Comparative evaluation of sequence analysis of 16S rRNA and rpoB genes for identification of aquatic mycobacteria

The nucleotide sequences of partial 16S rRNA and bacterial RNA polymerase ß-subunit (rpoB) genes for 57 mycobacterial strains were determined. Compared to the 16S rRNA gene sequences, variable regions were scattered along the whole fragment sequence, indicating that therpoBgene is more polymorphic. Unlike 16S rRNA sequences, species variation was observed within M. fortuitum strains. The topology of therpoB-based phylogenetic tree was almost the same as that of the 16S rRNA sequence analysis. These results suggest that therpoBgene is a highly conserved gene, and taxonomical studies based on this gene may be comparable with similar studies based on the 16S rRNA gene. The overall mean distance forrpoB-gene?based sequences was 2.5 times greater than that of the 16S rRNA gene for all 57 mycobacterial strains examined. However, some slowly growing mycobacteria could not be differentiated based onrpoBgene sequences. Moreover, a bootstrap value above 70% was observed for 13 nodes, while this value was 14 nodes in the case of 16S rRNA sequences. To the best of our knowledge, this is the first investigation evaluating the use of 16S rRNA andrpoBsequence analyses for identification of aquatic mycobacteria obtained from diverse geographical locations

Comparative evaluation of Polymerase Chain Reaction - Restriction Enzyme Analysis (PRA) and Sequencing of Heat shock protein 65 (hsp65) gene for identification of aquatic mycobacteria

Traditional identification of mycobacteria based on cultural and biochemical tests can take several weeks and may fail to provide a precise identification. Polymerase Chain Reaction-restriction analysis (PRA) of the gene encoding heat shock protein 65kDA (hsp65) gene has been proposed as a rapid and inexpensive alternative approach. Despite being widely used for differentiation of mammalian mycobacteria, this method has only been applied in the identification of a small number of aquatic mycobacteria. The present study aimed to evaluate the potential use of PRA of hsp65 for the identification of aquatic mycobacteria compared with sequence analysis. Seventy one mycobaterial isolates including, 10 type/reference strains and the remainder field isolates, were subjected to PRA of a 441 bp fragment of this gene. For 68 representative isolates, sequence analysis was performed. All rapidly and slow growing mycobacteria had best matches with 99.3% to 100% similarity with their corresponding species in the databanks. PRA proved to be a simple and rapid method for identifying aquatic mycobacteria. However, the incidence of similar or identical restriction patterns for some species of mycobacteria, and in particular, identification of new species of mycobacteria is a major problem using such a method. In contrast, the nucleic acid sequencing of the hsp65 gene yielded unambiguous results

Identification of aquatic mycobacteria based on sequence analysis of the 16S-23S rRNA internal transcribed spacer region

Purpose. Mycobacteria are common causative agents of bacterial infections in many species of freshwater and marine fish. Identification of mycobacteria to the species level based on phenotypic tests is inappropriate and time consuming. Molecular methods such as partial or entire gene sequence determination in mycobacteria have been employed to resolve these problems. The objective of this study was to assess the use of sequence analysis of the mycobacterial 16S–23S internal transcribed spacer (ITS) region for the identification of different aquatic mycobacteria species. Methodology. Using published primers, the ITS sequences of 64 field and reference strains were determined. Results/Key findings. The identity of all isolates previously identified as Mycobacterium marinum by RFLP was confirmed as M. marinum by sequence analysis. With the exception of five rapidly growing mycobacteria isolates, all other mycobacteria were easily identified by sequencing of the ITS region. Using this spacer region, it was possible to differentiate between slowly growing and rapidly growing mycobacteria, even before sequence analysis, by the size of the PCR product, although species identification could not be made by size alone. Conclusion. Overall, direct sequencing of this genetic element following PCR has been shown to be useful in the identification of aquatic mycobacteria species. With regard to the variability of the ITS region for different mycobacteria isolates, this may be a useful tool in epidemiological studies

Molecular detection and identification of aquatic mycobacteria

Abstract Mycobacteriosis (fish tuberculosis) is a progressive disease of a wide range of wild and captive marine and freshwater fish species. While Mycobacterium marinum, M. fortuitum and M. chelonae are the most frequently reported species to be involved in the disease, several new mycobacteria species have also recently been implicated. Conventional detection / identification of fish mycobacteria is based on histopathology, culture and biochemical characteristics. In this study complementary molecular approaches were developed to assist in Mycobacterium identification. First, a highly specific and sensitive multiplex PCR-based assay, targeting two genes (hsp65 and 16S RNA), was established to simultaneously detect the genus Mycobacterium and identify M. marinum, M. fortuitum or M. chelonae from culture or infected fish tissue, based on presence / absence of specific amplicons. In addition, PCR-restriction enzyme analysis (PRA) and DNA sequence analysis of the 16S-23S internal transcribed spacer (ITS) region and a 441 bp fragment of the hsp65 gene demonstrated the limitations of multiplex PCR (and commercial line probe assays) to differentiate among the species of the M. fortuitum complex. However DNA sequence analysis of the hsp65 gene fragment was found to reliably identify M. fortuitum from closely related species, M. conceptionense and M. senegalense. Reliable identification of novel species (or very similar species) of aquatic mycobacteria requires more extensive DNA sequence comparisons. Thus, multigene (polygenetic) analyses, as used here, provide rapid, accurate and reliable species identification of aquatic mycobacteria. Furthermore, a number of novel species of aquatic mycobacteria, M. stomatepiae, ‘M. angelicum’, ‘M. aemonae’ and M. salmoniphilum were discovered using the polygenetic analysis approach. Correct identification of Mycobacterium species by DNA sequence comparisons relies on accurate database information. Difficulties in this study in assigning M. marine and M. gordonae to their correct taxa suggest errors in the current public sequence repositories. The above methods were successfully applied to detect and identify mycobacteria in field samples including formalin-fixed, paraffin-embedded (FFPE) fish tissue, water and frozen fish tissue

Use of restriction enzyme fragment length polymorphism (RFLP) of the 16S-23S rRNA internal transcribed spacer region (ITS) for identification of fish mycobacteria

PCR targeting the 16S-23S rRNA gene internally transcribed spacer (ITS) region has been proposed as a rapid and reliable method for the detection of Mycobacterium species in human clinical specimens. Because of variation in ITS sequences amongst Mycobacterium species, a single PCR amplification can be used to differentiate slowly growing and rapidly growing species within this genus. In the present study, analysis by ITS-PCR and ITS-restriction fragment length polymorphism (RFLP) was found to be a useful, simple and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from 13 reference strains and 59 fish isolated mycobacteria using a set of published PCR primers. After PCR, the banding patterns generated allowed slowly growing mycobacteria to be differentiated from all other rapidly growing species, with the exception of Mycobacterium conceptionense. HaeIII was selected as one of two restriction enzymes that, together with the knowledge about amplicon sizes, would produce an acceptable level of discrimination in the resulting RLFP patterns, especially in the rapidly growing group of mycobacteria. After digestion with Sau96I, the amplified products of most isolates of Mycobacterium fortuitum, including subtypes II and V and those 2 isolates with new patterns (220, 100bp), presented identical or very similar patterns as obtained by HaeIII digestion. All isolates of Mycobacterium marinum, Mycobacterium chelonae and Mycobacterium gordonae, whose PCR products were not digested with HaeIII, produced two well-defined fragments with the Sau96I restriction enzyme

Comparison of three methods for detection of Mycobacterium marinum in goldfish (Carassius auratus)

Mycobacterium marinum is a well-recognised mycobacterial species which produces granulomatous reactions in a variety of aquatic organisms. Detection of this organism primarily relies on histological and bacteriological examination of infected fish tissues but recently a number of molecular methods have also been employed. In this study a comparison of three such methods in the detection of M. marinum in goldfish (Carassius auratus) was carried out. Histological analysis revealed that fish infected with M. marinum often developed granulomas in the absence of external lesions and clinical signs of mycobacteriosis. Approximately 40% of all infected fish had greater than/equal to 104 mycobacteria per gram of splenic tissue. Approximately 89% of samples (8/9) with < 104 CFU/g of spleen were positive by PCR. Statistical analysis revealed significant differences between the 3 methods of comparison for the entire sample (N = 15), but very significant differences between PCR and other detection methods (p = 0.0202). Overall, this study demonstrates that PCR is a rapid, specific, and sensitive method for detecting M. marinum in goldfish

Mycobacterium ahvazicum sp. nov., the nineteenth species of the Mycobacterium simiae complex

Abstract Four slowly growing mycobacteria isolates were isolated from the respiratory tract and soft tissue biopsies collected in four unrelated patients in Iran. Conventional phenotypic tests indicated that these four isolates were identical to Mycobacterium lentiflavum while 16S rRNA gene sequencing yielded a unique sequence separated from that of M. lentiflavum. One representative strain AFP-003T was characterized as comprising a 6,121,237-bp chromosome (66.24% guanosine-cytosine content) encoding for 5,758 protein-coding genes, 50 tRNA and one complete rRNA operon. A total of 2,876 proteins were found to be associated with the mobilome, including 195 phage proteins. A total of 1,235 proteins were found to be associated with virulence and 96 with toxin/antitoxin systems. The genome of AFP-003T has the genetic potential to produce secondary metabolites, with 39 genes found to be associated with polyketide synthases and non-ribosomal peptide syntases and 11 genes encoding for bacteriocins. Two regions encoding putative prophages and three OriC regions separated by the dnaA gene were predicted. Strain AFP-003T genome exhibits 86% average nucleotide identity with Mycobacterium genavense genome. Genetic and genomic data indicate that strain AFP-003T is representative of a novel Mycobacterium species that we named Mycobacterium ahvazicum, the nineteenth species of the expanding Mycobacterium simiae complex