An evaluation of the MicroSeq microbial identification system by nucleic acid sequencing and the Mayo Clinic experience with its integration into a routine clinical laboratory setting are described. Evaluation of the MicroSeq microbial identification system was accomplished with 59 American Type Culture Collection ATCC strains and clinical isolates of mycobacteria identified by conventional and 16S ribosomal DNA sequencing by using the MicroSeq microbial identification system.

Nucleic acid sequencing identified 58 of 59 The identification results for of clinical isolates All 85 isolates were determined to be mycobacterial species, either novel species or species that exhibited significant genotypic divergence from an organism in the database with the closest match. Integration of nucleic acid sequencing into the routine mycobacteriology laboratory and use of the MicroSeq microbial identification system and Mayo Clinic databases containing additional genotypes of common species and added species significantly reduced the number of organisms that could not be identified by phenotypic methods.

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The turnaround time was shortened to 24 h, and results were reported much earlier. A limited number of species could not be differentiated from one another by 16S ribosomal DNA sequencing; however, the method provides for the identification of unusual species and more accurate identifications and offers the promise of being the most accurate method available.

Abstract An evaluation of the MicroSeq microbial identification system by nucleic acid sequencing and the Mayo Clinic experience with its integration into a routine clinical laboratory setting are described. Publication types Evaluation Study.Determining the species of a Mycobacterium tuberculosis complex culture isolate. When this test is ordered, species identification will always be performed at an additional charge.

Identification of mycobacteria species by molecular methods

This assay provides a species-level identification of microbiologic culture isolates previously identified to be a member of the Mycobacterium tuberculosis complex. Species level identification can be important for patient care or for epidemiologic investigations.

For example, the species-level identification of M bovis bacillus Calmette-Guerin BCG can assist with identification of disseminated infections following use of the vaccine as an adjuvant during chemotherapy. This assay can differentiate the most common species within the Mycobacterium tuberculosis complex, M tuberculosisM bovisM bovis bacillus Calmette-Guerin BCG; the vaccine strainM canettiiM capraeM microtiand M pinnepedii to the species level.

mycobacterium species identification

Only isolates of Mycobacterium tuberculosis complex should be submitted and they must be in pure culture. Nontuberculous mycobacteria should not be submitted. Mixed cultures will result in a delay because the M tuberculosis complex organism must be isolated prior to performing the PCR assay. This assay has not been verified for the direct detection of M tuberculosis complex from clinical specimens. It is intended for use on microbiologic culture isolates already identified as M tuberculosis complex.

Type strains of Mycobacterium tuberculosis complex members were tested using the species identification PCR assay and all were identified correctly. In addition a clinical isolate of M canettii, identified by whole genome sequencing at the New York State Department of Health Wadsworth Center, was tested and confirmed to be M canettii by the M tuberculosis complex species identification PCR assay. As part of the verification of this assay, 78 M tuberculosis complex isolates with the species identified at a reference laboratory were tested using the species identification PCR assay.

All 78 isolates were correctly identified to the species level. Although the species identification test can be used only for mycobacterial isolates already identified as M tuberculosis complex, other Mycobacterium species isolates were tested to determine whether any nontuberculous mycobacteria would be positive in the test.

No nontuberculous mycobacteria were positive in the M tuberculosis complex species identification PCR assay. Skip to main content. Register Sign In. Test Catalog Account. Outreach Solutions Tactics Articles Events. Utilization Management Resource Center Algorithms. Test Catalog. Download Test. Infectious Specimen Shipping Guidelines. Species reported by reference laboratory.Universidad Km 1, Ex-Hda. Mycobacterium genus causes a variety of zoonotic diseases.

The best known example is the zoonotic tuberculosis due to M. Milk samples 35 and nasal exudate samples 68 were collected. From the strains isolated, 39 were selected for identification. Except M. The genus Mycobacterium causes a wide variety of zoonotic diseases.

Key biochemical methods used to distinguish Mycobacterial group

The best known example is zoonotic tuberculosis due to M. The NTM are found in various environmental sources such as soil, water, vegetation, animals, dairy products, and feces and may be transmitted inadvertently by inhalation, ingestion, or skin penetration [ 2 ]. The official diagnosis of bovine tuberculosis due to the presence of M.

Although used for several years, this test does not provide good sensitivity and specificity. Given the poor specificity and sensitivity of the tuberculin test, the actual presence of M. Thus, cattle breeders, veterinarians, technicians, and employees working in the livestock industry might be occupationally exposed to infections by M.

Very little is known about occupational exposure to zoonoses due to NTM species because the identification of these species was a rather difficult task prior to the development of identification techniques based on molecular biology.

Currently, the molecular biology techniques most commonly used for the diagnosis of diseases caused by mycobacteria are restriction fragment length polymorphism RFLP for the diagnosis of M. Among the NTM species identified by the aforementioned techniques are M. The largest percentage of the state inventory for heads of cattle in the State of Mexico in Mexico is concentrated in the southern region, and one of the main economic activities is cattle ranching [ 19 ].

Little is known about the presence of NTM in the cattle of the region. Given the possibility of identifying species of actinobacteria by detection of the base pair molecular marker on the 23S rRNA gene and the subsequent sequencing of the 16S rRNA gene, it is possible to identify the aforementioned NTM species. The objective of the present study was to isolate and identify NTM species from cattle of the south region of the State of Mexico. The Mycobacterium species were isolated from samples of nasal exudate and bovine milk and identified by detecting the base pair molecular marker in the 23S rRNA gene with subsequent sequencing of the 16S rRNA gene.

A sampling was performed based on the spatial distribution of herds positive for bovine tuberculosis in the state of Mexico conducted by Zaragoza et al. Four herds of cattle were selected in the south region of the State of Mexico, one herd belonging to the Municipality of Temascaltepec and three herds belonging to the municipality of Zacazonapan. A total of samples, 35 milk samples and 68 samples of nasal exudate, were collected. The distribution of the number and type of samples collected in each herd is shown in Table 1.

The milk samples were centrifuged at revolutions per minute rpm for 10 minutes. The isolated strains were distributed in groups according to the following characteristics: colony pigmentation, growth time, and colony characteristics shape, consistency, texture, and pigment production.

Isolated strains were stained with Ziehl-Neelsen to confirm the presence of acid-fast bacilli AFB [ 21 ]. Strains with microscopic characteristics similar to mycobacteria acid-fast positivity and two representative strains of each group were selected for identification. Then, the supernatant was removed and the pellet was transferred to 1.

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The obtained sequences were analyzed and corrected using the BioEdit program [ 22 ]. The sequences of the collection strains and those of the strains isolated in the present investigation were aligned with the BioEdit program [ 22 ].

Phylogenetic analysis was performed using the maximum parsimony method in MEGA software version 4 [ 25 ]. To form the root of the cladogram, the sequence of Pantoea agglomerans DSM was used. The strains isolated from the collected samples were distributed in 13 groups according to their macroscopic and microscopic morphological characteristics Table 2.Mycobacterium is a genus of Actinobacteriagiven its own family, the Mycobacteriaceae.

Over species are recognized in this genus. Mycobacteria are aerobic. They are bacillary in form, at least in most phases that have attracted human microbiological attention to date; they are straight or slightly curved rods between 0. They are generally nonmotile bacteria, except for the species Mycobacterium marinumwhich has been shown to be motile within macrophages.

They are characteristically acid-fast. The cell wall consists of the hydrophobic mycolate layer and a peptidoglycan layer held together by a polysaccharide, arabinogalactan. The cell wall makes a substantial contribution to the hardiness of this genus.

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The biosynthetic pathways of cell wall components are potential targets for new drugs for tuberculosis. Many Mycobacterium species adapt readily to growth on very simple substratesusing ammonia or amino acids as nitrogen sources and glycerol as a carbon source in the presence of mineral salts. Most Mycobacterium species, including most clinically relevant species, can be cultured in blood agar.

A natural division occurs between slowly - and rapidly-growing species. Mycobacteria that form colonies clearly visible to the naked eye within 7 days on subculture are termed rapid growers, while those requiring longer periods are termed slow growers. Some mycobacteria produce carotenoid pigments without light. Others require photoactivation for pigment production.

mycobacterium species identification

Mycobacteria are classical acid-fast organisms. Mycobacteria appear phenotypically most closely related to members of NocardiaRhodococcusand Corynebacterium. Mycobacteria are widespread organisms, typically living in water including tap water treated with chlorine and food sources.

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Some, however, including the tuberculosis and the leprosy organisms, appear to be obligate parasites and are not found as free-living members of the genus. Mycobacteria can colonize their hosts without the hosts showing any adverse signs.

For example, billions of people around the world have asymptomatic infections of M. Mycobacterial infections are notoriously difficult to treat. The organisms are hardy due to their cell wall, which is neither truly Gram negative nor positive.

In addition, they are naturally resistant to a number of antibiotics that disrupt cell-wall biosynthesis, such as penicillin.

mycobacterium species identification

Due to their unique cell wall, they can survive long exposure to acids, alkalis, detergents, oxidative bursts, lysis by complementand many antibiotics. Most mycobacteria are susceptible to the antibiotics clarithromycin and rifamycinbut antibiotic-resistant strains have emerged.

As with other bacterial pathogens, M. However, the mechanism by which these proteins contribute to virulence remains unknown. Mycobacteria can be classified into several major groups for purpose of diagnosis and treatment: M. Mycosides are glycolipid compounds isolated from Mycobacterium that contain varying lipid, carbohydrate, and amino acid moieties.COVID is an emerging, rapidly evolving situation.

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Unable to load your delegates due to an error Please try again. Create a file for external citation management software Selection: All results on this page All results Selection. Your saved search Name of saved search:. Search terms: The sustained increase in the incidence of nontuberculous mycobacterial NTM infection and the difficulty in distinguishing these infections from tuberculosis constitute an urgent need for NTM species-level identification.

The MeltPro Myco assay is the first diagnostic system that identifies 19 clinically relevant mycobacteria in a single reaction based on multicolor melting curve analysis run on a real-time PCR platform. The assay was comprehensively evaluated regarding its analytical and clinical performances. Clinical studies using 1, isolates collected from five geographically distinct health care units showed that the MeltPro Myco assay correctly identified 1, Further testing with 94 smear-positive sputum samples showed that all samples were correctly identified.

Additionally, the entire assay can be performed within 3 h. The results of this study confirmed the efficacy of this assay in the reliable identification of mycobacteria, suggesting that it might potentially be used as a screening tool in regions endemic for tuberculosis. Keywords: melting curve analysis; nontuberculous mycobacteria; real-time PCR; species identification. Abstract The sustained increase in the incidence of nontuberculous mycobacterial NTM infection and the difficulty in distinguishing these infections from tuberculosis constitute an urgent need for NTM species-level identification.The test is based on the nucleotide differences in the 16SS rRNA spacer region and can be performed starting from either liquid or solid culture.

The following Mycobacterium species can be detected simultaneously: M. Although more than 70 Mycobacterium species are currently described, relatively few are strictly pathogenic for men or animals. Most are harmless saprophytic bacteria; some are occasionally pathogenic However, opportunistic infections due to non-tuberculous mycobacteria NTM [also called mycobacteria other than tuberculosis MOTT or atypical mycobacteria] are on the increase, as a consequence of the AIDS pandemic Discrimination of M.

In general, the epidemiology of NTM infections is poorly understood 8. In clinical mycobacteriology laboratories identification of mycobacterial cultures is predominantly achieved by biochemical methods. Recently developed molecular methods, such as DNA probe tests, may simplify identification and may eventually lead to a better understanding of this complex group of organisms and their relevance in human disease Nolte FS, Metchock B.

Manual of Clinical Microbiology. Pitchenik AE, Fertel D. Tuberculosis and nontuberculosis mycobacterial disease.

Med Clin North Am ; Epidemiology of infection by nontuberculosis mycobacteria. Clin Microbiol Rev ; Mycobacterium avium complex in AIDS: who, when, where, why and how? Mycobacterium avium complex infection and AIDS: advances in theory and practice. Clin Infect Dis ; Semantide- and chemotaxonomy-based analyses of some problematic phenotypic clusters of slowly growing mycobacteria, a cooperative study of International Working Group on Mycobacterial Taxonomy.

Int J Syst Bacteriol ; Towards a phylogeny and definition of species at the molecular level within the genus Mycobacterium. J Clin Microbiol ; Performance assessment of new multiplex probe assay for identification of Mycobacteria. Product number. The provision of your data implies your consent to the processing of your data in our database and, depending on the option you choose, to receiving our monthly newsletter by e-mail.

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Click here to navigate Details Documentation Related products Product inquiry. Portaels F. Epidemiology of mycobacterial diseases. Clin Dermatol ; Doern GV. Diagnostic mycobacteriology: where are we today?