Mode and dynamics of vanA-type vancomycin resistance dissemination in Dutch hospitals

Background Enterococcus faecium is a commensal of the gastrointestinal tract of animals and humans but also a causative agent of hospital-acquired infections. Resistance against glycopeptides and to vancomycin has motivated the inclusion of E. faecium in the WHO global priority list. Vancomycin resistance can be conferred by the vanA gene cluster on the transposon Tn1546, which is frequently present in plasmids. The vanA gene cluster can be disseminated clonally but also horizontally either by plasmid dissemination or by Tn1546 transposition between different genomic locations. Methods We performed a retrospective study of the genomic epidemiology of 309 vancomycin-resistant E. faecium (VRE) isolates across 32 Dutch hospitals (2012-2015). Genomic information regarding clonality and Tn1546 characterization was extracted using hierBAPS sequence clusters (SC) and TETyper, respectively. Plasmids were predicted using gplas in combination with a network approach based on shared k-mer content. Next, we conducted a pairwise comparison between isolates sharing a potential epidemiological link to elucidate whether clonal, plasmid, or Tn1546 spread accounted for vanA-type resistance dissemination. Results On average, we estimated that 59% of VRE cases with a potential epidemiological link were unrelated which was defined as VRE pairs with a distinct Tn1546 variant. Clonal dissemination accounted for 32% cases in which the same SC and Tn1546 variants were identified. Horizontal plasmid dissemination accounted for 7% of VRE cases, in which we observed VRE pairs belonging to a distinct SC but carrying an identical plasmid and Tn1546 variant. In 2% of cases, we observed the same Tn1546 variant in distinct SC and plasmid types which could be explained by mixed and consecutive events of clonal and plasmid dissemination. Conclusions In related VRE cases, the dissemination of the vanA gene cluster in Dutch hospitals between 2012 and 2015 was dominated by clonal spread. However, we also identified outbreak settings with high frequencies of plasmid dissemination in which the spread of resistance was mainly driven by horizontal gene transfer (HGT). This study demonstrates the feasibility of distinguishing between modes of dissemination with short-read data and provides a novel assessment to estimate the relative contribution of nested genomic elements in the dissemination of vanA-type resistance.Peer reviewe

Genomic rearrangements uncovered by genome-wide co-evolution analysis of a major nosocomial pathogen, Enterococcus faecium

Enterococcus faecium is a gut commensal of the gastro-digestive tract, but also known as nosocomial pathogen among hospitalized patients. Population genetics based on whole-genome sequencing has revealed that E. faecium strains from hospitalized patients form a distinct Glade, designated Glade A1, and that plasmids are major contributors to the emergence of nosocomial E. faecium. Here we further explored the adaptive evolution of E faecium using a genome-wide co-evolution study (GWES) to identify co-evolving single-nucleotide polymorphisms (SNPs). We identified three genomic regions harbouring large numbers of SNPs in tight linkage that are not proximal to each other based on the completely assembled chromosome of the Glade A1 reference hospital isolate AUS0004. Close examination of these regions revealed that they are located at the borders of four different types of large-scale genomic rearrangements, insertion sites of two different genomic islands and an IS30-like transposon. In non-Glade A1 isolates, these regions are adjacent to each other and they lack the insertions of the genomic islands and IS30-like transposon. Additionally, among the Glade A1 isolates there is one group of pet isolates lacking the genomic rearrangement and insertion of the genomic islands, suggesting a distinct evolutionary trajectory. In silico analysis of the biological functions of the genes encoded in three regions revealed a common link to a stress response. This suggests that these rearrangements may reflect adaptation to the stringent conditions in the hospital environment, such as antibiotics and detergents, to which bacteria are exposed. In conclusion, to our knowledge, this is the first study using GWES to identify genomic rearrangements, suggesting that there is considerable untapped potential to unravel hidden evolutionary signals from population genomic data.Peer reviewe

A high-throughput multiplexing and selection strategy to complete bacterial genomes

Background: Bacterial whole-genome sequencing based on short-read technologies often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits those contiguous sequences to be unambiguously bridged into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced. Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose an isolate selection strategy that optimizes a representative selection of isolates for long-read sequencing considering as input large-scale bacterial collections. Results: Despite an uneven distribution of long reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98), which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library. Conclusions: The proposed long-read isolate selection ensures the completion of bacterial genomes that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.Peer reviewe

Comparative analysis of Mycobacterium tuberculosis pe and ppe genes reveals high sequence variation and apparent absence of selective constraints

CITATION: McEvoy, C. R. E. et al. 2012. Comparative analysis of Mycobacterium tuberculosis pe and ppe genes reveals high sequence variation and apparent absence of selective constraints. PLoS ONE, 7(4): e30593, doi:10.1371/journal.pone.0030593.The original publication is available at http://journals.plos.org/plosoneMycobacterium tuberculosis complex (MTBC) genomes contain 2 large gene families termed pe and ppe. The function of pe/ppe proteins remains enigmatic but studies suggest that they are secreted or cell surface associated and are involved in bacterial virulence. Previous studies have also shown that some pe/ppe genes are polymorphic, a finding that suggests involvement in antigenic variation. Using comparative sequence analysis of 18 publicly available MTBC whole genome sequences, we have performed alignments of 33 pe (excluding pe_pgrs) and 66 ppe genes in order to detect the frequency and nature of genetic variation. This work has been supplemented by whole gene sequencing of 14 pe/ppe (including 5 pe_pgrs) genes in a cohort of 40 diverse and well defined clinical isolates covering all the main lineages of the M. tuberculosis phylogenetic tree. We show that nsSNP's in pe (excluding pgrs) and ppe genes are 3.0 and 3.3 times higher than in non-pe/ppe genes respectively and that numerous other mutation types are also present at a high frequency. It has previously been shown that non-pe/ppe M. tuberculosis genes display a remarkably low level of purifying selection. Here, we also show that compared to these genes those of the pe/ppe families show a further reduction of selection pressure that suggests neutral evolution. This is inconsistent with the positive selection pressure of “classical” antigenic variation. Finally, by analyzing such a large number of genes we were able to detect large differences in mutation type and frequency between both individual genes and gene sub-families. The high variation rates and absence of selective constraints provides valuable insights into potential pe/ppe function. Since pe/ppe proteins are highly antigenic and have been studied as potential vaccine components these results should also prove informative for aspects of M. tuberculosis vaccine design.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030593Publisher's versio

Optimization of Standard In-House 24-Locus Variable-Number Tandem-Repeat Typing for Mycobacterium tuberculosis and Its Direct Application to Clinical Material

Variable-number tandem-repeat (VNTR) typing with a panel of 24 loci is the current gold standard in the molecular typing of Mycobacterium tuberculosis complex isolates. However, because of technical problems, a part of the loci often cannot be amplified by multiplex PCRs. Therefore, a considerable number of single-locus PCRs have to be performed for the loci with missing results, which impairs the laboratory work flow. Therefore, the original in-house method described by Supply et al. in 2006 was reevaluated. We modified seven primers and the PCR master mixture and obtained a strongly optimized in-house 24-locus VNTR typing method. The percentage of instantly complete 24-locus VNTR patterns detected in the routine flow of typing activities increased to 84.7% from the 72.3% obtained with the typing conducted with the commercially available Genoscreen MIRU-VNTR typing kit. The analytical sensitivity of the optimized in-house method was assessed by serial dilutions of M. tuberculosis in bronchoalveolar lavage fluid. A 1: 10 dilution of the different strains tested was the lowest dilution for the detection of a complete 24-locus VNTR pattern. The optimized in-house 24-locus VNTR typing method will reduce the turnaround time of typing significantly and also the financial burden of these activities

Evolution of Linked Avirulence Effectors in Leptosphaeria maculans Is Affected by Genomic Environment and Exposure to Resistance Genes in Host Plants

Brassica napus (canola) cultivars and isolates of the blackleg fungus, Leptosphaeria maculans interact in a ‘gene for gene’ manner whereby plant resistance (R) genes are complementary to pathogen avirulence (Avr) genes. Avirulence genes encode proteins that belong to a class of pathogen molecules known as effectors, which includes small secreted proteins that play a role in disease. In Australia in 2003 canola cultivars with the Rlm1 resistance gene suffered a breakdown of disease resistance, resulting in severe yield losses. This was associated with a large increase in the frequency of virulence alleles of the complementary avirulence gene, AvrLm1, in fungal populations. Surprisingly, the frequency of virulence alleles of AvrLm6 (complementary to Rlm6) also increased dramatically, even though the cultivars did not contain Rlm6. In the L. maculans genome, AvrLm1 and AvrLm6 are linked along with five other genes in a region interspersed with transposable elements that have been degenerated by Repeat-Induced Point (RIP) mutations. Analyses of 295 Australian isolates showed deletions, RIP mutations and/or non-RIP derived amino acid substitutions in the predicted proteins encoded by these seven genes. The degree of RIP mutations within single copy sequences in this region was proportional to their proximity to the degenerated transposable elements. The RIP alleles were monophyletic and were present only in isolates collected after resistance conferred by Rlm1 broke down, whereas deletion alleles belonged to several polyphyletic lineages and were present before and after the resistance breakdown. Thus, genomic environment and exposure to resistance genes in B. napus has affected the evolution of these linked avirulence genes in L. maculans

SNP/RD typing of Mycobacterium tuberculosis Beijing strains reveals local and worldwide disseminated clonal complexes

CITATION: Schurch, A. C. et al. 2011. SNP/RD typing of Mycobacterium tuberculosis Beijing strains reveals local and worldwide disseminated clonal complexes. PLoS ONE, 6(12): e28365, doi:10.1371/journal.pone.0028365.The original publication is available at http://journals.plos.org/plosoneThe Beijing strain is one of the most successful genotypes of Mycobacterium tuberculosis worldwide and appears to be highly homogenous according to existing genotyping methods. To type Beijing strains reliably we developed a robust typing scheme using single nucleotide polymorphisms (SNPs) and regions of difference (RDs) derived from whole-genome sequencing data of eight Beijing strains. SNP/RD typing of 259 M. tuberculosis isolates originating from 45 countries worldwide discriminated 27 clonal complexes within the Beijing genotype family. A total of 16 Beijing clonal complexes contained more than one isolate of known origin, of which two clonal complexes were strongly associated with South African origin. The remaining 14 clonal complexes encompassed isolates from different countries. Even highly resolved clonal complexes comprised isolates from distinct geographical sites. Our results suggest that Beijing strains spread globally on multiple occasions and that the tuberculosis epidemic caused by the Beijing genotype is at least partially driven by modern migration patterns. The SNPs and RDs presented in this study will facilitate future molecular epidemiological and phylogenetic studies on Beijing strains.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028365Publisher's versio

Mge-cluster : a reference-free approach for typing bacterial plasmids

Extrachromosomal elements of bacterial cells such as plasmids are notorious for their importance in evolution and adaptation to changing ecology. However, high-resolution population-wide analysis of plasmids has only become accessible recently with the advent of scalable long-read sequencing technology. Current typing methods for the classification of plasmids remain limited in their scope which motivated us to develop a computationally efficient approach to simultaneously recognize novel types and classify plasmids into previously identified groups. Here, we introduce mge-cluster that can easily handle thousands of input sequences which are compressed using a unitig representation in a de Bruijn graph. Our approach offers a faster runtime than existing algorithms, with moderate memory usage, and enables an intuitive visualization, classification and clustering scheme that users can explore interactively within a single framework. Mge-cluster platform for plasmid analysis can be easily distributed and replicated, enabling a consistent labelling of plasmids across past, present, and future sequence collections. We underscore the advantages of our approach by analysing a population-wide plasmid data set obtained from the opportunistic pathogen Escherichia coli, studying the prevalence of the colistin resistance gene mcr-1.1 within the plasmid population, and describing an instance of resistance plasmid transmission within a hospital environment.Peer reviewe

Apparent nosocomial adaptation of Enterococcus faecalis predates the modern hospital era

Enterococcus faecalis is a commensal and nosocomial pathogen, which is also ubiquitous in animals and insects, representing a classical generalist microorganism. Here, we study E. faecalis isolates ranging from the pre-antibiotic era in 1936 up to 2018, covering a large set of host species including wild birds, mammals, healthy humans, and hospitalised patients. We sequence the bacterial genomes using short- and long-read techniques, and identify multiple extant hospital-associated lineages, with last common ancestors dating back as far as the 19th century. We find a population cohesively connected through homologous recombination, a metabolic flexibility despite a small genome size, and a stable large core genome. Our findings indicate that the apparent hospital adaptations found in hospital-associated E. faecalis lineages likely predate the "modern hospital" era, suggesting selection in another niche, and underlining the generalist nature of this nosocomial pathogen.Enterococcus faecalis is a commensal microorganism of animals, insects and humans, but also a nosocomial pathogen. Here, the authors analyse genomic sequences from E. faecalis isolates from animals and humans, and find that the last common ancestors of multiple hospital-associated lineages date to the pre-antibiotic era.Peer reviewe

Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data.

Contains fulltext : 125977.pdf (publisher's version ) (Open Access)BACKGROUND: Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of the rate of change in the genome over relevant timescales is required. METHODS: We attempted to estimate a molecular clock by sequencing 199 isolates from epidemiologically linked tuberculosis cases, collected in the Netherlands spanning almost 16 years. RESULTS: Multiple analyses support an average mutation rate of ~0.3 SNPs per genome per year. However, all analyses revealed a very high degree of variation around this mean, making the confirmation of links proposed by epidemiology, and inference of novel links, difficult. Despite this, in some cases, the phylogenetic context of other strains provided evidence supporting the confident exclusion of previously inferred epidemiological links. CONCLUSIONS: This in-depth analysis of the molecular clock revealed that it is slow and variable over short time scales, which limits its usefulness in transmission studies. However, the superior resolution of whole genome sequencing can provide the phylogenetic context to allow the confident exclusion of possible transmission events previously inferred via traditional DNA fingerprinting techniques and epidemiological cluster investigation. Despite the slow generation of variation even at the whole genome level we conclude that the investigation of tuberculosis transmission will benefit greatly from routine whole genome sequencing