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ABSTRACTTuberculosis (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.

ObjectiveOsteoarticular tuberculosis (OATB) is one of the most common forms of extrapulmonary tuberculosis; however, limited epidemiological data are available on this public health concern worldwide, especially in developing countries. This study aimed to analyze the clinical epidemiology and drug resistance characteristics of OATB cases in Hunan province which located in South-central China.MethodsWe retrospectively enrolled OATB patients with Mycobacterium tuberculosis culture positive at Hunan Chest Hospital from January 2013 through March 31, 2022. The multiple demographic, clinical variables and drug susceptibility data of the patients were collected from the hospital’s electronic patient records. Descriptive statistical methods, Chi-square test and logistic regression analysis were employed as statistical methods.ResultsOf the 269 OATB cases, 197 (73.23%) were males, 206 (76.85%) were farmers; patients’ ages ranged from 5 to 85 years, 57 (21.19%) aged at 20–29 years old and 52 (19.33%) aged at 60–69 years old. In terms of the disease, 177 (65.80%) had spinal TB with most occurrence in lumbar vertebrae (26.02%, 70/269), multiple spinal sites (18.96%, 51/269) and thoracic vertebrae (15.24%, 41/269). Outside of the spine, OATB mainly occurred in the lower limb (13.38%, 36/269). In terms of drug resistance, 40 (14.87%) and 72 (26.77%) were resistant to rifampicin (RFP) and isoniazid (INH) respectively; 38 (14.13%) were multi-drug resistant (MDR), and a total of 78 (29.00%) isolates were drug resistant. OATB patients aged 40–49 years old (compared to those aged ≥70 years) and from the west of Hunan province, China (compared to those from the center of Hunan) were at risk for developing RR/MDR (ORs were 5.057 and 4.942, respectively; 95% CIs were 1.009–25.342 and 1.458–16.750, respectively).ConclusionIn South-central China, OATB mainly affected males, farmers and those aged 20–29 and 60–69 years old. Spinal TB is prone to occur in the lumbar and multiple spinal sites. The resistance situation of OATB was serious, and people aged 40–49 years old and patients from the west of Hunan were risk factors of RR/MDR. All these findings will help to improve the prevention, diagnosis and treatment strategies of OATB.

ObjectivesThis study aimed to investigate the epidemiological and drug resistance (DR) characteristics of extrapulmonary tuberculosis (EPTB) in South-Central China.MethodsEPTB inpatients who were culture-positive for Mycobacterium tuberculosis were retrospectively included in a study at a provincial TB hospital in Hunan, a province in South-Central China, from January 2013 to December 2021. Demographic, clinical, and drug susceptibility data were retrieved from TB treatment records. Descriptive statistical methods and a Chi-squared test were used to analyze the epidemiological and DR characteristics of EPTB patients. A logistic regression model was used to explore the risk factors of rifampicin-resistant/multidrug-resistant (RR/MDR)-EPTB.ResultsA total of 1,324 cases were included. The majority of EPTB patients were in the age range of 20–29 years, were predominantly men (male-to-female ratio: 2.03), and were farmers (65.63%). Most EPTB cases were found in 2013 and 2017 from 2013 to 2021. The most prevalent subtypes of EPTB were lymphatic TB (29.83%, 395/1,324), multiple EPTB (20.85%, 276/1,324), and musculoskeletal TB (14.65%, 194/1,324). Musculoskeletal TB and genitourinary TB predominantly presented as exclusive EPTB forms, while lymphatic TB and pharyngeal/laryngeal TB often co-occurred with pulmonary TB (PTB). Drug susceptibility testing results showed that total DR rates (resistance to any of RFP, isoniazid [INH], streptomycin [STR], and/or ethambutol [EMB]) and RR/MDR rates in EPTB were 25.23% and 12.39%, respectively. Musculoskeletal TB exhibited the highest rates of total DR (31.40%), INH resistance (28.90%), STR resistance (20.10%), EMB resistance (6.20%), MDR (13.90%), and poly-DR (6.70%). The multivariable logistic regression model showed that patients aged from 20 to 59 years (compared to those aged 10 years), workers (compared to retirees), and EPTB patients from the south and west of Hunan (compared to those from the east of Hunan) were at an increased risk of developing RR/MDR EPTB (all OR values > 1).ConclusionOur study provided a detailed account of the epidemiological and DR characteristics of EPTB in Hunan province, China. The significant DR rates, particularly in musculoskeletal TB cases, highlight the need for timely diagnosis, effective drug susceptibility testing, and the development of more effective treatment regimens for EPTB, especially targeting musculoskeletal TB treatments.

ObjectivesTo investigate the clinical epidemiological and drug resistance (DR) characteristics of lymph node tuberculosis (LNTB) in Hunan Province which locates in South-central China, and to provide scientific clues for effective prevention and treatment of LNTB.MethodsWe retrospectively collected LNTB patients with Mycobacterium tuberculosis culture positive at Hunan Chest Hospital, the biggest TB reference hospital in South-central China, from January 2013 to December 2021. The multiple demographic, clinical and drug susceptibility data of patients were collected from the hospital’s electronic patient records. Descriptive statistical methods, Chi-square test and logistic regression analysis were employed as statistical methods.ResultsOf the 577 LNTB cases, 373 (64.64%) were males, 352 (61.01%) were farmers; majority (161, 33.10%) aged at 20–29 years old; 147 (25.48%) had simple LNTB, 350 (60.66%) had LNTB combined with pulmonary TB (PTB) (defined as LNTB-PTB), and 80 (13.86%) had LNTB combined with other extrapulmonary TB (EPTB) (defined as LNTB-EPTB). A total of 345 (59.79%, 345/577) LNTB patients had cervical node infection, and the simple LNTB patients (81.63%, 120/147) had higher proportion of this infection than LNTB-PTB (51.71%, 181/350) and LNTB-EPTB (55.00%, 44/80) (both p values

IntroductionStenotrophomonas is a prominent genus owing to its dual nature. Species of this genus have many applications in industry and agriculture as plant growth-promoting rhizobacteria and microbial biological control agents, whereas species such as Stenotrophomonas maltophilia are considered one of the leading gram-negative multi-drug-resistant bacterial pathogens because of their high contribution to the increase in crude mortality and significant clinical challenge. Pathogenic Stenotrophomonas species and most clinical isolates belong to the Stenotrophomonas maltophilia complex (SMc). However, a strain highly homologous to S. terrae was isolated from a patient with pulmonary tuberculosis (TB), which aroused our interest, as S. terrae belongs to a relatively distant clade from SMc and there have been no human association reports.MethodsThe pathogenicity, immunological and biochemical characteristics of 610A2T were systematically evaluated.Results610A2T is a new species of genus Stenotrophomonas, which is named as Stenotrophomonas pigmentata sp. nov. for its obvious brown water-soluble pigment. 610A2T is pathogenic and caused significant weight loss, pulmonary congestion, and blood transmission in mice because it has multiple virulence factors, haemolysis, and strong biofilm formation abilities. In addition, the cytokine response induced by this strain was similar to that observed in patients with TB, and the strain was resistant to half of the anti-TB drugs.ConclusionsThe pathogenicity of 610A2T may not be weaker than that of S. maltophilia. Its isolation extended the opportunistic pathogenic species to all 3 major clades of the genus Stenotrophomonas, indicating that the clinical importance of species of Stenotrophomonas other than S. maltophilia and potential risks to biological safety associated with the use of Stenotrophomonas require more attention.

BackgroundTuberculosis (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.MethodsThis 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.ResultsLineage 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.ConclusionThis 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.

IntroductionTuberculosis (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.MethodsIn 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.ResultsERA005f 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.ConclusionThe 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.

A multi-omic analysis of 11 Mycobacterium tuberculosis (Mtb) isolates with different drug resistance profiles leads to the discovery of Rv3094c, an enzyme capable of activating the clinically relevant Mtb drug, ethionamide.

ABSTRACT Tuberculosis (TB) is an important infectious disease suffered by many countries, including China. In this stage, accurate diagnosis and treatment are key measures for the prevention and control of TB. Stenotrophomonas maltophilia is a global emerging Gram-negative, multidrug-resistant (MDR) organism characterized by its high contribution to the increase in crude mortality rates. By single cell preparation and strain identification, we isolated S. maltophilia from stored cultures of Mycobacterium tuberculosis (Mtb). We found that S. maltophilia could not be removed from sputum by alkali treatment or inhibited by antibiotic mixture added to MGIT 960 indicator tubes. When co-cultured with Mtb on a Löwenstein-Jensen (L-J) slant, it could inhibit the growth of Mtb and liquefy the medium. More seriously, it was resistant to 10 of the 12 anti-TB drugs, including isoniazid and rifampin, and made the mixed samples display multidrug-resistant Mtb (MDR-TB) results in the drug sensitivity test, which might change a treatment regimen and increase disease burden. Following, we conducted a small-scale surveillance which showed that the isolation rate of S. maltophilia in TB patients was 6.74%, but these patients had no special characteristics and the presence of S. maltophilia was hidden. The effect of S. maltophilus on TB and its mechanism are unclear and require more attention. IMPORTANCE China is a high-burden country for tuberculosis (TB), multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB), and HIV-associated TB. Increasing the positive rate of culture and the accuracy of antibiotic susceptibility testing (AST) are important for diagnosis, treatment, and control of TB. In our study, we found that the isolation rate of Stenotrophomonas maltophilia in TB patients was not neglectable and that this bacterium affects the isolation and AST results of TB. Due to a lack of relevant research, the impact of S. maltophilia on the course and outcome of TB is unclear. However, the characteristics of S. maltophilia that increase disease mortality require attention. Therefore, in the clinical testing of TB, in addition to mycobacteria, it is recommended to increase the detection of co-infected bacteria and improve the awareness of TB clinicians of these bacteria.

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.

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.

Although members of the Mycobacterium tuberculosis complex (MTBC) exhibit high similarity, they are characterized by differences with respect to virulence, immune response, and transmissibility. To understand the virulence of these bacteria and identify potential novel therapeutic targets, we systemically investigated the total cell protein contents of virulent H37Rv, attenuated H37Ra, and avirulent M. bovis BCG vaccine strains at the log and stationary phases, based on tandem mass tag (TMT) quantitative proteomics. Data analysis revealed that we obtained deep-coverage protein identification and high quantification. Although 272 genetic variations were reported in H37Ra and H37Rv, they showed very little expression difference in log and stationary phase. Quantitative comparison revealed H37Ra and H37Rv had significantly dysregulation in log phase (227) compared with stationary phase (61). While BCG and H37Rv, and BCG and H37Ra showed notable differences in stationary phase (1171 and 1124) with respect to log phase (381 and 414). In the log phase, similar patterns of protein abundance were observed between H37Ra and BCG, whereas a more similar expression pattern was observed between H37Rv and H37Ra in the stationary phase. Bioinformatic analysis revealed that the upregulated proteins detected for H37Rv and H37Ra in log phase were virulence-related factors. In both log and stationary phases, the dysregulated proteins detected for BCG, which have also been identified as M. tuberculosis response proteins under dormancy conditions. We accordingly describe the proteomic profiles of H37Rv, H37Ra, and BCG, which we believe will potentially provide a better understanding of H37Rv pathogenesis, H37Ra attenuation, and BCG immuno protection.

ABSTRACT Our objective was to evaluate the performance of whole-genome sequencing (WGS) from early positive liquid cultures for predicting Mycobacterium tuberculosis complex (MTBC) drug resistance. Clinical isolates were obtained from tuberculosis patients at Shanghai Pulmonary Hospital (SPH). Antimicrobial susceptibility testing (AST) was performed, and WGS from early Bactec mycobacterial growth indicator tube (MGIT) 960-positive liquid cultures was performed to predict the drug resistance using the TB-Profiler informatics platform. A total of 182 clinical isolates were enrolled in this study. Using phenotypic AST as the gold standard, the overall sensitivity and specificity for WGS were, respectively, 97.1% (89.8 to 99.6%) and 90.4% (83.4 to 95.1%) for rifampin, 91.0% (82.4 to 96.3%) and 95.2% (89.1 to 98.4%) for isoniazid, 100.0% (89.4 to 100.0%) and 87.3% (80.8 to 92.1%) for ethambutol, 96.6% (88.3 to 99.6%) and 61.8% (52.6 to 70.4%) for streptomycin, 86.8% (71.9 to 95.6%) and 95.8% (91.2 to 98.5%) for moxifloxacin, 86.5% (71.2 to 91.5%) and 95.2% (90.3 to 98.0%) for ofloxacin, 100.0% (54.1 to 100.0%) and 67.6% (60.2 to 74.5%) for amikacin, 100.0% (63.1 to 100.0%) and 67.2% (59.7 to 74.2%) for kanamycin, 62.5% (24.5 to 91.5%) and 88.5% (82.8 to 92.8%) for ethionamide, 33.3% (4.3 to 77.7%) and 98.3% (95.1 to 99.7%) for para-aminosalicylic acid, and 0.0% (0.0 to 12.3%) and 100.0% (97.6 to 100.0%) for cycloserine. The concordances of WGS-based AST and phenotypic AST were as follows: rifampin (92.9%), isoniazid (93.4%), ethambutol (89.6%), streptomycin (73.1%), moxifloxacin (94.0%), ofloxacin (93.4%), amikacin (68.7%), kanamycin (68.7%), ethionamide (87.4%), para-aminosalicylic acid (96.2%) and cycloserine (84.6%). We conclude that WGS could be a promising approach to predict MTBC resistance from early positive liquid cultures. IMPORTANCE In this study, we used whole-genome sequencing (WGS) from early positive liquid (MGIT) cultures instead of solid cultures to predict drug resistance of 182 Mycobacterium tuberculosis complex (MTBC) clinical isolates to predict drug resistance using the TB-Profiler informatics platform. Our study indicates that WGS may be a promising method for predicting MTBC resistance using early positive liquid cultures.

Objectives: Pasiniazid is a chemical complex of isoniazid (INH) and para-aminosalicylic acid (PAS). The aim of this study was to explore the cross-resistance of INH, PAS and pasiniazid against INH-resistant Mycobacterium tuberculosis isolates in China. Methods: A Microplate alamarBlue® Assay was performed to determine the minimum inhibitory concentrations (MICs) of INH, PAS and pasiniazid against 109 INH-resistant M. tuberculosis isolates. A statistical analysis of the relationship between different genotypes, gene mutations, and INH, PAS or pasiniazid susceptibility was then performed. Results: Among the 109 INH-resistant isolates, 13 (11.9%) and 21 (19.3%) showed resistance to PAS and pasiniazid, respectively. Among the 13 PAS-resistant M. tuberculosis isolates, 11 remained susceptible to pasiniazid. Of 63 INH-resistant isolates harbouring mutations in katG, the inhA promoter or the oxyR–ahpC intergenic region, 52 remained susceptible to pasiniazid. Moreover, 11 of 13 pasiniazid-resistant isolates carried mutations in katG, the inhA promoter or the oxyR–ahpC intergenic region. Conclusion: Taken together, these results demonstrate that PAS resistance and mutations in thekatG gene, inhA promoter or oxyR–ahpC intergenic region in INH-resistant M. tuberculosis have little effect on pasiniazid susceptibility.

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.

ABSTRACT Mycobacterium kansasii, an important opportunistic pathogen of humans, causes serious pulmonary disease. Sixty M. kansasii isolates were collected for investigating the clinical characteristics of patients with M. kansasii infections as well as drug susceptibility and genotypes of M. kansasii. More than 90% of the patients infected with M. kansasii were from eastern China. According to the internal transcribed spacers (ITS), rpoB, hsp65, and tuf, all M. kansasii isolates were classified as molecular type I, irrespective of the disease manifestation. Sixty M. kansasii isolates from China were diverse and separated into four branches. Pairwise average nucleotide identity (ANI) values for M. kansasii isolates affiliated with different genotypes were more than 85%. The earliest isolate was isolated from Jiangsu in 1983. Of the isolates, 78.3% (47/60) were isolated since 1999. All isolates were sensitive to rifabutin. All but one isolate was sensitive to clarithromycin. Sensitivity rates to rifampin, amikacin, moxifloxacin, and linezolid were 80.0%, 90.0%, 88.3%, and 91.7%, respectively. A high rate of resistance was noted for ciprofloxacin (44 isolates, 73.3%) and ethambutol (46 isolates, 76.7%). Compared with M. tuberculosis H37Rv, 12 mutations of embCA were observed in all M. kansasii isolates. All these 60 M. kansasii isolates shared identical sequences of rpoB, inhA, katG, rrl, rrs, rpsL, gyrA, and gyrB. In conclusion, M. kansasii isolates are exhibiting greater genetic diversity globally. The resistance mechanism of M. kansasii is not necessarily related to gene mutation. IMPORTANCE M. kansasii type I is the main genotype spreading worldwide. The molecular history of the global spread of type I isolates remains largely unclear. We conducted a detailed analysis of genomic evolution of global M. kansasii isolates. Our results suggest that M. kansasii isolates exhibit greater genetic diversity globally.

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.

Development of modern genomics provides us an effective method to understand the molecular mechanism of drug resistance and diagnose drug-resistant Mycobacterium tuberculosis. In this study, mutations in 18 genes or intergenic regions acquired by whole-genome sequencing (WGS) of 183 clinical M. tuberculosis strains, including 137 multidrug-resistant and 46 pan-susceptible isolates from China, were identified and used to analyze their associations with resistance of isoniazid, rifampin, ethambutol, and streptomycin. Using the proportional method as the gold standard method, the accuracy values of WGS to predict resistance were calculated. The association between synonymous or lineage definition mutations with different genotypes were also analyzed. The results show that, compared to the phenotypic proportional method, the sensitivity and specificity of WGS for resistance detection were 94.2 and 100.0% for rifampicin (based on mutations in rpoB), 90.5 and 97.8% for isoniazid (katG), 83.0 and 97.8% for streptomycin (rpsL combined with rrs 530 loop and 912 loop), and 90.9 and 65.1% for ethambutol (embB), respectively. WGS data also showed that mutations in the inhA promoter increased only 2.2% sensitivity for INH based on mutations in katG. Synonymous mutation rpoB A1075A was confirmed to be associated with the Beijing genotype. This study confirmed that mutations in rpoB, katG, rrs 530 loop and 912 loop, and rpsL were excellent biomarkers for predicting rifampicin, isoniazid, and streptomycin resistance, respectively, and provided clues in clarifying the drug-resistance mechanism of M. tuberculosis isolates from China.

Background:Klebsiella pneumoniae is an opportunistic pathogen associated with community-acquired and nosocomial infections. Since 2010, K. pneumoniae testing has been included into an existing diarrhea-syndrome surveillance system for estimating the prevalence of K. pneumoniae in diarrhea-syndrome patients, assessing antibiotic susceptibility, and investigating molecular characteristics of K. pneumoniae.Methods:Klebsiella pneumoniae strains were isolated from stool specimens from diarrhea-syndrome outpatients in Beijing, China. Isolates were tested for antibiotic susceptibility, and phylogenetic relationships were explored though whole genome sequence analysis. Multi-locus sequence type (MLST) alleles were extracted from the whole genome sequence (WGS) data. A maximum likelihood tree was generated by MEGAX. Genomes were annotated by Prokka; core genes were produced by Roary; a maximum likelihood phylogenetic tree was generated using FastTree.Results: Forty-four K. pnuemoniae strains were isolated from 2010 to July 2019; of these 37 were K. pneumoniae and seven were K. variicola. Antibiotic susceptibility testing showed that all 44 strains were sensitive to gentamicin, imipenem, amikacin, meropenem, kanamycin; 97.73% were sensitive to cefoxitin andlavo-ofloxacin; the highest antibiotic resistance rate was 79.55%, which was to ampicillin. We found three extended-spectrum beta-lactamase (ESBL) producing strains; we identified high-virulence ST types, including ST307 and ST65; and we found that ST23 has been the epidemic clone since 2010. MLST and core genome sequence analysis showed two distinct clusters of 44 K. pnuemoniae; 40 alleles were identified in core genome sequence analysis, while 36 alleles were identified in MLST typing.Conclusions: There is an urgent need for epidemiological and molecular studies to understand the dynamics of antibiotic resistance and virulence gene transmission to guide strategies for K. pneumoniae surveillance. WGS analysis provided high discrimination power and reliable and robust data useful for molecular epidemiology.

There is an urgent need for new immunodominant antigens to improve the diagnosis of tuberculosis (TB) and the efficacy of the TB vaccine to control the disease worldwide. In this study, we evaluated the diagnostic potential of a novel Mycobacterium tuberculosis (MTB)-specific antigen, Rv2351c, from region of difference (RD) 7 of the MTB genome, and investigated the potency of the vaccine by identifying its immunological function in human and animal immunological experiments. Twenty T-cell epitopes were identified using TEpredict and prediction tools from the Immune Epitope Database and Analysis Resource. A total of 159 subjects, including 61 patients with pulmonary TB, 38 patients with no TB and 55 healthy donors, were recruited and analyzed with an enzyme-linked immunospot (ELISpot) assay. The ELISpot assay using Rv2351c to detect TB infection, as compared with bacteriological tests as the gold standard, had a sensitivity and specificity of 61.4% (35/57) and 91.4% (85/93), respectively. The ELISpot assay using Rv2351c had a good conformance (κ=0.554) as compared with the bacteriological test. Rv2351c also elicited a potent cellular immune response with a high expression of cytokines (IFN-γ (4978±596.7 μg/mL) and IL-4 (68.3±15.5 μg/mL)) and a potent humoral immune response with a high concentration of IgG (1:2.2 × 106), IgG1 (1:4.5 × 105) and IgG2a (1:1.6 × 106) in immunized BALB/c mice. In addition, the ratio of IgG2a/IgG1 indicated that Rv2351c induced cellular immunity in the mice. The results of this study indicated that Rv2351c is an antigen with good immunogenicity that may potentially be used to develop diagnostic techniques and new TB vaccines.Emerging Microbes & Infections (2017) 6, e48; doi:10.1038/emi.2017.34; published online 7 June 2017

Objective: To investigate the drug resistance characteristics of Mycobacterium tuberculosis (MTB) isolates from patients with tuberculosis to 12 antituberculous drugs in China.Methods: All clinical isolates of MTB were isolated from patients with tuberculosis in Shanghai Pulmonary Hospital (SPH) during the period from January 1st to December 31th, 2018. Drug susceptibility testing (DST) was performed in micro plates with 12 antituberculous drugs in accordance with relevant guideline. Demographic information, including sex, age, and treatment history was recorded.Results: A total of 1,950 MTB isolates were included in this retrospective study which were isolated from 1,950 patients from 29 regions in China. One thousand six hundred and forty-four were initial treated and 306 were re-treated in the hospital. Two hundred and eight (10.67%, 208/1,950) cases were diagnosed as multidrug-resistant tuberculosis (MDR-TB), from which 74 (4.50%, 74/1,644) cases were initial treated, and the remaining (43.79%, 134/306) were re-treated cases. Besides, the percentage of extensively drug-resistant tuberculosis (XDR-TB) varied in such 3 different groups: 1.64% (32/1,950) in total cases, 0.30% (5/1,644) in initial treated cases and 8.82% (27/306) in re-treated cases. The total resistance rates were as follows: isoniazid (361, 18.51%), streptomycin (302, 15.49%), rifampin (241, 12.36%), ofloxacin (239, 12.26%), moxifloxacin (232, 11.90%), rifabutin (195, 10.00%), ethambutol (100, 5.13%), cycloserine (55, 2.82%), kanamycin (48, 2.46%), ethionamide (40, 2.05%), amikacin (39, 2.00%), and aminosalicylic acid (21, 1.08%). Rates of resistance to any drug in re-treated cases were significantly higher than in initial treated cases. The drug resistance rates of the 12 drugs were higher in males than in females. Patients older than 60 years had significantly lower percentages of MDR/XDR-TB (7.11 and 0.65%) than in younger age groups. The proportion of re-treated cases in Shanghai (11.38%, 88/773) was lower than that in other regions. Meanwhile, the percentages of MDR/XDR-TB in Shanghai (4.79 and 0.65%) were significantly lower than in other regions.Conclusions: In this study, we found higher proportion of MDR/XDR-TB among re-treated cases than initial treated cases in China and the drug resistance rate of tuberculosis varied with age, sex, and region, indicating that standardized anti-tuberculosis treatment can reduce the incidence of drug-resistant tuberculosis and the recurrence of tuberculosis.

BackgroundTuberculosis (TB) and non-tuberculous mycobacteriosis are serious threats to health worldwide. A simple non-sequencing method is needed for rapid diagnosis, especially in less experienced hospitals, but there is no specific biomarker commonly used for all mycobacteria. The ku gene of the prokaryotic error-prone non-homologous end joining system (NHEJ) has the potential to be a highly specific detection biomarker for mycobacteria.MethodsA total of 7294 mycobacterial genomes and 14 complete genomes of other families belonging to Corynebacteriales with Mycobacteriaceae were downloaded and analyzed for the existence and variation of the ku gene. Mycobacterium tuberculosis complex (MTBC) and non-tuberculosis mycobacteria (NTM)- specific primers were designed and the actual amplification and identification efficiencies were tested with 150 strains of 40 Mycobacterium species and 10 kinds of common respiratory pathogenic bacteria.ResultsThe ku gene of the NHEJ system was ubiquitous in all genome sequenced Mycobacterium species and absent in other families of Corynebacteriales. On the one hand, as a single gene non-sequencing biomarker, its specific primers could effectively distinguish mycobacteria from other bacteria, MTBC from NTM, which would make the clinical detection of mycobacteria easy and have great clinical practical value. On the other hand, the sequence of ku gene can effectively distinguish NTM to species level with high resolution.ConclusionThe Ku protein existed before the differentiation of Mycobacterium species, which was an important protein involved in maintaining of the genome’s integrity and related to the special growth stage of mycobacteria. It was rare in prokaryotes. These features made it a highly special differential biomarker for Mycobacterium.

The complete genome of methanol-utilizing Amycolatopsis methanolica strain 239T was generated, revealing a single 7,237,391 nucleotide circular chromosome with 7074 annotated protein-coding sequences (CDSs). Comparative analyses against the complete genome sequences of Amycolatopsis japonica strain MG417-CF17T, Amycolatopsis mediterranei strain U32 and Amycolatopsis orientalis strain HCCB10007 revealed a broad spectrum of genomic structures, including various genome sizes, core/quasi-core/non-core configurations and different kinds of episomes. Although polyketide synthase gene clusters were absent from the A. methanolica genome, 12 gene clusters related to the biosynthesis of other specialized (secondary) metabolites were identified. Complete pathways attributable to the facultative methylotrophic physiology of A. methanolica strain 239T, including both the mdo/mscR encoded methanol oxidation and the hps/hpi encoded formaldehyde assimilation via the ribulose monophosphate cycle, were identified together with evidence that the latter might be the result of horizontal gene transfer. Phylogenetic analyses based on 16S rDNA or orthologues of AMETH_3452, a novel actinobacterial class-specific conserved gene against 62 or 18 Amycolatopsis type strains, respectively, revealed three major phyletic lineages, namely the mesophilic or moderately thermophilic A. orientalis subclade (AOS), the mesophilic Amycolatopsis taiwanensis subclade (ATS) and the thermophilic A. methanolica subclade (AMS). The distinct growth temperatures of members of the subclades correlated with corresponding genetic variations in their encoded compatible solutes. This study shows the value of integrating conventional taxonomic with whole genome sequence data.

Objective: To evaluate the performance of three interferon-gamma release assay (IGRA) kits in detecting Mycobacterium tuberculosis infection in China. Methods: A multicenter clinical trial was used to compare the effectiveness and application of the three kits. A total of 1026 participants were enrolled at three hospitals, including 597 tuberculosis (TB) patients diagnosed clinically (517 patients with pulmonary TB and 80 patients with extrapulmonary TB) and 429 negative controls (244 patients with pulmonary disease but not TB, or with non-tuberculosis mycobacterial lung diseases, and 185 healthy people). Detection performance indicators including sensitivity, specificity, and the Youden index (YI) were used to evaluate performance. Results: Through bacterial culture evaluation, 224 of the 517 pulmonary TB patients were positive and all 429 negative controls were negative. When the gold standard bacterial methods were used, the sensitivity, specificity, and YI were 89.7% (201/224), 91.1% (391/429), and 0.81 for T-SPOT.TB, 86.2% (193/224), 87.2% (374/429), and 0.73 for QB-SPOT, and 83.9% (188/224), 88.6% (380/429), and 0.73 for TB-IGRA, respectively. There were no significant differences in the sensitivity and specificity of the three kits. Conclusions: The results showed that the three kits had very high sensitivity and specificity and exhibited a good performance for the detection of M. tuberculosis infection.

[This corrects the article DOI: 10.1371/journal.pone.0143444.].

Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a "probe dropout" manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

BACKGROUND: Investigations of Mycobacterium tuberculosis genetic diversity in China have indicated a significant regional distribution. The aim of this study was to characterize the genotypes of clinical M. tuberculosis isolates obtained from Gansu, which has a special geographic location in China. METHODOLOGY/PRINCIPAL FINDINGS: A total of 467 clinical M. tuberculosis strains isolated in Gansu Province were genotyped by 15-locus mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) and spoligotyping. The results showed that 445 isolates belonged to six known spoligotype lineages, whereas 22 isolates were unknown. The Beijing genotype was the most prevalent (87.58%, n = 409), while the shared type 1 was the dominant genotype (80.94%, n = 378). The second most common lineage was the T lineage, with 25 isolates (5.35%), followed by the H lineage with 5 isolates (1.07%), the MANU family (0.64%, 3 isolates), the U family (0.43%, 2 isolates) and the CAS lineage with 1 isolate (0.21%). By using the VNTR15China method, we observed 15 groups and 228 genotypes among the 467 isolates. We found no association between the five larger groups (including the Beijing genotype) and sex, age, or treatment status, and there was no noticeable difference in the group analysis in different areas. In the present study, seven of the 15 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index. CONCLUSIONS/SIGNIFICANCE: The Beijing genotype is the predominant genotype in Gansu province. We confirm that VNTR15China is suitable for typing Beijing strains in China and that it has a better discriminatory power than spoligotyping. Therefore, the use of both methods is the most suitable for genotyping analysis of M. tuberculosis.

BACKGROUND: Although the Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis (TB) has been available for more than 75 years, one third of the world's population is still infected with Mycobacterium tuberculosis and approximately 2 million people die of TB every year. To reduce this immense TB burden, a clearer understanding of the functional genes underlying the action of BCG and the development of new vaccines are urgently needed. METHODS AND FINDINGS: Comparative genomic analysis of 19 M. tuberculosis complex strains showed that BCG strains underwent repeated human manipulation, had higher region of deletion rates than those of natural M. tuberculosis strains, and lost several essential components such as T-cell epitopes. A total of 188 BCG strain T-cell epitopes were lost to various degrees. The non-virulent BCG Tokyo strain, which has the largest number of T-cell epitopes (359), lost 124. Here we propose that BCG strain protection variability results from different epitopes. This study is the first to present BCG as a model organism for genetics research. BCG strains have a very well-documented history and now detailed genome information. Genome comparison revealed the selection process of BCG strains under human manipulation (1908-1966). CONCLUSIONS: Our results revealed the cause of BCG vaccine strain protection variability at the genome level and supported the hypothesis that the restoration of lost BCG Tokyo epitopes is a useful future vaccine development strategy. Furthermore, these detailed BCG vaccine genome investigation results will be useful in microbial genetics, microbial engineering and other research fields.

In this study, 24 standard nontuberculous mycobacteria (NTM) species strains including 12 slowly growing mycobacteria strains and 12 rapidly growing mycobacteria strains were subjected to drug susceptibility testing using microplate Alamar Blue assay-based 7H9 broth. The most active antimicrobial agents against the 24 NTM strains were streptomycin, amikacin, the fluoroquinolones, and the tetracyclines. Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium simiae are resistant to most antimicrobial agents. The susceptibility results of this study from 24 NTM standard strains can be referenced by clinicians before susceptibility testing for clinical isolates is performed or when conditions do not allow for susceptibility testing. The application of broth-based methods is recommended by the Clinical and Laboratory Standards Institute, and the documentation of the susceptibility patterns of standard strains of mycobacteria can improve the international standardization of susceptibility testing methods.

Streptococcussuis is an important zoonotic agent causing severe diseases in pigs and humans. To date, 33 serotypes of S. suis have been identified based on antigenic differences in the capsular polysaccharide. The capsular polysaccharide synthesis (cps) locus encodes proteins/enzymes that are responsible for capsular production and variation in the capsule structures are the basis of S. suis serotyping. Multiplex and/or simplex PCR assays have been developed for 15 serotypes based on serotype-specific genes in the cps gene cluster. In this study, we developed a set of multiplex PCR (mPCR) assays to identify the 33 currently known S. suis serotypes. To identify serotype-specific genes for mPCR, the entire genomes of reference strains for the 33 serotypes were sequenced using whole genome high-throughput sequencing, and the cps gene clusters from these strains were identified and compared. We developed a set of 4 mPCR assays based on the polysaccharide polymerase gene wzy, one of the serotype-specific genes. The assays can identify all serotypes except for two pairs of serotypes: 1 and 14, and 2 and 1/2, which have no serotype-specific genes between them. The first assay identifies 12 serotypes (serotypes 1 to 10, 1/2, and 14) that are the most frequently isolated from diseased pigs and patients; the second identifies 10 serotypes (serotypes 11 to 21 except 14); the third identifies the remaining 11 serotypes (serotypes 22 to 31, and 33); and the fourth identifies a new cps cluster of S. suis discovered in this study in 16 isolates that agglutinated with antisera for serotypes 29 and 21. The multiplex PCR assays developed in this study provide a rapid and specific method for molecular serotyping of S. suis.