Resuscitation of latent M. tuberculosis?
Creators: name: Panaiotov, Stefan
name: Filevski, Georgi
name: Equestre, Michele
name: Nikolova, Elena
name: Levterova, Victoria
name: Simeonovski, Ivan
name: Bachiyska, Elizabeta
name: Nikolova, Maria
name: Kalfin, Reni
Publisher: name: F1000Research
Year: 2017

description: Current assays for determining the presence of <i>M. tuberculosis</i> infection, such as an interferon gamma release assay (IGRA) or tuberculin skin test (TST), cannot predict which infected individuals will develop active tuberculosis. Dormant latent bacteria remain undetected by conventional culture methods. Therefore, these “invisible”  bacteria represent a diagnostic target, which if successfully detected may improve tuberculosis control hindered by LTBI.Our previous studies identified that high concentration of vitamin K led to explosive resuscitation of the dormant blood microbiota.<br><br>Aim of our study was to test a novel approach based on resuscitation of dormant <i>M. tuberculosis</i> in blood samples.<br><br>Methods: Blood was collected using Vacutainer K3E 5 ml tubes from 28 healthy LTBI negative volunteers and two volunteers with confirmed LTBI by Mantoux tuberculin skin test and Interferon-gamma release assay (IGRA) and no previous treatment for TB. Blood cultivation was performed in BHI broth supplemented with vitamin K 1 mg/ml, 2% sucrose, 0.25% sodium citrate and 0.2% yeastolate at 43<sup>0</sup>C for 72 h. Isolated blood microbiota were confirmed by Gram staining, TEM and 16S rRNA genes PCR analysis. Eight commercial kits were tested for DNA isolation of the cultured blood microbiota. For DNA analysis we applied 16S rRNA genes, 18S rRNA genes and ITS2 targeted sequencing on Illumina MiSeq. The obtained sequences were clustered (≥97% identity) in Operational Taxonomic Units (OTUs). OTUs were analyzed for sequence similarities against reference sequence databases; Greengenes for 16S rDNA, Silva for 18S rDNA and UNITE for ITS2.<br><br>Results: Isolation of blood microbiota was equally effective from lysed or whole blood, as confirmed by Gram staining and TEM. TEM images demonstrated well defined cell structures. Most promising result for DNA isolation regarding purity and quantity was obtained with the RIBO-prep kit optimized with steps of freeze-thawing and DNA repurification with Chelex 100. On the bases of the 16S rRNA genes, 18S rRNA genes and ITS2 sequencing results we identified OTUs similarity with 25 bacterial genera belonging to 4 phyla and 10 fungi genera (predominantly <i>Alternaria, Epicoccum, Monographella, Malassezia, Filobasidium</i> and others) belonging to 2 phyla. Optimization of the conditions for molecular confirmation of resuscitated LTBI dormant bacteria in blood samples is ongoing.<br><br>Conclusions: Resuscitation of the blood <i>M. tuberculosis</i> of LTBI is highly challenging. Preliminary results suggest that using vitamin K to reanimate the dormant bacteria may enable quantitative detection of the dormant <i>M</i><i>. tuberculosis</i> population in patients with LTBI and holds promise for becoming a predictive tool for LTBI dynamics. We hypothesize that the amount of dormant MTB bacteria in LTB patients is related to the level of risk for TB activation.<br> <br><i>Acknowledgements</i>: This study was supported by Grant ДH-01-4/16.12.2016 from the National Science Fund of Bulgaria.