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

Background Diagnostics 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. Methods DNA 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 & conclusion Antibiotics 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 summary Tuberculosis (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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by (1) South African National Research Foundation Grant number UID 127338; (2) CRDFGlobal Grant number DAA9-20-66880-1. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval was provided by the Human Research Ethics Committee, Faculty of Health Sciences, University of Pretoria, South Africa. All protocols and consent forms were executed according to the agreed ethical approval terms and conditions. All clinical samples were obtained directly from patients, who agreed to our using their specimens for this research. The guidelines stated by the Declaration of Helsinki for involving human participants were followed in the study. All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the Bioproject accession PRJNA673633 and Biosample accessions JAFMQ-JAFJNR000000000. The versions described in this paper is version JAFMQ-JAFJNR 010000000.