In this study, we assessed the associated risk factors for rpoB gene mutation in data sourced areas. The rpoB gene mutation rate of retreated TB patients (83.41%) was higher than that of new cases (62.32%), it has been proved that the RIF resistance rate of retreated tuberculosis is higher than that of new cases in previous studies [13, 14], the higher rate may since that patients with retreated pulmonary tuberculosis often fail in the initial treatment due to unreasonable or irregular anti-tuberculosis treatment, resulting in the dominant growth of drug-resistant tuberculosis bacteria, and it’s drug resistance mechanism is related to the mutation of rpoB gene which coding RNA polymerase β-subunit . Notably, we found no rpoB gene mutation in partial RR-TB strains, but mutations in sensitive strains, the inconsistency between gene resistance and phenotype resistance may be caused by heterogeneity of Mtb. The presence of low-frequency RR-TB and the predominance of sensitive Mtb in the specimen may result in ineffective extraction of drug-resistant DNA if the specimen is not handled properly, while the proportional method of drug sensitivity suggested that it was RR-TB . Patients may have been treated with multiple anti-tuberculosis drugs before sputum specimens were sent for testing, resulting in multiple Mtb states in sputum specimens, which can also lead to this result . And mutations in the rpoB gene leading to low levels of rifampicin resistance may be the reason that these strains with mutations in the rpoB gene were detected as sensitive .
Different VNTR loci always has different discrimination ability between Beijing and non-Beijing genotype Mtb . In our study, MIRU10, MIRU39, QUB4156 and MIRU16 all showed a difference in allellic diversity between the Beijing and non-Beijing genotype strains, but only MIRU39 showed remarkable difference (△HGDI > 0.2).
VNTR is a highly polymorphic and highly repetitive DNA fragment, which is characterized by variety and wide distribution. The distribution of VNTR in Mtb showed high individual specificity . In recent years, MIRU-VNTR had been widely used in the typing of tuberculosis, some loci, such as MIRU10, MIRU39 and QUB4156 could genotype Mtb with high discriminatory power [20,21,22]. In this study, we found that strains with high MIRU10, MIRU39 QUB4156 or MIRU16 repetitive numbers may often have a high rpoB gene mutation rate, but only the repetitive number of MIRU10, MRIU39 and QUB4156 were risk factors for rpoB gene mutation after adjusting by category (retreated or new cases) and MIRU23.
MIRU loci are located in the spacer of DNA coding genes, and their specific functions are not clear. Some scholars [23,24,25] believed that the difference in the copy number of MIRU sites upstream of the coding gene will lead to the difference in the number of ribosomal binding sites (RBS), thus affecting the transcription and expression level of the gene. The coding product encoded by the fadB gene downstream of MIRU10 is an oxidoreductase that binds to flavin adenine dinucleotide (FAD), the oxidative stress response induced by this gene may be one of the mechanisms of anti-tuberculosis drugs killing bacteria . With the increase of MIRU10 loci repetitive number, it may increase the inhibition of fadB gene expression , finally resulting in RIF resistance. EccCa1 gene which downstream of MIRU39 is part of the ESX-1 specialized secretion system, which delivers several virulence factors to host cells during infection, including the key virulence factors ESAT-6 and CFP-10 [28, 29]. The increase of MIRU39 repetitive number may target up-regulation of eccCa1 gene expression, resulting in increased bacterial virulence. The coding product encoded by the murT gene downstream of QUB4156 is involved in the pathway peptidoglycan biosynthesis, which is part of cell wall biogenesis . The increase of QUB4156 repetitive number may enhance the virulence of Mtb by promoting the synthesis of cell wall.
The mutation rate of rpoB gene increased with the addition of MIRU10, MRIU39 and QUB4156 repetitive numbers, we speculated that these MIRU loci caused the RIF resistance in Mtb respectively through different ways. Repetitive numbers of MIRU loci are relatively easy to detect in the laboratory . We hope that this upward trend can deepen the understanding of the function of MIRU10, QUB4156 and MIRU39 loci and the mechanism of RIF resistance. However, this experiment is limited to the research characteristics of molecular epidemiology, which need to be further verified by experimental research.