An up-to-date review of biomedical applications of serratiopeptidase and its biobetter derivatives as a multi-potential metalloprotease.

Serratiopeptidase is a bacterial metalloprotease used in a variety of medical applications. The multidimensional properties of serratiopeptidase make it noticeable as a miraculous enzyme. Anti-coagulant, anti-inflammatory and anti-biofilm activity of serratiopeptidase making it useful in reducing pain and swelling associated with various conditions including arthritis, diabetes, cancer, swelling, pain and also thrombolytic disorders. It breaks down fibrin, thins the fluids formed during inflammation and due to its anti-biofilm activity, can be used in the combination of antibiotics to reduce development of antibiotic resistance. However, some drawbacks like sensitivity to environmental conditions and low penetration into cells due to its large size have limited its usage as a potent pharmaceutical agent. To overcome such limitations, improved versions of the enzyme were introduced using protein engineering in our previous studies. Novel functional serratiopeptidases with shorter length and higher stability have seemingly created a hope for using this enzyme as a more effective therapeutic enzyme. This review explains the structural properties and functional aspects of serratiopeptidase, its main characteristics and properties, pre-clinical and clinical applications of the enzyme, improved qualities of the modified forms, different formulations of the enzyme, and the potential future developments.

Interaction between Clostridium species and microbiota to progress immune regulation.

Gut microbiota can interact with the immune system through direct or indirect pathways. In the indirect pathway, gut microbiota produces metabolites such as short chain fatty acids (SCFAs), which may modulate the immune response. SCFAs reduce inflammation, repair intestinal barrier, and induce propagation of specific immune cells, e.g., T regulatory cells (Treg), which can suppress reactive cells such as macrophage and dendritic cells (DCs). As one of the most dominant members of microbiota, Clostridium produces SCFAs. As one of SCFA members, butyrate plays an important role in the modulation of immune cells. Through butyrate production, Clostridium helps to generate aryl hydrocarbon receptor (AhR). AhR interacts with many proteins inside the cytoplasm including Heat Shock Protein 90 (HSP 90), HSP 23, and chaperone. Activation of AhR leads to its translocation inside the nucleus and gene expression, which yields cell differentiation, energy metabolism, microbial defense, and immune cell propagation. Moreover, it may interact with other cells like B-cell and epithelial cell, which are responsible for modulation and maturation, respectively. AhR causes upregulation in the co-stimulatory marker in the DCs and interacts with nuclear factor KB (NF-ĸB) to modulate cell function. Butyrate induces Treg (iTreg) propagation and upregulates the Forkhead box p3 (FOXP3) as a special marker of Treg cell. It may also yield signaling through G-protein coupled receptors (GPRs) which, in turn, facilitates polymorphonuclear (PMN) chemotaxis.The interaction between microbiota and non-immune cells, such as Paneth cells, leads to the secretion of antimicrobial substance, erection of barriers against bacterial pathogens, and regulation of microbiota composition via feedback effect. In addition, the components released from microbiota, such as peptidoglycan, reinforce the maturation of both the immune system and non-immune tissue development. Moreover, microbiota can directly activate the effector cells, e.g., macrophage, to secrete cytokines and propagate Treg cells.

Studies in lung cancer cytokine proteomics: a review.

INTRODUCTION: Proteins are molecules that have role in the progression of the diseases. Proteomics is a tool that can play an effective role in identifying diagnostic and therapeutic biomarkers for lung cancer. Cytokines are proteins that play a decisive role in activating body's immune system in lung cancer. They can increase the growth of the tumor (oncogenic cytokines) or limit tumor growth (anti-tumor cytokines) by regulating related signaling pathways such as proliferation, growth, metastasis, and apoptosis. AREAS COVERED: In the present study, a total of 223 papers including 196 research papers and 27 review papers, extracted from PubMed and Scopus and published from 1997 to present, are reviewed. The most important involved-cytokines in lung cancer including TNF-α, IFN- γ, TGF-ß, VEGF and interleukins such as IL-6, IL-17, IL-8, IL-10, IL-22, IL-1ß and IL-18 are introduced. Also, the pathological and biological role of such cytokines in cancer signaling pathways is explained. EXPERT OPINION: In lung cancer, the cytokine expression changes under the physiological conditions of the immune system, and inflammatory cytokines are associated with the progression of lung cancer. Therefore, the cytokine expression profile can be used in the diagnosis, prognosis, prediction of therapeutic responses, and survival of patients with lung cancer.

Molecular mechanisms of azole resistance in Candida glabrata isolated from oropharyngeal candidiasis in head and neck cancer patients.

OBJECTIVE: The aim of the current work was to assess the molecular mechanisms of fluconazole-resistant Candida glabrata strains isolated from oropharyngeal candidiasis (OPC) in head and neck patients, as well as evaluation of virulence factors. DESIGN: Antifungal susceptibility pattern of sixty six clinical isolates of C. glabrata were evaluated by broth-microdilution method. The expression of ERG11, CDR1, CDR2, PDR1 genes as well as ERG11 gene capable of possible mutations was also detected in 21 fluconazol-resistant C. glabrata isolates. Phospholipase and proteinase activity of these isolates was estimated, too. The correlation between the virulence factors, antifungal susceptibility patterns and cancer type was also analyzed. RESULTS: Seven synonymous and four non-synonymous mutations were found in 21 fluconazole-resistant C. glabrata isolates; subsequently, four amino acid substitutions including H257P, Q47H, S487Y and I285N were then reported for the first time. High expression of CDR1 and PDR1 in related to other gene findings were tested in these isolates. Additionally, there was no significant difference between stage of cancer and MIC of all antimicrobial drugs. Significant differences between MIC of fluconazole, voriconazole and cancer types were also, found. The proteinase activity (92.4%) was higher than phospholipase activity in the isolates. Further, no significant difference between proteinase (rs: 0.003), phospholipase (rs: -0.107) activity and fluconazole MICs was observed. CONCLUSION: C. glabrata isolated from OPC in head and neck patients represented high capacities for proteolytic enzymes activity and high mRNA level of CDR1 and PDR1 gene and ERG11 mutations play an important role in azole drug resistance.

Shortening the sulfur cell cycle by a green approach for bio-production of extracellular metalloid-sulfide nanoparticles.

In the present study, a new approach was introduced regarding the extracellular synthesis of selenium sulfide micro/nano-particles using Saccharomyces cerevisiae in different ammonium sulfate supplementation and in the presence of sodium selenosulfate precursors (S1) and a blend of selenous acid and sodium sulfite (S2). In S1, only cell supernatant exposed to ammonium sulfate was able to reduce sodium selenosulfate. Whereas, in S2, cell supernatant in both pre-conditions of with or without ammonium sulfate (S2 + or S2-) were able to reduce selenous acid and sodium sulfite. Electron microscopy, also indicated that selenium sulfide NPs were successfully synthesized with average size of 288 and 332 nm for S2 + and S2- in SEM and 268 and 305 nm in TEM. Additionally, elemental mapping by energy-dispersive x-ray analysis confirmed the presence of sulfur/selenium elements in the particles in a proportion of 24.50 and 23.31 for S2- and S2 + , respectively. The mass spectrometry indicated the probability of Se2S2, SeS1.1, Se2, Se, SeS5, SeS3, Se3S5/Se5, Se3/SeS5, Se6, Se4/SeS7, Se2.57S5.43/Se2S2 and Se4S/Se2S6 molecules for S2 + and of Se, Se2, Se3S5/Se5, Se6 and Se4 species for S2-. In FTIR spectra, primary (i.e. 1090-1020 and 1650-1580 cm-1) and secondary (1580-1490 cm-1) amine bands duly confirmed the protein corona around the NPs.

PASylated Urate Oxidase Enzyme: Enhancing Biocatalytic Activity, Physicochemical Properties, and Plasma Half-Life.

Recombinant urate oxidase (UOX, E.C.1.7.3.3) is an important therapeutic enzyme used in preventing and treating chemotherapy-induced hyperuricemia and severe gout. However, UOX use is limited due to the poor stability and short plasma half-life. To solve this problem, we designed three PASylated variants of Aspergillus flavus UOX with different PAS sequences at the C- or N-terminus. The genes of native and PASylated variants (UOX-PAS20, PAS24-UOX, and UOX-PAS100) were designed and produced in Escherichia coli strain BL21 (DE3). The expressed recombinant native and PASylated enzymes were compared in terms of biophysical properties, kinetics parameters, and pharmacokinetics behavior using standard methods. PASylation of UOX with PAS100 polymer caused a 1.24-fold reduction in K m to 52.61 µM, and a 3.87-fold increase in K cat/K m for uric acid compared to the native variant. UOX-PAS100 retained its activity in different temperatures (20-55 °C); however, other variants lost nearly 50% of their original activity at 55 °C. UOX-PAS100 exhibited a 1.78-fold increase in hydrodynamic radius and a 1.64-fold larger apparent molecular size in comparison to the native UOX. Circular dichroism (CD) spectroscopy demonstrated that the addition of the PAS tag does not change the secondary structure of the fusion enzyme. The tryptophan fluorescence emission spectra for PASylated enzymes showed a significant modification in the conformational state of UOX by the PAS polymer presence. UOX-PAS100 retained 89.0% of the original activity following 72 h incubation in the presence of plasma at 37 °C. However, only about 61.0%, 57.0%, 50.0%, and 52.0% of activity from PAS24-UOX, UOX-PAS20, native UOX, and rasburicase (Fasturtec, Italy) remained, respectively, at the identical time. UOX-PAS100 had an increased biological half-life (8.21 h) when compared with the rasburicase (3.12 h) and native UOX (2.87 h) after being injected into a rat. Having considering everything, our results suggest that the UOX-PAS100, an A. flavus UOX fused with a C-terminally 100 amino acid PAS-residue, is a proper candidate with enhanced biological activity and extended plasma half-life for clinical therapy in patients suffering from hyperuricemia.

Bacillales: From Taxonomy to Biotechnological and Industrial Perspectives.

For a long time, the genus Bacillus has been known and considered among the most applicable genera in several fields. Recent taxonomical developments resulted in the identification of more species in Bacillus-related genera, particularly in the order Bacillales (earlier heterotypic synonym: Caryophanales), with potential application for biotechnological and industrial purposes such as biofuels, bioactive agents, biopolymers, and enzymes. Therefore, a thorough understanding of the taxonomy, growth requirements and physiology, genomics, and metabolic pathways in the highly diverse bacterial order, Bacillales, will facilitate a more robust designing and sustainable production of strain lines relevant to a circular economy. This paper is focused principally on less-known genera and their potential in the order Bacillales for promising applications in the industry and addresses the taxonomical complexities of this order. Moreover, it emphasizes the biotechnological usage of some engineered strains of the order Bacillales. The elucidation of novel taxa, their metabolic pathways, and growth conditions would make it possible to drive industrial processes toward an upgraded functionality based on the microbial nature.

Microbiological insights into anaerobic digestion for biogas, hydrogen or volatile fatty acids (VFAs): a review.

In the past decades, considerable attention has been directed toward anaerobic digestion (AD), which is an effective biological process for converting diverse organic wastes into biogas, volatile fatty acids (VFAs), biohydrogen, etc. The microbial bioprocessing takes part during AD is of substantial significance, and one of the crucial approaches for the deep and adequate understanding and manipulating it toward different products is process microbiology. Due to highly complexity of AD microbiome, it is critically important to study the involved microorganisms in AD. In recent years, in addition to traditional methods, novel molecular techniques and meta-omics approaches have been developed which provide accurate details about microbial communities involved AD. Better understanding of process microbiomes could guide us in identifying and controlling various factors in both improving the AD process and diverting metabolic pathway toward production of selective bio-products. This review covers various platforms of AD process that results in different final products from microbiological point of view. The review also highlights distinctive interactions occurring among microbial communities. Furthermore, assessment of these communities existing in the anaerobic digesters is discussed to provide more insights into their structure, dynamics, and metabolic pathways. Moreover, the important factors affecting microbial communities in each platform of AD are highlighted. Finally, the review provides some recent applications of AD for the production of novel bio-products and deals with challenges and future perspectives of AD.

High level isoniazid resistance correlates with multiple mutation in the katG encoding catalase proxidase of pulmonary tuberculosis isolates from the frontier localities of Iran.

The aim of this study was to investigate the significance of multiple-mutations in the katG gene, predominant nucleotide changes and its correlation with high level of resistance to isoniazid in Mycobacterium tuberculosis isolates that were randomly collected from sputa of 42 patients with primary and secondary active pulmonary tuberculosis from different geographic regions of Iran. Drug susceptibility testing was determined using the CDC standard conventional proportional method. DNA extraction, katG gene amplification, and DNA sequencing analysis were performed. Thirty four (80%) isolates were found to have multiple-mutations (composed of 2-5 mutations) in the katG gene. Increased number of predominant mutations and nucleotide changes were demonstrated in codons 315 (AGC-->ACC), 316 (GGC-->AGC), 309 (GGT-->GTT) with a higher frequency among patients bearing secondary tuberculosis infection with elevated levels of resistance to isoniazid (MIC ≥ 5-10 µg/mL). Furthermore, it was demonstrated that the combination of mutations with their predominant nucleotide changes were also observed in codons 315, 316, and 309 indicating higher frequencies of mutations among patients with secondary infection respectively. In this study, 62% (n= 21) of multi-mutated isolates found to have combination of mutations with predominant nucleotide changes in codons 315 (AGC-->ACC), 316 (GGC-->GTT), 309 (GGT-->GGT), and also demonstrated to be more frequent in isolates of patients with secondary infections, bearing higher level of resistance to isoniazid (≥ 5-10 µg/mL).

Effects of Akkermansia muciniphila and Faecalibacterium prausnitzii on serotonin transporter expression in intestinal epithelial cells.

PURPOSE: The highest level of peripheral serotonin in the body can be found in the gastrointestinal (GI) tract as its reservoir. There is complete interaction between human gastrointestinal microbiota and serotonin system. Serotonin in the GI is transferred by serotonin transporters (SERTs), which play a crucial role in the bioavailability of serotonin in the GI. SERT impairment is associated with the pathology of GI disorders. It is known that intestinal microbiota can regulate the SERT function. Therefore, it may be useful to regulate of SERT expression by modulation of microbiota and improvement of intestinal motility and GI sensation. In this study, we aimed to evaluate the effects of two next-generation probiotics, including Akkermansia muciniphila and Faecalibacterium prausnitzii, and their supernatants on SERT gene expression in human epithelial colorectal adenocarcinoma cells (Caco-2). METHODS: The Caco-2 cells were treated with multiplicity of infection (MOI) ratio of 100 of A. muciniphila and F. prausnitzii, as well as their supernatants. After 24 h, SERT gene expression was examined by quantitative real-time polymerase chain reaction (qRT-PCR) assay. RESULTS: A. muciniphila up-regulated the SERT mRNA level by 3.01 folds, compared to the control group. F. prausnitzii, similar to A. muciniphila, increased the expression of SERT gene in Caco-2 cells by 3.43 folds (P < 0.001). Moreover, the supernatants of A. muciniphila and F. prausnitzii significantly up-regulated the expression of SERT gene in the cell line by 2.4 and 5.7 folds, respectively, compared to the control group (P < 0.001). CONCLUSIONS: The present results showed that A. muciniphila and F. prausnitzii, as well as their supernatants, increased the expression of SERT gene in Caco-2 cells. Therefore, they might be helpful in the microbiota-modulating treatment of inflammatory bowel diseases.

Effect of Akkermansia muciniphila, Faecalibacterium prausnitzii, and Their Extracellular Vesicles on the Serotonin System in Intestinal Epithelial Cells.

The gastrointestinal (GI) tract is an essential reservoir of serotonin or 5-hydroxytryptamine (5-HT), which possesses a set of bacterial species communities. Intestinal microbiota has the ability to modulate the host's serotonin system. In this regard, we evaluated the effect of Akkermansia muciniphila and Faecalibacterium prausnitzii along with their extracellular vesicles (EVs) on serotonin system-related genes in human epithelial colorectal adenocarcinoma (Caco-2) cells. The differentiated Caco-2 cells were treated with A. muciniphila and F. prausnitzii with the multiplicity of infection ratio of 1 and 10 and the EV concentration of 1 µg/mL and 50 µg/mL, respectively. After 24 h, the serotonin level was quantified using an ELISA kit and also the gene expression of serotonin system-related genes was examined using the quantitative real-time PCR method. According to the results, treatment with A. muciniphila and F. prausnitzii-derived EVs increased the serotonin level, while none of the bacteria could affect the serotonin level in the Caco-2 cells. Both bacteria had significant effects on the mRNA expression of serotonin system-related genes in the Caco-2 cells. Moreover, we observed that A. muciniphila and F. prausnitzii-derived EVs could impact the expression of major genes involved in the serotonin system. Our findings showed that A. muciniphila and F. prausnitzii along with their EVs could modulate serotonin system-related genes; hence, they may be useful in microbiota modulation therapies to maintain the homeostasis of the serotonin system.

Computational Approach for Rational Design of Fusion Uricase with PAS Sequences.

Tumor lysis syndrome is a life-threatening condition for humans due to the lack of urate oxidase. In this study, several variants of PASylated uricase from the Aspergillus flavus species were analyzed computationally to find the appropriate fusions to solve short half-life and stability concern. The Ab initio method was performed using Rosetta software to structurally characterize the PAS sequences. The 3D structures of fusions were predicted for fused C- or N-terminally PAS sequences in different length to the uricase. The refinement and energy minimization steps revealed that physicochemical and conformational properties of fusions improved while the structures possessed prolonged PAS sequences. Molecular docking results showed that the highest binding affinity to uric acid belonged to uricase-PAS1-100 by the formation of six hydrogen and four non-hydrogen bonds. Altogether, the results indicated that the PASylation process would be promising upon the production of urate oxidase with improved solubility and stability.

Modulation of serotonin signaling/metabolism by Akkermansia muciniphila and its extracellular vesicles through the gut-brain axis in mice.

Several studies have reported that the host-microbe interactions in the gut modulate the host serotonin or 5-hydroxytryptamine (5-HT) system. Here, we evaluated the effects of Akkermansia muciniphila and its extracellular vesicles (EVs) on genes pertaining to the serotonergic system in the colon and hippocampus of mice. Male C57BL/6J mice were administered viable A. muciniphila and its EVs for 4 weeks. The serotonin levels in the colon, hippocampus, and serum of mice, as well as the human colon carcinoma cells (Caco-2), were measured by ELISA assays. Also, the effects of A. muciniphila and its EVs on the expression of serotonin system genes in the colon and hippocampus were examined. A. muciniphila and its EVs may have a biological effect on the induction of serotonin levels in the colon and hippocampus of mice. Also, EVs increased the serotonin level in the Caco-2 cell line. In contrast, both treatments decreased the serotonin level in the serum. Both the bacterium and its EVs had significant effects on the mRNA expression of genes, involved in serotonin signaling/metabolism in the colon and hippocampus of mice. Moreover, A. muciniphila and its EVs affected the mRNA expression of inflammatory cytokines (Il-10 and Tnf-α) in the colon, however, there is no significant difference in inflammatory cell infiltrate in the histopathology of the colon. The presence of A. muciniphila and its EVs in the gut promotes serotonin concentration, they also affect serotonin signaling/metabolism through the gut-brain axis and may be considered in new therapeutic strategies to ameliorate serotonin-related disorders.

MLVA typing of Haemophilus influenzae isolated from two Iranian university hospitals.

BACKGROUND AND OBJECTIVES: Different serotypes of Haemophilus influenzae is now divided into 2 divisions: encapsulated and unencapsulated. Multiple locus variable number tandem repeat analysis (MLVA) includes such specifications as the extra power of separation, ease of data interpretation, and epidemiological data accordance, which have made it an appropriate molecular device for good typing and phylogenetic analysis of bacterial pathogens. MATERIALS AND METHODS: In this research, cultured samples were studied and strains identified through biochemical tests were recognized. Moreover, DNA was extracted and studied qualitatively and quantitatively. Four pairs of specialized primers related to H. influenzae variable number tandem repeats (VNTR) and preparation of PCR were designed according to the regulated program. Also, electrophoresis of PCR products was performed. Finally, the interpretation of electrophoresis gel was done with respect to the observable bands showing the presence or absence of the required sequence in the samples related to every primer. RESULTS: This study was the first MLVA typing of the unencapsulated H. influenzae in Iran. In this research, the VNTR sequences were tested in 30 strains without the unencapsulated H. influenzae. Among 30 mentioned strains, for which MLVA profile was obtained in this research, 25 different MLVA types were observed. Likewise, there was no repetition in VNTR sequences resulting from PCR in few H. influenzae. In all these cases, the number of repetitions in MLVA profile was determined as 0, except for one of the primers in 4 strains, which was 16%. However, this did not occur for the other VNTRs. CONCLUSION: The highest diversity of the repeats was for VNTR5 (7 types), followed by VNTR6 with 6 types of repeats, and VNTR12-1 and VNTR12-2 with 3 different types.

Setting research priorities to achieve long-term health targets in Iran.

BACKGROUND: In 2015, it was estimated that the burden of disease in Iran comprised of 19 million disability-adjusted life years (DALYs), 74% of which were due to non-communicable diseases (NCDs). The observed leading causes of death were cardiovascular diseases (41.9%), neoplasms (14.9%), and road traffic injuries (7.4%). Even so, the health research investment in Iran continues to remain limited. This study aims to identify national health research priorities in Iran for the next five years to assist the efficient use of resources towards achieving the long-term health targets. METHODS: Adapting the Child Health and Nutrition Research Initiative (CHNRI) method, this study engaged 48 prominent Iranian academic leaders in the areas related to Iran's long-term health targets, a group of research funders and policy makers, and 68 stakeholders from the wider society. 128 proposed research questions were scored independently using a set of five criteria: feasibility, impact on health, impact on economy, capacity building, and equity. FINDINGS: The top-10 priorities were focused on the research questions relating to: health insurance system reforms to improve equity; integration of NCDs prevention strategy into primary health care; cost-effective population-level interventions for NCDs and road traffic injury prevention; tailoring medical qualifications; epidemiological assessment of NCDs by geographic areas; equality in the distribution of health resources and services; current and future common health problems in Iran's elderly and strategies to reduce their economic burden; the status of antibiotic resistance in Iran and strategies to promote rational use of antibiotics; the health impacts of water crisis; and research to replace the physician-centered health system with a team-based one. CONCLUSIONS: These findings highlight consensus amongst various prominent Iranian researchers and stakeholders over the research priorities that require investment to generate information and knowledge relevant to the national health targets and policies. The exercise should assist in addressing the knowledge gaps to support both the National General Health Policies by 2025 and the health targets of the United Nations' Sustainable Development Goals by 2030.

The Increasing Trend in Global Ranking of Websites of Iranian Medical Universities during January 2012-2015.

BACKGROUND: Researchers and academic institutions need assessment and rating to measure their performance. The criteria are designed to evaluate quality and adequacy of research and welcome by most universities as an international process to increase monitoring academic achievements. The study aimed to evaluate the increasing trend in global ranking of Iranian medical universities websites emphasizing on comparative approach. METHODS: This is a cross-sectional study involving websites of Iranian medical universities. Sampling was conducted by census selecting universities affiliated to the Ministry of Health in webometrics rating system. Web sites of Iranian medical universities were investigated based on the webometrics indicators, global ranking as well as the process of changing their rating. Universities of medical sciences were associated with improved ratings in seven periods from Jan 2012 until Jan 2015. RESULTS: The highest rank was in Jan 2014. Tehran University of Medical Sciences ranked the first in all periods. The highest ratings were about impact factor in universities of medical sciences reflecting the low level of this index in university websites. The least ranking was observed in type 1 universities. CONCLUSION: Despite the criticisms and weaknesses of these webometrics criteria, they are critical to this equation and should be checked for authenticity and suitability of goals. Therefore, localizing these criteria by the advantages model, ranking systems features, continuous development and medical universities evaluation based on these indicators provide new opportunities for the development of the country especially through online media.

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.

Study of Genetic Evolution in Mycobacterium tuberculosis Isolates from Patients with Active Pulmonary Tuberculosis in the Iran and Belarus.

OBJECTIVE: This is the new comparative geogenetic molecular evolution research of M. tuberculosis in Iran and Belarus. Thus, we researched the genetic patterns of samples collected in the first survey of anti-tuberculosis drug-resistance by gene coding of RNA polymerase as part of the international project of on tuberculosis. METHOD: DNA extraction and amplification of rpoB gene was performed. All PCR products of gene were sequenced using the Amersham auto sequencer. For analysing phenogram has been demonstrated by method UPGMA and Neighbour-Joining. Clinical isolates (70/473) were analyzed by using sequencing gene rpoB and genotyped by program DNAMAN and MEGA. RESULTS: The all data were compared with the international database of national center for biotechnology information website. Multi drug resistant of tuberculosis patient (MDR-TB) was 92% in never treated and 8% in previously treated. Mutations in rpoB gene and katG genes were showed in 95% and 84% of the MDR isolates, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis. The other isolates are divided in Iran into 2 groups: group A - similar to the Eastern strains (China, Taiwan) and group B - strains of another genotype. And 3 groups in Belarus: group A - Strains of the first group are more similar to the standard European and Eastern ones China and Taiwan) which diverged in the last 10 years (Genetic evolution rate), i.e. they are relatively new ones, and that is confirmed by the mutations, group B - Strains of the second group diverged earlier; they are older than the strains of the first group (16 years old- time and rate of evolution) and group C - Strains of the third group are similar to European strains and only circulate in Brest region. They are grouped separately on the phenogram and became prevalent in Iran (they are called Iranian residential strains and also is genetic analogy between group A from Iran and Belarusian isolates. CONCLUSION: This research gives a first result on genetic evolution of the M. tuberculosis strains distributing in the Iran and Belarus during the first survey of anti-tuberculosis drug-resistance and is homologies between groups A from Iran with group A from Belarus. It may aid in the creation of a national database that will be a valuable support for further studies.