No abstract

Lineage 4/X-family of Mycobacterium tuberculosis is not very notorious, except for the CDC1551 strain. One strain of this family, named Ara50, caused one of the largest tuberculosis outbreaks of the Aragon region, Spain, during the 1990s and remained until 2018. These X-strains are characterised by high transmissibility and by carrying a low copy number of IS6110 in their genomes. Epidemiological data of the 61 patients consisted of inmates, HIV seropositives, intravenous drug users and the homeless. The application of whole-genome sequencing (WGS) to 36 out of 61 isolates, selected by IS6110-RFLP, allowed to confirm 32 as recent transmissions. We

The purpose of this study was to increase our knowledge about Mycobacterium africanum and report the incidence and characteristics of tuberculosis (TB) due to their lineages in Aragon, Spain, over the period 2003–2019. The study includes all the cases in our region, where all the M. tuberculosis complex isolates are systematically characterised. We detected 31 cases of M. africanum among 2598 cases of TB in the period studied. TB caused by M. africanum is rare (1.19%) in our population, and it affects mainly men of economically productive age coming from West African countries. Among the isolates, Lineage (L) 6 was more frequent than L5. The genotyping of these strains identified five clusters and 13 strains with a unique pattern. The isolates’ characterisation identified a copy of IS6110 within the moaX gene, which turned out to be specific for L6. It will allow the differentiation of this lineage from the rest of MTBC with a simple PCR reaction. It remains to be established whether this polymorphism may limit M. africanum transmission. Furthermore, a mutation in the mutT2 promoter was found as specific for L6 strains, which could be related to the high variability found for L6 compared to L5.

Since 2004, a tuberculosis surveillance protocol has been carried out in Aragon, thereby managing to detect all tuberculosis outbreaks that take place in the community. The largest outbreak was caused by a strain named Mycobacterium tuberculosis Zaragoza (MtZ), causing 242 cases as of 2020. The main objective of this work was to analyze this outbreak and the molecular characteristics of this successful strain that could be related to its greater transmission. To do this, we first applied whole-genome sequencing to 57 of the isolates. This revealed two principal transmission clusters and six subclusters arising from them. The MtZ strain belongs to L4.8 and had eight specific single nucleotide polymorphisms (SNPs) in genes considered to be virulence factors [ptpA, mc3D, mc3F, VapB41, pks15 (two SNPs), virS, and VapC50]. Second, a transcriptomic study was carried out to better understand the multiple IS6110 copies present in its genome. This allowed us to observe three effects of IS6110: the disruption of the gene in which the IS6110 is inserted (desA3), the overexpression of a gene (ppe38), and the absence of transcription of genes (cut1:Rv1765c) due to the recombination of two IS6110 copies. Finally, because of the disruption of ppe38 and ppe71 genes by an IS6110, a study of PE_PGRS secretion was carried out, showing that MtZ secretes these factors in higher amounts than the reference strain, thereby differing from the hypervirulent phenotype described for the Beijing strains. In conclusion, MtZ consists of several SNPs in genes related to virulence, pathogenesis, and survival, as well as other genomic polymorphisms, which may be implicated in its success among our population.

The study of tuberculosis latency is problematic due to the difficulty of isolating the bacteria in the dormancy state. Despite this, several in vivo approaches have been taken to mimic the latency process. Our group has studied the evolution of the bacteria in 18 cases of recurrent tuberculosis. We found that HIV positive patients develop recurrent tuberculosis earlier, generally in the first two years (p value = 0.041). The genome of the 36 Mycobacterium tuberculosis paired isolates (first and relapsed isolates) showed that none of the SNPs found within each pair was observed more than once, indicating that they were not directly related to the recurrence process. Moreover, some IS6110 movements were found in the paired isolates, indicating the presence of different clones within the patient. Finally, our results suggest that the mutation rate remains constant during all the period as no correlation was found between the number of SNPs and the time to relapse.

Molecular epidemiology of circulating clinical isolates is crucial to improve prevention strategies. The Spanish Working Group on multidrug resistant tuberculosis (MDR-TB) is a network that monitors the MDR-TB isolates in Spain since 1998. The aim of this study was to present the study of the MDR-TB and extensively drug-resistant tuberculosis (XDR-TB) patterns in Spain using the different recommended genotyping methods over time by a national coordinated system. Based on the proposed genotyping methods in the European Union until 2018, the preservation of one method, MIRU-VNTR, applied to selected clustered strains permitted to maintain our study open for 20 years. The distribution of demographic, clinical and epidemiological characteristics of clustered and non-clustered cases of MDR/XDR tuberculosis with proportion differences as assessed by Pearson’s chi-squared or Fisher’s exact test was compared. The differences in the quantitative variables using the Student's-t test and the Mann–Whitney U test were evaluated. The results obtained showed a total of 48.4% of the cases grouped in 77 clusters. Younger age groups, having a known TB case contact (10.2% vs 4.7%) and XDR-TB (16.5% vs 1.8%) were significantly associated with clustering. The largest cluster corresponded to a Mycobacterium bovis strain mainly spread during the nineties. A total of 68.4% of the clusters detected were distributed among the different Spanish regions and six clusters involving 104 cases were grouped in 17 and 18 years. Comparison of the genotypes obtained with those European genotypes included in The European Surveillance System (TESSy) showed that 87 cases had become part of 20 European clusters. The continuity of MDR strain genotyping in time has offered a widespread picture of the situation that allows better management of this public health problem. It also shows the advantage of maintaining one genotyping method over time, which allowed the comparison between ancient, present and future samples.

Infectious disease outbreaks represent a significant problem for public health. Tracing outbreak expansion and understanding the main factors behind emergence and persistence remain critical to effective disease control.

The insertion sequence (IS) 6110 is a repetitive mobile element specific for the Mycobacterium tuberculosis complex (MTBC) used for years to diagnose and genotype this pathogen. It contains the overlapping reading frames orfA and orfB that encode a transposase. Its genetic variability is difficult to study because multiple copies are present in the genome. IS6110 is randomly located, nevertheless some preferential locations have been reported, which could be related to the behaviour of the strains. The aim of this work was to determine the intra- and inter-strain genetic conservation of this element in the MTBC. For this purpose, we analysed 158 sequences of IS6110 copies from 55 strains. Eighty-four copies were from 17 strains for which we knew all the locations in their genome. In addition, we studied 74 IS6110 copies in 38 different MTBC strains in which the location was characteristic of different families including Haarlem, LAM, S, and L6 strains. We observed mutation in 13.3% of the copies studied and we found 10 IS6110 variants in 21 copies belonging to 16 strains. The high copy number strains showed 6.2% of their IS6110 copies mutated, in contrast with the 31.1% in the low-copy-number strains. The apparently more ancient copy localised in the DR region was that with more variant copies, probably because this was the most studied location. Notably, all Haarlem and X family strains studied have an IS6110 in Rv0403c, suggesting a common origin for both families. Nevertheless, we detected a variant specific for the X family that would have occurred in this location after the phylogenetic separation. This variant does not prevent transposition although it may occur at a lower frequency, as X strains remain with low copy number (LCN) of IS6110.