Epidemiological investigation of Pseudomonas aeruginosa isolates from a six-year-long hospital outbreak using high-throughput whole genome sequencing

ArticleinEurosurveillance: bulletin europeen sur les maladies transmissibles = European communicable disease bulletin 18(42) · October 2013with17 Reads
DOI: 10.2807/1560-7917.ES2013.18.42.20611 · Source: PubMed
Abstract
Although previous bacterial typing methods have been informative about potential relatedness of isolates collected during outbreaks, next-generation sequencing has emerged as a powerful tool to not only look at similarity between isolates, but also put differences into biological context. In this study, we have investigated the whole genome sequence of five Pseudomonas aeruginosa isolates collected during a persistent six-year outbreak at Nottingham University Hospitals National Health Service (NHS) Trust - City Campus, United Kingdom. Sequencing, using both Roche 454 and Illumina, reveals that most of these isolates are closely related. Some regions of difference are noted between this cluster of isolates and previously published genome sequences. These include regions containing prophages and prophage remnants such as the serotype-converting bacteriophage D3 and the cytotoxin-converting phage phi CTX. Additionally, single nucleotide polymorphisms (SNPs) between the genomic sequence data reveal key single base differences that have accumulated during the course of this outbreak, giving insight into the evolution of the outbreak strain. Differentiating SNPs were found within a wide variety of genes, including lasR, nrdG, tadZ, and algB. These have been generated at a rate estimated to be one SNP every four to five months. In conclusion, we demonstrate that the single base resolution of whole genome sequencing is a powerful tool in analysis of outbreak isolates that can not only show strain similarity, but also evolution over time and potential adaptation through gene sequence changes.
    • "SNPs/year during a hospital outbreak [16]; Markussen et al. found the DK01 lineage to accumulate 1.3 SNPs/year in a chronically infected Danish CF patient; and Cramer et al. estimated the mutation rate of lineage PA14 to be ~1 SNP/year over the course of infection of a German CF patient [3]. Mutation rates of the other lineages (IT02, IT03, IT05, IT06) could not be estimated since the number and temporal distribution of isolates were insufficient to obtain proper estimates of the mutation rates (effective sample sizes (ESS) of modeled parameters were below 10). "
    [Show abstract] [Hide abstract] ABSTRACT: Background Chronic infection with Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients, and a more complete understanding of P. aeruginosa within-host genomic evolution, transmission, and population genomics may provide a basis for improving intervention strategies. Here, we report the first genomic analysis of P. aeruginosa isolates sampled from Italian CF patients. Results By genome sequencing of 26 isolates sampled over 19 years from four patients, we elucidated the within-host evolution of clonal lineages in each individual patient. Many of the identified mutations were located in pathoadaptive genes previously associated with host adaptation, and we correlated mutations with changes in CF-relevant phenotypes such as antibiotic resistance. In addition, the genomic analysis revealed that three patients shared the same clone. Furthermore, we compared the genomes of the Italian CF isolates to a panel of genome sequenced strains of P. aeruginosa from other countries. Isolates from two of the Italian lineages belonged to clonal complexes of P. aeruginosa that have previously been identified in Danish CF patients, and our genomic comparison showed that clonal isolates from the same country may be more distantly related than clonal isolates from different countries. Conclusions This is the first whole-genome analysis of P. aeruginosa isolated from Italian CF patients, and together with both phenotypic and clinical information this dataset facilitates a more detailed understanding of P. aeruginosa within-host genomic evolution, transmission, and population genomics. We conclude that the evolution of the Italian lineages resembles what has been found in other countries.
    Full-text · Article · Oct 2015
    • "The advances of next-generation sequencing progressively turned genome sequence of bacteria and microbial eukaryotes somewhat feasible and inexpensive. In addition, it allows the analysis of several thousand of SNPs and may link epidemiology to pathogen biology, genome evolution, gene characterization in terms of resistance and virulence, gather information from noncoding and coding regions across the entire genome, give relevant evidence regarding linkage, recombination, chromosomal aberrations, and low frequency events such as horizontal gene transfer (Araujo 2014; Snyder et al. 2013). Microbial identification, SNP genotyping, resequencing, and analyses of difficult DNA secondary structures are some of the fields under exploitation by next-generation sequencing (Schuster 2008). "
    [Show abstract] [Hide abstract] ABSTRACT: Practical schemes based on single nucleotide polymorphisms (SNP) have been proposed as alternatives to simplify and replace the molecular methodologies based on the extensive sequencing analysis of genes. SNaPshot mini-sequencing has been progressively experienced during the last decade and represents a fast and robust strategy to analyze critical polymorphisms. Such assays have been proposed to characterize some bacteria and microbial eukaryotes, and its feasibility was now reviewed in the present manuscript. The mini-sequencing schemes showed high discriminatory power and competence for identification of microorganisms, but some specificity errors were still found, particularly for species of the Burkholderia cepacia complex and mycobacteria. SNP assays designed for other goals, e.g., comparison of strains, detection of serotypes, virulence, epidemic, and phylogenetic-related subgroups of isolates, can be very useful by facilitating the investigation of large collections of isolates. The next-generation of SNP assays might consider the inclusion of large number of markers to fully characterize microbial taxonomy and strains; nevertheless, these new technologies are still prone to errors and can largely benefit from integration with well-established mini-sequencing assays. Newly proposed molecular tools should be systematically tested in collections of isolates with high indexes of diversity and guarantee interlaboratorial validation.
    Full-text · Article · Feb 2015
    • "New benchtop devices are providing WGS capability to routine microbiological laboratories, and WGS is now becoming an important part of clinical microbiology and molecular epidemiology. This emerging field of " genomic epidemiology "4567891011 already comprises numerous studies that have evaluated the potential of WGS to trace the transmission of pathogens such as Clostridium difficile, Pseudomonas aeruginosa, Klebsiella pneumonia, Neisseria meningitidis, Staphylococcus aureus and Mycobacterium tuberculosis12131415161718192021. Moreover, WGS is playing an increasing role in molecular drug susceptibility testing (DST) and the identification of drug resistance mutations22232425262728. "
    [Show abstract] [Hide abstract] ABSTRACT: High-throughput DNA sequencing produces vast amounts of data, with millions of short reads that usually have to be mapped to a reference genome or newly assembled. Both reference-based mapping and de novo assembly are computationally intensive, generating large intermediary data files, and thus require bioinformatics skills that are often lacking in the laboratories producing the data. Moreover, many research and practical applications in microbiology require only a small fraction of the whole genome data. We developed KvarQ, a new tool that directly scans fastq files of bacterial genome sequences for known variants, such as single nucleotide polymorphisms (SNP), bypassing the need of mapping all sequencing reads to a reference genome and de novo assembly. Instead, KvarQ loads “testsuites” that define specific SNPs or short regions of interest in a reference genome, and directly synthesizes the relevant results based on the occurrence of these markers in the fastq files. KvarQ has a versatile command line interface and a graphical user interface. KvarQ currently ships with two “testsuites” for Mycobacterium tuberculosis, but new “testsuites” for other organisms can easily be created and distributed. In this article, we demonstrate how KvarQ can be used to successfully detect all main drug resistance mutations and phylogenetic markers in 880 bacterial whole genome sequences. The average scanning time per genome sequence was two minutes. The variant calls of a subset of these genomes were validated with a standard bioinformatics pipeline and revealed >99% congruency. KvarQ is a user-friendly tool that directly extracts relevant information from fastq files. This enables researchers and laboratory technicians with limited bioinformatics expertise to scan and analyze raw sequencing data in a matter of minutes. KvarQ is open-source, and pre-compiled packages with a graphical user interface are available at http://www.swisstph.ch/kvarq.
    Full-text · Article · Oct 2014
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