International Journal of Molecular and Clinical Microbiology
Volume:3 Issue: 1, Winter and Spring 2013

New approaches for treatment of infectious diseases in aquatic animals have important roles in aquaculture technology progress. In the present study, In vitro effects of different extracts of propolis, royal jelly and pollen obtained from beehives have been investigated on aquatic pathogenic bacterial isolates. The isolated bacteria identified on the basis of their biochemical properties and sequence alignment of the amplified genome fragments. Antimicrobial activities of ethanol extracts of royal jelly, propolis and pollen, and acetone extract of propolis were determined through well diffusion and microdilution methods. The isolated bacteria identified as Aeromonas and Vibrio spp., based on biochemical characterization. Alignments of the amplified sequences showed most similarites to Vibrio cholerae and Aeromonas hydrophila. The results obtained from antibacterial effects of extracts showed that the acetone extract of propolis as well as the ethanol extract of royal jelly, had the greatest effect on Aeromonas hydrophila (MIC=25 mg ml-1); and the ethanol extracts of pollen and royal jelly as well as the acetone extract of propolis, showed the greatest effect on Vibrio cholerae (MIC=50 mg ml-1). The results of present in vitro study propose the beehive compounds (royal jelly, propolis and pollen) as powerful natural products to control pathogenic bacteria in aquaculture systems.

Aquatics are a source of bioactive compounds that these compounds have different properties such as antimicrobial activity. In this study, antibacterial activity of methanol, chloroform and hexane extracts from body wall, gonad and intestine of sea cucumber Holothuria leucospilota was evaluated against Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus. Bioactive compounds of body wall, gonad and intestine of the sea cucumber Holothuria leucospilota collected from the north coast of the Persian Gulf were extracted using methanol, chloroform and hexane. The antibacterial activity was determined using the serial dilution method. The minimum inhibitory concentration and minimum bactericidal concentration was evaluated by broth micro dilution method. Results demonstrated that the P.aeruginosa was shown to be the most sensitive microorganism. All concentrations of methanol extracts from body wall, gonad and intestine did not show antibacterial activity against B.subtilis and S.aureus. Methanol extracts of gonad and intestine and chloroform extract from body wall showed no antibacterial activity against P. aeruginosa. Hexane extract from gonad had no inhibitory effect on the growth of B. subtilis in any of the concentrations. Other extracts had antibacterial effect in certain concentrations studied. None of the extracts showed any bactericidal effect against B. subtilis. Based on findings of this study, sea cucumber extracts can be considered as a natural antibiotic in the future research.

Bacterial vaginosis or non-specific vaginitis describes the disease caused by a change in the normal Flora of the vagina, which leads to the elimination of Lactobacilli, generating hydrogen peroxide and excess growth of bacteria, particularly anaerobic bacteria. This disease is the most prevalent infection of the female genital tract, and the rate of frequency of anaerobic bacteria, specifically vaginal species of Gardnerella and Mycoplasma, is 100 to 1,000 times higher than that of healthy individuals. To determine the rate of frequency of Gardnerella vaginalis, Mycoplasma genitalium and Neisseria gonorrhoeae, which are present in bacterial vaginosis. samples of vaginal secretions of pregnant women referred to the Women’s Clinic in the Tonekabon Township were obtained. In order to detect the presence of Gardnerella vaginalis, Mycoplasma genitalium and Neisseria gonorrhoeae, the samples were studied using the Polymerase Chain Reaction (PCR) method. After obtaining the data, the results were analysed using the Chi-square (x2) test. Of the 44 samples tested, 3 cases were found to contain Gardnerella vaginalis (6.81 percent), 2 cases to contain Neisseria gonorrhoeae (4.54 percent), and 10 cases to contain Mycoplasma genitalium (22.72 percent). Statistical analysis showed that Mycoplasma genitalium was significantly related to the consequence of abortion. However, there was no relationship between infections caused by Gardernerella vaginalis, Mycoplasma genitalium and Neisseria gonorrhoeae with premature delivery and hospitalization of the newborn in the Neonatal Intensive Care Unit (NICU). Considering the findings, it seems that a low percentage of the studied populations were afflicted by the bacterial vaginosis.

Viruses are the most common causes of aseptic meningitis. Early detection, treatment and management of viral meningitis are priority. This study aimed to evaluate common viral meningitis in children referred to Taleghani pediatrics hospital in Gorgan, south east of Caspian Sea, Iran. In this descriptive study CSF and blood samples were taken from 40 children with negative bacterial culture who were referred with meningitis symptoms since Jun 2008 till Sep 2010. Samples were used for viral, biochemical and cytological assays. DNA extraction was done by high pure viral nucleic acid kit of viral nucleic acid from CSF. PCR and Real-time PCR were performed for detection of viruses. Demographic, clinical, biochemical and cytological data were collected and entered in SPSS version 18. All cases with p<0.05 were considered as significant. In overall 12 (30%) viruses were detected by distribution of 5 (41/7%) Enterovirus, 4 (33/3%) Herpes simplex virus-1 (HSV-1) and 3 (25%) Mumps virus. Patients aged between 1 month to 10 years old with mean of 3 years old of which 92/5% were living in urban area. All positive cases showed fever and CSF Pleosytosis with no bacterial growth, gram staining and urinary tract infection. In conclusion, the results showed that clinical and biochemical analyses are not sufficient for certain diagnosis of meningitis in children and molecular assay is recommended to apply for early detection, treatment and management of viral meningitis.

The ability of uropathogenic E. coli (UPEC) to cause symptomatic UTI is associated with the expression of a variety of virulence factors. Biofilm formation enables UPEC to resist the flow of urine and increases its tolerance to antimicrobials and the host immune response. The measurement of biofilm formation in vitro is affected by the type of culture medium used. The aim of this study was to evaluate biofilm-formation capabilities of UPEC in microtiter plate using two different culture media. A total of 170 isolates of E. coli were isolated from patients with symptomatic UTI in Gorgan, north of Iran. Biofilm formation of the strains was examined in LB and BHI broth with the addition of 1% sucrose. The quantitative analysis of biofilm formation was performed using crystal violet staining followed by spectrophotometry measurement after addition of decoloring solution. The biofilm formation of UPEC isolates in LB broth (20 isolates; 11.8%) was significantly (p <0.001) lower than those grown in BHI broth (105 isolates; 61.8%). All isolates that formed biofilm in LB broth also formed biofilm in BHI broth. Whilst 36 (21.2%) isolates grown in BHI broth formed strong biofilm, only one (0.6%) isolate grown in LB broth exhibited a similar result (P<0.007). Our data suggest that the process of biofilm formation by UPEC is strongly modulated by culture conditions and the method employed. In our study the use of BHI broth supplemented with 1% sucrose proved to be superior to the LB broth and can be employed for measurement of biofilm formation in UPEC.

Fusarium is a fungus that is commonly found as saprophytes with parasitic life on some living organisms. Mycoprotein or fungus proteins with physical and chemical features are known as food in recent years. When they are involved in temperature and wet stress, release materials such as mycotoxins or fungus toxins remained in food. Zearalenone and dependent compounds have distinct role in many fungus toxicity and beings especially despite acute toxicity. Also the global allowed rate of zearalenone in foods is 30-1000 nano gr/ppb. The submerged culture environment to produce fungus biomass was Vogel basic medium.Then, the produced fungus biomasses were harvested at 4500rpm for about 20 minutes by centrifugation, washed and rinsed twice and desiccated overnight in room temperature. The dry weight biomass was measured and used in order to measure protein rate extracts for toxin estimating based on producer instructions. Then, 50ml substrate was added and after 5 minutes stopper enzyme was added. Finally, the zearalenone toxin amount was measured by ELISA reader system. The results show that after nitrogen resource, the carbon resource play the second role in production and increase of toxin rate in mycoprotein biomass extracts. Verifying the optimum submerged environment in which all optimal conditions of each steps were applied (the first priority of optimal environment include 0.25% of Starch originated carbon resource and 0.25% of Urea originated nitrogen resource) showed the rate of protein production was 0.642% that is have more increased about 0.207% in relation to basic submerged environment. In this environment the amount of zearalenone toxin was 0.99 ppb/gr. In the other way, when we use 2.64% of Rice bran as replacement carbon resource, using 0.75% of Urea in compare with 0.50% of Soy bean peptone as nitrogen resources we could observe an increase of toxin rate in biomass about 0.66 %. While the amount of Urea reduced to 33% and even we use 0.25% of Starch instead of Rice bran, the toxin in biomass reduced into 71% and reached to 0.99 ppb/gr. When we use 2.30% of Meat peptone, applying 0.50% of Urea in compares with 0.50% of Soy bean peptone increase up to 66% of zearalenone toxin in biomass and reached to 0.77 ppb/gr.

Salmonella enterica is considered as one of the major pathogens in public health, worldwide. Diseases caused by Salmonella enterica serovars are especially prevalent in developing countries. It is one of the emerging pathogen in food-borne diseases, which is often found in contaminated chicken eggs. The aim of this study was to apply a PCR–RFLP method using Sau3AI and HhaI restriction endonucleases on the fliC gene to identify the serotypes of Salmonella isolates in avian living in Arak area in central Iran. Serotypes of 75 isolates included Salmonella enteritidis (45.33%), Salmonella infantis (44%), Salmonella typhimurium (5.33%), Salmonella bardo (2.67%) and Salmonella bacongo (2.67%). All the isolates showed fliC gene (1500bp) by using specific primers. The results of PCR-RFLP with restriction enzymes HhaI and Sau3AI for gene fliC showed 4 and 5 patterns. It was observed that Sau3AI is able to discriminate all serotypes of Salmonella but HhaI could not distinguish Salmonella infantis and Salmonella typhimurium. The prevalent Salmonella isolated in this study belonged to serotype enteritidis and infantis. The combination of the data obtained with both restriction enzymes could differentiate the serovars completely. These results showed that fliC gene is a suitable target gene for discriminating among these Salmonella serotypes by PCR-RFLP.

Atypical Mycobacterium granulomatous skin infections are often accured by Mycobacterium marinum, M. ulcerans, M. fortuitum, and M. avium colonies. Skin infections probably originate from an environmental source such as contacting with aquatic animals, fish farming and swimming in the pools, and inoculate into skin through skin wounds, scratches, trauma, and surgery. The lesions appear as purple papules, nodules in hands and feet, plaque blisters wart ulcers and markers transmission (sporotrichosis) in the path of lymph nodes. They have granulomatous accumulation with giant cells, and abscess pus appears, and sometimes in the form of ulcerative. Infection is limited to the skin, while in immunosuppressed cases it would be able to infect the whole body. To determine if Mycobacteria were present in granulomatose skin lesion, a total of 58 paraffine embedded tissue blocks were obtained and their DNA was extracted. The polymerase chain reaction (PCR) was used to amplify the HSP-65 gene. PCR amplification demonstrated the presence of Mycobacterium spp. In 18 blocks (31%). Among these 18 blocks, 8 (44%) were positive for M. marinum, 3 (17%) for M. ulcerans, 5 (27%) for M. fortuitum and M. chelonae, and 2 (12%) for M. avium. We conclude that Mycobacteria ought to be considered in the treatment of skin granulomas in Iran.

The introduction of newly devised wound dressing has been a major breakthrough in the management of wounds or infections. The aims of this paper are to isolate and identify bacterial species causing burn wound infections from a University-related Iranian hospital as well as determination of the antimicrobial susceptibility of the isolated microorganisms to newly devised nanocomposite materials for developing efficient wound dressing. The NiO nanoparticles were generated in situ and subsequently impregnated on the surface of cotton fabrics using ultrasound irradiation. Then, surface modification was performed to reduce initial bacterial attachment using polyethylene glycol. Cotton fabric was characterized by measuring scanning electron microscope (SEM), X-ray diffraction (XRD) and antibacterial properties. Disk diffusion method was used to quantify the efficacy of NiO-based wound dressing against the most common burn wound pathogen, Pseudomonas aeruginosa, isolated from burns and wound swabs patients of Emam Burn and Accidents hospital in Isfahan province, Iran. All isolates showed high resistance to the commonly used antibiotic (Ampicillin, Gentamicin, Cephalexin, Co-trimoxazole and Amoxicillin). In vitro evaluation showed that the modified cottons exhibited excellent biocidal action against high-resistant isolated Gram-negative bacteria compared to unmodified ones. The results suggested that NiO nanoparticles may be considered as an effective component of therapy for burn infections and in the combination with different antibacterial agents to overcome the resistance of the microorganisms and to obtain synergic antibacterial activity.

Streptococcus iniae is one of the most important agents that causes high mortality and losses in rainbow trout farms in Iran and the world. These bacteria can affects the consumers and people who deal with aquaculture via affected fish. So, viability of this bacterium in frozen fish and fish product can be useful for health care of consumers. A total of 90 rainbow trout, with average weight of 50 ± 3 g, were supplied from a fish farm and were transferred to six 200- liter tanks (15 fish per tank). The tanks were divided in two groups as control and treatment. The treatment group fish were experimentally challenged to Streptococcus iniae at dose of 3.6 × 105 cell/fish via intraperitoneal injection. Control group fish just received 0.1 ml of normal saline (0.9 % NaCl). In treatment group after 120 hours 91% of the fish were died and then mortality remained constant up to 14 days post challenge. Moribund and dead fish of treatment group and fish of control group were frozen and then kidney, liver and brain of fresh dead and frozen fish were used for bacterial culture after 8, 14, 20, 26, 30, 34, 36 and 38 months post freezing, to investigate the viability of these bacteria in different organs at freezing temperature (-20˚C). Streptococcus iniae were re-isolated from all of fresh and frozen tissue specimens within 38 month after freezing. All bacterium species confirmed as Streptococcus iniae using polymerase chain reaction (PCR) method. According to this study this bacterium as a zoonisis can be survived at least 38 month in freezing temperature (at -20˚C) in fish products, so the potential pathogenicity of this bacterium should be concerned in affected area as well as imported frozen fish.