Outbreak Investigation Using High-Throughput Genome Sequencing within a Diagnostic Microbiology Laboratory

Abstract

f Next-generation sequencing (NGS) of bacterial genomes has recently become more accessible and is now available to the routine diagnostic microbiology laboratory. However, questions remain regarding its feasibility, particularly with respect to data analysis in nonspecialist centers. To test the applicability of NGS to outbreak investigations, Ion Torrent sequencing was used to investigate a putative multidrug-resistant Escherichia coli outbreak in the neonatal unit of the Mercy Hospital for Women, Melbourne, Australia. Four suspected outbreak strains and a comparator strain were sequenced. Genome-wide single nucleotide polymorphism (SNP) analysis demonstrated that the four neonatal intensive care unit (NICU) strains were identical and easily differentiated from the comparator strain. Genome sequence data also determined that the NICU strains belonged to multilocus sequence type 131 and carried the bla CTX-M-15 extended-spectrum beta-lactamase. Comparison of the outbreak strains to all publicly available complete E. coli genome sequences showed that they clustered with neonatal meningitis and uropathogenic isolates. The turnaround time from a positive culture to the completion of sequencing (prior to data analysis) was 5 days, and the cost was approximately $300 per strain (for the reagents only). The main obstacles to a mainstream adoption of NGS technologies in diagnostic microbiology laboratories are currently cost (although this is decreasing), a paucity of user-friendly and clinically focused bioinformatics platforms, and a lack of genomics expertise outside the research environment. Despite these hurdles, NGS technologies provide unparalleled high-resolution genotyping in a short time frame and are likely to be widely implemented in the field of diagnostic microbiology in the next few years, particularly for epidemiological investigations (replacing current typing methods) and the characterization of resistance determinants. Clinical microbiologists need to familiarize themselves with these technologies and their applications. R esearch in the field of pathogen biology has been transformed over the last several decades with the introduction of wholegenome sequencing, beginning with the complete sequencing of the Haemophilus influenzae genome in 1995 (1). This has led to significant developments in the study of molecular epidemiology, virulence, antimicrobial resistance, and vaccinology and in understanding complex microbial communities. More recently, the development of high-throughput (or "next-generation") sequencing technologies has meant, for the first time, that these methods fall within the financial and technical grasp of a medium or large diagnostic microbiology laboratory (2).Next-generation sequencing (NGS) methods are also being used in smaller-scale local projects to determine epidemiology in outbreak settings (2, 3), examine the development of resistance mutations during antibiotic use in a single patient (4, 5), and identify bacteria in place of using 16S rRNA sequencing (6). However, the up...

Precise Dissection of an Escherichia coli O157:H7 Outbreak by Single Nucleotide Polymorphism Analysis

“…therefore, offers a precise and durable way to differentiate isolates. Recent reports demonstrated the value of whole-pathogen sequencing in controlling nosocomial antibiotic-resistant Klebsiella pneumoniae and Staphylococcus aureus and nosocomial neonatal E. coli outbreaks (20)(21)(22)(23). We now demonstrate that whole bacterial genome sequencing can dissect E. coli O157:H7 infection clusters and reduce the proportion of variant epidemiologic histories by refining the assignment of isolates as being outbreak or nonoutbreak.…”

Utility of Whole-Genome Sequencing in Characterizing Acinetobacter Epidemiology and Analyzing Hospital Outbreaks

“…These benefits of WGS must be balanced against the cost of commercially available next-generation sequencing instruments or services and the current complexity of sequence analysis. Published estimates of per-strain WGS costs approach those for conventional typing techniques (WGS, $35 to 300; PFGE, $150; MLST, $65 to 120; and Rep-PCR, $26 [79,94,113]), although these estimates are highly dependent upon how instrument costs are amortized. WGS may allow additional cost savings resulting from earlier control of hospital outbreaks and infection prevention.…”

Predicting the virulence of MRSA from its genome sequence

“…Since the first whole-genome sequence of a free-living organism, Haemophilus influenzae, was published (Fleischmann et al 1995), sequencing technology has advanced to a stage where a bacterial genome can be sequenced in a matter of hours (Parkhill and Wren 2011;Didelot et al 2012a;Eyre et al 2012;Köser et al 2012a). This has led to an explosion of genomic data that has allowed us to monitor outbreaks in hospitals (Köser et al 2012b;Young et al 2012;Harris et al 2013;Sherry et al 2013;Walker et al 2013), track strains transitioning from carrier to invasive status , and perform detailed epidemiological studies to understand aspects of pathogen biology (Castillo-Ramírez et al 2011Didelot et al 2012b;McAdam et al 2012;Holden et al 2013). While some success has also been made in predicting phenotype from genotype, such as the antimicrobial resistance (Farhat et al 2013;Holden et al 2013), for more complex phenotypes, such as virulence, involving the contribution of several genes, this has not yet been possible.…”