Agnieszka Napiórkowska

Screening for Streptomycin Resistance-Conferring Mutations in Mycobacterium tuberculosis Clinical Isolates from Poland

Currently, mutations in three genes, namely rrs, rpsL, and gidB, encoding 16S rRNA, ribosomal protein S12, and 16S rRNA-specific methyltransferase, respectively, are considered to be involved in conferring resistance to streptomycin (STR) in Mycobacterium tuberculosis. The aim of this study was to investigate the spectrum and frequency of these mutations in M. tuberculosis clinical isolates, both resistant and susceptible to STR. Sixty-four M. tuberculosis isolates recovered from as many TB patients from Poland in 2004 were included in the study. Within the sample were 50 multidrug-resistant (32 STR-resistant and 18 STR-susceptible) and 14 pan-susceptible isolates. Preliminary testing for STR resistance was performed with the 1% proportion method. The MICs of STR were determined by the Etest method. Mutation profiling was carried out by amplifying and sequencing the entire rrs, rpsL, and gidB genes. Non-synonymous mutations in either rrs or rpsL gene were detected in 23 (71.9%) of the STR-resistant and none of the STR-susceptible isolates. Mutations in the gidB gene were distributed among 12 (37.5%) STR-resistant and 13 (40.6%) STR-susceptible isolates. Four (12.5%) STR-resistant isolates were wild-type at all three loci examined. None of the rrs, rpsL or gidB mutations could be linked to low, intermediate or high level of STR resistance. In accordance with previous findings, the gidB 47T→G (L16R) mutation was associated with the Latin American-Mediterranean genotype family, whereas 276A→C (E92D) and 615A→G (A205A) mutations of the gidB gene were associated with the Beijing lineage. The study underlines the usefulness of rrs and rpsL mutations as molecular markers for STR resistance yet not indicative of its level. The gidB polymorphisms can serve as phylogenetic markers.

Detection of mutations associated with isoniazid resistance in multidrug-resistant Mycobacterium tuberculosis clinical isolates

OBJECTIVES To determine the prevalence of isoniazid resistance-conferring mutations among multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis from Poland. METHODS Nine genetic loci, including structural genes (katG, inhA, ahpC, kasA, ndh, nat and mshA) and regulatory regions (i.e. the mabA-inhA promoter and oxyR-ahpC intergenic region) of 50 MDR M. tuberculosis isolates collected throughout Poland were PCR-amplified in their entirety and screened for mutations by direct sequencing methodology. RESULTS Forty-six (92%) MDR M. tuberculosis isolates had mutations in the katG gene, and the katG Ser315Thr substitution predominated (72%). Eight (16%) isolates (six with a mutated katG allele) had mutations in the inhA promoter region and two such isolates also had single inhA structural gene mutations. Mutations in the oxyR-ahpC locus were found in five (10%) isolates, of which all but one had at least one additional mutation in katG. Mutations in the remaining genetic loci (kasA, ndh, nat and mshA) were detected in 12 (24%), 4 (8%), 5 (10%) and 17 (34%) MDR isolates, respectively. All non-synonymous mutants for these genes harboured mutations in katG. One isolate had no mutations in any of the analysed loci. CONCLUSIONS This study accentuates the usefulness of katG and inhA promoter mutations as predictive markers of isoniazid resistance. Testing only for katG 315 and inhA -15 mutations would detect isoniazid resistance in 84% of the MDR M. tuberculosis sample. This percentage would increase to 96% if the sequence analysis was extended to the entire katG gene. Analysis of the remaining genetic loci did not contribute greatly to the identification of isoniazid resistance.

Synthesis, Anticancer and Antibacterial Activity of Salinomycin N-Benzyl Amides

A series of 12 novel monosubstituted N-benzyl amides of salinomycin (SAL) was synthesized for the first time and characterized by NMR and FT-IR spectroscopic methods. Molecular structures of three salinomycin derivatives in the solid state were determined using single crystal X-ray method. All compounds obtained were screened for their antiproliferative activity against various human cancer cell lines as well as against the most problematic bacteria strains such as methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE), and Mycobacterium tuberculosis. Novel salinomycin derivatives exhibited potent anticancer activity against drug-resistant cell lines. Additionally, two N-benzyl amides of salinomycin revealed interesting antibacterial activity. The most active were N-benzyl amides of SAL substituted at -ortho position and the least anticancer active derivatives were those substituted at the -para position.

Mutations in the embB Gene and Their Association with Ethambutol Resistance in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates from Poland

Ethambutol (EMB) continues to be used as part of a standard drug regimen for the treatment of tuberculosis (TB). Mutations in the embB gene and those within its conserved EMB resistance determining region (ERDR) in particular have repeatedly been associated with resistance to EMB in Mycobacterium tuberculosis. The aim of this study was to examine the mutational “hot spots” in the embB gene, including the ERDR, among multidrug-resistant (MDR) M. tuberculosis clinical isolates and to find a possible association between embB mutations and resistance to EMB. An 863-bp fragment of the embB gene was sequenced in 17 EMB-resistant and 33 EMB-susceptible MDR-TB isolates. In total, eight embB mutation types were detected in 6 distinct codons of 27 (54%) M. tuberculosis isolates. Mutations in codon 306 were most common, found in both EMB-resistant (9) and EMB-susceptible (11) isolates. Only mutations in codons 406 and 507 were found exclusively in four and one EMB-resistant isolates, respectively. Sequence analysis of the ERDR in the embB gene is not sufficient for rapid detection of EMB resistance, and the codon 306 mutations are not good predictive markers of resistance to EMB.

Synthesis of novel 3-cyclohexylpropanoic acid-derived nitrogen heterocyclic compounds and their evaluation for tuberculostatic activity.

A series of novel 3-cyclohexylpropanoic acid derivatives and 3-cyclohexylpropanoic acid-derived nitrogen heterocyclic compounds (1-8) have been synthesized and evaluated for tuberculostatic activity. Compounds 1a, 1c, 1e and 1f bearing benzimidazole or benzimidazole-like systems showed the most potent tuberculostatic activity against Mycobacterium tuberculosis strains with MIC values ranging from 1.5 to 12.5μg/mL. More importantly 1a (6-chloro-2-(2-cyclohexylethyl)-4-nitro-1H-benzo[d]imidazole) and 1f (2-(2-cyclohexylethyl)-1H-imidazo[4,5-b]phenazine) appeared selective for M. tuberculosis as compared with eukaryotic cells (human fibroblasts), and other antimicrobial strains. These compounds may thus represent a novel, selective class of antitubercular agents. Additionally compound 1a stimulated type I collagen output by fibroblasts, in vitro.

Synthesis and Antimicrobial Activity of 4-Chloro-3-Nitrophenylthiourea Derivatives Targeting Bacterial Type II Topoisomerases.

A series of novel 4‐chloro‐3‐nitrophenylthiourea derivatives were synthesized and evaluated for their antimicrobial, antibiofilm and tuberculostatic activities. Most of compounds exhibited high antibacterial activity against both standard and hospital strains (MIC values 0.5–2 μg/mL), as compared to Ciprofloxacin. Derivatives with 3,4‐dichlorophenyl (11) and 3‐chloro‐4‐methylphenyl (13) substituents were the most promising towards Gram‐positive pathogens. Both of them exhibited antibiofilm potency and effectively inhibited the formation of biofilms of methicillin‐resistant and standard strains of Staphylococcus epidermidis. Two N‐alkylthioureas (20, 21) showed twofold to fourfold increase in in vitro potency against isolates of Mycobacterium tuberculosis, as compared to Isoniazid. An action of 7, 10, 11, 13, 20 and 21 against activity of topoisomerases isolated from Staphylococcus aureus was studied. Synthesized compounds were found as non‐genotoxic.

Novel 2-(2-phenalkyl)-1H-benzo[d]imidazoles as antitubercular agents. Synthesis, biological evaluation and structure-activity relationship.

A series of novel 2-(2-phenalkyl)-1H-benzo[d]imidazole derivatives and analogues (2a-3l) have been synthesized and evaluated for tuberculostatic activity. Benzimidazoles substituted at the C-2 position with phenethyl, styryl and 3,5-dichlorophenethyl moiety were obtained. Compounds 2g, 2h and 2i bearing methyl groups at the benzimidazole system and phenalkyl substituent at the C-2 position showed high tuberculostatic activity against Mycobacterium tuberculosis strains with MIC values ranging from 0.8 to 6.2 μg/mL (2.5-25 μM). More importantly, derivatives 2g (5,6-dimethyl-2-phenethyl-1H-benzo[d]imidazole) and 2i (2-(3,5-dichlorophenethyl)-5,6-dimethyl-1H-benzo[d]imidazole) appeared selective for M. tuberculosis as compared with eukaryotic cells: non-malignant (neonatal human dermal fibroblasts) and malignant (mouse melanoma B16-F10 cell line). These compounds may thus represent a novel, selective class of anti-tubercular agents. SAR studies resulted in interesting conclusions on structural factors affecting tuberculostatic activity.

Synthesis, structure, and antimicrobial activity of heterocyclic phenylsulfonyl- and 4-aminophenylsulfonyl-carboximidamides

A series of novel phenylsulfonyl- and 4-aminophenylsulfonyl-carboximidamides were synthesized by condensation of sulfonamides with heterocyclic methyl carbimidates obtained from heterocyclic carbonitriles and used ‘at its inception.’ The molecular structure of the obtained compounds is discussed. Compounds possessing heterocyclic systems with a nitrogen atom in the α position to the functional group showed a different single-crystal structure than expected. The synthesized derivatives were evaluated for antimicrobial activities: tuberculostatic, antibacterial, and antifungal.Graphical Abstract.

Synthesis, characterization, and tuberculostatic activity of novel 2-(4-nitrobenzoyl)hydrazinecarbodithioic acid derivatives

A series of novel S-esters of 2-(4-nitrobenzoyl)hydrazinecarbodithioic acid and S,S′-diesters of (4-nitrobenzoyl)carbonohydrazonodithioic acid were synthesized by reaction of 4-nitrobenzohydrazide and N-methyl-4-nitrobenzohydrazide with carbon disulfide and alkyl halides in the presence of triethylamine. Novel 5-(4-nitrophenyl)-1,3,4-oxadiazoles were also obtained. The structures were confirmed by IR, NMR, and mass spectroscopy, and by elemental analysis. All the compounds obtained were screened in vitro for their tuberculostatic activity. Promising preliminary results were obtained for some of the compounds. The crystal structure of the most active compound was determined.Graphical abstract

Synthesis and biological activity of novel 3-heteroaryl-2H-pyrido[4,3-e][1,2,4]thiadiazine and 3-heteroaryl-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxides

A series of novel 1,2,4-thiadiazine 1,1-dioxides were synthesized by condensation of 2-chlorobenzenesulfonamide and 4-chloropyridine-3-sulfonamide with heterocyclic methyl carbimidates obtained from heterocyclic carbonitriles and used at the time of their creation. Substituted amidines were isolated as the intermediates in the reaction with 2-chlorobenzenesulfonamide. Those intermediates were successfully cyclized to corresponding 1,2,4-thiadiazine 1,1-dioxides in pyridine with the addition of DBU. The newly synthesized compounds were evaluated for their tuberculostatic and anticancer activities. Eight compounds were able to inhibit the growth of some renal and non-small cell lung cancer cell lines.Graphical Abstract