Comprehensive Assessment of CFTR Modulators' Therapeutic Efficiency for N1303K Variant.

p.Asn1303Lys (N1303K) is a common missense variant of the CFTR gene, causing cystic fibrosis (CF). In this study, we initially evaluated the influence of CFTR modulators on the restoration of N1303K-CFTR function using intestinal organoids derived from four CF patients expressing the N1303K variant. The forskolin-induced swelling assay in organoids offered valuable insights about the beneficial effects of VX-770 + VX-661 + VX-445 (Elexacaftor + Tezacaftor + Ivacaftor, ETI) on N1303K-CFTR function restoration and about discouraging the prescription of VX-770 + VX-809 (Ivacaftor + Lumacaftor) or VX-770 + VX-661 (Ivacaftor + Tezacaftor) therapy for N1303K/class I patients. Then, a comprehensive assessment was conducted on an example of one patient with the N1303K/class I genotype to examine the ETI effect on the restoration of N1303K-CFTR function using in vitro the patient's intestinal organoids, ex vivo the intestinal current measurements (ICM) method and assessment of the clinical status before and after targeted therapy. All obtained results are consistent with each other and have proven the effectiveness of ETI for the N1303K variant. ETI produced a significant positive effect on forskolin-induced swelling in N1303K/class I organoids indicating functional improvement of the CFTR protein; ICM demonstrated that ETI therapy restored CFTR function in the intestinal epithelium after three months of treatment, and the patient improved his clinical status and lung function, increased his body mass index (BMI) and reduced the lung pathogenic flora diversity, surprisingly without improving the sweat test results.

Advances in the Study of Common and Rare CFTR Complex Alleles Using Intestinal Organoids.

Complex alleles (CAs) arise when two or more nucleotide variants are present on a single allele. CAs of the CFTR gene complicate the cystic fibrosis diagnosis process, classification of pathogenic variants, and determination of the clinical picture of the disease and increase the need for additional studies to determine their pathogenicity and modulatory effect in response to targeted therapy. For several different populations around the world, characteristic CAs of the CFTR gene have been discovered, although in general the prevalence and pathogenicity of CAs have not been sufficiently studied. This review presents examples of using intestinal organoid models for assessments of the two most common and two rare CFTR CAs in individuals with cystic fibrosis in Russia.

Clinical and Functional Characteristics of the E92K CFTR Gene Variant in the Russian and Turkish Population of People with Cystic Fibrosis.

The pathogenic variant E92K (c.274G > A) of the CFTR gene is rare in America and Europe, but it is common for people with cystic fibrosis from Russia and Turkey. We studied the effect of the E92K genetic variant on the CFTR function. The function of the CFTR channel was studied using the intestinal current measurements (ICM) method. The effects of CFTR modulators on the restoration of the CFTR function were studied in the model of intestinal organoids. To assess the effect of E92K on pre-mRNA splicing, the RT-PCR products obtained from patients' intestinal organoid cultures were analyzed. Patients with the genetic variant E92K are characterized by an older age of diagnosis compared to homozygotes F508del and a high frequency of pancreatic sufficiency. The results of the sweat test and the ICM method showed partial preservation of the function of the CFTR channel. Functional analysis of CFTR gene expression revealed a weak effect of the E92K variant on mRNA-CFTR splicing. Lumacaftor (VX-809) has been shown to restore CFTR function in an intestinal organoid model, which allows us to consider the E92K variant as a promising target for therapy with CFTR correctors.

The Effect of Complex Alleles of the CFTR Gene on the Clinical Manifestations of Cystic Fibrosis and the Effectiveness of Targeted Therapy.

The authors of this article analyzed the available literature with the results of studying the prevalence of complex alleles of the CFTR gene among patients with cystic fibrosis, and their pathogenicity and influence on targeted therapy with CFTR modulators. Cystic fibrosis (CF) is a multisystemic autosomal recessive disease caused by a defect in the expression of the CFTR protein, and more than 2000 genetic variants are known. Clinically significant variants are divided into seven classes. Information about the frequency of complex alleles appears in a number of registers, along with the traditional presentation of data on genetic variants. Complex alleles (those with the presence of more than two nucleotide variants on one allele) can complicate the diagnosis of the disease, and change the clinical manifestations of cystic fibrosis and the response to treatment, since each variant in the complex allele can contribute to the functional activity of the CFTR protein, changing it both in terms of increasing and decreasing function. The role of complex alleles is often underestimated, and their frequency has not been studied. At the moment, characteristic frequently encountered complex alleles have been found for several populations of patients with cystic fibrosis, but the prevalence and pathogenicity of newly detected complex alleles require additional research. In this review, more than 35 complex alleles of the CFTR gene from existing research studies were analyzed, and an analysis of their influence on the manifestations of the disease and the effectiveness of CFTR modulators was also described.

Composition of nutrient media and temperature of cultivation imposes effect on the content of secondary metabolites of Nocardiopsis sp. isolated from a Siberian Cave.

Growth of human population leads to many global and medical problems. The problems include the crisis of health, antibiotic resistance, drug discovery, etc. Increasing antimicrobial resistance of microorganisms results in the need to screen natural products (incl. antibiotics and antimicrobial peptides) and their producers in different ecological niches. The purpose of this study was to estimate antibiotic activity and biotechnological potential of rare actinobacteria Nocardiopsis sp. The strain was isolated from Okhotnichya cave located in Siberia. Here, we cultivated the strain at 3 temperature modes (13 °C, 28 °C, 37 °C) in 11 liquid nutrient (rich and poor) media. Using modern assays of liquid chromatography and high-resolution mass spectrometry, we estimated the content and number of produced natural products, distribution of their masses, and potential rate of novel secondary metabolites. We demonstrated that minimal nutrient media with l-asparagine and SM25 media with malt extract were less productive at current experimental parameters. As it was shown, this strain was characterized by antibiotic properties against Bacillus subtilis when cultivated at 28 °C. Also, weak antibiotic activity of crude extracts was found in strain cultivation at 13 °C. Also, we detected a high number of novel amphiphilic and hydrophobic NPs produced by this strain. We demonstrated both the influence of the nutrient media composition and cultivation temperature on biosynthetic capabilities of rare strain Nocardiopsis sp. Finally, high level of natural products that were predicted as novel confirms high biotechnological value of rare genera of Actinobacteria that could be explained by the evolution of microorganisms in the isolated environment of cave ecosystem.

Cultivable Actinobacteria First Found in Baikal Endemic Algae Is a New Source of Natural Products with Antibiotic Activity.

Inadequate use of antibiotics has led to spread of microorganisms resistant to effective antimicrobial compounds for humans and animals. This study was aimed to isolate cultivable strains of actinobacteria associated with Baikal endemic alga Draparnaldioides baicalensis and estimate their antibiotic properties. During this study, we isolated both widespread and dominant strains related to the genus Streptomyces and representatives of the genera Saccharopolyspora, Nonomuraea, Rhodococcus, and Micromonospora. For the first time, actinobacteria belonging to the genera Nonomuraea and Saccharopolyspora were isolated from Baikal ecosystem. Also, it was the first time when actinobacteria of the genus Nonomuraea were isolated from freshwater algae. Some rare strains demonstrated activity inhibiting growth of bacteria and yeasts. Also, it has been shown that the strains associated with Baikal alga D. baicalensis are active against both Gram-positive and Gram-negative bacteria. According to this study and previously published materials, diversity of cultivable actinobacteria and rare strains isolated from D. baicalensis is comparable to that of cultivable actinobacteria previously isolated from plant sources of Lake Baikal. Also, it exceeds the cultivable actinobacteria diversity previously described for macroinvertebrates, water, or sediments of Lake Baikal. The large number of rare and active strains associated with the endemic alga D. baicalensis could be the promising sources for biopharmaceutical and biotechnological developments and discovery of new natural compounds.

Drug susceptibility testing of slowly growing non-tuberculous mycobacteria using slomyco test-system.

OBJECTIVE: The objective of the research was to assess the susceptibility of the slowly growing nontuberculous mycobacteria strains to the antimicrobial drugs used for mycobaterioses treatment using SLOMYCO test system. MATERIALS AND METHODS: We assessed 363 NTM strains: 177 MAC (161 M. avium, 16 M. intracellulare), 112 M. kansasii and 74 M. xenopi collected from the respiratory material of the patients were under the treatment or under diagnostic procedures at our Center, affiliates and the diagnostic department in 2010-2016. Drug sucseptibility for NTM was tested using the Sensititre SLOWMYCO system (TREK DIAGNOSTIC Systems Ltd., UK). MICs were established by microdilutions in Mueller-Hinton broth on polystyrene 96-well plates. The statistical analysis was done using the StatGraphics Plus 5.0 software. The data were compared pairwise using Pearson χ2 test with Yates correction. 95% confidence interval (CI) were calculated. Statistically significant differences were considered for p <0.05. Log-rank test and Kaplan-Meier curves were used to assess the concentration-dependent surveillance probability. RESULTS: The statistically significant differences were revealed in sensitivity/resistance isolates of M. avium and M. intracellulare: M. avium strains were resistant to higher concentrations of amikacin, clarithromycin, linezolid and streptomycin (p <0.01); M. intracellulare strains were resistant to higher concentrations of ethionamide (p <0.05). The isolates of M. avium were significantly more resistant than M. kansasii to amikacin, doxycycline, isoniazid, clarithromycin, linezolid, moxifloxacin, rifabutin, rifampicin, streptomycin, trimethoprim/sulfamethoxazole, ciprofloxacin, ethambutol, ethionamide (visible growth of M. avium were inhibited by higher drug concentrations, p <0.01). The isolates of M. avium showed significantly higher resistance than M. xenopi to amikacin, doxycycline, isoniazid, clarithromycin, linezolid, moxifloxacin, rifampicin, streptomycin, trimethoprim/sulfamethoxazole, ciprofloxacin, ethambutol, and ethionamide (visible growth of M. avium were inhibited by higher drug concentrations, p <0.01). Statistically significant differences in the dynamics of the response to the antibacterial effects of isoniazid, linezolid, moxifloxacin, rifampicin, trimethoprim/sulfamethoxazole, ethambutol, and ethionamide were found for M. intracellulare and M. xenopi (complete inhibition of the visible growth of M. intracellulare required higher drugs concentrations, p <0, 05). Comparison of the Kaplan-Meyer curves revealed statistically significant differences in survialence probability of M. kansasii and M. xenopi for amikacin, doxycycline, rifampicin, trimethoprim/sulfamethoxazole, ciprofloxacin, ethambutol, and ethionamide (a higher number of isolates of M. xenopi were inhibited by low drugs concentrations, p <0.05). CONCLUSIONS: Our data show that M. avium and M. intracellulare were more resistant to the majority of the studied drugs than M. kansasii and M. xenopi.

A Comparison of the Sensititre MycoTB Plate, the Bactec MGIT 960, and a Microarray-Based Molecular Assay for the Detection of Drug Resistance in Clinical Mycobacterium tuberculosis Isolates in Moscow, Russia.

BACKGROUND: The goal of this study was to compare the consistency of three assays for the determination of the drug resistance of Mycobacterium tuberculosis (MTB) strains with various resistance profiles isolated from the Moscow region. METHODS: A total of 144 MTB clinical isolates with a strong bias toward drug resistance were examined using Bactec MGIT 960, Sensititre MycoTB, and a microarray-based molecular assay TB-TEST to detect substitutions in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis, and embB genes that are associated with resistance to rifampin, isoniazid, fluoroquinolones, second-line injectable drugs and ethambutol. RESULTS: The average correlation for the identification of resistant and susceptible isolates using the three methods was approximately 94%. An association of mutations detected with variable resistance levels was shown. We propose a change in the breakpoint minimal inhibitory concentration for kanamycin to less than 5 µg/ml in the Sensititre MycoTB system. A pairwise comparison of the minimal inhibitory concentrations (MICs) of two different drugs revealed an increased correlation in the first-line drug group and a partial correlation in the second-line drug group, reflecting the history of the preferential simultaneous use of drugs from these groups. An increased correlation with the MICs was also observed for drugs sharing common resistance mechanisms. CONCLUSIONS: The quantitative measures of phenotypic drug resistance produced by the Sensititre MycoTB and the timely detection of mutations using the TB-TEST assay provide guidance for clinicians for the choice of the appropriate drug regimen.

Evaluation of a low-density hydrogel microarray technique for mycobacterial species identification.

In addition to the obligatory pathogenic species of the Mycobacterium tuberculosis complex and Mycobacterium leprae, the genus Mycobacterium also includes conditionally pathogenic species that in rare cases can lead to the development of nontuberculous mycobacterial diseases. Because tuberculosis and mycobacteriosis have similar clinical signs, the accurate identification of the causative agent in a clinical microbiology laboratory is important for diagnostic verification and appropriate treatment. This report describes a low-density hydrogel-based microarray containing oligonucleotide probes based on the species-specific sequences of the gyrB gene fragment for mycobacterial species identification. The procedure included the amplification of a 352-nucleotide fragment of the gene and its hybridization on a microarray. The triple-species-specific probe design and the algorithm for hybridization profile recognition based on the calculation of Pearson correlation coefficients, followed by the construction of a profile database, allowed for the reliable and accurate identification of mycobacterial species, including mixed-DNA samples. The assay was used to evaluate 543 clinical isolates from two regions of Russia, demonstrating its ability to detect 35 mycobacterial species, with 99.8% sensitivity and 100% specificity when using gyrB, 16S, and internal transcribed spacer (ITS) fragment sequencing as the standard. The testing of clinical samples showed that the sensitivity of the assay was 89% to 95% for smear-positive samples and 36% for smear-negative samples. The large number of identified species, the high level of sensitivity, the ability to detect mycobacteria in clinical samples, and the up-to-date profile database make the assay suitable for use in routine laboratory practice.