Vaping during pregnancy: a systematic review of health outcomes.

INTRODUCTION: Smoking during pregnancy is harmful to maternal and child health. Vaping is used for smoking cessation but evidence on health effects during pregnancy is scarce. We conducted a systematic review of health outcomes of vaping during pregnancy. METHODS: We searched six databases for maternal/fetal/infant outcomes and vaping, including quantitative, English language, human studies of vaping during pregnancy, to November 10th, 2023. We assessed study quality with the Mixed-Methods Appraisal Tool. We focused on comparisons of exclusive-vaping with non-use of nicotine and tobacco products and with smoking. Presentation is narrative as the studies were of insufficient quality to conduct meta-analysis. RESULTS: We included 26 studies, with 765,527 women, with one randomised controlled trial (RCT) comparing vaping and nicotine replacement therapy for smoking cessation, 23 cohort studies and two case-control studies. While the RCT met 4/5 quality criteria, the quality of the cohort studies and case-control studies was poor; none adequately assessed exposure to smoking and vaping. For studies comparing exclusive-vaping with 'non-use', more reported no increased risk for vaping (three studies) than reported increased risk for maternal pregnancy/postpartum outcomes (one study) and for fetal and infant outcomes (20 studies no increased risk, four increased risk), except for birth-weight and neurological outcomes where two studies each observed increased and no increased risk. When the RCT compared non-users with those not smoking but vaping or using NRT, irrespective of randomisation, they reported no evidence of risk for vaping/NRT. For studies comparing exclusive-vaping and exclusive-smoking, most studies provided evidence for a comparable risk for different outcomes. One maternal biomarker study revealed a lower risk for vaping. For small-for-gestational-age/mean-birth-centile equal numbers of studies found lower risk for vaping than for smoking as found similar risk for the two groups (two each). CONCLUSIONS: While more studies found no evidence of increased risk of exclusive-vaping compared with non-use and evidence of comparable risk for exclusive-vaping and exclusive-smoking, the quality of the evidence limits conclusions. Without adequate assessment of exposure to vaping and smoking, findings cannot be attributed to behaviour as many who vape will have smoked and many who vape may do so at low levels. STUDY REGISTRATION: https://osf.io/rfx4q/ .

Genetic factors associated with acquired phenotypic drug resistance and its compensatory evolution during tuberculosis treatment.

OBJECTIVES: We elucidated the factors, evolution, and compensation of antimicrobial resistance (AMR) in Mycobacterium tuberculosis (MTB) isolates under dual pressure from the intra-host environment and anti-tuberculosis (anti-TB) drugs. METHODS: This retrospective case-control study included 337 patients with pulmonary tuberculosis from 15 clinics in Tianjin, China, with phenotypic drug susceptibility testing results available for at least two time points between January 1, 2009 and December 31, 2016. Patients in the case group exhibited acquired AMR to isoniazid (INH) or rifampicin (RIF), while those in the control group lacked acquired AMR. The whole-genome sequencing (WGS) was conducted on 149 serial longitudinal MTB isolates from 46 patients who acquired or reversed phenotypic INH/RIF-resistance during treatment. The genetic basis, associated factors, and intra-host evolution of acquired phenotypic INH/RIF-resistance were elucidated using a combined analysis. RESULTS: Anti-TB interruption duration of ≥30 days showed association with acquired phenotypic INH/RIF resistance (aOR = 2·2, 95% CI, 1·0-5·1) and new rpoB mutations (p = 0·024). The MTB evolution was 1·2 (95% CI, 1·02-1·38) single nucleotide polymorphisms per genome per year under dual pressure from the intra-host environment and anti-TB drugs. AMR-associated mutations occurred before phenotypic AMR appearance in cases with acquired phenotypic INH (10 of 16) and RIF (9 of 22) resistances. DISCUSSION: Compensatory evolution may promote the fixation of INH/RIF-resistance mutations and affect phenotypic AMR. The TB treatment should be adjusted based on gene sequencing results, especially in persistent culture positivity during treatment, which highlights the clinical importance of WGS in identifying reinfection and AMR acquisition before phenotypic drug susceptibility testing.

EspB and HtpG interact with the type III-A CRISPR/Cas system of Mycobacterium tuberculosis.

Introduction: Mycobacterium tuberculosis (MTB) has a type III-A clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas) system consisting of a Csm1-5 and CRISPR RNA (crRNA) complex involved in the defense against invading nucleic acids. However, CRISPR/Cas system in the MTB still is clearly unknown and needs to be further explored. Methods: In our work, two non-Cas system proteins EspB and HtpG protein were found and identified by LC-MS/MS. The effect of EspB and HtpG on Type III-A CRISPR/Cas System of M. tuberculosis was examined by using Plasmid interference assay and Co-immunoprecipitation analyses. We explored that EspB could interact with the crRNA RNP complex, but HtpG could inhibit the accumulation of the MTB Csm proteins and defense the mechanism of CRISPR/Cas system. Results: The proteins ESAT-6 secretion system-1(Esx-1) secreted protein B (EspB) and high-temperature protein G (HtpG), which were not previously associated with CRISPR/Cas systems, are involved in mycobacterial CRISPR/Cas systems with distinct functions. Conclusion: EspB is a novel crRNA-binding protein that interacts directly with the MTB crRNP complex. Meanwhile, HtpG influences the accumulation of MTB Csm proteins and EspB and interferes with the defense mechanism of the crRNP complex against foreign DNA in vivo. Thereby, our study not only leads to developing more precise clinical diagnostic tool to quickly detect for MTB infection, but also knows these proteins merits for TB biomarkers/vaccine candidates.

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.

CD84 is a Suppressor of T and B Cell Activation during Mycobacterium tuberculosis Pathogenesis.

Interest in host-directed therapies as alternatives/adjuncts to antibiotic treatment has resurged with the increasing prevalence of antibiotic-resistant tuberculosis (TB). Immunotherapies that reinvigorate immune responses by targeting immune checkpoints like PD-1/PD-L1 have proved successful in cancer therapy. Immune cell inhibitory receptors that trigger Mycobacterium tuberculosis-specific immunosuppression, however, are unknown. Here, we show that the levels of CD84, a SLAM family receptor, increase in T and B cells in lung tissues from M. tuberculosis-infected C57BL/6 mice and in peripheral blood mononuclear cells (PBMCs) from pulmonary TB patients. M. tuberculosis challenge experiments using CD84-deficient C57BL/6 mice suggest that CD84 expression likely leads to T and B cell immunosuppression during M. tuberculosis pathogenesis and also plays an inhibitory role in B cell activation. Importantly, CD84-deficient mice showed improved M. tuberculosis clearance and longer survival than M. tuberculosis-infected wild-type (WT) mice. That CD84 is a putative M. tuberculosis infection-specific inhibitory receptor suggests it may be a suitable target for the development of TB-specific checkpoint immunotherapies. IMPORTANCE Immune checkpoint therapies, such as targeting checkpoints like PD-1/PD-L1, have proved successful in cancer therapy and can reinvigorate immune responses. The potential of this approach for treating chronic infectious diseases like TB has been recognized, but a lack of suitable immunotherapeutic targets, i.e., immune cell inhibitory receptors that trigger immunosuppression specifically during Mycobacterium tuberculosis pathogenesis, has limited the application of this strategy in the development of new TB therapies. Our focus in this study was to address this gap and search for an M. tuberculosis-specific checkpoint target. Our results suggest that CD84 is a putative inhibitory receptor that may be a suitable target for the development of TB-specific checkpoint immunotherapies.

M. tuberculosis CRISPR/Cas proteins are secreted virulence factors that trigger cellular immune responses.

The role of prokaryotic CRISPR/Cas system proteins as a defensive shield against invasive nucleic acids has been studied extensively. Non-canonical roles in pathogenesis involving intracellular targeting of certain virulence-associated endogenous mRNA have also been reported for some Type I and Type II CRISPR/Cas proteins, but no such roles have yet been established for Type III system proteins. Here, we demonstrate that M. tuberculosis (Type III-A system) CRISPR/Cas proteins Csm1, Csm3, Csm5, Csm6, and Cas6 are secreted and induce host immune responses. Using cell and animal experiments, we show that Cas6, in particular, provokes IFN-γ release from PBMCs from active tuberculosis (TB) patients, and its deletion markedly attenuates virulence in a murine M. tuberculosis challenge model. Recombinant MTBCas6 induces apoptosis of macrophages and lung fibroblasts, and interacts with the surface of cells in a caspase and TLR-2 independent manner. Transcriptomic and signal pathway studies using THP-1 macrophages stimulated with MTBCas6 indicated that MTBCas6 upregulates expression of genes associated with the NF-κB pathway leading to higher levels of IL-6, IL-1ß, and TNF-α release, cytokines known to activate immune system cells in response to M. tuberculosis infection. Our findings suggest that, in addition to their intracellular shielding role, M. tuberculosis CRISPR/Cas proteins have non-canonical extracellular roles, functioning like a virulent sword, and activating host immune responses.

relA Inactivation Converts Sulfonamides Into Bactericidal Compounds.

Folates are required for the de novo biosynthesis of purines, thymine, methionine, glycine, and pantothenic acid, key metabolites that bacterial cells cannot survive without. Sulfonamides, which inhibit bacterial folate biosynthesis and are generally considered as bacteriostats, have been extensively used as broad-spectrum antimicrobials for decades. Here we show that, deleting relA in Escherichia coli and other bacterial species converted sulfamethoxazole from a bacteriostat into a bactericide. Not as previously assumed, the bactericidal effect of SMX was not caused by thymine deficiency. When E. coli ∆relA was treated with SMX, reactive oxygen species and ferrous ion accumulated inside the bacterial cells, which caused extensive DNA double-strand breaks without the involvement of incomplete base excision repair. In addition, sulfamethoxazole showed bactericidal effect against E. coli O157 ∆relA in mice, suggesting the possibility of designing new potentiators for sulfonamides targeting RelA. Thus, our study uncovered the previously unknown bactericidal effects of sulfonamides, which advances our understanding of their mechanisms of action, and will facilitate the designing of new potentiators for them.

Judicious use of low-dosage corticosteroids for non-severe COVID-19: A case report.

Inflammation-mediated lung injury in severe cases of infection with SARS-CoV-2, the aetiological agent of Coronavirus disease 2019 (COVID-19), can lead to respiratory failure and death, and therapies that block or ameliorate lung injury-associated inflammatory "cytokine storms" and progression to acute respiratory distress syndrome (ARDS) are urgently needed. Therapeutic use of corticosteroids for this purpose has been controversial because of conflicting reports on their efficacy and immunosuppressive behaviour. The WHO has strongly recommended treating critical COVID-19 patients with systemic corticosteroid therapy, but recommends against corticosteroid therapy in non-severe COVID-19 disease because of a lack of strong evidence on its efficacy. This retrospective case report describing the successful treatment of a non-severe COVID-19 case in Changchun, China, by judicious administration of corticosteroids using a personalized therapeutic approach was recorded to strengthen the evidence base showing how corticosteroid use in non-severe COVID-19 cases can be safe and efficacious. Alongside supportive care and lopinavir/ritonavir antiviral drugs, a low dosage of methylprednisolone was administered over a short period to attenuate lung inflammation. Regular chest CT scans guided dosage reduction in response to lesion absorption and improved lung condition. Judicious use of corticosteroids safely attenuated disease progression and facilitated rapid and complete recovery.

Mycobacterium tuberculosis CRISPR/Cas system Csm1 holds clues to the evolutionary relationship between DNA polymerase and cyclase activity.

Prokaryotic CRISPR/Cas systems confer immunity against invading nucleic acids through effector complexes. Csm1, the signature protein of Type III effector complexes, catalyses cyclic oligoadenylate synthesis when in the effector complex, but not when alone, activating the Csm6 nuclease and switching on the antiviral response. Here, we provide biochemical evidence that M. tuberculosis Csm1 (MtbCsm1) has ion-dependent polymerase activity when independent of the effector complex. Structural studies provide supporting evidence that the catalytic core of the MtbCsm1 palm2 domain is almost identical to that of classical DNA polymerase Pol IV, and that the palm1 and B domains function as the other structural elements required (thumb and fingers) for DNA polymerase activity. MtbCsm1 polymerase activity is relatively weak in vitro and its functional relevance in vivo is unknown. Our structural and mutagenesis data suggest that residue K692 in the palm2 domain has been significant in the evolution of Csm1 from a polymerase to a cyclase, and support the notion that the cyclase activity of Csm1 requires the presence of other elements provided by the CRISPR/Cas effector complex. This structural rationale for Csm1 polymerase (alone) and cyclase (within the effector complex) activity should benefit future functional investigations and engineering.

Systematic Evaluation of Mycobacterium tuberculosis Proteins for Antigenic Properties Identifies Rv1485 and Rv1705c as Potential Protective Subunit Vaccine Candidates.

The lack of efficacious vaccines against Mycobacterium tuberculosis (MTB) infection is a limiting factor in the prevention and control of tuberculosis (TB), the leading cause of death from an infectious agent. Improvement or replacement of the BCG vaccine with one that reliably protects all age groups is urgent. Concerns exist that antigens currently being evaluated are too homogeneous. To identify new protective antigens, we screened 1,781 proteins from a high-throughput proteome-wide protein purification study for antigenic activity. Forty-nine antigens (34 previously unreported) induced antigen-specific gamma interferon (IFN-γ) release from peripheral blood mononuclear cells (PBMCs) derived from 4,452 TB and suspected TB patients and 167 healthy donors. Three (Rv1485, Rv1705c, and Rv1802) of the 20 antigens evaluated in a BALB/c mouse challenge model showed protective efficacy, reducing lung CFU counts by 66.2%, 75.8%, and 60%, respectively. Evaluation of IgG2a/IgG1 ratios and cytokine release indicated that Rv1485 and Rv1705c induce a protective Th1 immune response. Epitope analysis of PE/PPE protein Rv1705c, the strongest candidate, identified a dominant epitope in its extreme N-terminal domain accounting for 90% of its immune response. Systematic preclinical assessment of antigens Rv1485 and Rv1705c is warranted.

Extending health messaging to the consumption experience: a focus group study exploring smokers' perceptions of health warnings on cigarettes.

Introduction: While most countries require health warnings on cigarette packs, the Scottish and Canadian Governments are considering requiring health warnings on cigarette sticks. Methods: Twenty focus groups were conducted in Glasgow and Edinburgh (Scotland) with smokers (n = 120) segmented by age (16-17, 18-24, 25-35, 36-50, >50), gender and social grade, to explore perceptions of cigarettes displaying the warning 'Smoking kills' on the cigarette paper and any demographic differences in how smokers responded to these. Results: A warning on each cigarette was thought to prolong the health message, as it would be visible when a cigarette was taken from a pack, lit, left in an ashtray, and with each draw, and make avoidant behavior more difficult. That it would be visible to others was perceived as off-putting for some. It was felt that a warning on each cigarette would create a negative image and be embarrassing. Within several female groups they were viewed as depressing, worrying and frightening, with it suggested that people would not feel good smoking cigarettes displaying a warning. Within every group there was mention of warnings on cigarettes potentially having an impact on themselves, others or both. Some, mostly younger groups, mentioned stubbing cigarettes out early, reducing consumption or quitting. The consensus was that they would be off-putting for young people, nonsmokers and those starting to smoke. Conclusions: Including a warning on each cigarette stick is a viable policy option and one which would, for the first time, extend health messaging to the consumption experience.

Mycobacterium smegmatis MSMEG_0129 is a nutrition-associated regulator that interacts with CarD and ClpP2.

Mycobacterium smegmatis MSMEG_0129 and Rv0164, its homologue in Mycobacterium tuberculosis, are single START-domain proteins essential for bacterial growth and survival, but their biochemical activities and biological roles remain undetermined. Here, we probed the possible functions of MSMEG_0129 and its underlying mechanisms by determining its cellular location, searching for its interaction partners and monitoring its transcription profile. MSMEG_0129, and Rv0164 by extension, were found to be cytosolic proteins rather than secreted components as previously understood. Increases in MSMEG_0129 expression at physiological levels accelerated bacterial growth in a proportional manner, but additional growth acceleration was not observed when MSMEG_0129 was overexpressed up to 20 fold. MSMEG_0129 is a short-lived protein, unstable at both the mRNA and protein levels. Co-IP and GST pull-down assays showed that MSMEG_0129 interacts with the ClpP2 protease and a global transcription factor, CarD, their expression being correlated with that of MSMEG_0129. Nutrient deficiency led to the downregulation of MSMEG_0129 but upregulation of CarD. However, in the context of constitutive MSMEG_0129 overexpression under nutrient-rich or starvation conditions, the mRNA level of CarD was reduced 3 fold. Conversely, expression of ClpP2 decreased with MSMEG_0129 downregulation under starvation conditions, but increased 4-8 fold when MSMEG_0129 was overexpressed. Our data suggest that MSMEG_0129, and Rv0164 by analogy, are likely to be nutrition sensing factors that regulate mycobacterial growth and may be involved in signal transfer under nutrient deficiency, possibly via physical and regulatory interactions with CarD and ClpP2.

Royal jelly-derived proteins enhance proliferation and migration of human epidermal keratinocytes in an in vitro scratch wound model.

BACKGROUND: Skin injury is inevitable in daily life. In recent years, with the increasing morbidity of diseases such as diabetes and metabolic disorders, chronic wounds have become a considerable challenge in clinical practice. Royal jelly, reported to have multifarious biological and physiological properties, has been used as a remedy for a variety of wounds since ancient times. However, the active components and mechanisms underlying the wound-healing properties of royal jelly are still largely unknown. METHODS: Water-soluble proteins of royal jelly were fractionated and investigated for the proliferative and migratory effects on human epidermal keratinocytes (HaCaT) in an in vitro wound healing model. The proteins present in bioactive fractions were characterised and quantified using Label-free protein quantification method. The potential functions of these proteins in biological systems were further analysed using bioinformatic tools. RESULTS: A protein fraction, mainly containing major royal jelly proteins 2 (MRJP2), MRJP3 and MRJP7, stimulated proliferative and migratory activities in HaCaT cells without visible cytotoxicity. It exerted the greatest effects on the growth of HaCaT cells in the first 48 h. Furthermore, when treated with this protein fraction, the closure rates of the in vitro scratch wound were significantly increased. Functional analysis indicated that MRJP2, MRJP3 and MRJP7 were associated with carbohydrate transport and metabolism. CONCLUSIONS: We fractionated the water-soluble proteins of royal jelly and identified one fraction (Fraction 2) that induced both proliferative and migratory effects on a human epidermal keratinocyte cell line. Major royal jelly proteins (MRJP2, MRJP3 and/or MRJP7) were speculated to possess potential wound-healing bioactivity. This is the first report that royal jelly may improve wound closure via MRJP-induced cellular proliferation and migration. These proteins may be valuable lead compounds for the development of novel wound healing medications. Our findings would facilitate better understanding of the wound repair mechanisms of royal jelly.

Retrospective Analysis of False-Positive and Disputed Rifampin Resistance Xpert MTB/RIF Assay Results in Clinical Samples from a Referral Hospital in Hunan, China.

Concerns about the specificity of the Xpert MTB/RIF (Xpert) assay have arisen, as false-positive errors in the determination of Mycobacterium tuberculosis complex (MTBC) infection and rifampin (RIF) resistance in clinical practice have been reported. Here, we investigated 33 cases where patients were determined to be RIF susceptible using the Bactec MGIT 960 (MGIT) culture system but RIF resistant using the Xpert assay. Isolates from two of these patients were found not to have any mutations in the rifampin resistance determining region (RRDR) region of rpoB and had good treatment outcomes with first-line antituberculosis (anti-TB) drugs. The remaining 31 patients included 5 new cases and 26 previously treated patients. A large number of well-documented disputed mutations, including Leu511Pro, Asp516Tyr, His526Asn, His526Leu, His526Cys, and Leu533Pro, were detected, and mutations, including a 508 to 509 deletion and His526Gly, were described here as disputed mutations for the first time. Twenty-one (81%) of the 26 previously treated patients had poor treatment outcomes, and isolates from 19 (90%) of these 21 patients were resistant to isoniazid (INH) as determined using the MGIT culture system. Twenty-seven of the 31 isolates with disputed rpoB mutations were phenotypically resistant to INH, 21 (78%) being predicted by GenoType MTBDRplus to have a high level of INH resistance. Most (77.4%) of the isolates with disputed mutations were of the Beijing lineage. These findings have implications for the interpretation of false-positive and disputed rifampin resistance Xpert MTB/RIF results in clinical samples and provide guidance on how clinicians should manage patients carrying isolates with disputed rpoB mutations.

Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus variability has been exploited in evolutionary and epidemiological studies of Mycobacterium tuberculosis, the causative agent of tuberculosis, for over 20 yr, yet the biological function of this type III-A system is largely unexplored. Here, using cell biology and biochemical, mutagenic, and RNA-seq approaches, we show it is active in invader defense and has features atypical of type III-A systems: mature CRISPR RNA (crRNA) in its crRNA-CRISPR/Cas protein complex are of uniform length (∼71 nt) and appear not to be subject to 3'-end processing after Cas6 cleavage of repeat RNA 8 nt from its 3' end. crRNAs generated resemble mature crRNA in type I systems, having both 5' (8 nt) and 3' (28 nt) repeat tags. Cas6 cleavage of repeat RNA is ion dependent, and accurate cleavage depends on the presence of a 3' hairpin in the repeat RNA and the sequence of its stem base nucleotides. This study unveils further diversity among CRISPR/Cas systems and provides insight into the crRNA recognition mechanism in M. tuberculosis, providing a foundation for investigating the potential of a type III-A-based genome editing system.-Wei, W., Zhang, S., Fleming, J., Chen, Y., Li, Z., Fan, S., Liu, Y., Wang, W., Wang, T., Liu, Y., Ren, B., Wang, M., Jiao, J., Chen, Y., Zhou, Y., Zhou, Y., Gu, S., Zhang, X., Wan, L., Chen, T., Zhou, L., Chen, Y., Zhang, X.-E., Li, C., Zhang, H., Bi, L. Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features.

Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing.

BACKGROUND: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear. METHODS: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance. RESULTS: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted. CONCLUSIONS: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.).

Structural and genetic analysis of START superfamily protein MSMEG_0129 from Mycobacterium smegmatis.

Mycobacterium tuberculosis is a notorious pathogen that continues to threaten human health. Rv0164, an antigen of both T- and B cells conserved across mycobacteria, and MSMEG_0129, its close homolog in Mycobacterium smegmatis, are predicted members of the START domain superfamily, but their molecular function is unknown. Here, gene knockout studies demonstrate MSMEG_0129 is essential for bacterial growth, suggesting Rv0164 may be a potential drug target. The MSMEG_0129 crystal structure determined at 1.95 Å reveals a fold similar to that in polyketide aromatase/cyclases ZhuI and TcmN from Streptomyces sp. Structural comparisons and docking simulations, however, infer that MSMEG_0129 and Rv0164 are unlikely to catalyze polyketide aromatization/cyclization, but probably play an irreplaceable role during mycobacterial growth, for example, in lipid transfer during cell envelope synthesis.

The mechanism of NDM-1-catalyzed carbapenem hydrolysis is distinct from that of penicillin or cephalosporin hydrolysis.

New Delhi metallo-ß-lactamases (NDMs), the recent additions to metallo-ß-lactamases (MBLs), pose a serious public health threat due to its highly efficient hydrolysis of ß-lactam antibiotics and rapid worldwide dissemination. The MBL-hydrolyzing mechanism for carbapenems is less studied than that of penicillins and cephalosporins. Here, we report crystal structures of NDM-1 in complex with hydrolyzed imipenem and meropenem, at resolutions of 1.80-2.32 Å, together with NMR spectra monitoring meropenem hydrolysis. Three enzyme-intermediate/product derivatives, EI1, EI2, and EP, are trapped in these crystals. Our structural data reveal double-bond tautomerization from Δ2 to Δ1, absence of a bridging water molecule and an exclusive ß-diastereomeric product, all suggesting that the hydrolytic intermediates are protonated by a bulky water molecule incoming from the ß-face. These results strongly suggest a distinct mechanism of NDM-1-catalyzed carbapenem hydrolysis from that of penicillin or cephalosporin hydrolysis, which may provide a novel rationale for design of mechanism-based inhibitors.

A standardised method for interpreting the association between mutations and phenotypic drug resistance in Mycobacterium tuberculosis.

A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence.Raw genotype-phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance.We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6-90.9%), while for isoniazid it was 78.2% (77.4-79.0%) and their specificities were 96.3% (95.7-96.8%) and 94.4% (93.1-95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1-70.6%) for capreomycin to 88.2% (85.1-90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1-92.5%) for moxifloxacin to 99.5% (99.0-99.8%) for amikacin.This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis.