Genetic evaluation of relationship between mutations in rpoB and resistance of Mycobacterium tuberculosis to rifampin

<p>Abstract</p> <p>Background</p> <p>Rifampin is a first line antituberculosis drug active against bacilli in logarithmic and stationary phase, which interferes with RNA synthesis by binding to bacterial RNA polymerase. Tubercle bacilli achieve resistance to rifampin by accumulation of mutations in a short-81 bp region of the <it>rpoB </it>gene. Among many mutations identified in the <it>rpo</it>B gene, few were verified by molecular genetic methods as responsible for resistance to rifampin (RMP).</p> <p>Results</p> <p>In this study eight different mutations identified in an 81 bp section of a "hot spot" region of the <it>rpo</it>B gene of RMP resistant <it>Mycobacterium tuberculosis </it>clinical strains were evaluated in respect to drug resistance. It was found that: mutations in positions 526 (H/D), 516 (D/V) and 531 (S/L) result in high level resistance to rifampin; mutations in positions 516 (D/Y), 515 (M/I), 510 (Q/H) or a double mutation in codons 512 (S/I) and 516 (D/G) relate to low level of resistance. Gene <it>rpo</it>B carrying mutations in codon 513 (Q/L) introduced into an <it>M. tuberculosis </it>laboratory strain did not cause resistance to rifampin, however the same gene introduced into two different clinical strains did, with the level of resistance depending on the host strain.</p> <p>Conclusion</p> <p>Mutations in an 81 bp "hot spot" region of the <it>rpoB </it>of <it>M. tuberculosis </it>lead to different levels of resistance to rifampin. Some mutations in this "hot spot" region of <it>rpoB </it>require a specific genetic background for the host strain to develop resistance to rifampin. Therefore, the identification of such mutations in a clinical <it>M. tuberculosis </it>strain is not enough to classify the given strain as resistant to rifampin.</p

Biopsy and tracheobronchial aspirates as additional tools for the diagnosis of bovine tuberculosis in living European bison (bison bonasus)

CITATION: Didkowska, Anna et al. 2020. Biopsy and tracheobronchial aspirates as additional tools for the diagnosis of bovine tuberculosis in living European bison (bison bonasus). Animals, 10(11):2017, doi:10.3390/ani10112017.The original publication is available at: https://www.mdpi.comENGLISH ABSTRACT: The diagnosis of bovine tuberculosis (BTB) in living wildlife remains a complex problem, and one of particular importance in endangered species like European bison (Bison bonasus). To identify infection and avoid the unnecessary culling of such valuable individuals, current best practice requires the collection and culture of material from living animals, as mycobacteria isolation remains the gold standard in BTB diagnosis. However, such isolation is challenging due to the need for the immobilization and collection of appropriate clinical material, and because of the sporadic shedding of mycobacteria. In the present study, we evaluated the potential of sampling for the detection of BTB in a group of seven living European bison suspected of being infected with Mycobacterium caprae. The specimens were collected both as swabs from the nasal and pharyngeal cavities, tracheobronchial aspirates (TBA), ultrasound-guided biopsies from lateral retropharyngeal lymph nodes, and post mortem, from mandibular, retropharyngeal and mediastinal lymph nodes. Clinical samples were tested for mycobacterial species via mycobacteriological culture and PCR. M. caprae was isolated from collected material in two out of four living infected individuals (TBA, biopsy) and mycobacterialDNAwas detected in three out of four (TBA, pharyngeal swab) bison. This is the first report of isolation of M. caprae in living European bison. Our findings demonstrate the value of diagnostic tests based on both molecular testing and culture in European bison and confirm the respiratory shedding of viable M. caprae in this host species.Publisher's versio

Mycobacterium szulgai Lung Disease or Breast Cancer Relapse—Case Report

Cancers are one of the risk factors of non-tuberculous mycobacterial (NTM) lung disease. The majority of data in this group of patients concern infections caused by Mycobacterium avium&mdash;the most prevalent NTM species worldwide. In contrast, limited information can be found regarding the uncommon NTM such as Mycobacterium szulgai. We present the case of M. szulgai lung disease in a patient with a history of breast cancer. Coexistence of NTM lung disease and breast cancer lung metastasis as well as primary lung cancer was suspected. Finally, neoplastic disease was ruled out based on negative results of endobronchial biopsy and negative tumor markers for lung and breast cancer. M. szulgai lung disease was successfully treated with rifampicin, ethambutol and clarithromycin

The Effect of Combining Natural Terpenes and Antituberculous Agents against Reference and Clinical Mycobacterium tuberculosis Strains

Background: On account of emergence of multi- and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains, combinations of drugs with natural compounds were tested to search for antibiotic activity enhancers. In this work we studied terpenes (α-pinene, bisabolol, β-elemene, (R)-limonene, (S)-limonene, myrcene, sabinene), which are the main constituents of essential oil obtained from Mutellina purpurea L., a plant with described antitubercular activity, to investigate their interactions with antibiotics against reference Mtb strains and multidrug-resistant clinical isolates. Methods: The serial dilution method was used to evaluate the minimal inhibitory concentration (MIC) of tested compounds, while the fractional inhibitory concentration index (FICI) was calculated for characterization of interactions. Moreover, IC50 values of tested compounds were determined using monkey kidney epithelial cell line (GMK). Results: The combinations of all studied terpenes with ethambutol or rifampicin resulted in a synergistic interaction. Bisabolol and (R)-limonene decreased the MIC for rifampicin at least two-fold for all tested strains, however no synergistic action was observed against virulent strains. The tested terpenes showed slight (bisabolol) or no cytotoxic effect against normal eukaryotic cells in vitro. Conclusions: The obtained enhanced activity (FICI &lt; 0.5) of ethambutol and rifampicin against H37Ra strain under the influence of the studied terpenes may be correlated to the capability of essential oil constituents to modify bacterial resistance mechanisms in general. The observed differences in avirulent and virulent bacteria susceptibility to terpenes tested separately and in combinations with antibiotics can be correlated with the differences in the cell wall structure between H37Ra mutant and all virulent strains

Synthesis, cytotoxicity and antimicrobial activity of thiourea derivatives incorporating 3-(trifluoromethyl)phenyl moiety

A total of 31 of thiourea derivatives was prepared reacting 3-(trifluoromethyl)aniline and commercial aliphatic and aromatic isothiocyanates. The yields varied from 35% to 82%. All compounds were evaluated in vitro for antimicrobial activity. Derivatives 3, 5, 6, 9, 15, 24 and 27 showed the highest inhibition against Gram-positive cocci (standard and hospital strains). The observed MIC values were in the range of 0.25e16 μg/ml. Inhibitory activity of thioureas 5 and 15 against topoisomerase IV isolated from Staphylococcus aureus was studied. Products 5 and 15 effectively inhibited the formation of biofilms of methicillin-resistant and standard strains of Staphylococcus epidermidis. Moreover, all obtained thioureas were evaluated for cytotoxicity and antiviral activity against a large panel of DNA and RNA viruses. Compounds 5, 6, 8e12, 15 resulted cytotoxic against MT-4 cells (CC50≤ 10μM)

Synthesis, Structural Characterization and Biological Activity Evaluation of Novel Cu(II) Complexes with 3-(trifluoromethyl)phenylthiourea Derivatives

Copper complexes with 1,3-disubstituted thiourea derivatives, all containing 3-(trifluoromethyl)phenyl tail and 1-alkyl/halogen-phenyl substituent, were synthesized. The experimental spectroscopic studies and theoretical calculation revealed that two ligands coordinate to Cu(II) in a bidentate fashion via thiocarbonyl S and deprotonated N atoms of thiourea moiety. Such monomers are characteristic of alkylphenylthiourea complexes, whereas the formation of a sandwich-type dimer is observed for halogeno derivatives. For the first time, the structural identifications of CuN2S2-based complexes using experimental and theoretical X-ray absorption near edge structure are demonstrated. The dimeric halogeno derivatives showed higher antimicrobial activity in comparison with alkylphenylthiourea complexes. The Cu(II) complex of 1-(4-chloro-3-nitrophenyl)-3-[3-(trifluoromethyl)phenyl]thiourea was active against 19 strains of methicillin-resistant Staphylococci (MIC = 2 µg/mL). This derivative acted as a dual inhibitor of DNA gyrase and topoisomerase IV isolated from Staphylococcus aureus. Additionally, complexes of halogenphenylthiourea strongly inhibited the growth of mycobacteria isolated from tuberculosis patients, even fourfold stronger than the reference isoniazid. The complexes exerted weak to moderate antitumor activity (towards SW480, SW620, and PC3) being non-toxic towards normal HaCaT cells