Lactococcus-based vaccine against brucellosis: IgG immune response in mice with rOmp16-IL2 fusion protein.

This study was designed to introduce the recombinant Lactococcus lactis MG1363 as a cell factory candidate for production of recombinant Brucella melitensis Omp16-Human IL2 (r-Omp16-IL2) and to suggest it as a promising safe, non-pathogenic mucosal live vaccine against brucellosis. Three groups of BALB/c mice (10 mice per group) were intragastrically administrated with phosphate-buffered saline (PBS), L. lactis harboring the empty pAMJ2008 plasmid and with L. lactis expressing rOmp-IL2. The first two groups were classified as control groups and the third one is indicated as treatment group. Another group was injected by the intraperitoneal (i.p.) route with purified rOmp16-IL2 protein. The total serum IgG of each group was assessed with indirect ELISAs at two days before immunization and also two weeks after the last immunization. Results showed that BALB/c mice intragastrically administrated with L. lactis expressing rOmp-IL2 had dominant IgG response compared to the control (PBS administrated) group (P < 0.05). The level of IgG was significantly increased by intraperitoneally injection of recombinant Omp-IL2 in adjuvant compared to the intragastrically administration of PBS and L. lactis/pAMJ2008 as control groups, and also compared to L. lactis/pAMJ2008-rOmp-IL2 (P < 0.05). Our findings provide the use of L. lactis rOmp16-IL2 as a new promising alternative safe strategy than presently live attenuated vaccines toward developing an oral vaccine or subunit-based vaccine against brucellosis.

Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis.

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.

Effect of supplements: Probiotics and probiotic plus honey on blood cell counts and serum IgA in patients receiving pelvic radiotherapy.

BACKGROUND: Radiotherapy is frequently used in treatment approaches of pelvic malignancies. Nevertheless, it has some known systemic effects on blood cells and the immune system that possibly results in their susceptibility to infection. Probiotics are live microbial food ingredients that provide a health advantage to the consumer. Honey has prebiotic properties. The aim of this clinical trial was to investigate probable effects of probiotic or probiotics plus honey on blood cell counts and serum IgA levels in patients receiving pelvic radiotherapy. MATERIALS AND METHODS: Sixty-seven adult patients with pelvic cancer were enrolled. Patients were randomized to receive either: (1) Probiotic capsules (including: Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Bifidobacterium breve, Bifidobacterium longum, and Streptococcus thermophiles) (n = 22), (2) probiotic capsules plus honey (n = 21) or (3) placebo capsules (n = 24) all for 6 weeks. Blood and serum samples were collected for one week before radiotherapy and 24-72 h after the end of radiotherapy. RESULTS: White blood cells (WBC), red blood cells (RBC), platelet counts, and serum IgA level were not significantly changed in patients taking probiotic (alone or plus honey) during pelvic radiotherapy. The mean decrease in RBC count was 0.52, 0.18, and 0.23 × 10(6) cells/µL, WBC count was 2.3, 1.21, and 1.34 × 10(3) cells/µL and platelet count was, 57.6, 53.3, and 66.35 × 10(3) cells/µL for the probiotic, probiotic plus honey, and placebo groups, respectively. The mean decrease of serum IgA was 22.53, 29.94, and 40.73 mg/dL for the probiotic, probiotic plus honey, and placebo groups, respectively. CONCLUSION: The observed nonsignificant effect of probiotics may be in favor of local effects of this product in the gut rather than systemic effects, however, as a trend toward a benefit was indicated, further studies are necessary in order to extract effects of probiotics or probiotic plus honey on hematologic and immunologic parameters in patients receiving pelvic radiotherapy.

Isolation of copper oxide (CuO) nanoparticles resistant Pseudomonas strains from soil and investigation on possible mechanism for resistance.

The present study deals with isolation and characterization of copper oxide nanoparticles resistant Pseudomonas strains that were isolated from the soil collected from mining and refining sites of Sarcheshmeh copper mine in the Kerman Province of Iran. The three isolates were selected based on high level of copper oxide nanoparticles (CuO NPs) resistance. The isolates were authentically identified as Pseudomonas fluorescens CuO-1, Pseudomonas fluorescens CuO-2 and Pseudomonas sp. CuO-3 by morphological, biochemical and 16S rDNA gene sequencing analysis. The growth pattern of these isolates with all the studied CuO NPs concentrations was similar to that of control (without CuO NPs) indicating that CuO NPs would not affect the growth of isolated strains. A reduction in the amount of exopolysaccharides was observed after CuO NPs-P. fluorescens CuO-1 culture supernatant interaction. The Fourier transform infrared spectroscopy (FT-IR) peaks for the exopolysaccharides extracted from the bacterial culture supernatant and the interacted CuO NPs were almost similar. The exopolysaccharide capping of the CuO NPs was confirmed by FT-IR and X-ray diffraction analysis. The study of bacterial exopolysaccharides capped CuO NPs with E. coli PTCC 1338 and S. aureus PTCC 1113 showed less toxicity compared to uncoated CuO NPs. Our study suggests that the capping of nanoparticles by bacterially produced exopolysaccharides serve as the probable mechanism of tolerance.

Isolation and characterization of novel Bacillus strains with superior probiotic potential: comparative analysis and safety evaluation.

Despite the current use of some Bacillus spp. as probiotics, looking for and introducing new efficient and safe potential probiotic strains is one of the most important topics in both microbiology and food industry. This study aimed to isolate, identify, and evaluate the probiotic characteristics and safety of some Bacillus spp. from natural sources. Thirty-six spore-forming, Gram-positive, and catalase-positive Bacillus isolates were identified in 54 samples of soil, feces and dairy products. Bacterial identification was performed using 16S rDNA sequencing. To evaluate the probiotic potential of isolates, the resistance of bacterial cells to simulated gastrointestinal tract (GIT) conditions, the presence of enterotoxin genes, their susceptibility to antibiotics, antimicrobial and hemolytic activities and biochemical profiles were investigated. The results revealed that eight sporulating Bacillus spp. isolates fulfilled all tested probiotic criteria. They showed a high growth rate, non-hemolytic and lecithinase activity, and resistance to simulated GIT conditions. These strains exhibited broad-spectrum antibacterial activity against pathogenic bacteria. In addition, they did not exhibit antibacterial resistance to the 12 tested antibiotics. The results of this study suggest that these isolates can be considered as candidates for functional foods and as safe additives to improve diet quality.

Functional and chemical properties of Phoenix dactylifera l. Polysaccharides and the effect of date flesh and seed intervention on some blood biomarkers: A contrastive analysis.

The chemical structure and bioactivity of ultrasonic-assisted alkaline extracted polysaccharides of date seed (DSP) and date flesh (DFP) were investigated. In addition, a crossover clinical trial was conducted to evaluate the effects of 28 days of date seed powder and date flesh consumption on blood biomarkers. Xylose (72.2 %) and galactose (41.6 %) were the most abundant monosaccharides in DSP and DFP, also DFP had a higher uronic acid content (12.16 ± 2.13 g/100 g) compared to DSP (5.57 ± 1.2 g/100 g). DSP had higher proliferation and antibacterial effects compared to DFP and inulin. Bifidobacterium animalis produced a higher short-chain fatty acid concentration during fermentation of DSP (66.98 ± 4.33 mM) and DFP (58.58 ± 5.57 mM) than inulin (19.68 ± 3.73 mM). Date seed powder could significantly reduce C-reactive proteins and triglycerides and increase red blood cell count (p < 0.05). DSP showed considerable prebiotic capability, antibacterial activity, and health-promoting effect; therefore, it could be considered for further investigation as nutraceuticals.

Bacillus subtilis isolates from camel milk as probiotic candidates.

Recently Bacillus spp. has gained much attention as potential probiotics due to the production of resistant cells. So, this research is purposeful for evaluation of probiotic characteristics of Bacillus isolates from camel milk as a suitable source for growth and isolation of microorganisms that can be candidate to be used as probiotic. First, forty-eight colonies were screened by using morphological and biochemical analysis. Among the isolates, two of them were recognized as Bacillus subtilis CM1 and CM2 by partial 16SrRNA sequencing that, probiotic potentials of them were evaluated. Both of them, in the preliminary safety screening, were found negative for hemolysis and lecithinase activity. Also, in vitro characteristics such as acid, bile salts and artificial gastric juice resistant, cell surface hydrophobicity, auto-aggregation, antioxidant characteristics, and adherent capability to HT-29 cells were determined for them approximately in the range of other probiotic strains. Two strains were susceptible to various antibiotics and enterotoxigenic activities were not detected by PCR which means isolated Bacillus strains could be classified as safe. Altogether, results demonstrate that Bacillus CM1 and CM2 strains could have the potential of consideration as probiotics, however more extensive in vitro/vivo studies are needed.

The effects of lactobacilli (L. rhamnosus, L. reuteri, L. Plantarum) on LPS-induced memory impairment and changes in CaMKII-α and TNF-α genes expression in the hippocampus of rat.

In recent years, some investigations have focused on the relationship between gut microbiota and brain function in healthy and disease conditions. Moreover, changes in the gut microbiome may affect memory, behavior, and cognition. This study aimed to evaluate the effect of lactobacilli on passive avoidance learning, hippocampal CaMKII-α, and TNF-α genes expression in one experimental model of neuroinflammtion. Male wistar rats (220-240 g) received daily oral gavage of different lactobacilli including L. rhamnosus, L. reuteri, and L. plantarum for 21 days. At the next step, behavioral test was performed and 4 hours after acute injection of LPS (1mg/kg, ip) retrieval was evaluated. Then the hippocampi were extracted and kept at _80°C. Finally CaMKII-α and TNF-α mRNA levels were evaluated by Real-time PCR. Our results showed that consumption of L. rhamnosus, L.reuteri, or L. plantarum significantly prevented LPS-induced enhancement of TNF-α mRNA and memory deterioration. Furthermore, L. rhamnosus significantly prevented changes induced by LPS on CaMKII-α mRNA levels. It seems the lactobacilli used in this study can affect brain function and memory performance by their immune-modulatory properties.

Anti-MRSA activity of a bioactive compound produced by a marine Streptomyces and its optimization using statistical experimental design.

OBJECTIVES: To address the alarming problem of methicillin-resistant Staphylococcus aureus (MRSA), herein, a marine Streptomyces capable of producing an anti-MRSA compound has been studied. MATERIALS AND METHODS: Strain MN41 was morphologically and physiologically characterized and then, molecularly identified using 16SrRNA analysis. To produce the bioactive compound in large scale, a kind of submerged liquid fermentation was adopted. The antibacterial agent was purified using a silica gel column followed by a semi-preparative HPLC and the isolated metabolite was identified using mass spectrometry, Nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR). Finally, the production process was subjected to a two steps optimization using Plackett-Burman design (PBD) and Response Surface Method (RSM), respectively. In addition, the antitumor activity of the active agent was studied. RESULTS: The purified compound with a molecular weight of 421.2 was identified as a natural pyrrole-derivative. The optimization revealed a significant effect for starch, pH, calcium carbonate and peptone on the production of this anti-MRSA compound and resulted in a 218% increase in the production yield. CONCLUSION: The isolated pyrrole-derivative showed a remarkable activity against MRSA and also showed some promising anti-tumor activity.

Prediction of the Omp16 Epitopes for the Development of an Epitope-based Vaccine Against Brucellosis.

BACKGROUND: Brucellosis is an infectious disease caused by Brucella bacteria that cause disease in animals and humans. Brucellosis is one of the most common zoonotic diseases transmitted from animals-to-human through direct contact with infected animals and also consumption of unpasteurized dairy products. Due to the wide incidence of brucellosis in Iran and economical costs in industrial animal husbandry, Vaccination is the best way to prevent this disease. All of the available commercial vaccines against brucellosis are derived from live attenuated strains of Brucella but because of the disadvantage of live attenuated vaccines, protective subunit vaccine against Brucella may be a good candidate for the production of new recombinant vaccines based on Brucella Outer Membrane Protein (OMP) antigens. In the present study, comprehensive bioinformatics analysis has been conducted on prediction software to predict T and B cell epitopes, the secondary and tertiary structures and antigenicity of Omp16 antigen and the validation of used software confirmed by experimental results. CONCLUSION: The final epitope prediction results have proposed that the three epitopes were predicted for the Omp16 protein with antigenicity ability. We hypothesized that these epitopes likely have the protective capacity to stimulate both the B-cell and T-cell mediated immune responses and so may be effective as an immunogenic candidate for the development of an epitope-based vaccine against brucellosis.

Marine Actinomycetes with Probiotic Potential and Bioactivity against Multidrug-resistant Bacteria.

Considering antimicrobial resistance problem, marine microorganisms with the bioactivity against multi-drug resistant (MDR) pathogens have attracted many scientific interests. To address this issue, a total of 21 marine actinomycetes isolated from the Caspian Sea have been screened out. Primary screening via cross-streak method revealed that 3 strains: MN2, MN39, and MN40 produce antimicrobial agents with wide spectrum activity. In the second step, the potent strains were characterized morphologically, and then identified genetically using 16S rRNA analysis. After that, the bioactivity of the ethyl acetate extracts of liquid culture against some MDR bacteria has been studied using disc diffusion method. Finally, the exoenzymatic activity of the strains, and the anti-vibrio activity of the extracts have been evaluated. The nucleotide sequence of the 16S rRNA gene (1.5 kb) showed that the potent strains belong to the genus Streptomyces. The results of disk diffusion method indicated that among the 3 potent isolates, MN39 and MN2 produce biomolecules with antibacterial activity against MDR bacteria specially methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). In addition, potent strains showed remarkable anti-vibrio activity as well as extracellular enzyme production including amylase and protease. The results of this study revealed that the marine actinomycetes isolated from the sediments of Caspian Sea produce biomolecules effective against MDR bacteria, and suggested that these strains deserve to be studied as potential probiotics due to their anti-vibrio activity besides exoenzyme production.

Modification of IgE binding to αS1-casein by proteolytic activity of Enterococcus faecium isolated from Iranian camel milk samples.

Milk is a perfect source of nutrients for neonates. When breast feeding cannot be done, an infant's alimentation is usually initiated to cow's milk, among the primary foods. It has been reported that about 2.5% of juveniles under the age of 3 years manifest allergic reactions to cow's milk proteins. Among the cow's milk proteins, casein fractions are considered as the strongest allergenic proteins. The proteolytic enzymes of lactic acid bacteria (LAB), during fermentation of dairy products, can break down milk proteins especially caseins and subsequently reduce the immune reactivity of allergenic proteins. In this research, raw bovine and camel milk samples were screened for cocci LAB strains and after isolation, their proteolytic activity against bovine milk caseins were evaluated by SDS-PAGE and RP-HPLC. The potential of cocci LAB strains on αS1-casein degradation and their potential to break down the principle allergenic epitopes of this protein was detected using indirect competitive ELISA. Molecular identification of the best proteolytic strain was fulfilled by 16S rDNA fragment sequencing with universal primers. The obtained results demonstrated that Enterococcus faecium isolated from raw camel milk samples was the most efficient isolate in hydrolyzing Na-caseinate and αS1-casein. Hydrolysated αS1-casein by Enterococcus faecium was also less recognized by IgE of bovine milk allergic patients' sera in comparison with native αS1-casein. It has been proposed that Enterococcus faecium could be an efficient strain in allergenicity reduction of cow's milk proteins. So it could be an excellent candidate to be potentially used in dairy industries.

Resistotyping, phenotyping and genotyping of New Delhi metallo-ß-lactamase (NDM) among Gram-negative bacilli from Iranian patients.

PURPOSE: The purpose of this study was to investigate New Delhi metallo-ß-lactamase (NDM) production among Gram-negative bacilli. METHODOLOGY: Antibiogram-resistotyping and detection of New Delhi metallo-ß-lactamase (NDM) in clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa and comparative evaluation of the diagnostic performance of three phenotypic methods for NDM detection, with PCR considered as the gold standard, were performed. Minimum inhibitory concentration (MIC) of antibiotics against NDM-positive strains using E-tests and clonal relationship analysis using enterobacterial repetitive intergenic consensus (ERIC)-PCR in these strains were determined. RESULTS: The most effective antibiotics against strains of the species K. pneumoniae were Colistin, Chloramphenicol and Tigecycline; against P. aeruginosa were Fosfomycin and Polymyxins, and against A. baumannii were Polymyxins, Ampicillin/Sulbactam and Minocycline. Overall, 66, 31 and 40 different resistotypes were observed among K. pneumoniae, A. baumannii and P. aeruginosa strains, respectively. The blaNDM-1 gene was detected in 28 (8.5 %) strains of the bacteria investigated. The sensitivities and specificities of the Meropenem-EDTA combined disk test, the meropenem-dipicolinic acid combined disk test and the modified Hodge test methods for NDM detection were 96.43, 55.15; 96.43, 54.85; and 89.29, 35.15, respectively. Additionally, in spite of the low positive predictive values of these tests, their negative predictive values were high. ERIC-PCR results revealed two main clusters in NDM-positive strains of each of the species P. aeruginosa and A. baumannii, and ten main clusters in K. pneumoniae. In all the NDM-positive strains maximum MIC rates (>256) were observed for all beta-lactam antibiotics. CONCLUSION: There were high levels of antibiotic resistance and a high frequency of multi-drug resistance and extensive-drug resistance profiles, as well as highly prevalent blaNDM-1 genes in the bacteria investigated.

Evaluation of antibacterial efficiency of chitosan and chitosan nanoparticles on cariogenic streptococci: an in vitro study.

BACKGROUND AND OBJECTIVES: The most prevalent and worldwide oral disease is dental caries that affects a significant proportion of the world population. There are some classical approaches for control, prevention and treatment of this pathologic condition; however, the results are still not completely successful. Therefore new methods are needed for better management of this important challenge. Chitosan is a natural and non-toxic polysaccharide with many biological applications, particularly as an antimicrobial agent. Chitosan nanoparticle is a bioactive and environment friendly material with unique physicochemical properties. The aim of the present study was to investigate the antimicrobial effect of chitosan and nano-chitosan on the most important cariogenic streptococci. MATERIALS AND METHODS: For evaluation of antimicrobial effect of chitosan and nano-chitosan against oral streptococci broth micro-dilution method was carried out for four bacterial species; Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguis and Streptococcus salivarius. Also the effect of these materials on adhesion of above bacteria was evaluated. One-way ANOVA and post hoc Tukey test were used for statistical analysis. RESULTS: The MICs of chitosan for S. mutans, S. sanguis, S. salivarius and S. sobrinus were 1.25, 1.25, 0.625 and 0.625 mg/mL, respectively. The MIC of chitosan nanoparticle for S. mutans, S. salivarius and S. sobrinus was 0.625 mg/mL and for S. sanguis was 0.312 mg/mL. Chitosan and chitosan nanoparticles at a concentration of 5 mg/mL also reduced biofilm formation of S. mutans up to 92.5% and 93.4%, respectively. CONCLUSION: The results of this study supported the use of chitosan and chitosan nanoparticles as antimicrobial agents against cariogenic Streptococci.

Determination of Acquired Resistance Profiles of Pseudomonas aeruginosa Isolates and Characterization of an Effective Bacteriocin-Like Inhibitory Substance (BLIS) Against These Isolates.

BACKGROUND: The emergence of pan-drug resistant strains (PDR) of Pseudomonas aeruginosa has led to renewed efforts to identify alternative agents, such as bacteriocins and bacteriocin-like inhibitory substances (BLISs). OBJECTIVES: The aims of this study were to determine the acquired resistance profiles of multidrug-resistant (MDR), extensively drug-resistant (XDR), and PDR P. aeruginosa isolates based on the revised definitions of the CDC and ECDC and to screen and characterize effective BLISs against these isolates. PATIENTS AND MATERIALS: In a cross-sectional study, 96 P. aeruginosa strains were isolated during a 12-month period. The resistance profiles of these isolates were determined as MDR, XDR, and PDR, and the data were analyzed using WHONET5.6 software. A BLIS against the P. aeruginosa strains was characterized based on its physicochemical properties, size, growth curves, and production profiles. RESULTS: Among the 96 isolates of P. aeruginosa, 2 (2.1%), 94 (97.9%), and 63 (65.6%) were non-MDR, MDR, and XDR, respectively, and 1 (1.1%) was PDR. The most effective antibiotics against these isolates were polymyxins and fosfomycin. A BLIS isolated from the P. aeruginosa DSH22 strain had potent activity against 92 (95.8%) of the 96 isolates. The BLIS was heat stable, (up to 100°C for 10 min), UV stable, and active within a pH range of 3 - 9. The activity of BLIS disappeared when treated with trypsin, proteinase K, and pepsin, indicating its proteinous nature. Based on its size (25 kDa), the BLIS may belong to the large colicin-like bacteriocin family. BLIS production started in the midexponential phase of growth, and the maximum level (2700 AU/mL) occurred in the late-stationary phase after 25 hours of incubation at 30°C. CONCLUSIONS: This BLIS with broad-spectrum activity may be a potential agent for the treatment or control of drug-resistant strains of P. aeruginosa infection.

Application of Chitosan/PVA Nano fiber as a potential wound dressing for streptozotocin-induced diabetic rats.

Diabetes mellitus is a worldwide health problem affecting 1-2% of the population of world with noticeable morbidity and mortality. Vascular events such as hypertension, nephropathy, neuropathy and retinopathy are happened in diabetic patients. Decline in tissue blood circulation may causes hypoxia and finally may leads to slow wound healing and amputation. PVA/Chitosan Nano fiber wound dressings have high moisture vapor transmission rate and good antimicrobial activity1 PCNWD substrate does not have any recognized cytotoxicity effects and has excellent odor absorbing capability. In the present study, Streptozotocin (STZ) is used to induce diabetes in rats, Skin ulcers are produced experimentally in the experimentally induced diabetic and non-diabetic rats. Then PCNWD used as wound dressing for 2 weeks period to evaluate its macroscopic and microscopic effects on wound healing in comparison with untreated diabetic and non-diabetic rats experimental ulcers. The findings of current study indicate significant acceleration in diabetes wound healing on the rats treated by PVA/Chitosan Nano fiber.

T cell polarizing properties of probiotic bacteria.

Different commensal bacteria employed as probiotics have been shown to be endowed with immunomodulatory properties and to actively interact with antigen presenting cells, such as dendritic cells and macrophages. In particular, different strains of probiotic bacteria may induce the secretion of a discrete cytokine profile able to induce divergent T cell polarization. Here, we briefly review current knowledge regarding the effects of different species and strains of probiotic bacteria on T cell polarization. Given that the loss of intestinal homeostasis is frequently associated with an aberrant T cell polarization profile, a comprehensive knowledge of the immunomodulatory potential of these bacteria is crucial for their employment in the management of human immune-mediated pathologies, such as allergies or inflammatory bowel diseases.