Preliminary Report
hsp65 Phylogenetic Assay for Molecular Diagnosis of Nontuberculous Mycobacteria Isolated in Mexico

https://doi.org/10.1016/j.arcmed.2013.12.004Get rights and content

Background and Aims

Nontuberculous mycobacteria (NTM) are mainly distributed as important emerging pathogens in patients with chronic or immunosuppressive diseases. Accurate identification of causative species is crucial for proper treatment and patient follow-up. However, several difficulties are associated with phenotypic and molecular diagnostic methods for precise identification at the species level due to shared metabolic and genetic characteristics. We undertook this study to evaluate the application of the phylogenetic method based on hsp65 gene into Telenti’s PCR-restriction enzyme analysis (PRA) for molecular identification of NTM.

Methods

The study population was comprised of 1646 Mycobacterium clinical isolates (AFB positive) collected from 2008–2011, of which 537 (32.6%) were MNT identified by PRA analysis. DNA sequencing of hsp65 in 53 isolates (10%) was performed. Sequence identification through NCBI-Basic Local Alignment Search Tool (BLAST) achieved correct identification in 23 isolates. Phylogenetic trees including hsp65 available GenBank sequences for all described genres of NTM and hsp65 obtained sequences were constructed using Mega 5.05 software. We compared sequence identification based on phylogenetic clustering and BLAST similarity search.

Results

Phylogenetic clustering allowed more specific differentiation of closely related species and clearer identification in comparison with BLAST; 30 Mycobacterium species (this is the first report of isolation of some of these from clinical samples in Mexico) were identified in this way.

Conclusions

The proposed 440 bp hsp65 phylogenetic method allows a better identification tool to differentiate Mycobacterium species and is useful to complement diagnosis and epidemiological surveillance of NTM.

Introduction

Nontuberculous mycobacteria (NTM) are Mycobacterium species different from M. tuberculosis complex (1). NTM are widely distributed in the natural environment but may be important pathogens in immune-compromised individuals, patients with congenital and chronic degenerative diseases (2) and females who have chronic difficulty for expectoration (3). Progressive lung disease and extrapulmonary localizations such as skin and soft tissue infections, lymphadenitis and dissemination are clinically recognized forms (2), although there is no evidence of animal-to-human and human-to human transmission (4). Diseases caused by NTM are considered emerging, first because of the difficulty to control infection sources and the intrinsic pyrazinamide, rifampicin or isoniazid resistance (5) and second because the prevalence of NTM disease is increasing in the non-AIDS population 6, 7. Proper identification of etiologic species is crucial for patient follow-up (1). Classic identification based on morphology, culture and biochemical tests may take several weeks and sometimes identification is difficult. Commercially available molecular methods represent a high cost for most laboratories (8). The 65 kDa heat-shock protein encoded by hsp65 gene is found in the cell wall of all species of Mycobacterium (9). In 1993, Telenti and colleagues (10) developed a molecular method for NTM differentiation based on amplification of a 440-bp fragment of hsp65 by polymerase chain reaction and restriction enzyme analysis (PRA). This method has been accepted by increased specificity and reduction in process time (11). Restriction patterns are continually updated 12, 13, 14, 15; however, some limitations for Telenti’s method are as follows (13). Up to six different restriction patterns for species with clinical significance such as M. avium and M. kansasii, species with the same restriction pattern and the reported comparison tables, may differ in the range of 5–15 bp. It is possible to infer clustering of similar DNA or protein sequences by the study of phylogenetic relationship (16). Phylogenetic analysis has been employed to describe genetic diversity and differentiation of the genus Mycobacterium (17). However, analysis of hsp65 sequences generates a more useful divergence pattern than 16S rRNA and rpoB coding sequences (18).

In this study we developed a phylogenetic method that included available NTM hsp65 nucleotide sequences from the National Center for Biotechnology Information (NCBI) GenBank (19) and obtained hsp65 sequences from clinical isolates in a national reference center (InDRE). Moreover, we compared this phylogenetic assay with classical NCBI Basic Local Alignment Search Tool (BLAST) (20) sequence identification in order to purpose a specific molecular method for NTM diagnosis.

Section snippets

Clinical Isolates, Amplification and Sequencing

Clinical isolates collected from 2008–2011 were processed by Telenti’s PRA method: DNA was extracted with phenol–chloroform. PCR was performed as described (10) using Tb11 (5'-ACCAACGATGGTGTGTCCAT) and Tb12 (5'-CTTGTCGAACCGCATACCCT) primers to amplify a 440 bp fragment. Amplicons were digested with BstEII and HaeIII enzymes. The obtained restriction patterns were compared in a public database (21) and in reported algorithms 12, 13, 14, 15 for NTM PRA–based identification. The hsp65 sequencing

Results

GenBank 440 bp hsp65 sequences allowed phylogenetic clustering (Figure 1A and B), which represented differentiation among Mycobacterium species. Similar phylogenetic behavior was observed in trees constructed including correct BLAST-based identification sequences (second phylogram built not shown). For 30 isolates (Table 2), sequence BLAST-based identification could not be established. However, identification according to phylogenetic grouping defined proper identification (Figure 2A and B).

Phylogenetic Relationship among hsp65 Sequences

hsp65 has been used for differentiation and classification of Mycobacterium species due to degree of sequence conservation in each (18). In this study we present a phylogenetic grouping pattern for all NTM 440 bp hsp65 available sequences, including the most recently reported (Figure 1A and B) where there is grouping of associated species. At the same time, the distinction between those closely related in a manner comparable to the mentioned report is observed in the same way as using a larger

Conflict of Interest

The authors declare no conflict of interest regarding the development of this work. There are no economic, political, personal or academic situations that could generate any bias in the results of the research, writing the report or decision to submit for publication.

Acknowledgments

The authors express their gratitude to the working team from the Laboratorio de Micobacterias and Laboratorio de Genoma de Patógenos at InDRE for the mycobacterial cultures and DNA sequencing, respectively.

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