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.

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.

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.

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.

Long-term molecular surveillance of multidrug-resistant tuberculosis in Spain.

The data presented here span 11 years (1998-2008) of monitoring of multidrug-resistant tuberculosis (MDR-TB) clustering through molecular typing techniques in Spain. The molecular and epidemiological data of 480 multidrug-resistant Mycobacterium tuberculosis complex isolates were analyzed. Thirty-one clusters involving 157 (32.7%) patients were identified. The proportion of immigrants increased substantially over the study period reaching 65% in 2008; however, the clustering rate remained stable indicating that local transmission was little influenced by imported MDR-TB. The three major clusters respond to the persistence of two autochthonous strains throughout the study period and an extensively drug-resistant (XDR) Mycobacterium bovis outbreak with only two cases was reported since 2002. Molecular and epidemiological evidence for the importation of new strains and their spread within the community was found. Immigrant-only clusters most often grouped patients infected abroad with strains belonging to rare spoligotypes. Conversely, widespread spoligotypes of the Latin-American and Mediterranean (LAM) and Haarlem families were responsible for the majority of the MDR-TB local transmission. The demonstration of clusters spanning several Spanish regions that have been ongoing throughout the study period makes it advisable to maintain a continuous molecular surveillance in order to monitor the spread of MDR-TB.