GenoType Mycobacterium CM Research Articles

Evaluation of the FluoroType Mycobacteria assay for identification of mycobacteria.

This study is the first clinical evaluation report of the novel FluoroType Mycobacteria assay. The assay has the potential to replace the established GenoType NTM product family in identification of culture-enriched mycobacteria. However, our research results suggest that the assay performs suboptimally and may not be feasible for use in all clinical settings.

Description of Mycobacterium pinniadriaticum sp. nov., isolated from a noble pen shell (Pinna nobilis) population in Croatia.

Shortly before the mass mortality event of the noble pen shell (Pinna nobilis) population in the south-eastern Adriatic coast, two rapidly growing Mycobacterium strains CVI_P3T (DSM 114013 T, ATCC TSD-295 T) and CVI_P4 were obtained from the organs of individual mollusks during the regular health status monitoring. The strains were identified as members of the genus Mycobacterium using basic phenotypic characteristics, genus-specific PCR assays targeting the hsp65 and 16S rRNA genes and the commercial hybridization kit GenoType Mycobacterium CM (Hain Lifescience, Germany). MALDI-TOF mass spectrometry did not provide reliable identification using the Bruker Biotyper Database. Genome-wide phylogeny and average nucleotide identity (ANI) values confirmed that the studied strains are clearly differentiated from their closest phylogenetic relative Mycobacterium aromaticivorans and other validly published Mycobacterium species (ANI ≤ 85.0%). The type strain CVI_P3T was further characterized by a polyphasic approach using both phenotypic and genotypic methods. Based on the phenotypic, chemotaxonomic and phylogenetic results, we conclude that strains CVI_P3T and CVI_P4 represent a novel species, for which the name Mycobacterium pinniadriaticum sp. nov. is proposed.

First isolation of Mycobacterium saskatchewanense from medical devices

Mycobacterium saskatchewanense is a species of pigmented slow-growing Non-Tuberculous Mycobacteria (NTM), positive for Mycobacterium avium complex (MAC) by AccuProbe system. MAC organisms have frequently been isolated from different medical devices. This is the first study reporting isolation of M. saskatchewanense from medical devices and highlights the importance of correctly identifying the NTMs that often colonize sanitary water. GenoType Mycobacterium CM CE-IVD kit (CM) was used as the first step of NTM strain identification, and all positive cultures were found to be components of MAC. Then, GenoType NTM-DR CE-IVD kit (NTM-DR) was used to differentiate the different species. Sub-culture on solid media were used for: (i) phenotypical confirmation by colony morphology and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry; (ii) molecular confirmation by Next Generation Sequencing. All positive cultures were identified as M. intracellulare by CM and NTM-DR assays, whereas colony morphology showed bright yellow scotochromogenic growth. MALDI-TOF analyses identified the strains as M. saskatchewanense with a high score, and identification was confirmed by NGS analysis based on the hsp-65 region. This paper suggests that it is important to actively monitor NTM contamination in medical devices that use sanitary water, to prevent the possibility of patients becoming infected.

Diagnostic performance of two commercial MPT64 assays for the identification of Mycobacterium tuberculosis complex from liquid culture

BackgroundTuberculosis (TB) is a chronic infectious disease caused by bacteria of the Mycobacterium tuberculosis complex (MTBC). Early diagnosis of TB is the most essential component to control TB. In this study, we evaluated the diagnostic performance of SD Bioline TB Ag MPT64 (SD Bioline) and BD MGIT TBc Identification (BD TBc ID) kits for the detection of MTBC from MGIT (Mycobacteria Growth Indicator Tube) 960 culture tube, positive for acid-fast bacilli (AFB). MethodsA total of 198 AFB positive MGIT cultures [MTBC, 150 and non-tuberculous mycobacteria (NTM), 48] were collected during the study period (April 2021 to March 2023) and subjected to SD Bioline and BD TBc ID assays, respectively. All the 150 MTBC positive cultures were confirmed by multiplex PCR targeting MPB64, protein B and IS6110 genes, and the 48 NTM cultures were speciated by using line probe assays technique- Genotype Mycobacterium CM assay. ResultsSD Bioline and BD TBc ID kits detected all 150 MTBC strains correctly from MGIT positive cultures and no false negative was observed. However, there was one false positive for M. abscessus by SD Bioline and two false positive results for M. abscessus and M. intracellulare each by BD TBc ID. Overall, the sensitivity of both SD Bioline and BD TBc ID tests was 100 % and specificity was 97.9 % and 95.8 %, respectively. ConclusionOur results highlighted that SD Bioline and BD TBc ID assays played a promising role in the earlier identification of MTBC present in liquid culture and may act as a potential alternative to biochemical and expensive molecular techniques.

Trends from the Last Decade with Nontuberculous Mycobacteria Lung Disease (NTM-LD): Clinicians' Perspectives in Regional Center of Pulmonology in Bydgoszcz, Poland.

Nontuberculous mycobacteria (NTM) are the cause of chronic lung disease called NTM lung disease (NTM-LD). There are about 180 known species of NTM. Nowadays the number of NTM-LD is increasing. To evaluate the clinical significance of NTM isolated from specimens and assess the frequency and clinical relevance of isolation of NTM in the Regional Center of Pulmonology in Bydgoszcz, hospital of Northern Poland. Clinical, radiological, and microbiological data were collected from all patients from whom NTM was isolated between 2013 and 2022. Data were reviewed retrospectively. Diagnostic criteria for NTM-LD published by the American Thoracic Society (ATS) were used to determine clinical relevance. The study comprised 81,985 clinical specimens submitted for mycobacterial culture in the Department of Microbiology at the Regional Center of Pulmonology in Bydgoszcz between 2013 and 2022. Clinical specimens were processed according to the standard procedure in mycobacteria laboratories in Poland. NTM strains were identified using analysis of mycolic acids by chromatography as well as GenoType NTM-DR, GenoType Mycobacterium AS, and GenoType Mycobacterium CM. There were 395 patients with NTM strains between 2013 and 2022. Out of them, 149 cases met the diagnostic criteria of NTM-LD and were classified as definite cases. M. kansasii (n = 77) was the most common species in the group (51.68%), followed by M. avium complex (n = 46). Patients with NTM-LD were 22-88 years old (median age was 60 years). There were 81 men and 68 women. The most common symptoms were cough, hemoptysis, and fever. Radiological X-ray images were dominated by infiltrative lesions in the upper and middle lobe of the right lung with cavities; the changes were in the upper lobe of the left lung and on both sides of the chest. They were smokers in 61%. The most common concomitant diseases were chronic obstructive pulmonary disease (COPD), diabetes mellitus, pulmonary carcinoma, and human immunodeficiency virus (HIV) infection, and other immunodeficiencies. The most common treatment was isoniazid, ethambutol, rifampicin, and ofloxacin for 18 months with a minimum of 12 months of culture negativity. NTM-LD infections are present with other pulmonary illnesses and extrapulmonary diseases and may be connected to primary immunologic deficiencies. These diseases concern patients of all ages and have various clinical manifestations. M. kansasii and MAC are the most prevalent NTM isolates among respiratory samples in Northern Poland. In addition, an increase in MAC and a decrease in M. kansasii both in cultivation and the cause of NTM-LD were reported.

Aspects of Stage-by-Stage Diagnosis of Pulmonary Mycobacteriosis in Real Clinical Practice

The objective: to analyze the effectiveness of etiological verification of pulmonary mycobacteriosis when using various biological materials at different diagnostic stages.Subjects and Methods. In Center for Diagnosis and Rehabilitation of Respiratory Diseases, CTRI, the results of stage-by-stage examinations of 142 patients (from 2018 to 2022) were analyzed. A retrospective, prospective, cohort, open study was conducted. Inclusion criteria: pulmonary mycobacteriosis etiologically verified by comprehensive microbiological examinations (molecular genetic and cultural tests) (with retrospective analysis after all stages of examination). Exclusion criteria: HIV infection. 115 women and 27 men were enrolled in the study, the age ranged from 21 to 86 years, the mean age was 57,9 years.All patients referred to CTRI for examination due to newly detected changes in the lungs (bronchiectasis, focal changes, and cavities). For etiological verification of the diagnosis, microbiological sputum testing was performed (Stage 1 of the examination); when negative results were obtained, bronchoscopy was prescribed and bronchobiopsies were used for microbiological and molecular genetic diagnostics – Stage 2. In case of negative results of Stage 2, the patient was recommended to undergo a diagnostic resection of the lung – Stage 3 of the examination. At all stages, specimens were sent for comprehensive microbiological testing: fluorescent microscopy to detect acid-fact mycobacteria, real-time polymerase chain reaction (PCR-RV) to detect NTBM DNA. Identification of NTBMB was carried out by molecular genetic methods (PCR for the presence of MTB DNA/NTBM (SYNTOL, Russia), and on DNA strips (GenoType Mycobacterium CM\AS Hain Livescience, Germany)). Specimens were cultured on a liquid medium in the ВАСТЕС MGIT960 (BD, USA).The diagnosis of mycobacteriosis was established in accordance with the diagnostic criteria proposed in the guidelines of the American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA) (2020): detection of NTBM in 2 sputum samples or in 1 sample of bronchobiopsy specimens or in 1 surgical specimens [15].The studied data were entered to the Microsoft Office Excel sheet (Microsoft, USA) for statistical processing; the level of statistical significance of differences was taken as 0.05.Results. It was found that slow-growing NTBM were identified as etiologically significant in 93.0%, of which 78.1% was M. avium, fast-growing NTBM were represented only by M. abscessus, 7% of observations. During X-ray examination, the bronchiectatic form – 52.1% (74/142) was detected more often compared to the abdominal – 30.9% (44/142) and focal forms – 16.9% (24/142) (p<0.001). When verifying mycobacteriosis in patients with a negative sputum test – Stage 2 of the examination – the effectiveness was 91.0% (80/88). Regardless of the radiological form of the disease and dissemination of the disease, bronchoscopy revealed pathology of the tracheobronchial tree in 85.2% of cases, mainly in the form of bronchitis with purulent secretions.

Identification Of Nontuberculous Mycobacterium Isolates in Suspected Pulmonary Tuberculosis Patients

According to World Health Organization, in the global tuberculosis ranking Pakistan is in 5th position. Mycobacterium tuberculosis bacterium is responsible for this dreadful disease, but there are other nontuberculous mycobacteria species that could also be the possible cause of this disease. Scanty data is available on the incidence and distribution of species responsible for this infection. Local studies on non-tuberculous mycobacterium species would be of great support in targeted therapy. Methods: This study was designed to investigate the incidence and distribution of non-tuberculous mycobacteria-associated infection in pulmonary suspected tuberculosis patients. Sputum samples were processed for microscopy and culturing on Lowenstein–Jensen regardless of age and gender suspected TB patients. Positive cultures were then processed for detection of non tuberculous mycobacteria species using commercially available Geno Type Mycobacterium CM hybridization strips. Results: A total of 1560 sputum samples were tested for Mycobacteria by culturing, 215 were positive, 71 contaminated and 1274 were negative. All 215 culture positive isolates were exposed to Geno Type Mycobacterium CM kit revealed, 55 cultures as Non-tuberculous mycobacteria and 160 as Mycobacterium complex. Adults between aged 40 to 60 years and male were predominantly (61.81%) infected than females (38.18%). Conclusions: Scanty data is available about the contributions of non tuberculous mycobacteria to tuberculosis-like disease, and noteworthy geographical distribution, clinical and molecular epidemiology-related knowledge gaps exist in the areas with a high burden of disease caused by mycobacterium tuberculosis complex. Isolation of nontuberculous mycobacteria from clinical specimens should promptly be evaluated for their clinical significance.

Could the type and severity of gross lesions in pig lymph nodes play a role in the detection of Mycobacterium avium?

The Mycobacterium avium complex (MAC) comprises a widespread group of slowly-growing bacteria from the Mycobacteriaceae. These bacteria are responsible for opportunistic infections in humans and animals, including farm animals. The aim of the study was to determine whether it is possible to predict the presence of M. avium in pig lymph nodes based on the size and type of lesions found during post-mortem examination at a slaughterhouse. Lymph nodes were collected from 10,600 pigs subjected to such post-mortem examination. The nodes were classified with regard to their quality, and the number of tuberculosis-like lesions; following this, 86 mandibular lymph nodes with lesions and 113 without visible macroscopic lesions were selected for further study. Cultures were established on Löwenstein-Jensen and Stonebrink media, and a commercial GenoType Mycobacterium CM test was used to identify and differentiate M. avium species. The prevalence of M. avium was 56.98% in the lymph nodes with lesions and 19.47% in the unchanged ones. Statistical analysis indicated that visual assessment of lesions in the mandibular lymph nodes, in particular the number of tuberculous lesions, is a highly-efficient diagnostic tool. Similar results were obtained for estimated percentage area affected by the lesion, i.e. the ratio of the changed area of the lymph node in cross-section to the total cross-sectional area of the lymph node; however, this method is more laborious and its usefulness in slaughterhouse conditions is limited. By incising the lymph nodes and assessing the number of tuberculosis-like lesions, it is possible to limit the inclusion of meat from pigs infected with M. avium into the human food chain.

Evaluation of the MeltPro Myco Assay for the Identification of Non-Tuberculous Mycobacteria.

PurposeThe purpose of this study was to evaluate a new commercial kit for Mycobacterium species identification and compare its results with that of the commonly used GenoType Mycobacterium CM assay. In addition, we were committed to identifying the main frequent species of nontuberculous mycobacteria (NTM) in Fujian.MethodsA total of 261 clinical strains, collected at the Center for Disease Control and Prevention of Fujian Province, China, were preliminarily identified as NTM based on p-nitrobenzoic acid (PNB) growth test. The genomic DNA of all 261 strains was extracted and subjected to species identification using MeltPro Myco assay and GenoType Mycobacterium CM assay. The results of the latter were used as a control to calculate the positive agreement, negative agreement, agreement and the total agreement of the former. For samples with inconsistent detection results, sequencing was performed for verification.ResultsCompared to GenoType Mycobacterium CM assay, the total agreement of MeltPro Myco assay was 96.55% (252/261 strains). Both the positive and negative agreement of MeltPro Myco assay in identifying M. intracellulare, M. abscessus, M. kansasii, M. fortuitum, M. avium, M. gordonae, M. scrofulaceum, and mixed infections were higher than 99.00%, but the positive agreement of M. chelonae was relatively low at only 33.33%. In Fujian, the predominant strain of NTM was M. intracellulare (64.36%, 130/202 strains), followed by M. abscessus (19.31%, 39/202 strains), M. kansasii (4.46%, 9/202 strains), and M. fortuitum (3.96%, 8/202 strains).ConclusionThe reliability of MeltPro Myco assay for identifying mycobacterium species was strongly demonstrated in this study, which greatly supports its usage for the clinical identification of mycobacteria. The present study also showed that the distribution of mycobacteria in Fujian, China, was significantly different from that in other regions and provided important data for future epidemiological study of NTM.

Non-tuberculous mycobacteria, not Mycobacterium bovis, are a significant cause of TB-like lesions observed in slaughtered cattle in Ghana

ObjectivesThe aim was to isolate and identify the species of mycobacteria causing tuberculous-like (TB-like) lesions in cattle in Ghana. MethodsBetween 2019 and 2020, 68 bovine tissue samples with TB-like lesions, identified during post slaughter examination, were obtained from four major abattoirs close to border towns in Ghana. The samples were cultured on Lowenstein–Jensen medium. Isolated bacteria were characterized by Ziehl–Neelsen staining and observation for acid-fast bacilli (AFB) under a microscope. DNA was extracted from AFB-positive isolates, and mycobacterial speciation was performed by line probe assay using GenoType Mycobacterium CM and also with mycobacterial 16S rRNA gene amplification and sequencing. ResultsNo Mycobacterium bovis was identified; however 53 bacterial isolates were obtained, of which 41 were non-tuberculous mycobacteria (NTM) strains and 12 were gram-positive bacteria. The predominant NTM species was M. fortuitum (43.9%, 18/41), with the rest being M. novocastrense, M. terrae, M. flavescens, M. holsaticum, M. cosmeticum, M. virginiense, M. intracellulare, M. mageritense, M. minnesotensis, M. duvalii, M. lehmannii, and M. koreense. ConclusionsIn cattle, NTM contribute significantly to lesions observed during slaughter examination and may be an important cause of zoonotic tuberculosis. A One Health surveillance of NTM in Ghana would provide insights into their clinical significance.

Bovine mastitis caused by rapid-growth environmental mycobacteria

Rapid-growth mycobacteria were isolated from two cases of cow mastitis with similar clinical appearance and within a narrow time frame. Mycobacteria were isolated on blood esculine agar. The isolated mycobacteria were Gram stained, Ziehl-Nielsen stained and tested for growth at 25°C, 37°C and 42°C, iron uptake, growth on Löwenstein-Jensen (LJ) agar with and without 5% NaCl, arylsulphatase (3 days), tween 80 hydrolysis, tellurite reduction, nitrate reductase and niacin synthesis. Molecular identification was performed using the Mycobacteria GenoType CM and AS tests (Hain Diagnostika, Nehren, Germany). One isolate was additionally sequenced for the hsp65, rpoB, 16S rRNA gene sequence and transcribed spacer sequence (ITS) DNA. Susceptibility testing of isolates was performed on the Sensititre Rapmycol plate (TREK Diagnostic Systems Ltd.) for trimethoprim/sulfamethoxasole, linezolid, ciprofloxacin, imipenem, moxifloxacin, cefepime, cefoxitin, amoxicillin / clavulanic acid, amikacin, ceftriaxone, doxycycline, minocycline, tigecycline, tobramycine and clarythromycine. Gram-positive acid-resistant rods were observed in stained smears. Both strains grew at 25°C, 37°C and 42°C on LJ medium, and on LJ medium containing 5 % NaCl. The conventional biochemical tests for iron uptake, arylsulphatase (3 days), Tween 80 hydrolysis, tellurite reduction and nitrate reductase were positive, while the niacin test was negative. Both isolates were identified by the GenoType Mycobacterium CM as Mycobacterium fortuitum II/ Mycobacterium mageritense, while application of the GenoType Mycobacterium AS kit identified both isolates as belonging to the species Mycobacterium smegmatis. Analysis of the isolate sequences (strain DS) for 16S ribosomal RNA confirmed a 100% identical result with Mycobacterium smegmatis strain INHR2. According to the CLSI criteria, both strains were sensitive to sulfametoxazole/trimethoprim, linezolid, doxicycline, amikacin and tobramycin. The strains differed in their sensitivity to cefoxitim, and both strains were resistant to clarithromycin. There was a strong difference between the isolates in sensitivity toward cefoxitime and tigecycline.

Evaluation of MALDI Biotyper Interpretation Criteria for Accurate Identification of Nontuberculous Mycobacteria.

Identification of mycobacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) requires not only a good protein extraction protocol but also an adequate cutoff score in order to provide reliable results. The aim of this study was to assess the cutoff scores proposed by the MALDI-TOF MS system for mycobacterial identification. A total of 693 clinical isolates from a liquid medium and 760 from a solid medium were analyzed, encompassing 67 different species of nontuberculous mycobacteria (NTM). MALDI-TOF MS identified 558 (80.5%) isolates from the liquid medium and 712 (93.7%) isolates from the solid medium with scores of ≥1.60. Among these, four (0.7%) misidentifications were obtained from the liquid medium and four (0.5%) from the solid medium. With regard to species diversity, MALDI-TOF MS successfully identified 64 (95.5%) different species, while PCR-reverse hybridization (GenoType Mycobacterium CM and AS assays) identified 24 (35.8%) different species. With MALDI-TOF MS scores of ≥2, all isolates were correctly identified, and with scores in the range from 1.60 to 1.99, most isolates were correctly identified, except for Mycobacterium angelicum, M. parascrofulaceum, M. peregrinum, M. porcinum, and M. gastri In conclusion, MALDI-TOF MS is a useful method for identifying a large diversity of NTM species. A score threshold of 1.60 proved useful for identifying almost all the isolates tested; only a few species required a higher score (≥2.00) to obtain a valid definitive identification.

Mycobacteriosis in a Zoo Population of Chinese Gliding Frogs (Rhacophorus dennysi) Due to Mycobacterium marinum

Mycobacteriosis was implicated in the deaths of eight Chinese gliding frogs (Rhacophorus dennysi) in a zoo population over a 3 yr period. Clinical signs included nonhealing skin lesions, cloacal prolapse, hind limb weakness, weight loss, and sudden death. Abnormalities on postmortem were proliferative or ulcerative skin lesions in four of eight, pneumonia in three of eight, and gall bladder empyema in two of eight cases. All eight clinical cases had multisystemic granulomas containing acid-fast bacilli. Tissues most commonly affected were lung (seven of eight), liver (six of eight), kidney (six of eight), spleen (five of eight), and heart (five of eight). The remaining eight clinically normal frogs in the population were euthanized: eight of eight had granulomatous lesions, with acid-fast bacilli in three of eight cases. A mycobacterial species was cultured from four of the clinical cases by the Public Health Ontario Laboratory and was initially misidentified as Mycobacterium tuberculosis complex by a commercial line-probe assay (GenoType Mycobacterium CM, Hain Lifesciences, Nehren, Germany). Further diagnostic testing using 16S rRNA gene sequencing ultimately identified the mycobacterial species as Mycobacterium marinum. The correct identification of mycobacterial species is essential in epidemiological investigations at zoological facilities, and in assessing health risks to staff and to other animals in the zoo population.

An injection abscess due to M. fortuitum: A rare case report

An iatrogenic injection abscess is usually easy to treat if caused by aerobic bacteria but some rapidly growing mycobacteria (RGM), namely, Mycobacterium fortuitum, M. chelonae, and M. abscessus are associated with postinjection abscess and may cause delayed wound healing. RGM can cause mild localized cellulitis or abscess to osteomyelitis following penetration injuries or unsafe injection practices. A 7-year-old girl was presented to pediatric surgery OPD with abscess formation over the right buttock. Incision and drainage from abscess were performed in OPD and pus sample was sent for aerobic bacterial culture and sensitivity. On gram stain plenty of pus cells with no microorganism were seen and growth on blood agar after 48 h of aerobic incubation at 37°C showed small off-white pinpoint, smooth butyrous waxy colonies. Smear prepared from blood agar showed uniformly stained short, slender, faintly stained gram-positive bacilli, for which acid-fast staining (1% and 20% H2SO4) was performed that showed acid-fast bacilli. The isolate was further identified by the molecular method and was confirmed to be Mycobacterium fortuitum by genotype Mycobacterium CM VER 1.0 (HAIN LIFESCIENCE, BioMerieux India Pvt. Ltd.).

Mycobacterium arosiense, an unexpected cause of osteomyelitis in a patient with sarcoidosis: a case report

BackgroundNontuberculous mycobacteria belonging to the Mycobacterium avium complex are recognized as opportunistic pathogens to humans. Mycobacterium arosiense is one of the novel members of the Mycobacterium avium complex. The organism has only rarely been reported in human clinical cases and may be routinely misidentified.Case presentationAn adult male with a history of a discus prolapse and sarcoidosis presented with high fever and a strong back pain with projection to the extremities. A Magnetic Resonance Imaging scan of columna revealed a tumor suspect process at thoracic vertebrae 11/12 with changes at the second lumbar vertebra, which was partly removed by laminectomy. Biopsy smears revealed acid-fast bacilli and turned out to be Mycobacterium tuberculosis complex PCR negative. The routine line probe assay INNO-LiPa v2 (INNOGENETICS NV, Gent), which differentiates 16 mycobacterial species indicated the presence of a not readily identifiable NTM species. Whereas, the GenoType Mycobacterium CM v2.0 (HAIN Lifescience GmbH) that routinely differentiates 14 clinically relevant mycobacteria revealed a Mycobacterium intracellulare species. However, additional diagnostic sequencing of the 16S rRNA gene confirmed the presence of a Mycobacterium arosiense species.ConclusionsThis is the second unusual case of osteomyelitis with clinical significance ever to be reported, caused by Mycobacterium arosiense and complicated by an underlying sarcoidosis. Mycobacterium arosiense has rarely been reported clinically and the first description of the species was identified as the cause of osteomyelitis in a child with a hereditary partial interferon gamma deficiency. Symptoms attributed to sarcoidosis waned on Mycobacterium arosiense treatment and it is inconclusive whether the patient ever suffered from sarcoidosis. Mycobacterium arosiense was misidentified by the GenoType as Mycobacterium intracellulare and implicates that the diagnosis requires supplemental sequencing of the 16S rRNA gene.

ВЫЯВЛЕНИЕ НЕТУБЕРКУЛЕЗНЫХ МИКОБАКТЕРИЙ, ЦИРКУЛИРУЮЩИХ В РАЗНЫХ РЕГИОНАХ СИБИРИ, И АНАЛИЗ ИХ ЛЕКАРСТВЕННОЙ УСТОЙЧИВОСТИ

The objective of the study: to identify the frequency of various species of non-tuberculous mycobacteria (NTM) isolated in the patients of TB services in Siberian Region during 2014-2018, to determine the correlation between rates of resistance to different antibacterial drugs. Subjects and methods . PCR hybridization on strip strips (Hain Lifescience GenoType Mycobacterium CM / AS) was used to genotype 475 NTM isolates of patients from various TB hospitals inSiberia. Drug susceptibility was tested by minimum inhibitory concentrations (MIC) on culture with serial micro-dilutions in a liquid medium using TREK Diagnostic Systems kits. 64 panels were used for slow-growing mycobacteria (SLOWMYCO), 46 - for fast-growing ones(RAPMYCO). Data processing was performed using the APL of 3.5.1 and the data analysis software of Rstudio, version 1.1.442. Results of the study 1. Tomsk Region differs from the other considered regions ofSiberiadue to higher frequency of M. intracellulare among isolates identified by PCR hybridization. In other regions, M. fortuitum prevails among isolated. 2. Incomparison with the reference data (CLSI M62, 2018), differences in the MIC of some NTM species isolated from patients in Siberia and theFar Eastwere observed, most likely related to the regional characteristics of these isolates. 3. HTM isolates resistant to moxifloxacin are also resistant to linezolid, trimethoprim, and ciprofloxacin. 4. Fast-growing clarithromycin resistant HTM isolates are predominantly sensitive to moxifloxacin, ciprofloxacin, and trimethoprim.

A case of bovine tuberculosis in pigs in Poland - a country free from the disease.

Bovine tuberculosis is a chronic contagious disease caused by Mycobacterium bovis or Mycobacterium caprae. Before widespread action conducted in Poland between 1959-1975 to combat bovine tuberculosis (BTB), about 40% of all tuberculosis cases in pigs was caused by the bovine bacillus. At the present time, correctly carried out, long-term control of cattle has resulted in cases of bovine tuberculosis in pigs and humans being extremely rare and sporadic. In pigs, tuberculosis is most often caught in a slaughterhouse during slaughter. Samples came from pigs kept on the farm. Traditional bacteriological methods on solid media (Stonebrink, LJ with pyruvate) supported by the semi-automatic, liquid indicative culture method (MGIT) and PCR test were applied in targeted studies. The GenoType Mycobacterium MTBC and CM tests (Hain Lifescience, Germany) were used to additionally confirm that isolated strains classification was used. Strains of mycobacteria were isolated from all examined pigs. Mycobacterium bovis was determined by real time PCR and Hain Genotype methods. In order to effectively fight against BTB, all animals on farms should be tested, regardless of species, while the milk of suspected cows should be utilized without being used for feed. It is important to adapt the current legal regulations to the current epidemiological situation.

Mycobacterial infections in carcasses of ruminants slaughtered at the two slaughterhouses of Kassala, Sudan

Tuberculosis (TB) is a chronic bacterial disease of humans and animals. It is characterized by the progressive development of specific granulomatous lesions in affected organs. Human TB is endemic in Eastern Sudan. However, knowledge on the epidemiology of TB in ruminants is scarce. In a six-month study from June to November 2014, a total of 2304 carcasses of cattle, sheep, goats and camels slaughtered at the East and West Gaash slaughterhouses of Kassala were inspected to investigate TB prevalence. Only 0.1% (n = 2) of the carcasses had suspicious TB lesions. These lesions were solely found in carcasses of sheep, in the liver, lungs, and peritoneal cavity. The samples collected from the lesions were investigated for the presence of mycobacteria, which were found in one of the two carcasses. The grown bacteria were subjected to a line probe assay (GenoType Mycobacterium CM), and to 16S rDNA and ITS gene sequencing, and whole genome sequencing (WGS). However, none of these methods identified this isolate as a valid Mycobacterium species. Nevertheless, 16S sequence allocated this isolate to slow growing mycobacteria. Neither Mycobacterium bovis nor M. caprae nor M. tuberculosis were found in the collected granulomatous lesions. In conclusion, the overall prevalence of TB-suggestive lesions in ruminants in Kassala was very low. Extended studies combining the use of a tuberculin skin test and slaughterhouse-based investigations should be adopted for TB surveillance in ruminants in Sudan.

Nontuberculosis mycobacteria are the major causes of tuberculosis like lesions in cattle slaughtered at Bahir Dar Abattoir, northwestern Ethiopia

BackgroundThe main cause of bovine tuberculosis (bTB) is believed to be Mycobacterium bovis (M. bovis). Nontuberculosis mycobacteria (NTM) are neglected but opportunistic pathogens and obstacles for bTB diagnosis. This study aimed to isolate and characterize the mycobacteria organisms involved in causing TB-like lesions in cattle in northwestern Ethiopia.ResultsA total of 2846 carcasses of cattle were inspected for TB lesions. Ninety six tissues (including lymph nodes such as submandibular, retropharyngeal, tonsilar, mediatinal, bronchial and mesenteric, and organs such as lung, liver and kidney) with suspicious TB lesion(s) were collected and cultured on Lowenstein-Jensen medium. Twenty one showed culture growth, of which only 17 were identified containing acid fast bacilli (AFB) by Ziehl–Neelsen staining. Among the 17 AFB isolates 15 generated a polymerase chain reaction product of 1030 bp by gel electrophoresis based on the 16S ribosomal RNA gene amplification. No M. tuberculosis complex species were isolated. Further characterization by Genotype Mycobacterium CM assay showed 6 isolates identified as M. peregrinum. Eight isolates represented by mixed species, which includes M. fortuitum-peregrinum (3 isolates), M. gordonae-peregrinum (3 isolates) and M. fortuitum-gordonae-peregrinum (2 isolates). One NTM could not be interpreted.ConclusionA significant number of NTM species were isolated from TB-like lesions of grazing cattle slaughtered at Bahir Dar Abattoir. Such finding could suggest the role of NTM in causing lesions in cattle. Further investigations are recommended on the pathogenesis of the reported NTM species in cattle, and if they have public health significance.

Comparison of methods available for identification of Mycobacterium chimaera

ObjectivesMycobacterium chimaera is a recently described nontuberculous mycobacterium belonging to the Mycobacterium avium complex (MAC). Because this species is implicated in a worldwide outbreak due to contaminated heater–cooler unit water tanks during open-heart surgery, it has become mandatory for clinical microbiology laboratories to be able to differentiate M. chimaera from the other MAC species, especially M. intracellulare. Such identification has so far been restricted to specialized laboratories because it required the analysis of several gene sequences. The aim of this study was to evaluate commercial methods for identifying M. chimaera with regard to the reference gene sequencing ITS, the internal transcribed spacer 16–23S. MethodsForty-seven clinical and environmental isolates including 41 MAC were identified by (a) PCR sequencing of the ITS and hsp65 genes, (b) three molecular biology kits (INNO-LiPA Mycobacteria, GenoType Mycobacterium CM and GenoType NTM-DR) and (c) matrix-assisted desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) using Microflex LT. ResultsThere was a high concordance for species determination between the reference ITS sequencing and the GenoType NTM-DR test (39/41, 95%), the INNO-LiPA Mycobacteria test (38/41, 93%) and the hsp65 sequencing (38/41, 93%). The GenoType Mycobacterium CM test did not distinguish M. chimaera from M. intracellulare. MALDI-TOF MS distinguished two M. chimaera–M. intracellulare groups separated from M. avium and from the other mycobacterial species on a score-oriented dendrogram, but it also failed to differentiate the two species. ConclusionsINNO-LiPA Mycobacteria and GenoType NTM-DR are efficient assays for M. chimaera identification in clinical microbiology laboratories.