Evolution and emergence of multidrug-resistant Mycobacterium tuberculosis in Chisinau, Moldova

Abstract

The evolution and emergence of drug-resistant tuberculosis (TB) has been studied extensively in some contexts, but the ecological drivers of these two processes remain poorly understood. This study sought to describe the joint evolutionary and epidemiological histories of a novel multidrug-resistant Mycobacterium tuberculosis strain recently identified in the capital city of the Republic of Moldova (MDR Ural/4.2), where genomic surveillance of drug-resistant M. tuberculosis has been limited thus far. Using whole genome sequence data and Bayesian phylogenomic methods, we reconstruct the stepwise acquisition of drug resistance mutations in the MDR Ural/4.2 strain, estimate its historical bacterial population size over time, and infer the migration history of this strain between Eastern European countries. We infer that MDR Ural/4.2 likely evolved (via acquisition of rpoB S450L, which confers resistance to rifampin) in the early 1990s, during a period of social turmoil following Moldovan independence from the Soviet Union. This strain subsequently underwent substantial population size expansion in the early 2000s, at a time when national guidelines encouraged inpatient treatment of TB patients. We infer exportation of this strain and its isoniazid-resistant ancestral precursor from Moldova to neighbouring countries starting as early as 1985. Our findings suggest temporal and ecological associations between specific public health practices, including inpatient hospitalization of drug-resistant TB cases from the early 2000s until 2013, and the evolution of drug-resistant M. tuberculosis in Moldova. These findings underscore the need for regional coordination in TB control and expanded genomic surveillance efforts across Eastern Europe.

Keywords: Mycobacterium tuberculosis; antimicrobial resistance; epidemiology; outbreaks; phylogenomics.

Fig. 1.

Bayesian phylogenetic analysis for n=293 M. tuberculosis isolates collected in the Republic of Moldova in 2013–2014. (a) Dated phylogenetic reconstruction for all n=293 sequences collected in Chisinau and included in phylogenetic analysis. Tips of the tree are annotated by SNP barcode-based sublineage assignment [12]. Selected mutations associated with antimicrobial resistance (specifically those associated with resistance to isoniazid, streptomycin, rifampin, and fluoroquinolones) are annotated in the adjacent heatmap. (b) Distribution of terminal branch lengths (in years) by sublineage. Lineages 4.4 and 4.7 are not included in panel (b) given the small number of sequences in each clade. The phylogenetic tree was estimated using BEAST v1.10 with the HKY nucleotide substitution model, constant-size coalescent population model, strict molecular clock, and an informative prior on the mutation rate (Table S1).

Fig. 2.

Bayesian phylogenetic reconstruction for n=1451 M. tuberculosis isolates from Eastern Europe. Tips of the phylogeny are coloured by the country of origin for each isolate. Annotations denote M. tuberculosis sequences collected in Chisinau during this study (inner ring, red) versus sequences from prior studies (inner ring, grey), sequences with genotypic multidrug-resistance (middle ring, blue), and sequences with any fluoroquinolone resistance-associated gyrA polymorphism (outer ring, blue). The phylogenetic tree was estimated using the IQtree and timetree tools in Nextstrain Augur 6.2.0 [11]. Additional details on this group of samples is available in Table S1. Clades of the tree are annotated with SNP barcode-based sublineage assignments [12].

Fig. 3.

Bayesian phylogenetic reconstruction with discrete trait analysis for country of origin for n=205 M. tuberculosis isolates from the Ural/4.2.1 lineage, including the multidrug resistant outbreak strain and closely related isolates. (a) Phylogenetic reconstruction and estimated TMRCA for isolates carrying known M. tuberculosis drug resistance mutations. Nodes corresponding to most recent common ancestor for relevant drug resistance mutations are labelled A-O, with the 95 % highest probability density for node age displayed the blue bar on each labelled node. Tips are labelled with the country of origin for each isolate (Azerbaijan, Georgia, Moldova, Romania, and Russia). Median node ages and 95%HPD (listed in parentheses are as follows: A: katG S315T 1984 (1970–1995); B: fabG C [15]T 1987 (1976-1997); C: rpoB S450L 1990 (1979–1999); D: rpoB H455N 2001 (1992–2009); E: rpoB S450W 1998 (1990–2005) F: rpoB S450L 1995 (1986–2003); G: rpoB S450L 1999 (1991–2007); H: rpoB S450L 2011 (1995–2006); I: rpoB D435Y 2001 (1996–2006); J: rpoB S450L 1993 (1983–2000); K: embA C(16T) 2001 (1996–2006) L: embA C(16T) 1996 (1989–2003); M: gyrA D94G 2008 (2005–2011); N: gyrA D94A 2005 (1999–2010; O: gyrA A90V 2009 (2005–2011). (b) Discrete trait analysis for migration events. Boxes show counts for estimated number of migration events between countries by year and are coloured according to pairs of sending and receiving countries. (c) Estimated effective population size over time with exponential demographic model for Bayesian phylogenetic reconstruction. Outer lines show the 95 % highest posterior density interval for the effective population size.