Osteoblastic Differentiation of Stem Cells from Human Exfoliated Deciduous Teeth by Probiotic Hydroxyapatite.

OBJECTIVE: Multipotent cells derived from human exfoliated deciduous teeth (SHED) possess the ability to differentiate into various cell types, including osteoblasts. This study aims to simulate the growth induction and osteogenic differentiation of SHED cells using probiotics and their resultant biomaterials. MATERIALS AND METHODS: This experimental study proceeded in two stages. Initially, we evaluated the effect of autoclaved nutrient agar (NA) grown probiotic Bacillus coagulans (B. coagulans) on the SHED and MG-63 cell lines. Subsequently, probiotics grown on the Pikovskaya plus urea (PVKU) medium and their synthesised hydroxyapatite (HA) were identified using X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and Fourier transform infrared spectroscopy (FTIR), and then used to stimulate growth and osteogenic differentiation of the SHED cell line. Osteoblast cell differentiation was assessed by morphological changes, the alkaline phosphatase (ALP) assay, and alizarin red staining. RESULTS: There was a substantial increase in SHED cell growth of about 14 and 33% due to probiotics grown on NA and PVKU medium, respectively. The PVKU grown probiotics enhanced growth and induced stem cell differentiation due to HA content. Evidence of this differentiation was seen in the morphological shift from spindle to osteocyte-shaped cells after five days of incubation, an increase in ALP level over 21 days, and detection of intracellular calcium deposits through alizarin red staining-all indicative of osteoblast cell development. CONCLUSION: The osteogenic differentiation process in stem cells, improved by the nano-HA-containing byproducts of probiotic bacteria in the PVKU medium, represents a promising pathway for leveraging beneficial bacteria and their synthesised biomaterials in tissue engineering.

Simultaneous Production of Antibacterial Protein and Lipopeptides in Bacillus tequilensis, Detected by MALDI-TOF and GC Mass Analyses.

As antibiotic resistance is nowadays one of the important challenges, efforts are crucial for the discovery of novel antibacterial drugs. This study aimed to evaluate antimicrobial/anticancerous activities of halophilic bacilli from the human microbiota. A spore-forming halotolerant bacterium with antibacterial effect against Staphylococcus aureus was isolated from healthy human feces. The antibacterial protein components of the extracted supernatant were identified by SDS-PAGE and zymography. The MALDI-TOF, GC mass, and FTIR analyses were used for peptide and lipopeptide identification, respectively. The stability, toxicity, and anticancerous effects were investigated using MTT and Flow cytometry methods. According to the molecular analysis, the strain was identified as Bacillus tequilensis and showed potential probiotic properties, such as bile and acid resistance, as well as eukaryotic cell uptake. SDS-PAGE and zymography showed that 15 and 10-kDa fragments had antibacterial effects. The MALDI-TOF mass analysis indicated that the 15-kDa fragment was L1 ribosomal protein, which was the first report of the RpL1 in bacilli. GC-mass and FTIR analyses confirmed the lipopeptide nature of the 10-kDa fragment. Both the extracted fractions (precipitation or "P" and chloroform or "C" fractions) were stable at < 100 °C for 10 min, and their antibacterial effects were preserved for more than 6 months. Despite its non-toxicity, the P fraction had anticancer activities against MCF7 cells. The anticancer and antibacterial properties of B. tequilensis, along with its non-toxicity and stability, have made it a potential candidate for studying the beneficial probiotic properties for humans and drug production.

Novel Chronic Wound Healing by Anti-biofilm Peptides and Protease.

Chronic wounds have made a challenge in medical healthcare due to their biofilm infections, which reduce the penetrance of the antibacterial agents in the injury site. In infected wounds, the most common bacterial strains are Staphylococcus aureus and Pseudomonas aeruginosa. Biofilm disruption in chronic wounds is crucial in wound healing. Due to their broad-spectrum antibacterial properties and fewer side effects, anti-biofilm peptides, especially bacteriocins, are promising in the healing of chronic wounds by biofilm destruction. This study reviews the effects of antimicrobial and anti-biofilm agents, including bacteriocins and protease enzymes as a novel approach, on wound healing, along with analyzing the molecular docking between a bacterial protease and biofilm components. Among a large number of anti-biofilm bacteriocins identified up to now, seven types have been registered in the antimicrobial peptides (AMPs) database. Although it is believed that bacterial proteases are harmful in wound healing, it has recently been demonstrated that these proteases like the human serine protease, in combination with AMPs, can improve wound healing by biofilm destruction. In this work, docking results between metalloprotease from Paenibacillus polymyxa and proteins of S. aureus and P. aeruginosa involved in biofilm production, showed that this bacterial protease could efficiently interact with biofilm components. Infected wound healing is an important challenge in clinical trials due to biofilm production by bacterial pathogens. Therefore, simultaneous use of proteases or anti-biofilm peptides with antimicrobial agents could be a promising method for chronic wound healing.

Predatory and biocontrol potency of Bdellovibrio bacteriovorus toward phytopathogenic strains of Pantoea sp. and Xanthomonas campestris in the presence of exo-biopolymers: in vitro and in vivo assessments.

Bdellovibrios are predatory bacteria that invade other live Gram-negative bacterial cells for growth and reproduction. They have recently been considered as potential living antibiotics and biocontrol agents. In this study, the predatory activity and biocontrol potency of Bdellovibrio bacteriovorus strain SOIR-1 against Pantoea sp. strain BCCS and Xanthomonas campestris, two exo-biopolymer-producing phytopathogens, was evaluated. Plaque formation assays and lysis analysis in the broth co-cultures were used for the in vitro evaluation of bacteriolytic activity of strain SOIR-1. The in vivo biocontrol potential of strain SOIR-1 was evaluated by pathogenicity tests on the onion bulbs and potato tuber slices. The phytopathogens were also recovered from the infected plant tissues and confirmed using biochemical tests and PCR-based 16S rRNA gene sequence analysis. Typical bdellovibrios plaques were developed on the lawn cultures of Pantoea sp. BCCS and X. campestris. The killing rate of strain SOIR-1 toward Pantoea sp. BCCS and X. campestris was 84.3% and 76.3%, respectively. Exo-biopolymers attenuated the predation efficiency of strain SOIR-1 up to 10.2-18.2% (Pantoea sp. BCCS) and 12.2-17.3% (X. campestris). The strain SOIR-1 significantly reduced rotting symptoms in the onion bulbs caused by Pantoea sp. BCCS (69.0%) and potato tuber slices caused by X. campestris (73.1%). Although more field assessments are necessary, strain SOIR-1 has the preliminary potential as a biocontrol agent against phytopathogenic Pantoea sp. BCCS and X. campestris, especially in postharvest storage. Due to the particular physicochemical properties of evaluated exo-biopolymers, they can be used in the designing encapsulation systems for delivery of bdellovibrios.

A Novel Approach on Leukodepletion Filters: Investigation of Synergistic Anticancer Effect of Purified α-Defensins and Nisin.

Purpose: There are number of reports available regarding defensins activity against mammalian cells besides their antimicrobial and immune regulatory activities. This study aims to investigate anticancer and apoptosis activity of the purified defensins from leukodepletion filters alone or in synergism with bacterial peptide, nisin, on prostate and colorectal cancer. Methods: Leucoflex LCR-5 filters were backflushed by an optimized elution system. Isolated granulocytes were sonicated and the supernatant treated before further purification by high performance liquid chromatography (HPLC). SDS-PAGE and western blot testing verified the fraction. Cell culture on PC-3 (human prostate adenocarcinoma), and HCT-116 (human colorectal carcinoma) were conducted following by MTT assays in addition to annexin flow cytometry for sole and synergistic effects with peptide nisin. Results: Viable and active neutrophils could recover, and α-defensins were extracted and purified. Combinations of an optimal dose of α-defensins and nisin showed a remarkable synergistic effect on cancer cell lines (over 90% and 70% for PC-3 and HCT-116, respectively). Conclusion: It also observed that less than 40% of both cells could survive after co-treatment with optimal dose. Also, apoptosis was increased after treatment by these peptides together. Annexin Vpositive populations significantly increased in percentage in comparison with control.

Characterization of the first highly predatory Bdellovibrio bacteriovorus from Iran and its potential lytic activity against principal pathogenic Enterobacteriaceae.

OBJECTIVES: Bdellovibrio-and-like organisms (BALOs) are predatory prokaryotes that attack and kill other Gram-negative bacteria for growth and reproduction. This study describes the isolation, identification, biological properties, and bacteriolytic activity of the first Bdellovibrio bacteriovorus with a broad prey range from Iran. MATERIALS AND METHODS: One BALO strain with high predatory potency was isolated from the rhizosphere soil using Enteropathogenic Escherichia coli as prey. It was identified and designated as Bdellovibrio bacteriovorus strain SOIR-1 through plaque assays, transmission electron microscopy (TEM), Bdellovibrio-specific PCRs, and 16S rRNA gene sequence analysis. Biological characterization and analysis of bacteriolytic activity were also performed. RESULTS: TEM and Bdellovibrio-specific PCRs confirmed that the strain SOIR-1 belongs to the genus Bdellovibrio. Analysis of the 16S rRNA gene sequence revealed its close phylogenetic relationship with strains of Bdellovibrio bacteriovorus. The strain SOIR-1 grew within the temperature range of 25-37 °C and the pH range of 6.0-8.0, with the optimal predatory activity at 30 °C and pH 7.4. It had the highest and lowest bacteriolytic activity toward Shigella dysenteriae and Pseudomonas aeruginosa with a killing rate of 89.66% and 74.83%, respectively. CONCLUSION: Considering the hypothesis of bdellovibrios heterogeneity, identification of new isolates contributes to a deeper understanding of their diversity, their ecological roles, and their promising potential as living antibiotics or biocontrol agents. Bdellovibrios with broad bacteriolytic nature has not previously been reported in sufficient detail from Iran. The results of this study showed the great potential of native B. bacteriovorus strain SOIR-1 in the control and treatment of diseases caused by pathogenic Enterobacteriaceae.

Inhibition of quorum sensing-controlled virulence factors with natural substances and novel protease, obtained from Halobacillus karajensis.

INTRODUCTION: In recent years, a challenge in clinical treatment has developed due to bacterial resistance to antibiotics. One of the new mechanisms against infections is virulence factor inhibition. Many virulence factors are controlled by quorum sensing pathways such as biofilm formation and pyocyanin production. The goal of the present study was to investigate the effect of an obligate halophilic bacterial strain on Pseudomonas aeruginosa and Staphylococcus aureus, due to its halo-tolerant substances and enzymes. METHODS: The effect of Halobacillus karajensis on bacterial growth and production of virulence factors was studied in this work. The obligate halophile cells and supernatant fractions were extracted by the methanol/chloroform method and characterized by Fourier Transform Infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Gas Chromatography-Mass Spectrometry (GC-MS), and zymography. The effects of these fractions were studied on biofilm formation in P. aeruginosa and S. aureus as well as on pyocyanin production in P. aeruginosa. The effective protein in the fraction was analyzed by the SDS-PAGE method, and all protein fragments were studied for pyocyanin inhibition. RESULTS: The crude supernatant extract, MMS fraction, from H. karajensis was effective for the biofilm reduction in S. aureus (74%) and P. aeruginosa (27%). Two proteases in this fraction, which were recognized by zymography on skim milk, were the probable causes for extracellular polymeric substances (EPS) hydrolysis in the biofilm matrix. Also, halide crystals and branched fatty acids, 12methyl-tetradecanoic acid, in the other fractions decreased the biofilm by 18% in S. aureus. The results showed that a new 25 kD protein, which was obtained from MMS fraction, inhibited pyocyanin production by 60% in P. aeruginosa. The zymogram and bioinformatics studies showed that this protein was a serine alkaline metalloprotease and had an interaction with AHL molecules. CONCLUSION: The inhibitory effects of the non-toxic natural substances and proteases on biofilm formation and pyocyanin production, specifically the 25 kD protease, are novel in this study and make them a good candidate for infected wound healing and inhibiting the virulence factors.

Bacterial Natural Compounds with Anti-Inflammatory and Immunomodulatory Properties (Mini Review).

Inflammation is part of the body's complex biological response to harmful stimuli such as damaged cells, pathogens, or irritants. It is a protective response involving blood cells, immune cells, and molecular mediators. The inflammation not only can eliminate the primary cause of cell injury but also clears out necrotic cells, tissue damaged from the original insults and inflammatory process. Furthermore, it can initiate tissue repair. Pro-inflammatory cytokines are produced predominantly by activated macrophages and are involved in the up-regulation of inflammatory reactions. They are involved in further regulating inflammatory reactions. There is ample evidence that some pro-inflammatory cytokines, such as interleukin 1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α), are involved in the pathological pain process. Some of the natural compounds promote cytokines production and inhibit inflammatory responses. The natural compounds which are produced from microorganisms such as omega-3 fatty acid, cyclic peptide, antimicrobial peptide, oligosaccharides, and polysaccharides can reduce inflammation and could be easily incorporated into the diet without any adverse effects. For example, SCFA (short-chain fatty acids), peptide bacteriocin, and polycyclic peptide bacteriocin (nisin) could be used in the treatment of atherosclerosis, orthopedic postoperative infections, and mycobacterium tuberculosis infection, respectively. Also, fatty acids (saturated and unsaturated fatty acids) can be introduced as anti-inflammatory drugs. This review article summarizes bacterial natural compounds with modulating effects on cytokines that are surveyed which may have potential anti-inflammatory drug-like activity.

The Efficacy of Electrospun PAN/Kefiran Nanofiber and Kefir in Mammalian Cell Culture: Promotion of PC12 Cell Growth, Anti-MCF7 Breast Cancer Cells Activities, and Cytokine Production of PBMC.

BACKGROUND: Kefiran is a useful polysaccharide made of branched glucogalactose which is produced by microorganisms. Here the anti-MCF-7 breast cancer cells activity of kefiran and cytokine productions (IL-6) of peripheral blood mononuclear cells (PBMC) treated by kefiran was studied. Also, the effect of using kefiran as a useful and cost-effective scaffold in neural stem cell culture (PC12 cell culture) was investigated. MATERIAL AND METHODS: Kefiran was produced from raw milk with 0.5% fat and 10 g of kefir grains. After incubation for 48 hrs at room temperature, the solvent collected (crude kefiran). These samples were kept at 100°C for 1 hr (boiled kefiran) and the supernatant was precipitated by ethanol (pure kefiran). Then, the electrospun nanofibers, pure polyacrylonitrile (PAN), PAN/kefiran 5%, and PAN/kefiran 10% were fabricated and used as scaffolds in the cell culture. The structure of fabricated was studied by SEM and the cytokine production (IL-6) in vitro in the cell culture supernatant of PBMC line after treatment with kefiran (1mg/mL, 5 mg/mL) and kefiran-PAN 5% and 10% were carried out by enzyme-linked immunosorbent assay (ELISA). The attachment of PC12 cells was examined by inverted microscope. Also, cytotoxicity of kefiran for PC12 and MCF7 cells and morphological changes of PC12 cells were evaluated by MTT and Cresyl violet staining (Nissl staining) respectively. RESULTS: The mean diameter of fabricated PAN/kefiran 5% and 10% nanofibers were 310.2±43.97 nm. The contact angle measurement results (26.9± 1.9 for the pure PAN scaffold vs 12.3± 1.13 for the PAN/kefiran) revealed enhanced hydrophilicity of scaffolds upon the incorporation of kefiran and PAN. Seeding of PC12 cells on the scaffolds showed that fabrication of kefiran into PAN led to the enhancement of cell attachment, proliferation, and morphological changes. Also, the promotion of PBMC growth and decreasing of MCF7 cell lines viability were shown through MTT assay. No significant changes were measured for the level of IL-6 in PAN/kefiran 5% treated cells compared to the control (p ≥ 0.05). CONCLUSION: These results suggest superior properties of kefiran/PAN nanofibrous scaffolds for the neural stem cell culture especially for repairing injured spinal cord. Also, the pure kefiran could be used for the enhancement of PBMC growth and reducing the MCF7 cancerous cells growth. So, using biocompatible, anti-bacterial, and anti-tumor kefiran/PAN nanofibers for regenerative medicine seems promising.

Multifunctional Polymeric Nanoplatforms for Brain Diseases Diagnosis, Therapy and Theranostics.

The blood-brain barrier (BBB) acts as a barrier to prevent the central nervous system (CNS) from damage by substances that originate from the blood circulation. The BBB limits drug penetration into the brain and is one of the major clinical obstacles to the treatment of CNS diseases. Nanotechnology-based delivery systems have been tested for overcoming this barrier and releasing related drugs into the brain matrix. In this review, nanoparticles (NPs) from simple to developed delivery systems are discussed for the delivery of a drug to the brain. This review particularly focuses on polymeric nanomaterials that have been used for CNS treatment. Polymeric NPs such as polylactide (PLA), poly (D, L-lactide-co-glycolide) (PLGA), poly (ε-caprolactone) (PCL), poly (alkyl cyanoacrylate) (PACA), human serum albumin (HSA), gelatin, and chitosan are discussed in detail.

Characterization and In Silico Analysis of The Structural Features of G-CSF Derived from Lysates of Escherichia coli.

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) has a wide variety of functions including stimulation of hematopoiesis and proliferation of granulocyte progenitor cells. Recombinant human G-CSF (rh-G-CSF) is used for treatment of neutropenia in patients receiving chemotherapy. The mature bloodstream neutrophils express G-CSF receptor (G-CSFR), presenting a significant and specific mechanism for circulating G-CSF clearance. Computational studies are essential bioinformatics methods used for characterization of proteins with regard to their physicochemical properties and 3D configuration, as well as protein-ligand interactions for recombinant drugs. We formerly produced rh-G-CSF in E. coli and showed that the isolated protein had unacceptable biological activity in mice. In the present paper, we aimed to characterize the purified rh-G-CSF by analytical tests and developed an in vivo model by computational modelling of G-CSF. MATERIALS AND METHODS: In this experimental study, we analyzed the purified G-CSF using the analytical experiments. Then, the crystalline structure was extracted from Protein Data Bank (PDB) and molecular dynamics (MD) simulation was performed using Gromacs 5.1 package under an Amber force field. The importance of amino acid contents of G-CSF, to bind the respective receptor was also detected; moreover, the effect of dithiothreitol (DTT) used in G-CSF purification was studied. RESULTS: The results revealed that characteristics of the produced recombinant G-CSF were comparable with those of the standard G-CSF and the recombinant G-CSF with the residual amino acid was stable. Also, purification conditions (DTT and existence of extra cysteine) had a significant effect on the stability and functionality of the produced G-CSF. CONCLUSION: Experimental and in silico analyses provided good information regarding the function and characteristics of our recombinant G-CSF which could be useful for industrial researches.

Molecular Evidence of Chlamydia trachomatis Infection and Its Relation to Miscarriage.

BACKGROUND: Chlamydia trachomatis (CT) infection is the most common sexually transmitted disease in the world that can persist and also ascend in the genital tract. This intracellular and silent infection is related to some adverse pregnancy outcomes, such as miscarriage. The aims of this study were to explore the best CT screening tests using blood and vaginal samples and to investigate the correlation between CT infection and the incidence of miscarriage. MATERIALS AND METHODS: This case-control study was done in October 2013 through June 2014, using purposive sampling from 157 female participants with or without a history of miscarriage. The samples were taken after each participant had signed a letter of consent and had completed a questionnaire. To achieve the objectives of this study, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) tests were performed on vaginal swabs and blood samples, respectively. RESULTS: PCR results showed a significantly higher CT infection rate in the miscarriage group compared to the control group (11.3 vs. 0%, P=0.007). Anti-CT IgG and IgA antibodies were found in 4.2 and 2.1% of cases in the miscarriage group, and in 1.7 and 6.7% of cases in the control group, respectively (P>0.05). Despite lower humoral responses in this study, positive samples were detected only by one of the following techniques; PCR, ELISA IgA and ELISA IgG. It also should be noted that PCR worked best in terms of detection. CONCLUSION: Based on the obtained data, there is a strong association between molecular evidence of CT infection and miscarriage. A higher rate of CT detection in molecular tests compared to serological assays suggests that PCR could be used as the first-choice assay for detection of C. trachomatis. However, the importance of serological tests in detecting potential past CT infection or upper genital infection not amenable to sampling is undeniable.

The interaction of beta-lactoglobulin with ciprofloxacin and kanamycin; a spectroscopic and molecular modeling approach.

A vast research has been conducted to find suitable and safe carriers for vital and pH-sensitive drugs including antibiotics. This article reports the use of easily accessible and abundant purified beta-lactoglobulin (ß-LG) protein as the potential carrier of widely used Kanamycin (Kana) and Ciprofloxacin (Cip) antibiotics. Spectroscopic techniques (Fluorescence, UV-vis, Circular Dichroism) combined with molecular docking were used to determine the binding mechanism of these drugs. Fluorescence studies showed moderate binding affinity with the calculated binding constants KCip = 60.1 (±0.2) × 103 M-1 and Kkana = 2.5 (±0.6) × 103 M-1 with the order of Cip > Kana. Results of UV-vis were consistent with fluorescence measurements and demonstrated a stronger complexation for Cip rather than Kana. The secondary structure of ß-LG was preserved upon interaction with Kana; however, a reduction in ß-sheet content from 39.1 to 31.9% was convoyed with an increase in α-helix from 12.8 to 20.5% due to complexation of Cip. Molecular docking studies demonstrated that preferred binding sites of these drugs are not the same and several amino acids are involved in stabilizing the interaction. Based on the achieved results, Kana and Cip can spontaneously bind to ß-LG and this protein may serve as their transport vehicle.

Comparison of serological and molecular test for diagnosis of infectious mononucleosis.

BACKGROUND: Epstein-Bar virus (EBV) is the main etiology of infectious mononucleosis (IM) syndrome that is characterized by fever, sore throat, and lymph adenopathy. Since, this virus could be associated with a number of malignancies, some hematologic disorders, and chronic fatigue syndrome, identification of IM is very important. The aim of study was to evaluate the specificity, as well as sensitivity of the two different methods that is, serology versus molecular diagnosis that are currently used for diagnosis of IM. MATERIALS AND METHODS: In this study, during a period of 3.5 years, 100 suspected patients as case group and 100 healthy individuals as a control group were studied. Fifty samples in each group were tested by polymerase chain reaction (PCR) and all the samples including case group and control group were carried out by enzyme-linked immunosorbent assay (ELISA). RESULTS: In 76% of patients and in 20% of the healthy individuals, samples were detected EBV DNA by PCR. On the other hand, 68.5% of the samples belong to the case group and 46% in the control group showed positivity by ELISA. CONCLUSION: By comparing the two methods, since PCR is very expensive and time consuming, and the percentages of difference ranges are narrow, ELISA could be applied as a first, easiest, and preliminary diagnostic test for IM. In addition, this test could be applied in various phases of the disease with a higher sensitivity comparing to PCR. Although PCR is routinely used for diagnosis of various infectious agents, it is considered as an expensive test and merely could be used after 1-2 weeks from the onset of the illness.

The beneficial effects of vitamin D3 on reducing antibody titers against Epstein-Barr virus in multiple sclerosis patients.

Recently, the relationship between immunoreactivity to Epstein-Barr virus (EBV) and hypo-vitamin D in multiple sclerosis (MS) patients has been described. The aim of this study was to investigate whether vitamin D3 supplementation in MS patients could influence the immune response against latent EBV infection. Forty MS patients were recruited in this study. Twenty-seven patients were supplemented with 50,000 IU/week of vitamin D3 for 6 months and thirteen enrolled as controls. 25-Hydroxyvitamin D (25OHD) levels and IgG titers against EBNA1 and VCA were determined pre- and post-supplementation. All the patients were seropositive for EBV prior to vitamin D supplementation. In this cohort, 22.5% and 47.5% of the MS patients had deficient and insufficient levels of 25OHD, respectively. Our findings confirm that antibody titers against EBV in MS patients rise after the onset of the disease and indicate that vitamin D3 supplementation could limit augmentation of these titers in MS patients.

Production of nano zinc, zinc sulphide and nanocomplex of magnetite zinc oxide by Brevundimonas diminuta and Pseudomonas stutzeri.

ZnO (Zincite) nanoparticle has many industrial applications and is mostly produced by chemical reactions, usually prepared by decomposition of zinc acetate or hot-injection and heating-up method. Synthesis of semi-conductor nanoparticles such as ZnS (Sphalerite) by ultrasonic was previously reported. In this work, high-zinc tolerant bacteria were isolated and used for nano zinc production. Among all isolated microorganisms, a gram negative bacterium which was identified as Brevundimonas diminuta could construct nano magnetite zinc oxide on bacterial surface with 22 nm in size and nano zinc with 48.29 nm in size. A piece of zinc metal was immersed in medium containing of pure culture of B. diminuta. Subsequently, a yellow-white biofilm was formed which was collected from the surface of zinc. It was dried at room temperature. The isolated biofilm was analysed by X-ray diffractometer. Interestingly, the yield of these particles was higher in the light, with pH 7 at 23°C. To the best of the authors knowledge, this is the first report about the production of nano zinc metal and nano zinc oxide that are stable and have anti-bacterial activities with magnetite property. Also ZnS (sized 12 nm) produced by Pseudomonas stutzeri, was studied by photoluminescence and fluorescent microscope.

Surveillance of single-cell behavior in different subpopulations of Ralstonia pickettii AR1 during growth and polyhydroxybutyrate production phases by flow cytometry.

Most bacterial strains accumulate intracellular polyhydroxybutyrate (PHB) granules as an energy reservoir, in response to fluctuations in their microenvironment. Flow cytometry was applied for the analysis of single cells of Ralstonia pickettii AR1 in response to changes in the culture conditions. Two parameters, the PHB production-related FL2 and side scatter (SSC) parameters, were used to monitor, distinguish and characterize different subpopulations in the growth and PHB production phases. A high SSC level was observed in the mid-log exponential growth phase. When the nitrogen source reached a limiting level, the SSC started to decrease, in contrast to the intracellular PHB granules-related FL2 parameter. The results show that ammonium limitation is a critical and important factor for the accumulation of reserve compounds. Four subpopulations were observed and distinguished upon entrance of the cells into the exponential growth phase. When the cells entered the late exponential growth or early stationary phase, two subpopulations had disappeared and only two, different subpopulations were dominant. One of the subpopulations with changed SSC and PHB production activity switched to another subpopulation that was only active in PHB production in the stationary phase. The whole cells of R. pickettii AR1 tended to form a homogeneous population at the end of the stationary phase. In fact, the changes in the subpopulations of a single strain are related to different physiological states of the cells. The observation of different subpopulations suggests that each subpopulation shows a specific response to changes in the surrounding microenvironment, nutrients and limiting factors.

Genotyping of Endocervical Chlamydia trachomatis Strains and Detection of Serological Markers of Acute and Chronic Inflammation in Their Host.

BACKGROUND: Chlamydia trachomatis (C. trachomatis) is the most prevalent cause of bacterial sexually transmitted infections (STI) recognized throughout the world. The aim of this study is to determine different genotypes of genital C. trachomatis and the association between the serological markers of inflammation and genotypes of C. trachomatis in sexually active women (n=80) attending Shahid Beheshti Hospital in Isfahan, Iran. MATERIALS AND METHODS: In this descriptive study, endocervical swabs were collected from 80 women. There were 17 endocervical samples that showed positivity for C. trachomatis by plasmid polymerase chain reaction (PCR) using KL1 and KL2 primers. The omp1 gene was directly amplified in 17 plasmid PCR positive samples and was used to differentiate the clinical genotypes by omp1 gene PCR-restriction fragment length polymorphism (PCR-RFLP). The levels of IgG and IgA specific to C. trachmatis and C-reactive protein (CRP) were evaluated. RESULTS: Based on restriction-digestion patterns, four genotypes were identified. Genotypes E (35.3%) and F (35.3%) were the most prevalent, followed by D/Da (23.5%) and K (5.9%). There was no significant association between genotypes and the presence of IgG and CRP. Patients infected with genotype E showed a serological marker of chronic inflammation, i.e. IgA seropositivity, significantly more than patients infected with other genotypes (p=0.042). CONCLUSION: Nested PCR could increase the sensitivity of omp1 amplification. Based on the presence of IgA, chronic C. trachomatis infections were observed more frequently among genotype E-infected patients in our population.

Comparison of three methods of DNA extraction in endocervical specimens for Chlamydia trachomatis infection by spectrophotometry, agarose gel, and PCR.

Chlamydia trachomatis is the major cause of sexually transmitted disease in the world. The aim of this study was to determine the best method of DNA extraction for detecting C. trachomatis by polymerase chain reaction (PCR) in sexually active women (n = 80) attending Shahid Beheshti Hospital in Isfahan, Iran. Endocervical swabs were collected from 80 women, 22 of whom were asymptomatic and 58 symptomatic. Three different DNA extraction methods were used in this study (phenol-chlorophorm, proteinase K, and boiling). DNA yield was evaluated by spectrophotometry, agarose gel, and PCR. The internal control was assayed by beta-globin primers (PCO4, GH20). The DNA cryptic plasmid was selected as the target for C. trachomatis and samples were examined by PCR using specific KL1 and KL2 primers. It was shown that DNA extraction by boiling was the most sensitive with the highest yield of DNA. Of the 80 samples, 17 (21.25%) showed positivity for C. trachomatis by PCR. The highest rate of C. trachomatis infection was found in the group aged between 35 and 45 years old and those who used withdrawal or an intrauterine device as methods of contraception. It was demonstrated that DNA extraction by boiling was the least expensive and a very rapid method that gave the highest DNA yield. The infection rate in the sexually active women, including symptomatic and asymptomatic, was 21.25%, with a presumably high prevalence compared with other studies done in this field.