Clinical Isolates Of Mycobacteria Research Articles

Evaluation of nucleotide MALDI-TOF-MS for the identification of Mycobacterium species.

The accurate identification of the Mycobacterium tuberculosis complex (MTBC) and different nontuberculous mycobacteria (NTM) species is crucial for the timely diagnosis of NTM infections and for reducing poor prognoses. Nucleotide matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been extensively used for microbial identification with high accuracy and throughput. However, its efficacy for Mycobacterium species identification has been less studied. The objective of this study was to evaluate the performance of nucleotide MALDI-TOF-MS for Mycobacterium species identification. A total of 933 clinical Mycobacterium isolates were preliminarily identified as NTM by the MPB64 test. These isolates were identified by nucleotide MALDI-TOF-MS and Sanger sequencing. The performance of nucleotide MALDI-TOF MS for identifying various Mycobacterium species was analyzed based on Sanger sequencing as the gold standard. The total correct detection rate of all 933 clinical Mycobacterium isolates using nucleotide MALDI-TOF-MS was 91.64% (855/933), and mixed infections were detected in 18.65% (174/933) of the samples. The correct detection rates for Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium avium, MTBC, Mycobacterium gordonae, and Mycobacterium massiliense were 99.32% (585/589), 100% (86/86), 98.46% (64/65), 94.59% (35/37), 100.00% (34/34), 95.65% (22/23), and 100% (19/19), respectively. For the identification of the MTBC, M. intracellulare, M.abscessus, M. kansasii, M. avium, M. gordonae, and M. massiliense, nucleotide MALDI-TOF-MS and Sanger sequencing results were in good agreement (k > 0.7). In conclusion, nucleotide MALDI-TOF-MS is a promising approach for identifying MTBC and the most common clinical NTM species.

MIXED POPULATIONS OF MYCOBACTERIA IN PATIENTS WITH TUBERCULOSIS AND MYCOBACTERIOSIS: FREQUENCY OF DETECTION AND SPECTRUM OF SPECIES

Introduction. Mixed mycobacterial infections are an urgent problem for phthisiology and pulmonology, which has not been studied enough. The aim of the study was to assess the frequency of detection of mixed populations of mycobacteria in patients with tuberculosis and mycobacteriosis and to determine the species composition of these populations. Materials and methods. Clinical isolates of mycobacteria isolated from 5531 patients who were treated or who sought advice from the FSBI «TSNIIT» were studied. The culture was isolated in the BACTEC MGIT 960 automatic crop growth registration system, species identification was carried out by molecular genetic methods using the GenoType Mycobacterium CM/AS kit (Hain Livescience, Germany) and/or a set based on multiplex PCR in real time. The conclusion about the presence of a mixed population was made if a mixture of two species of mycobacteria was isolated during the entire observation period or the species structure of the population changed dynamically. Descriptive statistics were used to evaluate the results of the study, the chi-squared criterion was used to determine significant differences between groups; differences were considered statistically significant at p < 0.05. The entire analysis was carried out using Microsoft Excel (Microsoft, USA). Results. The frequency of detection of mixed mycobacterial infection by culture method in patients of phthisiological institutions for the period 2011-2023 was 1.16%. The most common combinations of species were M. tuberculosis + M. avium and M. avium + M. intracellulare.

New design of multilocus sequence analysis of rpoB, ssrA, tuf, atpE, ku, and dnaK for identification of Mycobacterium species.

Differentiating Mycobacterium tuberculosis (MTB) from nontuberculous mycobacteria (NTM) is very important in the treatment process of patients. According to the American Thoracic Society guideline (ATS), NTM clinical isolates should be identified at the species level proper treatment and patient management. This study aimed to identify NTM clinical isolates by evaluationg rpoB, ssrA, tuf, atpE, ku, and dnaK genes, and use multilocus sequence analysis (MLSA) to concatenate the six genes. Ninety-six Mycobacterium isolates, including 86 NTM and 10 MTB isolates, from all the patients referred to the certain TB Reference Centres were included. All isolates were evaluated by PCR amplification of rpoB, ssrA, tuf, ku, atpE, and dnaK genes and MLSA. Out of 96 isolates, 91 (94.8%), 87 (90.6%), 72 (75%), 84 (87.5%) and 79 (82.3%) were differentiated to the species level by rpoB, tuf, ssrA, dnaK and atpE genes, respectively. The ku gene was able to identify 69 (80.2%) isolates of the 86 NTM isolates to the species level. We could identify 100% of the isolates to the species level by MLSA. None of the PCR targets used in this study were able to completely differentiate all species. The MLSA technique used to concatenate the six genes could increase the identification of clinical Mycobacterium isolates and all 16 species were well-differentiated.

Mycobacterium avium complex infected cells promote growth of the pathogen Pseudomonas aeruginosa

Bronchiectasis is considered a consequence of the neutrophilic inflammatory response to infection. Mycobacterial infections, mainly from the Mycobacterium avium complex and M. abscessus, have been inextricably linked to bronchiectasis development. The most important pathogen that infect patients with bronchiectasis is Pseudomonas aeruginosa, associated with an increased risk of death. Patients with bronchiectasis are often co-infected with P. aeruginosa and M. avium complex, and it was studied whether they interacted in immune cell cultures. Peripheral blood mononuclear cells from healthy volunteers were infected overnight with clinical isolates of mycobacteria, 18 h later co-infected with P. aeruginosa and Pseudomonas multiplication was quantified. Inoculated P. aeruginosa multiply faster when cells were previously infected in vitro with M. avium complex or M. tuberculosis, but not with M. kansasii or M. gordonae, mycobacteria not regularly isolated from patients with bronchiectasis. The interaction between mycobacteria and P. aeruginosa also takes place in the absence of cells, but to a lower degree. Growth of Staphylococcus aureus, less frequently co-isolated with mycobacteria, was not affected by previous infection with mycobacteria. Surprisingly, multiplication of P. aeruginosa in neutrophil cultures did not vary in the presence of mycobacteria. Nevertheless, co-infection of mycobacteria and P. aeruginosa induced the production of IL-1β, a mediator of neutrophilic inflammation. P. aeruginosa stimulation by mycobacteria provides evidence for explaining their common clinical association. Strategies to control mycobacteria may be useful to impair P. aeruginosa colonization.

The Mycobacterial Efflux Pump EfpA Can Induce High Drug Tolerance to Many Antituberculosis Drugs, Including Moxifloxacin, in Mycobacterium smegmatis

Active efflux of drugs across the membrane is a major survival strategy of bacteria against many drugs. In this work, we characterize an efflux pump, EfpA, from the major facilitator superfamily, that is highly conserved among both slow-growing and fast-growing Mycobacterium species and has been found to be upregulated in many clinical isolates of Mycobacterium tuberculosis. The gene encoding EfpA from Mycobacterium smegmatis was overexpressed under the control of both a constitutive and an inducible promoter. The expression of the efpA gene under the control of both promoters resulted in >32-fold-increased drug tolerance of M. smegmatis cells to many first-line (rifampicin, isoniazid, and streptomycin) and second-line (amikacin) antituberculosis drugs. Notably, the drug tolerance of M. smegmatis cells to moxifloxacin increased by more than 180-fold when efpA was overexpressed. The increase in MICs correlated with the decreased uptake of drugs, including norfloxacin, moxifloxacin, and ethidium bromide, and the high MIC could be reversed in the presence of an efflux pump inhibitor. A correlation was observed between the MICs of drugs and the efflux pump expression level, suggesting that the latter could be modulated by varying the expression level of the efflux pump. The expression of high levels of efpA did not impact the fitness of the cells when supplemented with glucose. The efpA gene is conserved across both pathogenic and nonpathogenic mycobacteria. The efpA gene from Mycobacterium bovis BCG/M. tuberculosis, which is 80% identical to efpA from M. smegmatis, also led to decreased antimicrobial efficacy of many drugs, although the fold change was lower. When overexpressed in M. bovis BCG, 8-fold-higher drug tolerance to moxifloxacin was observed. This is the first report of an efflux pump from Mycobacterium species that leads to higher drug tolerance to moxifloxacin, a promising new drug for the treatment of tuberculosis.

Characterization of Clinical Isolates of Mycobacterium simiae Using Drug Susceptibility Tests and Molecular Analyses.

Mycobacterium simiae is an emerging nontuberculous mycobacterium (NTM) and an opportunistic pathogen which is described mainly in Asia and presents in the environment that can cause pulmonary infection. The objective of this study is to characterize M. simiae clinical isolates using mycobacterial interspersed repetitive unit variable-number tandem repeats (MIRU-VNTR) typing for the differentiation of the strains. A total of 169 clinical isolates of NTM were recovered from patients suspected of having tuberculosis (TB)-like and related infections. After isolation and identification of mycobacterial strains by conventional biochemical and PCR-based tests, M. simiae strains were confirmed using restriction fragment length polymorphism (RFLP)-based identification assay. Furthermore, drug susceptibility and MIRU-VNTR typing was performed using on the clinical isolates of M. simiae. Out of 169 NTM strains, 92 (54.4%) isolates were identified as M. simiae. Antibiotic susceptibility experiments indicated that all 92 M. simiae isolates were resistant to first line antimycobacterial agents. Moreover, 8 (8.6%) M. simiae isolates were resistant to ciprofloxacin; and 6 (6.5%) were resistant to both amikacin and kanamycin, while the remaining were susceptible to second line antimycobacterial agents. MIRU-VNTR analysis showed that the M. simiae isolates were classified in four distinct M. simiae clusters and two single types. The minimum spanning analysis revealed that the isolates were grouped in three complexes. The data suggested that MIRI-VNTR typing is useful for typing of M. simiae isolates, however, MIRU-16 locus was absolutely absent in M. simiae.

Resistance To First-Line Antituberculosis Drugs And Prevalence Of pncA Mutations In Clinical Isolates Of Mycobacterium tuberculosis From Zunyi, Guizhou Province Of China.

BackgroundChina is one of the high-burden countries for multidrug-resistant tuberculosis (MDR-TB), and pyrazinamide is one of the anti-TB drugs used for the shorter MDR-TB treatment regimen. The aim of this study was to determine the correlation between pncA gene mutations and resistance to four first-line anti-TB drugs as well as treatment history in clinical isolates of Mycobacterium tuberculosis.Patients and methodsM. tuberculosis clinical isolates were collected from 318 in-patients with smear-positive TB between October 2008 and September 2016 at a major hospital in Zunyi, Guizhou Province of China, and used for drug susceptibility testing against four first-line anti-TB drugs. Genomic DNA extracted from clinical isolates was used for PCR amplification and DNA sequencing of the pncA gene.ResultsAmong 318 clinical isolates, 129 (40.6%), 170 (53.5%), 66 (20.8%) and 109 (34.3%) were resistant to rifampicin, isoniazid, ethambutol and streptomycin respectively. In addition, 124 clinical isolates were MDR-TB and 71.8% of them were previously treated cases. Sequencing results showed that 46.8% of MDR-TB and 2.2% of drug susceptible isolates harbored a pncA mutation, and 52 types of pncA mutations were detected from 64 isolates. The prevalence of pncA mutations in isolates resistant to first-line anti-TB drugs and previously treated TB cases was significantly higher than that in drug-susceptible isolates and new cases of TB.ConclusionHigh prevalence of pncA mutations in clinical isolates of M. tuberculosis from Zunyi, Guizhou Province of China, is correlated with resistance to four first-line anti-TB drugs, MDR-TB and previously treated TB cases.

Experience of selection, indication and identication of non-tuberculous mycobacteria, got during I National research on study of distribution of drugresistant of tuberculosis in Ukraine

Objective — a study of presence of non-tuberculosis mycobacteriumin a circulatory mycobacterial population among patients by a white plague in Ukraine, finding out of etiologic role of non-tuberculosis mycobacterium, their selection and identification, study of some morphological and biological properties.Materials and methods. The strokes of sputum were investigational in the presence of acid proof bacteria by a direct microscopy after coloring on the method of Ziehl—Neelsen. For all standards with the positive results of microscopy all spectrum of bacteriologicexaminations was executed on diagnostics of tubercu­losis, according to the algorithm ratified by the order of MH of Ukraine N 620. Bacteriological studies were undertaken on identification of clinical isolates on standard methodologies that is expounded in the order of MH of Ukraine N 45. Specific identification of the got cultures consisted of two stages: primary and final.In process for sowing environments were used: liquid — broth of Middlebrook 7Н9 and dense egg Lowenstein—Jensen medium. Pressed treatment of clinical material was carried out by means of a 0,5 % sterile solution of N-acetyl-L-of cystein from 1.0 % gidroxid of natrium (BBL Mycoprep NALC-NAOH). Test tubes placed in the system ВАСТЕС МGIТ 960 and did incubation. Sowing was carried out in parallel on the dense environment of Lowenstein—Jensen medium. For identification of the distinguished strains ID МТВ MGIT test used and determination of ability of height of clinical isolates of mycobacterium on an environment with a salicylic sour natrium and p-nitrodracylic acid with the purpose of determination of belonging of distinguished mycobacterium to the tuberculosis complex.For realization of final identification of mycobacterium used the system GenoType® MTB DR (Нain, Laifsains), that allowed to define the type of non-tuberculosis mycobacterium.Results and discussion. During Ist of National research on the study of distribution of drug resistant of tuberculosis1598 clinical isolates of mycobacterium were got in Ukraine. The analysis of results of bacteriologicexamination educed, that 48 (2.9 %) clinical isolates had been identified as non-tuberculosis mycobacterium. A chart over of primary authentication of non-tuberculosis mycobacteriumis brought.Nonphotochromogen mycobacterium prevailed in the structure of distinguished non-tuberculosis mycobacterium– 77.1 %. Photochromogen mycobacterium distinguished at level — 16.7 %, fast-growing mycobacterium was made the least — 6.3 %.For determination of specific belonging of non-tuberculosis mycobacterium used the system GenoType® MTB DR (Нain, Laifsains). Next non-tuberculosis mycobacterium were distinguished: M. kansasii, M. xenopi, M. avium, M. fortuitum. From 48 distinguished strains 20 strains were the clean cultures of non-tuberculosis mycobacterium (41.7 %), 28 strains (58.3 %) were in mix (М. tuberculosis + non-­tuberculosis mycobacterium). The results of researches showed that more often from the sputum of patients M. avium was distinguished in mix with М. tuberculosis — 39.6 %, in the clean culture of M. avium was distinguished in 20.8 % cases. Minimum was specific gravity of selection of M. fortuitum — 6.25 %. Here found out reliable difference was between prevalence of the distinguished strains of M. avium and other strains of non-tuberculosis mycobacterium.Conclusions. During Ist of National research on study of distribution of drug resistant of tuberculosis 48 strains of mycobacterium (2.9 %) that were identified as non-tuberculosis mycobacterium were got in Ukraine. Thus in 20 cases (1.2 %) were found out the clean cultures of non-tuberculosis mycobacterium. In 28 (1.7 %) cases were discovered in mix, that contained as M. tuberculosis, so non-tuberculosis mycobac­terium. Authors are bringing a chart over of primary identification of non-tuberculosis mycobacterium. Nonphotochromogen mycobacterium prevailed in the structure of non-tuberculosis mycobacterium — 77.1 %. Photochromogen mycobacterium distinguished at level — 16.7 %, fast-growing mycobacterium was made the least — 6.3 %. For determination of specific belonging of non-tuberculosis mycobacterium used the system GenoType® MTB DR (Нain, Laifsains). Next non-tuberculosis mycobacterium were distinguished: M. kansasii, M. xenopi, M. avium, M. fortuitum. The results of researches showed that more often from the patient’s sputum M. avium was distinguished. In mix with М. tuberculosis – 39.6 %, in the clean culture of M. avium was distinguished in 20.8 % cases. Minimum was specific gravity of selection of M. fortuitum — 6.25 %. Here found out a reliable difference was between prevalence of the distinguished strains of M. avium and other strains of non-tuberculosis mycobacterium.

Simple Identification of Mycobacterial Species by Sequence-Specific Multiple Polymerase Chain Reactions.

Several species of mycobacteria cause infections in humans. Species identification of clinical isolates of mycobacteria is very important for the decision of treatment and in choosing the appropriate treatment regimen. We have developed a multiplex PCR method that can identify practically all known species of mycobacteria, by determination of single-nucleotide differences at a total of 13 different polymorphic regions in the genes of rRNA and hsp65, in four PCR mixes. To achieve this goal, single-nucleotide differences in these polymorphic regions were used to divide mycobacterial species into two groups, than four, eight, etc., in an algorithmic manner. It was sufficient to reach single species level by evaluating 13 polymorphic regions. Evaluation of the multiplex PCR patterns by observable real-time electrophoresis (ORTE) simplified species identification. This new method may enable easy, rapid, and cost-effective identification of all species of mycobacteria.

Matrix Assisted Laser Desorption Ionization-Time-of-Flight mass spectrometry identification of Mycobacterium bovis in Bovinae.

In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.

In Vitro and In Vivo Activities of Contezolid (MRX-I) against Mycobacterium tuberculosis.

The in vitro activity of contezolid (MRX-I) against clinical isolates of Mycobacterium tuberculosis was evaluated using a microtiter broth dilution assay. MRX-I was as effective as linezolid (LZD) in vitro MRX-I and LZD were subsequently studied in BALB/c mice infected intranasally with M. tuberculosis Erdman. MRX-I and LZD at 100 mg/kg significantly reduced the bacterial load in lungs compared to the untreated early and late controls.

Evaluation of the Quantamatrix Multiplexed Assay Platform system for simultaneous detection of Mycobacterium tuberculosis and the rifampicin resistance gene using cultured mycobacteria

BackgroundThe differentiation of Mycobacterium tuberculosis complex (MTBC) from non-tuberculous mycobacteria (NTM) is of primary importance for infection control and the selection of anti-tuberculosis drugs. Up to date data on rifampicin (RIF)-resistant tuberculosis (TB) is essential for the early management of multidrug-resistant TB. The aim of this study was to evaluate the usefulness of a newly developed multiplexed, bead-based bioassay (Quantamatrix Multiplexed Assay Platform, QMAP) for the rapid differentiation of 23 Mycobacterium species including MTBC and RIF-resistant strains. MethodsA total of 314 clinical Mycobacterium isolates cultured from respiratory specimens were used in this study. ResultsThe sensitivity and specificity of the QMAP system for Mycobacterium species were 100% (95% CI 99.15–100%, p<0.0001) and 97.8% (95% CI 91.86–99.87%, p<0.0001), respectively. The results of conventional drug susceptibility testing and the QMAP Dual-ID assay were completely concordant for all clinical isolates (100%, 95% CI 98.56–100%). Out of 223 M. tuberculosis (MTB) isolates, 196 were pan-susceptible and 27 were resistant to RIF according to QMAP results. All of the mutations in the RIF resistance-determining region detected by the QMAP system were confirmed by rpoB sequence analysis and a REBA MTB-Rifa reverse blot hybridization assay. The majority of the mutations (n=26, 96.3%), including those missing wild-type probe signals, were located in three codons (529–534, 524–529, and 514–520), and 17 (65.4%) of these mutations were detected by three mutation probes (531TTG, 526TAC, and 516GTC). ConclusionsThe entire QMAP system assay takes about 3h to complete, while results from the culture-based conventional method can take up to 48–72h. Although improvements to the QMAP system are needed for direct respiratory specimens, it may be useful for rapid screening, not only to identify and accurately discriminate MTBC from NTM, but also to identify RIF-resistant MTB strains in positive culture samples.

Molecular Investigation of Resistance to Second-Line Injectable Drugs in Multidrug-Resistant Clinical Isolates of Mycobacterium tuberculosis in France.

The second-line injectable drugs (SLID, i.e., amikacin, kanamycin, capreomycin) are key drugs for the treatment of multidrug-resistant tuberculosis. Mutations in rrs region 1400, tlyA, and eis promoter are associated with resistance to SLID, to capreomycin, and to kanamycin, respectively. In this study, the sequencing data of SLID resistance-associated genes were compared to the results of phenotypic drug susceptibility testing by the proportion method for the SLID in 206 multidrug-resistant clinical isolates of Mycobacterium tuberculosis collected in France. Among the 153 isolates susceptible to the 3 SLID, 145 showed no mutation, 1 harbored T1404C and G1473A mutations in rrs, and 7 had an eis promoter mutation. Among the 53 strains resistant to at least 1 of the SLID, mutations in rrs accounted for resistance to amikacin, capreomycin, and kanamycin for 81%, 75%, and 44% of the isolates, respectively, while mutations in eis promoter were detected in 44% of the isolates resistant to kanamycin. In contrast, no mutations in tlyA were observed in the isolates resistant to capreomycin. The discrepancies observed between the genotypic (on the primary culture) and phenotypic drug susceptibility testing were explained by (i) resistance to SLID with MICs close to the critical concentration used for routine DST and not detected by phenotypic testing (n = 8, 15% of SLID-resistant strains), (ii) low-frequency heteroresistance not detected by sequencing of drug resistance-associated genes on the primary culture (n = 8, 15% of SLID-resistant strains), and (iii) other resistance mechanisms not yet characterized (n = 7, 13% of SLID-resistant strains).

Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis.

Drug-resistant Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a “probe dropout” manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.

Performance Assessment of the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Method for Rapid Detection of Susceptibility to Ethambutol and Molecular Prediction of Extensively Drug-resistant Tuberculosis in Clinical Isolates of Mycobacterium tuberculosis.

The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was employed for rapid detection of ethambutol (EMB) resistant clinical isolates of Mycobacterium tuberculosis. From 182 clinical isolates of M tuberculosis collected from different regions, 103 strains were entered in the investigation. DNA was extracted by Chelex 100 method and PCR was performed using specific primers for embB gene. Polymerase chain reaction products were digested with HaeIII and NlaII restriction endonucleases and the patterns of restriction fragments were analysed. Some randomly selected samples were sequenced. Out of 103 studied strains, 52 were resistant to EMB. The cases of secondary tuberculosis were 53 (51.50 ± 1.77%), and primary cases 50 (48.50 ± 1.77%; p > 0.05). From 63 extensively drug-resistant (XDR), pre-XDR and multidrug-resistant (MDR) isolates, 27 (87%), 18 (81.8%) and 7 (70%) strains were resistant to EMB, respectively. Results of PCR-RFLP method showed that from 27R EMB XDR isolates, 13 (sensitivity 48% with CI: 0.307, 0.66 and specificity 100%), from 18R EMB pre-XDR strains, 4 (sensitivity 22% with CI: 0.09, 0.45 and specificity 100%) and of 7R EMB MDR, 2 (sensitivity 28% with CI: 0.082, 0.64 and specificity 100%) had mutation in ATG-Met codon 306. Results of sequencing were concordant with RFLP method. Overall, sensitivity of the molecular method was 36.5% (CI: 0.09, 0.45) and specificity 100%. None of the 40 pansusceptible strains was embB306 mutants. Extensively drug-resistant strains had a higher proportion of embB306 mutants (43%) than pre-XDR and MDR isolates (odds ratio 6.78; p < 0.001). Fast detection of susceptibility to EMB drug is possible by PCR-RFLP. The embB306 locus is a candidate marker for rapid prediction of high resistance consisting of MDR and XDR forms to anti-tuberculosis drugs using this method.

Molecular Analysis of the embCAB Locus and embR Gene Involved in Ethambutol Resistance in Clinical Isolates of Mycobacterium tuberculosis in France

Modification of codon 306 in embB is regarded as the main mechanism leading to ethambutol (ETB) resistance in clinical isolates of Mycobacterium tuberculosis. However, numerous mutations elsewhere in the embCAB locus and in embR, a putative transcriptional activator of this locus, have been reported to be involved in ETB resistance. Here, we investigated the diversity of nucleotide variations observed in embCAB and embR in M. tuberculosis complex isolates from France. These regions were sequenced in 71 ETB-resistant (ETB-R) and 60 ETB-susceptible (ETB-S) clinical isolates of known phylogenetic lineages. The 131 isolates had 12 mutations corresponding to phylogenetic markers. Among the 60 ETB-S isolates, only 3 (5%) had nonsynonymous mutations that were not phylogenetic markers. Among the 71 ETB-R isolates, 98% had mutations in embCAB that likely contribute to ETB resistance: 70% had mutations located in embB codon 306, 406, or 497; 13% had mutations located outside these three positions between codons 296 and 426; and 15% had mutations corresponding to mutations in the embC-embA intergenic region. We found a strong association between resistance to ETB and the presence of mutations in embB and the embC-embA intergenic region (P < 0.001). In contrast, the mutations detected in embC and embA were not involved in ETB resistance, and no mutation was detected in embR. These results strongly suggest that the sensitivity of diagnostic assays for detecting ETB resistance based on testing of embB codon 306 can be increased by testing of the embB region between codons 296 and 497 and by including the embC-embA intergenic region between positions -8 and -21.

Draft Genome Sequences of Two Clinical Isolates of Mycobacterium tuberculosis from Sputum of Kazakh Patients.

Here, we report the draft genome sequences of two clinical isolates of Mycobacterium tuberculosis (MTB-476 and MTB-489) isolated from sputum of Kazakh patients.

Evaluation of Peptide Nucleic Acid Probe-Based Fluorescence In Situ Hybridization for the Detection ofMycobacterium tuberculosisComplex and Nontuberculous Mycobacteria in Clinical Respiratory Specimens

Background: Tuberculosis is globally the most important cause of death from single pathogen. Rapid and accurate identification of mycobacteria is essential for the control of tuberculosis. We evaluated a fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for the differentiation of Mycobacterium tuberculosis complex (MTB) and nontuberculous mycobacteria (NTM) in direct smears of sputum specimens. Methods: The cross-reactivity of MTBand NTM-specific PNA probes was examined with reference strains of M. tuberculosis ATCC 13950, Mycobacterium kansasii ATCC 12479, Mycobacterium fortuitum ATCC 6841, several clinical isolates of mycobacteria (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium gordonae and Mycobacterium chelonae), and 11 frequently isolated respiratory bacterial species other than mycobacteria. A series of 128 sputa (89 MTB culture positive, 29 NTM culture positive, and 10 under treatment culture negative) with grades of trace to 4+ were used to evaluate the performance of the method. Results: The MTBand NTM-specific PNA probes showed specific reactions with the reference strains of MTB and M. kansasii and clinical isolates of mycobacteria except M. fortuitum ATCC 6841, and no cross-reactivity with other tested bacteria. The PNA probe-based FISH assay for detection of MTB had a sensitivity and specificity of 100%, respectively. The sensitivity and specificity of the NTM-specific PNA probe was 100%. The smear grades of the PNA FISH test were same as with those of the fluorescence AFB stain in 2+ or higher grade. Conclusion: Detection and differentiation based on PNA FISH is sensitive and accurate for detecting mycobacteria and for differentiating MTB from NTM in clinical sputum smears. (Ann Clin Microbiol 2015;18:37-43)

MPT64 Antigen Simple and Rapid Test for Identification and Discrimination of Mycobacterium tuberculosis Complex from Nontuberculous Mycobacteria

Tuberculosis is a global pandemic and the incidence of nontuberculous mycobacteria (NTM) appears to be increasing worldwide. In this sense it is necessary that laboratories to use a rapid, accurate test, not laborious which does not require biosecurity measures. Molecular tests are accurate method, but is needs special equipments and lots labour, time and not economically. The aim for this study was to characterization the isolated of mycobacteria by immunochromatographic test, SD BIOLINE TB Ag MPT64 for differentiation into Mycobacterium tuberculosis (MTBC) complex and NTM. The immunochromatographic assay was evaluated with four reference strains of mycobacterial species and other clinical isolates of mycobacteria using the conventional methods as the gold standard. The results indicate that Ag MPT64 can be easily used for rapid discrimination between MTBC and NTM to confirm Mycobacterium tuberculosis specifically obtained from early stages of subculture without any technical complexity.

In vitro susceptibility of Mycobacterium tuberculosis isolates to an oral carbapenem alone or in combination with β-lactamase inhibitors.

We evaluated the antituberculosis (anti-TB) activity of five β-lactams alone or in combination with β-lactamase inhibitors against 41 clinical isolates of Mycobacterium tuberculosis, including multidrug-resistant and extensively drug-resistant strains. Of those, tebipenem, an oral carbapenem, showed the most potent anti-TB activity against clinical isolates, with a MIC range of 0.125 to 8 μg/ml, which is achievable in the human blood. More importantly, in the presence of clavulanate, MIC values of tebipenem declined to 2 μg/ml or less.