Species spectrum of nontuberculous mycobacteria isolated from suspected tuberculosis patients, identification by multi locus sequence analysis.

Identification of Mycobacterium species is difficult due to a complex and rapidly changing taxonomy, the failure of 16S rRNA to discriminate many closely related species and the unreliability of phenotypic testing. We investigated a collection of nontuberculous mycobacteria (NTM) strains isolated from suspected tuberculosis patients at Tuberculosis Reference Centre (Ahvaz, Iran) and Masoud Laboratory (Tehran, Iran) during 2008-2012 to evaluate the species spectrum of NTM isolates. Based on phenotypic tests, the isolates were identified up to species or complex level; however they were heterogonous by hsp65-PCR restriction fragment length polymorphism analysis (PRA) method. Representative isolates from each hsp65-PRA pattern, were subjected to identification using single locus and multi locus sequence analysis (MLSA) based on 16S rRNA, rpoB, hsp65 and 16S-23S internal transcribes spacer (ITS) fragments to determine their taxonomic affiliations. All 92 NTM isolates from different clinical specimens were considered as etiological agents causing disease according to American Thoracic Society (ATS) guideline. Phenotypic evaluation alone assigned 66 (72%) isolates to a species or complex level and consequently 76 (82%) isolates showed previously reported hsp65-PRA patterns. Although sequence base identification using single locus such as 16S rRNA, rpoB, hsp65 or ITS identified the isolates up to species level, MLSA correctly identified 16 different species of NTM from clinical isolates. In summary, four-locus MLSA is a reliable method for elucidating taxonomic data and reliable species identification of Mycobacterium isolates and therefore, would be more feasible for routine use in Tuberculosis (TB) reference laboratory.

Low rates of synonymous mutations in sequences of Mycobacterium tuberculosis GyrA and KatG genes.

Partial sequences of KatG and GyrA genes have been obtained from multi and extensively drug-resistant (MDR and XDR) clinical isolates of Mycobacterium tuberculosis. Nonsynonymous (DN) and synonymous (DS) distances between those sequences have been calculated by Kumar method. Results revealed that DN is significantly higher than DS between some pairs of partial GyrA sequences. We found out that DN is higher than DS in many other partial and complete sequences of KatG and GyrA coding regions deposited in GenBank. The cause of the DN > DS situation is in several nonsynonymous substitutions occurrence (which may be associated with drug-resistance or not) in the absence of synonymous substitutions. Low rates of synonymous mutations occurrence is a consequence of the strong mutational GC-pressure. Due to the high saturation of third codon positions by guanine and cytosine (78.81 ± 0.17% for all the genes from M. tuberculosis H37Rv genome), the probability to be synonymous for the nucleotide mutation of preferable (AT to GC) direction is low. Fixation of a single nonsynonymous mutation leading to drug-resistance is a consequence of Darwinian selection. This clear example of Darwinian selection on the molecular level can be confirmed by selection test (DN > DS) only in case of DN and DS calculation in pairs of sequences possessing at least two additional nonsynonymous mutations which may be neutral or excessive.

Growth and cell-division in extensive (XDR) and extremely drug resistant (XXDR) tuberculosis strains: transmission and atomic force observation.

The ultra-structure of Mycobacterium tuberculosis (MTB) was examined by transmission electronic (TEM)) and atomic force microscopy (AFM). The study was performed to describe the morphology of susceptible, multidrug-resistant (MDR), extensively drug-resistant (XDR) and extremely drug-resistant tuberculosis isolates (XXDR-TB) during their exponential growth phase. Four types of cell division were observed and described. While three of them (symmetrical, asymmetrical and branching type) occurred in all isolates studied, the fourth one (adapted type) was seen only in XDR and XXDR-TB bacilli. In the fourth type of cell division, a rod shaped mother cell produced a small round shape bacillus (0.3-0.5 µm). These round cells were different from buds or polar division, but similar to terminal endospores without showing the typing heat resistance. Based on the present observation, we suggest that XDR-and XXDR-TB bacilli accommodate changes helping them to overcome the hostile environment. Viewed under AFM, the other frequently detected shapes in MTB isolates were oval, V, Y and multi-branching filaments. These shape variation confirmed pleomorphic phenomena in MTB populations and the specific features of pan-resistant strains.

Frequency and molecular characterization of isoniazid resistance in katG region of MDR isolates from tuberculosis patients in southern endemic border of Iran.

The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis collected from patients in southern endemic region of Iran. Drug susceptibility testing was determined by using the BACTEC system and CDC standard conventional proportional method. All 28 isolates were resistant to isoniazid (100%), rifampicine (14%) streptomycin (82%) and 8 (28%) was resistant to ethambutol. Mono-resistance to isoniazid was observed in four isolates (14%). In 21 isolates three types of mutations were found in codon 315: AGC-->ACC (Ser-->Thr) (61%), AGC-->AGG (Ser-->Arg) (3.5%) and AGC-->AAC (Ser-->Asn) (11%). Moreover, one type of mutation was seen in codons 311 (32%), 299 (28.5%), and 322 (21.5%). Of 28 isolates 12 (46%) found with one mutation, 7 (27%) had two, 5 (19%) had three and 2 (8%) revealed to have four mutations in katG gene. In nine isolates single mutation was observed in codon 311 (GAC-->TAC). This study indicates presence of multiple mutations in codon 315 and 299 among multiple drug-resistant (MDR) strains of M. tuberculosis collected from patients sputum having secondary infections in adjacent southern endemic border of Iran.