Antimicrobial and anti-biofilm properties of oleuropein against Escherichia coli and fluconazole-resistant isolates of Candida albicans and Candida glabrata.

BACKGROUND: Side effects associated with antimicrobial drugs, as well as their high cost, have prompted a search for low-cost herbal medicinal substances with fewer side effects. These substances can be used as supplements to medicine or to strengthen their effects. The current study investigated the effect of oleuropein on the inhibition of fungal and bacterial biofilm in-vitro and at the molecular level. MATERIALS AND METHODS: In this experimental study, antimicrobial properties were evaluated using microbroth dilution method. The effect of oleuropein on the formation and eradication of biofilm was assessed on 96-well flat bottom microtiter plates and their effects were observed through scanning electron microscopy (SEM). Its effect on key genes (Hwp1, Als3, Epa1, Epa6, LuxS, Pfs) involved in biofilm formation was investigated using the quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) method. RESULTS: The minimum inhibitory concentration (MIC) and minimum fungicidal/bactericidal concentration (MFC/MBC) for oleuropein were found to be 65 mg/ml and 130 mg/ml, respectively. Oleuropein significantly inhibited biofilm formation at MIC/2 (32.5 mg/ml), MIC/4 (16.25 mg/ml), MIC/8 (8.125 mg/ml) and MIC/16 (4.062 mg/ml) (p < 0.0001). The anti-biofilm effect of oleuropein was confirmed by SEM. RT-qPCR indicated significant down regulation of expression genes involved in biofilm formation in Candida albicans (Hwp1, Als3) and Candida glabrata (Epa1, Epa6) as well as Escherichia coli (LuxS, Pfs) genes after culture with a MIC/2 of oleuropein (p < 0.0001). CONCLUSIONS: The results indicate that oleuropein has antifungal and antibacterial properties that enable it to inhibit or destroy the formation of fungal and bacterial biofilm.

Ulcerative dermatitis in barramundi due to coinfection with Streptococcus iniae and Shewanella algae.

Streptococcus iniae is a pathogenic bacterium which causes septicaemia, while Shewanella algae is an opportunistic pathogen found in marine environments. In this study, we investigated an uncommon coinfection of these 2 bacterial species which resulted in systemic disease and cutaneous ulcers in a barramundi Lates calcarifer farm in the Persian Gulf, Iran. Culture, molecular and histopathological specimens were taken from different organs. In histopathology, results indicated deep bacterial ulceration of skin and subcutaneous muscles. Haemorrhage and hyperaemia were the most common signs observed in visceral organs. In culture, Gram-positive cocci were grown from visceral organs while Gram-negative bacilli were isolated from ulcers. In molecular examination, Streptococcus iniae and Shewanella algae were identified from visceral and ulcer samples, respectively, by PCR of the 16S rRNA gene. The disk diffusion method was used to determine antimicrobial susceptibility of isolated bacteria, with Shewanella algae being resistant to most routinely used antibiotics. In this study, a mixed infection of 2 bacterial species was found; we conclude that systemic streptococcosis could act as a predisposing factor for Shewanella penetration into skin and subsequent ulcer formation. Coinfections are very common in mammals; however, this subject has received little attention in other species, such as fish, and particularly in aquaculture. This study highlights the potential significance of coinfections in barramundi, the effect on the severity of the disease and the potential for new opportunistic pathogens arising.

The inhibitory effects of zinc oxide nanoparticles on clinical isolates of Microsporum canis in dogs and cats.

INTRODUCTION: Microsporum canis is the most common dermatophyte infecting pets and their owners, and its long duration of treatment and increasing rate of drug resistance have caused the attention of researchers to be directed towards the use of nanoparticles and new alternatives for treatment. This study investigated the antifungal effects of zinc oxide (ZnO) nanoparticles on clinical isolates of M. canis in dogs and cats and subtilisin 1 (SUB1) gene expression. MATERIALS AND METHODS: Zinc oxide nanoparticles were prepared using the wet chemical method at a concentration of 4000 ppm. Its antifungal potential was evaluated at concentrations of 62.5-4000 ppm by disk diffusion and microdilution methods against 10 isolates of M. canis. The effect of this product on SUB1 gene expression was investigated by quantitative real-time PCR method. RESULTS: The results of the disk diffusion test showed that the highest inhibitory diameter was at the highest concentration of ZnO nanoparticles (34 mm), and the inhibitory zone was observed in dilutions up to 250 ppm. The minimum inhibitory concentration (MIC) of ZnO nanoparticles was between 250 and 500 ppm, and the minimum fungicidal concentration was between 500 and 1000 ppm. There was a significant reduction in SUB1 gene expression in sub-MIC concentration (125-250 ppm) (p < 0.05). CONCLUSION: This study showed that ZnO nanoparticles have a concentration-dependent inhibitory effect on M. canis. Moreover, ZnO nanoparticles could decrease the expression of SUB1, an enzyme involved in fungi adhesion to the epidermis. Nevertheless, more studies must be done in the future to determine the possible side effects and safety of ZnO nanoparticles along with their efficacy in vivo.

Antifungal susceptibility profile of yeasts isolated from the oral cavity of cats.

BACKGROUND: Microorganisms living in the oral cavity play an important role in health and disease of the host. Cats are susceptible to oral infections, and it is documented that fungi in the oral cavity could impact these infections. Antifungal resistance has been increasing in recent years. OBJECTIVES: This study was designed to identify yeast isolates from the oral cavity of healthy cats and to evaluate their antifungal susceptibility pattern. METHODS: Oral specimens were collected from 60 cats and cultured at 37°C for 10 days. Yeasts were isolated and identified. Their antifungal susceptibility pattern was determined according to CLSI M44-A. RESULTS: Three yeast genera were isolated, including Candida spp (55.5%), Rhodotorula spp (33.3%) and Hanseniaspora spp (11.1%). Antifungal susceptibility profiling showed that, apart from a dose-dependent effect of itraconazole, Hanseniaspora spp was susceptible to all seven drugs studied. The Candida species were susceptible to all drugs except ketoconazole (sensitivity 80%) and caspofungin (sensitivity 40%). In R. glutinis and R. minuta, 100% sensitivity was observed for amphotericin B, posaconazole, ketoconazole and voriconazole. CONCLUSIONS: The results suggest that, in comparison with humans and other animals, cats have a different oral mycoflora in terms of species, number and diversity. However, these isolates have similar susceptibility patterns to those seen in isolates from other animals and humans. More studies should be done to further characterize the oral mycobiota of cats and its role in oral infections.

Descriptive epidemiology of dermatophytosis in rodents.

INTRODUCTION: Dermatophytosis is a zoonotic disease caused by a group of keratinophilic fungi called dermatophytes. OBJECTIVES: Since the epidemiology of diseases revolves over time, this research studies the incidence of dermatophytosis among rodents referred to mycology laboratory during 2019-2021. METHODS: A total of 163 rodents including rabbits, guinea pigs, and hamsters suspecting having dermatophytosis were sampled by scraping lesions. Direct microscopic examination, culture, and polymerase chain reaction were done for diagnosis of dermatophytosis and identification of the etiologic agent. RESULTS: The results of this study showed that 37.4% of rodents were involved with dermatophytosis, among which 41.13% of rabbits, 25% of guinea pigs, and 26.3% of hamsters were included. Microsporum canis (52.7%) was the most isolated agent. Incidence of dermatophytosis was higher in female in rabbits while in hamsters and guinea pigs male were mostly infected. Rodents less than 6 months were more susceptible for dermatophytosis except for hamsters in which 6-12 months animals had a higher prevalence. CONCLUSION: In conclusion, it is significant to update our knowledge about the epidemiology of dermatophytosis in rodents and other animals every few years to define valid preventive strategies. Moreover, since dermatophytes are contagious and zoonotic, it is also a priority to apply preventing methods for dermatophytosis and treat infected rodents with appropriate antifungal agents to decrease the risk of infection.

Effect of yeast probiotic Saccharomyces boulardii cell wall extract on Aspergillus fumigatus allergenicity in A549 cells.

Background and Purpose: Interest in probiotic use for respiratory allergies has increased. In this regard, the present study aimed to evaluate the effect of cell wall extract of Saccharomyces boulardii on Aspergillus fumigatus as an allergenic fungus and its effectiveness in reducing inflammatory cytokines in A549 cells sensitized with A. fumigatus conidia. Materials and Methods: Cell wall of S. boulardii was prepared and challenged by A. fumigatus conidia at various concentrations. Secretory protease activity was tested using the Casein method. The A. fumigatus allergen 1 (Asp f1) gene expression was calculated by quantitative real-time polymerase chain reaction (qRT-PCR). In another experiment, qRT-PCR was used to examine gene expression of interleukin 13 and interleukin 17 by A549 lung epithelial cells exposed to A. fumigatus conidia and treated with different concentrations of S. boulardii cell wall extract. Results: Saccharomyces boulardii cell wall extract significantly reduced the protease activity of A. fumigatus at concentrations of 10 and 20 mg/ml (P<0.05). The Asp f1 gene expression was significantly down-regulated in each concentration of S. boulardii cell wall extract (P<0.05). Aspergillus fumigatus conidia upregulated the expression of IL-13 and IL-17 in A549 cells, and S. boulardii cell wall extract could downregulate the expression of the mentioned cytokines at concentrations of 10 and 20 mg/ml (P<0.05). Conclusion: According to the results, it can be concluded that S. boulardii cell wall extract could be a candidate for IL-13- and IL-17-induced Aspergillus-mediated allergy and asthma therapies. Nevertheless, future studies need to be conducted on the safety of S. boulardii cell wall extract in vivo and its effects on other arms of allergic hypersensitivity.

Efficacy of Syzygium aromaticum essential oil on the growth and enzymatic activity of pathogenic Candida albicans strains.

Background and Purpose: Candida albicans (C. albicans) is the most common human pathogen owing to the most virulence factors. It seems that extracellular hydrolytic enzymes play a key role in C. albicans pathogenicity. The present study aimed to assess the susceptibility and enzymatic activity of pathogenic C. albicans isolates exposed to the Syzygium aromaticum (S. aromaticum) essential oil. Materials and Methods: S. aromaticum oil was characterized using gas chromatography-mass spectrometry (GC-MS). The broth microdilution technique (CLSI, M27-A3) was used to determine the minimum inhibitory concentration (MIC) of test compounds. Furthermore, before and after treatment with S. aromaticum essential oil, the yeasts were analyzed regarding the proteinase (Prz), hemolysin (Hz), and phospholipase (Phz) production/activity. Results: ß-caryophyllene (12.76%) was found to be the major constituent in the essential oil after eugenol (84.64%). Only one isolate of C. albicans showed the antifungal resistance to fluconazole. All isolates were susceptible to S. aromaticum essential oil with MIC of 625-1250 µg/ml. S. aromaticum oil represented the best antifungal effect against C. albicans at MIC 1000 µg/ml. The mean±SD enzyme activity of C. albicans not exposed to S. aromaticum essential oil was obtained at 0.55±0.03, 0.73±0.04, and 0.61±0.05 for proteinase, hemolysin, and phospholipase, respectively. The activities of these enzymes were reduced significantly (P<0.05) to 0.33±0.06, 0.40±0.04, and 0.16±0.03 for phospholipase, proteinase, and hemolysin, respectively, after the yeasts were subjected to S. aromaticum essential oil. Conclusion: The present study aimed to determine the ability of S. aromaticum essential oil to prevent the growth of C. albicans and decrease their enzymatic activity. As a natural antifungal agent, S. aromaticum can be utilized in pharmaceutical systems.

Phylogenetic analysis and genotyping of Iranian infectious haematopoietic necrosis virus (IHNV) of rainbow trout (Oncorhynchus mykiss) based on the glycoprotein gene.

BACKGROUND: Infectious haematopoietic necrosis (IHN) is known as one of the most contagious systemic viral diseases in salmonids which can lead to significant mortality rates and negative impacts on the salmonid farming industry. Infectious haematopoietic necrosis virus (IHNV) was first detected in rainbow trout (Oncorhynchus mykiss) farms in Iran in 2003. OBJECTIVES: We conducted the present study to determine the detection of IHN genotypes in rainbow trout (O. mykiss) in farms in the central parts of Iran, using molecular and phylogenetic techniques. METHODS: Samples were collected from fries exhibiting clinical signs such as darkening of the skin, abdominal swelling, and loss of appetite. Phylogenetic analysis was performed by the neighbour-joining method, using MEGA 5.1 software. For phylogenetic analysis and genotyping of IHNV from central parts of Iran, the sequences of the glycoprotein gene were determined for two Iranian isolates (Jahad-UT1 and Jahad-UT2). RESULTS: Phylogenetic analysis revealed that the detected strains (Jahad-UT1 and Jahad-UT2 isolates) are closely related (97.23%-100%) to European isolates within genogroup 'E'. CONCLUSIONS: This finding indicates that Jahad-UT1 and Jahad-UT2 isolates have been widely transferred to Iran from European countries. Moreover, the nucleotide diversity of these Iranian isolates showed a close relationship with the North American and Asian isolates, although the Iranian isolates were collected from a smaller geographical area and within a shorter time period between 2014 and 2015.

Evaluation of inactivated vaccine of the variant 2 (IS-1494 /GI-23) genotype of avian infectious bronchitis.

The infectious bronchitis virus (IBV) is the cause of avian infectious bronchitis (IB). IB is one of the most highly contagious diseases, which results in many economic losses in the poultry industry worldwide. The nature of this virus is such that it generates new genotypes continuously. Proper vaccination is the most suitable way of combatting IB. One of the novel genotypes of IBV, which has been circulating in the Middle Eastern countries, is the variant 2 (IS-1494/GI-23) genotype. This study aims to design and produce an autogenous variant 2 vaccines. After isolation and characterization of the Iranian variant 2, the inactivated vaccine was formulated according to the OIE guidelines, and its different aspects (Purity, titration, inactivation, immunization) were evaluated. The designed vaccine passed all of OIE quality control standards. In the assessment process, the protection rate in the groups receiving the variant 2 and commercial vaccines was 67 % and 60 %, respectively. Although the differences were not significant, they indicated better protection, and the viral load in the feces and the kidney of the group receiving the variant 2 vaccine was lower than that in the commercial vaccine. It is suggested that the variant2 strain should be added as one of the local strains to the commercial inactivated vaccines in areas affected by this genotype. The use of this vaccine in layer and breeder flocks can help to protect them against variant 2 during the production phase. Also, the transfer of maternal antibodies to offspring can provide strain-specific immunity for one-day-old chickens.

Effect of microencapsulation on Saccharomyces cerevisiae var. boulardii viability in the gastrointestinal tract and level of some blood biochemical factors in wistar rats.

BACKGROUND AND OBJECTIVES: Probiotics are live microorganisms that, when administered in an adequate amount, confer a health benefit on the host through the gut. Saccharomyces cerevisiae is a widespread yeast found in nature. This microorganism has been used as a probiotic agent in recent years. In this study, the effect of microencapsulation on survival rate of S. cerevisiae var. boulardii in the simulated gastrointestinal tract medium and the impact of microencapsulated S. cerevisiae var. boulardii on some serum biochemical factors in a rat model was evaluated. MATERIALS AND METHODS: 30 male wistar rats were divided into three groups (control, rats receiving microencapsulated S. cerevisiae var. boulardii, and rats receiving S. cerevisiae var. boulardii alone). The probiotic was gavaged at a dosage of 2 gr/kg BW for 8 weeks. Blood was collected from rats at the end of the treatment period and biochemical factors were measured using Mancompany kits. RESULTS: The results showed a significant increase in viability of microencapsulated S. cerevisiae var. boulardii in comparison with free S. cerevisiae var. boulardii (p<0.05). Weight of rats in probiotic treated groups was significantly higher in comparison with the control group (p<0.05). Moreover, probiotic treatment reduced mean levels of triglycerides, cholesterol, free blood sugar and liver enzymes in rats. CONCLUSION: Microencapsulation could increase the survival rate of yeast probiotics in the gastrointestinal tract; however, more studies are needed for better understanding of the exact effect of microencapsulation on probiotics' function.

Role of pathogen-associated molecular patterns (PAMPS) in immune responses to fungal infections.

Recent years have seen the rise of invasive fungal infections, which are mostly due to the increase in patients. Three major opportunistic fungal species in human are Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans that pose the biggest concern for these immunocompromised patients' mortality. The growing occurrence of opportunistic fungal infections has sparked the interest to understand defense mechanisms against pathogenic fungi. Toll-like receptors (TLRs), as a part of innate immune system, play an important role for recognizing the invading microorganisms and initiating sufficient immune responses. Recent studies have revealed an integrated role for TLR, signaling inactivating immune defense mechanisms against exact fungi. Among TLRs, TLR2 and TLR4 are the major participants in fungi recognition. The present paper highlights the role of TLR participants in fungal recognition as well as their mechanisms.

The comparison of five low cost liquid formulations to preserve two phosphate solubilizing bacteria from the genera Pseudomonas and Pantoea.

BACKGROUND AND OBJECTIVES: Phosphorus is one of the low bioavailable macroelements. Use of microorganisms in biofertilizers could release phosphorus from insoluble compounds. Pseudomonas putida P13 and Pantoea agglomerans P5 are well recognized for application as phosphate solubilizing bioinoculants and are used as solid carrier based. Liquid bioinoculants are preferred for economizing production process and longer shelf-life. MATERIALS AND METHODS: Five low cost liquid formulations were examined. Formulations 1, 2 and 3, were phosphate buffer, 0.2% and 0.5% KNO3 dissolved in phosphate buffer, respectively. Formulation 4 was nutrient broth containing 4% glycerol and formulation 5 was diluted nutrient broth containing 4% glycerol. Survival (cfu) and phosphate solubilization index (SI) were evaluated after 3 months. RESULTS: Considering strain P5, increase in KNO3 concentration decreased preserving ability. While using KNO3 at 0.2% was accompanied with reaching maximum SI level. Overall, less nutritious formulations (1 and 5) provided maximum preserving ability without bioactivity loss. In the case of strain P13, maximum survival obtained in formulations 2 and 3, whereas SI level decreased. Preserving ability in formulations 1, 4 and 5 was similar but less nutritious formulations (1 and 5), improved bioactivity. CONCLUSION: The results introduced two formulations of 1 and 5 as economically efficient liquid bioinoculants for Pseudomonas putida and Pantoea agglomerans.

Virtual Dual inhibition of COX-2 / 5-LOX enzymes based on binding properties of alpha-amyrins, the anti-inflammatory compound as a promising anti-cancer drug.

Hydro-alcoholic fruit extract of Cordia myxa was considerably effective on curing acute inflammation in mouse model. Previous studies suggested significant anti-inflammatory activities as well as potential anticancer agent of α-amyrins in seeds. Inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipooxygenase (5-LOX) is significant in cancer prevention and therapeutics although this inhibition with chemo-drugs has its own side-effects. It is shown that these enzymes pathways are related to several cancers including colon, breast and lung cancer. This study was conducted based on Cordia species' α-amyrins as a safer natural anti-cancer compound for inhibition of COX-2 and 5-LOX enzymes by molecular docking. The X-ray crystal structure of COX2 / 5-LOX enzymes and α-amyrins was retrieved and energetically minimized respectively. The binding site and surface of enzymes were detected. Docking studies were performed by AutoDock 4.2 using Lamarckian genetic algorithm (LGA). Finally drug likeness, molecular pharmacokinetic properties and toxicity of α-amyrins was calculated. Molecular Docking revealed hydrogen and hydrophobic interactions between α-amyrins with both active sites of COX-2 and 5-LOX enzymes. Interestingly, it covalently bonded to Fe cofactor of 5-LOX enzyme and chelated this molecule. Base on binding energies (∆G) α-amyrin has more inhibitory effects on 5-LOX (-10.45 Kcal/mol) than COX-2 (-8.02 Kcal/mol). Analysis of molecular pharmacokinetic parameters suggested that α-amyrins complied with most sets of Lipinski's rules, and so it could be a suitable ligand for docking studies. Eventually, bioactivity score showed α-amyrins possess considerable biological activities as nuclear receptor, enzyme inhibitor, GPCR and protease inhibitor ligand. These results clearly demonstrate that α-amyrins could act as potential highly selective COX-/5-LOX inhibitor. Also, it is a safe compound in comparison with classical non-steroidal anti-inflammatory drugs (NSAIDs) that are known as cancer preventive agents, since it is free of side effects on human body and it can be a promising drug for cancer therapeutics.

Microencapsulation of Saccharomyces cerevisiae and its evaluation to protect in simulated gastric conditions.

BACKGROUND AND OBJECTIVES: Probiotic yeasts are used in production of functional foods and pharmaceutical products. They play an important role in promoting and maintaining human health. Until now, little work has been published on improving the survival of Saccharomyces in stimulated gastrointestinal condition. MATERIAL AND METHODS: In this study the exposure of the yeast in the capsulate and free forms to artificial gastrointestinal conditions was assessed and the number of viable Saccharomyces cerevisiae cells during 0 to 120 mines in these conditions was evaluated by a pour plate method using sabouraud dextrose agar. RESULTS: Results showed the shape of the beads was generally spherical, sometimes elliptical with a mean diameter of about 50-90 µm. Also count of viable probiotic cells obtained for all the microcapsules were above the recommended levels for a probiotic food. Also decrease of approximately 4 logs was noted in the number of free cells after 2 h of incubation at pH 2 and 8, when compared to decreases of about 2 logs in the all microencapsulated S. cerevisiae under similar conditions. CONCLUSION: It is concluded that microencapsulation process was significantly able to increase the survival rate of Saccharomyces in a simulated gastrointestinal condition (p<0.05)..

Yeasts as important agents of onychomycosis: in vitro activity of propolis against yeasts isolated from patients with nail infection.

OBJECTIVE: The purposes of this study were to determine the frequency of the yeast species obtained from patients with clinical features of onychomycosis and the in vitro antifungal susceptibility of the yeast species to propolis. METHODS: A prospective study was carried out at the Mycology Research Center in Iran from 2010 to 2011. Clinical diagnosis was performed by direct microscopic examination and culture. Different yeast species were identified by morphological and biochemical tests. An antifungal susceptibility test to fluconazole (FLU) and propolis by the broth microdilution method was performed on each isolate. RESULTS: One hundred and twenty-eight fungal isolates were obtained. The most prevalent fungi were yeasts (81, 63.2%), dermatophytes (36, 28.1%), and nondermatophyte fungi (11, 8.6%). Fingernails were more affected than toenails (65.4% vs. 19.8%, respectively). The most frequently found species was Candida albicans (38.5%), followed by Candida spp. (23.1%), C. tropicalis (10.8%), C. kefyr (6.2%), C. krusei (3.1%), Malassezia globosa (4.6%), M. slooffiae (4.6%), and M. pachydermatis (1.5%). Of all yeast isolates (65), seven showed resistance to FLU. The average MIC of propolis for FLU-susceptible isolates was 5.8 µg/mL, whereas this value was 12.25 µg/mL for FLU-resistant isolates. CONCLUSION: Our results proved that the propolis inhibits the growth of pathogenic yeasts and confirmed the efficiency of propolis as an anti-Candida and anti-Malassezia agent.