An Oxford Nanopore-based Characterisation of Sputum Microbiota Dysbiosis in Patients with Tuberculosis: from baseline to 7 days after Antibiotic Treatment

Abstract

AbstractBackgroundDiagnostics for tuberculosis (TB) and treatment monitoring remains a challenge, particularly in less-resourced laboratories. Further, the comprehensive sputum microbiota of TB patients during treatment are less described, particularly using long-read sequencers.MethodsDNA from sputum samples collected from newly-diagnosed TB patients were sequenced with Oxford Nanopore’s MinION. MG-RAST and R packages (Phyloseq, α/β diversities, functional components, OTUs networks and ordination plots. Statistical significance of the generated data was determined using GraphPad.Results & conclusionAntibiotics reduced the abundance and functional subsystems of each samples’ microbiota from baseline until day 7, when persistent, tolerant, and resistant microbiota, including fungi, grew back again. Variations in microbiota abundance and diversity were patient-specific. Closer microbiome network relationships observed in baseline samples reduced until day 7, when it became closer again. Bacterial microbiota networks and spatial ordination relationships were closer than that of other kingdoms. Actinobacteria phylum and Mycobacterium were more affected by antibiotics than other phyla and genera. Parasites, viruses, and fungi were less affected by antibiotics than bacteria in a descending order. Resistance genes/mechanisms to important antibiotics, plasmids, transposons, insertion sequences, integrative conjugative elements were identified in few samples.MinION can be adopted clinically to monitor treatment and consequent dysbiosis, and identify both known and unknown pathogens and resistance genes to inform tailored treatment choices, specifically in TB.Author summaryTuberculosis (TB), one of the major killers of mankind, continually remains elusive as challenges with early diagnosis and treatment monitoring remain. Herein, we use a single portable sequencer from Oxford Nanopore, the minION, to diagnose TB and monitor its treatment with antibiotics using routine sputum samples. In addition, the presence of other pathogens, important commensals, antibiotic resistance genes, mobile genetic elements, and the effect of the antibiotic treatment on the sputum microbiota were determined from the same data. This makes the minION an important tool that can be used in clinical laboratories to obtain data that can inform live-saving decisions.