found E. coli ST58, ST69, ST162, ST212, ST446, ST906, ST2520, ST6096 and ST6241, and a novel phylogroup D strain, ST10208. Notably, the ST58 strain hosted significant virulence gene carriage. The sequences of two plasmids hosting putative virulence-associated factors with incompatibility groups I1 and Y, an extrachromosomal integrative/conjugative element, and a variant of a large Escherichia phage of the family Myoviridae, were additionally characterized. We identified multiple epidemiologically relevant gene signatures that link the ibis isolates to sequences from international sources, plus novel variants of IS26 across different sequence types and in different contexts.
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- Microbe-Niche Interactions
- Host Adaptation
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Comparative genomic analysis of three intestinal species reveals reductions in secreted pathogenesis determinants in bovine-specific and non-pathogenic Cryptosporidium species
More LessThe three common intestinal Cryptosporidium species in cattle differ significantly in host range, pathogenicity and public health significance. While Cryptosporidium parvum is pathogenic in pre-weaned calves and has a broad host range, C. bovis and C. ryanae are largely non-pathogenic and bovine-specific species in post-weaned calves. Thus far, only the genome of C. parvum has been sequenced. To improve our understanding of the genetic determinants of biological differences among Cryptosporidium spcies, we sequenced the genomes of C. bovis and C. ryanae and conducted a comparative genomics analysis. The genome of C. bovis has a gene content and organization more similar to C. ryanae than to other Cryptosporidium species sequenced to date; the level of similarity in amino acid and nucleotide sequences between the two species is 75.2 and 69.4 %, respectively. A total of 3723 and 3711 putative protein-encoding genes were identified in the genomes of C. bovis and C. ryanae, respectively, which are fewer than the 3981 in C. parvum. Metabolism is similar among the three species, although energy production pathways are further reduced in C. bovis and C. ryanae. Compared with C. parvum, C. bovis and C. ryanae have lost 14 genes encoding mucin-type glycoproteins and three for insulinase-like proteases. Other gene gains and losses in the two bovine-specific and non-pathogenic species also involve the secretory pathogenesis determinants (SPDs); they have lost all genes encoding MEDLE, FLGN and SKSR proteins, and two of the three genes for NFDQ proteins, but have more genes encoding secreted WYLE proteins, secreted leucine-rich proteins and GPI-anchored adhesin PGA18. The only major difference between C. bovis and C. ryanae is in nucleotide metabolism. In addition, half of the highly divergent genes between C. bovis and C. ryanae encode secreted or membrane-bound proteins. Therefore, C. bovis and C. ryanae have gene organization and metabolic pathways similar to C. parvum, but have lost some invasion-associated mucin glycoproteins, insulinase-like proteases, MEDLE secretory proteins and other SPDs. The multiple gene families under positive selection, such as helicase-associated domains, AMP-binding domains, protein kinases, mucins, insulinases and TRAPs could contribute to differences in host specificity and pathogenicity between C. parvum and C. bovis. Biological studies should be conducted to assess the contribution of these copy number variations to the narrow host range and reduced pathogenicity of C. bovis and C. ryanae.
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- Genomic Methodologies
- Novel Phylogenetic Methods
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Deciphering the role of insertion sequences in the evolution of bacterial epidemic pathogens with panISa software
More LessNext-generation sequencing (NGS) is now widely used in microbiology to explore genome evolution and the structure of pathogen outbreaks. Bioinformatics pipelines readily detect single-nucleotide polymorphisms or short indels. However, bacterial genomes also evolve through the action of small transposable elements called insertion sequences (ISs), which are difficult to detect due to their short length and multiple repetitions throughout the genome. We designed panISa software for the ab initio detection of IS insertions in the genomes of prokaryotes. PanISa has been released as open source software (GPL3) available from https://github.com/bvalot/panISa. In this study, we assessed the utility of this software for evolutionary studies, by reanalysing five published datasets for outbreaks of human major pathogens in which ISs had not been specifically investigated. We reanalysed the raw data from each study, by aligning the reads against reference genomes and running panISa on the alignments. Each hit was automatically curated and IS-related events were validated on the basis of nucleotide sequence similarity, by comparison with the ISFinder database. In Acinetobacter baumannii , the panISa pipeline identified ISAba1 or ISAba125 upstream from the ampC gene, which encodes a cephalosporinase in all third-generation cephalosporin-resistant isolates. In the genomes of Vibrio cholerae isolates, we found that early Haitian isolates had the same ISs as Nepalese isolates, confirming the inferred history of the contamination of this island. In Enterococcus faecalis , panISa identified regions of high plasticity, including a pathogenicity island enriched in IS-related events. The overall distribution of ISs deduced with panISa was consistent with SNP-based phylogenic trees, for all species considered. The role of ISs in pathogen evolution has probably been underestimated due to difficulties detecting these transposable elements. We show here that panISa is a useful addition to the bioinformatics toolbox for analyses of the evolution of bacterial genomes. PanISa will facilitate explorations of the functional impact of ISs and improve our understanding of prokaryote evolution.
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