Bacteriomes in lesions of pulmonary tuberculosis and its association with status of Mycobacterium tuberculosis excretion.

BACKGROUND: Bacteria in lung play an important role in sustaining lung health. Understanding the characteristics of bacteriomes in lesions of pulmonary tuberculosis (TB) patients, who excrete Mycobacterium tuberculosis (MTB), is important for TB prevention and effective treatment.  METHODS: In this study, bacteriomes in lesions from TB patients excreting bacteria (TB-E) and those from TB patients not excreting bacteria (TB-NE) with matched normal lung tissues (NT) were compared by 16S rRNA sequencing. Bacterial MetaCyc functions in TB lesions were also predicted by PICRUSt2 tool. RESULTS: Alpha diversity of bacteria, including Chao 1 and Shannon indexes, for TB-E was significantly higher than those in TB-NE and NT; while for TB-NE group, Chao 1 index was higher than that in NT group. Predominant phyla in TB lesions and NT were Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes, but analysis of similarity (ANOSIM, p < 0.001) revealed significantly different bacterial compositions among TB-E, TB-NE and NT samples. As for bacteriomes in TB lesions, a strong association (ANOSIM, p < 0.001) was observed with the status of MTB excretion. Indicator genera identified in TB-E and TB-NE demonstrated distinctive micro-ecological environments of TB lesions from patients with different clinical manifestations. Co-occurrence analysis revealed a densely-linked bacterial community in TB-NE compared to that in TB-E. MetaCyc functions responsible for menaquinone synthesis and chorismate metabolism that could potentially impact the persistent-state and nutrient metabolism of MTB were enriched in TB-E samples. While in TB-NE samples, enrichment of bacterial MetaCyc function responsible for heme b synthesis might contribute to TB pathology through ferroptosis. CONCLUSION: Bacteriomes and their MetaCyc functions in TB lesions are elucidated, and they are associated with status of MTB excretion among pulmonary TB patients. These results serve as a basis for designing novel strategies for preventing and treating pulmonary TB disease.

Application of Amplicon-Based Targeted NGS Technology for Diagnosis of Drug-Resistant Tuberculosis Using FFPE Specimens.

Next-generation sequencing (NGS) enables rapid identification of common and rare drug-resistant genetic variations from tuberculosis (TB) patients' sputum samples and MTB isolates. However, whether this technology is effective for formalin-fixed and paraffin-embedded (FFPE) tissues remains unclear. An amplicon-based targeted NGS sequencing panel was developed to predict susceptibility to 9 antituberculosis drugs, including 3 first-line drugs, by directly detecting FFPE tissues. A total of 178 tissue samples from TB patients who underwent phenotypic drug susceptibility test were retrospectively tested from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. Phenotypic drug susceptibility test results were used as the reference standard. We identified 22 high-quality mutations from 178 FFPE tissue samples, including 15 high+moderate+minimal confidence-level mutations associated with drug resistance (rpoB D435V, S450F/L; KatG S315T; inhA-fabG promoter c-15t; embB G406S, M306V; rpsL K43R, K88R, rrs a1401g, a514c; gyrA D94G/Y/A, A90V), 6 mutations not associated with resistance (rpoB D435Y, H445S, L430P, L452P; embB G406A/D), and one mutation site embB M306I defined as indeterminate. Compared to the phenotypic method, sensitivities (95% CI) for rifampicin, isoniazid, and ethambutol were 96% (79.65-99.90%), 93.55% (78.58-99.21%), and 71.43% (35.24-92.44%), respectively; while for second-line drugs, it varied from 23.53% (9.05-47.77%) for capreomycin to 86.84% (72.20-94.72%) for streptomycin. Specificities for all drugs were satisfactory (>94.51%). Therefore, important pathological FFPE tissue samples, despite partially degraded DNA, can be used as essential specimens for molecular diagnosis of drug resistant TB by amplicon-based targeted NGS technology. IMPORTANCE Amplicon-based targeted NGS technology focuses on a set of gene mutations of known or suspected associations with drug susceptibility in Mycobacterium tuberculosis (MTB). This method offers many benefits, such as low sequencing cost, easy customization, high throughput, shorter testing time and not culture dependent. Formalin-fixed and paraffin-embedded (FFPE) tissues are important pathological specimen in diagnosing tuberculous disease because they are noninfectious and provide excellent preservation of tissue morphology with low storage cost. However, the performance of amplicon-based targeted NGS method on FFPE samples has not been reported yet. Therefore, we evaluated the performance of this method using FFPE samples collected from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. We demonstrate that the amplicon-based targeted NGS method performs excellent on FFPE samples, and it can be applied to pathological diagnosis of drug resistant tuberculosis.