Rifampicin (RIF) and isoniazid (INH) Mycobacterium tuberculosis isolates were characterized from south-central China and transmission patterns within the Beijing genotype were detected in multidrug-resistant isolates. Six genetic regions, including rpoB for RIF, and katG, inhA, ahpC, mabA-inhA promoter and oxyR-ahpC intergenic region for INH were analyzed by DNA sequencing in 60 multidrug-resistant isolates, including 7 extensively drug-resistant isolates. The genomic deletion RD105 was characterized by genotyping. The results showed that 91.7% of MDR isolates carried mutations in the rpoB gene and 85.0% of the MDR isolates had at least one mutation in the INH resistance-associated loci detected. In total, these six genetic regions are responsible for 95.0% of MDR isolates. Mutations in the XDR isolates were focused on rpoB 531 or rpoB 526, and katG 315, correlating to a higher frequency level of resistance to RIF MIC ⩾8 μg ml⁻¹ and INH MIC ⩾4 μg m⁻¹. Three novel katG mutants (G273S, I266T and P232S) and three new alleles (E458A, S509R and P535S) in the rpoB gene were identified. Among the 85 clinical isolates, 78 are Beijing genotypes and the other 7 are non-Beijing genotypes. The results present the identification of genetic markers in M. tuberculosis isolates, some of which may be unique to this particular geographic niche. An understanding of the mutations in these drug-resistant strains may aid in choosing the appropriate chemotherapy regimens on the pharmacogenetic properties of the mutations for the prevention and control of tuberculosis.

With mycobacteriosis increasing, the study of non-tuberculous mycobacteria is imperative for clinical therapy and management. Nontuberculous mycobacteria are naturally resistant to most anti-tuberculosis drugs. Accordingly, it is important to decipher the biology of the novel non-tuberculous mycobacteria through complete genomic analysis of novel pathogenic mycobacteria. We describe Mycobacterium sinense JDM601, a novel, slow-growing mycobacterium of the Mycobacterium terrae complex resistant to nine antibiotics, by clinical presentation, cultural and biochemical characteristics, minimal inhibitory concentrations, and genome-sequencing analysis. JDM601 is closest to Mycobacterium nonchromogenicum according to mycolic acid composition, but closest to Mycobacterium algericum sp. nov according to 16S rDNA. JDM601 is resistant to isoniazid, streptomycin, rifampin, euteropas, protionamide, capromycin, ciprofloxacin, amikacin and levofloxacin but not ethambutol. The clinical information, mycolic acid composition, and virulence genes indicate that JDM601 is an opportunistic pathogen.

Mycobacteriosis is on the increase. Nontuberculous mycobacteria (NTM) are resistant to most antituberculosis drugs naturally. We determined the complete genome sequence of a novel NTM strain, JDM601, of the Mycobacterium terrae complex, which was isolated from a patient with tuberculosis-like disease and with various antibiotic resistances.Mycobacterium terrae was first isolated in 1950 from radish washings and was described as an acid-fast saprophyte and nonpathogenic mycobacterium (8). Despite the common opinion that M. terrae complex (MTEC) isolates are nonpathogenic, these organisms are occasionally identified in the clinical laboratory in the setting of clinical disease (1, 2, 4, 10). In the past century, because of the presumed nonpathogenic nature of MTEC, there has been little effort to distinguish the species of this complex in the clinical setting. However, MTEC infection can cause debilitating disease that is relatively resistant to antibiotic therapy. M. senuense and M. paraterrae are two novel MTEC species that were recently isolated from patients with pulmonary infection (3, 6). However, the genomic knowledge of MTEC is limited. In this study, we determined the whole genome sequence, by the 454 GS FLX system (5), of a novel pathogenic mycobacterium belonging to MTEC, which was isolated from a patient with tuberculosis-like disease and with a high level of resistance to various antibiotics.The complete genome of Mycobacterium strain JDM601 contains a single, circular chromosome of 4,643,668 bp without any plasmid. The overall GC content of the chromosome is 68.4%. The entire genome of JDM601 contains 4,346 proteincoding genes, two sets of ribosome RNA operons, and 46 tRNA-encoding genes. It was possible to assign a biological function to 65.8% (2,862) of the coding sequence on the JDM601 chromosome; 28.5% (1,238) were found to be conserved hypothetical proteins, and 5.7% (246) were unique. The protein coding percentage is 92.2%.The genes of JDM601 were most similar to those of M. marinum M (11) among all mycobacteria with complete genome sequences determined, and they had common insertion sequences. Sixteen insertion sequences were found in the genome of JDM601, with five groups, four ISMyma01_aa1-like proteins, four ISMyma01_aa2-like proteins, five IS265 family transposases, an ISMsm5, and two putative transposases. No prophage was found in JDM601. There were only four integrase genes related to prophage and a gp49 without any other characteristic functional prophage genes.Most of the slowly growing mycobacteria (SGM) had only one rRNA operon, compared with the two found in rapidly growing mycobacteria (RGM). However, M. terrae and JDM601, which belong to the SGM, had two rRNA operons. The two operons of the former strain were different (7), and the operons of the latter strain were the same as each other. Interestingly, the sequencing coverage of the operon contig was close to the genome average coverage, which may mean this contig had one copy in the whole genome. However, there were two copies i...

Background: Failure to early detect multidrug-resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes, and highlights the need to rapidly detect resistance to rifampicin (RIF) and isoniazid (INH).Methods: In Multi-Fluorescence quantitative Real-Time PCR (MF-qRT-PCR) assay, 10 probes labeled with four kinds of fluorophores were designed to detect the mutations in regions of rpoB, katG, mabA-inhA, oxyR-ahpC, and rrs.The efficiency of MF-qRT-PCR assay was tested using 261 bacterial isolates and 33 clinical sputum specimens. Among these samples, 227 Mycobacterium tuberculosis isolates were analyzed using drug susceptibility testing (DST), DNA sequencing and MF-qRT-PCR assay.Results: Compared with DST, MF-qRT-PCR sensitivity and specificity for RIF-resistance were 94.6 and 100%, respectively. And the detection sensitivity and specificity for INH-resistance were 85.9 and 95.3%, respectively. Compared with DNA sequencing, the sensitivity and specificity of our assay were 97.2 and 100% for RIF-resistance and 97.9 and 96.4% for INH-resistance. Compared with Phenotypic strain identification, MF-qRT-PCR can distinguish 227 M. tuberculosis complexes (MTC) from 34 Non-tuberculous mycobacteria (NTM) isolates with 100% accuracy rate.Conclusions: MF-qRT-PCR assay was an efficient, accurate, reliable, and easy-operated method for detection of RIF and INH-resistance, and distinction of MTC and NTM of clinical isolates.

The Beijing genotype of Mycobacterium tuberculosis (MTB) is one of the most successful MTB lineages that has disseminated in the world. In China, the rate of multidrug-resistant (MDR) tuberculosis is significantly higher than the global average rate, and the Beijing genotype strains take the largest share of MDR strains. To study the genetic basis of the epidemiological findings that Beijing genotype has often been associated with tuberculosis outbreaks and drug resistance, we determined the genome sequences of four clinical isolates: two extensively drug resistant (XDR1219, XDR1221) and two multidrug resistant (WX1, WX3), using whole-genome sequencing. A large number of individual and shared SNPs of the four Beijing strains were identified. Our isolates harbored almost all classic drug resistance-associated mutations. The mutations responsible for drug resistance in the two XDR strains were consistent with the clinical quantitative drug resistance levels. COG analysis revealed that Beijing strains have significantly higher abundances of the mutations responsible for cell wall/membrane/envelope biogenesis (COG M), secondary metabolites biosynthesis, transport and catabolism (COG Q), lipid transport and metabolism (COG I) and defense mechanisms (COG V). The shared mutated genes of the four studied Beijing strains were significantly overrepresented in three DNA repair pathways. Our analyses promote the understanding of the genome polymorphism of the Beijing family strains and provide the molecular genetic basis for their wide dissemination capacity and drug resistance.

Tuberculosis (TB) is still one of the greatest health care problems in the world. In order to identify antigens that may be used in the serodiagnosis of active tuberculosis, a short fragment from Rv3391 (fRv3391) of Mycobacterium tuberculosis was cloned and expressed. Its molecular weight and secondary structure elements were identified by mass spectrometry and circular dichroism. And its immunological nature was also evaluated by enzyme-linked immunosorbent assay (ELISA). The fRv3391 was expressed in Escherichia coli BL21 (DE3) with the molecular weight of 42.5 kDa and the secondary structure elements 36.2% α-helix, 0.0% β-sheet, 32.6% β-turn, and 31.3% random coil. Evaluation of fRv3391 as an ELISA solid-phase antigen on a set of human sera from well-characterized TB cases and healthy subjects revealed that there was strong serum antibody reactivity to fRv3391 in many human TB patients. Taken together, a short fragment from Rv3391 of Mycobacterium tuberculosis was cloned and expressed and the ELISA results showed that the protein may be useful as an immunodominant antigen for the serodiagnosis of active TB.

HIV-infected individuals are susceptible to Mycobacterium tuberculosis (M.tb) infection and are at high risk of developing active tuberculosis (TB). Interferon-gamma release assays (IGRAs) are auxiliary tools in the diagnosis of TB. However, the performance of IGRAs in HIV-infected individuals is suboptimal, which limits clinical application. Interferon-inducible protein 10 (IP-10) is an alternative biomarker for identifying M.tb infection due to its high expression after stimulation with M.tb antigens. However, whether IP-10 mRNA constitutes a target for the diagnosis of TB in HIV-infected individuals is unknown. Thus, we prospectively enrolled HIV-infected patients with suspected active TB from five hospitals between May 2021 and May 2022, and performed the IGRA test (QFT-GIT) alongside the IP-10 mRNA release assay on peripheral blood. Of the 216 participants, 152 TB patients and 48 non-TB patients with a conclusive diagnosis were included in the final analysis. The number of indeterminate results of IP-10 mRNA release assay (13/200, 6.5%) was significantly lower than that of the QFT-GIT test (42/200, 21.0%) (P = 0.000026). IP-10 mRNA release assay had a sensitivity of 65.3% (95%CI 55.9% – 73.8%) and a specificity of 74.2% (95%CI 55.4% – 88.1%), respectively; while the QFT-GIT test had a sensitivity of 43.2% (95%CI 34.1% – 52.7%) and a specificity of 87.1% (95%CI 70.2% – 96.4%), respectively. The sensitivity of the IP-10 mRNA release assay was significantly higher than that of QFT-GIT test (P = 0.00062), while no significant difference was detected between the specificities of these two tests (P = 0.198). The IP-10 mRNA release assay showed a lower dependence on CD4+ T cells than that of QFT-GIT test. This was evidenced by the fact that the QFT-GIT test had a higher number of indeterminate results and a lower sensitivity when the CD4+ T cells counts were decreased (P < 0.05), while no significant difference in the number of indeterminate results and sensitivity were observed for the IP-10 mRNA release assay among HIV-infected individuals with varied CD4+T cells counts (P > 0.05). Therefore, our study suggested that M.tb specific IP-10 mRNA is a better biomarker for diagnosis of TB in HIV-infected individuals.