Omadacycline drug susceptibility testing for non-tuberculous mycobacteria using oxyrase to overcome challenges with drug degradation.

BACKGROUND: Drug susceptibility testing (DST) protocol of omadacycline against non-tuberculous mycobacteria has not yet been established. We developed a method to accurately determine MIC omadacycline MIC against Mycobacterium abscessus (Mab), Mycobacterium avium-complex (MAC), and Mycobacterium kansasii (Mkn). METHODS: First, we identified the oxyrase concentration not affecting Mab, MAC, and Mkn growth followed by omadacycline MIC experiments with and without oxyrase using reference and clinical strains. RESULTS: Oxyrase 0.5 % (v/v) stabilized omadacycline in the culture medium. The median omadacycline MIC was 1 mg/L for Mab and 8 mg/L for Mkn. For MAC, the median omadacycline MIC was 2 mg/L for M. avium, 256 mg/L for M. intracellulare, and 4 mg/L for M. chimaera (p < 0.0001). Wilcoxon matched-pairs signed rank test revealed statistically lower MICs with oxyrase for all MAC subspecies (p < 0.0001), all Mab subspecies (p < 0.0001), and Mkn (p = 0.0002). The decrease in MICs with oxyrase was 17/18 of Mab, 14/19 of Mkn, 8/8 of M. avium, 4/5 M. chimera, but only 11/18 of M. intracellulare (p < 0.013). CONCLUSION: Use of 0.5 % oxyrase could be a potential solution to reliable and reproducible omadacycline MIC of Mab. However, oxyrase demonstrated a variable effect in reducing MICs against MAC and Mkn.

Clinical and Microbiological Characteristics of Mycobacterium kansasii Pulmonary Infections in China.

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.

Copper(II) complexes based on thiosemicarbazone ligand: Preparation, crystal structure, Hirshfeld surface, energy framework, antiMycobacterium activity, in silico and molecular docking studies.

Considering the promising previous results on the remarkable activity exhibited by cobalt(III) and manganese(II) thiosemicarbazone compounds as antibacterial agents, the present study aimed to prepare and then evaluate the antibacterial activity of two different types of Cu(II) complexes based on a 2-acetylpyridine-N(4)-methyl-thiosemicarbazone ligand (Hatc-Me), a monomer complex [CuCl(atc-Me)] and a novel dinuclear complex [{Cu(µ-atc-Me)}2µ-SO4]. The compounds were characterized by infrared spectra, ultraviolet visible and CHN elemental analysis. In addition, the crystalline structures of the complexes were determined by single-crystal X-ray diffraction. In both cases, the Schiff base ligand coordinated in a tridentate mode via the pyridine nitrogen, imine nitrogen and sulfur atoms. The two Cu(II) atoms in the dimer are five coordinate, consisting of three NNS-donor atoms from the thiosemicarbazone ligand connected by a sulfate bridge. The Hirshfeld surface and energy framework of the complexes were additionally analyzed to verify the intermolecular interactions. The biological activity of the Cu(II) salts, the free ligand and its Cu(II) complexes was evaluated against six strains of mycobacteria including Mycobacterium tuberculosis. The complexes showed promising results as antibacterial agents for M. avium and M. tuberculosis, which ranged from 6.12 to 12.73 µM. Furthermore, molecular docking analysis was performed and the binding energy of the docked compound [{Cu(µ-atc-Me)}2µ-SO4] with M. tuberculosis and M. avium strains were extremely favorable (-11.11 and - 14.03 kcal/mol, respectively). The in silico results show that the complexes are potential candidates for the development of new antimycobacterial drugs.

[Nontuberculous mycobacterial pulmonary disease - The new ATS/ERS/ESCMID/IDSA Guideline]. / Lungenerkrankung durch nicht-tuberkulöse Mykobakterien ­ Die neue ATS/ERS/ESCMID/IDSA-Leitlinie.

The new ATS/ERS/ESCMID/IDSA guideline answers 22 PICO questions on the treatment of lung diseases caused by Mycobacterium avium complex (MAC), M. kansasii, M. xenopi and M. abscessus. NON-TUBERCULOUS MYCOBACTERIA (NTM) LUNG DISEASE: Especially in patients with microscopic detection of acid-fast bacteria in sputum or with cavernous disease manifestation, the start of treatment should not be delayed. Treatment should be based on species-specific resistance testing (according to the CLSI guidelines). In selected patients, adjuvant surgical resection after consultation with an expert is recommended. MAC LUNG DISEASE: Therapy is based on a regimen with at least three drugs including a macrolide (rather azithromycin than clarithromycin) and ethambutol. For patients with cavitation, with pronounced nodular bronchiectatic disease or with macrolide resistance, daily oral therapy should be expanded by parenteral amikacin or streptomycin. Liposomally encapsulated amikacin for inhalation is recommended in patients with treatment failure. Patients with nodular-bronchiectatic disease manifestation should receive oral macrolide-based therapy, which - depending on the extent - can be given 3 times a week. The recommended duration is 12 months after conversion of the sputum culture. M. KANSASII LUNG DISEASE: The triple combination of rifampicin, ethambutol and macrolide (or isoniazid) is recommended for at least 12 months. In patients with rifampicin resistance or intolerance, moxifloxacin is recommended as a replacement. M. XENOPI LUNG DISEASE: The combination of rifampicin, ethambutol and macrolide (and/or moxifloxacin) is recommended for at least 12 months after conversion of the sputum culture. For patients with cavernous disease manifestation, it is recommended to add at least parenteral amikacin and to consult experts. M. ABSCESSUS LUNG DISEASE: At least 3, in the beginning rather 4 drugs are recommended for therapy. The choice of substance should be based on a in vitro resistance test. Macrolides are the basis, but should not be counted in patients with strains with inducible macrolide resistance. Due to the lack of data, no explicit recommendations are made regarding the duration of therapy; a consultation of experts is recommended.

Transposon mutagenesis in Mycobacterium kansasii links a small RNA gene to colony morphology and biofilm formation and identifies 9,885 intragenic insertions that do not compromise colony outgrowth.

Mycobacterium kansasii (Mk) is a resilient opportunistic human pathogen that causes tuberculosis-like chronic pulmonary disease and mortality stemming from comorbidities and treatment failure. The standard treatment of Mk infections requires costly, long-term, multidrug courses with adverse side effects. The emergence of drug-resistant isolates further complicates the already challenging drug therapy regimens and threatens to compromise the future control of Mk infections. Despite the increasingly recognized global burden of Mk infections, the biology of this opportunistic pathogen remains essentially unexplored. In particular, studies reporting gene function or generation of defined mutants are scarce. Moreover, no transposon (Tn) mutagenesis tool has been validated for use in Mk, a situation limiting the repertoire of genetic approaches available to accelerate the dissection of gene function and the generation of gene knockout mutants in this poorly characterized pathogen. In this study, we validated the functionality of a powerful Tn mutagenesis tool in Mk and used this tool in conjunction with a forward genetic screen to establish a previously unrecognized role of a conserved mycobacterial small RNA gene of unknown function in colony morphology features and biofilm formation. We also combined Tn mutagenesis with next-generation sequencing to identify 12,071 Tn insertions that do not compromise viability in vitro. Finally, we demonstrated the susceptibility of the Galleria mellonella larva to Mk, setting the stage for further exploration of this simple and economical infection model system to the study of this pathogen.

Septic Arthritis Caused by Mycobacterium Kansasii in a Bone Marrow Transplant Recipient.

We report an 18-year-old female individual with septic arthritis due to Mycobacterium kansasii. Three years and 6 months before arthritis, the patient underwent bone marrow transplantation and developed severe chronic graft-versus-host disease. The arthritis was refractory to medication, and she underwent joint lavage of the right foot, hip joint, and elbow joint. After surgery, her joint symptoms were relieved, and chronic graft-versus-host disease remitted more easily. It is important that we maintain a high index of suspicion for mycobacterial arthritis and diagnose it early when immunosuppressed patients experience chronic pain and joint swelling.

Bioactivity of Methoxylated and Methylated 1-Hydroxynaphthalene-2-Carboxanilides: Comparative Molecular Surface Analysis.

A series of twenty-six methoxylated and methylated N-aryl-1-hydroxynaphthalene- 2-carboxanilides was prepared and characterized as potential anti-invasive agents. The molecular structure of N-(2,5-dimethylphenyl)-1-hydroxynaphthalene-2-carboxamide as a model compound was determined by single-crystal X-ray diffraction. All the analysed compounds were tested against the reference strain Staphylococcus aureus and three clinical isolates of methicillin-resistant S. aureus as well as against Mycobacterium tuberculosis and M. kansasii. In addition, the inhibitory profile of photosynthetic electron transport in spinach (Spinacia oleracea L.) chloroplasts was specified. In vitro cytotoxicity of the most effective compounds was tested on the human monocytic leukaemia THP-1 cell line. The activities of N-(3,5-dimethylphenyl)-, N-(3-fluoro-5-methoxy-phenyl)- and N-(3,5-dimethoxyphenyl)-1-hydroxynaphthalene-2-carbox- amide were comparable with or even better than the commonly used standards ampicillin and isoniazid. All promising compounds did not show any cytotoxic effect at the concentration >30 µM. Moreover, an in silico evaluation of clogP features was performed for the entire set of the carboxamides using a range of software lipophilicity predictors, and cross-comparison with the experimentally determined lipophilicity (log k), in consensus lipophilicity estimation, was conducted as well. Principal component analysis was employed to illustrate noticeable variations with respect to the molecular lipophilicity (theoretical/experimental) and rule-of-five violations. Additionally, ligand-oriented studies for the assessment of the three-dimensional quantitative structure-activity relationship profile were carried out with the comparative molecular surface analysis to determine electron and/or steric factors that potentially contribute to the biological activities of the investigated compounds.

In Vitro Activity of Bedaquiline and Delamanid against Nontuberculous Mycobacteria, Including Macrolide-Resistant Clinical Isolates.

We evaluated the in vitro activities of the antimicrobial drugs bedaquiline and delamanid against the major pathogenic nontuberculous mycobacteria (NTM). Delamanid showed high MIC values for all NTM except Mycobacterium kansasii However, bedaquiline showed low MIC values for the major pathogenic NTM, including Mycobacterium avium complex, Mycobacterium abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. kansasii Bedaquiline also had low MIC values with macrolide-resistant NTM strains and warrants further investigation as a potential antibiotic for NTM treatment.

Drug susceptibility profile of Mycobacterium kansasii clinical isolates from Brazil.

OBJECTIVES: Mycobacterium kansasii (M. kansasii) pulmonary infection can cause disease with clinical and radiological features similar to tuberculosis. Failure to treat M. kansasii infection is usually associated with resistance; to increase the chance of successful treatment it is important to identify the species and know the susceptibility profile. This study aimed to evaluate the antimycobacterial susceptibility profiles of M. kansasii isolates from Brazil. METHODS: Sixty-nine M. kansasii isolates from 69 patients were identified by partial sequencing of the hsp65 gene, and their susceptibility profiles were analysed by minimal inhibitory concentration (MIC) assays. RESULTS: From 69 isolates, 68 showed susceptibility to clarithromycin, amikacin, and moxifloxacin. Most strains showed high rates of resistance to trimethoprim-sulfamethoxazole and ciprofloxacin. Resistance to rifampicin and ethambutol was found in 12% and 25% of isolates, respectively. CONCLUSIONS: Worrying results were found regarding susceptibility to some drugs used as first-line agents in the treatment of diseases caused by M. kansasii.

Recent advances in molecular diagnostics and understanding mechanisms of drug resistance in nontuberculous mycobacterial diseases.

Accumulating evidence suggests that human infections caused by nontuberculous mycobacteria (NTM) are increasing worldwide, indicating that NTM disease is no longer uncommon in many countries. As a result of an increasing emphasis on the importance of differential identification of NTM species, several molecular tools have recently been introduced in clinical and experimental settings. These advances have led to a much better understanding of the diversity of NTM species with regard to clinical aspects and the potential factors responsible for drug resistance that influence the different outcomes of NTM disease. In this paper, we review currently available molecular diagnostics for identification and differentiation of NTM species by summarizing data from recently applied methods, including commercially available assays, and their relevant strengths and weaknesses. We also highlight drug resistance-associated genes in clinically important NTM species. Understanding the basis for different treatment outcomes with different causative species and drug-resistance mechanisms will eventually improve current treatment regimens and facilitate the development of better control measures for NTM diseases.

Dibasic Derivatives of Phenylcarbamic Acid against Mycobacterial Strains: Old Drugs and New Tricks?

In order to provide a more detailed view on the structure⁻antimycobacterial activity relationship (SAR) of phenylcarbamic acid derivatives containing two centers of protonation, 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(dipropylammonio)propyl]pyrrolidinium oxalates (1a⁻d)/dichlorides (1e⁻h) as well as 1-[2-[({[2-/3-(alkoxy)phenyl]amino}carbonyl)oxy]-3-(di-propylammonio)propyl]azepanium oxalates (1i⁻l)/dichlorides (1m⁻p; alkoxy = butoxy to heptyloxy) were physicochemically characterized by estimation of their surface tension (γ; Traube's stalagmometric method), electronic features (log ε; UV/Vis spectrophotometry) and lipophilic properties (log kw; isocratic RP-HPLC) as well. The experimental log kw dataset was studied together with computational logarithms of partition coefficients (log P) generated by various methods based mainly on atomic or combined atomic and fragmental principles. Similarities and differences between the experimental and in silico lipophilicity descriptors were analyzed by unscaled principal component analysis (PCA). The in vitro activity of compounds 1a⁻p was inspected against Mycobacterium tuberculosis CNCTC My 331/88 (identical with H37Rv and ATCC 2794, respectively), M. tuberculosis H37Ra ATCC 25177, M. kansasii CNCTC My 235/80 (identical with ATCC 12478), the M. kansasii 6509/96 clinical isolate, M. kansasii DSM 44162, M. avium CNCTC My 330/80 (identical with ATCC 25291), M. smegmatis ATCC 700084 and M. marinum CAMP 5644, respectively. In vitro susceptibility of the mycobacteria to reference drugs isoniazid, ethambutol, ofloxacin or ciprofloxacin was tested as well. A very unique aspect of the research was that many compounds from the set 1a⁻p were highly efficient almost against all tested mycobacteria. The most promising derivatives showed MIC values varied from 1.9 µM to 8 µM, which were lower compared to those of used standards, especially if concerning ability to fight M. tuberculosis H37Ra ATCC 25177, M. kansasii DSM 44162 or M. avium CNCTC My 330/80. Current in vitro biological assays and systematic SAR studies based on PCA approach as well as fitting procedures, which were supported by relevant statistical descriptors, proved that the compounds 1a⁻p represented a very promising molecular framework for development of 'non-traditional' but effective antimycobacterial agents.

In Vitro Activity of Clofazimine against Nontuberculous Mycobacteria Isolated in Beijing, China.

Due to the natural resistance of nontuberculous mycobacteria (NTM) to many antibiotics, the treatment of diseases caused by NTM is often long-term but unsuccessful. The main goal of this study was to evaluate the in vitro susceptibilities to clofazimine of 209 isolates consisting of different NTM species isolated in Beijing, China. Furthermore, 47 reference strains were also tested, including 30 rapidly growing mycobacterium (RGM) species and 17 slowly growing mycobacterium (SGM) species. The potential molecular mechanism contributing to clofazimine resistance of NTM was investigated as well. Clofazimine exhibited excellent activity against both reference strains and clinical isolates of different SGM species, and most of the strains had MICs far below 1 µg/ml. Although the majority of the clinical isolates of Mycobacterium abscessus and Mycobacterium fortuitum had MICs higher than 2 µg/ml, 17 out of the 30 reference strains of different RGM species had MICs below 1 µg/ml in vitro According to the MIC distributions, the tentative epidemiological cutoff (ECOFF) values for Mycobacterium kansasii, Mycobacterium avium, and Mycobacterium intracellulare were defined at 0.5 µg/ml, 1 µg/ml, and 2 µg/ml, respectively. Intriguingly, single-direction cross-resistance between bedaquiline- and clofazimine (Cfz)-resistant isolates was observed among the tested NTM species. This study demonstrates that clofazimine had strong activity against most SGM species in vitro, as well as some RGM species. The data provide important insights into the possible clinical application of Cfz to treat NTM infections.

Clofazimine for the Treatment of Mycobacterium kansasii.

Mycobacterium kansasii pulmonary infection is a global problem. Standard combination therapy consists of isoniazid at 300 mg/day, rifampin at 600 mg/day, and ethambutol at 15 mg/kg of body weight/day for 18 months. Coincubation of M. kansasii with different clofazimine concentrations over 7 days in test tubes resulted in a maximal kill (maximum effect [Emax]) of 2.03 log10 CFU/ml below the day 0 bacterial burden. The concentration associated with Emax was 110 times the MIC. Next, the effects of human-like concentration-time profiles of clofazimine human-equivalent doses ranging from 0 to 200 mg daily for 21 days were examined in the hollow-fiber model of intracellular M. kansasii (HFS-Mkn). On day 14, when the clofazimine microbial effect was maximal, the Emax was 2.57 log10 CFU/ml, while the dose associated with Emax was 100 mg/day. However, no dose killed M. kansasii to levels below the day 0 bacterial burden. Thus, the antimicrobial effect of clofazimine monotherapy in the HFS-Mkn was modest. Human-equivalent concentration-time profiles of standard combination therapy and doses were used as comparators in the HFS-Mkn On day 14, standard therapy killed to a level 2.32 log10 CFU/ml below the day 0 bacterial burden. The effect of standard therapy was consistent with a biexponential decline, with kill rate constants of 1.85 per day (half-life = 0.37 days) and 0.06 per day (half-life = 12.76 days) (r2 > 0.99). This means that standard therapy would take 9.3 to 12 months to completely eliminate M. kansasii in the model, which is consistent with clinical observations. This observation for standard therapy means that the modest to poor effect of clofazimine on M. kansasii identified here is likely to be the same in the clinic.

Drug Susceptibility Profiling and Genetic Determinants of Drug Resistance in Mycobacterium kansasii.

Very few studies have examined drug susceptibility of Mycobacterium kansasii, and they involve a limited number of strains. The purpose of this study was to determine drug susceptibility profiles of M. kansasii isolates representing a spectrum of species genotypes (subtypes) with two different methodologies, i.e., broth microdilution and Etest assays. To confirm drug resistance, drug target genes were sequenced. A collection of 85 M. kansasii isolates, including representatives of eight different subtypes (I to VI, I/II, and IIB) from eight countries, was used. Drug susceptibility against 13 and 8 antimycobacterial agents was tested by using broth microdilution and Etest, respectively. For drug-resistant or high-MIC isolates, eight structural genes (rrl, katG, inhA, embB, rrs, rpsL, gyrA, and gyrB) and one regulatory region (embCA) were PCR amplified and sequenced in the search for resistance-associated mutations. All isolates tested were susceptible to rifampin (RIF), amikacin (AMK), co-trimoxazole (SXT), rifabutin (RFB), moxifloxacin (MXF), and linezolid (LZD) according to the microdilution method. Resistance to ethambutol (EMB), ciprofloxacin (CIP), and clarithromycin (CLR) was found in 83 (97.7%), 17 (20%), and 1 (1.2%) isolate, respectively. The calculated concordance between the Etest and dilution method was 22.6% for AMK, 4.8% for streptomycin (STR), 3.2% for CLR, and 1.6% for RIF. For EMB, INH, and SXT, not even a single MIC value determined by one method equaled that by the second method. The only mutations disclosed were A2266C transversion at the rrl gene (CLR-resistant strain) and A128G transition at the rpsL gene (strain with STR MIC of >64 mg/liter). In conclusion, eight drugs, including RIF, CLR, AMK, SXT, RFB, MXF, LZD, and ethionamide (ETO), showed high in vitro activity against M. kansasii isolates. Discrepancies of the results between the reference microdilution method and Etest preclude the use of the latter for drug susceptibility determination in M. kansasii Drug resistance in M. kansasii may have different genetic determinants than resistance to the same drugs in M. tuberculosis.

Extended spectrum of antibiotic susceptibility for tuberculosis, Djibouti.

In the Horn of Africa, there is a high prevalence of tuberculosis that is reported to be partly driven by multidrug-resistant (MDR) Mycobacterium tuberculosis strictu sensu strains. We conducted a prospective study to investigate M. tuberculosis complex species causing tuberculosis in Djibouti, and their in vitro susceptibility to standard anti-tuberculous antibiotics in addition to clofazimine, minocycline, chloramphenicol and sulfadiazine. Among the 118 mycobacteria isolates from 118 successive patients with suspected pulmonary tuberculosis, 111 strains of M. tuberculosis, five Mycobacterium canettii, one 'Mycobacterium simulans' and one Mycobacterium kansasii were identified. Drug-susceptibility tests performed on the first 78 isolates yielded nine MDR M. tuberculosis isolates. All isolates were fully susceptible to clofazimine, minocycline and chloramphenicol, and 75 of 78 isolates were susceptible to sulfadiazine. In the Horn of Africa, patients with confirmed pulmonary tuberculosis caused by an in vitro susceptible strain may benefit from anti-leprosy drugs, sulfamides and phenicol antibiotics.

Synthesis and biological evolution of hydrazones derived from 4-(trifluoromethyl)benzohydrazide.

Reflecting the known biological activity of isoniazid-based hydrazones, seventeen hydrazones of 4-(trifluoromethyl)benzohydrazide as their bioisosters were synthesized from various benzaldehydes and aliphatic ketones. The compounds were screened for their in vitro activity against Mycobacterium tuberculosis, nontuberculous mycobacteria (M. avium, M. kansasii), bacterial and fungal strains. The most antimicrobial potent derivatives were also investigated for their cytostatic and cytotoxic properties against three cell lines. Camphor-based molecule, 4-(trifluoromethyl)-N'-(1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene)benzohydrazide, exhibited the highest and selective inhibition of M. tuberculosis with the minimum inhibitory concentration (MIC) of 4 µM, while N'-(4-chlorobenzylidene)-4-(trifluoromethyl)benzohydrazide was found to be superior against M. kansasii (MIC = 16 µM). N'-(5-Chloro-2-hydroxybenzylidene)-4-(trifluoromethyl)benzohydrazide showed the lowest MIC values for gram-positive bacteria including methicillin-resistant Staphylococcus aureus as well as against two fungal strains of Candida glabrata and Trichophyton mentagrophytes within the range of ≤0.49-3.9 µM. The convenient substitution of benzylidene moiety at the position 4 or the presence of 5-chloro-2-hydroxybenzylidene scaffold concomitantly with a sufficient lipophilicity are essential for the noticeable antimicrobial activity. This 5-chlorosalicylidene derivative avoided any cytotoxicity on two mammalian cell cultures (HepG2, BMMΦ) up to the concentration of 100 µM, but it affected the growth of MonoMac6 cells.

Biochemical Validation of a Third Guanidine Riboswitch Class in Bacteria.

Recently, it was determined that representatives of the riboswitch candidates called ykkC and mini-ykkC directly bind free guanidine. These riboswitches regulate the expression of genes whose protein products are implicated in overcoming the toxic effects of this ligand. Thus, the relevant ykkC motif and mini-ykkC motif RNAs have been classified as guanidine-I and guanidine-II riboswitch RNAs, respectively. Moreover, we had previously noted that a third candidate riboswitch class, called ykkC-III, was associated with a distribution of genes similar to those of the other two motifs. Therefore, it was predicted that ykkC-III motif RNAs would sense and respond to the same ligand. In this report, we present biochemical data supporting the hypothesis that ykkC-III RNAs represent a third class of guanidine-sensing RNAs called guanidine-III riboswitches. Members of the guanidine-III riboswitch class bind their ligand with an affinity similar to that observed for members of the other two classes. Notably, there are some sequence similarities between guanidine-II and guanidine-III riboswitches. However, the characteristics of ligand discrimination by guanidine-III RNAs are different from those of the other guanidine-binding motifs, suggesting that the binding pockets have distinct features among the three riboswitch classes.

Factors which influence treatment initiation for pulmonary non-tuberculous mycobacterium infection in HIV negative patients; a multicentre observational study.

BACKGROUND: Clinical, radiological and microbiological criteria inform diagnosis of pulmonary Non-Tuberculous Mycobacteria (NTM) disease and treatment decisions. This multicentre, review aims to characterise NTM disease meeting ATS/IDSA criteria and define factors associated with initiation of treatment. METHODS: Sputum samples growing NTM from 5 London hospitals between 2010 and 2014 were identified. Data for HIV-negative individuals meeting ATS/IDSA guidelines for pulmonary NTM disease were extracted. Associations between clinical variables and treatment decision were investigated using Chi-squared, Fishers-exact or Mann Whitney tests. Factors associated with treatment in univariate analysis (p < 0.150) were included in a multivariate logistic regression model. RESULTS: NTM were identified from 817 individuals' sputum samples. 108 met ATS/IDSA criteria. 42/108 (39%) were initiated on treatment. Median age was 68 (56-78) in the cohort. On multivariate analysis, factors significantly associated with treatment of pulmonary NTM infection were: Cavitation on HRCT (OR: 6.49; 95% CI: 2.36-17.81), presenting with night sweats (OR 4.18; 95% CI: 1.08-16.13), and presenting with weight loss (OR 3.02; 95% CI: 1.15-7.93). Of those treated, 18(43%) have completed treatment, 9(21%) remain on treatment, 10(24%) stopped due to side effects, 5(12%) died during treatment. Mortality was 31% (n = 13) in treated versus 21% (n = 14) in the non-treated cohort. Subgroup analysis of individual NTM species did not observe any differences in treatment initiation or outcomes between groups. DISCUSSION: Decision to treat pulmonary NTM infection requires clinical judgement when interpreting clinical guidelines. Factors independently associated with decision to treat in this HIV-negative cohort include cavitation on HRCT and presenting with night sweats or weight loss.