Nontuberculous Mycobacteria Strains Research Articles

The biofilm formation of nontuberculous mycobacteria and its inhibition by essential oils.

Nontuberculous mycobacteria (NTM) form two types of biofilms: Bottom biofilm and pellicle. The spatial distribution of cells between these types of biofilms and their dispersion into the liquid medium depends on the ratio of the nutrient components of the growth medium. The inhibition of biofilm formation by NTM can be achieved through the use of lipophilic compounds, such as essential oils (EOs). The biofilm and pellicle formation of Mycobacterium smegmatis and Mycobacterium avium on four nutrient media under static conditions and in the vapors of six EOs was evaluated by conventional method. The antimycobacterial effect of EOs was also studied by the disc diffusion method. The bottom biofilm and pellicle formation of NTM largely depended on the composition and availability of nutrients. Nutrient media in which NTM form powerful bottom biofilm or pellicle or both have been determined. The growth of studied NTM strains on agar was highly sensitive to the EOs of Scots pine, Atlas cedar, bergamot, and a mixture of EO of different plants. The cultivation of bacteria in the EO vapors also resulted in total suppression of the pellicle for all studied NTM strains. Our data clearly indicate that the carbon-nitrogen ratio is involved in the regulation of the spatial distribution of the biofilm. The preventing effect of EOs vapors, especially the synergistic action of mixture of EOs on the biofilm and pellicle formation by NTMs can be observed.

1637. SPR720, A Novel Benzamidazole Gyrase Inhibitor, Demonstrates Potent Efficacy Against Mycobacterium avium ATCC 700898 in a Chronic C3HeBFeJ Mouse Infection Model

BackgroundSPR719 (the active metabolite of phosphate prodrug SPR720) belongs to a novel class which targets the ATPase subunits of gyrase by a mechanism distinct from fluoroquinolones. SPR719 has potent antibacterial activity against nontuberculous mycobacteria strains (NTM), including Mycobacterium avium, and is under development for treatment of NTM pulmonary disease. Oral efficacy of SPR720 was evaluated alone and in combination treatment in the C3HeBFeJ chronic mouse infection model which produces necrotic granulomas, similar to humans.MethodsMice were infected with a pulmonary aerosol of 1x108.5 CFU of M. avium ATCC 700898, (SPR719 MIC = 2 mg/mL). Treatment started on day 28 for 8 weeks with: saline, clarithromycin 250 mg/kg (CLR) QD, SPR720 at 10, 30 and 100 mg/kg QD, or SPR720 at 50 mg/kg BID. SPR720 at 30 mg/kg QD was also combined with CLR +/- ethambutol at 100 mg/kg (EMB), or CLR + rifabutin at 100 mg/kg (RFB) +/- EMB. Mice were evaluated for bacterial burden (CFU) on days 1, 27 and 60 after infection by plating serial dilutions of organ homogenates on nutrient 7H11 and charcoal agar plates. Lung pathology was evaluated by assessing prevalence and size of pulmonary lesions.ResultsCLR treatment for 28 days showed a significant reduction in the bacterial burden in the lung, spleen, and liver compared to the untreated control. SPR720 demonstrated a dose dependent reduction in bacterial burden, including at 100 mg/kg which showed a statistically significant reduction in the bacterial burden in the lung, spleen, and liver. CLR + EMB + SPR720 at 30 mg/kg reduction in the bacterial burden in the lung, spleen, and liver. RFB when added to the treatment regimen did not demonstrate enhanced efficacy compared the additive effect of EMB + CLR +/- SPR720. Lung pathology showed that lesions were less numerous and smaller in infected mice treated with all regimens.ConclusionOral administration of SPR720 demonstrated a statistically significant reduction in the bacterial burden in all tissues with concomitant improvement in lung pathology, both alone and in combination with standard of care agents. These results support the continued development of SPR720 for treatment of NTM pulmonary infections.DisclosuresNicole S. Cotroneo, BS, Spero Therapeutics (Employee, Shareholder) Suzanne Stokes, PhD, Spero Therapeutics (Employee, Shareholder)

Pilot Study on non-tuberculous mycobacteria infections in Italy

Abstract Background The rise in non-tuberculous mycobacteria (NTM) diseases has been reported worldwide, but no major information is known for Italy. The purpose of this pilot study is to release nationwide data on distribution of NTM in our country. Methods In 2016, the Istituto Superiore di Sanità (National Institute of Health) began to collect microbiological, clinical and minimum inhibitory concentration (MIC) data on NTM infections, in collaboration with hospital laboratories located in 15 out of 20 regions (Studio Multicentrico Italiano NTM, SMI-NTM), which routinely isolate and characterize NTM by commercial identification (Genotype) and MIC (Sensititre) assays. Results In 2016-2018, 32 labs reported data on 4169 NTM strains, including 644 rapid growers (15%) and 3525 slow growers (85%). The most frequent species were Mycobacterium avium (MA) (29.2%), M. intracellulare (MI) (21.5%), M. xenopi (MX) (10.6%), M. gordonae (10.6%), M. abscessus (5.9%), M. chimaera (MC) (5.1%). Overall, 88% NTM strains were isolated from pulmonary sites, 84% from Italians, 51% from men. NTM infections in Italians occurred in 75-84 years old patients, while in foreign-born people were observed in 15-44 years old patients. Strains from cystic fibrosis were 11.9%. The MICs of clarithromycin (CLA) for MA or MI peaked at 2 µg/ml, while for amikacin (AK) peaked at 16 µg/ml. When MICs were interpreted according to the 2018 Clinical and Laboratory Institute Standards (CLSI) breakpoints, MA or MI resistances for CLA were 2.6% and 2.6% respectively, while for AK they were 7.2% and 4.5%, respectively. Higher resistance rates for MA and MI were observed for moxifloxacin and linezolid. MICs of MC, MX, M. kansasii, M. marinum and rapid grower NTM were also determined and interpreted on the basis of CLSI breakpoints. Conclusions This 3-years pilot study is the basis for a future multiannual national strategic plan for surveillance of NTM infections in Italy (collection of 2019 data is in progress). Key messages This 3-years pilot study is the basis for a future multiannual national strategic plan for surveillance of NTM infections in Italy. The purpose of this pilot study is to release nationwide data on distribution of NTM in our country.

Comprehensive Determination of Mycobacterium tuberculosis and Nontuberculous Mycobacteria From Targeted Capture Sequencing.

Infection of Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) challenges effective pulmonary infectious disease control. Current phenotypic and molecular assays could not comprehensively and accurately diagnose MTB, NTM, and drug resistance. Next-generation sequencing allows an “all-in-one” approach providing results on expected drug susceptibility testing (DST) and the genotype of NTM strains. In this study, targeted capture sequencing was used to analyze the genetic backgrounds of 4 MTB strains and 32 NTM pathogenic strains in 30 clinical samples, including 14 sputum specimens and 16 bronchoalveolar lavage fluid samples. Through comparing with other TB diagnostic tests, we proved that targeted capture sequencing could be used as a highly sensitive (91.3%) and accurate (83.3%) method to diagnose TB, as well as MGIT 960. Also, we identified 7 NTM strains in 11 patients; among them, seven patients were MTB/NTM co-affected, which indicated that it was a meaningful tool for the diagnosis and treatment of NTM infection diseases in clinic. However, based on a drug-resistant mutation library (1,325 drug resistance loci), only 9 drug resistance strains and 22 drug resistance loci were discovered, having considerable discordance with the drug-resistant results of MGIT 960. Our finding indicated that targeted capture sequencing approach was applicable for the comprehensive and accurate diagnosis of MTB and NTM. However, from data presented here, the DST results identified by next-generation sequencing (NGS) showed a relatively low consistency with MGIT 960, especially in sputum samples. Further work should be done to explore the reasons for low drug-resistance detection rate of NGS.

A Comparative Genomics Approach for Shortlisting Broad-Spectrum Drug Targets in Nontuberculous Mycobacteria.

Many members of nontuberculous mycobacteria (NTM) are opportunistic pathogens causing several infections in animals. The incidence of NTM infections and emergence of drug-resistant NTM strains are rising worldwide, emphasizing the need to develop novel anti-NTM drugs. The present study is aimed to identify broad-spectrum drug targets in NTM using a comparative genomics approach. The study identified 537 core proteins in NTM of which 45 were pathogen specific and essential for the survival of pathogens. Furthermore, druggability analysis indicated that 15 were druggable among those 45 proteins. These 15 proteins, which were core proteins, pathogen-specific, essential, and druggable, were considered as potential broad-spectrum candidates. Based on their locations in cytoplasm and membrane, targets were classified as drug and vaccine targets. The identified 15 targets were different enzymes, carrier proteins, transcriptional regulator, two-component system protein, ribosomal, and binding proteins. The identified targets could further be utilized by researchers to design inhibitors for the discovery of antimicrobial agents.

The Use of Amikacin Liposome Inhalation Suspension (Arikayce) in the Treatment of Refractory Nontuberculous Mycobacterial Lung Disease in Adults.

Nontuberculous mycobacteria (NTM) can cause and perpetuate chronic inflammation and lung infection. Despite having the diagnostic criteria, as defined by the American Thoracic Society (ATS) and Infectious Diseases Society of America (IDSA), clinicians find it challenging to diagnose and treat NTM-induced lung disease. Inhaled antibiotics are suitable for patients with lung infection caused by Pseudomonas aeruginosa and other organisms, but until recently, their utility in NTM-induced infection was not established. The most common NTM pathogens identified are the slow-growing Mycobacterium avium complex (MAC) and the rapid-growing M. abscessus complex (MABSC), both of which include several subspecies. Other less commonly isolated species include M. kansasii, M. simiae, and M. fortuitum. NTM strains are frequently more resistant than what is found in bacterial sputum cultures. Until recently, there was no approved inhaled antibiotic therapy for patients who were culture positive for pulmonary NTM infection. Of late, inhaled amikacin has been under investigation for the treatment of NTM-induced pulmonary infection. The FDA approved Arikayce (amikacin liposome inhalation suspension or ALIS) based on results from the ongoing Phase 3 CONVERT trial. In this study, the use of Arikayce met its primary endpoint of sputum culture conversion by the sixth month of treatment. The addition of Arikayce to guideline-based therapy led to negative sputum cultures for NTM by month 6 in 29% of patients compared to 8.9% of patients treated with guideline-based therapy alone. The effectiveness of Arikayce holds promise. However, due to limited data on Arikayce’s safety, it is currently useful only for a specific population, particularly patients with refractory NTM-induced lung disease. Future trials must verify the target group and endorse the clinical benefits of Arikayce.

Typing and classification of non-tuberculous mycobacteria isolates

Background: There are a large and growing number of non-tuberculous mycobacteria (NTM) species that have been isolated, identified, and described in the literature, yet there are many clinical isolates which are not assignable to known species even when the genome has been sequenced. Additionally, a recent manuscript has proposed the reclassification of the Mycobacterium genus into five distinct genera. Methods: We describe using a community standard fast average nucleotide identity (ANI) approximation method, MASH, for classifying NTM genomes by comparison to a resource of type strain genomes and proxy genomes. We evaluate the genus reclassification proposal in light of our ANI, MLST, and pan-genome work. Results: We describe here a sequencing study of hundreds of clinical NTM isolates. To aid in characterizing these isolates we defined a multi-locus sequence typing (MLST) schema for NTMs which can differentiate strains at the species and subspecies level using eight ribosomal protein genes. We determined and deposited the allele profiles for 2,802 NTM and Mycobacterium tuberculosis complex strains in PubMLST. Conclusions: The MLST schema and our pan-genome analysis of Mycobacteria can help inform the design of marker-gene diagnostics. The ANI comparisons likewise can assist in the classification of unknown genomes, even from previously unknown species.

Whole genome sequencing of Nontuberculous Mycobacterium (NTM) isolates from sputum specimens of co-habiting patients with NTM pulmonary disease and NTM isolates from their environment

BackgroundNontuberculous mycobacterium (NTM) species are ubiquitous microorganisms. NTM pulmonary disease (NTM-PD) is thought to be caused not by human-to-human transmission but by independent environmental acquisition. However, recent studies using next-generation sequencing (NGS) have reported trans-continental spread of Mycobacterium abscessus among patients with cystic fibrosis.ResultsWe investigated NTM genomes through NGS to examine transmission patterns in three pairs of co-habiting patients with NTM-PD who were suspected of patient-to-patient transmission. Three pairs of patients with NTM-PD co-habiting for at least 15 years were enrolled: a mother and a daughter with M. avium-PD, a couple with M. intracellulare-PD, and a second couple, one of whom was infected with M. intracellulare and the other of whom was infected with M. abscessus. Whole genome sequencing was performed using patients’ NTM isolates as well as environmental specimens. Genetic distances were estimated based on single nucleotide polymorphisms (SNPs). By comparison with the genetic distances among 78 publicly available NTM genomes, NTM isolates derived from the two pairs of patients infected with the same NTM species were not closely related to each other. In phylogenetic analysis, the NTM isolates from patients with M. avium-PD clustered with isolates from different environmental sources.ConclusionsIn conclusion, considering the genetic distances between NTM strains, the likelihood of patient-to-patient transmission in pairs of co-habiting NTM-PD patients without overt immune deficiency is minimal.

Mycobacterium abscessus complex representatives in patients with bronchopulmonary pathology: prevalence, peculiarities of cultivation and identification.

More and more publications appear in the modern literature on the increase in the prevalence of non-tuberculous mycobacteria (NTMs), in particular, representatives of M. chelonae / Mycobacterium abscessus complex (MABSc). The paper presents data on the current classification of M. chelonae / Mycobacterium abscessus complex and its main representatives. The main data on the possible sources and ways of infection of MABSc patients in hospital are presented. The main features of cultivation on various nutrient media and their possible identification using modern methods are also indicated. The main risk factors for the development of mycobacteriosis in patients and the possible clinical picture are described. The prevalence of MABSc representatives in the structure of non-tuberculous mycobacteria isolated from clinical material from 483 patients from the Samara region was assessed for examination for tuberculosis, and the prevalence from 933 patients with cystic fibrosis (CF) from 55 regions of the Russian Federation from 2016 to 2019 was estimated. In total, as a result of the study, 316 NTM strains (65.4%) were isolated and identified in the first group of patients. M.abscessus was isolated and identified 10 strains and 5 strains - M.chelonae, which amounted to 3.2% and 1.6%, respectively, of all NTMs. In general, MABSc representatives were isolated in 3.1% of the examined patients. As a result of a screening study of patients with CF, 14194 microorganism strains from 933 patients were isolated and identified. Altogether M. abscessus was isolated and confirmed from 14 patients of different ages. Thus, the prevalence of MABSc among the examined patients with CF in the Russian Federation was 1.5%.

Typing and classification of non-tuberculous mycobacteria isolates

Background: There are a large and growing number of non-tuberculous mycobacteria (NTM) species that have been isolated, identified, and described in the literature, yet there are many clinical isolates which are not assignable to known species even when the genome has been sequenced. Additionally, a recent manuscript has proposed the reclassification of the Mycobacterium genus into five distinct genera. Methods: We describe using a fast average nucleotide identity (ANI) approximation method, MASH, for classifying NTM genomes by comparison to a resource of type strain genomes and proxy genomes. We evaluate the genus reclassification proposal in light of our ANI, MLST, and pan-genome work. Results: We describe here a sequencing study of hundreds of clinical NTM isolates. To aid in characterizing these isolates we defined a multi-locus sequence typing (MLST) schema for NTMs which can differentiate strains at the species and subspecies level using eight ribosomal protein genes. We determined and deposited the allele profiles for 2,802 NTM and Mycobacterium tuberculosis complex strains in PubMLST. Conclusions: The MLST schema and our pan-genome analysis of Mycobacteria can help inform the design of marker-gene diagnostics. The ANI comparisons likewise can assist in the classification of unknown genomes, even from previously unknown species.

Rare species in the structure of acid-resistent members of the order Actinomycetales isolated from clinical material.

The article presents data on the structure of acid-resistant members of the order Actinomycetales and rare species that have been isolated and identified using various methods. The study included strains of non-tuberculous mycobacteria (NTM) isolated from clinical material during examination for tuberculosis in the period from 2016 to 2019. The total number of samples with signs of NTMs growth that were included in the study was 316 samples. Primary isolation on Levenshtein-Jensen, Finn II, and MGIT media and NTMs identification by DNA-hybridization. All strains that were not identified prior to the species and culture, identified as microorganisms with a high G+C content (High GC GR +) were re-identified using a MALDI-ToF Microflex LT mass spectrometer (Bruker®). By the method of DNA-hybridization, 188 strains isolated by NTM were successfully identified to form 58.5% of all selected cultures. Among the selected species, representatives of slowly growing NTMs (M. avium complex, M. gordonae, M. kansasii) predominated, which amounted to 67.0% of all NTM strains identified to the species. Among the cultures for which DNA hybridization failed to carry out acceptable identification, predominantly NTMs were found, among which M. gordonae, M. avium, M. kansasii dominated. A number of NTMs were represented by rare species: M. iranicum and M. pseudoshottsii. Among this group of microorganisms, other acid-resistant aerobic actinomycetes were isolated, including those of potential clinical significance: Gordonia spp., Tsukamurella spp., Rhodococcus spp., Nocardia spp. When identifying cultures containing high concentrations of G+C, the maximum number of microbial associations was revealed, including those consisting of two types of NTMs (M. monacense + M. flavescens, M. avium + M. kansasii), as well as associations of M. gordonae with staphylococci. The same group included rare NTM species: M. fredericbergense, M. szulgai, M. malmoense, M. bohemicum, M. septicum, as well as representatives of the genera Nocardia, Gordonia, Tsukamurella.

Port-site infection due to nontuberculous mycobacteria following laparoscopic surgery.

Nontuberculous mycobacteria (NTM) are commonly found in soil and water and can cause nosocomial infections by contaminating equipment and disinfectants solution used in hospitals. NTM port-site infection after laparoscopic surgery is increasingly observed, but its clinical features, management, and prevention have not been reviewed adequately. We performed a comprehensive literature review of reports that described the clinical manifestation and management of NTM port-site infections following laparoscopic surgery. The perceived increase in NTM port-site infections is likely multifactorial, influenced by greater awareness, better diagnostics, changes in medical practice, increased prevalence of immunosuppression, and potential pathogen spread. Widespread resistance to common disinfectants is a major concern. Patients with NTM port-site infections typically present 1-3 months after the laparoscopic intervention with chronic local and minimal systemic symptoms. Surgical excision plays an important role in localized or refractory cases. Medical treatment should be guided by species identification and in vitro drug-susceptibility testing (DST) of the infecting NTM strain, with a combination of second-line antituberculosis agents, given for a prolonged duration. NTM port site infection is best prevented by meticulous skin preparation and infection control, using only sterilized supplies for laparoscopic surgery.

VapBC and MazEF toxin/antitoxin systems in the regulation of biofilm formation and antibiotic tolerance in nontuberculous mycobacteria.

Mycobacterium smegmatis and other nontuberculous mycobacteria (NTM) are widely distributed in the environment, but a significant increase of NTM infections has taken place in the last few decades. The objective of this study was to determine the role of toxin-antitoxin (TA) vapBC and mazEF systems that act as effectors of persistence in the stress response of NTM. The growth ability and the biofilm formation of NTM were evaluated by conventional methods. Bacterial cell viability was determined using MTT staining, agar plating, or the method of limiting dilutions. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antibiotics were estimated using broth and agar dilution methods. Despite a comparable growth dynamics and biofilm formation on solid/liquid interface with the wild type, a M. smegmatis vapBC, mazEF, and vapBC × mazEF deletion mutant produced more abundant pellicle and were more susceptible to heat shock. Significant differences were also found in the resistance wild type of NTM to isoniazid and ciprofloxacin reflected by higher MBC/MIC ratios. The proposed method of cultivation of agar blocks without visible growth after MIC determination into a liquid medium allows us to detect transition of all wild type of NTM strains to a dormant state in the presence of subMICs of isoniazid and ciprofloxacin while all deletion mutants failed to form dormant cells. Our data suggest that both vapBC and mazEF TA systems putatively involved in the heat and antibiotic stress response of NTM via their key role in transition to the dormant state.

Investigation of non-tuberculous mycobacteria in a primary hospital from southeastern China.

Non-tuberculous mycobacterium (NTM) can colonize the human body, leading to opportunistic infection. This study was conducted to analyze the NTM species composition in a primary hospital and investigate the potential features of the patients with different NTM species. Mycobacterial strains were collected from the patients admitted at the hospital from January 2016 to May 2019. MPB64 assay was used to screen NTM strains and confirmed by Rv0577 amplification. The species were identified by hsp65 sequencing. The clinical records of patients with NTM were retrospectively reviewed. Among the 122 identified NTM isolates, the most common strains were Mycobacterium avium complex (MAC, n = 102, 83.6%), Mycobacterium abscessus (n = 9, 7.4%) and Mycobacterium lentiflavum (n = 5, 4.1%). The predominant species among MAC were Mycobacterium chimaera (n = 57, 46.7%), followed by Mycobacterium intracellulare (n = 25, 20.5%) and Mycobacterium colombiense (n = 17, 13.9%). A significantly lower percentage of positive acid-fast assay was observed in Mycobacterium colombiense positive patients than in those with Mycobacterium intracellulare and Mycobacterium chimaera. Mycobacterium intracellulare was more frequently isolated in patients from the infectious department than in other MAC members. A predominant prevalence of Mycobacterium chimaera in Dongyang of Zhejiang Province was different from other regions in China, indicating that its prevalence has been likely underestimated. The heterogeneity in clinical features, caused by different MAC members, required an accurate species identification of the NTM isolated in the primary hospitals.

Phenotypic characterization of mycobacteria isolates from tuberculosis patients in Kaduna State, Nigeria

Background: Tuberculosis (TB) remains one of the leading public health challenges in Nigeria and the burden is still high. There is hence a need for continuous characterization of mycobacteria to obtain current data that will aid the ongoing TB prevention and control programme. The aim of this study was to phenotypically characterize mycobacteria isolates recovered from clinical specimens of patients with tuberculosis in Kaduna State, Nigeria.Methods: Two thousand, two hundred and twelve (2212) sputum samples were collected from patients clinically suspected to have TB in three different zones of Kaduna State, Nigeria, between May 2017 and October, 2018. Samples were processed by decontaminating with NaOH-Citrate N-acetyl-L-Cystein method for Ziehl Neelsen (ZN) AFB microscopy and culture on Lowenstein Jensen (LJ) slants which were incubated at 37ᵒC for 8 weeks. Positive LJ cultures were further analyzed with a rapid TB antigen assay (SD-Bioline) to differentiate Mycobacterium tuberculosis complex (MTBC) from Non Tuberculous Mycobacteria (NTM).Results: Out of the 2212 patients with suspected TB, 300 (13.6%) were positive for AFB by microscopy with Zone A (Kaduna North) having the highest AFB positive cases of 169 (15.2%). Of the 300 AFB positive samples, 272 (91.0%) were culture positive on LJ medium, 18 (6.0%) were culture negative and 10 (3.0%) were culture contaminated. Result of the distribution of mycobacteria among infected patients within the study area revealed that 219 (80.5%) were infected with MTBC, 42 (15.4%) with NTM and 11 (4.0%) with both MTBC and NTM.Conclusion: A relatively high number of TB in the study area was caused by NTM. There is need for advanced diagnostic tools that can differentiate MTBC and NTM strains among TB patients in all TB Reference Laboratories in Nigeria.Keywords: Phenotypic, Characterization, Tuberculosis, Mycobacteria

Frequently Isolated Slow Growing Nontuberculous Mycobacteria from Pulmonary Samples and Evaluation of Drug Susceptibility Testing Results in a Referral Hospital in Turkey

Most of the nontuberculous mycobacteria (NTM) are opportunistic pathogenic microorganisms and free-living in nature. NTM can cause a wide range of infections. However, pulmonary NTM disease is the most frequent clinical picture. The aim of this study was to identify and evaluate drug susceptibility of slow growing NTM isolated from pulmonary samples of patients prediagnosed as tuberculosis between 2014 and 2018 in Atatürk Chest Diseases and Chest Surgery Training and Research Hospital Microbiology Laboratory by a commercial microtube dilution plaque method. A total of 435 NTM strains obtained from suspected TB patients were included in the study. After the samples were processed by homogenization and decontamination and acid-fast staining, culture in two solid media (Löwenstein-Jensen, Ogawa) and in MGIT-BACTEC960 automated system were performed. Acid-fast bacilli isolated from culture media were identified by using cart test (MPB64, Capilla TB-Neo) and polymerase chain reaction (PCR) based reverse hybridization "line probe assay (LPA)" method (GenoType MycobacteriumCM/AS, Hain Lifescience, GmbH, Germany). After DNA isolation from the culture, PCR was performed by using the primers specific for mycobacterial 23S rRNA spacer region. PCR products were then hybridized with the probes specific for Mycobacterium species on nitrocellulose strips according to the recommendations of the manufacturer and the results were evaluated. In this study, Mycobacterium avium (n= 77, 17.7%), Mycobacterium intracellulare (n= 70, 16.1%), Mycobacterium szulgai (n= 19, 4.4%), Mycobacterium kansasii (n= 10, 2.3%) ve Mycobacterium smiae (n= 9, 2.1%) were isolated as slowly growing mycobacteria from the pulmonary patients. Susceptibility testing was performed in cation-adjusted Mueller-Hinton broth (CAMH), supplemented with "oleic acid, albumin dextrose catalase" according to CLSI/ M24-A2 guideline recommendations. For the antibiotic susceptibility test, ready-to-use plaque drugs for slow-growing mycobacteria (SLOMYCO-Sensititre, TREK Diagnostic Systems Ltd, UK), were used. M.intracellulare, M.avium, M.kansasii and M.smiae isolates were found to be sensitive to clarithromycin %100, %99, %100 and %100, respectively. For M.intracellulare and M.avium isolates, moxifloxacin and linezolid sensitivity values were found to be 91%, 64% and 80%, 74% respectively. M.kansasii isolates were more sensitive than M.simiae isolates to the most of the drugs. M.kansasii isolates, were susceptible to rifabutin, rifampin, moxifloxacin, amikacin, linezolid, trimethoprim-sulfamethoxazole (TMP-SMX), ciprofloxacin and etambutol, with the frequencies of 100%, 90%, 100%, 100%, 80%, 70% and 50%, respectively. The study showed that the species identification and drug susceptibility testing of frequently isolated slow-growing NTM's from pulmonary specimens could guide for the treatment.

Drug resistance and pathogenic spectrum of patients coinfected with nontuberculous mycobacteria and human-immunodeficiency virus in Chengdu, China.

Background:Human-immunodeficiency virus (HIV) infection is increasing worldwide and nontuberculous mycobacteria (NTM) is an established microbiologic cause of pulmonary disease, lymphadenitis, and disseminated disease in cases of advanced immune suppression. Data on patients coinfected with HIV and NTM are limited. Thus, this study aimed to analyze the clinical characteristics, drug resistance, and pathogen spectrum of patients coinfected with both HIV and NTM in the Chengdu area of China.Methods:Data of 59 patients coinfected with both HIV and NTM collected from the Public Health Clinical Center of Chengdu, between January 2014 and December 2018, were analyzed. NTM drug sensitivity testing was performed using the microporous plate ratio method. Data were analyzed using SPSS 19.0, and the change in drug resistance rate was analyzed using the chi-square (χ2) test.Results:Seven species/complex of NTM were identified from patients coinfected with HIV and NTM in this study, with Mycobacterium avium–intracellulare complex (52.5%) and M. kansasii (27.1%) as the predominant species. Male patients were more affected 50/59 (84.7%); the mean age of the 59 cases was 45 years. The clinical characteristics mainly included anemia (86.4%), cough and expectoration (79.7%). The baseline CD4 count was <50 cells/μL (84.7%). Patients were mainly in advanced acquired immunodeficiency syndrome (AIDS) stage. Chest imaging mainly showed patchy shadows (42.4%) and nodules (32.2%), with various degrees of AIDS-defining diseases. The drug resistance of NTM was severe, and the rate of isoniazid resistance (100.0%) was the highest, followed by rifampicin (94.9%), streptomycin (94.9%), ofloxacin (93.2%), and others. Ethambutol (52.5%) and clarithromycin (33.9%) were relatively low. No significant difference was found in the drug resistance rate of NTM strain against nine antituberculosis drugs in 5 years (P > 0.05).Conclusions:The immune level of patients coinfected with HIV and NTM is low in advanced AIDS stage; more male are affected in patients who are mainly infected with MAC and M. kansasii and with serious drug resistance. The drug resistance rate of ethambutol and clarithromycin is relatively low.

Recovery of Non-tuberculous Mycobacteria from Water is Influenced by Phenotypic Characteristics and Decontamination Methods.

Infections related to non-tuberculous mycobacteria (NTM) have recently increased worldwide. The transmission of these microorganisms from the environment has been suggested as the main source for human infections. To elucidate the epidemiological aspects and distribution of these pathogens, many studies have evaluated several decontamination methods and protocols to properly isolate NTM from environmental samples, mainly from water. However, no satisfactory strategy has been found for isolation of most of the NTM species harboring different phenotypic characteristics. Here, we evaluated the susceptibility of 23 NTM strains presenting variable growth rate and pigmentation patterns to eight different methods: oxalic acid (2.5% and 5%), cetylpyridinium chloride (CPC) (0.0025% and 0.005%), sodium hydroxide (NaOH) (2% and 4%), and sodium dodecyl sulfate (SDS) plus NaOH (SDS 1.5%-NaOH 0.5% and SDS 3%-NaOH 1%). It was found that the viability of NTM exposed to different decontamination methods varies according to their phenotypic characteristics and two methods (SDS 1.5% plus NaOH 0.5% and CPC 0.0025%) were necessary for effective isolation of all of the species tested. These findings supply important insights for future studies on the environmental occurrence of mycobacteria and improving the sensibility of traditional strategies.

The importance of multiple samples in mycobacterial recovery: A 10-year retrospective study

Instead of the three samples previously recommended, the World Health Organization considered that two sputum samples are sufficient for the diagnosis of pulmonary tuberculosis. Our goal is to evaluate the value of multiple samples in the microscopic and cultural examination of Mycobacterium tuberculosis complex (MTBc) and nontuberculous mycobacteria (NTM). Through a 10-year retrospective study, including 38,887 sputum samples from 11,676 patients, the relative contribution of each sputum specimen for acid-fast bacilli detection and mycobacteria isolation was determined by calculating the yield of the first sample and the incremental yield of the second, third, and more than three samples. With regard to patients with MTBc isolation, we found that 95.2% (435/457) of the cases could be detected in the first and second smear and 97.9% (659/673) in the first and second culture, with a very low increment of the third sample, corresponding to 3.1% (14/457) in smear and 1.3% (9/673) in culture. Among patients with an NTM strain, the percentage of cases detected in the first and second smear and culture was 69% (20/29) and 86.5% (316/365), respectively. In these patients, we found an increment yield of the third sample of 20.7% (6/29) for smear and 10.5% (38/365) for culture. The incremental yield of third and above-third sample for NTM detection was of 31% (9/29) in smear and 13.5% (49 of 365) in culture. We found an added value of multiple samples in NTM recovery. However, our findings suggest that only two samples are required for the diagnosis of pulmonary tuberculosis.

The importance of non-tuberculous mycobacteria identification in Chinese patients infected with HIV.

The increased co-incidence of tuberculosis (TB) and AIDS is compounded by the emergence of opportunistic infections with non-tuberculous mycobacteria (NTM) in patients with HIV/AIDS, and the treatment for these infections differs from that for TB. The high frequency of NTM strains found in patients infected with HIV raises concerns about accurate species identification before deciding proper treatment. A total of 101 isolates from 2014, 137 from 2015, and 162 from 2016 were subjected to 16S rDNA sequencing to identify the species. Forty-one (41/101, 40.6%) were identified as NTM in 2014, 64 (64/137, 46.7%) were identified as NTM in 2015, and 72 (72/162, 44.4%) were identified as NTM in 2016 in Chinese patients infected with HIV. The species of Mycobacteria isolates needs to be rapidly and accurately identified to determine appropriate antibiotic therapy, and this is especially true for patients infected with HIV.