Evolution of Mycobacterium tuberculosis drug resistance in the genomic era.

Mycobacterium tuberculosis has acquired drug resistance to all drugs that have been used against it, including those only recently introduced into clinical practice. Compared to other bacteria, it has a well conserved genome due to its role as an obligate human pathogen that has adapted to a niche over five to ten thousand years. These features facilitate reconstruction and dating of M. tuberculosis phylogenies, giving key insights into how resistance has been acquired and spread globally. Resistance to each new drug has occurred within five to ten years of clinical use and has occurred even more rapidly with recently introduced drugs. In most cases, resistance-conferring mutations come with a fitness cost, but this can be overcome by compensatory mutations which restore fitness to that of wild-type bacteria. It is likely that M. tuberculosis acquires drug resistance while maintaining limited genomic variability due the generation of low frequency within-host variation, combined with ongoing purifying selection causing loss of variants without a clear fitness advantage. However, variants that do confer an advantage, such as drug resistance, can increase in prevalence amongst all bacteria within a host and become the dominant clone. These resistant strains can then be transmitted leading to primary drug resistant infection in a new host. As many countries move towards genomic methods for diagnosis of M. tuberculosis infection and drug resistance, it is important to be aware of the implications for the evolution of resistance. Currently, understanding of resistance-conferring mutations is incomplete, and some targeted genetic diagnostics create their own selective pressures. We discuss an example where a rifampicin resistance-conferring mutation which was not routinely covered by standard testing became dominant. Finally, resistance to new drugs such as bedaquiline and delamanid is caused by individually rare mutations occurring across a large mutational genomic target that have been detected over a short time, and do not provide statistical power for genotype-phenotype correlation - in contrast to longer-established drugs that form the backbone of drug-sensitive antituberculosis therapy. Therefore, we need a different approach to identify resistance-conferring mutations of new drugs before their resistance becomes widespread, abrogating their usefulness.

Recurrences of multidrug-resistant tuberculosis: Strains involved, within-host diversity, and fine-tuned allocation of reinfections.

Recurrent tuberculosis occurs due to exogenous reinfection or reactivation/persistence. We analysed 90 sequential MDR Mtb isolates obtained in Argentina from 27 patients with previously diagnosed MDR-TB that recurred in 2018 (1-10 years, 2-10 isolates per patient). Three long-term predominant strains were responsible for 63% of all MDR-TB recurrences. Most of the remaining patients were infected by strains different from each other. Reactivation/persistence of the same strain caused all but one recurrence, which was due to a reinfection with a predominant strain. One of the prevalent strains showed marked stability in the recurrences, while in another strain higher SNP-based diversity was observed. Comparisons of intra- versus inter-patient SNP distances identified two possible reinfections with closely related variants circulating in the community. Our results show a complex scenario of MDR-TB infections in settings with predominant MDR Mtb strains.

Five-year microevolution of a multidrug-resistant Mycobacterium tuberculosis strain within a patient with inadequate compliance to treatment.

BACKGROUND: Whole-genome sequencing has shown that the Mycobacterium tuberculosis infection process can be more heterogeneous than previously thought. Compartmentalized infections, exogenous reinfections, and microevolution are manifestations of this clonal complexity. The analysis of the mechanisms causing the microevolution -the genetic variability of M. tuberculosis at short time scales- of a parental strain into clonal variants with a patient is a relevant issue that has not been yet completely addressed. To our knowledge, a whole genome sequence microevolution analysis in a single patient with inadequate adherence to treatment has not been previously reported. CASE PRESENTATION: In this work, we applied whole genome sequencing analysis for a more in-depth analysis of the microevolution of a parental Mycobacterium tuberculosis strain into clonal variants within a patient with poor treatment compliance in Argentina. We analyzed the whole-genome sequence of 8 consecutive Mycobacterium tuberculosis isolates obtained from a patient within 57-months of intermittent therapy. Nineteen mutations (9 short-term, 10 fixed variants) emerged, most of them associated with drug resistance. The first isolate was already resistant to isoniazid, rifampicin, and streptomycin, thereafter the strain developed resistance to fluoroquinolones and pyrazinamide. Surprisingly, isolates remained susceptible to the pro-drug ethionamide after acquiring a frameshift mutation in ethA, a gene required for its activation. We also found a novel variant, (T-54G), in the 5' untranslated region of whiB7 (T-54G), a region allegedly related to kanamycin resistance. Notably, discrepancies between canonical and phage-based susceptibility testing to kanamycin were previously found for the isolate harboring this mutation. In our patient, microevolution was mainly driven by drug selective pressure. Rare short-term mutations fixed together with resistance-conferring mutations during therapy. CONCLUSIONS: This report highlights the relevance of whole-genome sequencing analysis in the clinic for characterization of pre-XDR and MDR resistance profile, particularly in patients with incomplete and/or intermittent treatment.

Screening of inmates transferred to Spain reveals a Peruvian prison as a reservoir of persistent Mycobacterium tuberculosis MDR strains and mixed infections.

It is relevant to evaluate MDR-tuberculosis in prisons and its impact on the global epidemiology of this disease. However, systematic molecular epidemiology programs in prisons are lacking. A health-screening program performed on arrival for inmates transferred from Peruvian prisons to Spain led to the diagnosis of five MDR-TB cases from one of the biggest prisons in Latin America. They grouped into two MIRU-VNTR-clusters (Callao-1 and Callao-2), suggesting a reservoir of two prevalent MDR strains. A high-rate of overexposure was deduced because one of the five cases was coinfected by a pansusceptible strain. Callao-1 strain was also identified in 2018 in a community case in Spain who had been in the same Peruvian prison in 2002-5. A strain-specific-PCR tailored from WGS data was implemented in Peru, allowing the confirmation that these strains were currently responsible for the majority of the MDR cases in that prison, including a new mixed infection.

Trends of Two Epidemic Multidrug-Resistant Strains of Mycobacterium tuberculosis in Argentina Disclosed by Tailored Molecular Strategy.

Two Mycobacterium tuberculosis strains-M (sublineage 4.1) and Ra (sublineage 4.3)-have long prevailed in Argentina among patients with multidrug-resistant tuberculosis (MDR-TB). Recently, budget constraints have hampered the surveillance of MDR-TB transmission. Based on whole-genome sequence analysis, we used M- and Ra-specific single nucleotide polymorphisms to tailor two multiplex allele-specific polymerase chain reactions (PCRs), which we applied to 252 stored isolates (95% of all newly diagnosed MDR-TB cases countrywide, 2015-2017). Compared with the latest data available (2007-2009), the M strain has receded (80/324 to 20/252, P < 0.0001), particularly among cross-border migrants (12/58 to 0/53, P = 0.0003) and HIV-infected people (30/97 to 7/74, P = 0.0007), but it still accounts for 4/12 new cases of extensively drug-resistant TB. Differently, the Ra strain remained stable in frequency (39/324 to 33/252) and contributed marginally to the extensive drug-resistance load (1/12). Our novel strategy disclosed recent trends of the two major MDR-TB strains, providing meaningful data to allocate control interventions more efficiently.

Survival of an epidemic MDR strain of Mycobacterium tuberculosis and its non-prosperous variant within activated macrophages.

The fitness of a pathogen results from the interaction of multiple factors favoring either epidemiological success or failure. Herein, we studied the performance of the M strain, a highly successful multidrug resistant Mycobacterium tuberculosis genotype, and its non-prosperous variant, the 410 strain, in activated human monocyte-derived macrophages. Both strains showed comparable ability to induce necrotic cell death and to survive in apoptotic macrophages. Of the various macrophage activation conditions tested, none led to an enhanced control of the outbreak strain. The combination of 1,25(OH)2 vitaminD3 and IFN-γ favored significantly the control of the non-prosperous 410 strain. These observations indicate that the ability of the M strain to survive within the hostile intracellular milieu is conserved, and the overall fitness cost paid by this genotype would be low. Our results provide additional evidence on bacterial traits that may have contributed to the epidemiological success of the M strain.

Endocarditis infecciosa por Mycobacterium mageritense asociada a dispositivo cardíaco electrónico / Electronic cardiovascular device-associated to infective endocarditis caused by Mycobacterium mageritense

Las micobacterias de crecimiento rápido son una rara causa de endocarditis bacteriana. Durante las últimas décadas han aumentado las infecciones debido a este tipo de micobacterias, en especial las postraumáticas y las posquirúrgicas. Estas infecciones pueden ser localizadas o diseminadas, y también pueden producir brotes nosocomiales debido a la contaminación del equipamiento médico. Por lo general, las tinciones para bacterias ácido-alcohol resistentes no se emplean de rutina en el procesamiento de hemocultivos positivos. Sin embargo, el microbiólogo debe estar atento al ver un bacilo gram positivo, ya que podría tratarse de una micobacteria de crecimiento rápido. Describimos un caso de endocarditis por de Mycobacterium mageritense en una paciente con parche pericárdico autógeno y un catéter para medir la presión en la aurícula izquierda. La bacteria fue identificada por espectrometría de masas (MALDI-TOF MS), score 2,3, y luego confirmada por secuenciación y análisis del gen ARNr 16s con las bases de datos del NCBI y EzTaxon, con una concordancia del 99,8 y el 100%, respectivamente.

Rapidly growing non-tuberculosis mycobacteria are a rare cause of bacterial endocarditis. During the last decades, there has been an increase in infections due to rapidly growing mycobacteria, mainly after trauma and post-surgical procedures, both localized and disseminated, as well as nosocomial outbreaks due to contamination of medical equipment. Routine acid-fast staining for blood culture bottles is not always performed; however, the microbiologist should be aware of potential RGM infections especially when gram positive bacilli are observed. We describe a case of endocarditis caused by Mycobacterium mageritense in a patient with an autologous pericardial patch and a pressure catheter in the left auricle. The bacterial species was identified as Mycobacterium mageritense by mass spectrometry (MALDI-TOF MS), score 2.3, and confirmed by 16S rRNA analysis with 99.8 and 100% agreement, respectively.

[Electronic cardiovascular device-associated to infective endocarditis caused by Mycobacterium mageritense]. / Endocarditis infecciosa por Mycobacterium mageritense asociada a dispositivo cardíaco electrónico.

Rapidly growing non-tuberculosis mycobacteria are a rare cause of bacterial endocarditis. During the last decades, there has been an increase in infections due to rapidly growing mycobacteria, mainly after trauma and post-surgical procedures, both localized and disseminated, as well as nosocomial outbreaks due to contamination of medical equipment. Routine acid-fast staining for blood culture bottles is not always performed; however, the microbiologist should be aware of potential RGM infections especially when gram positive bacilli are observed. We describe a case of endocarditis caused by Mycobacterium mageritense in a patient with an autologous pericardial patch and a pressure catheter in the left auricle. The bacterial species was identified as Mycobacterium mageritense by mass spectrometry (MALDI-TOF MS), score 2.3, and confirmed by 16S rRNA analysis with 99.8 and 100% agreement, respectively.

Global expansion of Mycobacterium tuberculosis lineage 4 shaped by colonial migration and local adaptation.

On the basis of population genomic and phylogeographic analyses of 1669 Mycobacterium tuberculosis lineage 4 (L4) genomes, we find that dispersal of L4 has been completely dominated by historical migrations out of Europe. We demonstrate an intimate temporal relationship between European colonial expansion into Africa and the Americas and the spread of L4 tuberculosis (TB). Markedly, in the age of antibiotics, mutations conferring antimicrobial resistance overwhelmingly emerged locally (at the level of nations), with minimal cross-border transmission of resistance. The latter finding was found to reflect the relatively recent emergence of these mutations, as a similar degree of local restriction was observed for susceptible variants emerging on comparable time scales. The restricted international transmission of drug-resistant TB suggests that containment efforts at the level of individual countries could be successful.

Genotypic diversity of Mycobacterium tuberculosis in Buenos Aires, Argentina.

Buenos Aires is an overpopulated port city historically inhabited by people of European descent. Together with its broader metropolitan area, the city exhibits medium tuberculosis rates, and receives migrants, mainly from tuberculosis highly endemic areas of Argentina and neighboring countries. This work was aimed to gain insight into the Mycobacterium tuberculosis population structure in two suburban districts of Buenos Aires which are illustrative of the overall situation of tuberculosis in Argentina. The Lineage 4 Euro-American accounted for >99% of the 816 isolates analyzed (one per patient). Frequencies of spoligotype families were T 35.9%, LAM 33.2%, Haarlem 19.5%, S 3.2%, X 1.5%, Ural 0.7%, BOV 0.2%, Beijing 0.2%, and Cameroon 0.2%. Unknown signatures accounted for 5.3% isolates. Of 55 spoligotypes not matching any extant shared international type (SIT) in SITVIT database, 22 fitted into 15 newly-issued SITs. Certain autochthonous South American genotypes were found to be actively evolving. LAM3, which is wild type for RDrio, was the predominant LAM subfamily in both districts and the RDrio signature was rare among autochthonous, newly created, SITs and orphan patterns. Two genotypes that are rarely observed in neighboring countries ̶ SIT2/H2 and SIT159/T1 Tuscany ̶ were conspicuously represented in Argentina. The infrequent Beijing patterns belonged to Peruvian patients. We conclude that the genotype diversity observed reflects the influence of the Hispanic colonization and more recent immigration waves from Mediterranean and neighboring countries. Unlike in Brazil, the RDrio type does not play a major role in the tuberculosis epidemic in Buenos Aires.

Performance of a highly successful outbreak strain of Mycobacterium tuberculosis in a multifaceted approach to bacterial fitness assessment.

Determining bacterial fitness represents a major challenge and no single parameter can accurately predict the ability of a certain pathogen to succeed. The M strain of Mycobacterium tuberculosis managed to spread and establish in the community and caused the largest multidrug-resistant tuberculosis outbreak in Latin America. We have previously shown that the M strain can manipulate the host immune response, but we still have no direct evidence, other than epidemiology, that can account for the enhanced fitness of the M strain. Our objective was to further characterize the performance of the outbreak strain M in different fitness assays. Two main aspects were evaluated: (1) molecular characterization of selected isolates from the M outbreak and related strains and (2) comparative fitness and in vivo performance of representative M strain isolates vs. the non-prosperous M strain variant 410. Our approach confirmed the multifaceted nature of fitness. Altogether, we conclude that the epidemiologically abortive strain 410 was vulnerable to drug-driven pressure, a weak competitor, and a stronger inductor of protective response in vivo. Conversely, the isolate 6548, representative of the M outbreak peak, had a growth disadvantage but performed very well in competition and induced lung damage at advanced stages in spite of reaching relatively low CFU counts. Integration of these observations supports the idea that the M strain managed to find a unique path to success.

Relation of Mycobacterium tuberculosis mutations at katG315 and inhA-15 with drug resistance profile, genetic background, and clustering in Argentina.

We analyzed 362 isoniazid-resistant clinical isolates of Mycobacterium tuberculosis obtained countrywide for the presence of mutation at katG315 and inhA-15 in relation to genotype, pattern of phenotypic resistance to other drugs, and ability to spread. We found the following mutation frequencies: katG315MUT/inhA-15wt 53.0%, katG315wt/inhA-15MUT 27.4%, katG315wt/inhA-15wt 19.3%, and katG315MUT/inhA-15MUT only 0.3%. Mutation at katG315 associated with the LAM superfamily; mutation at inhA-15 associated with the S family and the T1 Tuscany genotype; the combination katG315wt/inhA-15wt associated with the T1 Ghana genotype. Isolates harboring katG315MUT/inhA-15wt tended to accumulate resistance to other drugs and were more frequently found in cluster; isolates harboring katG315wt/inhA-15wt were more frequently found as orphan isolates. Although epidemiological and host factors could also be modulating the events observed, in Argentina, the systematic genotyping of drug resistant clinical isolates could help to predict an enhanced risk of transmission and a propensity to develop resistance to increasing numbers of drugs.

Impact of HIV co-infection on the evolution and transmission of multidrug-resistant tuberculosis.

The tuberculosis (TB) epidemic is fueled by a parallel Human Immunodeficiency Virus (HIV) epidemic, but it remains unclear to what extent the HIV epidemic has been a driver for drug resistance in Mycobacterium tuberculosis (Mtb). Here we assess the impact of HIV co-infection on the emergence of resistance and transmission of Mtb in the largest outbreak of multidrug-resistant TB in South America to date. By combining Bayesian evolutionary analyses and the reconstruction of transmission networks utilizing a new model optimized for TB, we find that HIV co-infection does not significantly affect the transmissibility or the mutation rate of Mtb within patients and was not associated with increased emergence of resistance within patients. Our results indicate that the HIV epidemic serves as an amplifier of TB outbreaks by providing a reservoir of susceptible hosts, but that HIV co-infection is not a direct driver for the emergence and transmission of resistant strains.

Four decades of transmission of a multidrug-resistant Mycobacterium tuberculosis outbreak strain.

The rise of drug-resistant strains is a major challenge to containing the tuberculosis (TB) pandemic. Yet, little is known about the extent of resistance in early years of chemotherapy and when transmission of resistant strains on a larger scale became a major public health issue. Here we reconstruct the timeline of the acquisition of antimicrobial resistance during a major ongoing outbreak of multidrug-resistant TB in Argentina. We estimate that the progenitor of the outbreak strain acquired resistance to isoniazid, streptomycin and rifampicin by around 1973, indicating continuous circulation of a multidrug-resistant TB strain for four decades. By around 1979 the strain had acquired additional resistance to three more drugs. Our results indicate that Mycobacterium tuberculosis (Mtb) with extensive resistance profiles circulated 15 years before the outbreak was detected, and about one decade before the earliest documented transmission of Mtb strains with such extensive resistance profiles globally.

Genotypes of Mycobacterium tuberculosis in patients at risk of drug resistance in Bolivia.

Bolivia ranks among the 10 Latin American countries with the highest rates of tuberculosis (TB) and multidrug resistant (MDR) TB. In view of this, and of the lacking information on the population structure of Mycobacterium tuberculosis in the country, we explored genotype associations with drug resistance and clustering by analyzing isolates collected in 2010 from 100 consecutive TB patients at risk of drug resistance in seven of the nine departments in which Bolivia is divided. Fourteen isolates were MDR, 29 had other drug resistance profiles, and 57 were pansusceptible. Spoligotype family distribution was: Haarlem 39.4%, LAM 26.3%, T 22.2%, S 2.0%, X 1.0%, orphan 9.1%, with very low intra-family diversity and absence of Beijing genotypes. We found 66 different MIRU-VNTR patterns; the most frequent corresponded to Multiple Locus Variable Analysis (MLVA) MtbC15 patterns 860, 372 and 873. Twelve clusters, each with identical MIRU-VNTR and spoligotypes, gathered 35 patients. We found no association of genotype with drug resistant or MDR-TB. Clustering associated with SIT 50 and the H3 subfamily to which it belongs (p<0.0001). The largest cluster involved isolates from three departments and displayed a genotype (SIT 50/MLVA 860) previously identified in Bolivian migrants into Spain and Argentina suggesting that this genotype is widespread among Bolivian patients. Our study presents a first overview of M. tuberculosis genotypes at risk of drug resistance circulating in Bolivia. However, results should be taken cautiously because the sample is small and includes a particular subset of M. tuberculosis population.

Conspicuous multidrug-resistant Mycobacterium tuberculosis cluster strains do not trespass country borders in Latin America and Spain.

Multidrug-resistant Mycobacterium tuberculosis strain diversity in Ibero-America was examined by comparing extant genotype collections in national or state tuberculosis networks. To this end, genotypes from over 1000 patients with multidrug-resistant tuberculosis diagnosed from 2004 through 2008 in Argentina, Brazil, Chile, Colombia, Venezuela and Spain were compared in a database constructed ad hoc. Most of the 116 clusters identified by IS6110 restriction fragment length polymorphism were small and restricted to individual countries. The three largest clusters, of 116, 49 and 25 patients, were found in Argentina and corresponded to previously documented locally-epidemic strains. Only 13 small clusters involved more than one country, altogether accounting for 41 patients, of whom 13 were, in turn, immigrants from Latin American countries different from those participating in the study (Peru, Ecuador and Bolivia). Most of these international clusters belonged either to the emerging RD(Rio) LAM lineage or to the Haarlem family of M. tuberculosis and four were further split by country when analyzed with spoligotyping and rifampin resistance-conferring mutations, suggesting that they did not represent ongoing transnational transmission events. The Beijing genotype accounted for 1.3% and 10.2% of patients with multidrug-resistant tuberculosis in Latin America and Spain, respectively, including one international cluster of two cases. In brief, Euro-American genotypes were widely predominant among multidrug-resistant M. tuberculosis strains in Ibero-America, reflecting closely their predominance in the general M. tuberculosis population in the region, and no evidence was found of acknowledged outbreak strains trespassing country borders.