The Inhibitory Effects of Silver Nanoparticles on Bap Gene Expression in Antibiotic-Resistant Acientobacter bumanni Isolates using Real-Time PCR

Introduction: Acinetobacter bomanni is one of the most common opportunistic pathogens in hospitals that are resistant to many antibiotics due to biofilm production. Nowadays, silver nanoparticles(AgNPs) have received wide attention in medicine due to their physical and chemical properties. This study aimed to evaluate the anti-biofilm activity of silver nanoparticles on antibiotic-resistant A. bumanni strains.   Materials & Methods: In this experimental study, A. bumanni was isolates from 100 clinical samples. After identification of A. bummani strains and determination of antibiotic resistant profiles, biofilm-producing isolates were determined using Congo red agar at 37°C after 24 hours. The minimum inhibitory concentration (MIC) of the strains against AgNPs was determined. After 24 hours of exposure of the strains to sub-MIC concentration of AgNPs, RNA extraction and cDNA synthesis were performed. Finally, Bap gene expression was measured using real time PCR method. Finding: Out of 100 clinical isolates, 12 belonged to A. bummani, and all the strains were resistant to antibiotics, except for colistin. Real-time PCR results show that all the strains had a significant down-regulation in Bap gene expression compared to the control gene (P<0.05). Discussion & Conclusions: According to the anti-biofilm effects of AgNPs, it seems that AgNPs can be used as drug candidates in pharmaceutical industries

Fabrication and Characterization of pcDNA3.1(+) Location within Chitosan/Nanoparticles Complexes for Enhanced Gene Delivery

Background: Chitosan nanoparticles (CSNP) are becoming a popular alternative for delivering nucleic acids to tissues for gene transfer (gene therapy). The size and morphology of these biodegradable nano-carriers are adjustable, and their positive charge allows them to interact strongly with negatively charged nucleic acids. Objective: This study aimed to fabricate and characterize pcDNA3.1 (+) plasmid (pDNA) and CSNP complexes and determine the plasmid location in these vehicles. Materials and Methods: The characteristics of the pDNA/CSNP complex after production were investigated by SEM, XRD, DLS, TGA, and FTIR. The capacity of CSNP to form complexes with pDNA was investigated by labeling free plasmids with the fluorescent intercalating dye OliGreen. The stability of pDNA/CSNP in the presence of chitosanase was evaluated. Surface-Enhanced Raman Spectroscopy (SERS) for pDNA localization was performed, and absorption rate in BALB/c mice was assessed by real-time PCR. Results: The optimum pDNA/CSNP ratio for plasmid complex formation was established to be 1:2 (w.w) by measuring spectroscopy. At these optimum complex formation ratios, spectroscopy, and gel digest experiments, SERS indicated that a part of the pDNA was present on the complex outer surface. The findings of plasmid absorption in mouse thigh tissue by real-time PCR revealed that the rate of gene uptake was significantly greater at a dose of 1:2 (w.w) of pDNA/CSNP than in other groups (P< 0.001). Conclusions: The findings of this study reveal exactly pDNA fits into polymer nanostructured delivery systems, allowing the formulation to be adjusted for selective distribution. This understanding will aid future research into the system’s functioning in vitro and in vivo

Novel adjuvant nano-vaccine induced immune response against Acinetobacter baumannii

Developing adjuvant vaccines to combat rising multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) infections is a promising and cost-effective approach. The aim of this analysis was to construct a pDNA-CPG C274-adjuvant nano-vaccine and investigate its immunogenicity and protection in BALB/c mice. The CPG ODN C274 adjuvant was chemically synthesized and cloned into pcDNA3.1(+), and the cloning was verified using PCR and BamHI/EcoRV restriction enzyme digestion. Then, utilizing a complex coacervation approach, pDNA-CPG C274 was encapsulated by chitosan (CS) nanoparticles (NPs). TEM and DLS are used to explore the properties of the pDNA/CSNP complex. TLR-9 pathway activation was investigated in human HEK-293 and RAW 264.7 mouse cells. The vaccine's immunogenicity and immune-protective effectiveness were investigated in BALB/c mice. The pDNA-CPG C274/CSNPs were small (mean size 79.21 ± 0.23 nm), positively charged (+ 38.87 mV), and appeared to be spherical. A continuous slow release pattern was achieved. TLR-9 activation was greatest in the mouse model with CpG ODN (C274) at concentrations of 5 and 10 μg/ml with 56% and 55%, respectively (**P < 0.01). However, in HEK-293 human cells, by increasing the concentration of CpG ODN (C274) from 1 to 50 μg/ml, the activation rate of TLR-9 also increased, so that the highest activation rate (81%) was obtained at the concentration of 50 μg/ml (***P < 0.001). pDNA-CPG C274/CSNPs immunized BALB/c mice produced increased amounts of total-IgG, as well as IFN-γ and IL-1B in serum samples, compared to non-encapsulated pDNA-CPG C274. Furthermore, liver and lung injuries, as well as bacterial loads in the liver, lung, and blood, were reduced, and BALB/c mice immunized with pDNA-CPG C274/CSNPs showed potent protection (50–75%) against acute fatal Intraperitoneal A. baumannii challenge. pDNA-CPG C274/CSNPs evoked total-IgG antibodies, Th1 cellular immunity, and the TLR-9 pathway, as well as protection against an acute fatal A. baumannii challenge. Our findings suggest that this nano-vaccine is a promising approach for avoiding A. baumannii infection when used as a powerful adjuvan

Identification of Antigenic Properties of Acinetobacter baumannii Proteins as Novel Putative Vaccine Candidates Using Reverse Vaccinology Approach

Multidrug-resistant Acinetobacter baumannii (A. baumannii) infections are becoming more prevalent all over the world. As a cost-effective and preventative method, vaccination seems to be required against this bacterium. In the present study, subtractive proteomics along with reverse vaccinology approaches was used to predict suitable therapeutics against A. baumannii. Using the Vaxign online tool, we studied over 35 genomes of A. baumannii strains and chose outer membrane and secreted proteins of A. baumannii 1656-2 as possible vaccine candidates. Then, investigations were performed on the immunogenicity, antigenic characteristics, physicochemical properties, B-cell and MHC class I, and MHC class II molecules epitope densities of proteins. After optimizing the codon of the proteins, the pcDNA3.1( +) expression construct was designed and the immunogenicity, allergenicity, and physicochemical properties of the vaccine construct were predicted. Hcp and OmpC proteins were predicted as extracellular and outer membrane proteins, respectively. These proteins interact with 10 other proteins to form a network of protein interactions with virulence properties. Immunoassays of Hcp and OmpC proteins showed antigenicity of 0.88 and 0.79, respectively. These proteins have 5 structural cell epitope points and 5 linear B epitope points. They are also able to bind to different HLA alleles of MCH class I/class II as selected immunogenic proteins and designed non-allergenic structures with solubility of 0.650 and immunogenicity score of 0.91. The results of this "in silico" study indicate high specificity and the development of a significant humoral and cellular immune response. It can be concluded that the Hcp and OmpC dual vaccine construct is one of the promising candidates against A. baumannii. The findings of this "in silico" study show excellent specificity and the emergence of a substantial humoral and cellular immune response. This is a computer-based study that needs to be tested in vitro and in vivo to corroborate the conclusions of the vaccine design procedures

Oral administration of DNA alginate nanovaccine induced immune-protection against Helicobacter pylori in Balb/C mice

Abstract Background Helicobacter pylori (H. Pylori), is an established causative factor for the development of gastric cancer and the induction of persistent stomach infections that may lead to peptic ulcers. In recent decades, several endeavours have been undertaken to develop a vaccine for H. pylori, although none have advanced to the clinical phase. The development of a successful H. pylori vaccine is hindered by particular challenges, such as the absence of secure mucosal vaccines to enhance local immune responses, the absence of identified antigens that are effective in vaccinations, and the absence of recognized indicators of protection. Methods The DNA vaccine was chemically cloned, and the cloning was verified using PCR and restriction enzyme digestion. The efficacy of the vaccination was investigated. The immunogenicity and immune-protective efficacy of the vaccination were assessed in BALB/c mice. This study demonstrated that administering a preventive Alginate/pCI-neo-UreH Nanovaccine directly into the stomach effectively triggered a robust immune response to protect against H. pylori infection in mice. Results The level of immune protection achieved with this nano vaccine was similar to that observed when using the widely accepted formalin-killed H. pylori Hel 305 as a positive control. The Alginate/pCI-neo-UreH Nanovaccine composition elicited significant mucosal and systemic antigen-specific antibody responses and strong intestinal and systemic Th1 responses. Moreover, the activation of IL-17R signaling is necessary for the defensive Th1 immune responses in the intestines triggered by Alginate/pCI-neo-UreH. Conclusion Alginate/pCI-neo-UreH is a potential Nanovaccine for use in an oral vaccine versus H. pylori infection, according to our findings

Retracted: Novel NAD‐independent Avibacterium paragallinarum: Isolation, characterization and molecular identification in Iran

Abstract Background Infectious coryza (IC) is an invasive upper respiratory disease caused by Avibacterium paragallinarum that affects birds, particularly chickens. The objective of this study is to isolate, characterize and molecularly identify the bacterium A. paragallinarum in poultry birds, as well as to determine its antibiotic sensitivity and resistance. Methods A total of 10 chickens from four different Iranian farms with typical IC symptoms were used in this study. The nasal swabs were streaked onto chocolate agar plates and blood agar plates and incubated at 37°C in 5% CO2 for 24 to 48 h. As part of the identification of bacteria, bacteriological observations and polymerase chain reaction (PCR) testing are conducted. The antibiotic sensitivity tests were also performed using the disk diffusion method against A. paragallinarum and the prevalence in different farms was determined. Results By using biochemical assays and PCR analyses, seven strains of A. paragallinarum were isolated from samples of four chicken farms with typical IC clinical signs. Most isolates (4/7) showed the typical requirement for nicotinamide adenine dinucleotide (NAD) and an enriched CO2 atmosphere for growth. Three of the seven strains of A. paragallinarum were found to be novel NAD‐independent under anaerobic conditions. There was one biochemical biovar identified in terms of carbohydrate fermentation patterns, although changes in maltose carbohydrate fermentation patterns were detected in the No. 5 strain. All isolates were sensitive to gentamicin and spectinomycin. Three novel NAD‐independent strains (Nos.1, 5 and 7) were found to be multidrug‐resistant (MDR) and resistant to at least three classes of antibiotics. There was greater antibiotic resistance in the three NAD‐independent isolates than in normal NAD‐dependent bacteria. Conclusion By discovering NAD‐independent forms of A. paragallinarum, these species have a greater range than previously believed. A clear, cautious approach should be taken in diagnosing and possibly controlling IC

Additional file 1 of Oral administration of DNA alginate nanovaccine induced immune-protection against Helicobacter pylori in Balb/C mice

Supplementary Material

Simultaneous and rapid detection of avian respiratory diseases of small poultry using multiplex reverse transcription-Polymerase Chain Reaction assay

ABSTRACT: Major viral infections, such as Newcastle disease virus, infectious bronchitis virus, avian influenza virus, and infectious bursal disease virus, inflict significant injury to small poultry and tremendous economic damage to the poultry sector. This research aims to develop a multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) approach to simultaneously determine these important viral pathogens. The conserved segment of various viral genetic sequences was used to design and synthesize specific primers. Moreover, as positive controls, recombinant vectors were synthesized in this investigation. The d-optimal approach was used to improve PCR conditions in this investigation. Positive controls and clinical samples were used to assess the m-PCR assay's specificity, sensitivity, repeatability, and reproducibility. According to the sensitivity test findings, the m-PCR technique could generate the 8 target genes from viral genomes using 1 × 102. In addition, 8 viral pathogens were detected from the infected samples. The findings also suggest that live animal oral swabs were not significantly different from tissue sampling of a dead animal (P < 0.05), and this kit had a high sensitivity for analyzing both types of samples. The suggested m-PCR test may detect and evaluate viral infection in birds with excellent specificity, sensitivity, and throughput