Transcriptomic profile of the most successful Mycobacterium tuberculosis strain in Aragon, the MtZ strain, during exponential and stationary growth phases.

IMPORTANCE: Aragon Community suffered, during the first years of the beginning of this century, a large outbreak caused by the MtZ strain, producing more than 240 cases to date. MtZ strain and the outbreak have been previously studied from an epidemiological and molecular point of view. In this work, we analyzed the transcriptomic profile of the strain for better understanding of its success among our population. We have discovered that MtZ has some upregulated virulence pathways, such as the ESX-1 system, the cholesterol degradation pathway or the peptidoglycan biosynthesis. Interestingly, MtZ has downregulated the uptake of iron. Another special feature of MtZ strain is the interruption of desA3 gene, essential for producing oleic acid. Although the strain takes a long time to grow in the initial culture media, eventually it is able to reach normal optical densities, suggestive of the presence of another route for obtaining oleic acid in Mycobacterium tuberculosis.

Rapid Identification of Lineage and Drug Resistance in Clinical Samples of Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis is a slow-growing bacterium, which could delay its diagnosis and, therefore, promote the spread of the disease. Whole-genome sequencing allows us to obtain the complete drug-resistance profile of the strain; however, bacterial cultivation of clinical samples, along with complex processing, is required. METHODS: In this work, we explore AmpliSeq, an amplicon-based enrichment method for preparing libraries for targeted next-generation sequencing, to identify lineage and drug resistance directly from clinical samples. RESULTS: In our study, 111 clinical samples were tested. The lineage was identified in 100% of the culture-derived samples (52/52), in 95% of the smear (BK)-positive clinical samples (38/40) and in 42.1% of the BK-negative clinical samples (8/19). The drug-resistance profile was accurately identified in all but 11 samples, in which some phenotypic and genotypic discrepancies were found. In this respect, our panels were not exact in the detection of streptomycin resistance for isolates derived from clinical samples, as an extremely high number of SNPs in the rrs and rrl genes were detected due to cross-contamination. CONCLUSION: This technique has demonstrated high sensitivity in obtaining the drug-resistance profile of the isolates, as even those samples with DNA concentrations below the detection limit of Qubit produced a result. AmpliSeq technology is cheaper than whole-genome sequencing, easy to perform by laboratory technicians and applicable to any microorganism using the Ion Torrent platform.

Deciphering the Tangible Spatio-Temporal Spread of a 25-Year Tuberculosis Outbreak Boosted by Social Determinants.

Outbreak strains of Mycobacterium tuberculosis are promising candidates as targets in the search for intrinsic determinants of transmissibility, as they are responsible for many cases with sustained transmission; however, the use of low-resolution typing methods and restricted geographical investigations represent flaws in assessing the success of long-lived outbreak strains. We can now address the nature of outbreak strains by combining large genomic data sets and phylodynamic approaches. We retrospectively sequenced the whole genome of representative samples assigned to an outbreak circulating in the Canary Islands (the GC strain) since 1993, which accounts for ~20% of local tuberculosis cases. We selected a panel of specific single nucleotide polymorphism (SNP) markers for an in-silico search for additional outbreak-related sequences within publicly available tuberculosis genomic data. Using this information, we inferred the origin, spread, and epidemiological parameters of the GC strain. Our approach allowed us to accurately trace the historical and more recent dispersion of the GC strain. We provide evidence of a highly successful nature within the Canarian archipelago but limited expansion abroad. Estimation of epidemiological parameters from genomic data disagree with a distinctive biology of the GC strain. With the increasing availability of genomic data allowing for the accurate inference of strain spread and critical epidemiological parameters, we can now revisit the link between Mycobacterium tuberculosis genotypes and transmission, as is routinely carried out for SARS-CoV-2 variants of concern. We demonstrate that social determinants rather than intrinsically higher bacterial transmissibility better explain the success of the GC strain. Importantly, our approach can be used to trace and characterize strains of interest worldwide. IMPORTANCE 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. Our study allows researchers and public health authorities to use Whole-Genome Sequencing-based methods to trace outbreaks, and shows how available epidemiological information helps to evaluate the factors underpinning outbreak persistence. Taking advantage of all the freely available information placed in public repositories, researchers can accurately establish the expansion of an outbreak beyond original boundaries, and determine the potential risk of a strain to inform health authorities which, in turn, can define target strategies to mitigate expansion and persistence. Finally, we show the need to evaluate strain transmissibility in different geographic contexts to unequivocally associate spread to local or pathogenic factors, an important lesson taken from genomic surveillance of SARS-CoV-2.

Analysis of the twenty-six largest outbreaks of tuberculosis in Aragon using whole-genome sequencing for surveillance purposes.

The incidence of tuberculosis in Aragon, Spain, is around ten cases per 100,000 inhabitants. Since 2004, a molecular surveillance protocol has been carried out; therefore, all M. tuberculosis strains are genotyped. Recently, whole-genome sequencing has been implemented for relevant isolates. The aim of this work is to characterise at the molecular level the causative strains of the 26 largest outbreaks of the community (including ten or more cases), genotyped by IS6110-RFLP and causing 26% of tuberculosis cases. To achieve this objective, two or three isolates of each IS6110-cluster belonging to different years were selected for sequencing. We found that strains of lineages L4.8, L4.3 and L4.1.2 were the most frequent. The threshold of 12 SNPs as the maximum distance for confirming the belonging to an outbreak was met for 18 of the 26 IS6110-clusters. Four pairs of isolates with more than 90 SNPs were identified as not belonging to the same strain, and four other pairs were kept in doubt as the number of SNPs was close to 12, between 14 and 35. The study of Regions of Difference revealed that they are lineage conserved. Moreover, we could analyse the IS6110 locations for all genome-sequenced isolates, finding some frequent locations in isolates belonging to the same lineage and certain IS6110 movements between the paired isolates. In the vast majority, these movements were not captured by the IS6110-RFLP pattern. After classifying the genes containing SNP by their functional category, we could confirm that the number of SNPs detected in genes considered as virulence factors and the number of cases the strain produced were not related, suggesting that a particular SNP is more relevant than the number. The characteristics found in the most successful strains in our community could be useful for other researchers in epidemiology, virulence and pathogenesis.

Estimation of the mutation rate of Mycobacterium tuberculosis in cases with recurrent tuberculosis using whole genome sequencing.

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.

The MtZ Strain: Molecular Characteristics and Outbreak Investigation of the Most Successful Mycobacterium tuberculosis Strain in Aragon Using Whole-Genome Sequencing.

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.

In-depth Analysis of IS6110 Genomic Variability in the Mycobacterium tuberculosis Complex.

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.

Analysis of Mycobacterium africanum in the last 17 years in Aragon identifies a specific location of IS6110 in Lineage 6.

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.

A whole-genome sequencing study of an X-family tuberculosis outbreak focus on transmission chain along 25 years.

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 found 10 SNPs in genes considered as virulence factors, five of them specific of this strain. WGS identified three sub-clusters (CLSs). The largest one, sub-CLS 1, included 10 cases. Seven of them shared a SNP in the mce3C gene, considered a virulence factor gene. Sub-CLS 2 involved familiar cases, and no link was known for sub-CLS 3. Finally, the strain showed efficacy in latency as a confirmed epidemiological link was established between two cases, with 6 years of distance in their diagnosis. This outbreak study combined epidemiological and molecular analyses in order to elucidate tuberculosis transmission.

The value of the continuous genotyping of multi-drug resistant tuberculosis over 20 years in Spain.

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.

Investigation of a rapidly spreading tuberculosis outbreak using whole-genome sequencing.

This paper describes the application of whole-genome sequencing (WGS) to investigate an outbreak of Mycobacterium tuberculosis occurring in Aragon, Spain, where strains have been submitted to genotyping since 2004. The responsible outbreak strain appeared in our region first in 2014 and it spread to 14 patients in the following three years. WGS found low variability between the isolates with none of the SNPs differences detected more than once, all of which were attributed to a recent transmission. Although two ambiguous bases linked two cases with those who presented the SNP in the same position, the establishment of a definitive transmission route was not possible. The epidemiological data supported the existence of a super-spreader, probably responsible for the majority of the cases involved since there was a two-year delay in diagnoses among cases. This fact would also help explaining the low variability found. The index case was not identified, possibly because it was not diagnosed in Aragon. In addition WGS characterised the strain as a Linage 4.3.3/LAM family and corroborated the susceptibility to anti-tuberculosis drugs observed by the clinical laboratories. This work shows the need to have epidemiological data to support the genomic data in order to clarify the evolution of tuberculosis outbreaks.