Identification of nontuberculous mycobacteria using commercial DNA probes and gene sequencing

Diagnosis of NonTuberculous Mycobacteria (NMT) infection frequently runs into difficulties regarding a precise definition of the strains.The use of molecular assays is the technique of choice for the identification of species, but commercial methods recognize only a limitednumber of species.Aim of this study was molecular identification of NonTuberculous Mycobacteria (NTM) in clinical specimens using commercial methodsand automated sequencing.We analyzed 6192 clinical specimens for the isolation of Mycobacteria. One hundred and twelve strains of NTM were previously analyzedwith GenoType Mycobacteria CM/AS kit and then with entire 16S rDNA and partial hsp65 sequencing.100/112 NMT strains were identified with GenoType Mycobacteria CM/AS kit as: M. gordonae (n°25), M. xenopi (n°24), M. fortuitum (n°15),M. avium (n°12), M. intracellulare (n°7), M. chelonae (n°6), M. malmoense (n°4), M. peregrinum (n°3), M. mucogenicum (n°1), M. kansasii (n°1), M.abscessus (n°1), M. heckeshornense (n°1).Twelve unidentified strains were subjected to the entire 16S rDNA gene sequencing and nine wereidentified as M. arupense (n°7), M. avium complex (n°1), M. kumamotonense (n°1).Three unidentified strains were subjected to partial hsp65gene sequencing and one was identified as M. arupense.Conclusions. Direct sequencing of entire 16SrDNA gene and of partial hsp65 gene appears to be a useful tool for the study of strains that arenot identifiable by commercial methods.This new approach, applied to clinical diagnostic, allows also recognition of unusual strains or new species

Identification of nontuberculous mycobacteria using commercial DNA probes and gene sequencing

Diagnosis of NonTuberculous Mycobacteria (NMT) infection frequently runs into difficulties regarding a precise definition of the strains.The use of molecular assays is the technique of choice for the identification of species, but commercial methods recognize only a limitednumber of species.Aim of this study was molecular identification of NonTuberculous Mycobacteria (NTM) in clinical specimens using commercial methodsand automated sequencing.We analyzed 6192 clinical specimens for the isolation of Mycobacteria. One hundred and twelve strains of NTM were previously analyzedwith GenoType Mycobacteria CM/AS kit and then with entire 16S rDNA and partial hsp65 sequencing.100/112 NMT strains were identified with GenoType Mycobacteria CM/AS kit as: M. gordonae (n°25), M. xenopi (n°24), M. fortuitum (n°15),M. avium (n°12), M. intracellulare (n°7), M. chelonae (n°6), M. malmoense (n°4), M. peregrinum (n°3), M. mucogenicum (n°1), M. kansasii (n°1), M.abscessus (n°1), M. heckeshornense (n°1).Twelve unidentified strains were subjected to the entire 16S rDNA gene sequencing and nine wereidentified as M. arupense (n°7), M. avium complex (n°1), M. kumamotonense (n°1).Three unidentified strains were subjected to partial hsp65gene sequencing and one was identified as M. arupense.Conclusions. Direct sequencing of entire 16SrDNA gene and of partial hsp65 gene appears to be a useful tool for the study of strains that arenot identifiable by commercial methods.This new approach, applied to clinical diagnostic, allows also recognition of unusual strains or new species

Molecular investigations applied to nontuberculous mycobacteria identification

Objective. Aim of this study was molecular identification in clinical specimens of NonTuberculousMycobacteria (NTM) with commercial methods and automated sequencing.Materials and methods. Three thousand clinical specimens were analyzed for the isolation of Mycobacteria.Forty strains of NTM were previously analyzed with GenoType Mycobacteria CM/AS kit and then with partial16S rDNA sequencing.Results. 38/40 NMT strains were identified with GenoType Mycobacteria CM/AS kit as: M. gordonae (15), M.intracellulare (8), M. xenopi (8), M. mucogenicum (2), M. kansasii (2), M. chelonae (1), M. avium (1), M. lentiflavum (1).Two unidentified strains were subjected to 16S rDNA sequencing and were identified as M. kumamotonense.Conclusions. Partial 16SrDNA sequencing is a valid assay to study NTM strains unidentified with commercial assays.This new approach, applied to clinical diagnostic, also permits the recognition of unusual strains or new species

The TIGR4 strain of Streptococcus pneumoniae is phagocytosed but resists intracellular killing by microglia in experimental pneumococcal meningitis

The TIGR4 strain of Streptococcus pneumoniae is phagocytosed but resists intracellular killing by microglia in experimental pneumococcal meningiti

Interaction of pneumococcal surface structures with microglia

Interaction of pneumococcal surface structures with microgli

The encapsulated strain TIGR4 of Streptococcus pneumoniae is phagocytosed but is resistant to intracellular killing by mouse microglia

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD(50) of 2 × 10² CFU, whereas the LD(50) for the unencapsulated FP23 was greater than 10⁷ CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing

The encapsulated strain TIGR4 of Streptococcus pneumoniae is phagocytosed but is resistant to intracellular killing by mouse microglia

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae as it confers resistance to phagocytosis. The encapsulated serotype 4 TIGR4 strain was shown to be efficiently phagocytosed by the mouse microglial cell line BV2, whereas the type 3 HB565 strain resisted phagocytosis. Comparing survival after uptake of TIGR4 or its unencapsulated derivative FP23 in gentamicin protection and phagolysosome maturation assays, it was shown that TIGR4 was protected from intracellular killing. Pneumococcal capsular genes were up-regulated in intracellular TIGR4 bacteria recovered from microglial cells. Actual presence of bacteria inside BV2 cells was confirmed by transmission electron microscopy (TEM) for both TIGR4 and FP23 strains, but typical phagosomes/phagolysosomes were detected only in cells infected with the unencapsulated strain. In a mouse model of meningitis based on intracranic inoculation of pneumococci, TIGR4 caused lethal meningitis with an LD(50) of 2 × 10(2) CFU, whereas the LD(50) for the unencapsulated FP23 was greater than 10(7) CFU. Phagocytosis of TIGR4 by microglia was also demonstrated by TEM and immunohistochemistry on brain samples from infected mice. The results indicate that encapsulation does not protect the TIGR4 strain from phagocytosis by microglia, while it affords resistance to intracellular killing