Borrelia theileri infections in Rhipicephalus annulatus ticks from the north of Iran.

Ticks serve as vectors and reservoirs of various Borrelia species, potentially causing diseases in humans and animals. Mazandaran, a fertile green land in northern Iran, provides ample grazing grounds for livestock and harbors at least 26 hard tick species. This study investigated Borrelia infection in hard ticks from forest areas in this region and compared their genetic identity with the species data in the GenBank database. A total of 2,049 ticks were collected manually from mammalian hosts or using dragging and flagging methods. These ticks were then grouped into 190 pools and 41 individuals based on host, species, developmental stage, and gender. A real-time PCR (qPCR) detected Borrelia DNA in 26 pools from female, male, and nymph of Rhipicephalus annulatus (n = 17) and Ixodes ricinus (n = 9) ticks and one individual female Haemaphysalis punctata tick. The generated partial flaB and glpQ sequences from qPCR-positive Rh. annulatus ticks exhibited the highest identities of 98.1-100% and 98.2% with Borrelia theileri and closely related undefined isolates. Additionally, in phylogenetic analysis, these sequences clustered within well-supported clades with B. theileri and the closely related undefined isolates from various geographic regions, confirming the presence of B. theileri in the north of Iran. Divergence in B. theileri flaB and glpQ sequences across various geographical areas suggests potential subspeciation driven by adaptations to different tick species. This divergence in our flaB sequences implies the possible introduction of B. theileri-infected ticks from different geographical origins into Iran.

Cloning and expression of recombinant arazyme with anti-inflammatory and anti-breast cancer potential.

Arazyme is an extracellular metalloprotease which is secreted by a Gram-negative symbiotic bacterium called Serratia proteomaculans. There are limited studies on various biological activities of arazyme. This preliminary study was designed to investigate the anti-cancer and anti-inflammatory capacities of recombinant arazyme (rAra) in vitro and in vivo. Arazyme gene, araA was cloned and expressed in E. coli BL21 (DE3) using pET-28a as a vector. Nickel column purification was used to obtain pure rAra. SDS-PAGE and protein assay were used to identify the product and to measure protein content, respectively. Skimmed milk test and casein assay were carried out to assess protease activity. MCF7 cells as a breast cancer cell model were exposed to different concentrations of rAra to study anti-breast cancer potentials using MTT assay. The anti-inflammatory property of rAra was investigated using a murine air-pouch model. PCR and SDS-PAGE data showed that cloning and expression of rAra was successful and the enzyme of interest was observed at 52 KDa. Protein assay indicated that 1 mg/ml of rAra was obtained through purification. A clear zone around the enzyme on skimmed milk agar confirmed the proteolytic activity of rAra and the enzymatic activity was 320 U/mg protein in the casein assay. Cytotoxic effects of rAra reported as IC50 were 16.2 µg/ml and 13.2 mg/ml after 24 h and 48 h, respectively. In the air-pouch model, both the neutrophil count and myeloperoxidase activity, which are measures of inflammation, were significantly reduced. The results showed that rAra can be used in future mechanistic studies and R&D activities in the pharmaceutical industry to investigate the safety and efficacy of the recombinant arazyme.

A new probiotic Lactobacillus plantarum strain isolated from traditional dairy together with nanochitosan particles shows the synergistic effect on aflatoxin B1 detoxification.

The present study aimed to evaluate the effects of new Lactobacillus plantarum strain isolated from dairy products as well as chitosan nanoparticles on reducing aflatoxin B1 (AFB1) toxicity In vitro. After collection and preparation of yogurt, cheese, milk, and whey products, lactic acid bacteria (LABs) were isolated and identified using biochemical and molecular methods. pH, bile, and salt tolerance tests were used to measure probiotic activity. Then, the antimicrobial activity of LABs against gastrointestinal pathogens was studied. The strain isolated from cheese (C1) was selected as the appropriate strain and antibiotic susceptibility test was performed for this strain. Then, the effect of C1 isolate and chitosan nanoparticles on reducing aflatoxin B1 (AFB1) in the medium was studied by measuring AFB1 using the enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC). The results of biochemical evaluations indicated the separation of different strains of L. plantarum. Antimicrobial activity test showed extensive antimicrobial activity of C1 isolate. The results showed that this strain has good probiotic activities. This strain was shown to be resistant to erythromycin, fusidic acid, gentamicin, kanamycin, nalidixic acid, neomycin, ofloxacin, and vancomycin antibiotics. C1 strain together with chitosan nanoparticles was able to reduce AFB1 in the medium and, when both were used simultaneously, a synergistic effect was seen in reducing AFB1 from the medium. Based on the findings, it can be concluded that the new C1 L. plantarum strains together with chitosan nanoparticles had synergistic effects on reducing AFB1 toxin in food products.

Investigating the effect of nanoparticle on phenanthrene biodegradation by Labedella gwakjiensis strain KDI.

Polycyclic aromatic hydrocarbons (PAHs), as persistent organic contaminants, are a major source of concern due to their toxic effect on ecosystems and human health. This study attempted to isolate halotolerant PAHs degrading bacteria from saline oil-contaminated soils. Among the isolates, strain KDI with the highest 16S rRNA gene sequence similarity to Labedella gwakjiensis was able to reduce surface tension (ST) from 65.42 to 26.60 mN m-1 and increase the emulsification index to 81.04%, as a result of significant biosurfactant production. Response Surface Methodology (RSM) analysis was applied to optimize the factors, i.e. PAHs concentration and NaCl concentration as well as to determine the effect of these important variables on PAHs biodegradation. The Carbon Quantum Dots. Iron Oxide (CQDs.Fe3O4) nanoparticles were characterized by several popular analytical techniques, after which the effect of CQD.Fe3O4 nanoparticles on biodegradation was examined. PAHs biodegradation rate and efficiency of strain KDI to degrade PHE in the presence of CQD.Fe3O4 nanoparticles was analyzed by GC. According to the results during biodegradation both the concentration of PAHs and the amount of NaCl were effective. The biodegradation rate significantly increased in the presence of CQD.Fe3O4. The highest biodegradation of PHE occurred in the presence of 0.5 g/L of CQD.Fe3O4 which was 63.63% and 81.77% after 48 and 72 h of incubation. To the best of our knowledge, this is the first report on optimization of PAHs concentration and salinity by RSM and nanobioremediation of PHE using a bacterial strain in the presence of CQD.Fe3O4 nanoparticles.

Isolation and identification of carotenoid-producing Rhodotorula sp. from Pinaceae forest ecosystems and optimization of in vitro carotenoid production.

Yeasts are alternative source of natural carotenoids, a group of colored terpenoids with various market applications. Carotenoid Production by yeast fermentation technology is greatly effective and proposes considerable benefits with large scale production, cost effectiveness and safety. In this study, four pigment-producing yeasts were isolated from forest park soils with the potential to produce carotenoids. Morphological, physiological, biochemical and molecular characterization indicates the isolates belong to Rhodotorula mucilaginosa. Carotenoid production was optimized by small scale cultivation. The optimum condition was 120 h of incubation at pH 6.0, 28 °C, white light irradiation, Yeast Extract Peptone Glycerol medium composed of 10 g/L yeast extract, 20 g/L peptone, 20 ml/L glycerol, yielding maximum content of 223.5 µg/g dry weight. The ß-carotene content was confirmed by HPLC and FT-IR. The results suggested that soil yeasts are potential sources of carotenoids that could be utilized as a natural agent for industrial products.

Surfactin production in the bioreactor: Emphasis on magnetic nanoparticles application.

Surfactin is one of the main lipopeptide biosurfactants produced by different species of Bacillus subtilis. This study aims to analyze the effect of starch-coated Fe0 and Fe3+ nanoparticles on the biomass and biosurfactant production of Bacillus subtilis. Out of 70 soil samples, 20 Bacillus were isolated and genome sequenced by biochemical methods and 16S rRNA gene. Quantitative and qualitative screening methods were used to isolate and detect biosurfactant production. For the aim of this study, 61 and 63 (Bacillus subtilis subsp. Inaquosorum) were selected. Then, hemolytic activity, biomass amount, surfactant production, and reduction of surface tension in Minimal Salt Medium containing Fe0 and Fe3+ nanoparticles were examined after 48, 72, and 96 h of culture. Strain 61 was the best bacterium and Fe3+ was the best nanoparticle. The results were compared with the results of non-nanoparticle bioreactor. The results showed the amount of biomass, surfactin, and surface tension decrease, 72 h after growth in 61 strain containing Fe3+ reached the highest values. Surfactin from strain 61 culture in the Fe3+nanoparticle bioreactor after 72 h of growth showed higher production than the same strain culture after 72 h without Fe3+, if continuing the research, this strain can be commercialized in the future.

Using autochthonous Bdellovibrio as a predatory bacterium for reduction of Gram-negative pathogenic bacteria in urban wastewater and reuse it.

BACKGROUND AND OBJECTIVES: The microbial contamination of wastewater is associated with health risks. The aim of this study was to use the autochthonous Bdellovibrio potential to prey Gram-negative pathogenic bacteria as a bio-control agent to treat urban wastewater. MATERIALS AND METHODS: Thirty-six raw sewage samples were collected for isolation of Bdellovibrio. Double layer plaque assay was used for isolation and the isolates were identified by microscopic examination and molecular analysis. To evaluate the predatory potential for decrease number of Gram-negative pathogenic bacteria, plaque perdition assay, reduction in host cells viability by colony-forming unit (CFU) counting, reduction in optical density (OD) in co-cultures and assay of killing efficiency were carried out. Also, the raw wastewater was treated by Bdellovibrio then the reduction in CFU counting and reduction in OD was evaluated. RESULTS: Four strains of Bdellovibrio were isolated and were registered in Gene Bank. Clear plaques were observed after 3-6 days of incubation for all prey cells. The CFU enumerations of all preys were decreased after 48 hrs in co-cultures and raw wastewater. Also, OD was decreased down to 0.2 nm after 48 hrs. CONCLUSION: These autochthonous Bdellovibrio strains are proposed to use for bio-control of Gram-negative pathogenic bacteria in wastewater and reuse it for irrigation in arid regions.

Purification and study of anti-cancer effects of Serratia marcescens serralysin.

BACKGROUND AND OBJECTIVES: Serralysin is an extracellular metalloprotease from Serratia marcescens which has been the subject of extensive biological investigations. The goal of this study was to extract and purify serralysin from S. marcescens and to investigate its cytotoxic activity on the colorectal cancer cell line. MATERIALS AND METHODS: The presence of the serralysin gene was confirmed using PCR. The supernatant of bacterial culture was collected and precipitated using ammonium sulfate. The precipitated protein was dialyzed and subjected to ion exchange chromatography for further purification. Casein assay and skim milk assay was used to confirm the enzymatic activity. SDS-PAGE was used to visualize the presence of serralysin. Metalloprotease inhibition activity was performed using 50 mM EDTA. Cytotoxic activity of serralysin was assessed on MTT assay. RESULTS: The PCR product corresponding to serralysin was estimated to be approximately 1500 bp. A transparent zone around the bacterial colonies on skim milk agar and casein digestion confirmed the proteolytic activity of serralysin. A 52 kDa band in SDS-PAGE corresponding to serralysin was observed before and after purification processes. MTT assay showed IC50 values 24.78 µg/ml and 19.16 µg/ml after 24 h and 48 h exposure of Caco-2 cells to serralysin, respectively. CONCLUSION: Our results showed that native serralysin has anticancer potential and may be a candidate for further pharmaceutical research and development. Further in vivo and in vitro mechanistic studies are suggested to confirm the biological activities.

Simulation-based protein engineering of R. erythropolis FMN oxidoreductase (DszD).

The sulfur contents of fossil fuels have negative impacts on the environment and human health. The bio-catalytic desulfurization strategies and the biological refinement of fossil fuels are a cost-effective process compared to classical chemistry desulfurization. Rhodococcus erythropolis IGTS8 is able to metabolize the organic sulfur compound by the unique genes cluster (i.e. DszA, B, C and D genes) in the 4S metabolic pathway. The dszD gene codes a key enzyme for sulfur reduction in the gene cluster. In this study, the structure of the DszD enzyme was predicted and then the key residues toward FMN binding were identified which were Thr62, Ser63, Asn77, and Ala79. To investigate the effect of manipulation in key residues on the enzymatic activity of the DszD, different mutations were performed on key residues. The molecular docking simulation showed that A79I and A79N mutants have the lowest binding free energies compared to the wild-type enzyme in binding with FMN substrate. A 50 ns molecular dynamics (MD) simulation performed using GROMACS software. The RMSD and RMSF analysis showed that two mutants are more stable than the wild-type enzyme during MD simulation. The binding free energies between FMN substrate and complexes were calculated and analyzed by the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) method. The experimental results showed that the enzyme activity for the oxidoreductase process toward biodesulfurization increased 1.9 and 2.3 fold for A79I and A79N mutants, respectively.

The gut microflora assay in patients with colorectal cancer: in feces or tissue samples?

Gut microbiota is the complex community of microorganisms that live in the digestive tracts of humans and other animals, including insects. The relationship between gut microbiota and human health is mutualistic and altered bacterial compositions in fecal and mucosal specimens of colon in patients with cancer compared to healthy subjects were observed. Thereby, studying the gut microbiota, their interactions with the host and their alterations in colorectal cancer (CRC) patients could be helpful to diagnose and treat the disease in earlier stages. In CRC research, the most common samples are feces and tumor tissues. Interestingly, scientists have quite different views regarding gut microbiota composition of feces and tissues. Some believe bacterial populations in feces and mucosa are completely distinct and differ in composition and diversity while some others declare similar variations. Actually, both types of specimens have some advantages and disadvantages in survey of gut microbiota. Fecal samples serve as a noninvasive approach for screening tests while mucosal associated samples are more powerful for identification of bacteria with adenoma and CRC initiation and growth. Here we have discussed the advantages and disadvantages of two type of specimens in CRC investigations and also discussed the similarities and differences of microbial composition between stool and tissue specimens.

Genetic Diversity and Prevalence of Nontuberculous Mycobacteria Isolated from Clinical Samples in Tehran, Iran.

The prevalence of nontuberculous mycobacteria (NTM) has increased in tuberculosis (TB)-suspected clinical samples. These bacteria are now recognized as important emerging pathogens, which affect both immunocompetent and immunocompromised individuals. The aim of this study was to evaluate the frequency of NTM in clinical samples and to efficacy of genomic loci as targets for detection of NTM species. This cross-sectional study was performed on 8166 clinical samples to determine the presence of NTM species from April 2013 to December 2015. The phenotypic methods were applied for preliminary NTM identification. The PCR-RFLP assay of heat shock protein-65 (hsp-65) gene and multilocus sequence analysis based on 16S-23S internal transcribes spacer (ITS), rpoB, and 16S rRNA genes were applied for species identification. In a total of 520 isolates from TB-suspected cases, 61 samples (11.7%) were identified as NTM. Overall, Mycobacterium (M.) fortuitum (63.9%) was the most frequently encountered species, followed by M. kansasii (9.8%), M. simiae (9.8%), M. abscessus (6.7%), M. gordonae (4.9%), M. flavescens (3.3%), and M. setense (1.6%). Moreover, sequencing of 16S rRNA and rpoB genes could identify all NTM species. In conclusion, we showed that the samples were infected by six NTM species, and M. fortuitum was the most frequent NTM strain. Based on the findings, 16S rRNA and rpoB genes were superior to ITS gene in identification of NTM species.

Evaluation of TRIM5 and TRIM22 polymorphisms on treatment responses in Iranian patients with chronic hepatitis C virus infection.

The tripartite motif (TRIM)-5 and TRIM22 are involved in innate immune response and show anti-viral activities. The current study aimed at evaluating the association of TRIM5 and TRIM22 polymorphisms with treatment outcomes in patients with chronic hepatitis C virus (CHC). TRIM5 rs3824949 and TRIM22 polymorphisms (rs7113258, rs7935564, and rs1063303) were genotyped using TaqMan polymerase chain reaction (PCR) assay in 425 treatment-naïve CHC patients. Rapid virological response (RVR), early virological response (EVR), and sustained virological response (SVR) were found in 54.1%, 74.8%, and 67.1% of the patients, respectively. RVR and SVR were associated with TRIM5 rs3824949 (GG), TRIM22 rs1063303 (GC), and TRIM22 rs7113258 (AA), while there was a relationship between TRIM5 rs3824949 (GG) and EVR. TRIM5 and TRIM22 single nucleotide polymorphisms (SNPs) were strongly associated with increased odds of RVR, EVR, and SVR after an interferon-based therapy in patients with CHC.

Comparative antibacterial effects of cellulose nanofiber, chitosan nanofiber, chitosan/cellulose combination and chitosan alone against bacterial contamination of Iranian banknotes.

The aims of the current study were to evaluate the antibacterial effects of cellulose nanofiber (CNF), chitosan nanofiber (ChNF), CNF/ChNF combination and chitosan alone (Ch) against the isolated bacterial contaminations from the surface of Iranian banknotes using biochemical, disc diffusion and molecular analyses. The results unveiled that the CNF did not show the significant antibacterial effect against isolated bacterial strains, whereas the combination of CNF/ChNF (at concentration 100 µg/disc 1:1) exhibited the synergistic effects against Stenotrophomonas maltophilia. Also, ChNF (100 µg/disc) and Ch (100 and 200 µg/disc) displayed dose-dependent antibacterial effects against the sensitive bacteria including Bacillus subtilis, Bacillus pumilus, Micrococcus sp. Kosakonia cowanii, Brevibacterium frigoritolerans, Escherichia coli standard ATCC 25922, Staphylococcus aureus standard ATCC 25923. On the other hand, chitosan displayed the highest inhibitory effects against Gram-positive bacteria. Our results showed that the coating of banknotes with these compounds is a novel strategy to reduce the bacterial contaminations and increase the durability (or quality) of banknotes, without being toxic to the surrounding environment.

Novel mutant of Escherichia coli asparaginase II to reduction of the glutaminase activity in treatment of acute lymphocytic leukemia by molecular dynamics simulations and QM-MM studies.

L-Asparaginases (ASNase) belong to a family of amidohydrolases, have both asparaginase and glutaminase activity. Acute lymphocytic leukemia (ALL) is an outrageous disease worldwide. Bacterial ASNase has been used for the treatment of ALL. Glutaminase activity of enzyme causes some side effect and it is not essential for anticancer activity. The aim of this study was engineering of Escherichia coli asparaginase II to find a mutant with reduced glutaminase activity by molecular docking, molecular dynamics (MD) and QM-MM (Quantum mechanics molecular dynamics) simulations. Residues with low free energy of binding to Asn and high free binding energy to Gln were chosen for mutagenesis. Then, a mutant with higher glutaminase free binding energy was selected for further studies. Additionally, the MD simulation and QM-MM computation of wild type (WT) were employed and the selected mutated ASNase were analyzed and discussed. Our data showed that V27T is a good candidate to reduction the glutaminase activity, while has no remarkable effect on asparaginase activity of the enzyme. The simulation analysis revealed that V27T mutant is more stable than WT and mutant simulation was successful completely. QM-MM results confirmed the successfulness of our mutagenesis.

The combined effects of ethanolic extract of Artemisia aucheri and Artemisia oliveriana on biofilm genes expression of methicillin resistant Staphylococcus aureus.

BACKGROUND AND OBJECTIVES: One of the most important antibiotic-resistant bacteria is methicillin-resistant Staphylococcus aureus (MRSA) biofilm that has caused significant problems in treating the patients. Therefore, the aim of this study was to evaluate the levels of expression of genes involved in biofilm formation in MRSA (ATCC 33591) while being treated by a combination of Artemisia aucheri and Artemisia oliveriana. MATERIALS AND METHODS: The minimum inhibitory concentration (MIC) of ethanolic extract of A. aucheri and A. oliveriana and also the minimum inhibitory concentration of combination of both extracts were 512, 1024 and 256 µg/ml, respectively; then at concentrations lower than the MIC, expression levels of the desired genes were determined by Real Time PCR. RESULTS: Based on results, using a combination of two ethanolic extracts had a significant effect on expression of genes involved in biofilm formation in MRSA. The expression level of icaA at 4, 8, 16 h after being treated by herbal extracts of A. aucheri and A. oliveriana was 0.293, 0.121, 0.044, respectively. The expression level of icaD was 0.285, 0.097, 0.088, respectively, while that of ebps was 0.087, 0.042, 0.009 at 4, 8 and 16 h, respectively. CONCLUSION: This study provided evidence that ethanol extract of A. oliveriava and A. aucheri can inhibit the biofilm formation of S. aureus. As a traditional Iranian medicine, A. oliveriava and A. aucheri extracts have a potential antibiofilm formation against MRSA strains.

A novel recombinant vaccine candidate comprising PBP2a and autolysin against Methicillin Resistant Staphylococcus aureus confers protection in the experimental mice.

The methicillin-resistant Staphylococcus aureus infection is a hot topic area in microbiology research. Here a novel vaccine candidate consisting of recombinant PBP2a and autolysin proteins were used. The proteins over expressed in E.coli BL21 (DE3) cells, and purified by the Ni-NTA affinity column and conjugated using EDAC and ADH as a linker and spacer, respectively. To investigate the immunogenicity and protective effects of recombinant proteins, 5 and 20µg of proteins in various formulations were subcutaneously injected in different groups. Two booster vaccinations were carried out in three-week intervals and blood samples were collected three weeks after each injection. To evaluate the immune response, total IgG, IgG1, IgG2a, and IgG2b were analyzed. Immunization of mice with r-autolysin and r-autolysin-PBP2a mixture raised total IgGantibody. Additionally, both IgG1 and IgG2a responses induced. Opsonophagocytosis assay showed that anti r-PBP2a and r-autolysin IgG not only promoted phagocytosis of S.aureus, but also decreased the number of viable bacterial cells. Furthermore, survival rate of experimental mice increased in the bacteremia infection. Our results demonstrated that active vaccination with a mixture of r-PBP2a/r-autolysin and conjugate form vaccine reduced the mortality rate and protected mice against lethal MRSA challenge as well as single proteins.

Cloning, expression and purification of autolysin from methicillin-resistant Staphylococcus aureus: potency and challenge study in Balb/c mice.

Staphylococcus aureus (MRSA) is an opportunistic pathogen which causes a variety of clinical diseases and leads to high rates of morbidity and mortality. Development of an effective vaccine appears to be a useful strategy to control the infection. Here, the internal region of atl was cloned into the pET24a plasmid and expressed in E. coli BL21 (DE3). Cloning of atl was confirmed by colony-PCR, enzymatic digestion and sequencing. Protein expressed in E coli, BL21 DE3 and was confirmed with SDS-PAGE and western blot analysis. Subsequently, BALB/c mice were injected subcutaneously three times with 20µg of the recombinant autolysin. After Bleeding, autolysin-specific total IgG antibodies and isotypes were evaluated using ELISA. Opsonophagocytic killing assay was performed and experimental challenge was done by intraperitoneal injection with sub lethal doses of MRSA in mice and also survival rate was regularly monitored. Results showed that vaccinated mice could exhibit higher levels of autolysin-specific antibodies (P<0.0001) with a predominant IgG1 response versus control group. Results from in vitro experiments indicated that S. aureus opsonized with immunized-mice sera displayed significantly increased phagocytic uptake and effective intracellular killing versus non-immunized mice. The number of viable bacteria in the kidney of immunized mice showed 1000 times less than the control mice; additionally, an increased survival rate was found after immunization with the candidate vaccine versus control group. Results from this study demonstrated that the autolysin is a valuable target for the development of immunotherapeutic strategies against S. aureus and candidate vaccines.

Canthaxanthin biosynthesis by Dietzia natronolimnaea HS-1: effects of inoculation and aeration rate.

The interest in production of natural colorants by microbial fermentation has been currently increased. The effects of D-glucose concentration (3.18-36.82 g/L), inoculum size (12.5 × 10(9)-49.5 × 10(9) cfu cells/mL) and air-flow rate (1.95-12.05 L/L min) on the biomass, total carotenoid and canthaxanthin (CTX) accumulation of Dietzia natronolimnaea HS-1 in a batch bioreactor was scrutinized using a response surface methodology-central composite rotatable design (RSM-CCRD). Second-order polynomial models with high R (2) values ranging from 0.978 to 0.990 were developed for the studied responses using multiple linear regression analysis. The models showed the maximum cumulative amounts of biomass (7.85 g/L), total carotenoid (5.48 mg/L) and CTX (4.99 mg/L) could be achieved at 23.38 g/L of D-glucose, 31.2 × 10(9) cfu cells/mL of inoculation intensity and air-flow rate of 7.85 L/L min. The predicted values for optimum conditions were in good agreement with experimental data.

Canthaxanthin biosynthesis by Dietzia natronolimnaea HS-1: effects of inoculation and aeration rate

The interest in production of natural colorants by microbial fermentation has been currently increased. The effects of D-glucose concentration (3.18-36.82 g/L), inoculum size (12.5 x 10(9)-49.5 x 10(9) cfu cells/mL) and air-flow rate (1.95-12.05 L/L min) on the biomass, total carotenoid and canthaxanthin (CTX) accumulation of Dietzia natronolimnaea HS-1 in a batch bioreactor was scrutinized using a response surface methodology-central composite rotatable design (RSM-CCRD). Second-order polynomial models with high R² values ranging from 0.978 to 0.990 were developed for the studied responses using multiple linear regression analysis. The models showed the maximum cumulative amounts of biomass (7.85 g/L), total carotenoid (5.48 mg/L) and CTX (4.99 mg/L) could be achieved at 23.38 g/L of D-glucose, 31.2 x 10(9) cfu cells/mL of inoculation intensity and air-flow rate of 7.85 L/L min. The predicted values for optimum conditions were in good agreement with experimental data.

Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus: A Study on Immunoreactivity in Balb/C Mouse.

BACKGROUND: Staphylococcus aureus (S. aureus) is a major nosocomial pathogen and the infection with this organism in human is increasing due to the spread of antibiotic resistant strains. One of the resistance mechanisms of S. aureus comprises modification in binding proteins to penicillin. Vaccine strategy may be useful in controlling the infections induced by this organism. This study aimed at developing and producing the recombinant protein PBP2a as a vaccine candidate and evaluating the related humoral immune response in a murine model. METHODS: A 242 bp fragment of mecA gene was amplified by PCR from S. aureus COL strain and then cloned into prokaryotic expression vector pET-24a. For expression of recombinant protein, pET24a-mec plasmid was transformed into competent E. coli BL21 (DE3) cells. Recombinant protein was over expressed with 1 mM isopropythio-ß-D-galctoside (IPTG) and purified using Ni-NTA agarose. SDS-PAGE and western blotting were carried out to confirm protein expression. For immunization of experimental groups, Balb/c mice were injected subcutaneously with 20 µg of recombinant PBP2a three times with three weeks intervals. The sera of experimental groups were collected three weeks after the last immunization and then specific antibodies were evaluated by ELISA method. RESULTS: Successful cloning of mecA was confirmed by colony-PCR, enzymatic digestion, and sequencing. SDS-PAGE and western blot analysis showed that recombinant protein with molecular weight of 13 kDa is over expressed. In addition, high titer of specific antibody against PBP2a in vaccinated mice was developed as compared with the control group and confirmed the immunogenicity of the vaccine candidate. CONCLUSION: Results suggest that PBP2a recombinant induced specific antibodies and can be used as Staphylococcal vaccine candidate after further studies.