Reliable identification of mycobacterial species by PCR-restriction enzyme analysis (PRA)-hsp65 in a reference laboratory and elaboration of a sequence-based extended algorithm of PRA-hsp65 patterns.
ABSTRACT: BACKGROUND:Identification of nontuberculous mycobacteria (NTM) based on phenotypic tests is time-consuming, labor-intensive, expensive and often provides erroneous or inconclusive results. In the molecular method referred to as PRA-hsp65, a fragment of the hsp65 gene is amplified by PCR and then analyzed by restriction digest; this rapid approach offers the promise of accurate, cost-effective species identification. The aim of this study was to determine whether species identification of NTM using PRA-hsp65 is sufficiently reliable to serve as the routine methodology in a reference laboratory. RESULTS:A total of 434 NTM isolates were obtained from 5019 cultures submitted to the Institute Adolpho Lutz, Sao Paulo Brazil, between January 2000 and January 2001. Species identification was performed for all isolates using conventional phenotypic methods and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing a 441 bp fragment of hsp65. Phenotypic evaluation and PRA-hsp65 were concordant for 321 (74%) isolates. These assignments were presumed to be correct. For the remaining 113 discordant isolates, definitive identification was based on sequencing a 441 bp fragment of hsp65. PRA-hsp65 identified 30 isolates with hsp65 alleles representing 13 previously unreported PRA-hsp65 patterns. Overall, species identification by PRA-hsp65 was significantly more accurate than by phenotype methods (392 (90.3%) vs. 338 (77.9%), respectively; p < .0001, Fisher's test). Among the 333 isolates representing the most common pathogenic species, PRA-hsp65 provided an incorrect result for only 1.2%. CONCLUSION:PRA-hsp65 is a rapid and highly reliable method and deserves consideration by any clinical microbiology laboratory charged with performing species identification of NTM.
Project description:Species identification of isolates of the Mycobacterium avium complex (MAC) remains a difficult task. Although M. avium and Mycobacterium intracellulare can be identified with expensive, commercially available probes, many MAC isolates remain unresolved, including those representing Mycobacterium lentiflavum as well as other potentially undefined species. PCR restriction analysis (PRA) of the hsp65 gene has been proposed as a rapid and inexpensive approach. We applied PRA to 278 MAC isolates, including 126 from blood of human immunodeficiency virus (HIV)-infected patients, 59 from sputum of HIV-negative patients with chronic obstructive pulmonary disease, 88 from environmental sources, and 5 pulmonary isolates from a different study. A total of 15 different PRA patterns were observed. For 27 representative isolates, a 441-bp fragment of the hsp65 gene was sequenced; based on 54 polymorphic sites, 18 different alleles were defined, including 12 alleles not previously reported. Species and phylogenetic relationships were more accurately defined by sequencing than by PRA or commercial probe. The distribution of PRA types and, by implication, phylogenetic lineages among blood isolates was significantly different from that for pulmonary and environmental isolates, suggesting that particular lineages have appreciably greater virulence and invasive potential.
Project description:The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.
Project description:Here we describe a novel duplex PCR method which can differentiate Mycobacterium tuberculosis and nontuberculosis mycobacteria (NTM) strains by amplifying hsp65 DNAs of different sizes (195 and 515 bp, respectively). The devised technique was applied to 54 reference and 170 clinical isolates and differentiated all strains into their respective groups with 100% sensitivity and specificity. Furthermore, a duplex PCR-restriction analysis (duplex PRA) and a direct sequencing protocol were developed to differentiate NTM strains at the species and subspecies levels based on previously reported hsp65 DNA sequences (H. Kim et al., Int. J. Syst. Evol. Microbiol. 55:1649-1656, 2005) and then applied to 105 NTM clinical isolates. All NTM isolates were clearly differentiated at the species and subspecies levels by subsequent procedures (PRA or direct sequencing) targeting 515-bp NTM duplex PCR amplicons. Our results suggest that novel duplex PCR-based methods are sensitive and specific for identifying mycobacterial culture isolates at the species level.
Project description:The limitations of conventional methods of identification of Mycobacterium tuberculosis have led to the development of several nucleic acid amplification techniques which have the advantage of being rapid, sensitive, and specific. However, their expense or the need for technical expertise makes it difficult to use them in regions in which tuberculosis is endemic. A novel PCR restriction analysis (PRA) of the hsp65 gene was therefore developed for rapid screening of clinical isolates to identify Mycobacterium spp. The restriction enzymes NruI and BamHI were selected to obtain a limited number of restriction patterns to further differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). Three hundred ten isolates from clinical specimens and 24 reference strains were tested. The assay correctly identified 295 of the 310 culture isolates as MTBC, while the remaining 15 isolates were identified as NTM. Of the isolates tested, 135 MTBC strains and all 15 NTM were also confirmed by PRA using Sau96I and CfoI. Thirty-eight randomly selected MTBC strains and all 15 NTM were further confirmed by sequencing. The NruI/BamHI PRA was simple, as it did not require any elaborate analyses. It was cost-effective, rapid, highly sensitive, and specific and did not require technical expertise. The assay can, therefore, be used as a simple screening test not only to detect Mycobacterium spp. but also to differentiate MTBC from NTM in peripheral laboratories with minimal availability of funds.
Project description:Pulmonary diseases caused by nontuberculous mycobacteria (NTM) are increasing in incidence and prevalence worldwide. In this study, we identified NTM species of the clinical isolates from 8 provinces in China, in order to preliminarily provide some basic scientific data in the different species and distribution of NTM related to pulmonary disease in China. A total of 523 clinical isolates from patients with tuberculosis (TB) diagnosed clinically from 2005 to 2012 were identified to the species using conventional and molecular methods, including multilocus PCR, rpoB and hsp65 PCR-PRA, hsp65, rpoB, and 16S-23S internal transcribed spacer region sequencing. The isolates were identified into 3 bacterium genera, including NTM, Gordonia bronchialis, and Nocardia farcinica, and, for the 488 NTM isolates, 27 species were identified. For all the 27 species of NTM which were found to cause pulmonary infections in humans, the most prevalent species was M. intracellulare, followed by M. avium and M. abscessus. And seven other species were for the first time identified in patients with TB in China. NTM species identification is very important for distinguishing between tuberculosis and NTM pulmonary diseases, and the species diversity drives the creation of diverse and integrated identification methods with higher accuracy and efficacy.
Project description:Nocardia identification required laborious and time-consuming phenotypic and chemotaxonomic methods until molecular methods were developed in the mid-1990s. Here we reassessed the capacity of PCR-restriction enzyme pattern analysis (PRA) of the hsp65 gene to differentiate Nocardia species, including 36 new species. Our results confirm that hsp65 PRA must no longer be used for Nocardia species identification, as many species have the same restriction pattern. We then compared sequencing-based strategies using an hsp65 database and a 16S rRNA database and found that the hsp65 region contained sufficient polymorphisms for comprehensive Nocardia species identification.
Project description:Recent molecular studies have shown Mycobacterium porcinum, recovered from cases of lymphadenitis in swine, to have complete 16S rDNA sequence identity and >70% DNA-DNA homology with human isolates within the M. fortuitum third biovariant complex. We identified 67 clinical and two environmental isolates of the M. fortuitum third biovariant sorbitol-negative group, of which 48 (70%) had the same PCR restriction enzyme analysis (PRA) profile as the hsp65 gene of M. porcinum (ATCC 33776(T)) and were studied in more detail. Most U.S. patient isolates were from Texas (44%), Florida (19%), or other southern coastal states (15%). Clinical infections included wound infections (62%), central catheter infections and/or bacteremia (16%), and possible pneumonitis (18%). Sequencing of the 16S rRNA gene (1,463 bp) showed 100% identity with M. porcinum ATCC 33776(T). Sequencing of 441 bp of the hsp65 gene showed four sequevars that differed by 2 to 3 bp from the porcine strains. Clinical isolates were positive for arylsulfatase activity at 3 days, nitrate, iron uptake, D-mannitol, i-myo-inositol, and catalase at 68 degrees C. They were negative for L-rhamnose and D-glucitol (sorbitol). Clinical isolates were susceptible to ciprofloxacin, sulfamethoxazole, and linezolid and susceptible or intermediate to cefoxitin, clarithromycin, imipenem, and amikacin. M. porcinum ATCC 33776(T) gave similar results except for being nitrate negative. These studies showed almost complete phenotypic and molecular identity between clinical isolates of the M. fortuitum third biovariant D-sorbitol-negative group and porcine strains of M. porcinum and confirmed that they belong to the same species. Identification of M. porcinum presently requires hsp65 gene PRA or 16S rRNA or hsp65 gene sequencing.
Project description:It has been reported that lateral gene transfer (LGT) events among Mycobacteroides abscessus strains are prevalent. The hsp65 gene, a chronometer gene for bacterial phylogenetic analysis, is resistant to LGT events, particularly among mycobacterial strains, rendering the hsp65-targeting method the most widely used method for mycobacterial detection. To determine the prevalence of M. abscessus strains that are subject to hsp65 LGT, we applied rpoB typing to 100 clinically isolated Korean strains of M. abscessus that had been identified by hsp65 sequence analysis. The analysis indicated the presence of 2 rough strains, showing a discrepancy between the 2 typing methods. MLST analysis based on the partial sequencing of seven housekeeping genes, erm(41) PCR and further hsp65 PCR-restriction enzyme and polymorphism analysis (PRA) were conducted to identify the two strains. The MLST results showed that the two strains belong to M. abscessus subsp. massiliense and not to M. abscessus subsp. abscessus, as indicated by the rpoB-based analysis, suggesting that their hsp65 genes are subject to LGT from M. abscessus subsp. abscessus. Further analysis of these strains using the hsp65 PRA method indicated that these strains possess a PRA pattern identical to that of M. abscessus subsp. abscessus and distinct from that of M. abscessus subsp. massiliense. In conclusion, we identified two M. abscessus subsp. massiliense rough strains from Korean patients with hsp65 genes that might be laterally transferred from M. abscessus subsp. abscessus. To the best of our knowledge, this is the first demonstration of possible LGT events associated with the hsp65 gene in mycobacteria. Our results also suggest that there is the potential for misidentification when the hsp65-based protocol is used for mycobacterial identification.
Project description:Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. For evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. The species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. The PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.
Project description:Studies have shown that certain opportunistic pathogenic species of nontuberculous mycobacteria (NTM) can be present in distributed drinking water. However, detailed information about NTM population composition in drinking water is lacking. Therefore, NTM communities in unchlorinated drinking water from the distribution system of five treatment plants in the Netherlands were characterized using 454 pyrosequencing of the hsp65 gene. Results showed high diversities in unchlorinated drinking water, with up to 28 different NTM operational taxonomic units (OTUs) in a single sample. Each drinking water sample had a unique NTM community, and most (81.1%) OTUs were observed only once. One OTU was observed in 14 of 16 drinking water samples, indicating that this NTM species is well adapted to unchlorinated drinking water conditions. A clear influence of season, source type (groundwater, surface water), easily assimilable organic carbon (AOC) concentration, biofilm formation rate, and active biomass in treated water on the establishment of an NTM community in drinking water was not observed. Apparently, local conditions are more important for the development of a specific NTM community in the drinking water distribution system. A low (4.2%) number of hsp65 gene sequences showed more than 97% similarity to sequences of the opportunistic pathogens M. avium, M. genavense, and M. gordonae. However, most (95.8%) NTM hsp65 gene sequences were related to not-yet-described NTM species that have not been linked to disease, indicating that most NTM species in unchlorinated drinking water from distribution systems in the Netherlands have a low public health significance.