Identification of positively selected genes in Mycobacterium tuberculosis from southern Xinjiang Uygur autonomous region of China.

Background: Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), remains a serious public health problem. Increasing evidence supports that selective evolution is an important force affecting genomic determinants of Mtb phenotypes. It is necessary to further understand the Mtb selective evolution and identify the positively selected genes that probably drive the phenotype of Mtb. Methods: This study mainly focused on the positive selection of 807 Mtb strains from Southern Xinjiang of China using whole genome sequencing (WGS). PAML software was used for identifying the genes and sites under positive selection in 807 Mtb strains. Results: Lineage 2 (62.70%) strains were the dominant strains in this area, followed by lineage 3 (19.45%) and lineage 4 (17.84%) strains. There were 239 codons in 47 genes under positive selection, and the genes were majorly associated with the functions of transcription, defense mechanisms, and cell wall/membrane/envelope biogenesis. There were 28 codons (43 mutations) in eight genes (gyrA, rpoB, rpoC, katG, pncA, embB, gid, and cut1) under positive selection in multi-drug resistance (MDR) strains but not in drug-susceptible (DS) strains, in which 27 mutations were drug-resistant loci, 9 mutations were non-drug-resistant loci but were in drug-resistant genes, 2 mutations were compensatory mutations, and 5 mutations were in unknown drug-resistant gene of cut1. There was a codon in Rv0336 under positive selection in L3 strains but not in L2 and L4 strains. The epitopes of T and B cells were both hyper-conserved, particularly in the T-cell epitopes. Conclusion: This study revealed the ongoing selective evolution of Mtb. We found some special genes and sites under positive selection which may contribute to the advantage of MDR and L3 strains. It is necessary to further study these mutations to understand their impact on phenotypes for providing more useful information to develop new TB interventions.

Exploring the immunogenicity of Rv2201-519: A T-cell epitope-based antigen derived from Mycobacterium tuberculosis AsnB with implications for tuberculosis infection detection and vaccine development.

Research dedicated to diagnostic reagents and vaccine development for tuberculosis (TB) is challenging due to the paucity of immunodominant antigens that can predict disease risk and exhibit protective potential. Therefore, it is crucial to identify T-cell epitope-based Mycobacterium tuberculosis (MTB) antigens characterized by specific and prominent recognition by the immune system. In this study, we constructed a T-cell epitope-rich tripeptide-splicing fragment (nucleotide positions 131-194, 334-377, and 579-643) of Rv2201 (also known as the 72 kDa AsnB)from the MTB genome, ultimately yielding the recombinant protein Rv2201-519 in Escherichia coli BL21 (DE3). Subsequently, we gauged the recombinant protein's ability to detect tuberculosis infection through ELISpot and assessed its immunostimulatory effect on mouse models using flow cytometry and ELISA. Our results indicated that Rv2201-519 possessed promising sensitivity; however, the sensitivity was lower than that of a commercial diagnostic kit containing ESAT-6, CFP-10, and Rv3615c (80.56 % vs. 94.44 %). The Rv2201-519 group exhibited a propensity for a CD4+ Th1 cell immune response in inoculated BALB/c mice that manifested as higher levels of antigen-specific IgG production (IgG2a/IgG1 > 1). In comparison to Ag85B, Rv2201-519 induced a more robust Th1-type cellular immune response as evidenced by a notable rise in the ratio of IFN-γ/IL-4 and IL-12 cytokine production and increased CD4+ T cell activation with a higher percentage of CD4+IFN-γ+ T cells. Rv2201-519 also induced a higher level of IL-6 compared with Ag85B, a higher percentage of CD8+ T cells specific for Rv2201-519, and a lower percentage of CD8+IL-4+ T cells. Collectively, the current evidence suggests that Rv2201-519 could potentially serve as an immunodominant protein for tuberculosis infection screening, laying the groundwork for further evaluation in recombinant Bacillus Calmette-Guérin (BCG) and subunit vaccines against MTB challenges in future studies.

Prevalence, Transmission and Genetic Diversity of Pyrazinamide Resistance Among Multidrug-Resistant Mycobacterium tuberculosis Isolates in Hunan, China.

Background: China is a country with a burden of high rates of both TB and multidrug-resistant TB (MDR-TB). However, published data on pyrazinamide (PZA) resistance are still limited in Hunan province, China. This study investigated the prevalence, transmission, and genetic diversity of PZA resistance among multidrug-resistant Mycobacterium tuberculosis isolates in Hunan province. Methods: Drug susceptibility testing (DST) with the Bactec MGIT 960 PZA kit and pyrazinamidase (PZase) testing were conducted on all 298 MDR clinical isolates. Moreover, 24-locus MIRU-VNTR and DNA sequencing of pncA, rpsA, and panD genes were conducted on 180 PZA-resistant (PZA-R) isolates. Results: The prevalence of PZA resistance among MDR-TB strains reached 60.4%. Newly diagnosed PZA-R TB patients and clustered isolates with identical pncA, rpsA, and panD mutations showed that transmission of PZA-R isolates played a significant role in the formation of PZA-R TB. Ninety-eight mutation patterns were observed in the pncA among 180 PZA-R isolates, and seventy-one (72.4%) were point mutations. Twenty-four of these mutations are new, including 2 base substitutions (V93G and T153S) and 22 nucleotide deletions or insertions. The W119C was found in PZA-S isolates, on the other hand, F94L and V155A mutations were found in both PZA resistant and susceptible isolates with positive PZase activity, indicating that they were not associated with PZA resistance. This is not entirely in line with the WHO catalogue. Ten novel rpsA mutations were found in 10 PZA-R isolates, which all combined with mutations in pncA. Thus, it is unpredictable whether these mutations in rpsA can impact PZA resistance. No panD mutation was found in all PZA-R isolates. Conclusion: DNA sequencing of pncA and PZase activity testing have great potential in predicting PZA resistance.

Recombinant BCG vaccine expressing multistage antigens of Mycobacterium tuberculosis provides long-term immunity against tuberculosis in BALB/c mice.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) persistently kills nearly 1.5 million lives per year in the world, whereas the only licensed TB vaccine BCG exhibits unsatisfactory efficacy in adults. Taking BCG as a vehicle to express Mtb antigens is a promising way to enhance its efficacy against Mtb infection. In this study, the immune efficacy of recombination BCG (rBCG-ECD003) expressing specific antigens ESAT-6, CFP-10, and nDnaK was evaluated at different time points after immunizing BALB/c mice. The results revealed that rBCG-ECD003 induced multiple Th1 cytokine secretion including IFN-γ, TNF-α, IL-2, and IL-12 when compared to the parental BCG. Under the action of PPD or ECD003, rBCG-ECD003 immunization resulted in a significant increase in the proportion of IL-2+ and IFN-γ+IL-2+ CD4+T cells. Importantly, rBCG-ECD003 induced a stronger long-term humoral immune response without compromising the safety of the parental BCG vaccine. By means of the protective efficacy assay in vitro, rBCG-ECD003 showed a greater capacity to inhibit Mtb growth in the long term. Collectively, these features of rBCG-ECD003 indicate long-term protection and the promising effect of controlling Mtb infection.

Comparison of the Immunogenicity and Efficacy of rBCG-EPCP009, BCG Prime-EPCP009 Booster, and EPCP009 Protein Regimens as Tuberculosis Vaccine Candidates.

Bacillus Calmette-Guérin (BCG) is the only widely used prophylactic tuberculosis (TB) vaccine that can prevent severe TB in infants. However, it provides poor protection in adults, and therefore, there is ongoing research into new TB vaccines and immunization strategies with more durable immune effects. The recombinant BCG and BCG prime-protein booster are two important vaccine strategies that have recently been developed based on BCG and could improve immune responses. In this study, three immune strategies based on four protective antigens, namely, ESAT-6, CFP-10, nPPE18, and nPstS1, were applied to construct recombinant rBCG-EPCP009, EPCP009 subunit protein, and BCG prime-EPCP009 booster vaccine candidates. The short- and long-term immune effects after vaccination in Balb/c mice were evaluated based on humoral immunity, cellular immunity, and the ability of spleen cells to inhibit in vitro mycobacterial growth. At 8 and 12 weeks after the initial immunization, splenocytes from mice inoculated with the BCG prime-EPCP009 protein booster secreted higher levels of PPD- and EPCP009-specific IFN-γ, IL-2, TNF-α, IL-17, GM-CSF, and IL-12 and had a higher IFN-γ+CD4+ TEM:IL-2+CD8+ TCM cell ratio than splenocytes from mice inoculated with the rBCG-EPCP009 and EPCP009 proteins. In addition, the EPCPE009-specific IgG2a/IgG1 ratio was slightly higher in the BCG prime-EPCP009 protein booster group than in the other two groups. The in vitro mycobacterial inhibition assay showed that the splenocytes of mice from the BCG prime-EPCP009 protein booster group exhibited stronger inhibition of Mycobacterium tuberculosis (M. tuberculosis) growth than the splenocytes of mice from the other two groups. These results indicate that the BCG prime-EPCP009 protein booster exhibited superior immunogenicity and M. tuberculosis growth inhibition to the parental BCG, rBCG-EPCP009, and EPCP009 proteins under in vitro conditions. Thus, the BCG prime-EPCP009 protein booster may be important for the development of a more effective adult TB vaccine.

A novel multistage antigens ERA005f confer protection against Mycobacterium tuberculosis by driving Th-1 and Th-17 type T cell immune responses.

Introduction: Tuberculosis (TB) is a major threat to human health. In 2021, TB was the second leading cause of death after COVID-19 among infectious diseases. The Bacillus Calmette-Guérin vaccine (BCG), the only licensed TB vaccine, is ineffective against adult TB. Therefore, there is an urgent need to develop new effective vaccines. Methods: In this study, we developed a novel multistage subunit vaccine (ERA005f) comprising various proteins expressed in metabolic states, based on three immunodominant antigens (ESAT-6, Rv2628, and Ag85B). We utilized the E. coli prokaryotic expression system to express ERA005f and subsequently purified the protein using nickel affinity chromatography and anion exchange. Immunogenicity and protective efficacy of ERA005f and ERA005m were evaluated in BALB/c mice. Results: ERA005f was consistently expressed as an inclusion body in a prokaryotic expression system, and a highly pure form of the protein was successfully obtained. Both ERA005f and ERA005m significantly improved IgG titers in the serum. In addition, mice immunized with ERA005f and ERA005m generated higher titers of antigen-specific IgG2a than the other groups. Elispot results showed that, compared with other groups, ERA005f increased the numbers of IFN-γ-secreting and IL-4-secreting T cells, especially the number of IFN-γ-secreting T cells. Meanwhile, ERA005f induced a higher number of IFN-γ+ T lymphocytes than ERA005m did. In addition, ERA005f improved the expression of cytokines, including IFN-γ, IL-12p70, TNF-α, IL-17, and GM-CSF and so on. Importantly, both ERA005f and ERA005m significantly inhibited the growth of Mtb. Conclusion: The novel multistage antigen ERA005f elicited a strong antigen-specific humoral response and Th-1 and Th-17 cell-mediated immunity in mice. Meanwhile, it can effectively inhibit H37Rv growth in vitro, and represents a correlate of protection in vivo, indicating that ERA005f may exhibit excellent protective efficacy against Mycobacterium tuberculosis H37Rv infection. Our study suggests that ERA005f has the potential to be a promising multistage tuberculosis vaccine candidate.

Rapid detection of fluoroquinolone resistance in Mycobacterium tuberculosis using a novel multienzyme isothermal rapid assay.

Simple, rapid, and accurate detection of Fluoroquinolone (FQ) resistance is essential for early initiation of appropriate anti-tuberculosis treatment regimen among rifampicin-resistant tuberculosis (RR-TB). In this study, we developed a new assay, which combines multienzyme isothermal rapid amplification and a lateral flow strip (MIRA-LF), to identify the mutations on codons 90 and 94 of gyrA for detecting levofloxacin (LFX) resistance. Compared to conventional phenotypic drug susceptibility testing, the new assay detected fluoroquinolone resistance with a sensitivity, specificity, and accuracy of 92.4%, 98.5%, and 96.5%, respectively. Thus, these characteristics of the newly developed MIRA-LF assay make it particularly useful and accurate for detecting FQ resistance in Mycobacterium tuberculosis in resource-limited condition.

A novel multi-component protein vaccine ECP001 containing a protein polypeptide antigen nPstS1 riching in T-cell epitopes showed good immunogenicity and protection in mice.

Tuberculosis (TB) is an infectious disease that seriously affects human health. Until now, the only anti-TB vaccine approved for use is the live attenuated Mycobacterium bovis (M. bovis) vaccine - BCG vaccine, but its protective efficacy is relatively low and does not provide satisfactory protection against TB in adults. Therefore, there is an urgent need for more effective vaccines to reduce the global TB epidemic. In this study, ESAT-6, CFP-10, two antigens full-length and the T-cell epitope polypeptide antigen of PstS1, named nPstS1, were selected to form one multi-component protein antigens, named ECP001, which include two types, one is a mixed protein antigen named ECP001m, the other is a fusion expression protein antigen named ECP001f, as candidates for protein subunit vaccines. were prepared by constructing one novel subunit vaccine by mixing or fusing the three proteins and combining them with aluminum hydroxide adjuvant, and the immunogenicity and protective properties of the vaccine was evaluated in mice. The results showed that ECP001 stimulated mice to produce high titre levels of IgG, IgG1 and IgG2a antibodies; meanwhile, high levels of IFN-γ and a broad range of specific cytokines were secreted by mouse splenocytes; in addition, ECP001 inhibited the proliferation of Mycobacterium tuberculosis in vitro with a capacity comparable to that of BCG. It can be concluded that ECP001 is a novel effective multicomponent subunit vaccine candidate with potential as BCG Initial Immunisation-ECP001 Booster Immunisation or therapeutic vaccine for M. tuberculosis infection.

Genetic Characterization Conferred Co-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Isolates from Southern Xinjiang, China.

Background: Ethionamide (ETH), a structural analogue of isoniazid (INH), is used for treating multidrug-resistant tuberculosis (MDR-TB). Due to the common target InhA, INH and ETH showed cross-resistance in M. tuberculosis. This study aimed to explore the INH and ETH resistant profiles and genetic mutations conferring independent INH- or ETH-resistance and INH-ETH cross-resistance in M. tuberculosis circulating in south of Xinjiang, China. Methods: From Sep 2017 to Dec 2018, 312 isolates were included using drug susceptibility testing (DST), spoligotyping, and whole genome sequencing (WGS) to analyze the resistance characteristics for INH and/or ETH. Results: Among the 312 isolates, 185 (58.3%) and 127 (40.7%) belonged to the Beijing family and non-Beijing family, respectively; 90 (28.9%) were INH-resistant (INHR) with mutation rates of 74.4% in katG, 13.3% in inhA and its promoter, 11.1% in ahpC and its upstream region, 2.2% in ndh, 0.0% in mshA, whilst 34 (10.9%) were ETH-resistant (ETHR) with mutation rates of 38.2% in ethA, 26.2% in inhA and its promoter, and 5.9% in ndh, 0.0% in ethR or mshA; and 25 (8.0%) were INH-ETH co-resistant (INHRETHR) with mutation rates of 40.0% in inhA and its promoter, and 8% in ndh. katG mutants tended to display high-level resistant to INH; and more inhA and its promoter mutants showed low-level of INH and ETH resistance. The optimal gene combinations by WGS for the prediction of INHR, ETHR, and INHRETHR were, respectively, katG+inhA and its promoter (sensitivity: 81.11%, specificity: 90.54%), ethA+inhA and its promoter+ndh (sensitivity: 61.76%, specificity: 76.62%), and inhA and its promoter+ndh (sensitivity: 48.00%, specificity: 97.65%). Conclusion: This study revealed the high diversity of genetic mutations conferring INH and/or ETH resistance among M. tuberculosis isolates, which would facilitate the study on INHR and/or ETHR mechanisms and provide clues for choosing ETH for MDR treatment and molecular DST methods in south of Xinjiang, China.

Association of Single-Nucleotide Polymorphisms in the VDR Gene with Tuberculosis and Infection of Beijing Genotype Mycobacterium tuberculosis.

Background: The aim of the present study was to investigate the association between vitamin D receptor (VDR) gene polymorphism and tuberculosis susceptibility, as well as the potential interaction of host genetic factors with the heterogeneity of Mycobacterium tuberculosis in the population from Xinjiang, China. Methods: From January 2019 to January 2020, we enrolled 221 tuberculosis patients as the case group and 363 staff with no clinical symptoms as the control group from four designated tuberculosis hospitals in southern Xinjiang, China. The polymorphisms of Fok I, Taq I, Apa I, Bsm I, rs3847987 and rs739837 in the VDR were detected by sequencing. M. tuberculosis isolates were collected from the case group and identified as Beijing or non-Beijing lineage by multiplex PCR. Propensity score (PS), univariate analysis and multivariable logistic regression models were used to perform the analysis. Results: Our results showed that the allele and genotype frequencies of Fok I, Taq I, Apa I, Bsm I, rs3847987 and rs739837 in VDR were not correlated with tuberculosis susceptibility or lineages of M. tuberculosis. Two out of six loci of the VDR gene formed one haplotype block, and none of the haplotypes was found to correlate with tuberculosis susceptibility or lineages of M. tuberculosis infected. Conclusion: Polymorphisms in the VDR gene may not indicate susceptibility to tuberculosis. There was also no evidence on the interaction between the VDR gene of host and the lineages of M. tuberculosis in the population from Xinjiang, China. Further studies are nonetheless required to prove our conclusions.

Immunogenicity and efficacy analyses of EPC002, ECA006, and EPCP009 protein subunit combinations as tuberculosis vaccine candidates.

Tuberculosis (TB) is the leading cause of death from infectious diseases worldwide, and developing a new TB vaccine is a priority for TB control. Combining multiple immunodominant antigens to form a novel multicomponent vaccine with broad-spectrum antigens to induce protective immune responses is a trend in TB vaccine development. In this study, we used T-cell epitope-rich protein subunits to construct three antigenic combinations: EPC002, ECA006, and EPCP009. Fusion expression of purified protein EPC002f (CFP-10-linker-ESAT-6-linker-nPPE18), ECA006f (CFP-10-linker-ESAT-6-linker-Ag85B), and EPCP009f (CFP-10-linker-ESAT-6-linker-nPPE18-linker-nPstS1) and recombinant purified protein mixtures EPC002m (mix of CFP-10, ESAT-6, and nPPE18), ECA006m (mix of CFP-10, ESAT-6, and Ag85B), and EPCP009m (mix of CFP-10, ESAT-6, nPPE18, and nPstS1) were used as antigens, formulated with alum adjuvant, and the immunogenicity and efficacy were analyzed using immunity experiments with BALB/c mice. All protein-immunized groups elicited higher levels of humoral immunity, including IgG and IgG1. The IgG2a/IgG1 ratio of the EPCP009m-immunized group was the highest, followed by that of the EPCP009f-immunized group, which was significantly higher than the ratios of the other four groups. The multiplex microsphere-based cytokine immunoassay revealed that EPCP009f and EPCP009m induced the production of a wider range of cytokines than EPC002f, EPC002m, ECA006f, and ECA006m, which included Th1-type (IL-2, IFN-γ, TNF-α), Th2-type (IL-4, IL-6, IL-10), Th17-type (IL-17), and other proinflammatory cytokines (GM-CSF, IL-12). The enzyme-linked immunospot assays demonstrated that the EPCP009f- and EPCP009m-immunized groups had significantly higher amounts of IFN-γ than the other four groups. The in vitro mycobacterial growth inhibition assay demonstrated that EPCP009m inhibited Mycobacterium tuberculosis (Mtb) growth most strongly, followed by EPCP009f, which was significantly better than that of the other four vaccine candidates. These results indicated that EPCP009m containing four immunodominant antigens exhibited better immunogenicity and Mtb growth inhibition in vitro and may be a promising candidate vaccine for the control of TB.

A Multistage Antigen Complex Epera013 Promotes Efficient and Comprehensive Immune Responses in BALB/c Mice.

Tuberculosis (TB) remains a serious global health problem. Despite the widespread use of the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine, the primary factor for the TB pandemic and deaths is adult TB, which mainly result from endogenous reactivation of latent Mycobacterium tuberculosis (MTB) infection. Improved new TB vaccines with eligible safety and long-lasting protective efficacy remains a crucial step toward the prevention and control of TB. In this study, five immunodominant antigens, including three early secreted antigens and two latency associated antigens, were used to construct a single recombinant fusion protein (Epera013f) and a protein mixture (Epera013m). When formulated with aluminum adjuvant, the two subunit vaccines Epera013m and Epera013f were administered to BALB/c mice. The humoral immune responses, cellular responses and MTB growth inhibiting capacity elicited after Epera013m and Epera013f immunization were analyzed. In the present study, we demonstrated that both the Epera013f and Epera013m were capable of inducing a considerable immune response and protective efficacy against H37Rv infection compared with BCG groups. In addition, Epera013f generated a more comprehensive and balanced immune status, including Th1, Th2 and innate immune response, over Epera013f and BCG. The multistage antigen complex Epera013f possesses considerable immunogenicity and protective efficacy against MTB infection ex vivo indicating its potential and promising applications in further TB vaccine development.

Molecular Epidemiology of Clinical Mycobacterium tuberculosis Isolates from Southern Xinjiang, China Using Spoligotyping and 15-Locus MIRU-VNTR Typing.

Background: In the last decades, the molecular epidemiological investigation of Mycobacterium tuberculosis has significantly increased our understanding of tuberculosis epidemiology. However, few such studies have been done in southern Xinjiang, China. We aimed to clarify the molecular epidemic characteristics and their association with drug resistance in the M. tuberculosis isolates circulating in this area. Methods: A total of 347 isolates obtained from southern Xinjiang, China between Sep, 2017 and Sep, 2019 were included to characterize using a 15-locus MIRU-VNTR (VNTR-15China) typing and spoligotyping, and test for drug susceptibility profiles. Then the lineages and clustering of the isolates were analyzed, as well as their association with drug resistance. Results: Spoligotyping results showed that 60 spoligotype international types (SITs) containing 35 predefined SITs and 25 Orphan or New patterns, and 12 definite genotypes were found, and the top three prevalent genotypes were Beijing genotype (207, 59.7%), followed by CAS1-Delhi (46, 13.6%), and Ural-2 (30, 8.6%). The prevalence of Beijing genotype infection in the younger age group (≤30) was more frequent than the two older groups (30~59 and ≥60 years old, both P values <0.05). The Beijing genotype showed significantly higher prevalence of resistance to isoniazid, rifampicin, ethambutol, multi-drug or at least one drug than the non-Beijing genotype (All P values ≤0.05). The estimated proportion of tuberculosis cases due to transmission was 18.4% according to the cluster rate acquired by VNTR-15China typing, and the Beijing genotype was the risk factor for the clustering (OR 9.15, 95% CI: 4.18-20.05). Conclusion: Our data demonstrated that the Beijing genotype is the dominant lineage, associated with drug resistance, and was more likely to infect young people and contributed to tuberculosis transmission in southern Xinjiang, China. These findings will contribute to a better understanding of tuberculosis epidemiology in this area.

The Evolution and Transmission Dynamics of Multidrug-Resistant Tuberculosis in an Isolated High-Plateau Population of Tibet, China.

On the Tibetan Plateau, most tuberculosis is caused by indigenous Mycobacterium tuberculosis strains with a monophyletic structure and high-level drug resistance. This study investigated the emergence, evolution, and transmission dynamics of multidrug-resistant tuberculosis (MDR-TB) in Tibet. The whole-genome sequences of 576 clinical strains from Tibet were analyzed with the TB-profiler tool to identify drug-resistance mutations. The evolution of the drug resistance was then inferred based on maximum-likelihood phylogeny and dated trees that traced the serial acquisition of mutations conferring resistance to different drugs. Among the 576 clinical M. tuberculosis strains, 346 (60.1%) carried at least 1 resistance-conferring mutation and 231 (40.1%) were MDR-TB. Using a pairwise distance of 50 single nucleotide polymorphisms (SNPs), most strains (89.9%, 518/576) were phylogenetically separated into 50 long-term transmission clusters. Eleven large drug-resistant clusters contained 76.1% (176/231) of the local multidrug-resistant strains. A total of 85.2% of the isoniazid-resistant strains were highly transmitted with an average of 6.6 cases per cluster, of which most shared the mutation KatG Ser315Thr. A lower proportion (71.6%) of multidrug-resistant strains were transmitted, with an average cluster size of 2.9 cases. The isoniazid-resistant clusters appear to have undergone substantial bacterial population growth in the 1970s to 1990s and then subsequently accumulated multiple rifampicin-resistance mutations and caused the current local MDR-TB burden. These findings highlight the importance of detecting and curing isoniazid-resistant strains to prevent the emergence of endemic MDR-TB. IMPORTANCE Emerging isoniazid resistance in the 1970s allowed M. tuberculosis strains to spread and form into large multidrug-resistant tuberculosis clusters in the isolated plateau of Tibet, China. The epidemic was driven by the high risk of transmission as well as the potential of acquiring further drug resistance from isoniazid-resistant strains. Eleven large drug-resistant clusters consisted of the majority of local multidrug-resistant cases. Among the clusters, isoniazid resistance overwhelmingly evolved before all the other resistance types. A large bacterial population growth of isoniazid-resistant clusters occurred between 1970s and 1990s, which subsequently accumulated rifampicin-resistance-conferring mutations in parallel and accounted for the local multidrug-resistant tuberculosis burden. The results of our study indicate that it may be possible to restrict MDR-TB evolution and dissemination by prioritizing screening for isoniazid (INH)-resistant TB strains before they become MDR-TB and by adopting measures that can limit their transmission.

Omics analysis of Mycobacterium tuberculosis isolates uncovers Rv3094c, an ethionamide metabolism-associated gene.

Global control of the tuberculosis epidemic is threatened by increasing prevalence of drug resistant M. tuberculosis isolates. Many genome-wide studies focus on SNP-associated drug resistance mechanisms, but drug resistance in 5-30% of M. tuberculosis isolates (varying with antibiotic) appears unrelated to reported SNPs, and alternative drug resistance mechanisms involving variation in gene/protein expression are not well-studied. Here, using an omics approach, we identify 388 genes with lineage-related differential expression and 68 candidate drug resistance-associated gene pairs/clusters in 11 M. tuberculosis isolates (variable lineage/drug resistance profiles). Structural, mutagenesis, biochemical and bioinformatic studies on Rv3094c from the Rv3093c-Rv3095 gene cluster, a gene cluster selected for further investigation as it contains a putative monooxygenase/repressor pair and is associated with ethionamide resistance, provide insights on its involvement in ethionamide sulfoxidation, the initial step in its activation. Analysis of the structure of Rv3094c and its complex with ethionamide and flavin mononucleotide, to the best of our knowledge the first structures of an enzyme involved in ethionamide activation, identify key residues in the flavin mononucleotide and ethionamide binding pockets of Rv3094c, and F221, a gate between flavin mononucleotide and ethionamide allowing their interaction to complete the sulfoxidation reaction. Our work broadens understanding of both lineage- and drug resistance-associated gene/protein expression perturbations and identifies another player in mycobacterial ethionamide metabolism.

rpoB Mutations are Associated with Variable Levels of Rifampin and Rifabutin Resistance in Mycobacterium tuberculosis.

Objective: To assess the relationship between the variant rpoB mutations and the degree of rifampin (RIF)/rifabutin (RFB) resistance in Mycobacterium tuberculosis (M. tuberculosis). Methods: We analyzed the whole rpoB gene in 177 M. tuberculosis clinical isolates and quantified their minimum inhibitory concentrations (MICs) using microplate-based assays. Results: The results revealed that of the 177 isolates, 116 were resistant to both RIF and RFB. There were 38 mutated patterns within the sequenced whole rpoB gene of the 120 isolates. Statistical analysis indicated that mutations, S450L, H445D, H445Y, and H445R, were associated with RIF and RFB resistance. Of these mutations, S450L, H445D, and H445Y were associated with high-level RIF and RFB MIC. H445R was associated with high-level RIF MIC, but not high-level RFB MIC. D435V and L452P were associated with only RIF, but not RFB resistance. Q432K and Q432L were associated with high-level RFB MIC. Several single mutations without statistical association with rifamycin resistance, such as V170F, occurred exclusively in low-level RIF but high-level RFB resistant isolates. Additionally, although cross-resistance to RIF and RFB is common, 21 RIF-resistant/RFB-susceptible isolates were identified. Conclusion: This study highlighted the complexity of rifamycin resistance. Identification of the rpoB polymorphism will be helpful to diagnose the RIF-resistant tuberculosis that has the potential to benefit from a treatment regimen including RFB.

Whole-Genome Sequencing and Epidemiological Investigation of Tuberculosis Outbreaks in High Schools in Hunan, China.

Background: Tuberculosis (TB) seriously threatens individual and public health. Recently, TB outbreaks in schools have been reported more frequently in China and have attracted widespread attention. We reported three TB outbreaks in high schools in Hunan Province, China. Methods: When a tuberculosis patient was reported in a school, we carried out field epidemiological investigations, including tuberculin skin testing (TST), chest X-ray (CXR) and laboratory test for all close contacts, and whole-genome sequencing (WGS) analyses to understand the transmission patterns, the causes and the risk factors for the outbreaks, thereby providing a foundation for the control of TB epidemics in schools. Results: A total of 49 students with TB patients were identified in the three schools where TB outbreaks occurred, including nine patients in School A, 14 patients in School B, and 26 patients in School C. In Schools A, B and C, the putative attack rates in the classes of the index case were 13.8% (8/58), 7.6% (5/66), and 40.4% (21/52), while the putative attack rates of expanding screening in the school were 0.3% (1/361), 0.2% (9/3955), and 0.2% (5/2080), respectively. Thirteen patients had patient delay, with a median delay interval of 69 days (IQR 30.5-113 days). Twelve patients had a healthcare diagnostic delay with a median delay interval of 32 days (IQR 24-82 days). Phylogenetic analysis of culture-positive patients revealed that most of them shared a small genetic distance (≤12 SNPs), with three separate genetic clusters (including one MDR-TB genomic cluster), indicating the recent transmission of Mycobacterium tuberculosis strains. Conclusion: This combination of field investigation and WGS analysis revealed the transmission of three TB outbreaks in schools. Reinforced implementation is needed to improve timely case finding and reduce diagnosis delay in routine TB control in the school population.

Super Dominant Pathobiontic Bacteria in the Nasopharyngeal Microbiota Cause Secondary Bacterial Infection in COVID-19 Patients.

Coronavirus disease 2019 (COVID-19) is a respiratory infectious disease responsible for many infections worldwide. Differences in respiratory microbiota may correlate with disease severity. Samples were collected from 20 severe and 51 mild COVID-19 patients. High-throughput sequencing of the 16S rRNA gene was used to analyze the bacterial community composition of the upper and lower respiratory tracts. The indices of diversity were analyzed. When one genus accounted for >50% of reads from a sample, it was defined as a super dominant pathobiontic bacterial genus (SDPG). In the upper respiratory tract, uniformity indices were significantly higher in the mild group than in the severe group (P < 0.001). In the lower respiratory tract, uniformity indices, richness indices, and the abundance-based coverage estimator were significantly higher in the mild group than in the severe group (P < 0.001). In patients with severe COVID-19, SDPGs were detected in 40.7% of upper and 63.2% of lower respiratory tract samples. In patients with mild COVID-19, only 10.8% of upper and 8.5% of lower respiratory tract samples yielded SDPGs. SDPGs were present in both upper and lower tracts in seven patients (35.0%), among which six (30.0%) patients possessed the same SDPG in the upper and lower tracts. However, no patients with mild infections had an SDPG in both tracts. Staphylococcus, Corynebacterium, and Acinetobacter were the main SDPGs. The number of SDPGs identified differed significantly between patients with mild and severe COVID-19 (P < 0.001). SDPGs in nasopharyngeal microbiota cause secondary bacterial infection in COVID-19 patients and aggravate pneumonia. IMPORTANCE The nasopharyngeal microbiota is composed of a variety of not only the true commensal bacterial species but also the two-face pathobionts, which are one a harmless commensal bacterial species and the other a highly invasive and deadly pathogen. In a previous study, we found that the diversity of nasopharyngeal microbiota was lost in severe influenza patients. We named the genus that accounted for over 50% of microbiota abundance as super dominant pathobiontic genus, which could invade to cause severe pneumonia, leading to high fatality. Similar phenomena were found here for SARS-CoV-2 infection. The diversity of nasopharyngeal microbiota was lost in severe COVID-19 infection patients. SDPGs in nasopharyngeal microbiota were frequently detected in severe COVID-19 patients. Therefore, the SDPGs in nasopharynx microbiota might invade into low respiratory and be responsible for secondary bacterial pneumonia in patients with SARS-CoV-2 infection.

Association of Mannose-Binding Lectin 2 Gene Polymorphism with Tuberculosis Based on Mycobacterium tuberculosis Lineages.

Purpose: Polymorphisms in MBL2 may contribute to the susceptibility to tuberculosis. The aim of the present study was to determine the associations of the polymorphisms of five loci (rs1800450, rs1800451, rs7096206, rs7095891, and rs11003125) in the MBL2 gene with susceptibility to tuberculosis and specific lineages of Mycobacterium tuberculosis causing tuberculosis in the Uyghur population of Xinjiang, China. Methods: From January 2019 to January 2020, we enrolled 170 Uyghur tuberculosis patients as the case group and 147 Uyghur staff with no clinical symptoms as the control group from four designated tuberculosis hospitals in southern Xinjiang, China. The polymorphisms of five loci in MBL2 of human were detected by sequencing. Whole-genome sequencing was applied in 68 M. tuberculosis isolates from the case group and the data were used to perform genealogy analysis. Results: The distributions of allele and genotype frequencies of five loci in MBL2 varied little between the case and control groups and varied little among the groups, including those infected with different lineages of M. tuberculosis and the control (except those of rs11003125), the P values were all >0.05. The distribution of alleles of rs11003125 was statistically different between patients infected with lineages 3 and 4 M. tuberculosis (χ 2=7.037, P=0.008). The C allele and CC genotype of rs11003125 were found to be protective factors against lineage 4 infection when compared to lineage 3 (ORs were 0.190 and 0.158, respectively; 95% confidence intervals were 0.053~0.690 and 0.025~0.999, respectively). Conclusion: Our results suggested that human's susceptibility to tuberculosis is affected both by the host genetic polymorphisms and the lineage of the M. tuberculosis that people were exposed to. However, due to the limitation of the sample size in the present study, larger sample size and more rigorous design should be guaranteed in future studies.

High Immunogenicity of a T-Cell Epitope-Rich Recombinant Protein Rv1566c-444 From Mycobacterium tuberculosis in Immunized BALB/c Mice, Despite Its Low Diagnostic Sensitivity.

The discovery of immunodominant antigens is of great significance for the development of new especially sensitive diagnostic reagents and effective vaccines in controlling tuberculosis (TB). In the present study, we targeted the T-Cell epitope-rich fragment (nucleotide position 109-552) of Rv1566c from Mycobacterium tuberculosis (MTB) and got a recombinant protein Rv1566c-444 and the full-length protein Rv1566c with Escherichia coli expression system, then compared their performances for TB diagnosis and immunogenicity in a mouse model. The results showed that Rv1566c-444 had similar sensitivity with Rv1566c (44.44% Vs 30.56%) but lower sensitivity than ESAT-6&CFP-10&Rv3615c (44.4% Vs. 94.4%) contained in a commercial kit for distinguishing TB patients from healthy donors. In immunized BALB/c mice, Rv1566c-444 elicited stronger T-helper 1 (Th1) cellular immune response over Rv1566c with higher levels of Th1 cytokine IFN-γ and IFN-γ/IL-4 expression ratio by ELISA; more importantly, with a higher proliferation of CD4+ T cells and a higher proportion of CD4+ TNF-α+ T cells with flow cytometry. Rv1566c-444 also induced a higher level of IL-6 by ELISA and a higher proportion of Rv1566c-444-specific CD8+ T cells and a lower proportion of CD8 + IL-4 + T cells by flow cytometry compared with the Rv1566c group. Moreover, the Rv1566c-444 group showed a high IgG secretion level and the same type of CD4+ Th cell immune response (both IgG1/IgG2a >1) as its parental protein group. Our results showed the potential of the recombinant protein Rv1566c-444 enriched with T-Cell epitopes from Rv1566c as a host T cell response measuring biomarker for TB diagnosis and support further evaluation of Rv1566c-444 as vaccine antigen against MTB challenge in animal models in the form of protein mixture or fusion protein.