Claudio U. Köser

Routine Use of Microbial Whole Genome Sequencing in Diagnostic and Public Health Microbiology

Whole genome sequencing (WGS) promises to be transformative for the practice of clinical microbiology, and the rapidly falling cost and turnaround time mean that this will become a viable technology in diagnostic and reference laboratories in the near future. The objective of this article is to consider at a very practical level where, in the context of a modern diagnostic microbiology laboratory, WGS might be cost-effective compared to current alternatives. We propose that molecular epidemiology performed for surveillance and outbreak investigation and genotypic antimicrobial susceptibility testing for microbes that are difficult to grow represent the most immediate areas for application of WGS, and discuss the technical and infrastructure requirements for this to be implemented.

Whole-Genome Sequencing for Rapid Susceptibility Testing of M. tuberculosis

As reported here, whole-genome sequencing has the potential to rapidly facilitate the determination of antimicrobial susceptibility, especially for slower-growing pathogens, such as Mycobacterium tuberculosis.

Genomic Diversity among Drug Sensitive and Multidrug Resistant Isolates of Mycobacterium tuberculosis with Identical DNA Fingerprints

Background Mycobacterium tuberculosis complex (MTBC), the causative agent of tuberculosis (TB), is characterized by low sequence diversity making this bacterium one of the classical examples of a genetically monomorphic pathogen. Because of this limited DNA sequence variation, routine genotyping of clinical MTBC isolates for epidemiological purposes relies on highly discriminatory DNA fingerprinting methods based on mobile and repetitive genetic elements. According to the standard view, isolates exhibiting the same fingerprinting pattern are considered direct progeny of the same bacterial clone, and most likely reflect ongoing transmission or disease relapse within individual patients. Methodology/Principal Findings Here we further investigated this assumption and used massively parallel whole-genome sequencing to compare one drug-susceptible (K-1) and one multidrug resistant (MDR) isolate (K-2) of a rapidly spreading M. tuberculosis Beijing genotype clone from a high incidence region (Karakalpakstan, Uzbekistan). Both isolates shared the same IS6110 RFLP pattern and the same allele at 23 out of 24 MIRU-VNTR loci. We generated 23.9 million (K-1) and 33.0 million (K-2) paired 50 bp purity filtered reads corresponding to a mean coverage of 483.5 fold and 656.1 fold respectively. Compared with the laboratory strain H37Rv both Beijing isolates shared 1,209 SNPs. The two Beijing isolates differed by 130 SNPs and one large deletion. The susceptible isolate had 55 specific SNPs, while the MDR variant had 75 specific SNPs, including the five known resistance-conferring mutations. Conclusions Our results suggest that M. tuberculosis isolates exhibiting identical DNA fingerprinting patterns can harbour substantial genomic diversity. Because this heterogeneity is not captured by traditional genotyping of MTBC, some aspects of the transmission dynamics of tuberculosis could be missed or misinterpreted. Furthermore, a valid differentiation between disease relapse and exogenous reinfection might be impossible using standard genotyping tools if the overall diversity of circulating clones is limited. These findings have important implications for clinical trials of new anti-tuberculosis drugs.

Rapid Bacterial Whole-Genome Sequencing to Enhance Diagnostic and Public Health Microbiology

IMPORTANCE The latest generation of benchtop DNA sequencing platforms can provide an accurate whole-genome sequence (WGS) for a broad range of bacteria in less than a day. These could be used to more effectively contain the spread of multidrug-resistant pathogens. OBJECTIVE To compare WGS with standard clinical microbiology practice for the investigation of nosocomial outbreaks caused by multidrug-resistant bacteria, the identification of genetic determinants of antimicrobial resistance, and typing of other clinically important pathogens. DESIGN, SETTING, AND PARTICIPANTS A laboratory-based study of hospital inpatients with a range of bacterial infections at Cambridge University Hospitals NHS Foundation Trust, a secondary and tertiary referral center in England, comparing WGS with standard diagnostic microbiology using stored bacterial isolates and clinical information. MAIN OUTCOMES AND MEASURES Specimens were taken and processed as part of routine clinical care, and cultured isolates stored and referred for additional reference laboratory testing as necessary. Isolates underwent DNA extraction and library preparation prior to sequencing on the Illumina MiSeq platform. Bioinformatic analyses were performed by persons blinded to the clinical, epidemiologic, and antimicrobial susceptibility data. RESULTS We investigated 2 putative nosocomial outbreaks, one caused by vancomycin-resistant Enterococcus faecium and the other by carbapenem-resistant Enterobacter cloacae; WGS accurately discriminated between outbreak and nonoutbreak isolates and was superior to conventional typing methods. We compared WGS with standard methods for the identification of the mechanism of carbapenem resistance in a range of gram-negative bacteria (Acinetobacter baumannii, E cloacae, Escherichia coli, and Klebsiella pneumoniae). This demonstrated concordance between phenotypic and genotypic results, and the ability to determine whether resistance was attributable to the presence of carbapenemases or other resistance mechanisms. Whole-genome sequencing was used to recapitulate reference laboratory typing of clinical isolates of Neisseria meningitidis and to provide extended phylogenetic analyses of these. CONCLUSIONS AND RELEVANCE The speed, accuracy, and depth of information provided by WGS platforms to confirm or refute outbreaks in hospitals and the community, and to accurately define transmission of multidrug-resistant and other organisms, represents an important advance.

Whole-genome sequencing to control antimicrobial resistance

Highlights • Owing to improvements in sequencing technologies, microbial whole-genome sequencing (WGS) has emerged as a central tool to control antibiotic resistance.• WGS has been used to develop novel antibiotics and diagnostic tests.• WGS has been key to surveillance and the study of the emergence of antibiotic resistance.• Rapid WGS has the potential to be used as a tool for infection control in the clinic and, in some cases, as a primary diagnostic tool to detect resistance.

A pilot study of rapid whole-genome sequencing for the investigation of a Legionella outbreak

Objectives Epidemiological investigations of Legionnaires’ disease outbreaks rely on the rapid identification and typing of clinical and environmental Legionella isolates in order to identify and control the source of infection. Rapid bacterial whole-genome sequencing (WGS) is an emerging technology that has the potential to rapidly discriminate outbreak from non-outbreak isolates in a clinically relevant time frame. Methods We performed a pilot study to determine the feasibility of using bacterial WGS to differentiate outbreak from non-outbreak isolates collected during an outbreak of Legionnaires’ disease. Seven Legionella isolates (three clinical and four environmental) were obtained from the reference laboratory and sequenced using the Illumina MiSeq platform at Addenbrookes Hospital, Cambridge. Bioinformatic analysis was performed blinded to the epidemiological data at the Wellcome Trust Sanger Institute. Results We were able to distinguish outbreak from non-outbreak isolates using bacterial WGS, and to confirm the probable environmental source. Our analysis also highlighted constraints, which were the small number of Legionella pneumophila isolates available for sequencing, and the limited number of published genomes for comparison. Conclusions We have demonstrated the feasibility of using rapid WGS to investigate an outbreak of Legionnaires’ disease. Future work includes building larger genomic databases of L pneumophila from both clinical and environmental sources, developing automated data interpretation software, and conducting a cost–benefit analysis of WGS versus current typing methods.

The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria : report from the EUCAST Subcommittee

Whole genome sequencing (WGS) offers the potential to predict antimicrobial susceptibility from a single assay. The European Committee on Antimicrobial Susceptibility Testing established a subcommittee to review the current development status of WGS for bacterial antimicrobial susceptibility testing (AST). The published evidence for using WGS as a tool to infer antimicrobial susceptibility accurately is currently either poor or non-existent and the evidence / knowledge base requires significant expansion. The primary comparators for assessing genotypic-phenotypic concordance from WGS data should be changed to epidemiological cut-off values in order to improve differentiation of wild-type from non-wild-type isolates (harbouring an acquired resistance). Clinical breakpoints should be a secondary comparator. This assessment will reveal whether genetic predictions could also be used to guide clinical decision making. Internationally agreed principles and quality control (QC) metrics will facilitate early harmonization of analytical approaches and interpretive criteria for WGS-based predictive AST. Only data sets that pass agreed QC metrics should be used in AST predictions. Minimum performance standards should exist and comparative accuracies across different WGS laboratories and processes should be measured. To facilitate comparisons, a single public database of all known resistance loci should be established, regularly updated and strictly curated using minimum standards for the inclusion of resistance loci. For most bacterial species the major limitations to widespread adoption for WGS-based AST in clinical laboratories remain the current high-cost and limited speed of inferring antimicrobial susceptibility from WGS data as well as the dependency on previous culture because analysis directly on specimens remains challenging. For most bacterial species there is currently insufficient evidence to support the use of WGS-inferred AST to guide clinical decision making. WGS-AST should be a funding priority if it is to become a rival to phenotypic AST. This report will be updated as the available evidence increases.

Rapid Whole-Genome Sequencing for Investigation of a Suspected Tuberculosis Outbreak

ABSTRACT Two Southeast Asian students attending the same school in the United Kingdom presented with pulmonary tuberculosis. An epidemiological investigation failed to link the two cases, and drug resistance profiles of the Mycobacterium tuberculosis isolates were discrepant. Whole-genome sequencing of the isolates found them to be genetically identical, suggesting a missed transmission event.

Phylogenetic polymorphisms in antibiotic resistance genes of the Mycobacterium tuberculosis complex

OBJECTIVES Sequence analysis of known antibiotic resistance genes of the Mycobacterium tuberculosis complex (MTBC) is increasingly being used to infer phenotypic resistance to a variety of antibiotics. However, a clear understanding of the genotype-phenotype relationship is required to interpret genotypic susceptibility results accurately. In this context, it is particularly important to distinguish phylogenetically informative neutral polymorphisms from true resistance-conferring mutations. METHODS Using a collection of 71 strains that encompasses all major MTBC genotypes, we mapped the genetic diversity in 18 genes that are known to be involved or were previously implicated in antibiotic resistance to eight current as well as two novel antibiotics. This included bedaquiline, capreomycin, ethambutol, fluoroquinolones, isoniazid, PA-824, para-aminosalicylic acid, prothionamide, rifampicin and streptomycin. Moreover, we included data from one of our prior studies that focused on two of the three known pyrazinamide resistance genes. RESULTS We found 58 phylogenetic polymorphisms that were markers for the genotypes M. tuberculosis Beijing, Haarlem, Latin American-Mediterranean (LAM), East African Indian (EAI), Delhi/Central Asian (CAS), Ghana, Turkey (Tur), Uganda I and II, Ural and X-type, as well as for Mycobacterium africanum genotypes West African I (WA I) and II (WA II), Mycobacterium bovis, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium microti and Mycobacterium canettii. CONCLUSIONS This study represents one of the most extensive overviews of phylogenetically informative polymorphisms in known resistance genes to date, and will serve as a resource for the design and interpretation of genotypic susceptibility assays.

Importance of the Genetic Diversity within the Mycobacterium tuberculosis Complex for the Development of Novel Antibiotics and Diagnostic Tests of Drug Resistance

ABSTRACT Despite being genetically monomorphic, the limited genetic diversity within the Mycobacterium tuberculosis complex (MTBC) has practical consequences for molecular methods for drug susceptibility testing and for the use of current antibiotics and those in clinical trials. It renders some representatives of MTBC intrinsically resistant against one or multiple antibiotics and affects the spectrum and consequences of resistance mutations selected for during treatment. Moreover, neutral or silent changes within genes responsible for drug resistance can cause false-positive results with hybridization-based assays, which have been recently introduced to replace slower phenotypic methods. We discuss the consequences of these findings and propose concrete steps to rigorously assess the genetic diversity of MTBC to support ongoing clinical trials.