Bactericidal and sterilizing activity of sudapyridine-clofazimine-TB47 combined with linezolid or pyrazinamide in a murine model of tuberculosis.

As an obligate aerobe, Mycobacterium tuberculosis relies on its branched electron transport chain (ETC) for energy production through oxidative phosphorylation. Regimens targeting ETC exhibit promising potential to enhance bactericidal activity against M. tuberculosis and hold the prospect of shortening treatment duration. Our previous research demonstrated that the bacteriostatic drug candidate TB47 (T) inhibited the growth of M. tuberculosis by targeting the cytochrome bc1 complex and exhibited synergistic activity with clofazimine (C). Here, we found synergistic activities between C and sudapyridine (S), a structural analog of bedaquiline (B). S has shown similar anti-tuberculosis efficacy and may share a mechanism of action with B, which inhibits ATP synthesis and the energy metabolism of bacteria. We evaluated the efficacy of SCT in combination with linezolid (L) or pyrazinamide (Z) using a well-established murine model of tuberculosis. Compared to the BPa(pretomanid)L regimen, SCT and SCTL demonstrated similar bactericidal and sterilizing activities. There was no significant difference in activity between SCT and SCTL. In contrast, SCZ and SCTZ showed much higher activities, with none of the 15 mice experiencing relapse after 2 months of treatment with either SCZ or SCTZ. However, T did not contribute to the activity of the SCZ. Our findings emphasize the efficacy and the potential clinical significance of combination therapy with ETC inhibitors. Additionally, cross-resistance exists not only between S and B but also between S/B and C. This is supported by our findings, as spontaneous S-resistant mutants exhibited mutations in Rv0678, which are associated with cross-resistance to B and C.

From phenotyping to genetic mapping: identifying water-stress adaptations in legume root traits.

BACKGROUND: Climate change induces perturbation in the global water cycle, profoundly impacting water availability for agriculture and therefore global food security. Water stress encompasses both drought (i.e. water scarcity) that causes the drying of soil and subsequent plant desiccation, and flooding, which results in excess soil water and hypoxia for plant roots. Terrestrial plants have evolved diverse mechanisms to cope with soil water stress, with the root system serving as the first line of defense. The responses of roots to water stress can involve both structural and physiological changes, and their plasticity is a vital feature of these adaptations. Genetic methodologies have been extensively employed to identify numerous genetic loci linked to water stress-responsive root traits. This knowledge is immensely important for developing crops with optimal root systems that enhance yield and guarantee food security under water stress conditions. RESULTS: This review focused on the latest insights into modifications in the root system architecture and anatomical features of legume roots in response to drought and flooding stresses. Special attention was given to recent breakthroughs in understanding the genetic underpinnings of legume root development under water stress. The review also described various root phenotyping techniques and examples of their applications in different legume species. Finally, the prevailing challenges and prospective research avenues in this dynamic field as well as the potential for using root system architecture as a breeding target are discussed. CONCLUSIONS: This review integrated the latest knowledge of the genetic components governing the adaptability of legume roots to water stress, providing a reference for using root traits as the new crop breeding targets.

Synthesis and biological evaluation of 2-(Tetrazol-5-yl)sulfonylacetamides as inhibitors of Mycobacterium tuberculosis and Mycobacterium marinum.

A series of 2-(tetrazol-5-yl)sulfonylacetamide derivatives were synthesized and evaluated for their in vitro inhibitory activity against Mycobacterium tuberculosis (Mtb) and Mycobacterium marinum (Mm). The most active compounds exhibited in vitro MIC90 values of 1.25 µg/mL against Mtb, but they were less effective against Mm (MIC90 ≥ 10 µg/mL). Despite the most active compounds having favourable physicochemical properties and one of them having a half-life of ∼3 h when incubated with mouse liver microsomes, two representative highly active compounds showed strong chemical reactivity to cysteine derivatives, as surrogate in vivo sulfur-centred nucleophiles, indicating excessive electrophilicity, and therefore, likely indiscriminate chemical reactivity in vivo, representing an unacceptably high risk of general toxicity, and low likelihood of being therapeutically effective.

Genotypic variation in the tolerance to moderate cadmium toxicity among 20 maize genotypes with contrasting root systems.

BACKGROUND: Cadmium (Cd) contamination in farmland is a serious environmental and safety issue affecting plant growth, crop productivity, and human health. This study aimed to investigate genotypic variation in root morphology and Cd accumulations under moderate Cd stress among diverse maize genotypes. Twenty maize genotypes with contrasting root systems were assessed for Cd tolerance 39 days after transplanting (V6, six-leaf stage) under 20 µmol L-1 CdCl2 using a semi-hydroponic phenotyping platform in a glasshouse. RESULTS: Cadmium stress significantly inhibited plant growth across all genotypes. Genotypic variation in response to Cd toxicity was apparent: shoot dry weight varied from 0.13 (genotype NS2020) to 0.35 g plant-1 (Dongke301) with deductions up to 63% compared with non-Cd treatment (CK). Root dry weight of 20 genotypes ranged from 0.06 (NS2020) to 0.18 g plant-1 (Dongke301) with a deduction up to 56%. Root length ranged from 2.21 (NS590b) to 9.22 m (Dongke301) with a maximal decline of 76%. Cadmium-treated genotypes generally had thicker roots and average diameter increased by 34% compared with CK. Genotypes had up to 3.25 and 3.50 times differences in shoot and root Cd concentrations, respectively. Principal component and cluster analyses assigned the 20 genotypes into Cd-tolerant (five genotypes) and Cd-sensitive (15 genotypes) groups. CONCLUSIONS: Maize genotypes varied significantly in response to moderate Cd stress. Cadmium-tolerant genotypes optimized root morphology and Cd accumulation and distribution. This study could assist in the selection and breeding of new cultivars with improved adaptation to Cd-contaminated soil for food and feed or land remediation purposes. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Transcriptomic and Metabolomic Analyses of the Effects of Exogenous Trehalose on Heat Tolerance in Wheat.

Trehalose can improve the tolerance of plants to various types of environmental stress. Nonetheless, information respecting the molecular networks of wheat seedlings to exogenous trehalose under heat stress is limited. Here, two wheat varieties pretreated with exogenous trehalose were selected to explore the molecular mechanism by which trehalose improves the heat tolerance of wheat (Triticum aestivum L.). The results indicated that exogenous trehalose improved the physiological state of wheat seedlings under heat stress. Through RNA sequencing and metabolomics analysis, the genes and metabolites specifically expressed in trehalose pretreatment were identified. After heat stress, there were 18,352 differentially expressed genes (DEGs) in the control and trehalose-treated (H_vs_TreH) groups of Yangmai 18 and 9045 DEGs in Yannong 19. Functional annotation and enrichment analyses showed that the DEGs in the two wheat varieties were mainly involved in carbohydrate metabolism and biosynthesis of secondary metabolites. Through a liquid chromatography-mass spectrometry platform, 183 differential metabolites in H_vs_TreH groups of Yangmai 18 and 77 differential metabolites in Yannong 19 were identified. Compared with the control group, many protective metabolites, such as amino acids, purines, phenylpropanoids and flavonoids, showed significant differences under heat stress. The results indicated that exogenous trehalose protected the wheat biomembrane system, enhanced carbohydrate metabolism and signal transduction, strengthened the activity of the tricarboxylic acid cycle (TCA cycle), regulated purine metabolism, gene expression and metabolite accumulation in the phenylpropanoid biosynthesis and flavonoid biosynthesis pathways, thus improving the heat tolerance of wheat.

Discovery of meta-Amido Bromophenols as New Antitubercular Agents.

A series of meta-amido bromophenol derivatives were designed and synthesized. The compounds were found to potently inhibit the growth of Mycobacterium tuberculosis H37Ra. They also exhibited moderate inhibitory activity against Mycobacterium tuberculosis H37Rv and multidrug-resistant strains. The compounds did not show inhibitory activity against normal Gram-positive and Gram-negative bacteria. Moderate cytotoxicities and good metabolic stability were observed for the selected compounds. The results demonstrated meta-amido bromophenols as a new class of antitubercular agents with good potentials.

Design, synthesis, and antitubercular activity of 3-amidophenols with 5-heteroatomic substitutions.

A series of novel 3-amidophenols with 5-heteroatomic substitutions were designed and synthesized. Several compounds showed potent antitubercular activity against Mycobacterium tuberculosis H37Ra (MIC = 0.25-5 µg/mL). Compounds 12j and 14i also displayed good inhibitory activity against M. tuberculosis H37Rv and two clinically isolated multidrug-resistant M. tuberculosis strains (MIC = 0.39-3.12 µg/mL). The privileged compound 14i showed certain oral efficacy on a mouse infection model. The compounds are non-cytotoxic against L-O2 hepatocytes and RAW264.7 macrophagocytes. They did not exert inhibitory activity against representative Gram-positive and Gram-negative bacteria.

A novel TNKS/USP25 inhibitor blocks the Wnt pathway to overcome multi-drug resistance in TNKS-overexpressing colorectal cancer.

Modulating Tankyrases (TNKS), interactions with USP25 to promote TNKS degradation, rather than inhibiting their enzymatic activities, is emerging as an alternative/specific approach to inhibit the Wnt/ß-catenin pathway. Here, we identified UAT-B, a novel neoantimycin analog isolated from Streptomyces conglobatus, as a small-molecule inhibitor of TNKS-USP25 protein-protein interaction (PPI) to overcome multi-drug resistance in colorectal cancer (CRC). The disruption of TNKS-USP25 complex formation by UAT-B led to a significant decrease in TNKS levels, triggering cell apoptosis through modulation of the Wnt/ß-catenin pathway. Importantly, UAT-B successfully inhibited the CRC cells growth that harbored high TNKS levels, as demonstrated in various in vitro and in vivo studies utilizing cell line-based and patient-derived xenografts, as well as APCmin/+ spontaneous CRC models. Collectively, these findings suggest that targeting the TNKS-USP25 PPI using a small-molecule inhibitor represents a compelling therapeutic strategy for CRC treatment, and UAT-B emerges as a promising candidate for further preclinical and clinical investigations.

Indole Propionic Acid Disturbs the Normal Function of Tryptophanyl-tRNA Synthetase in Mycobacterium tuberculosis.

Tuberculosis (TB) is the leading infectious disease caused by Mycobacterium tuberculosis and the second-most contagious killer after COVID-19. The emergence of drug-resistant TB has caused a great need to identify and develop new anti-TB drugs with novel targets. Indole propionic acid (IPA), a structural analog of tryptophan (Trp), is active against M. tuberculosis in vitro and in vivo. It has been verified that IPA exerts its antimicrobial effect by mimicking Trp as an allosteric inhibitor of TrpE, which is the first enzyme in the Trp synthesis pathway of M. tuberculosis. However, other Trp structural analogs, such as indolmycin, also target tryptophanyl-tRNA synthetase (TrpRS), which has two functions in bacteria: synthesis of tryptophanyl-AMP by catalyzing ATP + Trp and producing Trp-tRNATrp by transferring Trp to tRNATrp. So, we speculate that IPA may also target TrpRS. In this study, we found that IPA can dock into the Trp binding pocket of M. tuberculosis TrpRS (TrpRSMtb), which was further confirmed by isothermal titration calorimetry (ITC) assay. The biochemical analysis proved that TrpRS can catalyze the reaction between IPA and ATP to generate pyrophosphate (PPi) without Trp as a substrate. Overexpression of wild-type trpS in M. tuberculosis increased the MIC of IPA to 32-fold, and knock-down trpS in Mycolicibacterium smegmatis made it more sensitive to IPA. The supplementation of Trp in the medium abrogated the inhibition of M. tuberculosis by IPA. We demonstrated that IPA can interfere with the function of TrpRS by mimicking Trp, thereby impeding protein synthesis and exerting its anti-TB effect.

Investigating the role of MAB_1915 in intrinsic resistance to multiple drugs in Mycobacterium abscessus.

The increasing clinical significance of Mycobacterium abscessus is owed to its innate high-level, broad-spectrum resistance to antibiotics and therefore rapidly evolves as an important human pathogen. This warrants the identification of novel targets for aiding the discovery of new drugs or drug combinations to treat M. abscessus infections. This study is inspired by the drug-hypersensitive profile of a mutant M. abscessus (U14) with transposon insertion in MAB_1915. We validated the role of MAB_1915 in intrinsic drug resistance in M. abscessus by constructing a selectable marker-free in-frame deletion in MAB_1915 and complementing the mutant with the same or extended version of the gene and then followed by drug susceptibility testing. Judging by the putative function of MAB_1915, cell envelope permeability was studied by ethidium bromide accumulation assay and susceptibility testing against dyes and detergents. In this study, we established genetic evidence of the role of MAB_1915 in intrinsic resistance to rifampicin, rifabutin, linezolid, clarithromycin, vancomycin, and bedaquiline. Disruption of MAB_1915 has also been observed to cause a significant increase in cell envelope permeability in M. abscessus. Restoration of resistance is observed to depend on at least 27 base pairs upstream of the coding DNA sequence of MAB_1915. MAB_1915 could therefore be associated with cell envelope permeability, and hence its role in intrinsic resistance to multiple drugs in M. abscessus, which presents it as a novel target for future development of effective antimicrobials to overcome intrinsic drug resistance in M. abscessus. IMPORTANCE: This study reports the role of a putative fadD (MAB_1915) in innate resistance to multiple drugs by M. abscessus, hence identifying MAB_1915 as a valuable target and providing a baseline for further mechanistic studies and development of effective antimicrobials to check the high level of intrinsic resistance in this pathogen.

Additive screening and formula optimization of microbial inhibitor having disease prevention and growth promotion effects on Avena sativa.

In order to develop environment friendly microbial inhibitor that can also control disease and promote oat (Avena sativa) growth, the growth rate method and response surface methodology were used to screen wetting agents, preservatives and protective agents at optimal concentrations in this study. Antagonistic activity of the tested bacterium and cell-free fermentation liquid against pathogenic fungi was evaluated on potato dextrose agar (PDA) substratum plates by dual culture technique. Oxford cup method was used to measure antagonistic reaction between screened bacteria. According to each screened bacteria with 50 mL were mixed and cultured in Luria-bertani (LB) substratum. Additives of Wetting agents, UV-protectors, and preservatives were screened by single factor test on the growth concentration of screened mixed bacteria. Afterwards, the optimal additives and concentrations were screened by Box-Behnken method. The microbial inhibitor was detected according to national standards GB20287-2006 and tested on oat in a pot experiment. The results showed that: (1) Functional bacteria which including Bacillus velezensis and Brevundimonas faecalis had control effects of 50.00% to 83.29% on three pathogenic fungi, and their cell free-fermentation liquid could inhibit the growth of pathogenic fungi from 23.51% to 39.90%; (2) Tween-80 was most suitable as wetting agents for Mix biocontrol bacteria (MBB) with 1.00% mass fraction; Sorbitol was selected as UV protective agents for MBB with 0.50% mass fraction. And methyl paraben was used as a preservative for MBB, with 0.50% mass fraction; (3) The most effective adjuvant contained 14.96 mL/L Tween-80, 5.12 g/L methylparaben and 5.6 g/L sorbitol; and (4) The microbial inhibitor controlled 45.57% of oat root rot and increased plant height, root length and seedling biomass. This study provides a suitable environment for the protection of mixed biocontrol bacteria, and lays a foundation for the prevention and control of oat diseases, the promotion of growth and the improvement of quality.

Variability in cadmium stress tolerance among four maize genotypes: Impacts on plant physiology, root morphology, and chloroplast microstructure.

Cadmium (Cd) is detrimental to both plants and humans. Maize (Zea mays L.) genotypes exhibit variations in Cd accumulations. This study examined variations in Cd accumulation and tolerance among four maize genotypes with contrasting root morphology. The four maize genotypes were cultivated in a semi-hydroponic system with three Cd concentrations (0, 10, 20 µmol L-1). The effects of Cd on plant growth and physiology were assessed 39 days after transplanting. Results showed that root characteristics were positively correlated with root Cd accumulation and the bioconcentration factor under Cd20 treatment. Genotypes Shengrui999 and Zhengdan958 exhibited higher total Cd content than Xundan29 and Zhongke11 under Cd20 conditions. Cd toxicity led to membrane degradation of chloroplast mesophyll cells, loosening and swelling of grana lamella, and reduced starch reserves. The greater tolerance of Shengrui999 and Zhengdan958 was contributed to factors such as root biomass, shallower root depth, higher Cd content, accumulation of osmolyte such as soluble protein, antioxidant activities such as catalase (CAT), and the presence of phytohormone gibberellic acid. The study establishes a link between root morphology, Cd accumulation, and tolerance in maize plants, as demonstrated by the higher Cd accumulation and shallower root system in Cd-tolerant genotypes. This research provides a foundation for breeding maize cultivars better suited for adaptation to moderate Cd-contaminated environments.

The role of arbuscular mycorrhizal fungi in the alleviation of cadmium stress in cereals: A multilevel meta-analysis.

The symbiotic relationships between crop species and arbuscular mycorrhizal fungi (AMF) are crucial for plant health, productivity, and environmental sustainability. The roles of AMF in reducing crop stress caused by cadmium (Cd) toxicity and in the remediation of Cd-contaminated soil are not fully understood. Here we report on a meta-analysis that sought to identify the functions of AMF in cereals under Cd stress. A total of 54 articles published between January 1992 and September 2022 were used to create the dataset, which provided 7216 data sets on mycorrhizal cereals under Cd stress examined. AMF effects on colonization rate, biomass, physiological level, nutritional level, and plant Cd level were measured using the logarithmic response ratio (Ln R). The results showed that AMF overall greatly reduced 5.14 - 33.6 % Cd stress on cereals in greenhouse experiments under controlled conditions. AMF colonization significantly stimulated crop biomass by 65.7 %, boosted the formation of photosynthetic pigments (23.2 %), and greatly increased plant nitrogen (24.8 %) and phosphorus (58.4 %) uptake. The dilution effect of mycorrhizal plants made the Cd concentration decline by 25.2 % in AMF plants compared to non-mycorrhizal ones. AMF also alleviated Cd stress by improving osmotic regulators (soluble protein, sugar, and total proline, from 14.8 to 36.0 %) and lowering the membrane lipid peroxidation product (MDA, 12.9 %). Importantly, the results from the random forest and model selection analysis demonstrated that crop type, soil characteristics, chemical form, and Cd levels were the main factors determining the function of AMF in alleviating Cd stress. Additionally, there was a significant interaction between AMF colonization rate and Cd addition, but their interactive effect was less than the colonization rate alone. This meta-analysis demonstrated that AMF inoculation could be considered as a promising strategy for mitigation of Cd stress in cereals.

3-Methoxy-2-phenylimidazo[1,2-b]pyridazines highly active against Mycobacterium tuberculosis and Mycobacterium marinum.

A series of 3-methoxy-2-phenylimidazo[1,2-b]pyridazine derivatives which were highly active against autoluminescent Mycobacterium tuberculosis (Mtb) and Mycobacterium marinum (Mm) in an in vitro assay were identified. SAR analysis showed that the most active compounds, which included a phenyl group bearing fluoro substituent(s) at C2, a methoxy function at C3, and a benzyl-heteroatom moiety at C6, exhibited in vitro MIC90 values generally around 0.63-1.26 µM against Mtb and Mm. However, these compounds were inactive against Mtb in vivo (mice), and investigations revealed very short metabolic half-lives (<10 min) when incubated with mouse liver microsomes. Multiple observations of side products produced from oxidative cleavage of the imidazole moiety during the chemical synthesis work suggested that this is a likely metabolic pathway leading to the lack of observed activity in vivo.

Structure-Activity Relationship of Novel Pyrimidine Derivatives with Potent Inhibitory Activities against Mycobacterium tuberculosis.

Discovery of novel antitubercular drugs is an effective strategy against drug-resistant tuberculosis (TB). Our previous study has identified LPX-16j as a novel antitubercular compound. Herein, we perform a comprehensive structure-activity relationship (SAR) based on LPX-16j, indicating that the central pyrimidine ring moiety was crucial for the antitubercular activities of its derivatives, and replacing the naphthyl group with hydrophobic substitutes was well tolerated. The representative derivative 5a exhibited potent activity against H37Ra, H37Rv, and clinical drug-resistant TB with minimum inhibitory concentration (MIC) values of 0.5-1.0 µg/mL. Meanwhile, 5a showed an acceptable safety in vivo and displayed a favorable oral bioavailability with a value of 40.7%. The differential scanning fluorescence, isothermal titration calorimetry, and molecular docking assays indicated that PknB could be one of the targets of compound 5a. Overall, this study identified 5a as a novel promising lead compound with the potential to develop candidates for the treatment of drug-resistant TB.

A Caps-Ubi Model for Protein Ubiquitination Site Prediction.

Ubiquitination, a widespread mechanism of regulating cellular responses in plants, is one of the most important post-translational modifications of proteins in many biological processes and is involved in the regulation of plant disease resistance responses. Predicting ubiquitination is an important technical method for plant protection. Traditional ubiquitination site determination methods are costly and time-consuming, while computational-based prediction methods can accurately and efficiently predict ubiquitination sites. At present, capsule networks and deep learning are used alone for prediction, and the effect is not obvious. The capsule network reflects the spatial position relationship of the internal features of the neural network, but it cannot identify long-distance dependencies or focus on amino acids in protein sequences or their degree of importance. In this study, we investigated the use of convolutional neural networks and capsule networks in deep learning to design a novel model "Caps-Ubi," first using the one-hot and amino acid continuous type hybrid encoding method to characterize ubiquitination sites. The sequence patterns, the dependencies between the encoded protein sequences and the important amino acids in the captured sequences, were then focused on the importance of amino acids in the sequences through the proposed Caps-Ubi model and used for multispecies ubiquitination site prediction. Through relevant experiments, the proposed Caps-Ubi method is superior to other similar methods in predicting ubiquitination sites.

Stichoposide C Exerts Anticancer Effects on Ovarian Cancer by Inducing Autophagy via Inhibiting AKT/mTOR Pathway.

PURPOSE: Stichoposide C (STC) is a triterpene glycoside isolated from Thelenota ananas, which is previously demonstrated to wide spectrum of anticancer effects against various tumor cells. However, the antitumor effects and underlying molecular mechanisms in ovarian cancer (OC) cells are not fully understood. Here, we examined if and through which mechanisms STC exerts anticancer effects on OC. METHODS: CCK-8 and colony formation assays were used to detect cell viability and proliferation. Flow cytometry was used to detect apoptosis and cell cycle arrest. Protein expression and phosphorylation were measured by Western blotting analysis. Confocal fluorescence microscopy was used to observe the autophagy flux. Autophagosome formation was observed via transmission electron microscopy. Antitumor effect of STC was investigated in patient-derived organoids (PDOs) and A2780 subcutaneous xenograft tumors. RESULTS: STC was found that not only exerted antiproliferation activity and apoptosis but also induced autophagy. Mechanistically, STC induced autophagy via inhibited the AKT/mTOR signaling pathway in ovarian cancer cells. In addition, STC and an autophagy inhibitor 3-methyladenine (3-MA) combination treatment showed significant synergetic effects on inhibiting proliferation and promoting apoptosis in vitro. Consistent with cell experiments, STC also inhibited the growth of two OC PDOs. Finally, STC markedly reduced the growth of A2780 subcutaneous xenograft tumors without organ toxicity and activated autophagy in vivo. CONCLUSION: Stichoposide C exerts in vitro and in vivo anticancer effects on ovarian cancer by inducing autophagy via inhibiting AKT/mTOR pathway. The findings warrant further prove for STC as a potential therapeutic agent for ovarian cancer.

Comparative genome analysis reveals high-level drug resistance markers in a clinical isolate of Mycobacterium fortuitum subsp. fortuitum MF GZ001.

Introduction: Infections caused by non-tuberculosis mycobacteria are significantly worsening across the globe. M. fortuitum complex is a rapidly growing pathogenic species that is of clinical relevance to both humans and animals. This pathogen has the potential to create adverse effects on human healthcare. Methods: The MF GZ001 clinical strain was collected from the sputum of a 45-year-old male patient with a pulmonary infection. The morphological studies, comparative genomic analysis, and drug resistance profiles along with variants detection were performed in this study. In addition, comparative analysis of virulence genes led us to understand the pathogenicity of this organism. Results: Bacterial growth kinetics and morphology confirmed that MF GZ001 is a rapidly growing species with a rough morphotype. The MF GZ001 contains 6413573 bp genome size with 66.18 % high G+C content. MF GZ001 possesses a larger genome than other related mycobacteria and included 6156 protein-coding genes. Molecular phylogenetic tree, collinearity, and comparative genomic analysis suggested that MF GZ001 is a novel member of the M. fortuitum complex. We carried out the drug resistance profile analysis and found single nucleotide polymorphism (SNP) mutations in key drug resistance genes such as rpoB, katG, AAC(2')-Ib, gyrA, gyrB, embB, pncA, blaF, thyA, embC, embR, and iniA. In addition, the MF GZ001strain contains mutations in iniA, iniC, pncA, and ribD which conferred resistance to isoniazid, ethambutol, pyrazinamide, and para-aminosalicylic acid respectively, which are not frequently observed in rapidly growing mycobacteria. A wide variety of predicted putative potential virulence genes were found in MF GZ001, most of which are shared with well-recognized mycobacterial species with high pathogenic profiles such as M. tuberculosis and M. abscessus. Discussion: Our identified novel features of a pathogenic member of the M. fortuitum complex will provide the foundation for further investigation of mycobacterial pathogenicity and effective treatment.

Characterization of Genetic Variants Associated with Rifampicin Resistance Level in Mycobacterium tuberculosis Clinical Isolates Collected in Guangzhou Chest Hospital, China.

Objective: Rifampicin (RIF)-resistance, a surrogate marker for multidrug-resistant tuberculosis (TB), is mediated by mutations in the rpoB gene. We aimed to investigate the prevalence of mutations pattern in the entire rpoB gene of Mycobacterium tuberculosis clinical isolates and their association with resistance level to RIF. Methods: Among 465 clinical isolates collected from the Guangzhou Chest Hospital, drug-susceptibility of 175 confirmed Mtb strains was performed via the proportion method and Bactec MGIT 960 system. GeneXpert MTB/RIF and sanger sequencing facilitated in genetic characterization, whereas the MICs of RIF were determined by Alamar blue assay. Results: We found 150/175 (85.71%) RIF-resistant strains (MIC: 4 to >64 µg/mL) of which 57 were MDR and 81 pre-XDR TB. Genetic analysis identified 17 types of mutations 146/150 (97.33%) within RRDR (codons 426-452) of rpoB, mainly at L430 (P), D435 (V, E, G, N), H445 (N, D, Y, R, L), S450 (L, F) and L452 (P). D435V 12/146 (8.2%), H445N 16/146 (10.9%), and S450L 70/146 (47.94%) were the most frequently encountered mutations. Mutations Q432K, M434V, and N437D are rarely identified in RRDR. Deletions at (1284-1289 CCAGCT), (1295-1303 AATTCATGG), and insertion at (1300-1302 TTC) were detected within RRDR of three RIFR strains for the first time. We detected 47 types of mutations and insertions/deletions (indels) outside the RRDR. Four RIFR strains were detected with only novel mutations/indels outside the RRDR. Two of the four had (K274Q + C897 del + I491M) and (A286V + L494P), respectively. The other two had (G1687del + P454L) and (TT1835-6 ins + I491L) individually. Compared with phenotypic characterization, diagnostic sensitivities of GeneXpert MTB/RIF and sequencing analysis were 95.33% (143/150), and 100% (150/150) respectively. Conclusion: Our findings underscore the key role of RRDR mutations and the contribution of non-RRDR mutations in rapid molecular diagnosis of RIFR clinical isolates. Such insights will support early detection of disease and recommend the appropriate anti-TB regimens in high-burden settings.

Arabinosyltransferase C Mediates Multiple Drugs Intrinsic Resistance by Altering Cell Envelope Permeability in Mycobacterium abscessus.

Mycobacterium abscessus is an emerging human pathogen leading to significant morbidity and even mortality, intrinsically resistant to almost all the antibiotics available and so can be a nightmare. Mechanisms of its intrinsic resistance remain not fully understood. Here, we selected and confirmed an M. abscessus transposon mutant that is hypersensitive to multiple drugs including rifampin, rifabutin, vancomycin, clofazimine, linezolid, imipenem, levofloxacin, cefoxitin, and clarithromycin. The gene MAB_0189c encoding a putative arabinosyltransferase C was found to be disrupted, using a newly developed highly-efficient strategy combining next-generation sequencing and multiple PCR. Furthermore, selectable marker-free deletion of MAB_0189c recapitulated the hypersensitive phenotype. Disruption of MAB_0189c resulted in an inability to synthesize lipoarabinomannan and markedly enhanced its cell envelope permeability. Complementing MAB_0189c or M. tuberculosis embC restored the resistance phenotype. Importantly, treatment of M. abscessus with ethambutol, a first-line antituberculosis drug targeting arabinosyltransferases of M. tuberculosis, largely sensitized M. abscessus to multiple antibiotics in vitro. We finally tested activities of six selected drugs using a murine model of sustained M. abscessus infection and found that linezolid, rifabutin, and imipenem were active against the MAB_0189c deletion strain. These results identified MAB_0189 as a crucial determinant of intrinsic resistance of M. abscessus, and optimizing inhibitors targeting MAB_0189 might be a strategy to disarm the intrinsic multiple antibiotic resistance of M. abscessus. IMPORTANCE Mycobacterium abscessus is intrinsically resistant to most antibiotics, and treatment of its infections is highly challenging. The mechanisms of its intrinsic resistance remain not fully understood. Here we found a transposon mutant hypersensitive to a variety of drugs and identified the transposon inserted into the MAB_0189c (orthologous embC coding arabinosyltransferase, EmbC) gene by using a newly developed rapid and efficient approach. We further verified that the MAB_0189c gene played a significant role in its intrinsic resistance by decreasing the cell envelope permeability through affecting the production of lipoarabinomannan in its cell envelope. Lastly, we found the arabinosyltransferases inhibitor, ethambutol, increased activities of nine selected drugs in vitro. Knockout of MAB_0189c made M. abscessus become susceptible to 3 drugs in mice. These findings indicated that potential powerful M. abscessus EmbC inhibitor might be used to reverse the intrinsic resistance of M. abscessus to multiple drugs.