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Understanding Mycobacterium tuberculosis (Mtb) transmission is essential to guide efficient tuberculosis control strategies. Traditional strain typing lacks sufficient discriminatory power to resolve large outbreaks. Here, we tested the potential of using next generation genome sequencing for identification of outbreak-related transmission chains. During long-term (1997 to 2010) prospective population-based molecular epidemiological surveillance comprising a total of 2,301 patients, we identified a large outbreak caused by an Mtb strain of the Haarlem lineage. The main performance outcome measure of whole genome sequencing (WGS) analyses was the degree of correlation of the WGS analyses with contact tracing data and the spatio-temporal distribution of the outbreak cases. WGS analyses of the 86 isolates revealed 85 single nucleotide polymorphisms (SNPs), subdividing the outbreak into seven genome clusters (two to 24 isolates each), plus 36 unique SNP profiles. WGS results showed that the first outbreak isolates detected in 1997 were falsely clustered by classical genotyping. In 1998, one clone (termed \"Hamburg clone\") started expanding, apparently independently from differences in the social environment of early cases. Genome-based clustering patterns were in better accordance with contact tracing data and the geographical distribution of the cases than clustering patterns based on classical genotyping. A maximum of three SNPs were identified in eight confirmed human-to-human transmission chains, involving 31 patients. We estimated the Mtb genome evolutionary rate at 0.4 mutations per genome per year. This rate suggests that Mtb grows in its natural host with a doubling time of approximately 22 h (400 generations per year). Based on the genome variation discovered, emergence of the Hamburg clone was dated back to a period between 1993 and 1997, hence shortly before the discovery of the outbreak through epidemiological surveillance. Our findings suggest that WGS is superior to conventional genotyping for Mtb pathogen tracing and investigating micro-epidemics. WGS provides a measure of Mtb genome evolution over time in its natural host context.

In genome evolution, genetic variants are the source of diversity, which natural selection acts upon. Treatment of human tuberculosis (TB) induces a strong selection pressure for the emergence of antibiotic resistance-conferring variants in the infecting Mycobacterium tuberculosis (MTB) strains. MTB evolution in response to treatment has been intensively studied and mainly attributed to point substitutions. However, the frequency and contribution of insertions and deletions (indels) to MTB genome evolution remains poorly understood. Here, we analyzed a multi-drug resistant MTB outbreak for the presence of high-quality indels and substitutions. We find that indels are significantly enriched in genes conferring antibiotic resistance. Furthermore, we show that indels are inherited during the outbreak and follow a molecular clock with an evolutionary rate of 5.37e-9 indels/site/year, which is 23 times lower than the substitution rate. Inherited indels may co-occur with substitutions in genes along related biological pathways; examples are iron storage and resistance to second-line antibiotics. This suggests that epistatic interactions between indels and substitutions affect antibiotic resistance and compensatory evolution in MTB.

Transmission-driven multi-/extensively drug resistant (M/XDR) tuberculosis (TB) is the largest single contributor to human mortality due to antimicrobial resistance. A few major clades of the Mycobacterium tuberculosis complex belonging to lineage 2, responsible for high prevalence of MDR-TB in Eurasia, show outstanding transnational distributions. Here, we determined factors underlying the emergence and epidemic spread of the W148 clade by genome sequencing and Bayesian demogenetic analyses of 720 isolates from 23 countries. We dated a common ancestor around 1963 and identified two successive epidemic expansions in the late 1980s and late 1990s, coinciding with major socio-economic changes in the post-Soviet Era. These population expansions favored accumulation of resistance mutations to up to 11 anti-TB drugs, with MDR evolving toward additional resistances to fluoroquinolones and second-line injectable drugs within 20 years on average. Timescaled haplotypic density analysis revealed that widespread acquisition of compensatory mutations was associated with transmission success of XDR strains. Virtually all W148 strains harbored a hypervirulence-associated ppe38 gene locus, and incipient recurrent emergence of prpR mutation-mediated drug tolerance was detected. The outstanding genetic arsenal of this geographically widespread M/XDR strain clade represents a “perfect storm” that jeopardizes the successful introduction of new anti-M/XDR-TB antibiotic regimens. An outbreak of the multidrug-resistant Mycobacterium tuberculosis lineage W148 has spread widely across Russia, Central Asia and Europe. Here, the authors use whole genome sequences of ~700 isolates of this lineage collected over ~20 years to analyze its spread, evolution of drug resistance, and impact of compensatory mutations.

Mycobacterium abscessus is an emerging multidrug-resistant non-tuberculous mycobacterium that causes a wide spectrum of infections and has caused several local outbreaks worldwide. To facilitate standardized prospective molecular surveillance, we established a novel core genome multilocus sequence typing (cgMLST) scheme. Whole genome sequencing data of 1991 isolates were employed to validate the scheme, re-analyze global population structure and set genetic distance thresholds for cluster detection and taxonomic identification. We confirmed and amended the nomenclature of the main dominant circulating clones and found that these also correlate well with traditional 7-loci MLST. Dominant circulating clones could be linked to a corresponding reference genome with less than 250 alleles while 99% of pairwise comparisons between epidemiologically linked isolates were below 25 alleles and 90% below 10 alleles. These thresholds can be used to guide further epidemiological investigations. Overall, the scheme will help to unravel the apparent global spread of certain clonal complexes and as yet undiscovered transmission routes. Mycobacterium abscessus is an emerging infection of increasing public health concern due to outbreaks and intrinsic multidrug-resistance. Here, the authors develop and evaluate a core-genome multilocus sequence typing scheme for this pathogen to facilitate standardised molecular surveillance.

Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains represent a major threat for tuberculosis (TB) control. Treatment of MDR-TB patients is long and less effective, resulting in a significant number of treatment failures. The development of further resistances leads to extensively drug-resistant (XDR) variants. However, data on the individual reasons for treatment failure, e.g. an induced mutational burst, and on the evolution of bacteria in the patient are only sparsely available. To address this question, we investigated the intra-patient evolution of serial MTBC isolates obtained from three MDR-TB patients undergoing longitudinal treatment, finally leading to XDR-TB. Sequential isolates displayed identical IS6110 fingerprint patterns, suggesting the absence of exogenous re-infection. We utilized whole genome sequencing (WGS) to screen for variations in three isolates from Patient A and four isolates from Patient B and C, respectively. Acquired polymorphisms were subsequently validated in up to 15 serial isolates by Sanger sequencing. We determined eight (Patient A) and nine (Patient B) polymorphisms, which occurred in a stepwise manner during the course of the therapy and were linked to resistance or a potential compensatory mechanism. For both patients, our analysis revealed the long-term co-existence of clonal subpopulations that displayed different drug resistance allele combinations. Out of these, the most resistant clone was fixed in the population. In contrast, baseline and follow-up isolates of Patient C were distinguished each by eleven unique polymorphisms, indicating an exogenous re-infection with an XDR strain not detected by IS6110 RFLP typing. Our study demonstrates that intra-patient microevolution of MDR-MTBC strains under longitudinal treatment is more complex than previously anticipated. However, a mutator phenotype was not detected. The presence of different subpopulations might confound phenotypic and molecular drug resistance tests. Furthermore, high resolution WGS analysis is necessary to accurately detect exogenous re-infection as classical genotyping lacks discriminatory power in high incidence settings.

Background A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret whole-genome sequencing data for genotypic drug susceptibility testing (DST). Methods We investigated mutations in 92 genes implicated in resistance to 21 anti-tuberculosis drugs using the genomes of 405 phylogenetically diverse MTBC strains. The role of phylogenetically informative mutations was assessed by routine phenotypic DST data for the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide from a separate collection of over 7000 clinical strains. Selected mutations/strains were further investigated by minimum inhibitory concentration (MIC) testing. Results Out of 547 phylogenetically informative mutations identified, 138 were classified as not correlating with resistance to first-line drugs. MIC testing did not reveal a discernible impact of a Rv1979c deletion shared by M. africanum lineage 5 strains on resistance to clofazimine. Finally, we found molecular evidence that some MTBC subgroups may be hyper-susceptible to bedaquiline and clofazimine by different loss-of-function mutations affecting a drug efflux pump subunit (MmpL5). Conclusions Our findings underline that the genetic diversity in MTBC has to be studied more systematically to inform the design of clinical trials and to define sound epidemiologic cut-off values (ECOFFs) for new and repurposed anti-tuberculosis drugs. In that regard, our comprehensive variant catalogue provides a solid basis for the interpretation of mutations in genotypic as well as in phenotypic DST assays.

Background The Mycobacterium avium complex (MAC) comprises the most frequent non-tuberculous mycobacteria (NTM) in Central Europe and currently includes twelve species. M. avium (MAV), M. intracellulare subsp. intracellulare (MINT), and M. intracellulare subsp. chimaera (MCH) are clinically most relevant. However, the population structure and genomic landscape of MAC linked with potential pathobiological differences remain little investigated. Methods Whole genome sequencing (WGS) was performed on a multi-national set of MAC isolates from Germany, France, and Switzerland. Phylogenetic analysis was conducted, as well as plasmids, resistance, and virulence genes predicted from WGS data. Data was set into a global context with publicly available sequences. Finally, detailed clinical characteristics were associated with genomic data in a subset of the cohort. Results Overall, 610 isolates from 465 patients were included. The majority could be assigned to MAV ( n  = 386), MCH ( n  = 111), and MINT ( n  = 77). We demonstrate clustering with less than 12 SNPs distance of isolates obtained from different patients in all major MAC species and the identification of trans-European or even trans-continental clusters when set into relation with 1307 public sequences. However, none of our MCH isolates clustered closely with the heater-cooler unit outbreak strain Zuerich-1. Known plasmids were detected in MAV (325/1076, 30.2%), MINT (62/327, 19.0%), and almost all MCH-isolates (457/463, 98.7%). Predicted resistance to aminoglycosides or macrolides was rare. Overall, there was no direct link between phylogenomic grouping and clinical manifestations, but MCH and MINT were rarely found in patients with extra-pulmonary disease (OR 0.12 95% CI 0.04–0.28, p  < 0.001 and OR 0.11 95% CI 0.02–0.4, p  = 0.004, respectively) and MCH was negatively associated with fulfillment of the ATS criteria when isolated from respiratory samples (OR 0.28 95% CI 0.09-0.7, p = 0.011). With 14 out of 43 patients with available serial isolates, co-infections or co-colonizations with different strains or even species of the MAC were frequent (32.6%). Conclusions This study demonstrates clustering and the presence of plasmids in a large proportion of MAC isolates in Europe and in a global context. Future studies need to urgently define potential ways of transmission of MAC isolates and the potential involvement of plasmids in virulence.

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) is an emerging threat to TB control in Ukraine, a country with the third highest XDR TB burden globally. We used whole-genome sequencing of a convenience sample to identify bacterial genetic and patient-related factors associated with MDR/XDR TB in this country. MDR/XDR TB was associated with 3 distinct Mycobacterium tuberculosis complex lineage 2 (Beijing) clades, Europe/Russia W148 outbreak, Central Asia outbreak, and Ukraine outbreak, which comprised 68.9% of all MDR/XDR TB strains from southern Ukraine. MDR/XDR TB was also associated with previous treatment for TB and urban residence. The circulation of Beijing outbreak strains harboring broad drug resistance, coupled with constraints in drug supply and limited availability of phenotypic drug susceptibility testing, needs to be considered when new TB management strategies are implemented in Ukraine.

Whole genome sequencing (WGS) is revolutionary for diagnostics of TB and its mutations associated with drug-resistances, but its uptake in low- and middle-income countries is hindered by concerns of implementation feasibility. Here, we provide a proof of concept for its successful implementation in such a setting. WGS was implemented in the Kyrgyz Republic. We estimated needs of up to 55 TB-WGS per week and chose the MiSeq platform (Illumina, USA) because of its capacity of up to 60 TB-WGS per week. The project’s timeline was completed in 93-weeks. Costs of large equipment and accompanying costs were 222,065 USD and 8462 USD, respectively. The first 174 WGS costed 277 USD per sequence, but this was skewed by training inefficiencies. Based on real prices and presuming optimal utilization of WGS capacities, WGS costs could drop to 167 and 141 USD per WGS using MiSeq Reagent Kits v2 (500-cycles) and v3 (600-cycles), respectively. Five trainings were required to prepare the staff for autonomous WGS which cost 48,250 USD. External assessment confirmed excellent performance of WGS by the Kyrgyz laboratory in an interlaboratory comparison of 30  M. tuberculosis genomes showing complete agreeance of results.