Antimicrobial and anti-biofilm effects of carotenoid pigment extracted from Rhodotorula glutinis strain on food-borne bacteria.

Background and Objectives: Carotenoid pigments are among the most important pigments and have many applications in various food, cosmetics, hygiene industries and biotechnology. These pigments are produced by plants and microorganisms including Rhodotorula spp. This research intended to study the antimicrobial and antibiofilm effects of the carotenoid pigment from Rhodotorula glutinis on food spoilage bacteria (Staphylococcus aureus and Salmonella Typhimurium). Materials and Methods: The R. glutinis was isolated from milk samples of cows with mastitis and ITS sequence-based typing was performed on them. After extracting the pigment from R. glutinis, its purity was examined using thin-layer chromatography. Following that, the broth microdilution method was used to evaluate antimicrobial effects of the pigment and MtP assay and subsequently scanning electron microscopy were used to assess the antibiofilm effects. In addition, the sub-MIC effects of the pigment on expression of quorum-sensing (QS) genes in S. Typhimurium isolates (sdiA and luxS) and S. aureus isolates (hld) were studied. Finally, the degree of toxicity of the pigment was analyzed using the MTT assay. Results: ITS sequence analysis of R. glutinis revealed that the recently separated isolates exhibited strong differences with the strains recorded in NCBI database in genetic structure. The pigment produced by R. glutinis had strong antimicrobial effects and its mean MIC against S. Typhimurium isolates (17.0 µl.ml-1) was higher than the mean MIC against the S. aureus isolates (4.1 µl.ml-1). Electron microscope images and real-time observations indicated that the sub-MIC values of the pigment suppressed biofilm formation by suppressing expression of QS genes. In addition, the mentioned pigment at high MIC concentrations did not have toxic effects on Vero cells. Conclusion: This research suggests that R. glutinis pigment is effective in destroying the planktonic form of food spoilage bacteria and degrading food spoilage biofilm-forming bacteria. Moreover, considering the low toxicity level of R. glutinis pigment for eukaryotic cells, we can suggest its use as a natural antibacterial preservative in various food materials.

Evaluation of viral load and transcriptome changes in tracheal tissue of two hybrids of commercial broiler chickens infected with avian infectious bronchitis virus: a comparative study.

Infectious bronchitis virus (IBV) is one of the major threats to the poultry industry, with significant economic consequences. Despite strict measures, the disease is difficult to control worldwide. Experimental evidence demonstrates that the severity of IBV is affected by the genetic background of the chicken, and the selection of appropriate breeds can increase production efficiency. Therefore, the aim of the present study was to assess the strength of the immune response to IBV in tracheal tissues of Ross 308 and Cobb 500 broiler chickens by evaluating transcriptome changes, focusing on immune responses and the viral load in tracheal tissues two days after IBV infection. We identified 899 and 1350 differentially expressed genes (DEGs) in the Cobb 500 and Ross 308 experimental groups compared to their respective control groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated the involvement of signaling pathways (Toll-like receptor [TLR], NOD-like receptor [NLR], and RIG-I-like receptor [RLR] signaling pathways). Interestingly, the RLR signaling pathway appears to be affected only in the Cobb hybrid. Furthermore, the viral loads in tracheal samples obtained from the Ross challenged group were significantly higher than those of the Cobb challenged group. The results of this study indicated that the host transcriptional response to IBV infection as well as the viral load can differ by hybrid. Furthermore, genes such as TLR-3, ChIFN-α, MDA5, LGP2, IRF-7, NF-κB, and TRIM25 may interfere with IBV proliferation.

Prevalence of main quinolones and carbapenems resistance genes in clinical and veterinary Escherichia coli strains.

Background and Objectives: Antibiotics-resistant Escherichia coli strains are considered one of the most important causes of human and animal infections worldwide. The aim of current study was to detect common resistance (carbapenems and quinolones) genes by PCR. Materials and Methods: A total of 100 E. coli strains isolated from human urinary tract infection and 20 isolated strains of aborted sheep embryos were collected. PCR was performed using specific primers to detect the resistance genes. Results: Overall, among the quinolones resistance genes, qnrS resistance gene had the highest frequency (48%) and among carbapenem resistance genes, imp resistance gene had the highest frequency (45%). The frequency of resistance genes, IMP (28.45%), KPC (9.5%), VIM (9.15%), NDM (7.20%) were observed in clinical and veterinary strains, respectively. According to the results, 38.6% of E. coli strains had at least one from five genes of resistance to quinolones. The lowest frequency of resistance gene was related to qnrA, which was observed in only 29 (24.2%) strains. Conclusion: Monitoring of carbapenem and quinolone resistance in pathogenic E. coli to humans and animals has an important value in revising treatment guidelines and the national public health, and plays an important role in preventing the spread of resistant strains.

Pathogenic Salmonella weakens avian enriched blood monocytes through ATP depletion, apoptosis induction and phagocytosis inefficiency.

Salmonella enterica Subsp enterica serovar Typhimurium (S. Typhimurium, ST) is one of the most important serovars of the genus Salmonella in human and animals. Because of its intracellular tropism, monocytes/macrophages are pivotal in killing of Salmonella serovars; they are also responsible for transporting of ST to extra-intestinal organs. To investigate the effect of the ST on the functions of avian innate immune cells, almost homogeneous enriched monocytes (EMo) were isolated from peripheral blood mononuclear cells of 2-3 weeks-old of healthy broilers. The EMo were then divided in three groups: control (media only), treatments (challenged with ST clinical isolates) and [doxorubicin (Dox), specifically as positive control for EMo apoptosis] groups. Cellular-molecular damage caused by ST in EMo was assessed with bioluminescence (for caspase-3, 7, and 9 activities and intracellular ATP content), chemiluminescence (for pro/anti-oxidant capacities) and flow cytometry (for apoptosis/necrosis). Further, phagocytosis capacity of post-ST challenged EMo was assessed using a flow cytometry-based internalisation of FITC-loaded polystyrene microparticles. Like the effects of Dox, in post-ST challenged EMo much higher caspase-3, 7 and 9 activities and ATP depletion along with decreased phagocytosis capacity and anti-oxidant load were observed. The results herein indicate that ST weakens EMo particularly through caspases activation/apoptosis. These findings can open a new window on the molecular aspects of Salmonella-macrophage interactions and immunopathology/pathogenicity of salmonellosis in animals especially avian species.

Induction of specific cell-mediated immune responses and protection in BALB/c mice by vaccination with outer membrane vesicles from a Brucella melitensis human isolate.

Brucellosis is a worldwide bacterial zoonosis caused by Brucella spp. No approved vaccine is available for human use against the disease. In this study, outer membrane vesicles (OMVs) from a Brucella melitensis biovar 1 human isolate obtained in Iran were used to immunize BALB/c mice (n = 12) by 2 intramuscular injections with a 2-week interval. Another group of 12 mice was used as non-vaccinated controls. Two weeks after the last vaccination, six mice of each group were sacrificed, and proliferation and interferon gamma (IFNγ) production responses of their splenocytes were evaluated following in vitro stimulation with killed Brucella cells. The other mice were challenged with the virulent B. melitensis isolate. Two weeks later, mice were killed and spleens were cultured to determine the number of the challenge strain. The results showed proliferative response and IFNγ production of splenocytes from vaccinated mice (stimulation index: 2.18 ± 0.57, and 1519.35 ± 10.70 pg/mL, respectively) were significantly higher than those of control mice (stimulation index: 1.02 ± 0.02, and 210.01 ± 17.58 pg/mL, respectively). Numbers of the challenge strain in spleens of vaccinated mice were also significantly less than those in the controls with 1.6 units of protection. Our study revealed vaccination with OMVs of the B. melitensis isolate could induce specific immune responses and protection against infection in the mouse model suggesting their potential application for active immunization against brucellosis.

Genotyping of Avian infectious bronchitis viruses in Iran (2015-2017) reveals domination of IS-1494 like virus.

Avian infectious bronchitis virus (IBV) is causing major economic losses to the poultry industry. The analysis of the S1 gene has been used to determine IBV genotype. The aim of this study was genotyping of IBVs circulating among the Iranian broiler flocks in the period between 2015 to 2017. Trachea samples from 278 broiler flocks were collected from broiler farms in eight provinces of Iran. After Real-time RT-PCR, IBV-positive samples were further characterized based on S1 gene. The results of the Real-time RT-PCR showed that 52.16% of flocks were IBV positive. Four genotypes were detected and the frequency of occurrence rates of IS-1494-like, 793/B, QX and Massachusetts IBV genotypes were 70.34%, 19.31%, 7.58% and 2.75%, respectively. Sequence analysis revealed that nucleotide identities within IS-1494-like group ranged between 98.86-100%, while each of the QX, Massachusetts and 793/B groups were 98.05-100%, 98.20-100% and 93.29-100% respectively. These results show that the IS-1494-like IBV is the dominant IBV genotype in Iran. Proper control strategies are essential to overcoming the high frequency of occurrence of IS-1494-like IBV. The phylogenetic relationship of the strains with respect to different sequences and geographical regions displayed complexity and diversity. Further studies are needed and should include the isolation and full-length molecular characterization of IBV in Iran.

A comparative study of the timecourse of the expression of the thermo­inducible HSP70 gene in clinical and environmental isolates of Aspergillus fumigatus.

The internal environment within animals or humans provides different conditions to invading saprophytic fungal pathogens, requiring the differential regulation of genes in comparison to environmental conditions. Understanding the mechanisms by which pathogens regulate genes within the host may be key in determining pathogen behavior within the host and may additionally facilitate further investigation into novel therapeutic agents. The heat shock protein (HSP)70 gene and its associated proteins have been frequently reported to be among the most highly expressed and dominant proteins present within various locations at physiological temperatures. The present study examined relative gene expression levels of the HSP70 gene in Aspergillus fumigatus isolates from both clinical and environmental origins, at a range of temperature points (20, 30, 37 and 42˚C) over five days, using reverse transcription­quantitative polymerase chain reaction, comparing with a standard A. fumigatus strain incubated at 25˚C. The results indicated a differential gene expression pattern for the environmental and clinical isolates. During the five days, the HSP70 expression levels in the clinical samples were higher than in the environmental samples. However, the difference in the expression levels between the two groups at 42˚C was reduced. The mean HSP70 expression level over the five incubation days demonstrated a gradual and continual increasing trend by temperature elevation in both groups at 30, 37 and 42˚C, however, at 20˚C both groups demonstrated reduced expression. The temperature shift from 20 to 42˚C resulted in HSP70 induction and up to a 10­ and 8.6­fold change in HSP70 expression levels on the fifth day of incubation in the clinical and environmental groups, respectively. In conclusion, incubation at 37 and 42˚C resulted in the highest expression levels in both experimental groups, with these temperature points important for the induction of HSP70 expression in A. fumigatus.

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)..