Analysis of rpsL and rrs genes mutations related to streptomycin resistance in Mdr and Xdr clinical isolates of Mycobacterium tuberculosis.

INTRODUCTION: Streptomycin is a bactericidal and aminoglycoside antibiotic. It is one of the most effective drugs for treatment of multi-drug Tuberculosis disease. Incidence of resistance is increasingly reported. Its action mechanism is by inhibition of binding aminoacyl tRNA to position "A" in elongation phase, which finally it causes to stop bacterial protein synthesis. In this study, resistance rapid investigation to streptomycin was conducted in clinical strains of Mycobacterium tuberculosis. MATERIALS AND METHODS: In this study, among 105 strains of phlegm-positive and culture-positive Mycobacterium tuberculosis, 45 strains of resistant and sensitive to streptomycin were selected for possible mutations examination in genes rrs and rpsL. Specific primers that used for PCR were named rpsL 1, rpsL 2 and rrsR, Frrs. PCR products were sequenced. RESULT: PCR Products represents 504 bp band for gene rpsL and 1027 bp for gene rrs that shows proper selection of primers and determining an amplification appropriate program. From 26 resistant strains to streptomycin 26 strain have mutation in rpsL gene and 1 strain have alteration in rrs gene. In this study 19 strains were sensitive to streptomycin that have no mutation in these gene. CONCLUSIONS: Streptomycin resistance is mainly related to mutation at codons 43 and 88 "rpsL" gene and to a lesser extent "rrs" that are the greatest cause of drug resistance to streptomycin.

Study of pyrazinamidase structural changes in pyrazinamide resistant and susceptible isolates of Mycobacterium tuberculosis.

INTRODUCTION: Pyrazinamide is one of the first line four drugs for treatment of tuberculosis. It was proved that mutations in two nucleotides of 359 and 374 pnc genes are highly associated with resistance to pyrazinamide. MATERIALS AND METHODS: In this study, mutations in these two codones in 30 clinical isolates of Mycobacterium tuberculosis were detected by means of sequencing. Protein structures encoded by this gene with and without mutation were investigated in resistant and susceptible isolates to pyrazinamide, respectively. RESULTS: Mutation in the positions 359 and 374 altered some parameters like change in electronic charge, distance change of mutated amino acids to situation of active enzyme and metal connection situation. In these conditions, structure and function of pyrozinamidase enzyme were changed and antibiotic was ineffective and consequently caused resistance to pyrazinamide in M. tuberculosis. CONCLUSION: This work was revealed protein changes in resistance to pyrazinamide in clinical isolates of M. tuberculosis.

Serum level of soluble interleukin-2 receptor alpha as a predictor of treatment response in brucellosis.

INTRODUCTION: Iran is one of the endemic regions with high prevalence of brucellosis. Several serological markers for diagnosis and response to treatment are available. Serum level of Soluble Interleukin-2 Receptor alpha (SIL-2Ralpha) is a new marker to assess response to therapy and clinical relapse of brucellosis. This study intends to investigate the serum levels of SIL-2Ralpha before and after treatment, to evaluate this marker for patients responding to treatment of brucellosis. METHODS: This study is an analytical cross-sectional study. Forty patients who had clinical signs of brucellosis and serological tests confirmed the disease have been treated with standard antibiotics for 6 weeks. 2ME and SIL-2Ralpha levels were measured before and after treatment and these values were compared. RESULTS: Among the 40 patients, 27 patients (67.5%) had improvement in symptoms and 13 patients (32.5%) had no symptoms after treatment. In Comparing serum levels of SIL-2Ralpha and 2ME before and after treatment, decreasing of both markers after treatment was significant (p < 0.001). In patients with false positive for 2ME, SIL-2Ralpha in 57% of patients had a reduction, but in patients with false negative for 2ME, SIL-2Ralpha in only 28% of patients increased. CONCLUSION: Not only is Serum level of SIL-2Ralpha useful for predicting response to treatment of brucellosis, but also in cases of false positive of 2ME can be helpful.

Determination of principal genotypic groups among susceptible, MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus and Iran.

INTRODUCTION: All members of the Mycobacterium tuberculosis complex were assigned to one of the three principle genetic groups based on KatG463/GyrA95 polymorphism. MATERIALS AND METHODS: A total of 202 isolates of M. tuberculosis consisting of 50 susceptible, 121 MDR (multidrug resistant) and 31 XDR (extensively drug resistant) isolated from culture-confirmed tuberculosis patients in different regions of Belarus and Iran (Tehran and Markazi province). Isolates were screened by sequencing and polymerase chain reaction restriction fragment length polymorphism (RFLP) assay, and were further divided into three principal genetic groups (PGG), based on Sreevatsan's pattern as polymorphisms in KatG463/GyrA95 codons. RESULTS: Among the 104 isolates, characterized as MDR from Belarus, 57 (54.8 ± 4.8%), 30 (28.8 ± 4.43%), 17 (16.3 ± 3.6), belonged to PGG 1, 2, and 3, respectively (p< 0.05). Thirty one XDR isolates from Belarus had a similar pattern as 15 (48.4%), 12 (38.7%), 4 (12.9%) PGG 1, 2, and 3, respectively. From Iranian samples, Markazi isolates (susceptible to drugs) had a pattern as 12 (36.5%), 15 (45.5%), 3 (6%), and Tehran samples were (selected MDR): 9 (53%), 6 (35.2%), 2 (11.8%) (PGG 1, 2, and 3, respectively). In a study of tuberculosis patients, who were in prison, no relation was found between PGG and resistance to isoniazid, but most of the identified isolates belonged to PGG 1 (45.5 ± 10.9%) (p< 0.05). Overall, the group 1 isolates showed more frequency in MDR and XDR rather than susceptible strains, and there aren't any relations to geographic region.

Biaxial electrospun nanofibers based on chitosan-poly (vinyl alcohol) and poly (Ɛ-caprolactone) modified with CeAlO3 nanoparticles as potential wound dressing materials.

A two-nozzle electrospinning method was employed to fabricate hybrid nanofibers based on chitosan/polyvinyl alcohol (CS/PVA), with a ratio of 50:50, and poly (Ɛ-caprolactone) (PCL). CeAlO3 nanoparticles were synthesized by combustion method and utilized to improve the nanofiber's properties for wound dressing application. Cephalexin (CFX), as an antibiotic model, was also incorporated into the hydrophilic nanofibers. X-ray diffraction showed an increase in crystallinity when CeAlO3-NPs were present in the nanofibers. Water vapor transmission rates in the samples were calculated as 2201-2627 g m-2 day-1, all within the normal range of ideal wound dressings. Mechanical studies revealed a 43 % and 85 % increase in tensile strength and modulus when CeAlO3-NPs were incorporated. In vitro drug release tests were conducted to simulate drug release, and the neat fibers showed faster release than the modified fibers. The MTT assay and cell morphology experiments showed that CeAlO3-NPs did not affect the nanofiber's biocompatibility and fibroblast cells could better grow, differentiate and cover the prepared hybrid scaffold surface compared to the neat fibers. Taking the results of our study into account, we believe the prepared nanofibrous has the potential for use as a low-cost, effective wound dressing.

In vitro and in vivo evaluation of antibacterial and anti-biofilm properties of five ethnomedicinal plants against oral bacteria by TEM.

OBJECTIVE: The aim of the present study was to investigate antibacterial and antibiofilm activity of a few medicinal plants against oral bacteria. MATERIALS AND METHODS: Salvia officinalis, Lippie citriodora, Mentha piperita, Echinacea purpurea and Matricaria chamomilla were extracted. Isolates from oral cavity were identified by microbiological and molecular methods. Minimum inhibitory concentration and minimum bactericidal concentration were determined by Broth microdilution method. The anti-biofilm activity of essential oils and extracts investigated and as a mixture by Broth dilution method. Toxicity of the herbal mixture was assayed by in Wistar rats treated with intradermal injection. Wound healing properties of the herbal mixture against infected wounds on the back of the rats were investigated. Anti-biofilm activity was investigated on tooth surfaces. Bacterial structure changes and fine- structure study were performed by light microscopy and Transmission electron microscopy. RESULTS: The lowest MIC and MBC for the plant mixtures was 0.0002 mg/ml belonged to Streptococcus pyogenes and the highest values (0.025 mg/ml) belonged to Eikenella corrodens. The essential oils of S.officinalis, L.citriodora and M.piperita, but not E.purpurea and M.chamomilla extracts, were able to remove the biofilms created by the studied bacteria. The herbal mixture was able to completely heal the wound skin of rats in 21 days (p<0.05 compared to control). The mixture was able to decompose the teeth biofilm in 45 seconds. The results of light and electron microscopy showed that the bacterial structure exposed to the herbal mixture was deformed. CONCLUSION: It was concluded that the essential oils of S.officinalis, L.citriodora and M.piperita had significant effects on inhibition of oral bacteria biofilm formation.

Nanofibrous cellulose acetate/gelatin wound dressing endowed with antibacterial and healing efficacy using nanoemulsion of Zataria multiflora.

In this paper, a multifunctional nanofibrous cellulose acetate/gelatin/Zataria multiflora-nanoemulsion (CA/Gel/ZM-nano) wound dressing was fabricated, in which nanoemulsion of a natural active antibacterial plant, by the scientific name of Zataria multiflora (ZM) was loaded into the nanofibrous mat. To fabricate the wound dressing, different weight ratios of CA/Gel (100, 0, 75, 25, 50, 50 and 25, 75) were selected, and solutions with concentrations of 16, 15, 14 and 12% w/v were prepared for each ratio, respectively to achieve smooth and uniform fibers by electrospinning. In vitro and in vivo analysis was taken for the samples. Nanofibrous mats with a lower ratio of CA/Gel and incorporated with ZM-nano promoted the adhesion and proliferation of L929 fibroblast cells significantly. Also, by lowering the ratio of CA/Gel, nanoemulsion drug-loading into nanofibers increased considerably, as the amount of ZM-nano loaded into CA/Gel = 50:50 was found to be 2.4-fold higher than CA/Gel = 100:0. Moreover, the rat model experiment in our study revealed that the nanofibrous samples incorporated with nanoemulsion drug (ZM-nano) accelerated the wound healing process so that the relative wound area for the nanoemulsion-loaded dressings was much smaller than the other samples after 22 days. Therefore, this multifunctional CA/Gel/ZM-nano wound dressing could be a promising and potential candidate for wound healing applications.

Molecular Detection of Campylobacter Species: Comparision of 16SrRNA with slyD, cadF, rpoA, and dnaJ Sequencing.

BACKGROUND: Campylobacter spp. are the main cause of human gastroenteritis. The 16SrRNA sequencing is one of fast molecualr method to detect this fastidious. In this study, we compared the sequencing of 16srRNA genewith four housekeeping genestodetect Campylobacter spp. in patients with diarrhea and healthy people. METHODS: 60 samples of Campylobacter DNA extracted from stool samples of 30 patients with diarrhea and 30 healthy people were used. In order to detect Campylobacter, we designed primers for proliferation of 16SrRNA, cadF, dnaJ, slyD , and rpoA genes using Primer 3, Mega 4.0 and Blast software. Then the PCR products were sequenced using ABI system. RESULTS: The sequencing showed concordance of PCR-products with deposited sequences in the Gene Bank. Among diarrhea patients, 53.3% of samples were significantly (p< 0.05) positive for slyD and cadF genes and 50% of samples were positive using 16SrRNA, rpoA, and dnaJ genes by PCR assay. The average of sensitivity and specificity were found 53.33% and 83.33%, respectively. CONCLUSION: Due to various copies of repeated sequences of 16SrRNA gene, analyzing its amplicons on electrophoresis may be more difficult than the slyD and cadF genes. According to our results, among the 5 studied genes; the highest detection rate was related to slyD and cadF genes. Although, dnaJ and rpoA genes,instead of 16SrRNA gene, can be considered as appropriate genes for molecular detection of Campylobacter bacteria.

Evaluation of Splicing on X-box Binding Protein Transcript in Tissue Samples of Colorectal Cancer.

Background The genetic etiology of colorectal cancer (CRC) is the occurrence of mutation in the genes involved in signal transduction pathways including that of cellular responses to endoplasmic reticulum (ER) stress. This study examines alterations of pre-messenger ribonucleic acid (pre-mRNA) splicing in X-box binding protein (XBP) transcripts related to the ER stress pathway in CRC. Materials and methods In this study, samples were deparaffinized and underwent RNA extraction. A total of 30 synthesized complementary deoxyribonucleic acid (cDNA) templates from the extracted RNAs related to tumor and non-tumor CRC samples, collected over three years and containing pathological data, were subjected to semi-quantitative reverse transcriptase polymerase chain reaction (sqRT-PCR). These cDNA templates were amplified in reaction tubes with specific primers for both spliced and non-spliced isoforms of XBP. Results with P< .05 were considered statistically significant. Results Microscopic assessment represented lymphocyte-rich effusion in tumor samples. sqRT-PCR electrophoresis results showed spliced and non-spliced forms of XBP messenger RNA in the studied samples. In addition, our data showed there were more than 7.8 times the total number of spliced variants in the marginal tumor samples than in the tumor tissue samples (P<.05). Conclusion Alterations of expression in genes involved in stress signaling pathways in cancer have been identified previously. Our results showed an inverse relationship between XBP splicing and CRC tumor tissue, possibly lead to the inactivation of apoptosis in the downstream response to ER stress. However, we propose that the remaining genes in this pathway should undergo gene expression analysis using a greater number of samples.

The Relationship Between rs3212986C>A Polymorphism and Tumor Stage in Lung Cancer Patients.

Background The nucleotide excision repair (NER) system is one of the most important deoxyribonucleic acid (DNA) repair mechanisms and is critical for chemotherapy resistance. We conducted the present study to investigate the association between two polymorphisms of excision of repair cross-complementing group 1 (ERCC1), the key component of the NER pathway, and the clinicopathological features of patients with non-small cell lung cancer (NSCLC). Methods A total of 38 patients with confirmed NSCLC were included in our study. DNA was extracted from peripheral blood. ERCC1 rs3212986 (8092) and rs11615 (118) were genotyped using molecular assays including polymerase chain reaction (PCR) with restriction fragment length polymorphism (by MboII and HpyCH4 enzymes) and sequencing. Results The PCR results indicated the correct performance of the genomics extraction and molecular protocols. The distribution of C/C, C/A and A/A genotypes at position 8092 was 42.10%, 47.36%, and 10.52% respectively (P=0.03). Multivariate regression analysis showed that there was a significant correlation between C8092A (rs3212986) polymorphism and metastasis, grade of the tumor, and response to treatment. Individuals carrying the rs3212986 CA genotype and A allele had a significantly worse response to the treatment. Also, the correlation between alteration at this genomics location and patients with NSCLC who used to smoke cigarettes was positive. However, no significant association was detected between rs11615 C118>T polymorphism and demographic characteristics of patients with NSCLC. Conclusion We concluded that in lung cancer patients there is a relationship between tumor stage and rs3212986C>A polymorphism.

Comparison of antibacterial effects of a carrier produced in microemulsion system from aqueous extract of Aloe vera with selected antibiotics on Enterobacteriacea.

BACKGROUND AND OBJECTIVES: Antibiotics resistance has recently increased. The aim of this study was the evaluation of antibacterial efficacy of Aloe vera carrier produced in microemulsion system in comparison with ordinary antibiotics against some Enterobacteriacea. MATERIALS AND METHODS: The aquatic extract of Aleo vera was produced by the Soxhlet method and a nonocarrier in the microemulsion system was prepared by two emulsifiers. The clinical isolates of Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Shigella dysenteriae, Salmonella Typhimurium, Salmonella Paratyphi, Serratia marcescens, Proteus mirabilis, Enterobacter aerogenes, Citrobacter freundii and Morganella morganii were obtained from patients and were identified by microbiological methods. Diffusion disk was used for evaluation of antibacterial properties in comparison with selected ordinary antibiotics. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for tested materials were determined using MTT in the Micro Broth dilution method. RESULTS: The results proved that effect of carrier on studied isolates is dependent on concentration level. The inhibitory effect of carrier in concentration of 15 µg/ml by 18 mm zone of inhibition for Klebsiella pneumoniae was comparable to Ceftazidime and Cefalothin. The lowest MIC and MBC determined by the Microbroth dilution method with MTT belonged to Klebsiella pneumoniae as 0.1 and 3 µg/ml and higher concentrations belonged to Enterobacter aerogenes at 7 and 15 µg/ml. The greatest effect of carrier of Aleo vera aquatic extract was observed for Klebsiella pneumoniae and the lowest effect belonged to Enterobacter aerogenes, Citrobacter freundii and Morganella morganii. CONCLUSION: It was concluded that the carrier of Aloe vera produced in microemulsion system was most effective and had equal effects in comparison with ordinary antibiotics against Enterobacteriacea.

Association of rs1042522 SNP with Clinicopathologic Factors of Breast Cancer Patients in the Markazi Province of Iran.

BACKGROUND: The nucleotide changes in different genetic loci increased the incidence risk of breast cancer. AIM: The aim of present study was to investigate genotype distribution at codon 72 of the TP53 gene (rs1042522) in breast cancer patients to achieve a potential diagnostic marker related to some demographic feathers. METHODS: In our case-control study, blood samples were collected from a total of 34 patients harboured breast cancer. DNA was extracted, and nested-PCR was performed. Products were digested with AccII and subsequently were sequenced. Results were compared with samples characteristics. RESULTS: The PCR results indicated the correct implementation of extraction and amplification protocol. The genotypic distribution at codon 72 of TP53 in control group was 20%, 62.4% and 16.6% for Arg (wildtype), Arg/Pro (heterozygous) and Pro (homozygous variant) respectively. Also, this distribution in the patient group was 23.52% homozygous, 50% heterozygous, and 26.47% another homozygous variant (Adjusted odds ratio: 1.12 and 95%CI = 0.57 to 2.2, P = 0.03). The absence of Arg at codon 72 of TP53 is relevant with age higher than 40 years and metastasis to other organs. CONCLUSION: Polymorphism at codon 72 of TP53 was associated with high-grades of breast cancer risk and different responses to chemotherapy treatment. It is recommended genotype distribution of codon 72 of TP53 before chemotherapy.

Novel Mutations in pncA Gene of Pyrazinamide Resistant Clinical Isolates of Mycobacterium tuberculosis.

In clinical isolates of Mycobacterium tuberculosis (MTB), resistance to pyrazinamide occurs by mutations in any positions of the pncA gene (NC_000962.3) especially in nucleotides 359 and 374. In this study we examined the pncA gene sequence in clinical isolates of MTB. Genomic DNA of 33 clinical isolates of MTB was extracted by the Chelex100 method. The polymerase chain reactions (PCR) were performed using specific primers for amplification of 744 bp amplicon comprising the coding sequences (CDS) of the pncA gene. PCR products were sequenced by an automated sequencing Bioscience system. Additionally, semi Nested-allele specific (sNASP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were carried out for verification of probable mutations in nucleotides 359 and 374. Sequencing results showed that from 33 MTB clinical isolates, nine pyrazinamide-resistant isolates have mutations. Furthermore, no mutation was detected in 24 susceptible strains in the entire 561 bp of the pncA gene. Moreover, new mutations of G→A at position 3 of the pncA gene were identified in some of the resistant isolates. Results showed that the sNASP method could detect mutations in nucleotide 359 and 374 of the pncA gene, but the PCR-RFLP method by the SacII enzyme could not detect these mutations. In conclusion, the identification of new mutations in the pncA gene confirmed the probable occurrence of mutations in any nucleotides of the pncA gene sequence in resistant isolates of MTB.

Evaluation of Antioxidant Activity and Growth Control Properties of Nonoscale Structure Produced from Aloe vera var. littoralis Extract on Clinical Isolates of Salmonella.

The aim of the study was to examine antibacterial properties of microemulsion structure produced from Aloe vera var. littoralis extract as a new tool of nanoscale drug-like materials. Aloe vera var. littoralis (A. littoralis) extract was prepared by distillation method. A nonocarrier structure in the microemulsion system was prepared from the extract. Serial concentrations were prepared from 8 mg/mL extract and the nonocarrier containing 0.1 mg/mL pure extract and were evaluated by a disk diffusion method for 35 Salmonella clinical isolates. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by microbroth dilution assay using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method by an enzyme-linked immunosorbent assay(ELISA) Microplate Reader apparatus. Antioxidant activity of the extract was determined by measuring the ferric reducing ability of plasma (FRAP) assay. From 35 clinical isolates of Salmonella, 17 isolates-including resistant isolates of S.E.1103 and S.E.49-had a zone of inhibition (ZI) of 7 to 32 mm in 0.007 mg/mL of the extract. S.E.76 isolate exposed to 30 µg/mL ceftazidime disk had a ZI of 12 mm but had 10 mm in 7µg/mL of A. littoralis extract. The inhibitory effect of a nanocarrier at a concentration of 25 µg/mL by 20 mm ZI was comparable by the ceftazidime (30 µg/mL) effect. MIC50 was 0.25 mg/mL and MBC50 was 0.5 mg/mL by MTT method for the extract. It was shown that A.littoralis extract had antioxidant activity of 31.67 µM/mg that could be increased based on concentration. It was concluded that the nanocarrier had a significant effect on the studied isolates in comparison with ordinary antibiotics and had potential for use as a natural antioxidant and antimicrobial material in complementary medicine.

Transmission Electron Microscopy of XDR Mycobacterium tuberculosis Isolates Grown on High Dose of Ofloxacin.

The aim of the study was to investigate behavior of resistant Mycobacterium tuberculosis (MTB) isolates under a high dose of ofloxacin and its morphological changes. 19 extensively drug resistant (XDR) clinical isolates of MTB were grown on Löwenstein-Jensen medium containing progressively increasing concentrations of ofloxacin (2, 4, 8, 16, 32 mg/L). Ultra-structure analyses of resistant isolates grown on ofloxacin were conducted by transmission electron microscopy (TEM). Fixation was carried out by 4% glutaraldehyde in 0.1 M sodium cacodylate buffer on 300 mesh carbon formvar copper grid. The samples were negatively stained with uranium acetate suspension. All19XDRMTBisolatesweregrownandformedcoloniessuccessfullyon2,4,8mg/L,sevenisolates on16mg/L,andfourisolateson32mg/Lofloxacin. Morphologicalchangesandunusualformswere detected in 8, 16 and 32 mg/L ofloxacin at 43%, 76.5% and 81% of cells, respectively. Swollen form (protoplast like), ghost-like cell, degraded forms, and in a few cases, detached cytoplasm from cell wall were clearly detected in high drug concentrations in comparison to control. Changes in morphology were increased with increasing ofloxacin concentrations (p < 0.05). Some XDR isolates could be successfully grown on high doses of ofloxacin (32 mg/L), but with changes in morphology. It was concluded that several magnitudes of the drug doses could not prevent growth of drug resistant forms.

trfA1 Gene in clinical isolates of Mycobacterium tuberculosis.

OBJECTIVE/BACKGROUND: The incidence of resistant strains of Mycobacterium tuberculosis (MTB), including multi-drug resistant, extensively drug resistant, or totally drug resistant, is one of the major problems of health policies worldwide. The accumulation of mutations causes multi-drug resistant strains. Mycobacterium abscessus has a plasmid called pMab2401 containing the trfA1 gene in its integron part. The aim of the present study was to investigate the possible existence of the trfA1 gene in clinical strains of MTB for the first time. METHODS: Bioinformatics analysis in GenBank revealed an absence of any integrons or internal components in MTB. Several specific primers for different genes and the trfA1 gene of M. abscessus were used in a touch-down (60-52) amplification program and followed by loading on gel electrophoresis. Products were extracted and were sequenced. Sequencing results were analyzed carefully and compared with those in the databank. RESULTS: Bands of 500bp were resulted with pair primers in an amplification reaction by clinical MTB that has 94% identity with a fragment in most plasmids and phages M13. It should be noted that such an independent replication system-like structure has not been reported to date in MTB strains. CONCLUSION: The trfA1 gene is depends on the replication process. It is necessary to investigate other probable new areas that may be of concern with drug resistance in clinical isolates of MTB.

The Optimization of Molecular Detection of Clinical Isolates of Brucella in Blood Cultures by eryD Transcriptase Gene for Confirmation of Culture-Negative Samples.

BACKGROUND: Brucellosis is a zoonosis disease which is widespread across the world. OBJECTIVES: The aim of the present study is the evaluation of culture-negative blood samples. MATERIALS AND METHODS: A total of 100 patients with suspected brucellosis were included in this experimental study and given positive serological tests. Diagnosis was performed on patients with clinical symptoms of the disease, followed by the detection of a titer that was equal to or more than 1:160 (in endemic areas) by the standard tube agglutination method. Blood samples were cultured by a BACTEC 9050 system, and subsequently by Brucella agar. At the same time, DNA from all blood samples was extracted by Qiagen Kit Company (Qia Amp Mini Kit). A molecular assay of blood samples was carried out by detection of eryD transcriptase and bcsp 31 genes in specific double PCR reactions. The specificity of the primers was evaluated by DNA from pure and approved Brucella colonies found in the blood samples, by DNA from other bacteria, and by ordinary PCR. DNA extraction from the pure colonies was carried out by both Qiagen Kit and Chelex 100 methods; the two were compared. RESULTS: 39 cases (39%) had positive results when tested by the BACTEC system, and 61 cases (61%) became negative. 23 culture-positive blood samples were randomly selected for PCR reactions; all showed 491 bp for the eryD gene and 223 bp for the bcsp 31 gene. Interestingly, out of 14 culture-negative blood samples, 13 cases showed positive bonds in PCR. The specificity of the PCR method was equal to 100%. DNA extraction from pure cultures was done by both Chelex 100 and Qiagen Kit; these showed the same results for all samples. CONCLUSIONS: The results prove that the presented double PCR method could be used to detect positive cases from culture-negative blood samples. The Chelex 100 method is simpler and safer than the use of Qiagen Kit for DNA extraction.

Molecular detection of fluoroquinolone resistance-associated gyrA mutations in ofloxacin-resistant clinical isolates of Mycobacterium tuberculosis from Iran and Belarus.

OBJECTIVE/BACKGROUND: Detection of mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene could determine resistance to fluoroquinolone antituberculosis drugs. The aim of this study was to detect mutations in QRDRs. METHODS: From 184 clinical isolates of Mycobacterium tuberculosis, ofloxacin resistance was proven in 42 isolates using the proportion method. The molecular basis of resistance to ofloxacin were investigated by the determination of mutations in the QRDR region of the gyrA gene. Extracted DNA fragments of 194bp from the gyrA gene were amplified and an automatic DNA sequencer was used for the sequencing process. RESULTS: Molecular genetic analysis of 42 resistant M. tuberculosis strains demonstrated that they belong to Principal Genetic Group (PGG) 1 in 19 cases (45.2±10.9%), to PGG2 in 15 cases (35.7±10.5%), and to PGG3 in eight cases (19.0±8.4%). Isolates from PGG1 were dominant among resistant isolates (P<.05). It was found that 24 (57%) resistant isolates carried mutations at codon 94 with five different amino acid changes: D94A (n=11), D94G (n=3), D94T (n=4), D94A (n=4), and D94Y (n=2). The remaining 18 (43%) isolates had mutations in codon A90V (GCG→GTG) and S91P (TCG→CCG). Five isolates had two mutations in codons 90 and 94. There was no difference between mutations at these two codons in resistant isolates of the two countries (P<.001). There was no polymorphism observed in codon 95 in any of the ofloxacin-susceptible isolates. CONCLUSION: It was concluded that the determination of nucleotide sequences of QRDRs can be used as a molecular test for the rapid detection of ofloxacin resistance. Furthermore, frequencies in gyrA codons in Belarus and Iran were similar, therefore it is not of geographical concern for the two countries.

Efficacy of Synbiotics for Treatment of Bacillary Dysentery in Children: A Double-Blind, Randomized, Placebo-Controlled Study.

Bacillary dysentery is a major cause of children's admission to hospitals. To assess the probiotic and prebiotic (synbiotics) effects in children with dysentery in a randomized clinical trial, 200 children with dysentery were studied in 2 groups: the synbiotic group received 1 tablet/day of synbiotic for 3-5 days and the placebo group received placebo tablets (identical tablet form like probiotics). The standard treatment was administered for all patients. Duration of hospitalization, dysentery, fever, and the weight loss were assessed in each group. It was concluded that there was no significant difference in both groups in the baseline characteristics. The mean duration of dysentery reduced (P < 0.05). The mean duration of fever has been significantly reduced in the synbiotic group (1.64 ± 0.87 days) in comparison to the placebo group (2.13 ± 0.94 days) (P < 0.001). Average amount of weight loss was significantly lower in the synbiotic group in comparison to that in the placebo group (129.5 ± 23.388 grams and 278 ± 28.385 grams, resp.; P < 0.001). There was no significant difference in the mean duration of hospitalization in both groups (P > 0.05). The use of synbiotics as an adjuvant therapy to the standard treatment of dysentery significantly reduces the duration of dysentery, fever, and rate of weight losses. The trial is registered with IRCT201109267647N1.