دو ماهنامه میکروب شناسی پزشکی ایران
سال سیزدهم شماره 1 (فروردین و اردیبهشت 1398)

Fermented foods containing probiotic bacteria have been used for many years to improve the health or treatment of some diseases. Nowadays, the Therapeutic properties of probiotics are becoming more and more obvious to everyone, insofar as in recent years, more attention has been paid to the potential relation between gut microbiota and mental health. Several studies have shown that intestinal microbiota play some roles in development and function of brain as well as psychiatric parameters such as sleep, appetite, mood and perception through production of active neural molecules. These findings, in turn had led to further research on new therapies for the resolution of mental disorders through changing and modification of intestinal microflora using a new therapeutic group called psychobiotics. Psychobiotics are probiotic bacteria that if consumed in adequate dosage, affect the function of the intestines and brain and reduce the symptoms of psychiatric illness.

Candida albicans as an opportunistic fungal pathogen in human is the cause of volvovaginitis candidiasis. Azole resistance is cinsiderable as worldwide problem in treatment of candidiasis. Azole resistance can occur through different mechanisms such as mutation in ERG11 gene. The aim of our study was evaluation of ERG11 gene mutations in fluconazole resistant islotes of C. albicans obtained from patients with volvovaginitis in west of Mazandaran. In this study, clinical specimens were obtained from vaginal mucosa of 120 individual. C. albicans isolates were identified by standard methods such as germ tubes and culture in chrome agar media. Susceptibility test to fluconazole in the isolates was evaluated by Disc diffusion and MIC methods. After DNA extraction, ERG11 gene mutations in clinical isolates were determined by PCR- sequencing method. From 45 isolates of C. albicans, 40 isolates were resistant to fluconazole. The MIC of fluconazole in isolates was determined between 2 to 64μg/ml. Also, sequencing analysis showed that 7 fluconazole resistant isolates had three missense mutations (D116E, K128T and A114S) in ERG11 gene. It apears that high frequency of floconazol resistance is the results of different reasons such as mutations in ERG11 gene in C. albicans isolates.

High prevalence of Methicillin Resistant Staphylococcus Aureus isolates (MRSA) as well as the multi-drug resistance in this bacterium causes difficulties in the treatment of infections due to these bacteria. Hence, detection of MRSA isolates by rapid and accurate methods is necessary. PCR-ELISA is an accurate and molecular technique that is used for the detection of several pathogens. The aim of this study is the detection of MRSA using PCR-ELISA. Specific primers for mecA gene were designed. Then, dNTP labeled with Digoxigenin was applied for amplifying mecA gene. DIG-labeled PCR products were seeded on the well coated streptoavidin and identified by anti-DIG–peroxidase conjugate. Furthermore, Biotin-labeled DNA probe specific for mecA gene was used. Sensitivity and specificity of the method was determined. Resistance to methicillin among 70 clinical isolates was determined by the disk diffusion, agar dilution and PCR-ELISA methods. MecA gene of S. aureus was amplified using gene specific primers resulted in a fragment with 310 bp length. Findings from the PCR-ELISA technic showed no cross-reactivity with Klebsiella Pneumoniae, Bacillus subtilis and Esheriashia coli as control bacteria and its sensitivity was 0.5 ng. The prevalence of MRSA clinical isolates by the disk diffusion, agar dilution and PCR-ELISA methods was 60%, 58.5% and 60%, respectively. The PCR-ELISA technique was known as an accurate and rapid test for the detection of infection agents using their specific gene. This technic can applied as an appropriate alternative method for time-consuming, less sensitive and expensive techniques such as Real-time PCR and differential biochemical tests which are currently used in laboratory.

The rate of variation in various genes of a bacterial species is different during evolution. Therefore, in systematic bacterial studies many researchers compare the phylogenetic tree of a particular gene to the standard tree of an rRNA gene. Regarding the importance of 16SrRNA gene and the evolutional process of RecA protein family, we investigated the changes in the selected phylum of bacterial RecA family in comparing with the 16SrRNA gene. For this purpose, sequences of the RecA protein family were extracted from Uniprot database and categorized by using CD-hit algorithm. One species was selected from each category. Then we found 16SrRNA complete sequences for same species. After determining the index, based on the Average Alignment Score (AAS), the 16s-taxonomic tree was obtained. Furthermore, Similar calculations were considered for corresponding RecA proteins phylums. By comparing amount of AAS in 16srRNA phylums and RecA phylums, we observed that the Actinobacteria phylum is the closest to the header phylum in the 16s-taxonomic tree, but the same phylum in the RecA is the most distant to the header phylum; and the position of the cyanobacteria phylum remains the same in both trees, which indicates the least amount of changes in the genus and species of this phylum. The 16s-taxonomic tree which is presented in this study for the first time is different from the available bioinformatics algorithms for tree drawing. Finding the species with the highest and lowest rates of changes, can be a type of prediction method for indicating the reasons why bacteria become resistant to drugs over a long period of time.

Microbial detoxification is one of the methods for eliminating of aflatoxins, including aflatoxin M1. Reports indicate that some strains of lactic acid bacteria family through surface adsorption of aflatoxin in their cellwall can be effective in removing them and as a primer culture. In this study, the ability of Bifidobacterium animalis and Lactobacillus delbrueckii in the adsorption of aflatoxin M1 in skim milk was assessed. For this purpose, about 108 and 109 cfu/ ml of B. animalis (Lactis) and L. delbrueckii (Blegaricus) were inoculated into skim milk without aflatoxin M1. Then, the samples were spiked by aflatoxin M1 in concentrations of 0.25, 0.5 and 0.75 ng/ ml. The concentration of the aflatoxin reside in supernatant of milk samples after different storage times (0.5, 1, 2 and 24 h) and temperatures of 4 and 37°C was measured by ELISA method, and the results were confirmed by HPLC. The results showed that the highest amount of aflatoxin M1 removal was respectively related to B. animalis (60 ± 2.5%) with a concentration of 108 cells/ ml and L. delbrueckii (58.5 ± 2.5%) with a concentration of 109 cells/ ml and a concentration of 0.5 ng/ml poison at 37°C for 30 minutes. By comparing the concentration of both bacteria, it also appeared that the B. animalis concentration at 37°C and L. delbrueckii concentration at 4°C were more effective. Also, the results indicate that the ability of bacteria to reduce the amount of poison in half an hour in milk samples with values of 0.75 ng/ml poison at 4°C and 0.5 ng/ml poison at 37°C is higher; but over time, contaminated milk at a concentration of 0.75 ng/ml poison compared to 0.5 ng/ml poison showed an increased amount of aflatoxin removal. B. animalis and L. delbrueckii can act as two useful probiotics to reduce the harmful effects of aflatoxin M1.

Colorectal cancer is considered to be an important cause of morbidity and mortality worldwide. Anti-cancer properties of probiotic lactic acid bacteria are considerable for treatment of cancers such as colorectal cancer. The aim of this study was to investigate the molecular characterization of probiotic bacterial isolates from collected stool specimens in west of Mazandaran province, in order to confirm the probiotic effect of isolated strains, so to compare their effect on cancer cell lineages, in future studies. In this study, out of 60 stool samples collected from healthy children and adult people, 50 bacterial strains were isolated. Antibiotic susceptibility was performed by 15 antibiotics for 7 strains. Then, pH and bile salts tolerance were measured in the strains. Molecular analysis of these strains was carried out by PCR-sequencing and their results were used to draw a phylogenetic tree. Of the 50 isolates, 7 isolates were lactic acid bacteria and two strains were completely probiotic. These strains were resistance to low pH and 0.3% bile salt. Molecular analysis showed that two strains SE and SG were probiotics related to Lactobacillus fermentum (86% homology) and Lactobacillus rhamnosus (61% homology) family, respectively, which were known as new strains. The results suggested that there are 7 strains with good tolerance in the gastrointestinal tract that have probiotic properties and this is the basis of future studies to evaluate their anti-tumor properties and ultimately oral administration.

The purpose of this study was to investigate the possibility of Listeria monocytogenes entering the VBNC state during the frozen storage and the expression of its pathogenic genes Bacteria in 106 Colony counts of in mid log phase were inoculated into three Culture medium including Normal Saline (NS), BHI Broth and Fish Broth (FB) and kept at -18ºCfor 2 months and examined. Then, bacteria were evaluated on enriched medium BHI agar using culture methods (colony count) over times 4 and 8 hours, 2, 4, 8, 20, 30 and 60 days after the freezing shock using the method of RT-PCR for investigating the expression of 16S rRNA, hly and inlA genes; they were evaluated before and after the freezing shock. This bacterium retained its ability to cultivate until the end of the shock, but reduced its number. Freezing stopped the expression of genes of hly and inlA, as these genes were not expressed in a rich culture medium either. By adding blood to the rich culture medium of this bacterium, only the hemolysin O pathogen gene was expressed. Although freezing does not lead to the introduction of this bacterium into the VBNC state, it is effective as an adverse environmental factor for the bacteria in the expression of its pathogenic genes. Blood and its agents can act as an agent for the induction and clarification of the hly gene, and the expression of pathogenic bacterial genes are independent of each other.

Helicobacter pylori is the main cause of various gastroduodenal diseases. It is estimated that app roximately, more than half of the adult population in developed countries and 90% of people in developing countries infected with H. pylori. H. pylori infection may be related to Genetic of virulence factors and environmental factors. The aim of this study was to assess of frequency cagA and vacA genes of H. pylori isolated from patients with Gastrointestinal Disorders. This cross-sectional descriptive study carried out on 120 patients with gastrointestinal diseases in Gorgan city in 2017. (40 patients of gastric cancer, 40 patients of peptic ulcer, 40 patients of without cancer and ulcer). After genomic DNA extraction PCR was carried out using specific H.pylori primers. Overall, 120 H.pylori strains were isolated. The frequency of cagA was %67.5 in gastric cancer, %60 in peptic ulcer and %45 in patiens without ulcer and gastric cancer. Also frequency of vacA gene was detected %55 in gastric cancer, %40 in peptic ulcer and %27.5 in patiens without ulcer and gastric cancer. Based on our findings it seems that the cag A and vac A genes were virulence among H. pylori isolated from studied patients. The frequency of cagA and vacA genes H. pylori were than in gastric cancer and peptic ulcer patients.