Jundishapur Journal of Microbiology
Volume:6 Issue: 9, Nov 2013

Helicobacter pylori is a human pathogen that causes chronic gastritis, which playsrole in gastric and duodenal ulcers, is also involved in gastric carcinogenesis and may be regarded as a possible important factor in at least a subset of patients with functional dyspepsia.. This study was aimed to construct a recombinant protein containing H. pylori antigenic CagA region and determine its antigenicity as a vaccine candidate against H. pylori.. The antigenic region of CagA gene was detected by bioinformatics techniques. In this study, the H. pylori antigenic CagA region was amplified by PCR and sub-cloned to prokaryotic expression vector pET32a. Escherichia coli BL21 (DE3) pLysS was transformed with pET32a- CagA and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography using Ni-NTA resin. The integrity of the product was confirmed by western-blot analysis using Sera of infected individual. Finally antigenicity was studied by western-blot analysis using human sera infected with H. pylori.. Enzyme digestion analysis, PCR and sequencing showed that the target gene (1245 bp) was correctly inserted into the recombinant vector. The expressed protein was purified using affinity chromatography by Ni-NTA resin. The data also indicated that CagA protein from H. pylori detected from patients'' sera.. Results indicates that antigenic region of recombinant CagA protein were recognized as an antigen, so it might be a candidate for the development of H. pylori vaccine, ELISA kit designs and serological diagnosis of H. pylori infections..

The clinical spectrum of Mycoplasma pneumoniae CNS diseases had a wide range. Encephalitis and meningoencephalitis are the most frequent neurological manifestations, but cases of optic neuritis, transverse myelitis, Guillain-Barré syndrome (GBS), etc. have been reported. To determine the role of M. pneumonia (using PCR and serologically test) in Cerebro Spinal Fluid (CSF) in febrile children with neurologic manifestations (in comparison with normal CSF).. This cross sectional study was done in pediatric wards of Rasoul hospital in Tehran between 2008 - 2010 upon 55 febrile cases with neurological signs and 10 controls with normal CSF (simple febrile seizure). The CSF samples tested for M. pneumonia DNA (PCR); and Immune Globulin G (IgG) level. Chi square values < 0.05 were considered statistically significant.. Positive PCR found in 1 case with Guillan Barre syndrome (1/53; 2%) and none of controls; IgG-CSF level (Cut off 0.0025) had significant difference between cases and controls (Kappa = 0.27, P Value = 0.000). The lowest and highest IgG level observed in aseptic meningitis, and convulsive cases respectively. 73% sensitivity; 90% specificity; 100% Variance (PPV); 28.8% Net Present Value (NPV) determined for IgG-CSF test.. Even, very low amount of CSF-IgG with good specificity (90%); could differentiate cases and controls (P = 0.000). The CSF-IgG test (sensitivity 70%, NPV 28.8%) was weak for ruling out the M. pneumonia in cases. positive PCR was rare (2%) in CSF of cases and controls but is more reliable for diagnosing the recent M. pneumonia infection. We prefer to assay the CSF for both serology and PCR in highly suspicious cases. Anti-microbial or immune modulating therapies had possible benefits..

Klebseilla pneumoniae causes urinary tract infections, nosocomial pneumonia and intra-abdominal infections. Capsular antigens are considered to be the ultimate virulence determinants. Among 77 capsular serotypes of K. pneumoniae, serotypes K1 and K2 are the most virulent ones in humans.. We designed a PCR method for detection of capsular serotypes K1 and K2 of K. pneumoniae using genes cps cluster wzc and orf10 which are required for biosynthesis of capsular polysaccharides in K1 and K2 types, respectively.. We collected 89 K. pneumoniae clinical isolates from the patients of Labafinejad Hospital located in Tehran. Clinical isolates were mostly chosen from urine. We used PCR technique to detect isolates possessing K1 and K2 capsular polysaccharides based on the wzc and orf10 genes which are required for biosynthesis of capsular polysaccharide in K1 and K2 types, respectively. The results of PCR were compared with those obtained by capsular serotyping using Quellung test.. Of 89 isolates of K. pneumoniae tested by both techniques, 10 (11.2%) belonged to K1 and 13 (14.6%) belonged to K2 serotypes, respectively.. We found that these serotypes are probably important in clinical specimens. PCR was a simple and inexpensive tool for identification of K. pneumoniae clinical isolates of K1 and K2 geno-serotypes..

Citric acid is produced in insignificant quantities in Iran. Despite its great range of utilizations, and from another aspect, high level of production of sugarcane bagasse, the related problems arising from maintenance of this acid require thinking of a measure in the direction of its optimal usage and production.. The objective of the present study is to obtain effectual variables in producing citric acid from sugarcane bagasse through Solid State Fermentation (SSF) method using Aspergillus niger mold and to optimize its mass production by employing Taguchi method.. The effective parameters such as spore inoculation level, methanol percentage, solvent type, spore age, humidity percentage, initial pH of substrate, fermentation period and temperature, initial sugar percentage, autoclaving duration, nitrogen source and etc. were studied for producing citric acid from sugarcane bagasse with respect to Tagouchi method.. By considering the findings obtained from the tests, the highest production rate of citric acid g/kg out of untreated sugarcane bagasse is 75.45 based on the consumed sugar and a yield of 15.1 g/kg of sugarcane was achieved per day. Application of sodium hydroxide and acid pretreated sugarcane bagasse increased the production of citric acid in such a fashion that the production rates were 97.81 g/kg and 87.32 g/kg of sugarcane bagasse, respectively, compared to sodium hydroxide and acid untreated sugarcane bagasse.. The obtained findings in the present study indicated that sugarcane bagasse is an ideal substrate in producing citric acid and the aforementioned process could be considered as a beneficial and cost-effective method in citric acid production..

Toxoplasmosis is an opportunistic infection caused by Toxoplasma gondii. That is asymptomatic in the majority of patients, but it can be life threatening in immune-compromised subjects, such as patients with HIV and organ transplant recipients.. This study aimed to determine the Toxoplasmosis in Renal Transplant Recipients in Ahvaz, South-West of Iran.. A total of 100 patients and 100 healthy subjects participated in this study. The specific IgG and IgM antibodies of T. gondii were detected in the two groups by ELISA and the presence of T. gondii in the blood samples was evaluated by GRA6 PCR.. 34 (34%) of the renal transplant recipients and 26 (26%) of the control group were positive for anti-Toxoplasma IgG. In addition 18 (18%) of the renal transplant recipients and 4 (4%) of the control group were positive for anti-Toxoplasma IgM. For both values of the antibodies, differences were statistically significant (P < 0.025 and P < 0.001 respectively). The PCR results indicated the presence of T. gondii parasite in 2 blood samples of recipients, but not in those of the healthy control.. According to the findings of the current study, renal transplant recipients have a higher prevalence of toxoplasmosis than healthy people and also there is disseminated infection in the patients. Therefore to prevent the severe complications screening and follow up of renal transplant recipients for toxoplasmosis should not be neglected..

Highly pathogenic H5N1 influenza virus is causing damages to the poultry industry and is responsible for loss of human lives. Vaccination is the most effective method to prevent and control influenza infections. Recombinant virus-like particles (VLPs) by baculovirus expression vector system have been suggested as a promising platform for new viral vaccines.. In this study we constructed a recombinant baculovirus that was potent to express influenza structural proteins; Haemagglutinin (HA) and Neuraminidase (NA) as well as matrix protein (M1) which is essential to generate immunogenic VLPs in insect cells.. A triplet cassette providing simultaneous and independent expression of HA, NA and M1 genes of avian influenza virus (A/Indonesia/5/05(H5N1)) was designed and subjected to synthesis. The cassette was then cloned into pFastBac1 plasmid and then transformed in to competent Escherichia coli DH10Bac cells and the recombinant bacmids were produced following the homologous Tn7 site-specific transposition. This construction was verified by polymerase chain reaction (PCR) and then transfected into Sf9 insect cells to package new recombinant baculovirus expressing complex proteins of the interest.. Restriction map of pFastBacIHNM1 plasmid confirmed the fidelity of the clone. The PCR carried out on the recombinant bacmids as template indicated that a proper homologous recombination has occurred between pFastBacIHNM1 donor plasmid and the bacmid. Following the transfection of new recombinant bacmids to Sf9 cells, cytopathic effects were observed which indicating the successful packaging of recombinant baculovirus. Protein analysis of the infected Sf9 cells showed that all target proteins were efficiently expressed at the same time.. The recombinant baculovirus constructed in this work has proper characteristics to produce H5N1 influenza virus-like particles in Sf9 cells..

Methicillin Resistant Staphylococcus aureus (MRSA) is a common major human pathogen that causes hospital-acquired infections. Characterization and typing of the staphylococcal cassette chromosome has led to a better understanding of MRSA infection cycle in hospital. The mecA-associated hypervariable region size classifies MRSA isolates that colonized in nasal carriers.. The aim of this study was to compare the genetic background of hypervariable region (HVR) of mecA gene in S. aureus isolated from nasal carriers and clinical samples.. A cross sectional study was performed on 261 nasal swabs collected from healthy health care workers (HCW) and 109 clinical samples from Tehran university hospitals. All the S. aureus isolates were identified by biochemical tests (Coagulase, Catalase, Manitol fermentation, and DNase tests). S. aureus isolates were investigated for the variability of HVR of mecA gene by PCR method.. Among 261 collected nasal swabs, 70 (27%) were S. aureus. Of these, 29(41%) isolates were resistant to Oxacillin and 32 (46%) of those had mec-HVR gene. The polymerase chain reaction (PCR) products showed five different patterns of HVR. Also among 109 clinical samples, 52 (48%) of them were S. aureus. Of these, 40 (77%) were resistant to Oxacillin and 45 (87%) of them carried the mec-HVR gene. The PCR products showed 11 different patterns of HVR.. Molecular typing of MRSA isolates by HVR amplification has shown a high diversity among the strains and can be used as a basis for tracking the contaminations and the source of hospital infections from staff to patients and vice versa..

Continuous researches on drugs that solve the problem of growing number of resistant bacteria are important.. This research aims to investigate the effectiveness of anti-bacterial properties shown by the extracts prepared from different parts of Punica granatum L. (punicaceae), grown in Syria, against Pasteurella haemolytica, which are resistant to all studied antibiotics.. A total number of 504 samples of dead sheep lungs were investigated for detection of P. haemolytica, using blood agar, blue methylene, and biochemical tests (oxidase, catalase, indole, urease). Different parts of P. granatum (pericarp, leaves, flowers, seeds) were extracted by water, absolute alcohol, and ether using soxhlet device and rotary vacuum evaporator. Antibiotic susceptibility testing for P. haemolytica by Kirby-Bauer disk diffusion method was conducted, then the extracts susceptibility test against P. haemolytica was conducted.. P. haemolytica has infected 20.63% of the total number of samples. The alcoholic extracts prepared from different parts of P. granatum showed the high antibacterial effectiveness, the pericarp extract was the best, whereas the water and ether petroleum extracts had no antibacterial effectiveness against resistant P. haemolytica.. Ethanol extracts of P. granatum (pericarp, leaves, flowers, seeds) have antibacterial effects against P. haemolytica which has shown resistance to all studied antibiotics..

Viral meningitis is an inflammation of the leptomeninges as a manifestation of central nervous system (CNS) infection. More than 85% of viral meningitis cases are caused by non-polio enteroviruses. This is the first study on the description of the enteroviruses and the related serotypes involved in viral meningitis in Iran.. This project was conducted to improve our knowledge about the role of enteroviruses and their circulating serotypes in viral meningitis in Iran..Patients and Cerebrospinal fluids from 118 children under 13 years old with primary clinical diagnosis of aseptic meningitis were collected in Children Medical Center in Tehran and sent to Department of Virology of Pasteur Institute of Iran. To investigate the enteroviruses, 5`-noncoding regions were amplified by Real-Time PCR method using Pan-EV primers and a specific probe. Serotype identification in Enterovirus positive samples was conducted by RT-PCR.. Enterovirus detection rate in all 118 Cerebrospinal fluid specimens was 10.16%. Most patients were 0 - 2 years old (40.67%). Serotyping was achieved from 6 specimens with two polio viruses type 1 (vaccine type), two echoviruses 14, one echovirus 5 and one echovirus 30.. Enteroviruses should be considered as the main cause of viral meningitis in Iran. Molecular detections of 5-''NCR and VP1-2A RT-PCR with sequence analysis were found to be better than the conventional methods, for direct diagnosis and EVs typing that may lead to decrease of the unnecessary hospitalizations..

Food born pathogenic bacteria are the most important agents of infections in humans, and food spoilage also results in economic losses in food industry.. The aim of this study was the evaluation of chemical components, total phenolic content, antioxidant and antibacterial activities of Artemisia dracunculus essential oil.. The essential oil of Tarragon was analyzed by gas chromatography-flame ionization detector (GC-FID) and gas chromatography/mass spectrometry (GC-MS). The antioxidant activity and total phenolic content were evaluated by bleaching of β-carotene and folin ciocalteu methods, respectively. The antibacterial effect of the essential oil was inspected on seven Gram- positive and negative bacteria using the microdilution method.. A total of 19 compounds were identified by GC-FID and GC-MS. The main compounds were methyl chavicol (84.83%), trans-ocimene (3.86%), z-β-ocimene (3.42%), limonene (1.79%) and α-pinene (0.57%). Total phenols were 10.16 ± 0.08 mg/g Gallic acid equivalent. The essential oil showed good antioxidant activity in bleaching of β-carotene method (50 ± 1.63%). The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) for essential oil ranged between 3.8 to 250 mg/mL, respectively.. The essential oil of Tarragon might be replaced by synthetic antioxidant and preservatives in food industry..

Bacteroides fragilis are among the most important anaerobic bacteria behind most of the anaerobic infections. They have acquired resistance to essential treatment antibiotics of anaerobic infections, more than other anaerobic bacteria.. The goal of this study is to determine the resistance of isolated B. fragilis against common anaerobic infections treatment antibiotics.. Patients and A total of 188 fecal samples including 59 samples from hospitalized patients, 84 samples from outpatients, and 45 samples from healthy individuals were collected. The samples were cultured in Bacteroides- Bile- Esculine agar and Kanamycin-Vancomycin-Laked Blood media and were incubated in anaerobic atmosphere at 37°C for at least 48 hours. Suspected one millimeter sized colonies with black surroundings, were selected and further studied using MID8, as well as biochemical tests. For MIC determination of antibiotics against isolated B. fragilis, Etest was used.. There wasn’t any difference between antibiotic resistance patterns of isolated B. fragilis from hospitalized patients or outpatients, including diarrheal and non-diarrheal cases, and resistant pattern of isolates from healthy individuals. All or most of isolated B. fragilis were susceptible to imipenem (100%), metronidazole (95%), rifampin (100%) and piperacillin/tazobactam (95%). On the contrary, they exhibited resistance to other antibiotics such as clindamycin (90%), and chloramphenicol (55%).. In the present study, we have figured out that a number of the important anaerobic infections treatment antibiotics have lost partly or totally their effectiveness on B. fragilis..

Dermatophytosis is a cutaneous fungal infection with a worldwide occurrence. In dermatophyte infections, the release of keratinocyte cytokines, in the presence of dermatophyte antigens, causes an acute phase response; subsequently, the acute-phase proteins are produced by hepatocytes. Mannose–binding lectin (MBL), an acute-phase protein, also acts as a kind of pattern recognition receptor. MBL deficiency plays a role in susceptible viral, bacterial, fungal and parasitic infections.. Some research has been conducted on the role of acute-phase proteins in dermatophyte infections. This study has been designed to determine the serum MBL levels in patients with dermatophytosis.. This cross-sectional study, included 96 healthy individuals and 105 patients with dermatophytosis, in access sampling procedure. Microscopic examinations were conducted and cultivated to detect dermatophytes, and in the cases that the identification of different dermatophyte species was necessary, complementary examinations were conducted. Additionally, the enzyme–linked immunosorbent assay (ELISA) was used to determine the serum MBL levels of healthy individuals and patients. Various tests (Chi-square, Fisher exact, Mann - Whitney, Kruskal Wallis, Kendal tau correlation coefficient and ROC curve analysis) were used to examine the relationships between variables, when the P < 0.05 were considered as significant level.. The mean serum MBL level of healthy individuals and patients, was 1.53 ± 1.87 µg/mL and 1.97±2.03 µg/mL (P = 0.039), respectively. Using ROC curve analysis, the MBL level was established as a significant predictor of dermatophytosis (P = 0.042). MBL deficiency (serum level < 1 µg/mL) was more common in healthy group (56.2%) than the patients with dermatophytosis (41.0%).. The findings showed that the increased concentrations of serum MBL in patients with dermatophytosis play a role in this fungal infection. The high frequency of MBL deficiency in healthy individuals was compared with patients indicated that MBL deficiency is not a predisposing factor of this type of infection..

Efficient isolation and detection of low pathogenic avian influenza viruses from surveillance samples continues to be a high priority. Currently, the new cell lines are considered for supporting the replication to high virus strains titers.. The replication efficiency of a low pathogenic avian influenza virus in different origin cells was evaluated under different conditions.. Chicken embryo fibroblast (CEF) cell and human alveolar epithelial cell line (A549) were infected with H9N2 at a multiplicity of infection of 0.1. The amount of infectious virus released into the cell culture supernatants at various post-infection time intervals were tittered by tissue culture infectious dose (TCID50) assay. The impact of these cells adaptation was investigated by determination the virus genes nucleotide sequences.. The influenza virus infectivity was not significant difference in these cells in the presence of trypsin. The results of fusion assay and determination of cellular protease confirmed that A549 cells support virus entry with or without supplemental trypsin. However, the H9N2 virus showed lower titer and infectivity in the trypsin–free infected A549 cells within longer time. The comparative sequence analysis indicated several simultaneously nucleotide substitutions were occurred in NA of the virus replicated in A549 cells resulted in two fixed amino acid changes at positions G320 to A and G414 to A up to the fifth passage.. After seven consecutive passages of both cell cultures, the H9N2 virus showed similar antigenicity, also no change on viral titer level and virus replication behavior in adaptation was found. The results highlighted the use of A549 cells for efficient virus isolation.

The search on natural plant compounds revealed that those compounds can be used for the elimination of oral biofilms as a substitute of current chemical based treatments and is becoming of increasing interest.. The objective of this study was to investigate the biofilm inhibitory effect and disruptive efficacy of Shiitake mushroom (Lentinula edodes) extracts.. Essential oils were extracted by hydrodistillation and the components were analyzed by gas chromatography mass-spectroscopy. Biofilm assessments were carried out on four oral pathogens and \a mixed culture containing Streptococcus mutans, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Minimum inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) were determined on planktonic cells and crystal violet assays performed to assess the biofilm inhibition and disruption amount.. No inhibitory effect was observed on F. nucleatum biofilms (P > 0.05). Significant disruptive properties on A. actinomycetemcomitans at values lower than the MIC (3.90 µg/mL) was detected (P < 0.05). Shiitake mushroom oil had significant inhibitory and disruptive effects on P. gingivalis (P < 0.05). Furthermore, S. mutans and multispecies biofilms were inhibited at the same concentrations (15.60 - 32.25 µg/mL).. Shiitake essential oil is an effective anti-bacterial and anti-biofilm agent on important oral pathogens and has a possible therapeutic potential as an oral anti-biofilm agent..

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) contaminated sediments is an effective remediation technique and its success depends on the optimal condition of PAH-degrading isolates.. The present study was conducted to isolate the PAHs-degrading bacteria from Naybandbay mangrove sediments and to investigate the effect of different variables on phenanthrene (Phe) biodegrading efficiency of the most effective isolated strains, by using response surface methodology (RSM).. Phe degrading bacteria were isolated from surface sediments. Isolated strains were then identified by biochemical and molecular (16S rDNA gene sequence) analysis. RSM was employed to evaluate the optimum biodegradation of Phe by the most effective isolated strain. The investigated parameters included the temperature, inoculum sizes, pH, NH4Cl concentration, and salinity.. One Gram-negative bacterium strain (SBU1) was isolated from enrichment consortium SBU. SBU1 have been identified by 16S rDNA sequence analysis and revealed 96% homology with Roseovarius sp., the biodegradation activity of the SBU1 was properly interpreted using a second-order polynomial regression model. Maximum biodegradation efficiency was predicted at pH = 8.2, temperature≈35˚C, salinity = 30 ppt, NH4Cl concentration = 0.13 g/L and inoculum size = 0.2 OD600nm. Under these conditions the aerobic biodegradation rate reached up to 28.4%.. Indigenous bacteria from mangrove surface sediments of Naybandbay were found to be able to degrade Phe. The similarity of the predicted and observed results confirmed the validity and applicability of RSM in optimization processes.