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Molecular and biological determinants associated with Mycobacterium tuberculosis strains prevalent in Portugal

Publication . Silva, Carla; Portugal, Isabel; Jordao, Luisa

Multidrug (MDR) and extensively drug resistant (XDR) tuberculosis (TB) cases constitute a serious health problem in Portugal due to the existence of two unusual phylogenetic clades, Lisboa3 and Q1 clades, identified in Lisbon Health Region in the1990’s. The high prevalence of these strains over the years, suggest the existence of molecular and biological determinants underlying adaptation and persistence in the Portuguese settings. In this research work we first sought to investigate the molecular basis of resistant TB, as well as to determine the prevalence of specific drug resistance mutations and its association with M/XDRITB. SeventyIfour M. tuberculosis clinical isolates collected in Lisbon Health Region were genotyped by 24&loci MIRUIVNTR, and the mutational profile associated with firstI and second line drug resistance was studied. Seven new mutations were found, while the remaining mutations (n=28) were previously associated with drug resistance. None of which was specifically associated with MDR-TB. We also establish that the 15 loci MIRU-VNTR was the most adequate genotyping technique, showing an adequate discriminatory power,comparable to the 24 loci set, allowing the clustering of 60% and 86% of the MDR and XDR-TB isolates, respectively, the majority of which belonging to the Lisboa3 and Q1 clusters. From an epidemiological standpoint, this study suggests that a combined mutational and genotyping analysis is a valuable tool for molecular epidemiological surveillance of drug resistance strains. Then, we analysed the morphological and structural features of the Lisboa family and Q1 cluster isolates by scanning and transmission electron microscopy techniques. These analyses allowed the identification of structural differences, namely the reduced cell length in Lisboa family strains, and a thickened cell envelope observed in drug resistant M. tuberculosis clinical isolates. Moreover, the infection of human monocyte derived macrophages allowed us todocument the relative selective advantage of the Lisboa family isolates over other circulating M. tuberculosis isolates, translated in a higher growth rate in vivo. Given the observations of the multiple advantages of the coccoid morphology regarding adaptation to different environmental stresses, and the contribute of the reduced cell size in the interactions between bacteria and the surrounding environment and the influence in complement evasion strategy as well, led us to hypothesized that the reduced size observed might confer a selective advantage, which could be the result of an adaptation process to the host or an advantage mechanism for host persistence and dissemination. Next, to enable the rapid characterization of Mycobacterium metabolism upon adaptation to adverse environmental conditions, we described a simple and rapid method to evaluate the metabolic rate of M. tuberculosis strains, based on the resazurin reduction kinetics. To optimize this technique, two parameters were analysed: mycobacterial concentration and time-point measure of resazurin reduction. We evaluate different bacterial concentration under standard (pH6.7), and different environmental conditions (pH4.5, pH5.5 and anaerobiosis), and the absorbances corresponding to the reduced and oxidized form of resazurin were measured at the following time-points: 0h, 6h, 24h and 48h. Three independent replicas of the entire procedure were performed. The method revealed to be reproducible with low standard deviations. The data generated showed that the 1x108 CFU/ml was the best bacterial suspension for this assay,considering measurements at the following time points: 0h, 6h and 24h. This methodology enables precise determination of mycobacterial metabolic activity in a given environmental condition, at several time points of incubation. Such precision allows comparison of the metabolic activity of Mycobacterium strains, from different genotypes or phenotypes. Subsequently, this methodology was used to analyse and compare the metabolic activity of 13 M. tuberculosis strains in response to low pH and anaerobiosis, which are known to trigger metabolic shift towards low metabolic states and non replicative persistance. Overall, isolates from Lisboa3 and Q1 clade showed low metabolic activity under pH5.5, pH4.5; Lisboa3 isolates presented lower metabolic rate under anaerobic conditions. No association was found between drug resistance and the metabolic rate of M. tuberculosis isolates. Overall, comparing metabolic activity, Lisboa3 isolates presented a very homogenous metabolic pattern, whereas isolates from other family or clade showed no similar metabolic patterns. This study provides important clues for the potential adaptation of M. tuberculosis strains from distinct phylogenetic clades, suggesting that the distinct metabolic profile found may underlie a more effective response under harsh conditions, such as the ones found during the in vivo infection. The data herein generated, provides insights into the morphological and biological features of M. tuberculosis Lisboa3 and Q1 isolates, highlighting specific characteristicts that might contribute to Lisboa strains prevalence in the Portuguese settings through selectively advantageous phenotypic traits.

2017-07Doctoral thesis

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Insights on the Mycobacterium tuberculosis population structure associated with migrants from Portuguese-speaking countries over a three-year period in greater Lisbon, Portugal: Implications at the public health level

Publication . Pereira, Catarina; Gomes, Pedro; Taveira, Ricardo; Silva, Carla; Maltez, Fernando; Macedo, Rita; Costa, Catarina; Couvin, David; Rastogi, Nalin; Viveiros, Miguel; Perdigão, João; Portugal, Isabel

Tuberculosis among foreign-born patients is a key indicator of country-level epidemiological profiles and, of an increasing concern in Europe given the more intensified migratory waves of refugees. Since Portugal presents a lower immigrant-associated TB incidence rate when compared to other European countries, we sought to characterize the epidemiology and transmission dynamics among the foreign-born population coming from Portuguese-speaking countries that are associated with higher TB incidences. In the present study we analyzed 133 Mycobacterium tuberculosis isolates obtained from foreign-born individuals over a three-year period in Lisbon, Portugal, using molecular epidemiological methods such as spoligotyping and 24-loci MIRU-VNTR. Moreover, all strains were subjected to drug susceptibility testing. The genetic profiles obtained suggest that strain importation from Portuguese speaking countries plays a less important role in TB epidemiology but instead argue in favor of a high degree of penetrance of Portuguese endemic strains to the migrant population, including multidrug resistant strains, which is particularly relevant to active screening programs.

2019-03-27Journal article

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