Discovery of a novel Mycobacterium asiaticum PRA-hsp65 pattern

PLOS Neglected Tropical Diseases

Background Nontuberculous mycobacterial (NTM) lung infections are a major public health concern. Diagnosis of NTM-pulmonary disease (NTM-PD) is difficult because its clinical, microbiological, and radiological features resemble to those of pulmonary tuberculosis (TB), leading to misdiagnosis. Identification at the species level is essential for diagnosis and determination of therapy, which is currently not performed routinely in Indonesian laboratories. Methodology and principal findings From January 2020 to May 2021, 94 NTM isolates were collected from three TB referral centers in Java Province. Species were identified using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). Tests were performed to determine antibiotic susceptibility, biofilm formation ability, sliding motility characteristics, and the ability to adhere to and invade pneumocytes. After identifying the species of all the isolates, we found nine groups of NTMs: M. fortuitum g...

Molecular and Cellular Probes, 2004

This report describes detailed taxonomic and phylogenetic analysis of 15 non-tuberculous mycobacteria (NTMs) isolated from human pathological specimens in a Caribbean setting (12 slow-growers and three rapid-growers) that were not identified by cultural and biochemical tests and drug-susceptibility results. These isolates were further studied using PCR restriction fragment length polymorphism analysis (PRA) of a 441 bp hsp65 fragment, as well as the sequencing of 16S rDNA and hsp65 DNA, and HPLC of the mycolic acids. Our results showed that taxonomic position of well-defined NTMs was resolved by PRA and sequencing of hsp65, nonetheless, it was not suitable to investigate rarely observed or new strains that required 16S rDNA sequencing and HPLC for a definite response. Unrooted neighbor-joining phylogenetic trees were drawn based upon the 16S rDNA and hsp65 sequences of the 15 NTMs compared with those from described species (73 for 16S rDNA and 45 for hsp65). For most of the NTMs not showing an exactly matching sequence with either hsp65 or 16S rDNA in the GenBank, the phylogenetic tree was able to provide with useful indications about their relatedness to known species. In such a case, a concording HPLC pattern with the sequence data and the place of the strain within the tree could lead to a potential identification. We also identified three identical isolates that define a new mycobacterial species within the group of M. simiae-related mycobacteria. The isolation and characterization of mycobacteria from new settings may lead to identify potential pathogens that may propogate in future because of increased human migration, travels, and climatic and ecological changes of the modern world.

European Journal of Molecular & Clinical Medicine, 2021

Mycobacterium is still is one of the major causes of mortality, since two million people die each year from this malady. Currently, there are over 170 recognized species of Mycobacterium, the only genus in the family Mycobacteriaceae. Organisms belonging to this genus are quite diverse with respect to their ability to cause disease in humans; some are strict pathogens, while others are opportunistic pathogens or nonpathogenic. Similar to other major groups of bacteria, the mycobacteria have undergone an extraordinary expansion in the number of different species over the last 2 decades, due in large part to the discriminatory power of gene sequencing, which phenotypic methods cannot achieve. This discriminatory power is such that phenotypic traits, i.e., biochemical and cultural characteristics, are no longer acceptable for the identification of mycobacteria. M. tuberculosis strains are reportedly more virulent than others, as defined by increased transmissibility as well as being associated with higher morbidity and mortality in infected individuals. As more clinical laboratories use molecular or other methods, such as mass spectrometry, for identification, our understanding of clinical significance will change and evolve as the number of case reports regarding the "new" species increase; it may well be that the role of many of the newly described mycobacterial species has been underestimated either because of misidentification or because the species were unrecognized. Molecular typing methods have greatly improved our understanding of the biology of mycobacteria and provide powerful tools to combat the diseases caused by these pathogens. The utility of various typing methods depends on the Mycobacterium species under investigation as well as on the research question.. Within this review, we summarize currently available molecular methods for strain typing of M. tuberculosis. For the various methods, technical practicalities as well as discriminatory power and accomplishments are reviewed. General Description of Taxonomy and Nomenclature The discovery of leprosy bacillus (originally named Bacillus leprosy) in 1880, and of tubercle bacillus (named Bacterium tuberculosis) in 1883, led to the first steps in the classification of mycobacteria. These organisms were renamed Mycobacterium leprae and Mycobacterium tuberculosis by Lehmann and Neumann and grouped within the genus Mycobacterium, which is the single genus within the Mycobacteriaceae family, in the Actinomycetales order and Actinomycetes class. Bacteria were first classified as plants constituting the class Schizomycetes, which along with the Schizophyceae (blue green algae/Cyanobacteria) formed the phylum Schizophyta. Despite there being little agreement

Diagnostic Microbiology and Infectious Disease, 2010

This study utilized the hsp65 polymerase chain reaction restriction analysis (PRA) method in the identification of nontuberculous mycobacteria (NTMs) isolated in a Brazilian mycobacteria laboratory. NTM isolates from clinical specimens collected from 192 patients were characterized using the hsp65 PRA method and analyzed using both 16S rRNA and hsp65 gene sequencing. Only 30% of the NTM strains were correctly identified through PRA, though the suggested inclusion of an additional restriction enzyme could increase the resolution to roughly 90%. A total of 17 NTM strains were not identified to species level and may represent a new taxonomic entity classified as belonging to the Mycobacterium simiae complex. This study demonstrates the applicability of hsp65 PRA in the identification of several NTM strains in a reference laboratory, though the results suggest that some modifications to the original PRA method could increase its resolution substantially.

Advances in Experimental Medicine and Biology

West Africa is the only region in the world where six out of seven mycobacterial lineages of human importance are endemic. In particular, two evolutionary ancient lineages, Mycobacterium africanum West Africa 1 (MTBC Lineage 5) and M. africanum West Africa 2 (MTBC Lineage 6) are of interest as they cause up to 40% of all pulmonary TB cases in some West African countries. Although these M. africanum lineages are closely related to M. tuberculosis sensu stricto lineages, they differ significantly in respect to biology, epidemiology and in their potential to cause disease in humans. Most importantly the M. africanum lineages are exclusive to West Africa. Although the exact mechanisms underlying this geographical restriction are still not understood, it is increasingly suspected that this is due to an adaptation of the bacteria to West African host populations. In this chapter, we summarize the geographical distribution of the M. africanum lineages within the region, describe biological and clinical differences and the consequent implications for TB control in West Africa. We also try to shed light on the geographical restriction, based on recently published analyses on whole genomes of M. africanum isolates.

Emerging Infectious Diseases, 2012

Journal of Clinical Microbiology, 2014

Species identification of nontuberculous mycobacteria (NTM) is challenging due to the increasing number of identified NTM species and the lack of standardized testing strategies. The objectives of this study were to investigate the distribution of NTM species recovered from respiratory specimens by multigene sequence-based typing and to evaluate the clinical significance of identified species. Two hundred thirty-two consecutive clinical NTM isolates were subjected to sequencing of multiple genes, including hsp65 , rpoB , and 16S-23S rRNA internal transcribed spacer (ITS) sequence. In addition, clinical data from all patients whose specimens had NTM isolates were analyzed to examine clinical virulence and treatment history. Eighteen strains from 227 isolates from 169 patients were successfully identified at the species level by multigene sequence-based typing. Mycobacterium avium complex and M. abscessus complex made up the majority of isolated NTM (88%; 199/227), followed by M. fort...

SUMMARY: Mycobacterium simiae has been reported worldwide, particularly from the Middle East. This organism has been recognized as a causative agent of pulmonary and disseminated infections. In this study, we used molecular methods to detect this organism from patients who were suspected of having tuberculosis. A total of 117 isolates of mycobacteria were evaluated from different regions of Iran. Isolates were identified using phenotypic methods and gene sequencing of 16S rRNA, rpoB, hsp65, and ITS. Of the 117 isolates, 12 M. simiae isolates (10.2z) were identified from different clinical samples, including bronchoalveolar lavage and sputum (n ＝ 8), blood (n ＝ 3), and lymph node biopsy (n ＝ 1). Three isolates (3/12, 25z) were recovered from blood samples of HIV cases when the CD4 ＋ count was less than 50/ml. There was no significant relationship between infection and age or gender. Infection with nontuberculous mycobacteria (NTM), including M. simiae, is the major problem among immunocompromised patients. The results of this study illustrated the importance of molecular methods for accurate and rapid detection of NTM infections in the treatment of nonresponding patients with suspected tuberculosis.

PLoS ONE, 2014

Introduction: While NTM infection is mainly acquired from environmental exposure, monitoring of environmental niches for NTM is not a routine practice. This study aimed to find the prevalence of environmental NTM in soil and water in four highly populated suburbs of Tehran, Iran. Material and Methods: A total of 4014 samples from soil and water resources were collected and studied. Sediments of each treated sample were cultured in Lowenstein-Jensen medium and observed twice per week for growth rate, colony morphology, and pigmentation. Colonies were studied with phenotypic tests. Molecular analysis was performed on single colonies derived from subculture of original isolates. Environmental samples were compared with 34 NTM isolates from patients who were residents of the study locations. Results: Out of 4014 samples, mycobacteria were isolated from 862 (21.4%) specimens; 536 (62.1%) belonged to slow growing mycobacteria (SGM) and 326 (37.8%) were rapid growing mycobacteria (RGM). The five most frequent NTM were M. farcinogens (105/862; 12.1%), M. fortuitum (72/862; 8.3%), M. senegalense (58/862; 6.7%), M. kansasii (54/862; 6.2%), and M. simiae (46/862; 5.3%). In total, 62.5% (539/862) of mycobacterial positive samples were isolated from water and only 37.4% (323/862) of them were isolated from soil samples (P,0.05). Out of 5314 positive clinical samples for mycobacteria, 175 (3.2%) isolates were NTM. The trend of NTM isolates increased from 1.2% (13 out of 1078) in 2004 to 3.8% (39 out of 1005) in 2014 (P50.0001). The major clinical isolates were M. simiae (51; 29.1%), M. kansasii (26; 14.8%), M. chelonae (28; 16%), and M. fortuitum (13; 7.4%).

Mycobacteria are aerobic, nonspore forming, non-motile,single-cell bacteria.Of more than 40 currently recognized species of mycobacteria, Mycobacterium tuberculosis, the causative agent of human TB is the commonest pathogen for pulmonary and extra pulmonary tuberculosis cases. The other members of the Mycobacterium tuberculosis complex (MTC) or the nontubercular mycobacterium (NTM) produces similar diseases which cannot be differentiated from tuberculosis by clinical symptoms and signs. But this differentiation is important as the chemotherapy varies widely according to the strain of mycobacterium. The burden of morbidity and mortality of tuberculosis is rapidly growing worldwide, particularly with the HIV/AIDS epidemic. The strain identification of Mycobacterium remains a cumbersome, labor intensive and expensive procedure, which requires 3 to 12 weeks of time. The conventional methods of strain identification lack proper standardization and precise diagnosis. The prime objective of this study is to overcome these problems.

Journal of clinical microbiology, 1997

The use of high-performance liquid chromatography (HPLC) revealed four previously unreported profiles within a group of mycobacteria consisting of 14 clinical isolates. These mycobacteria, whose identification by conventional tests appeared problematic, mostly resembled Mycobacterium avium complex or Mycobacterium simiae. Genetic analysis revealed, within this group, six different nucleic acid sequences in a hypervariable 16S rRNA segment, but all the isolates appeared to be phylogenetically related to M. simiae. Six isolates representing the largest of groups defined by means of genetic sequencing turned out to belong to the newly described species Mycobacterium lentiflavum. Furthermore, three such clusters precisely coincided with three of those defined by HPLC, while the three remaining clusters shared almost identical HPLC profiles. All but one strain (which, although clearly not belonging to the M. avium complex, hybridized with specific commercial DNA probes) showed high-grade...

2011

2004

Sylvia Cardoso LEAO Universidade Federal de Sao Paulo, Sao Paulo, Brazil Anandi MARTIN Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium Gloria Isabel MEJIA M. Corporacion para Investigaciones Biologicas and Escuela de Ciencias de la Salud, Universidad Pontificia Bolivariana, Medellin, Colombia Juan Carlos PALOMINO Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium Jaime ROBLEDO R. Corporacion para Investigaciones Biologicas and Escuela de Ciencias de la Salud, Universidad Pontificia Bolivariana, Medellin, Colombia Maria Alice da Silva TELLES Instituto Adolfo Lutz, Sao Paulo, Brazil Francoise PORTAELS Prince Leopold Institute of Tropical Medicine, Antwerp, Belgium

Annals of Clinical Microbiology and Antimicrobials, 2013

Aim: Mycobacteria other than tuberculosis (MOTT) cause increasingly serious infections especially in immunosuppressive patients by direct transmission from the environment or after colonization. However, identification of these species is difficult because of the cost and difficulties in defining to species level. Identification and distribution of these species can help clinician in the choice of treatment. Materials and methods: A total of 90 MOTT strains obtained from four different centers were included in the study. These strains were identified by sequence analysis of 16S rRNA and Hsp65 genetic regions. Results: Accordingly, within the 90 MOTT strains, 17 different species were identified. In order of frequency, these species were M. gordonea (n = 21), M. abscessus (n = 13), M. lentiflavum (n = 9), M. fortuitum (n = 8), M. intracellulare (n = 6), M. kumamotonense (n = 6), M. neoaurum (n = 5), M. chimaera (n = 5), M. alvei (n = 5), M. peregrinum (n = 3), M. canariasense (n = 3), M. flavescens (n = 1), M. mucogenicum (n = 1), M. chelona (n = 1), M. elephantis (n = 1), M.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has demonstrated its ability to promptly identify nontuberculous mycobacteria using the Mycobacteria Library v2.0. However, some species are particularly difficult to identify reliably using this database, providing a low log(score). In this study, the identification power of an updated Mycobacteria Library (v3.0) has been evaluated. Overall, 109 NTM isolates were analyzed with both databases. The v3.0 database allowed a high-level confidence in the identification [log(score) value, >1.8] of 91.7% of the isolates versus 83.5% with the v2.0 version (P < 0.01). R apid identification of nontuberculous mycobacteria (NTM) with matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) has outperformed molecular techniques, such as GenoType (Hain Lifescience GmbH, Nehren, Germany), and provides accurate identification that correlates well with 16S rRNA gene sequencing when applied to the most common species of NTM (1-3). The Mycobacteria Library database (Bruker Daltonik GmbH, Bremen, Germany) available so far (v2.0) provided low scores, particularly for NTM belonging to the slow-growing groups (3-5). In the present study, we assessed the power of a new database to identify NTM (i.e., Myco-bacteria Library v3.0) using 109 isolates from 26 NTM species (Table 1) and compared the identification scores of version v2.0 and version v3.0. Ninety-nine nonselected NTM isolates from clinical samples and 10 reference strains (Table 1) were collected in the clinical microbiology laboratory from the Hospital Gregorio Marañón (Madrid, Spain) between January 2011 and May 2015. These isolates were routinely identified by 16S rRNA hsp65 sequencing and, in parallel, by MALDI-TOF MS using a Microflex LT benchtop mass spectrometer (Bruker Daltonik) and the Mycobacteria Library v2.0, containing 313 Mycobacterium isolates from 131 species (Bruker Daltonik) (4). Sample preparation was described elsewhere (6). Briefly, colonies of NTM isolates grown on Lowen-stein-Jensen medium were harvested into a 1.5-ml Eppendorf tube with 300 l of deionized water and inactivated for 30 min at 95°C under biosafety level 3 conditions. Then, they were centrifuged at maximal speed and subsequently resuspended in 300 l of water and 900 l of absolute ethanol and centrifuged again at 13,000 rpm. The supernatant was discarded, and the pellet was taken to biosafety level 2 conditions in order to disrupt the myco-bacteria cell aggregates with silica bead vortexing and extract the bacterial proteins using formic acid and acetonitrile. In the end, 1 l of supernatant was placed onto a steel plate for MALDI-TOF MS analysis. Samples were analyzed in duplicates; the species identification, using the Mycobacteria Library v2.0, and the higher log(score) value result were recorded. For comparison reasons, all of the protein spectra from the 109 NTM isolates were reanalyzed using the new Mycobacteria Library v3.0, containing 853 references from 149 Mycobacterium species. Log(score) value differences of 0.