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

Abstract

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.

Figure 1

Dendrogram showing the IS6110-RFLP patterns of the selected isolates of the different outbreaks.

Figure 2

Number of cases of each IS6110-cluster vs. SNP number in genes considered as virulence factors. No cause–effect relationship is observed (p-value = 0.8). Clusters with more than 90 SNPs were not considered.

Figure 3

Specific SNPs in virulence genes for each WGS-cluster classified by lineage according to the categories of Forrellad et al. and Ramage et al.^,. The number of categories remains constant among the three main lineages studied (L4.8, L4.3 and L4.1.2.1). The oxidative and nitrossidative stress, other genes related to lipid synthesis, cell wall proteins, synthesis of complex lipids and toxin/antitoxin systems categories are present in the three lineages. For clusters belonging to the L4.4.1.1, L4.9, L4.6.1.1 and L4.1.1.3/X families, there were no common and specific SNPs as only one cluster of each lineage had been sequenced, so comparison was not possible. The categories with more SNPs for these lineages were cell wall proteins, the synthesis of complex lipids and toxin/antitoxin systems. Clusters with more than 90 SNPs were not considered.