Iraqi pre-XDR tuberculosis strains reveal diverse lineages and resistance mutations

Whole-genome sequencing of two pre-extensively drug-resistant Mycobacterium tuberculosis isolates from Iraqi patients has revealed genetically distinct tuberculosis lineages carrying a compact set of established drug-resistance mutations. The study, reported in BMC Infectious Diseases, combined phenotypic drug-susceptibility testing with genomic analysis to examine how resistance and evolutionary history coincide in a country that remains poorly represented in international tuberculosis datasets. One isolate, designated MH-11, was assigned to lineage 3, also known as the East African-Indian lineage, while the other, MM-22, belonged to lineage 4.3.2, part of the Euro-American or Latin American Mediterranean lineage. The result demonstrates that pre-XDR tuberculosis in Iraq is not confined to a single bacterial population and supports whole-genome sequencing as a tool for more precise surveillance.

The researchers began with 96 clinical M. tuberculosis isolates collected from tuberculosis patients in Iraq. Drug-susceptibility testing found that two strains met the study’s pre-XDR designation and showed resistance to a fluoroquinolone, prompting the researchers to take the two isolates forward for detailed whole-genome sequencing. This focused design allowed the team to connect laboratory resistance phenotypes with the underlying genetic variants, rather than inferring resistance solely from visible growth patterns. The selection also illustrates a practical challenge in tuberculosis research: although phenotypic testing remains essential for clinical decisions, identifying the mutations behind a resistance phenotype can reveal relationships among strains that routine susceptibility testing cannot detect.

For the two sequenced isolates, researchers used global reference genomes to construct a single-nucleotide-polymorphism-based phylogenetic tree. The comparison placed MH-11 in lineage 3 and MM-22 in lineage 4.3.2. These branches represent broad evolutionary groupings of M. tuberculosis, and their presence in the same collection of Iraqi patients points to substantial lineage diversity in the country. The isolates also differed from one another in a whole-genome comparison performed with BRIG, or BLAST Ring Image Generator, indicating notable genomic divergence beyond their shared resistance phenotype. The finding matters because tuberculosis control depends on interrupting transmission and selecting effective treatment, both of which require an understanding of which bacterial lineages are circulating and how closely related they may be.

Several well-characterized mutations were associated with drug resistance in the genomes. The researchers identified changes in gyrA, including p.Asp94Gly in one isolate and p.Ala90Val in the other, as well as the rpoB S450L mutation. They also detected the katG p.Ser315Thr mutation. The gyrA variants are especially important in the context of fluoroquinolone resistance, whereas resistance to rifampicin is commonly linked to rpoB, and katG variants can affect the activity of isoniazid. The presence of these mutations provides a genomic explanation for the resistant phenotype recorded by the laboratory. Because these are established resistance-associated variants, the findings are not limited to showing that the strains looked different from reference bacteria; they connect specific genome changes to the clinically important capacity to withstand multiple antimicrobial classes.

Both isolates also carried a shared upstream variant in the ribosomal RNA gene rrs, recorded as n-187 C>T. The study identifies this feature as part of their shared resistance-associated profile, although the upstream position means that its interpretation differs from the more familiar rrs mutations associated with aminoglycoside or fluoroquinolone resistance. Reporting the precise genomic context is therefore important. It allows the variant to be distinguished from well-established drug-resistance substitutions in the same gene and prevents it from being treated, without further evidence, as a simple marker equivalent to a canonical resistance mutation. The overall result is a detailed molecular portrait of two pre-XDR strains that nevertheless share a recognizable resistance architecture.

Beyond resistance genes, the investigators compared genomic features associated with virulence and compared the isolates with the reference strain H37Rv. Differences were observed in genes connected with secretion systems, lipid metabolism and modulation of host immune responses. Structural variations involving pks15 and plcA were specifically identified in comparison with H37Rv. These genes belong to a much larger network in which M. tuberculosis acquires nutrients, remodels its cell envelope, exports molecules and interferes with host defenses. The genomic differences do not by themselves establish that either isolate was more virulent or more transmissible, and the study did not make such a comparison. They do show, however, that sequencing can identify biologically meaningful variation that may inform future experimental studies of phenotype, immune interaction and strain behavior.

The results underscore why resistance surveillance benefits from a combined strategy. Phenotypic drug-susceptibility testing measures whether a bacterial isolate can grow under defined antimicrobial conditions, providing directly relevant information for treatment selection. Whole-genome sequencing adds a different layer by revealing the precise alleles responsible for resistance, the lineage background, and the broader genomic architecture of each organism. In this study, the two approaches reinforced one another: the isolates were selected because they displayed pre-XDR phenotypes, and sequencing then defined the mutations and evolutionary branches associated with those isolates. The authors describe the integration of these methods as a way to improve tuberculosis management and highlight the potential value of genomic surveillance in Iraq.

Iraq’s position in this research is especially significant because the country is inadequately represented in global tuberculosis genomic datasets. Without representative sequence data, international analyses may provide an incomplete picture of the lineages and resistance mechanisms present in the region. Limited representation can also make it harder to connect domestic patterns with broader international transmission, identify repeatedly circulating strain groups, or assess whether locally observed mutations occur alongside the diversity found elsewhere. The present study does not establish a complete national genetic map of Iraqi tuberculosis, because only two pre-XDR isolates from a set of 96 underwent detailed sequencing. Even so, it provides direct evidence that more than one major phylogenetic lineage is represented among the sampled resistant isolates and gives researchers a foundation for larger surveillance efforts.

For clinicians and public-health programs, the immediate value of these findings lies in the convergence of a rare and difficult-to-treat resistance pattern with detailed genomic evidence. Pre-XDR M. tuberculosis requires effective combination therapy selected through reliable susceptibility testing, while genomic analysis can help explain why an isolate has that phenotype and place it within a broader evolutionary context. Routine molecular surveillance could eventually make such characterization faster and more systematic, particularly if additional Iraqi isolates are sequenced and linked to demographic and epidemiological information. The study’s central conclusion is not that genomic sequencing replaces culture or phenotypic testing, but that the approaches are complementary. By combining them, researchers can follow resistance from the laboratory phenotype to the individual DNA changes, while also revealing the lineage diversity hidden beneath a shared clinical label.

Subject of Research: Whole-genome characterization of pre-XDR Mycobacterium tuberculosis isolates from Iraq

Article Title: Whole genomic sequencing of pre-XDR Mycobacterium tuberculosis isolates in iraq reveals resistance-associated mutations and distinct phylogenetic lineages

Article References: Hadi, M., & Al-Haideri, H. (2026). Whole genomic sequencing of pre-XDR Mycobacterium tuberculosis isolates in iraq reveals resistance-associated mutations and distinct phylogenetic lineages. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14402-3

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14402-3

Keywords: Mycobacterium tuberculosis, pre-XDR tuberculosis, whole-genome sequencing, drug resistance, fluoroquinolone resistance, bacterial genomics, phylogenetic lineages, antimicrobial resistance, tuberculosis surveillance, virulence genes, Whole, genomic