Protective Efficacy of the OprF/OprI/PcrV Recombinant Chimeric Protein Against Pseudomonas aeruginosa in the Burned BALB/c Mouse Model.

BACKGROUND: Pseudomonas aeruginosa infection is the major cause of death in burn patients. Thus, in this study, a chimeric vaccine harboring the OprF185-350-OprI22-83-PcrV was designed and expressed in Escherichia coli. The immunogenicity of the recombinant chimer, OprI, OprF, and PcrV was studied in a burned mouse model. METHODOLOGY: Recombinant proteins including the proposed chimer, OprF, OprI, and PcrV were expressed in the E.coli. Mice were immunized with the purified recombinant proteins, and the antibody titre was estimated in the sera obtained from immunized mice. Immunized and control mice were challenged with 2, 5, and 10xLD50 of the P. aeruginosa strains (PAO1, PAK, and R5), and microbial counts were measured in the skin, liver, spleen, and kidney of the studied mice. RESULTS: Results showed that the antibody titre (total IgG) was significantly increased by injection of 10 µg of chimeric protein in the experimental groups compared to the control groups. The antibody survival titre was high until 235 days after administration of the second booster. The survival rate of the mice infected with 10xLD50 was significantly increased and the number of bacteria was reduced, especially in the internal organs (kidney, spleen, and liver) compared to the mice immunized with any of the OprF, OprI, and PcrV proteins alone. CONCLUSION: The findings of our study revealed that the chimeric protein is a promising vaccine candidate for control of the P. aeruginosa infection.

In-Silico Analysis and Protective Efficacy of the PcrV Recombinant Vaccine against Pseudomonas Aeruginosa in the Burned and PA-Infected BALB/c Mouse Model.

BACKGROUND: Pseudomonas aeruginosa is considered as the most severe cause of infections in burn patients and pneumonia infections. OBJECTIVE: To study the protective effects of recombinant protein vaccine harboring the PcrV of P. aeruginosa in the mouse model of burn and respiratory infections. METHODS: Recombinant protein vaccine harboring the PcrV was expressed in the E. coli BL-21 strain. Mice were immunized with the purified recombinant protein, and the antibody titer was measured in the sera obtained from the immunized mice. Immunized and control mice were challenged by active and passive immunization. The microbial counts in the lung, skin, liver, spleen, and kidney were compared with the control mice. RESULTS: Bioinformatics analysis indicated that the PcrV protein was conserved in 1552 clinical and environmental isolates. Also, the isoelectric point (pI), molecular weight, and Grand Average of Hydropathy (GRAVY) score were analyzed. Mice were injected with recombinant protein, and serum from immunized mice reacted strongly with recombinant antigen at a dilution of 1:64000. The survival rate of mice infected with 5xLD50 of the P. aeruginosa increased significantly up to 75% in the standard strains (PAO1 and PAK), and the number of bacteria, especially in the internal organs (kidney, spleen, and liver) significantly reduced compared to the mice immunized with placebo. CONCLUSION: Our results demonstrated that the PcrV protein could be an effective candidate vaccine for the generation of antibody response against P. aeruginosa infection.

Evaluation of Three Different Laboratory Methods for Identification of Pneumocystis jirovecii Pneumonia (PCP) among HIV Positive Asymptomatic Prisoners.

BACKGROUND: Pneumocystis jirovecii pneumonia (PCP) remains a leading cause of mortality among HIV-infected patients. The aim of study was to find out P. jirovecii in versatile group of HIV-positive patients prisoners. METHODS: Overall, 102 HIV positive patients from Ghezel Hesar Prison, Karaj, Iran from October 2016 to March 2017 without any respiratory symptoms were selected with different medication histories against HIV and PCP. Microscopic and molecular (qualitative real-time PCR) examination were applied on sputum specimens and serological investigation (ß-D-glucan assay for fungal diseases) carried out on patient's sera. RESULTS: Only 3 and 1 patients were positive for PCP by microscopic and molecular testing, respectively. Twenty-four (23.5%) and 78 (76.5%) out of 102 patients were seropositive and seronegative for fungi disease, respectively. Seropositive patients were older than seronegative subjects (P<0.001). Most of seropositive individuals showed less mean value of CD4 counts compared to seronegative group (P<0.001). Of 54 patients who were under HIV therapy, 13 were seropositive compared to 11 out of 24 seropositives who were no adhere to treatment (P<0.001). In terms of prophylactic antibiotic therapy against PCP, of 24 patients who received prophylaxis, 3 (12.5%) and 21 (87.5%) were seropositive and seronegative, respectively (P<0.001). On the contrary, among 78 patients who did not receive prophylaxis, 21 (27%) and 57 (73%) belonged to seropositive and seronegative patients, respectively (P<0.001). CONCLUSION: There was no strong evidence for PCP infection/disease among symptomless, HIV positive patients. According to their mean CD4 counts, the hypothesis for being negative in a majority of applied tests would be the absence of severe immunosuppression in the patients.

The assessment of anti-tumoral activity of polysaccharide extracted from terrestrial filamentous fungus.

Fungal polysaccharides are well-known for the medicinal properties such as antitumor and immunomodulating effects. Hence, this study evaluated antitumor effects of polysaccharide extracted from Fusarium sp. isolated from soil samples of Karaj district, Alborz, Iran along with its taxonomic study. The filamentous fungus strain FK1 was isolated from the soil sample of Karaj, Iran. The strain was identified based on cultural, morphological and 18 S rRNA gene parameters as Fusarium. Further, the strain Fusarium was cultured in fermented broth of modified (PDB) for 10  days at 25 °C. The polysaccharide of strain FK1 was extracted from the mycelium free supernatant by boiling water method and evaluated for antitoxicity effect on two human cancer cell lines: HeLa cell line and Lymphoblastoid cell line (LCL) by MTT method. Findings revealed that water-extracted from mycelia polysaccharide of strain FK1 had the highest cytotoxicity effect against LCL which is the cause of B lymphocyte cancer, at 50  µg/ml concentration dose (114 ± 1.63) followed by 100  µg/ml (105 ± 0.57) and 10  µg/ml (104 ± 0.57), while it did not have a considerable effect on HeLa cell line. Fusarium could be alternative sources as an antitumor component.

Investigation of adherent-invasive E. coli in patients with Crohn's disease.

Background: Crohn's disease and Ulcerative colitis are known as inflammatory bowel disease with high morbidity which are as a result of increasing immune responses to intestinal microbiota in genetically susceptible individuals. The association of adherent invasive Escherichia coli with Crohn's disease in human has been discussed for decades. The principal aim of this study was to assess the relationship between adherent invasive Escherichia coli in Iranian patients with Crohn's disease. Methods: The presence of adherent invasive Escherichia coli DNA and viable adherent invasive Escherichia coli cells were identified through PCR and conventional culture methods, respectively. All the specimens were subsequently cultured in Hi Chrome Agar medium. Results: Using molecular assay, the invasive plasmid antigen H and invasion-association locus genes were detected from tissue samples confirming the presence of adherent-invasive Escherichia coli. The invasive plasmid antigen H was detected in 46.7% of CD and 13.3% of healthy peoples. The invasion-association locus gene was found in 36.7% of patients with Crohn's disease and 10% in individuals without IBD. Conclusion: This study demonstrated an increased frequency of adherent invasive E. coli with invasive plasmid antigen H and invasion-association locus genes from patients with CD in comparison to control individuals. Moreover, it was shown that adherent invasive E. coli with the invasive plasmid antigen H and invasion-association locus genes can act as a predisposing factor in the development of IBD.

RAPD PCR Profile, Antibiotic Resistance, Prevalence of armA Gene, and Detection of KPC Enzyme in Klebsiella pneumoniae Isolates.

The increasing prevalence of multidrug-resistant Klebsiella pneumoniae strains isolated from hospitals shows the limitation of recent antibiotics used for bacterial eradication. In this study, 81 K. pneumoniae isolates were collected from three hospitals in Tehran. Antibiotic susceptibility test showed the highest rates of resistance to cefotaxim (85.5%) and ceftazidime (78.3%), and the lowest rates of resistance were detected for colistin (16.9%), streptomycin (16.8%), and chloroamphenicol (21.7%). Eleven different resistance patterns were observed. Sixty-six out of 81 isolates (81.5%) were found to be multidrug resistant (MDR), and 35.8% of them belonged to A3 resistance pattern. 7.4% and 66.7% were KPC enzyme and armA gene positive, respectively. RAPD PCR assay of these bacteria showed 5 clusters, 16 single types, and 14 common types, and there was not any correlation between genetic patterns of the isolates and presence of resistance agents. Simultaneous detection of resistance-creating agents could be an important challenge for combination therapy of MDR K. pneumoniae-caused infections.

Growth Inhibitory Effect of Lactocare on Vibrio cholerae.

BACKGROUND AND OBJECTIVE: Acute microbial diarrheal diseases are the major public health problems in the developing countries. People affected by diarrheal diseases have the lowest financial resources and poorest hygienic facilities. Children under five, primarily in Asian and African countries, are mostly the subjects affected by microbial diseases transmitted through water.The current study aimed at investigating the comparative inhibitory effect of Lactocare (commercial probiotic) on clinical samples and standard strains of Vibrio cholerae. METHODS: A total of 20 clinical samples and a standard strain (ATCC 14035) were provided by Health Reference Laboratory and Biotechnology Institute, respective ly. In order to confirm the samples, biochemical analysis and the polymerase chain reaction (PCR) were performed on intergenic space. Afterward, agar well diffusion method was performed in order to measure the minimum inhibitory concentration to monitor the antimicrobial activity of Lactocare. RESULTS: Colony count of V. cholerae for the standard strain in 30% and mean for clinical samples in 50% concentration of Lactocare treatment revealed that it would propel to death phase. Since the number of colonies decreased to 100, it was considered that higher concentrations of Lactocare would completely inhibit the growth of V. cholera. CONCLUSION: Probiotics are employed to develop new pharmaceutical preparations and functional foods in order to promote the public health.

High prevalence of OXA-type carbapenemases among Acinetobacter baumannii strains in a teaching hospital of Tehran.

Nosocomial infection caused by carbapenem-resistant Acinetobacter baumannii (CRAB) has created a public health concern all around the world. In this study, 100 isolates of CRAB from hospitalized patients during 2015-2016 at Imam Khomeini Hospital were investigated to determine the rates of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains using Kirby-Bauer disk diffusion method. The minimum inhibitory concentrations (MICs) of six antibiotics were determined by broth microdilution method. Multiplex polymerase chain reaction (PCR) was performed to detect blaOXA-51 like and blaOXA-58 like, blaOXA-23 like, and blaOXA-24 like that are encoding resistance to carbapenems. All CRAB isolates were MDR and XDR and 2% of them were pandrug-resistant (PDR), whereas colistin, polymyxin B, and tigecycline were the most effective agents. All isolates were positive for blaOXA-51 like by PCR. The frequency of blaOXA-23 like and blaOXA-24 like was 81% and 22%, respectively. Findings of this study showed that very few therapeutic options remained for the treatment of CRAB infections and blaOXA-23 like is a dominant resistance gene in CRAB at this hospital.

Isolation of Stenotrophomonas maltophilia from clinical samples: An investigation of patterns motility and production of melanin pigment

Objectives:To investigate possible sources of Stenotrophomonas maltophilia (S.maltophilia) in the clinical environment.Methods:Different samples were collected from Amol City of Iran.Steps for the identification of S.maltophilia included culturing,biochemical tests,polymerase chain reaction (PCR) of 16S rRNA gene and 23S rRNA gene.In addition,production of melanin pigment and patterns of motility of the bacteria,were also investigated.Results:In our study,20 S.maltophilia strains were isolated from clinical sources,oxygen manometer apparatus of hospitals were 7/1 10 (6.36％),blood was 1/777 (0.13％),sputum was 4/40 (4％),urine was 1/2947 (0.03％),tap water was 1/240 (0.42％) and dental suction was 6/120 (5％).The isolated bacteria showed production of melanin pigment with rates of strong,moderate,weak,and lack of pigment.Types of motilities were seen in isolates.Conclusions:The highest percentage of bacteria is isolated of oxygen manometer system and dental suction,yet has not been reported from oxygen manometer system.These bacteria have also been associated with patients who have respiratory problems,so it is essential for staffs of hospitals to draw attention to this source of bacteria.

Microdilution in vitro Antifungal Susceptibility Patterns of Candida Species, From Mild Cutaneous to Bloodstream Infections.

BACKGROUND: Candida species, as opportunistic organisms, can cause various clinical manifestations, ranging from mild cutaneous infections to systemic candidiasis in otherwise healthy individuals. Remarkably, the incidence and mortality rates of candidemia have significantly increased worldwide, even after advances in medical interventions and the development of novel antifungal drugs. OBJECTIVES: Given the possible resistance to antifungal agents, susceptibility testing can be useful in defining the activity spectrum of antifungals and determining the appropriate treatment regime. MATERIALS AND METHODS: The in vitro susceptibilities of molecularly identified Candida strains (n = 150) belonging to seven species recovered from clinical specimens, including vaginal, cutaneous, sputum, bronchoalveolar lavage (BAL), and blood samples, were determined for six antifungal drugs (amphotericin B, fluconazole, itraconazole, voriconazole, posaconazole, and caspofungin), based on the clinical and laboratory standards institute's M27-A3 and M27-S4 documents. RESULTS: Candida albicans was the most frequently isolated species (44.66%), followed by non-albicans Candida, including C. glabrata (20%), C. parapsilosis (13.33%), C. krusei (8%), C. tropicalis (7.3%), C. dubliniensis (4%), and C. africana (3.33%). Posaconazole had the lowest geometric mean minimum inhibitory concentration (MIC) (0.0122 µg/ml), followed by amphotericin B (0.0217 µg/mL), voriconazole (0.1022 µg/mL), itraconazole (0.1612 µg/mL), caspofungin (0.2525 µg/mL), and fluconazole (0.4874 µg/mL) against all isolated Candida species. Candida africana and C. parapsilosis were significantly more susceptible to fluconazole, compared to C. albicans and other Candida species (P < 0.001). However, their clinical effectiveness in the treatment of Candida infections remains to be determined. CONCLUSIONS: These findings highlight the importance of precise and correct species identification of clinical yeast isolates via molecular approaches, and of monitoring the antifungal susceptibility of Candida species recovered from clinical sources. Laboratories should consider routine MIC testing of C. glabrata isolates collected from sterile sites. Surveillance studies of Candida species and new analyses of antifungal treatment outcomes will allow more informed determinations of the value of these drugs in the antifungal armamentarium.

Effect of polymer/nanosilver composite packaging on long-term microbiological status of Iranian saffron (Crocus sativus L.).

Crocus sativus L. (saffron) is a valuable plant which is native to Iran. Saffron is the dried stigmata of the flowering part of the plant that is usually contaminated with different bacteria and fungi through production process. Antimicrobial properties of silver nanoparticles are well recognized. To survey the effects of nanosilver packaging on microbiological status of spiked, saffron samples over a six month period were chosen. Saffron samples from five regions of Khorasan province were purchased and de novo frequencies of microbial contaminants were determined using standard procedures. Totally 35 g of saffron was spiked with known numbers of four bacterial and two fungal species and packaged into one gram packets. The packaging materials consisted of polyethylene polymers containing 0, 400, 800, 1200 or 4000 ppm nanosilver (as Ag). Total and differential numbers of spiked microorganisms in the packaged saffrons were enumerated at initial and at six time points of seven, 14, 28, 64, 90 and 180 days. Baird-Parker agar (BP agar), Kenner Fecal (KF), Salmonella-Shigella agar (SS agar), Violet Red Bile Glucose Agar (VRBGA), and Sabouraud Dextrose agar (SD agar) media were used for enumeration of the six spiked microorganisms including Staphylococcus aureus, Enterococcus faecalis, Salmonella Enteritidis, Enterobacter species and Escherichia coli, Fusarium oxysporum and Aspergillus flavus, respectively. Direct antibacterial activity of the composites was also determined. De novo frequencies of microorganisms in five saffron samples were at acceptable levels with dominance of fungi species. Nanosilver embedded packages accelerated the reduction in live microbial numbers in saffron samples and the efficacy was the best in packages containing 4000 ppm nanosilver particles. Nanosilver packaging can significantly reduce microbial burden of saffron.

Identification of Isolated Salmonella enterica Serotype gallinarum Biotype Pullorum and Gallinarum by PCR-RFLP.

BACKGROUND: Salmonella spp. is the major bacterial pathogen in poultry and is responsible for significant economic losses of the poultry industry in many parts of the world. Among Salmonella spp., Salmonella gallinarum and Salmonella. pullorum are the most common causative agents of chicken salmonellosis resulting in high mortality and morbidity. OBJECTIVES: The aim of this study was to identify S. gallinarum and S. pullorum by using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. MATERIALS AND METHODS: In this study, 13 samples of Salmonella, isolated from local poultry, were obtained from Razi Type Culture Collection (RTCC). For the PCR-RFLP method based on the fliC gene, extracted DNA was used as a template for amplifying of the fliC gene (197bp) using specific primers. PCR products were subjected to digestion using Hinp1I restriction endonuclease. RESULTS: For the PCR, 197 bp fliC fragment was amplified from all 13 isolates. Ten out of 13 were S. gallinarum and the other three were S. pullorum. As part of the PCR-RFLP, two fragments were obtained (82 bp and 115 bp) for all S. gallinarum, whereas no digestion was observed in S. pullorum, and 197 bp fragment was seen. CONCLUSIONS: PCR-RFLP with fliC gene and Hinp1I endonuclease were successfully applied to differentiate the two biotypes. The results suggested that this technique could be effective in detecting S. gallinarum and S. pullorum.

Comparison of the in vitro Effect of Chemical and Herbal Mouthwashes on Candida albicans.

BACKGROUND: During the recent decades research has focused to find scientific evidence for the effects of herbal medicines. Researchers are interested in herbal remedies for medication and aim to substitute herbal material instead of chemical formula with limited side effects for human being. OBJECTIVES: The aim of the current study was to compare the in vitro effect of herbal and chemical mouthwashes against Candida albicans. MATERIALS AND METHODS: In this research, we used a standard strain of C. albicans, PTCC 5027. The suspension was made by a fresh culture of C. albicans (24 hours) and the optical density (turbidity equating to a McFarland standard of 0.5) was read at 530 nm. The C. albicans suspension was cultured on Sabouraud dextrose agar plate. Next, two wells were filled with mouthwashes and after incubation at 30ºC for 24 hours, the inhibition zone was measured. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of mouthwashes were determined. Data were analyzed using the SPSS software, independent T-tests and one-sided variance analysis (ANOVA-one way). RESULTS: Based on these findings on agar diffusion with (P = 0.764), MIC and MFC tests (P = 0.879), there were no significant differences between the antifungal effect of herbal and chemical mouthwashes. CONCLUSIONS: This study showed that, chemical mouthwashes acted better than herbal mouthwashes and among different chemical mouthwashes, Oral B was most effective.

Evaluation of immunological parameters in purified protein derivative positive tuberculin workers.

OBJECTIVE(S): According to the occupationally risk of infection in staff workers who have direct contact with mycobacterium species, we investigated their immunological parameters and compared with healthy purified protein derivative (PPD) negative volunteers. Materials and Methods : We investigated 20 PPD positive volunteers working at Tuberculin Unit of Razi Vaccine and Serum Research Institute and PPD negative healthy controls with no exposure or history of active tuberculosis. The percentages of circulating lymphocyte subpopulations were detected by flowcytometry. IL-4 and IFN-γ production levels were measured by ELISA in supernatants of PPD-stimulated peripheral blood mononuclear cells (PBMCs) culture. Results : Tuberculin workers showed an increase in IFN-γ level and significant decrease of CD4+ T cells percentage and CD4/CD8 ratio compared to PPD negative normal individuals. However the IL-4 production and percentage of other lymphocyte population has been unchanged. DISCUSSION: These observations suggest that the immunological parameters of tuberculin workers with PPD positive reaction, who are occupationally exposed to mycobacterium antigens, could be changed. Future studies will be directed towards cytokine networking and regulatory lymphocytes, which will help us validate the significant data presented in this study.

Genetic variability of citrinin-producing Penicillium citrinum strains as occupational health hazards in northern Iran.

We evaluated the ability of randomly amplified polymorphic DNA (RAPD) to type citrinin-producing Penicillium citrinum (P. citrinum) strains recovered from the forest's air in northern Iran. A total of 12 P. citrinum strains (P1-P12) were characterised by citrinin production and random amplification of polymorphic DNA (RAPD) technique. All the strains produced citrinin with levels ranging from 1.5 µg mL(-1) to 39.6 µg mL(-1) (average value: 12.68 µg mL(-1)). Of 11 primers tested, eight primers produced polymorphic amplification patterns. These primers generated a total of 105 reproducible RAPD bands, averaging to 13.1 bands per primer. Dendrogram for each primer indicating the distance of the strains to each other was constructed. RAPD results showed that the collected strains constituted four different clusters. The first cluster included two isolates (P1 and P3). The second cluster included seven isolates (P2, P4, P5, P6, P7, P8, and P10). The third and fourth clusters included one isolate (P9) and two isolates (P11 and P12), respectively. We concluded that RAPD analysis might be used in providing genotypic characters for toxigenic P. citrinum strains typing in epidemiological investigations and public health related risk assessment.

Molecular typing of Epidermophyton floccosum isolated from patients with dermatophytosis by RAPD-PCR.

We evaluated the ability of randomly amplified polymorphic DNA (RAPD) to type Epidermophyton floccosum isolates recovered from patients with dermatophytosis originating from different regions of Iran. A total of 13 clinical isolates of E. floccosum obtained from Iranian patients were analyzed by RAPD with 7 arbitrary primers (OPN16, OPD18' OPU15, OPX19, R28, OPA04 and OPAA17). Among the applied primers, OPN16 produced banding patterns from all the isolates. In addition, some of the isolates had very close relation. The phenon line which represented the mean similarities was at the value of 73%. At this level, 4 groups were characterized. Two isolates of a patient had different molecular patterns, suggesting infection transmission from different sources in the case of a single patient. RAPD-PCR provided a rapid and practical tool for identification of E. floccosum isolates, which was independent of morphological characteristics, and enhanced laboratory diagnosis of dermatophytosis.