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Targeted deep sequencing of mycobacteria species from extrapulmonary sites not identified by routine line probe assays: a retrospective laboratory analysis of stored clinical cultures

ABSTRACT: Targeted deep sequencing of mycobacteria species from extrapulmonary sites

PROVIDER: PRJEB77459 | ENA |

REPOSITORIES: ENA

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Targeted deep sequencing of mycobacteria species from extrapulmonary sites not identified by routine line probe assays: A retrospective laboratory analysis of stored clinical cultures.

Project description:ObjectivesNontuberculous mycobacteria (NTM) infections present a global health challenge. This study describes unidentified mycobacteria species from extrapulmonary sites, using advanced identification and sequencing techniques.MethodsExtrapulmonary mycobacteria growth indicator tube primary cultures collected retrospectively between 2019 and 2023, featuring unidentified mycobacteria species detected by GenoType Mycobacterium line probe assays, underwent multilocus targeted next-generation sequencing using Oxford Nanopore Technology, polymerase chain reaction amplicon Sanger sequencing, and Deeplex Myc-TB analysis. Previously collected clinical and laboratory data were reported.ResultsA total of 28 cultures, collected from extrapulmonary sites, each from different patients, were included. Mycobacterial mixtures were identified in 19 of 28 (68%) cultures, with four of 28 (14%) showing unidentified species based on sequencing of rpoB and hsp65 targets. Mycobacterium monacense was present in 13 of 28 (46%) of the cultures. Culturable Mycobacterium tuberculosis complex (MTBC) was identified in five extrapulmonary specimens that previously tested negative for MTBC using Xpert MTB/RIF Ultra. The comparative analysis between Sanger and targeted next-generation sequencing using Oxford Nanopore Technology sequencing (for hsp65) demonstrated 27 of 28 (96%) agreement on the predominant strain. Deeplex Myc-TB could not identify NTM-MTBC co-infections in minor subpopulations.ConclusionsThis study highlights the role of advanced sequencing in identifying NTM mixtures and mycobacterial co-infections. It calls for ongoing efforts to integrate next-generation sequencing into mycobacteria testing algorithms.

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Project description:Some intracellular bacteria are known to cause long-term infections for periods of time that last decades without compromising the viability of the host. Although of critical importance, the changes that intracellular bacteria suffer during this long process of residence in a host cell environment remain obscure. Here, we report an experimental approach to study the adaptations of intracellular mycobacteria forced by a long-term intracellular lifestyle. Long-term infection of host macrophages with mycobacteria was maintained for a period of years. Mycobacteria in the long-term infected macrophages underwent an adaptation process leading to impaired phenolic glycolipids (PGL) synthesis, preference for glucose as a carbon source and neutral lipids accumulation. These changes correlated with increased survival of mycobacteria in macrophages and mice during re-infection and specific expression of stress- and survival-related genes. Our findings identify bacterial traits implicated in the establishment of long-term cellular infections and represent a tool for understanding the physiological states of bacteria living in fluctuating intracellular environments.

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