Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens.

/pdf/real-time-whole-genome-sequencing-for-routine-typing-4s0cecv3a6.pdf

Real-Time Whole-Genome Sequencing for Routine Typing, Surveillance, and Outbreak Detection of Verotoxigenic Escherichia coli

The Gram-negative bacteria Klebsiella pneumoniae is a major cause of nosocomial infections, primarily among immunocompromised patients. The emergence of strains resistant to carbapenems has left few treatment options, making infection containment critical. In 2011, the U.S. National Institutes of Health Clinical Center experienced an outbreak of carbapenem-resistant K. pneumoniae that affected 18 patients, 11 of whom died. Whole-genome sequencing was performed on K. pneumoniae isolates to gain insight into why the outbreak progressed despite early implementation of infection control procedures. Integrated genomic and epidemiological analysis traced the outbreak to three independent transmissions from a single patient who was discharged 3 weeks before the next case became clinically apparent. Additional genomic comparisons provided evidence for unexpected transmission routes, with subsequent mining of epidemiological data pointing to possible explanations for these transmissions. Our analysis demonstrates that integration of genomic and epidemiological data can yield actionable insights and facilitate the control of nosocomial transmission.

/pdf/tracking-a-hospital-outbreak-of-carbapenem-resistant-4wt5qr93c4.pdf

Tracking a Hospital Outbreak of Carbapenem-Resistant Klebsiella pneumoniae with Whole-Genome Sequencing

Rapid molecular typing of bacterial pathogens is critical for public health epidemiology, surveillance and infection control, yet routine use of whole genome sequencing (WGS) for these purposes poses significant challenges. Here we present SRST2, a read mapping-based tool for fast and accurate detection of genes, alleles and multi-locus sequence types (MLST) from WGS data. Using >900 genomes from common pathogens, we show SRST2 is highly accurate and outperforms assembly-based methods in terms of both gene detection and allele assignment. We include validation of SRST2 within a public health laboratory, and demonstrate its use for microbial genome surveillance in the hospital setting. In the face of rising threats of antimicrobial resistance and emerging virulence among bacterial pathogens, SRST2 represents a powerful tool for rapidly extracting clinically useful information from raw WGS data. Source code is available from http://katholt.github.io/srst2/.

https://minerva-access.unimelb.edu.au/bitstream/handle/11343/262273/PMC4237778.pdf

SRST2: Rapid genomic surveillance for public health and hospital microbiology labs

Summary Background Tuberculosis incidence in the UK has risen in the past decade. Disease control depends on epidemiological data, which can be difficult to obtain. Whole-genome sequencing can detect microevolution within Mycobacterium tuberculosis strains. We aimed to estimate the genetic diversity of related M tuberculosis strains in the UK Midlands and to investigate how this measurement might be used to investigate community outbreaks. Methods In a retrospective observational study, we used Illumina technology to sequence M tuberculosis genomes from an archive of frozen cultures. We characterised isolates into four groups: cross-sectional, longitudinal, household, and community. We measured pairwise nucleotide differences within hosts and between hosts in household outbreaks and estimated the rate of change in DNA sequences. We used the findings to interpret network diagrams constructed from 11 community clusters derived from mycobacterial interspersed repetitive-unit–variable-number tandem-repeat data. Findings We sequenced 390 separate isolates from 254 patients, including representatives from all five major lineages of M tuberculosis . The estimated rate of change in DNA sequences was 0·5 single nucleotide polymorphisms (SNPs) per genome per year (95% CI 0·3–0·7) in longitudinal isolates from 30 individuals and 25 families. Divergence is rarely higher than five SNPs in 3 years. 109 (96%) of 114 paired isolates from individuals and households differed by five or fewer SNPs. More than five SNPs separated isolates from none of 69 epidemiologically linked patients, two (15%) of 13 possibly linked patients, and 13 (17%) of 75 epidemiologically unlinked patients (three-way comparison exact p Interpretation Whole-genome sequencing can delineate outbreaks of tuberculosis and allows inference about direction of transmission between cases. The technique could identify super-spreaders and predict the existence of undiagnosed cases, potentially leading to early treatment of infectious patients and their contacts. Funding Medical Research Council, Wellcome Trust, National Institute for Health Research, and the Health Protection Agency.

https://www.thelancet.com/pdfs/journals/laninf/PIIS1473-3099(12)70277-3.pdf

Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study

Whole-genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for addressing many microbiological questions. Here, we review the current status of clinical microbiology and how it has already begun to be transformed by using next-generation sequencing. We focus on three essential tasks: identifying the species of an isolate, testing its properties, such as resistance to antibiotics and virulence, and monitoring the emergence and spread of bacterial pathogens. We predict that the application of next-generation sequencing will soon be sufficiently fast, accurate and cheap to be used in routine clinical microbiology practice, where it could replace many complex current techniques with a single, more efficient workflow.

/pdf/transforming-clinical-microbiology-with-bacterial-genome-4hty8gxowx.pdf

Transforming clinical microbiology with bacterial genome sequencing

Whole-genome sequencing (WGS) could potentially provide a single platform for extracting all the information required to predict an organism's phenotype. However, its ability to provide accurate predictions has not yet been demonstrated in large independent studies of specific organisms. In this study, we aimed to develop a genotypic prediction method for antimicrobial susceptibilities. The whole genomes of 501 unrelated Staphylococcus aureus isolates were sequenced, and the assembled genomes were interrogated using BLASTn for a panel of known resistance determinants (chromosomal mutations and genes carried on plasmids). Results were compared with phenotypic susceptibility testing for 12 commonly used antimicrobial agents (penicillin, methicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, vancomycin, trimethoprim, gentamicin, fusidic acid, rifampin, and mupirocin) performed by the routine clinical laboratory. We investigated discrepancies by repeat susceptibility testing and manual inspection of the sequences and used this information to optimize the resistance determinant panel and BLASTn algorithm. We then tested performance of the optimized tool in an independent validation set of 491 unrelated isolates, with phenotypic results obtained in duplicate by automated broth dilution (BD Phoenix) and disc diffusion. In the validation set, the overall sensitivity and specificity of the genomic prediction method were 0.97 (95% confidence interval [95% CI], 0.95 to 0.98) and 0.99 (95% CI, 0.99 to 1), respectively, compared to standard susceptibility testing methods. The very major error rate was 0.5%, and the major error rate was 0.7%. WGS was as sensitive and specific as routine antimicrobial susceptibility testing methods. WGS is a promising alternative to culture methods for resistance prediction in S. aureus and ultimately other major bacterial pathogens.

http://sro.sussex.ac.uk/49919/1/Prediction_of_Staphylococcus_aureus_Antimicrobial_Resistance.pdf

Prediction of Staphylococcus aureus Antimicrobial Resistance by Whole-Genome Sequencing

Objectives To investigate the prospects of newly available benchtop sequencers to provide rapid whole-genome data in routine clinical practice. Next-generation sequencing has the potential to resolve uncertainties surrounding the route and timing of person-to-person transmission of healthcare-associated infection, which has been a major impediment to optimal management. Design The authors used Illumina MiSeq benchtop sequencing to undertake case studies investigating potential outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile . Setting Isolates were obtained from potential outbreaks associated with three UK hospitals. Participants Isolates were sequenced from a cluster of eight MRSA carriers and an associated bacteraemia case in an intensive care unit, another MRSA cluster of six cases and two clusters of C difficile . Additionally, all C difficile isolates from cases over 6 weeks in a single hospital were rapidly sequenced and compared with local strain sequences obtained in the preceding 3 years. Main outcome measure Whole-genome genetic relatedness of the isolates within each epidemiological cluster. Results Twenty-six MRSA and 15  C difficile isolates were successfully sequenced and analysed within 5 days of culture. Both MRSA clusters were identified as outbreaks, with most sequences in each cluster indistinguishable and all within three single nucleotide variants (SNVs). Epidemiologically unrelated isolates of the same spa -type were genetically distinct (≥21 SNVs). In both C difficile clusters, closely epidemiologically linked cases (in one case sharing the same strain type) were shown to be genetically distinct (≥144 SNVs). A reconstruction applying rapid sequencing in C difficile surveillance provided early outbreak detection and identified previously undetected probable community transmission. Conclusions This benchtop sequencing technology is widely generalisable to human bacterial pathogens. The findings provide several good examples of how rapid and precise sequencing could transform identification of transmission of healthcare-associated infection and therefore improve hospital infection control and patient outcomes in routine clinical practice.

https://bmjopen.bmj.com/content/bmjopen/2/3/e001124.full.pdf

A pilot study of rapid benchtop sequencing of Staphylococcus aureus and Clostridium difficile for outbreak detection and surveillance

Whole-genome sequencing in hierarchy with pulsed-field gel electrophoresis: the utility of this approach to establish possible sources of MRSA cross-transmission.

N C Gordon

Papers

Prediction of Staphylococcus aureus Antimicrobial Resistance by Whole-Genome Sequencing

A pilot study of rapid benchtop sequencing of Staphylococcus aureus and Clostridium difficile for outbreak detection and surveillance

Whole-genome sequencing in hierarchy with pulsed-field gel electrophoresis: the utility of this approach to establish possible sources of MRSA cross-transmission.