Slow-growing Mycobacteria Research Articles

MmpL8MAB controls Mycobacterium abscessus virulence and production of a previously unknown glycolipid family

Mycobacterium abscessus is a peculiar rapid-growing Mycobacterium (RGM) capable of surviving within eukaryotic cells thanks to an arsenal of virulence genes also found in slow-growing mycobacteria (SGM), such as Mycobacterium tuberculosis A screen based on the intracellular survival in amoebae and macrophages (MΦ) of an M. abscessus transposon mutant library revealed the important role of MAB_0855, a yet uncharacterized Mycobacterial membrane protein Large (MmpL). Large-scale comparisons with SGM and RGM genomes uncovered MmpL12 proteins as putative orthologs of MAB_0855 and a locus-scale synteny between the MAB_0855 and Mycobacterium chelonae mmpL8 loci. A KO mutant of the MAB_0855 gene, designated herein as mmpL8MAB , had impaired adhesion to MΦ and displayed a decreased intracellular viability. Despite retaining the ability to block phagosomal acidification, like the WT strain, the mmpL8MAB mutant was delayed in damaging the phagosomal membrane and in making contact with the cytosol. Virulence attenuation of the mutant was confirmed in vivo by impaired zebrafish killing and a diminished propensity to induce granuloma formation. The previously shown role of MmpL in lipid transport prompted us to investigate the potential lipid substrates of MmpL8MAB Systematic lipid analysis revealed that MmpL8MAB was required for the proper expression of a glycolipid entity, a glycosyl diacylated nonadecyl diol (GDND) alcohol comprising different combinations of oleic and stearic acids. This study shows the importance of MmpL8MAB in modifying interactions between the bacteria and phagocytic cells and in the production of a previously unknown glycolipid family.

The Mycobacterial Cell Envelope: A Relict From the Past or the Result of Recent Evolution?

Mycobacteria are well known for their taxonomic diversity, their impact on global health, and for their atypical cell wall and envelope. In addition to a cytoplasmic membrane and a peptidoglycan layer, the cell envelope of members of the order Corynebacteriales, which include Mycobacterium tuberculosis, also have an arabinogalactan layer connecting the peptidoglycan to an outer membrane, the so-called “mycomembrane.” This unusual cell envelope composition of mycobacteria is of prime importance for several physiological processes such as protection from external stresses and for virulence. Although there have been recent breakthroughs in the elucidation of the composition and organization of this cell envelope, its evolutionary origin remains a mystery. In this perspectives article, the characteristics of the cell envelope of mycobacteria with respect to other actinobacteria will be dissected through a molecular evolution framework in order to provide a panoramic view of the evolutionary pathways that appear to be at the origin of this unique cell envelope. In combination with a robust molecular phylogeny, we have assembled a gene matrix based on the presence or absence of key determinants of cell envelope biogenesis in the Actinobacteria phylum. We present several evolutionary scenarios regarding the origin of the mycomembrane. In light of the data presented here, we also propose a novel alternative hypothesis whereby the stepwise acquisition of core enzymatic functions may have allowed the sequential remodeling of the external cell membrane during the evolution of Actinobacteria and has led to the unique mycomembrane of slow-growing mycobacteria as we know it today.

A definition of the Mycobacterium avium complex for taxonomical and clinical purposes, a review.

Nontuberculous mycobacteria, particularly the Mycobacterium avium complex (MAC) bacteria, are increasingly recognized as opportunistic pathogens of humans. As a result, studies on antibiotic treatment and taxonomy of the MAC are intensifying, but an updated definition of what constitutes the MAC, either for taxonomical studies or for clinical purposes, is lacking. On the basis of literature review and phylogenetic analyses, we propose to define the MAC as a grouping of slow-growing mycobacteria that show corresponding values in at least two of the following targets against either M. avium ATCC 25291T or Mycobacterium intracellulare ATCC 13950T: >99.4 % sequence identity for the full 16S rRNA gene, >98.7 % for the partial (5') 16S rRNA gene, >97.3 % for hsp65 and >94.4 % for rpoB region V. A >97.5 % value in concatenated analyses of >2500 bp that includes 16S rRNA, hsp65 and rpoB gene sequence data or ≥85 % average nucleotide identity to M. avium ATCC 25291T or M. intracellulare ATCC 13950T on basis of whole genome sequencing data is recommended. This molecular definition is based on the distances observed between the classical members of the MAC, M. avium and M. intracellulare. Applying this definition, the complex currently consists of 12 validly published species: Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium arosiense, Mycobacterium vulneris, Mycobacterium bouchedurhonense, Mycobacterium timonense, Mycobacterium marseillense, Mycobacterium yongonense, Mycobacterium paraintracellulare and Mycobacterium lepraemurium.

Oxadiazolone derivatives, new promising multi-target inhibitors against M. tuberculosis

A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc26230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC50, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.

Cutaneous Manifestations of Mycobacterium kansasii Infection.

Mycobacterium kansasii is a slow-growing member of the nontuberculous mycobacterial species that manifests clinically with pulmonary, cutaneous, or disseminated forms. Most cases of M. kansasii infection occur in immunocompromised hosts including HIV/AIDS, solid-organ transplantation, anti-interferon-γ antibody disorder, or among those with pulmonary disorders such as chronic obstructive pulmonary disease or cystic fibrosis. In this review, we aim to discuss relevant literature regarding the clinical manifestations of this infection with a particular emphasis on the clinical spectrum and treatment of the cutaneous manifestations of this mycobacterial infection. Infection caused by M. kansasii is associated with exposure to contaminated municipal water systems since this organism thrives in human-engineered environments. Chronic or subacute skin lesions may represent the primary source of entry of the organism due to open sores or traumatic injuries. Alternatively, cutaneous lesions may occur secondary to dissemination of the organism to the skin from pulmonary infection. The diagnosis of M. kansasii of the skin and soft tissues should be entertained among immunocompetent or immunocompromised individuals presenting with subacute or chronic lesions. The differential diagnosis of M. kansasii infection includes infections of skin and soft tissues caused by rapidly growing mycobacteria, subcutaneous mycosis, pyogenic bacterial infections, nocardiosis, free-living amoebas, and protothecosis. Cutaneous lesions caused by M. kansasii most commonly manifest in the setting of concomitant pulmonary disease or in the setting of disseminated infection among immunocompromised hosts. When clinicians suspect a cutaneous mycobacterial infection either due to slow-growing mycobacteria such as M. kansasii or due to rapidly growing mycobacteria, obtaining a skin biopsy of chronic nonhealing lesions for histopathologic evaluation and for microbiological assessment is crucial. Mycobacteriology assessment of clinical specimen includes acid-fast bacilli staining, culture in solid/liquid media, and molecular assays for confirmation. Treatment of M. kansasii infection of the skin and subcutaneous tissues follows the same management recommendations as those for the treatment of isolated pulmonary forms or disseminated disease.

Clinical, Radiological, and Microbiological Characteristics of Mycobacterium simiae Infection in 97 Patients.

Mycobacterium simiae is a rare species of slow-growing nontuberculous mycobacteria (NTM). From 2002 to 2017, we conducted a retrospective study that included all patients with NTM-positive respiratory samples detected in two university hospitals of the French overseas department of Reunion Island. We recorded the prevalence of M. simiae in this cohort, as well as the clinical, radiological, and microbiological features of patients with at least 1 sample positive for M. simiae In our cohort, 97 patients (15.1%) were positive for M. simiae Twenty-one patients (21.6%) met the American Thoracic Society (ATS) criteria for infection. M. simiae infection was associated with bronchiectasis, micronodular lesions, and weight loss. Antibiotic susceptibility testing was performed for 60 patients, and the isolates were found to have low susceptibility to antibiotics, except for amikacin, fluoroquinolones, and clarithromycin. Treatment failed for 4 of the 8 patients treated for M. simiae infection. Here, we describe a specific cluster corresponding to a large cohort of patients with M. simiae, a rare nontuberculous mycobacterium associated with low pathogenicity and poor susceptibility to antibiotics.

Mycobacterium shigaense sp. nov., a slow-growing, scotochromogenic species, is a member of the Mycobacterium simiae complex.

Among non-tuberculous mycobacteria (NTM), the Mycobacterium simiae complex is one of the largest groups, consisting of 18 species of slow-growing mycobacteria. In 2009, a case of NTM-associated infectious skin disease was reported in Shiga Prefecture, Japan. The patient presented with scattered nodules on the chest, back and extremities, and an M. simiae-like organism was isolated from skin biopsy specimens obtained from one of these lesions. Based on several assessments, including multiple-gene analyses, biochemical characterization and drug susceptibility testing, we concluded that this isolate represented a novel species of NTM, and proposed the name 'Mycobacterium shigaense'. Since 2009, five more cases of NTM-associated infectious disease in which there was a suspected involvement of 'M. shigaense' have been reported. Interestingly, four of these six cases occurred in Shiga Prefecture. Here we performed multiple-gene phylogenetic analyses, physiological and biochemical characterization tests, drug susceptibility tests, and profiling of proteins, fatty acids and mycolic acids of eight clinical isolates from the six suspected 'M. shigaense' cases. The results confirmed that all of the clinical isolates were 'M. shigaense', a slow-growing, scotochromogenic species. Here M. shigaense is validly proposed as a new member of the M. simiae complex, with the type strain being UN-152T (=JCM 32072T=DSM 46748T).

Preliminary Study on Drug Susceptibility Profile and Resistance Mechanisms to Macrolides of Clinical Isolates of Non-tuberculous Mycobacteria from China

ObjectiveMacrolide susceptibility and drug resistance mechanisms of clinical non-tuberculous mycobacteria (NTM) isolates were preliminarily investigated for more accurate diagnosis and treatment of the infection in China. MethodsFour macrolides, including clarithromycin (CLAR), azithromycin (AZM), roxithromycin (ROX), and erythromycin (ERY), were used to test the drug susceptibility of 310 clinical NTM isolates from six provinces of China with the broth microdilution method. Two resistance mechanisms, 23S rRNA and erm, were analyzed with nucleotide sequence analysis. ResultsVaried effectiveness of macrolides and species-specific resistance patterns were observed. Most Mycobacterium abscessus subsp. massiliense were susceptible and all M. fortuitum were highly resistant to macrolides. All the drugs, except for erythromycin, exhibited excellent activities against slow-growing mycobacteria, and drug resistance rates were below 22.2%. Only four highly resistant strains harbored 2,058/2,059 substitutions on rrl and none of other mutations were related to macrolide resistance. G2191A and T2221C on rrl were specific for the M. abscessus complex (MABC). Seven sites, G2140A, G2210C, C2217G, T2238C, T2322C, T2404C, and A2406G, were specifically carried by M. avium and M. intracellulare. Three sites, A2192G, T2358G, and A2636G, were observed only in M. fortuitum and one site G2152A was specific for M. gordonae. The genes erm(39) and erm(41) were detected in M. fortuitum and M. abscessus and inducible resistance was observed in relevant sequevar. ConclusionThe susceptibility profile of macrolides against NTM was demonstrated. The well-known macrolide resistance mechanisms, 23S rRNA and erm, failed to account for all resistant NTM isolates, and further studies are warranted to investigate macrolide resistance mechanisms in various NTM species.

Disseminated nontuberculous mycobacterial disease in a cat caused by Mycobacterium sp. strain MFM001

Mycobacterium sp. strain MFM001 (MFM001), a new strain of slow-growing nontuberculous mycobacteria not associated with the Mycobacterium avium complex, led to disseminated mycobacterial disease characterized by transmural granulomatous gastroenterocolitis of lepromatous type in a cat treated with immunosuppressive medication. MFM001 was found to be closely related to M. kyorinense or M. celatum by partial sequence of 16S rDNA, and identical with an unspecified M. sp. strain B10-07.09.0206 isolated from a human pulmonary infection in Germany. MFM001 should be considered as a pathogenic organism in cats, especially in immunocompromised animals.

Isolation of Mycobacterium arupense from pleural effusion: culprit or not?

BackgroundMycobacterium arupense, first identified in 2006, is a slow-growing nontuberculous mycobacterium (NTM) and an emerging cause of tenosynovitis, potentially associated with immunosuppression. However, unlike the diagnostic value of its isolation from osteoarticular specimens, the significance of detecting M. arupense in respiratory specimens is not yet clear.Case presentationTo our knowledge, we, for the first time, described the identification of M. arupense from the pleural effusion of an immunocompetent patient, who presented with fever and chylothorax. The symptoms resolved with doxycycline treatment for 45 days and a low-fat, high-protein diet. Follow-up at 14 months showed no relapse.ConclusionsBecause the patient fully recovered without combined anti-NTM treatment, we did not consider M. arupense the etiological cause in this case. This indicates that M. arupense detected in pleural effusion is not necessarily a causative agent and careful interpretation is needed in terms of its clinical relevance.

A Rapid Growth-Independent Antibiotic Resistance Detection Test by SYBR Green/Propidium Iodide Viability Assay.

Antibiotic-resistant bacteria have caused huge concerns and demand innovative approaches for their prompt detection. Current antimicrobial susceptibility tests (AST) rely on the growth of the organisms which takes 1–2 days for fast-growing organisms and several weeks for slow growing organisms. Here, we show for the first time the utility of the SYBR Green I/propidium iodide (PI) viability assay for rapidly identifying antibiotic resistance in less than 30 min for major, antibiotic-resistant, fast-growing bacteria, such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii for bactericidal and bacteriostatic agents and in 16 h for extremely rapid detection of drug resistance for isoniazid and pyrazinamide in slow-growing Mycobacterium tuberculosis. The SYBR Green I/PI assay generated rapid and robust results in concordance with traditional AST methods. This novel growth-independent methodology changes the concept of the current growth-based AST and may revolutionize current drug susceptibility testing for all cells of prokaryotic and eukaryotic origin and, subject to further clinical validation, may play a major role in saving lives and improving patient outcomes.

Impact of the epoxide hydrolase EphD on the metabolism of mycolic acids in mycobacteria

Mycolic acids are the hallmark of the cell envelope in mycobacteria, which include the important human pathogens Mycobacterium tuberculosis and Mycobacterium leprae Mycolic acids are very long C60-C90 α-alkyl β-hydroxy fatty acids having a variety of functional groups on their hydrocarbon chain that define several mycolate types. Mycobacteria also produce an unusually large number of putative epoxide hydrolases, but the physiological functions of these enzymes are still unclear. Here, we report that the mycobacterial epoxide hydrolase EphD is involved in mycolic acid metabolism. We found that orthologs of EphD from M. tuberculosis and M. smegmatis are functional epoxide hydrolases, cleaving a lipophilic substrate, 9,10-cis-epoxystearic acid, in vitro and forming a vicinal diol. The results of EphD overproduction in M. smegmatis and M. bovis BCG Δhma strains producing epoxymycolic acids indicated that EphD is involved in the metabolism of these forms of mycolates in both fast- and slow-growing mycobacteria. Moreover, using MALDI-TOF-MS and 1H NMR spectroscopy of mycolic acids and lipids isolated from EphD-overproducing M. smegmatis, we identified new oxygenated mycolic acid species that accumulated during epoxymycolate depletion. Disruption of the ephD gene in M. tuberculosis specifically impaired the synthesis of ketomycolates and caused accumulation of their precursor, hydroxymycolate, indicating either direct or indirect involvement of EphD in ketomycolate biosynthesis. Our results clearly indicate that EphD plays a role in metabolism of oxygenated mycolic acids in mycobacteria.

Clinical features of infection caused by non-tuberculous mycobacteria: 7years' experience.

Non-tuberculous mycobacteria (NTM) are ubiquitous organisms associated with various infections. The aim of the study was to determine the most relevant clinical characteristics of NTM during the 7-year period. A retrospective study of NTM infections was conducted between January 2009 and December 2016. The American Thoracic Society/Infectious Disease Society of America criteria were used to define cases of pulmonary or an extrapulmonary site. A total of 85 patients were included in the study. Pulmonary cases predominated 83/85 (98%), while extrapulmonary NTM were present in 2/95 (2%) patients. Overall, ten different NTM species were isolated. The most common organisms were slow-growing mycobacteria (SGM) presented in 70/85 (82.35%) patients. Isolated SGM strains were Mycobacterium avium complex (MAC) in 25/85 (29.41%) patients, M. xenopi in 20/85 (23.53%) patients, M. kansasii in 15/85 (17.65%) patients and M. peregrinum and M. gordonae in 5/85 (5.88%) patients each. Isolated rapid-growing mycobacteria (RGM) strains were M. abscessus in 8/85 (9.41%) patients, M. fortuitum in 4/85 (4.71%) patients and M. chelonae in 3/85 (3.53%) patients. Almost all patients (98%; 83/85) had comorbidities. Among 75 (88.24%) patients who completed follow-up, 59 (69.41%), 10 (11.76%) and 6 (7%), were cured, experienced relapse and died, respectively. In the present study, pulmonary NTM infections were more frequent compared to extrapulmonary disease forms. SGM were most common isolates with MAC pulmonary disease the most frequently found. Comorbidities have an important role in NTM occurrence. Further investigation should focus on an NTM drug susceptibility testing.

Late-onset postoperative Mycobacterium haemophilum endophthalmitis masquerading as inflammatory uveitis: a case report

BackgroundAlthough atypical mycobacteria had been increasingly found in various ocular infections in the past decades, a slow-growing Mycobacterium haemophilum (M. haemophilum) was scarcely reported. Similar to tuberculous infection, the presentation can masquerade as low-grade granulomatous intraocular inflammation with partial response to corticosteroids. Besides, the special requirements for culture make this pathogen difficult to diagnose. The study aims to report the clinical presentation and notify the awareness of NTM endophthalmitis among clinicians. This is the first case report of late-onset, postoperative M. haemophilum endophthalmitis in the literature.Case presentationA 66-year-old man with non-insulin-dependent diabetes mellitus (NIDDM) manifested chronic granulomatous inflammation in the left eye after multiple glaucoma surgeries. With a diagnosis of noninfectious panuveitis, he was treated with systemic corticosteroids. The inflammation initially responded to therapy although it subsequently worsened and became purulent endophthalmitis. The vitreous cultures grew M. haemophilum. Intraocular and systemic antimicrobial treatments were administered early, but the patient eventually turned blind.ConclusionsM. haemophilum endophthalmitis is a rare but serious intraocular complication leading to loss of vision or eyeball. Awareness of atypical mycobacterial infections is necessary especially in patients with impaired immune function, previous intraocular surgery, and corticosteroid resistance. Proper laboratory investigations and treatments should be performed. However, due to the rarity of the disease, the development of guidelines for its investigation and therapy is still challenging.

Quantification of the Metabolic Heterogeneity in Mycobacterial Cells Through the Measurement of the NADH/NAD+ Ratio Using a Genetically Encoded Sensor.

NADH/NAD+ levels are an indicator of the bacterial metabolic state. NAD(H) levels are maintained through coordination of pathways involved in NAD(H) synthesis and its catabolic utilization. Conventional methods of estimating NADH/NAD+ require cell disruption and suffer from low specificity and sensitivity and are inadequate in providing spatiotemporal resolution. Recently, genetically encoded biosensors of the NADH/NAD+ ratio have been developed. One of these sensors, Peredox-mCherry, was adapted for the measurement of cellular levels of NADH/NAD+ in the slow-growing Mycobacterium tuberculosis (Mtb) and the fast-growing Mycobacterium smegmatis. Importantly, the use of the engineered reporter strains of Mtb demonstrated a significantly higher heterogeneity among the bacteria residing in macrophages compared to the bacteria grown in synthetic media. Previous estimations of NADH/NAD+ levels have missed this important aspect of the biology of Mtb, which may contribute to the variable response of intracellular Mtb to different antimycobacterial agents. In this chapter, we describe the details of a method used in the generation of reporter strains for the measurement of the NADH/NAD+ ratio in mycobacteria. Importantly, once the reporter strains are created, they can be exploited with fluorescence spectroscopy, FACS, and confocal microscopy to access the dynamic changes in the NADH/NAD+ levels in intact individual bacterial cells. Although we have only described the method for the creation of reporter strains capable of measuring NADH/NAD+ in mycobacteria in this chapter, a similar method can be used for generating reporter strains for other bacterial species, as well. We believe that such reporter stains can be used in novel screens for small molecules that could alter the metabolism of bacterial cells and thus aid in the development of new class of therapeutic agents.

Cyclophostin and Cyclipostins analogues, new promising molecules to treat mycobacterial-related diseases

The progression of mycobacterial diseases requires the development of new therapeutics. This study evaluated the efficacy and selectivity of a panel of Cyclophostin and Cyclipostins analogues (CyCs) against various bacteria and mycobacteria. The activity 26 CyCs was first assayed by the agar plate method. Compounds exhibiting 50–100% growth inhibition were then selected to determine their minimum inhibitory concentrations (MICs) by the resazurin microtiter assay (REMA). The best drug candidate was further tested against clinical mycobacterial isolates and bacteria responsible for nosocomial infections, including 6 Gram-negative bacteria, 5 Gram-positive bacteria, 29 rapid-growing mycobacteria belonging to the Mycobacterium chelonae–abscessus clade and 3 slow-growing mycobacteria (Mycobacterium marinum, Mycobacterium bovis BCG and Mycobacterium tuberculosis). Among the 26 CyCs tested, 10 were active and their inhibitory activity was exclusively restricted to mycobacteria. The best candidate (CyC17) was further tested against 26 clinical strains and showed high selectivity for mycobacteria, with MICs (<2–40 µg/mL) comparable with those of most classical antimicrobials used to treat M. abscessus infections. Together, these results support the fact that such CyCs represent a new family of potent and selective inhibitors against mycobacteria. This is of particular interest for future chemotherapeutic developments against mycobacterial-associated infections, especially against M. abscessus, the most drug-resistant mycobacterial species.

Clinical characteristics of pulmonary Mycobacterium lentiflavum disease in adult patients

BackgroundMycobacterium lentiflavum is a slow-growing non-tuberculous Mycobacterium that is often associated with an immunocompromised state and cervical lymphadenitis in young children. However, little is known about the clinical importance of pulmonary infection with M. lentiflavum in adults. MethodsThe medical records of all adults who met the diagnostic criteria of pulmonary M. lentiflavum disease at Keio University Hospital and Fukujuji Hospital from 2001 to 2015 were reviewed. In addition, the PubMed database was searched to identify further reported cases in non-HIV adults. ResultsFive cases of pulmonary M. lentiflavum disease were identified in the medical records search and 11 additional cases were identified in the literature review. Eleven of the total 16 cases were female, and 15 of 16 cases showed a nodular/bronchiectatic pattern on chest computed tomography imaging. No cases showed an aggressive clinical course of pulmonary M. lentiflavum disease, although one patient died of an exacerbation of underlying vasculitis and bacterial pneumonia. ConclusionsThe clinical characteristics of pulmonary M. lentiflavum disease in adult patients were identified. This disease mainly affects females, displays a nodular/bronchiectatic pattern on chest computed tomography imaging, and does not demonstrate an aggressive clinical course. Further larger studies are needed to reveal detailed clinical features.

Impact of updating the MALDI-TOF MS database on the identification of nontuberculous mycobacteria.

Conventional identification of mycobacteria species is slow, laborious and has low discriminatory power. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proved highly effective for identifying conventional bacteria, and it may also be useful for identifying mycobacteria. The aim of this study was to evaluate and compare MALDI-TOF MS with currently recommended molecular methods for the identification of nontuberculous mycobacteria (NTM), applying Mycobacteria Libraries v3.0 (ML3.0) and v2.0 (ML2.0). A total of 240 clinical isolates of 41 NTM species grown on solid media were analysed: 132 isolates of slow-growing mycobacteria and 108 of rapid-growing mycobacteria. MALDI-TOF MS, using ML3.0, identified 192 (80%) NTM isolates with a score ≥1.7, encompassing 35 (85.4%) different species, that is, 17 (7.1%; p=0.0863) isolates and 15 (36.6%; p=0.0339) species more than currently recommended molecular techniques (polymerase chain reaction reverse hybridization). All these isolates were correctly identified according to molecular identification methods. The application of ML3.0 also identified 15 (6.2%) NTM isolates more than ML2.0 (p<0.01). The scores obtained with MALDI-TOF MS using ML3.0 (mean score: 1.960) were higher in 147 (61.2%) isolates than when using ML2.0 (mean score: 1.797; p<0.01). Three of the species analysed were not included in either database, so they were not recognized by this system. In conclusion, MALDI-TOF MS identified more isolates and species than the recommended polymerase chain reaction reverse hybridization assays. Although the new ML3.0 is not the definitive database, it yielded better results than ML2.0. This shows that the updating of the MALDI-TOF MS database plays an essential role in mycobacterial identification. Copyright © 2017 John Wiley & Sons, Ltd.

ОПЫТ ПРИМЕНЕНИЯ МЕТОДА ОПРЕДЕЛЕНИЯ МИНИМАЛЬНЫХ ИНГИБИРУЮЩИХ КОНЦЕНТРАЦИЙ В ЖИДКОЙ ПИТАТЕЛЬНОЙ СРЕДЕ ДЛЯ ВЫЯВЛЕНИЯ ЛЕКАРСТВЕННОЙ УСТОЙЧИВОСТИ НЕТУБЕРКУЛЕЗНЫХ МИКОБАКТЕРИЙ

Goal of the study : analysis of drug resistance of certain species of non-tuberculous mycobacteria through testing by minimum inhibitory concentrations on the fluid nutritional media. Materials and method : drug resistance of non-tuberculous mycobacteria was tested by the method of minimum inhibitory concentrations on the fluid nutritional media. 51 strains of non-tuberculous mycobacteria from the species of M. intracellulare, M. avium, M. kansasii, M. gordonae, M. fortuitum, M. abscessus were tested. The minimum inhibitory concentration of the drug for slow-growing non-tuberculous mycobacteria was defined using the panel of 13 drugs (SLOWMYCO) and for the fast-growing mycobacteria – the panel of 15 drugs RAPMYCO Sensititre (Trek Diagnostic System, Thermo Scientific, USA) was used. It was proved that the majority of tested fast growing mycobacteria were resistant to ceftriaxone, cefepime, cefoxitin but susceptible to amikacin; and slow growing non-tuberculous mycobacteria were susceptible to clarithromycin, linezolid and amikacin.

In vitro activity of bedaquiline against slow-growing nontuberculous mycobacteria.

Bedaquiline (BDQ) is a recently approved antibiotic for the treatment of multidrug-resistant tuberculosis, but its potential against slow-growing mycobacteria (SGM) is still unknown. The objective of this study was to determine the in vitro activity of BDQ on SGM by assessing their MIC and minimal bactericidal concentration (MBC). The MIC of BDQ against 17 clinical isolates including Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium kansasii and Mycobacterium simiae species was determined by the resazurin microtitre assay and the MBC by the c.f.u. determination on 7H10 agar plates. BDQ has a bacteriostatic activity on all SGM tested with a MIC range from 0.03 to 0.007 µg ml-1 and surprisingly a good bactericidal activity on the majority of the isolates tested with an MBC of 1-2 µg ml-1 . Based on these preliminary results BDQ seems to be very promising for treatment of diseases caused by SGM.