Thomas Abeel

Evolution of Extensively Drug-Resistant Tuberculosis over Four Decades: Whole Genome Sequencing and Dating Analysis of Mycobacterium tuberculosis Isolates from KwaZulu-Natal

Background The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents. Methods and Findings We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations, including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tuberculosis that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 y prior to the Tugela Ferry outbreak (katG S315T [isoniazid]; gidB 130 bp deletion [streptomycin]; 1957 [95% highest posterior density (HPD): 1937–1971]), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P [rifampicin]; pncA 1 bp insertion [pyrazinamide]; 1984 [95% HPD: 1974–1992]) and 10 y (rpoB D435G [rifampicin]; rrs 1400 [kanamycin]; gyrA A90V [ofloxacin]; 1995 [95% HPD: 1988–1999]) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe. Conclusions In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic era. Subsequent accumulation of stepwise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts but also those that date back 50 y. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance.

Genomic and functional analyses of Mycobacterium tuberculosis strains implicate ald in D-cycloserine resistance

A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic second-line drugs such as D-cycloserine. Here we used the whole-genome sequences from 498 strains of M. tuberculosis to identify new resistance-conferring genotypes. By combining association and correlated evolution tests with strategies for amplifying signal from rare variants, we found that loss-of-function mutations in ald (Rv2780), encoding L-alanine dehydrogenase, were associated with unexplained drug resistance. Convergent evolution of this loss of function was observed exclusively among multidrug-resistant strains. Drug susceptibility testing established that ald loss of function conferred resistance to D-cycloserine, and susceptibility to the drug was partially restored by complementation of ald. Clinical strains with mutations in ald and alr exhibited increased resistance to D-cycloserine when cultured in vitro. Incorporation of D-cycloserine resistance in novel molecular diagnostics could allow for targeted use of this toxic drug among patients with susceptible infections.

Genomic analysis of globally diverse Mycobacterium tuberculosis strains provides insights into the emergence and spread of multidrug resistance

Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

Whole Genome Sequencing of Mycobacterium africanum Strains from Mali Provides Insights into the Mechanisms of Geographic Restriction

Background Mycobacterium africanum, made up of lineages 5 and 6 within the Mycobacterium tuberculosis complex (MTC), causes up to half of all tuberculosis cases in West Africa, but is rarely found outside of this region. The reasons for this geographical restriction remain unknown. Possible reasons include a geographically restricted animal reservoir, a unique preference for hosts of West African ethnicity, and an inability to compete with other lineages outside of West Africa. These latter two hypotheses could be caused by loss of fitness or altered interactions with the host immune system. Methodology/Principal Findings We sequenced 92 MTC clinical isolates from Mali, including two lineage 5 and 24 lineage 6 strains. Our genome sequencing assembly, alignment, phylogeny and average nucleotide identity analyses enabled us to identify features that typify lineages 5 and 6 and made clear that these lineages do not constitute a distinct species within the MTC. We found that in Mali, lineage 6 and lineage 4 strains have similar levels of diversity and evolve drug resistance through similar mechanisms. In the process, we identified a putative novel streptomycin resistance mutation. In addition, we found evidence of person-to-person transmission of lineage 6 isolates and showed that lineage 6 is not enriched for mutations in virulence-associated genes. Conclusions This is the largest collection of lineage 5 and 6 whole genome sequences to date, and our assembly and alignment data provide valuable insights into what distinguishes these lineages from other MTC lineages. Lineages 5 and 6 do not appear to be geographically restricted due to an inability to transmit between West African hosts or to an elevated number of mutations in virulence-associated genes. However, lineage-specific mutations, such as mutations in cell wall structure, secretion systems and cofactor biosynthesis, provide alternative mechanisms that may lead to host specificity.

Mycobacterium tuberculosis Whole Genome Sequences From Southern India Suggest Novel Resistance Mechanisms and the Need for Region-Specific Diagnostics

Key points By sequencing 223 M. tuberculosis strains from Southern India, we expanded the studied genetic diversity of lineages 1 and 3. We observed local transmission of strains; unexplained resistance; potential novel resistance mutations; and that isoniazid resistance was gained first.

Fatal Nosocomial MDR TB Identified through Routine Genetic Analysis and Whole-Genome Sequencing

To the Editor: In November 2012, a 44-year-old HIV-negative white man (patient 1) with fever, fatigue, and breathlessness sought care at a hospital in the United Kingdom. He had never traveled abroad but had biopsy-proven alcoholic cirrhosis. No acid-fast bacilli were seen on multiple samples, including ascitic fluid, and he received treatment for presumptive abdominal tuberculosis (TB). Mycobacterium tuberculosis was subsequently cultured after 12 days. His clinical condition deteriorated, and he died of multiorgan failure 44 days after admission. The cultured M. tuberculosis was subsequently confirmed as multidrug resistant (Technical Appendix Table). n nRoutine mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) testing was performed (1) (Technical Appendix Table). A matching MIRU-VNTR profile was identified from a 42-year-old South African–born, HIV-positive health care worker (patient 2) who had died in 2008 after admission to the same hospital. She has been described previously in detail because she had worked at Tugela Ferry hospital in KwaZulu-Natal, South Africa, which was associated with a 2005 outbreak of multidrug-resistant TB (MDR TB) and extensively drug-resistant TB (2,3) (Technical Appendix Figure 1). To ascertain whether these isolates could have matching MIRU-VNTR patterns by chance alone, we compared the MIRU-VNTR results with a national database of ≈11,745 isolates typed since the UK typing service began in 2010. Only 2 other isolates matched (from patients 3 and 4), originating from a UK hospital ≈100 miles away. Although both patients were HIV-positive health care workers from sub-Saharan Africa, no history of contact could be established with patients 1 or 2. n nA review of admission records established that patients 1 and 2 were admitted to the same medical ward in 2008 for 8 days, suggesting a high probability of nosocomial transmission. The ward had a traditional “Nightingale” configuration with beds for male and female patients arranged dormitory-style. In 2009, patient 1 had been identified as a contact of patient 2 and was offered screening for latent infection but had failed to attend appointments and was not under regular medical follow-up. No other common contact was identified. The estimated time from known contact between patients 1 and 2 until the clinical presentation of patient 1 was 49 months. n nSequencing libraries from genomic DNA extracted from the 4 UK M. tuberculosis isolates that had matching MIRU-VNTR profiles were paired-end sequenced by using Illumina MiSeq (Illumina, San Diego, CA, USA). To investigate the origins of the infections, they were compared with 36 South Africa strains (including 1 from the Tugela Ferry outbreak [4]) sequenced by using Illumina HiSeq 2000 platforms. n nFor each sequenced strain, a random subset of reads was aligned at ≈100× coverage to the M. tuberculosis H37Rv reference genome by using BWA version 0.5.9-r16 (5). Pilon v1.5 (http://www.broadinstitute.org/software/pilon/) was run in variant discovery to generate a list of single-nucleotide polymorphisms (SNPs) and insertions and deletions. We estimated a phylogeny using RAxML v7.7.8 (6) using a general time reversible + gamma substitution model with 1,000 bootstrap replicates. n nPairwise comparison of whole-genome sequences from M. tuberculosis isolated from patients 1 and 2 found that the 2 sequences differed at only 4 SNPs (Table). Based on previous estimates of background mutations rates of 0.5 SNP/year (7), the pairwise distance between isolates from patient 1 and 2 increases confidence in the epidemiologic data implicating transmission >4 years earlier, although uncertainties exist around such estimates. Comparison between samples from patient pairs (1+2 vs. 3+4) found differences of 69–72 SNPs, which strongly argues against transmission between them. n n n nTable n nPairwise distances between 2 pairs of Mycobacterium tuberculosis isolates from patients in the United Kingdom, an isolate from the 2005 Tugela Ferry outbreak in KwaZulu-Natal, South Africa (KZN605), and reference strain H37Rv n n n nIn comparison with isolates sampled from KwaZulu-Natal (Technical Appendix Figure 1), isolates from patients 1 and 2 were closely related to a strain associated with the Tugela Ferry outbreak (KZN605; Technical Appendix Figure 2). Isolates from patients 3 and 4 were less closely related to isolates from the Tugela Ferry outbreak but were closely related to other isolates circulating within the region, consistent with the hypothesis that both infections originally occurred within South Africa. n nThis investigation illustrates the power of current technology to inform our understanding of the links in MDR TB transmission between low- and high-incidence areas. Whole-genome sequencing of pathogens is becoming part of routine practice for establishing transmission and resistance patterns (8). The greater certainty it brings to transmission data can provide evidence to justify more active policies of screening and isolation as part of infection control. The nosocomial transmission described here is consistent with the fact that a person with pulmonary TB (patient 2) was managed on an open ward before being put into respiratory isolation and had not been previously screened by occupational health services. n nRecent data reviewing MDR TB transmission in the United Kingdom before 2007 did not identify cases of nosocomial transmission during that period (9). However, the emergence of MDR TB in regions of high HIV prevalence is relatively recent (10), and the cases described here suggest that increased vigilance for TB and MDR TB among migrating health care workers might be required. n nTechnical Appendix: nCharacteristics of 4 clinical isolates of multidrug-resistant Mycobacterium tuberculosis; isolates sequenced from KwaZulu-Natal, South Africa; and phylogenetic representation of isolates collected from the United Kingdom and KwaZulu-Natal. n nClick here to view.(179K, pdf)

Nanopore sequencing enables near-complete de novo assembly of Saccharomyces cerevisiae reference strain CEN.PK113-7D

Abstract The haploid Saccharomyces cerevisiae strain CEN.PK113‐7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short‐read sequencing data scaffolded based on homology to strain S288C. However, these assemblies contain large sequence gaps, particularly in subtelomeric regions, and the assumption of perfect homology to S288C for scaffolding introduces bias. In this study, we obtained a near‐complete genome assembly of CEN.PK113‐7D using only Oxford Nanopore Technologys MinION sequencing platform. Fifteen of the 16 chromosomes, the mitochondrial genome and the 2‐&mgr;m plasmid are assembled in single contigs and all but one chromosome starts or ends in a telomere repeat. This improved genome assembly contains 770 Kbp of added sequence containing 248 gene annotations in comparison to the previous assembly of CEN.PK113‐7D. Many of these genes encode functions determining fitness in specific growth conditions and are therefore highly relevant for various industrial applications. Furthermore, we discovered a translocation between chromosomes III and VIII that caused misidentification of a MAL locus in the previous CEN.PK113‐7D assembly. This study demonstrates the power of long‐read sequencing by providing a high‐quality reference assembly and annotation of CEN.PK113‐7D and places a caveat on assumed genome stability of microorganisms.

Populations of latent Mycobacterium tuberculosis lack a cell wall: Isolation, visualization, and whole-genome characterization.

Objective/Background: Mycobacterium tuberculosis (MTB) causes active tuberculosis (TB) in only a small percentage of infected people. In most cases, the infection is clinically latent, where bacilli can persist in human hosts for years without causing disease. Surprisingly, the biology of such persister cells is largely unknown. This study describes the isolation, identification, and whole-genome sequencing (WGS) of latent TB bacilli after 782 days (26 months) of latency (the ability of MTB bacilli to lie persistent). Methods: The in vitro double-stress model of latency (oxygen and nutrition) was designed for MTB culture. After 26 months of latency, MTB cells that persisted were isolated and investigated under light and atomic force microscopy. Spoligotyping and WGS were performed to verify the identity of the strain. Results: We established a culture medium in which MTB bacilli arrest their growth, reduce their size (0.3–0.1 μm), lose their acid fastness (85–90%) and change their shape. Spoligopatterns of latent cells were identical to original H37Rv, with differences observed at spacers two and 14. WGS revealed only a few genetic changes relative to the already published H37Rv reference genome. Among these was a large 2064-bp insertion (RvD6), which was originally detected in both H37Ra and CDC1551, but not H37Rv. Conclusion: Here, we show cell-wall free cells of MTB bacilli in their latent state, and the biological adaptation of these cells was more phenotypic in nature than genomic. These cell-wall free cells represent a good model for understanding the nature of TB latency.

Normalizing alternate representations of large sequence variants across multiple bacterial genomes

Background and description Variant-focused comparative genomics enables researchers to study the evolution of distinct genetic characteristics in bacterial populations, while avoiding the difficulties of whole-genome assembly and alignment. A major challenge in using this method is that many variant detecting tools are largely limited to predicting single nucleotide variants (SNVs) and small indels. This is a challenge because bacterial organisms do not only possess SNVs but also harbor much larger sequence variants (LSVs), such as large indels and substitutions (>25 nt), when compared to a reference genome. LSVs have been shown to play a role in shaping important biological aspects such as virulence and drug resistance as well as reporting on population structure [1-3]. Recent variant callers, such as Pilon http://www.broadinstitute. org/software/pilon/, can identify LSVs with single nucleotide accuracy in microbial genomes. However, one remaining challenge is that identical LSVs can be represented non-identically by a single variant detecting tool; this generally results from similarity in the flanking sequence of the variant and variability of the read quality and alignment information in that region across the different strains. As a result, alternate representations of large variants make it difficult to perform downstream analyses such as association studies that depend on consistent representations of variants. We present Emu, an algorithm that resolves alternate representations of LSVs by comparing variant calls across genomes. Results To evaluate Emu’s ability to resolve alternate representations of LSVs, we introduced 179 simulated LSVs into the H37Rv genome–a carefully curated and finished reference genome for Mycobacterium tuberculosis (Mtb). We then used Pilon to identify variants in a set of 146 clinical samples of Mtb that were collected in China using the modified H37Rv genome as a reference [4]. We identified a total of 10,001 unique variant representations. The average number of non-identical representations of each simulated LSV was 56 (in the range of 1 to 145). We then applied Emu to identify the non-identical representations across the genomes of the 146 clinical samples and canonicalize them to a single form. Emu reduced the total number of non-identical representations to 676 LSVs bringing the average number of non-identical representations at each LSV to 4, with 15 LSVs reduced to a single representation and no LSV having more than 25 representations. We then investigated how Emu’s ability to resolve alternate representations might impact association analyses, e.g., associating LSVs with population structure. We ran Pilon again on the set of 161 clinical samples from China, but used the unmodified H37Rv genome. Pilon identified a total of 20,512 distinct LSVs when compared to the unmodified H37Rv genome. By applying Emu, the number of distinct LSVs decreased by almost 50% to 10,936 LSVs. Emu also increased the power of association tests on the LSVs. While we initially identified a total number of 69 LSVs that were significantly associated (p < 0.01) with membership to a specific clade, after processing with Emu that number increased to 94.

Schizophyllum commune has an extensive and functional alternative splicing repertoire

Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regions (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically.