Reduced susceptibility and resistance to bedaquiline in clinical M. tuberculosis isolates.

OBJECTIVES: Bedaquiline is an effective drug used to treat MDR and XDR tuberculosis, providing high cure rates in complex therapy. Mutations in the mmpR (rv0678) and atpE genes are associated with reduced susceptibility to bedaquiline and have been identified in both in vitro selected strains and clinical isolates. However, the phenotypic criteria used to detect bedaquiline resistance have yet to be established due to the collection of few clinical isolates from patients receiving bedaquiline-containing treatment regimens. METHODS: One hundred eighty-two clinical isolates from 74 patients receiving bedaquiline and 163 isolates from 107 patients not exposed to bedaquiline were analysed. The bedaquiline MICs were tested using serial dilutions on 7H11 agar plates and the Bactec MGIT 960 system. The mmpR and atpE genes were sequenced by Sanger sequencing. RESULTS: The 7H11 agar method allowed for rapid discrimination between mutated and wild-type isolates and between exposed and non-exposed isolates. Seventy-three percent of bedaquiline-exposed isolates, as well as 91% of isolates with mutations, had an elevated bedaquiline MIC (≥ 0.12 mg/L on 7H11 media) compared to the reference isolates (89% had an MIC ≤ 0.03 mg/L). Previously reported in vitro-selected mutants (E61D and A63P) and novel AtpE substitutions (G25S and D28G) were observed in the clinical isolates. Substitutions in codon 63 of AtpE were likely associated with a higher bedaquiline MIC. Five new cases of pre-existing reduced susceptibility to bedaquiline, accompanied by mmpR mutations in most isolates, without a history of bedaquiline treatment were identified. CONCLUSIONS: Bedaquiline treatment leads to an elevated bedaquiline MIC and the acquisition of mmpR and atpE gene mutations in tuberculosis strains. The standardisation of bedaquiline phenotypic susceptibility testing is urgently needed based on observed discrepancies between our study and previous studies and differences in solid and liquid media MIC determinations.

Redefining MDR-TB: Comparison of Mycobacterium tuberculosis clinical isolates from Russia and Taiwan.

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are global challenges due to the limited number of effective drugs for treatment. Treatment with less than 4-5 effective drugs might lead to the further emergence of drug resistance and poor clinical outcomes. For better prediction of treatment outcomes, we compared drug-resistance profiles of consecutive clinical MDR Mycobacterium tuberculosis isolates from high- and low-burden settings. This was a retrospective cohort study. We analysed 225 and 229 MDR isolates from Moscow (Russia) and Taiwan, respectively, obtained between 2014 and 2015. Drug susceptibility testing was performed by the Bactec MGIT 960 automated system and the agar proportion method. Detection of resistance-associated mutations in the M. tuberculosis genome was carried out by an array and/or sequencing of selected loci. The principal differences between resistance profiles of MDR isolates in the two countries were the percentages of pre-XDR (40.9% vs. 14.8%) and XDR (34.7% vs. 1.7%) isolates, both of which were significantly higher in Moscow isolates. Forty-eight (33%) of 147 MDR and pre-XDR Russian isolates fall into a group with less than four effective drugs, which accounts for 40% (N = 120) of these isolates. The other 60% in this group were XDR strains (N = 72). Consequently, the average number of effective anti-tuberculosis drugs for MDR-TB treatment was lower for Russian isolates (3 vs. 7). Furthermore, a notable percentage (9%) of isolates resistant to kanamycin harboured mutations in the whiB7 locus, which was not detected by molecular tests targeting common mutations in the rrs and eis loci. We found that 98.2% and 45.9% of MDR isolates from Moscow and Taiwan, respectively, were resistant to streptomycin. Molecular tests for detecting resistance to drugs other than rifampicin, isoniazid, fluoroquinolones, and second-line injectable drugs are needed for individualized therapy. The conventional MDR treatment schemes most probably fail in these cases due to the limited number of effective drugs.

Examination of bedaquiline- and linezolid-resistant Mycobacterium tuberculosis isolates from the Moscow region.

Objectives: To study the isolates with acquired resistance to bedaquiline and linezolid that were obtained from patients enrolled in a clinical study of a novel therapy regimen for drug-resistant TB in Moscow, Russia. Methods: Linezolid resistance was detected using MGIT 960 with a critical concentration of 1 mg/L. The MIC of bedaquiline was determined using the proportion method. To identify genetic determinants of resistance, sequencing of the mmpR ( Rv0678 ), atpE , atpC , pepQ , Rv1979c , rrl , rplC and rplD loci was performed. Results: A total of 85 isolates from 27 patients with acquired resistance to linezolid and reduced susceptibility to bedaquiline (MIC ≥0.06 mg/L) were tested. Most mutations associated with a high MIC of bedaquiline were found in the mmpR gene. We identified for the first time two patients whose clinical isolates had substitutions D28N and A63V in AtpE, which had previously been found only in in vitro -selected strains. Several patients had isolates with elevated MICs of bedaquiline prior to treatment; four of them also bore mutations in mmpR , indicating the presence of some hidden factors in bedaquiline resistance acquisition. The C154R substitution in ribosomal protein L3 was the most frequent in the linezolid-resistant strains. Mutations in the 23S rRNA gene (g2294a and g2814t) associated with linezolid resistance were also found in two isolates. Heteroresistance was identified in ∼40% of samples, which reflects the complex nature of resistance acquisition. Conclusions: The introduction of novel drugs into treatment must be accompanied by continuous phenotypic susceptibility testing and the analysis of genetic determinants of resistance.

Simultaneous drug resistance detection and genotyping of Mycobacterium tuberculosis using a low-density hydrogel microarray.

BACKGROUND: Nucleic acid amplification tests are widely used in TB diagnostics. Priority tasks in their development consist of increasing the specificity and sensitivity of the detection of resistance to a wide spectrum of anti-TB drugs. METHODS: We developed a multiplexed assay allowing the detection of 116 drug resistance-determining mutations in the rpoB, katG, inhA, ahpC, gyrA, gyrB, rrs, eis and embB genes in the Mycobacterium tuberculosis complex genome and six SNPs to identify the main lineages circulating in Russia. The assay is based on the amplification of 17 fragments of the genome using the universal primer adapter technique and heat pulses at the elongation step, followed by hybridization on a microarray. RESULTS: The method was evaluated using 264 pairs of clinical samples and corresponding isolates. A significant proportion (25%) of smear-negative samples were correctly analysed by microarray analysis in addition to 96% of smear-positive samples. The sensitivity and specificity of the assay exceeded 90% for rifampicin, isoniazid, ofloxacin and second-line injection drugs. In agreement with previous studies, the specificity of ethambutol resistance was as low as 57%, while the sensitivity was 89.9%. Strong association of the Beijing lineage with a resistant phenotype was observed. Euro-American lineage strains, excluding Ural and LAM, were predominantly associated with the susceptible phenotype. CONCLUSIONS: The developed test has a high sensitivity and specificity and can be directly applied to clinical samples. The combination of mutation-based drug resistance profiling and basic genotyping could be useful for clinical microbiology studies and epidemiological surveillance of the M. tuberculosis complex.

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.

Spoligotyping of Mycobacterium tuberculosis complex isolates using hydrogel oligonucleotide microarrays.

Mycobacterium tuberculosis remains a leading cause of morbidity and mortality worldwide. This circumstance underscores the relevance of population studies of tuberculosis for transmission dynamics control. In this study, we describe a conversion of the spoligotyping of M.tuberculosis complex isolates on a platform of custom designed hydrogel microarrays (biochips). An algorithm of automated data processing and interpretation of hybridization results using online database was proposed. In total, the 445 samples were tested. Initially, 97 samples representing multiple species of M.tuberculosis complex and nontuberculous mycobacteria were used for protocol optimization and cut-off settings. The developed assay was further evaluated on the out-group of the 348 mycobacterial samples. Results showed high concordance with the conventional membrane-based spoligotyping method. Diagnostic sensitivity and diagnostic specificity of the spoligo-biochip assay were 99.1% and 100%, respectively. The analytical sensitivity was determined to be 500 genomic equivalents of mycobacterial DNA. The high sensitivity and specificity, ease of operation procedures, and the automatic processing of measured data make the developed assay a useful tool for the rapid and accurate genotyping of M. tuberculosis.

Analysis of a novel 8.9kb cryptic plasmid from Bacteroides uniformis, its long-term stability and spread within human microbiota.

The analysis of plasmid content in dominant Bacteroidales order intestinal strains isolated from the same child at a 5 year interval identified a 8.9 kb plasmid in Bacteroides uniformis BUN24 strain isolated at age 6 and indistinguishably sized plasmids in the isolates of B. uniformis, B. vulgatus, B. intesinalis, and Parabacteroides distasonis at age 11. We sequenced a B. uniformis BUN24 plasmid, designated pBUN24, and using molecular surveys of diverse species we established that this 8944bp molecule (G+C content 43.5%) represents a novel family of small cryptic Bacteroidales plasmids. The replication region of pBUN24 was experimentally localized to a 1707-bp fragment that includes a putative repA gene, coding for a protein of Rep_3 superfamily of replication proteins of theta-type plasmids preceded by a putative iteron-containing origin of replication. The other open reading frames (ORFs) identified in pBUN24 sequence include a putative tad-ata-type toxin-antitoxin and mobA-mobB mobilization modules, as well as seven additional cryptic ORFs. The interaction of Tad and Ada components demonstrated by a pull-down assay and the toxicity of Tad in Escherichia coli host suggests the functionality of the plasmid addiction module. Re-sequencing of plasmids in two Bacteroides strains isolated at the age of 11 showed 100% nucleotide identity to pBUN24. This data supports the notion that this plasmid is transmissible to other Bacteroidales strains in the natural ecosystem. The possible roles of toxin-antitoxin system and other proteins encoded by pBUN24 in providing an apparent ecological advantage to the plasmid-harbouring strains of a bacterial symbiont in the human gut deserve further investigation.

Species composition of Bacteroidales order bacteria in the feces of healthy people of various ages.

A study of species distribution of numerically predominant Bacteroidales order isolates in feces of healthy people aged 1-33 years was accomplished using a combination of amplified ribosomal DNA restriction analysis (ARDRA) and 16S ribosomal DNA (rDNA) sequencing. It was found that the majority of isolates in all age groups belonged to species B. xylanisolvens, B. vulgatus, and B. uniformis. Members of genera Alistipes, Parabacteroides, Odoribacter, Barnesiella, and Prevotella were also detected frequently.

Application of several molecular techniques to study numerically predominant Bifidobacterium spp. and Bacteroidales order strains in the feces of healthy children.

Bifidobacteria and Bacteroides-like bacteria are strictly anaerobic nonpathogenic members of human intestinal microflora. Here we describe an analysis of the species and subspecies composition of these bacterial populations in healthy children using a combination of culture and molecular methods at two different time points. It was found that B. bifidum and B. longum are the most common dominant taxons in infants aged between 8 and 16 months. The majority of the infants carried several dominant Bifidobacterium strains belonging to different species. Examination of the dominant bifidoflora in some of these children after a 5-year period showed major shifts in both species and strain composition, but the dominant strains remained unchanged in two children. The majority of dominant Bacteroides-like isolates belonged to species B. vulgatus and B. uniformis, but members of genera Alistipes and Barnesiella were common too. In addition, a novel approach to species identification of Bacteroidales order bacteria using amplified ribosomal DNA restriction analysis (ARDRA) is described.