HIV co-infection is associated with reduced Mycobacterium tuberculosis transmissibility in sub-Saharan Africa.

Persons living with HIV are known to be at increased risk of developing tuberculosis (TB) disease upon infection with Mycobacterium tuberculosis (Mtb). However, it has remained unclear how HIV co-infection affects subsequent Mtb transmission from these patients. Here, we customized a Bayesian phylodynamic framework to estimate the effects of HIV co-infection on the Mtb transmission dynamics from sequence data. We applied our model to four Mtb genomic datasets collected in sub-Saharan African countries with a generalized HIV epidemic. Our results confirm that HIV co-infection is a strong risk factor for developing active TB. Additionally, we demonstrate that HIV co-infection is associated with a reduced effective reproductive number for TB. Stratifying the population by CD4+ T-cell count yielded similar results, suggesting that, in this context, CD4+ T-cell count is not a better predictor of Mtb transmissibility than HIV infection status alone. Together, our genome-based analyses complement observational household contact studies, and more firmly establish the negative association between HIV co-infection and Mtb transmissibility.

Back-to-Africa introductions of Mycobacterium tuberculosis as the main cause of tuberculosis in Dar es Salaam, Tanzania.

In settings with high tuberculosis (TB) endemicity, distinct genotypes of the Mycobacterium tuberculosis complex (MTBC) often differ in prevalence. However, the factors leading to these differences remain poorly understood. Here we studied the MTBC population in Dar es Salaam, Tanzania over a six-year period, using 1,082 unique patient-derived MTBC whole-genome sequences (WGS) and associated clinical data. We show that the TB epidemic in Dar es Salaam is dominated by multiple MTBC genotypes introduced to Tanzania from different parts of the world during the last 300 years. The most common MTBC genotypes deriving from these introductions exhibited differences in transmission rates and in the duration of the infectious period, but little differences in overall fitness, as measured by the effective reproductive number. Moreover, measures of disease severity and bacterial load indicated no differences in virulence between these genotypes during active TB. Instead, the combination of an early introduction and a high transmission rate accounted for the high prevalence of L3.1.1, the most dominant MTBC genotype in this setting. Yet, a longer co-existence with the host population did not always result in a higher transmission rate, suggesting that distinct life-history traits have evolved in the different MTBC genotypes. Taken together, our results point to bacterial factors as important determinants of the TB epidemic in Dar es Salaam.

Using population-specific add-on polymorphisms to improve genotype imputation in underrepresented populations.

Genome-wide association studies rely on the statistical inference of untyped variants, called imputation, to increase the coverage of genotyping arrays. However, the results are often suboptimal in populations underrepresented in existing reference panels and array designs, since the selected single nucleotide polymorphisms (SNPs) may fail to capture population-specific haplotype structures, hence the full extent of common genetic variation. Here, we propose to sequence the full genomes of a small subset of an underrepresented study cohort to inform the selection of population-specific add-on tag SNPs and to generate an internal population-specific imputation reference panel, such that the remaining array-genotyped cohort could be more accurately imputed. Using a Tanzania-based cohort as a proof-of-concept, we demonstrate the validity of our approach by showing improvements in imputation accuracy after the addition of our designed add-on tags to the base H3Africa array.

Model-based integration of genomics and metabolomics reveals SNP functionality in Mycobacterium tuberculosis.

Human tuberculosis is caused by members of the Mycobacterium tuberculosis complex (MTBC) that vary in virulence and transmissibility. While genome-wide association studies have uncovered several mutations conferring drug resistance, much less is known about the factors underlying other bacterial phenotypes. Variation in the outcome of tuberculosis infection and diseases has been attributed primarily to patient and environmental factors, but recent evidence indicates an additional role for the genetic diversity among MTBC clinical strains. Here, we used metabolomics to unravel the effect of genetic variation on the strain-specific metabolic adaptive capacity and vulnerability. To define the functionality of single-nucleotide polymorphisms (SNPs) systematically, we developed a constraint-based approach that integrates metabolomic and genomic data. Our model-based predictions correctly classify SNP effects in pyruvate kinase and suggest a genetic basis for strain-specific inherent baseline susceptibility to the antibiotic para-aminosalicylic acid. Our method is broadly applicable across microbial life, opening possibilities for the development of more selective treatment strategies.

Whole-Genome Sequencing Has the Potential To Improve Treatment for Rifampicin-Resistant Tuberculosis in High-Burden Settings: a Retrospective Cohort Study.

Treatment of multidrug-resistant or rifampicin-resistant tuberculosis (MDR/RR-TB), although improved in recent years with shorter, more tolerable regimens, remains largely standardized and based on limited drug susceptibility testing (DST). More individualized treatment with expanded DST access is likely to improve patient outcomes. To assess the potential of TB drug resistance prediction based on whole-genome sequencing (WGS) to provide more effective treatment regimens, we applied current South African treatment recommendations to a retrospective cohort of MDR/RR-TB patients from Khayelitsha, Cape Town. Routine DST and clinical data were used to retrospectively categorize patients into a recommended regimen, either a standardized short regimen or a longer individualized regimen. Potential regimen changes were then described with the addition of WGS-derived DST. WGS data were available for 1274 MDR/RR-TB patient treatment episodes across 2008 to 2017. Among 834 patients initially eligible for the shorter regimen, 385 (46%) may have benefited from reduced drug dosage or removing ineffective drugs when WGS data were considered. A further 187 (22%) patients may have benefited from more effective adjusted regimens. Among 440 patients initially eligible for a longer individualized regimen, 153 (35%) could have been switched to the short regimen. Overall, 305 (24%) patients had MDR/RR-TB with second-line TB drug resistance, where the availability of WGS-derived DST would have allowed more effective treatment individualization. These data suggest considerable benefits could accrue from routine access to WGS-derived resistance prediction. Advances in culture-free sequencing and expansion of the reference resistance mutation catalogue will increase the utility of WGS resistance prediction.

Transition bias influences the evolution of antibiotic resistance in Mycobacterium tuberculosis.

Transition bias, an overabundance of transitions relative to transversions, has been widely reported among studies of the rates and spectra of spontaneous mutations. However, demonstrating the role of transition bias in adaptive evolution remains challenging. In particular, it is unclear whether such biases direct the evolution of bacterial pathogens adapting to treatment. We addressed this challenge by analyzing adaptive antibiotic-resistance mutations in the major human pathogen Mycobacterium tuberculosis (MTB). We found strong evidence for transition bias in two independently curated data sets comprising 152 and 208 antibiotic-resistance mutations. This was true at the level of mutational paths (distinct adaptive DNA sequence changes) and events (individual instances of the adaptive DNA sequence changes) and across different genes and gene promoters conferring resistance to a diversity of antibiotics. It was also true for mutations that do not code for amino acid changes (in gene promoters and the 16S ribosomal RNA gene rrs) and for mutations that are synonymous to each other and are therefore likely to have similar fitness effects, suggesting that transition bias can be caused by a bias in mutation supply. These results point to a central role for transition bias in determining which mutations drive adaptive antibiotic resistance evolution in a key pathogen.

The Genetic Background Modulates the Evolution of Fluoroquinolone-Resistance in Mycobacterium tuberculosis.

Fluoroquinolones (FQ) form the backbone in experimental treatment regimens against drug-susceptible tuberculosis. However, little is known on whether the genetic variation present in natural populations of Mycobacterium tuberculosis (Mtb) affects the evolution of FQ-resistance (FQ-R). To investigate this question, we used nine genetically distinct drug-susceptible clinical isolates of Mtb and measured their frequency of resistance to the FQ ofloxacin (OFX) in vitro. We found that the Mtb genetic background led to differences in the frequency of OFX-resistance (OFX-R) that spanned two orders of magnitude and substantially modulated the observed mutational profiles for OFX-R. Further, in vitro assays showed that the genetic background also influenced the minimum inhibitory concentration and the fitness effect conferred by a given OFX-R mutation. To test the clinical relevance of our in vitro work, we surveyed the mutational profile for FQ-R in publicly available genomic sequences from clinical Mtb isolates, and found substantial Mtb lineage-dependent variability. Comparison of the clinical and the in vitro mutational profiles for FQ-R showed that 51% and 39% of the variability in the clinical frequency of FQ-R gyrA mutation events in Lineage 2 and Lineage 4 strains, respectively, can be attributed to how Mtb evolves FQ-R in vitro. As the Mtb genetic background strongly influenced the evolution of FQ-R in vitro, we conclude that the genetic background of Mtb also impacts the evolution of FQ-R in the clinic.

Expression Dysregulation as a Mediator of Fitness Costs in Antibiotic Resistance.

Antimicrobial resistance (AMR) poses a threat to global health and the economy. Rifampicin-resistant Mycobacterium tuberculosis accounts for a third of the global AMR burden. Gaining the upper hand on AMR requires a deeper understanding of the physiology of resistance. AMR often results in a fitness cost in the absence of drug. Identifying the molecular mechanisms underpinning this cost could help strengthen future treatment regimens. Here, we used a collection of M. tuberculosis strains that provide an evolutionary and phylogenetic snapshot of rifampicin resistance and subjected them to genome-wide transcriptomic and proteomic profiling to identify key perturbations of normal physiology. We found that the clinically most common rifampicin resistance-conferring mutation, RpoB Ser450Leu, imparts considerable gene expression changes, many of which are mitigated by the compensatory mutation in RpoC Leu516Pro. However, our data also provide evidence for pervasive epistasis-the same resistance mutation imposed a different fitness cost and functionally distinct changes to gene expression in genetically unrelated clinical strains. Finally, we report a likely posttranscriptional modulation of gene expression that is shared in most of the tested strains carrying RpoB Ser450Leu, resulting in an increased abundance of proteins involved in central carbon metabolism. These changes contribute to a more general trend in which the disruption of the composition of the proteome correlates with the fitness cost of the RpoB Ser450Leu mutation in different strains.

Rifampicin-Monoresistant Tuberculosis Is Not the Same as Multidrug-Resistant Tuberculosis: a Descriptive Study from Khayelitsha, South Africa.

Rifampin monoresistance (RMR; rifampin resistance and isoniazid susceptibility) accounts for 38% of all rifampin-resistant tuberculosis (RR-TB) in South Africa and is increasing. We aimed to compare RMR-TB with multidrug-resistant TB (MDR-TB) in a setting with high TB, RR-TB, and HIV burdens. Patient-level clinical data and stored RR Mycobacterium tuberculosis isolates from 2008 to 2017 with available whole-genome sequencing (WGS) data were used to describe risk factors associated with RMR-TB and to compare RR-conferring mutations between RMR-TB and MDR-TB. A subset of isolates with particular RR-conferring mutations were subjected to semiquantitative rifampin phenotypic drug susceptibility testing. Among 2,041 routinely diagnosed RR-TB patients, 463 (22.7%) had RMR-TB. HIV-positive individuals (adjusted odds ratio [aOR], 1.4; 95% confidence interval [CI], 1.1 to 1.9) and diagnosis between 2013 and 2017 versus between 2008 and 2012 (aOR, 1.3; 95% CI, 1.1 to 1.7) were associated with RMR-TB. Among 1,119 (54.8%) patients with available WGS data showing RR-TB, significant differences in the distribution of rpoB RR-conferring mutations between RMR and MDR isolates were observed. Mutations associated with high-level RR were more commonly found among MDR isolates (811/889 [90.2%] versus 162/230 [70.4%] among RMR isolates; P < 0.0001). In particular, the rpoB L430P mutation, conferring low-level RR, was identified in 32/230 (13.9%) RMR isolates versus 10/889 (1.1%) in MDR isolates (P < 0.0001). Among 10 isolates with an rpoB L430P mutation, 7 were phenotypically susceptible using the critical concentration of 0.5 µg/ml (range, 0.125 to 1 µg/ml). The majority (215/230 [93.5%]) of RMR isolates showed susceptibility to all other TB drugs, highlighting the potential benefits of WGS for simplified treatment. These data suggest that the evolution of RMR-TB differs from MDR-TB with a potential contribution from HIV infection.

Natural Polymorphisms in Mycobacterium tuberculosis Conferring Resistance to Delamanid in Drug-Naive Patients.

Mutations in the genes of the F420 signaling pathway of Mycobacterium tuberculosis complex, including dnn, fgd1, fbiA, fbiB, fbiC, and fbiD, can lead to delamanid resistance. We searched for such mutations among 129 M. tuberculosis strains from Asia, South America, and Africa using whole-genome sequencing; 70 (54%) strains had at least one mutation in one of the genes. For 10 strains with mutations, we determined the MIC of delamanid. We found one strain from a delamanid-naive patient carrying the natural polymorphism Tyr29del (ddn) that was associated with a critical delamanid MIC.

HIV Coinfection Is Associated with Low-Fitness rpoB Variants in Rifampicin-Resistant Mycobacterium tuberculosis.

We analyzed 312 drug-resistant genomes of Mycobacterium tuberculosis isolates collected from HIV-coinfected and HIV-negative TB patients from nine countries with a high tuberculosis burden. We found that rifampicin-resistant M. tuberculosis strains isolated from HIV-coinfected patients carried disproportionally more resistance-conferring mutations in rpoB that are associated with a low fitness in the absence of the drug, suggesting these low-fitness rpoB variants can thrive in the context of reduced host immunity.

Whole-Genome Sequencing for Drug Resistance Profile Prediction in Mycobacterium tuberculosis.

Whole-genome sequencing allows rapid detection of drug-resistant Mycobacterium tuberculosis isolates. However, the availability of high-quality data linking quantitative phenotypic drug susceptibility testing (DST) and genomic data have thus far been limited. We determined drug resistance profiles of 176 genetically diverse clinical M. tuberculosis isolates from the Democratic Republic of the Congo, Ivory Coast, Peru, Thailand, and Switzerland by quantitative phenotypic DST for 11 antituberculous drugs using the BD Bactec MGIT 960 system and 7H10 agar dilution to generate a cross-validated phenotypic DST readout. We compared DST results with predicted drug resistance profiles inferred by whole-genome sequencing. Classification of strains by the two phenotypic DST methods into resistotype/wild-type populations was concordant in 73 to 99% of cases, depending on the drug. Our data suggest that the established critical concentration (5 mg/liter) for ethambutol resistance (MGIT 960 system) is too high and misclassifies strains as susceptible, unlike 7H10 agar dilution. Increased minimal inhibitory concentrations were explained by mutations identified by whole-genome sequencing. Using whole-genome sequences, we were able to predict quantitative drug resistance levels for the majority of drug resistance mutations. Predicting quantitative levels of drug resistance by whole-genome sequencing was partially limited due to incompletely understood drug resistance mechanisms. The overall sensitivity and specificity of whole-genome-based DST were 86.8% and 94.5%, respectively. Despite some limitations, whole-genome sequencing has the potential to infer resistance profiles without the need for time-consuming phenotypic methods.

Treemmer: a tool to reduce large phylogenetic datasets with minimal loss of diversity.

BACKGROUND: Large sequence datasets are difficult to visualize and handle. Additionally, they often do not represent a random subset of the natural diversity, but the result of uncoordinated and convenience sampling. Consequently, they can suffer from redundancy and sampling biases. RESULTS: Here we present Treemmer, a simple tool to evaluate the redundancy of phylogenetic trees and reduce their complexity by eliminating leaves that contribute the least to the tree diversity. CONCLUSIONS: Treemmer can reduce the size of datasets with different phylogenetic structures and levels of redundancy while maintaining a sub-sample that is representative of the original diversity. Additionally, it is possible to fine-tune the behavior of Treemmer including any kind of meta-information, making Treemmer particularly useful for empirical studies.

Acquired Resistance to Bedaquiline and Delamanid in Therapy for Tuberculosis.

Treatment of multidrug-resistant Mycobacterium tuberculosis is a challenge. This letter describes the emergence of resistance to new therapies, bedaquiline and delamanid.

Molecular characterization of bovine tuberculosis strains in two slaughterhouses in Morocco.

BACKGROUND: Bovine tuberculosis (BTB) is caused by Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. Mycobacterium bovis have been described to be responsible of most cases of bovine tuberculosis. Although M. tuberculosis, M. africanum and non-complex mycobacteria were isolated from cattle. In Morocco, so far, no molecular studies were conducted to characterize the strains responsible of BTB. The present study aims to characterize M. bovis in Morocco. The present study was conducted in slaughterhouses in Rabat and El Jadida. Samples were collected from 327 slaughtered animals with visible lesions suggesting BTB. RESULTS: A total of 225 isolates yielded cultures, 95% (n = 215) of them were acid-fast (AF). Sixty eight per cent of the AF positive samples were confirmed as tuberculous mycobacteria (n = 147), 99% of these (n = 146) having RD9 and among the latter, 98% (n = 143) positive while 2% (n = 3) negative for RD4 A total of 134 samples were analyzed by spoligotyping of which 14 were in cluster and with 41 different spoligotypes, ten of them were new patterns (23%). The most prevalent spoligotypes were SB0121, SB0265, and SB0120, and were already identified in many other countries, such as Algeria, Spain, Tunisia, the United States and Argentina. CONCLUSION: The shared borders between Algeria and Morocco, in addition to the previous importation of cattle from Europe and the US could explain the similarities found in M. bovis spoligotypes. On the other hand, the desert of Morocco could be considered as an efficient barrier preventing the introduction of BTB to Morocco from West Central and East Africa. Our findings suggest a low level endemic transmission of BTB similar to other African countries. However, more research is needed for further knowledge about the transmission patterns of BTB in Morocco.

Strain Diversity and the Evolution of Antibiotic Resistance.

Drug resistance is best thought of as an ongoing biological process. Resistant bacteria must emerge, become established and ultimately transmit in order to be relevant to human health. In this context, genetic diversity can influence the rate and likelihood of resistance emerging; it can also modulate the net physiological impact of resistance and the propensity of an organism to improve any defects that arise from it. Combined, these effects can have an impact on a larger scale, with highly transmissible drug-resistant bacterial strains posing a formidable threat to global health. These considerations are pertinent to the future of tuberculosis control as well. In this chapter, we review our current understanding of the impact of genetic diversity in the broadest sense on the evolution of drug-resistant members of the Mycobacterium tuberculosis complex.

The heterogeneous evolution of multidrug-resistant Mycobacterium tuberculosis.

Recent surveillance data of multidrug-resistant tuberculosis (MDR-TB) reported the highest rates of resistance ever documented. As further amplification of resistance in MDR strains of Mycobacterium tuberculosis occurs, extensively drug-resistant (XDR) and totally drug-resistant (TDR) TB are beginning to emerge. Although for the most part, the epidemiological factors involved in the spread of MDR-TB are understood, insights into the bacterial drivers of MDR-TB have been gained only recently, largely owing to novel technologies and research in other organisms. Herein, we review recent findings on how bacterial factors, such as persistence, hypermutation, the complex interrelation between drug resistance and fitness, compensatory evolution, and epistasis affect the evolution of multidrug resistance in M. tuberculosis. Improved knowledge of these factors will help better predict the future trajectory of MDR-TB, and contribute to the development of new tools and strategies to combat this growing public health threat.

Standard Genotyping Overestimates Transmission of Mycobacterium tuberculosis among Immigrants in a Low-Incidence Country.

Immigrants from regions with a high incidence of tuberculosis (TB) are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520 Mycobacterium tuberculosis isolates from 2000 to 2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat [MIRU-VNTR] typing and spoligotyping). Here, we used whole-genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR typing clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of the clusters involving only foreign-born patients. The overall clustering proportion was 17% (90 patients; 95% confidence interval [CI], 14 to 21%) by standard genotyping but only 8% (43 patients; 95% CI, 6 to 11%) by WGS. The clustering proportion was 17% (67/401; 95% CI, 13 to 21%) by standard genotyping and 7% (26/401; 95% CI, 4 to 9%) by WGS among foreign-born patients and 19% (23/119; 95% CI, 13 to 28%) and 14% (17/119; 95% CI, 9 to 22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence of an association between birth origin and transmission (adjusted odds ratio of 2.2 and 95% CI of 0.9 to 5.5 comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland compared to WGS, particularly among immigrants from regions with a high TB incidence, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in settings with a low incidence of TB.

Molecular epidemiology of Mycobacterium africanum in Ghana.

BACKGROUND: Mycobacterium africanum comprises two phylogenetic lineages within the M. tuberculosis complex (MTBC) and is an important cause of human tuberculosis (TB) in West Africa. The reasons for this geographic restriction of M. africanum remain unclear. Here, we performed a prospective study to explore associations between the characteristics of TB patients and the MTBC lineages circulating in Ghana. METHOD: We genotyped 1,211 MTBC isolates recovered from pulmonary TB patients recruited between 2012 and 2014 using single nucleotide polymorphism typing and spoligotyping. Associations between patient and pathogen variables were assessed using univariate and multivariate logistic regression. RESULTS: Of the 1,211 MTBC isolates analysed, 71.9 % (871) belonged to Lineage 4; 12.6 % (152) to Lineage 5 (also known as M. africanum West-Africa 1), 9.2 % (112) to Lineage 6 (also known as M. africanum West-Africa 2) and 0.6 % (7) to Mycobacterium bovis. Univariate analysis revealed that Lineage 6 strains were less likely to be isoniazid resistant compared to other strains (odds ratio = 0.25, 95 % confidence interval (CI): 0.05-0.77, P < 0.01). Multivariate analysis showed that Lineage 5 was significantly more common in patients from the Ewe ethnic group (adjusted odds ratio (adjOR): 2.79; 95 % CI: 1.47-5.29, P < 0.001) and Lineage 6 more likely to be found among HIV-co-infected TB patients (adjOR = 2.2; 95 % confidence interval (CI: 1.32-3.7, P < 0.001). CONCLUSION: Our findings confirm the importance of M. africanum in Ghana and highlight the need to differentiate between Lineage 5 and Lineage 6, as these lineages differ in associated patient variables.

HIV infection disrupts the sympatric host-pathogen relationship in human tuberculosis.

The phylogeographic population structure of Mycobacterium tuberculosis suggests local adaptation to sympatric human populations. We hypothesized that HIV infection, which induces immunodeficiency, will alter the sympatric relationship between M. tuberculosis and its human host. To test this hypothesis, we performed a nine-year nation-wide molecular-epidemiological study of HIV-infected and HIV-negative patients with tuberculosis (TB) between 2000 and 2008 in Switzerland. We analyzed 518 TB patients of whom 112 (21.6%) were HIV-infected and 233 (45.0%) were born in Europe. We found that among European-born TB patients, recent transmission was more likely to occur in sympatric compared to allopatric host-pathogen combinations (adjusted odds ratio [OR] 7.5, 95% confidence interval [95% CI] 1.21-infinity, p = 0.03). HIV infection was significantly associated with TB caused by an allopatric (as opposed to sympatric) M. tuberculosis lineage (OR 7.0, 95% CI 2.5-19.1, p<0.0001). This association remained when adjusting for frequent travelling, contact with foreigners, age, sex, and country of birth (adjusted OR 5.6, 95% CI 1.5-20.8, p = 0.01). Moreover, it became stronger with greater immunosuppression as defined by CD4 T-cell depletion and was not the result of increased social mixing in HIV-infected patients. Our observation was replicated in a second independent panel of 440 M. tuberculosis strains collected during a population-based study in the Canton of Bern between 1991 and 2011. In summary, these findings support a model for TB in which the stable relationship between the human host and its locally adapted M. tuberculosis is disrupted by HIV infection.