Increasing numbers of individuals with cystic fibrosis are becoming infected with the multidrug-resistant non-tuberculous mycobacterium (NTM) Mycobacterium abscessus, which causes progressive lung damage and is extremely challenging to treat. How this organism is acquired is not currently known, but there is growing concern that person-to-person transmission could occur. We aimed to define the mechanisms of acquisition of M abscessus in individuals with cystic fibrosis. Whole genome sequencing and antimicrobial susceptibility testing were done on 168 consecutive isolates of M abscessus from 31 patients attending an adult cystic fibrosis centre in the UK between 2007 and 2011. In parallel, we undertook detailed environmental testing for NTM and defined potential opportunities for transmission between patients both in and out of hospital using epidemiological data and social network analysis. Phylogenetic analysis revealed two clustered outbreaks of near-identical isolates of the M abscessus subspecies massiliense (from 11 patients), differing by less than ten base pairs. This variation represents less diversity than that seen within isolates from a single individual, strongly indicating between-patient transmission. All patients within these clusters had numerous opportunities for within-hospital transmission from other individuals, while comprehensive environmental sampling, initiated during the outbreak, failed to detect any potential point source of NTM infection. The clusters of M abscessus subspecies massiliense showed evidence of transmission of mutations acquired during infection of an individual to other patients. Thus, isolates with constitutive resistance to amikacin and clarithromycin were isolated from several individuals never previously exposed to long-term macrolides or aminoglycosides, further indicating cross-infection. Whole genome sequencing has revealed frequent transmission of multidrug resistant NTM between patients with cystic fibrosis despite conventional cross-infection measures. Although the exact transmission route is yet to be established, our epidemiological analysis suggests that it could be indirect. The Wellcome Trust, Papworth Hospital, NIHR Cambridge Biomedical Research Centre, UK Health Protection Agency, Medical Research Council, and the UKCRC Translational Infection Research Initiative.

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis (M. tb). Whole genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike conventional molecular tests, this has only previously been achievable from cultured M. tb. Here we describe a method utilising biotinylated RNA baits, designed specifically for M. tb DNA to capture full M. tb genomes directly from infected sputum samples, allowing whole genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the UK and Lithuania where a matched culture sample was available, and two samplesthat had failed to grow in culture. M. tb sequencing data was obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were high quality (>20x depth and >90% of genome covered). Results were compared with conventional molecular and culture-based methods, and high levels of concordance were observed between phenotypical resistance and predicted resistance based on genotype. High quality sequence data was obtained from one smear positive culture negative case. This study demonstrates for the first time, the successful and accurate sequencing of M. tb genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalised, rather than empirical, treatment of drug resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.

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.

Efforts to contain drug-resistant tuberculosis depend on the rapid detection and effective treatment of cases, together with public health interventions to prevent and investigate ongoing transmission. The necessary laboratory support for these activities includes the identification of the Mycobacterium tuberculosis complex, antimicrobial susceptibility testing, and bacterial genotyping. However, even in well-resourced countries, it typically takes 1 to 2 months to achieve all these goals because of the slow growth rate of the M. tuberculosis complex.^1,2 Moreover, phenotypic susceptibility testing can be unreliable and is not performed for some agents. Molecular techniques have accelerated some of these diagnostic functions, but they only interrogate a small part of the microbial genome and do not provide all the clinically relevant information.^1-3 Whole-genome sequencing has not been used as a diagnostic tool for tuberculosis, in part because of the need to culture M. tuberculosis complex for several weeks, until sufficient DNA can be extracted.^2,4

Jump starting pathogen evolution: Mycobacteria are mostly environmental saprotrophs, but during human history, some have become our pathogens. In the past 50 years or so, intractable and virulent infections of Mycobacterium abscessus have emerged in people with cystic fibrosis. Bryant et al. investigated how these mycobacteria have evolved into human pathogens so quickly (see the Perspective by Brugha and Spencer). Chronic infections in the lung offer plenty of evolutionary scope for the emergence of virulent clones after horizontal gene transfer and hypermutation. Pathogens are acquired by environmental contamination, which leaves open a window for clinical control because the most virulent clones survive poorly outside the body. Therefore, immediate treatment and enhanced infection-control measures for M. abscessus cases could reduce opportunities for the evolution of direct person-to-person transmission. Science , this issue p. eabb8699 ; see also p. 465

Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.