A new candidate epitope-based vaccine against PspA PhtD of Streptococcus pneumoniae: a computational experimental approach.

Introduction: Pneumococcus is an important respiratory pathogen that is associated with high rates of death in newborn children and the elderly. Given the disadvantages of current polysaccharide-based vaccines, the most promising alternative for developing improved vaccines may be to use protein antigens with different roles in pneumococcus virulence. PspA and PhtD, highly immunogenic surface proteins expressed by almost all pneumococcal strains, are capable of eliciting protective immunity against lethal infections. Methods: In this study using immunoinformatics approaches, we constructed one fusion construct (called PAD) by fusing the immunodominant regions of PspA from families 1 & 2 (PA) to the immunodominant regions of PhtD (PD). The objective of this project was to test the immunogenicity of the fusion protein PAD and to compare its protective activity against S. pneumoniae infection with PA or PD alone and a combination of PA and PD. The prediction of physicochemical properties, antigenicity, allergenicity, toxicity, and 3D-structure of the constructs, as well as molecular docking with HLA receptor and immune simulation were performed using computational tools. Finally, mice were immunized and the serum levels of antibodies/cytokines and functionality of antibodies in vitro were evaluated after immunization. The mice survival rates and decrease of bacterial loads in the blood/spleen were examined following the challenge. Results: The computational analyses indicated the proposed constructs could be antigenic, non-allergenic, non-toxic, soluble and able to elicit robust immune responses. The results of actual animal experiments revealed the candidate vaccines could induce the mice to produce high levels of antibodies and cytokines. The complement-mediated bactericidal activity of antibodies was confirmed and the antibodies provided favorable survival in immunized mice after bacterial challenge. In general, the experimental results verified the immunoinformatics studies. Conclusion: For the first time this report presents novel peptide-based vaccine candidates consisting of immunodominant regions of PspA and PhtD antigens. The obtained findings confirmed that the fusion formulation could be relatively more efficient than the individual and combination formulations. The results propose that the fusion protein alone could be used as a serotype-independent pneumococcal vaccine or as an effective partner protein for a conjugate polysaccharide vaccine.

Identification of Putative Drug Targets in Highly Resistant Gram-Negative Bacteria; and Drug Discovery Against Glycyl-tRNA Synthetase as a New Target.

Background: Gram-negative bacterial infections are on the rise due to the high prevalence of multidrug-resistant bacteria, and efforts must be made to identify novel drug targets and then new antibiotics. Methods: In the upstream part, we retrieved the genome sequences of 4 highly resistant Gram-negative bacteria (e.g., Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter cloacae). The core proteins were assessed to find common, cytoplasmic, and essential proteins with no similarity to the human proteome. Novel drug targets were identified using DrugBank, and their sequence conservancy was evaluated. Protein Data Bank files and STRING interaction networks were assessed. Finally, the aminoacylation cavity of glycyl-tRNA synthetase (GlyQ) was virtually screened to identify novel inhibitors using AutoDock Vina and the StreptomeDB library. Ligands with high binding affinity were clustered, and then the pharmacokinetics properties of therapeutic agents were investigated. Results: A total of 6 common proteins (e.g., RP-L28, RP-L30, RP-S20, RP-S21, Rnt, and GlyQ) were selected as novel and widespread drug targets against highly resistant Gram-negative superbugs based on different criteria. In the downstream analysis, virtual screening revealed that Rimocidin, Flavofungin, Chaxamycin, 11,11'-O-dimethyl-14'-deethyl-14'-methylelaiophylin, and Platensimycin were promising hit compounds against GlyQ protein. Finally, 11,11'-O-dimethyl-14'-deethyl-14'-methylelaiophylin was identified as the best potential inhibitor of GlyQ protein. This compound showed high absorption capacity in the human intestine. Conclusion: The results of this study provide 6 common putative new drug targets against 4 highly resistant and Gram-negative bacteria. Moreover, we presented 5 different hit compounds against GlyQ protein as a novel therapeutic target. However, further in vitro and in vivo studies are needed to explore the bactericidal effects of proposed hit compounds against these superbugs.

Enhanced anti-biofilm activity of the minocycline-and-gallium-nitrate using niosome wrapping against Acinetobacter baumannii in C57/BL6 mouse pneumonia model.

Acinetobacter baumannii is a worldwide health issue in terms of its high antibiotic resistance and ability to form biofilms. Nanoparticles (NPs) with high biocompatibility, high penetrating ability, and low medication dose can successfully treat the antibiotic-resistant infections. In this research, the anti-biofilm activity of niosomes containing minocycline and gallium nitrate (GaN) against A. baumannii biofilm was determined. In order to improve their anti-biofilm properties, minocycline and GaN were encapsulated in niosomes as biocompatible drug carriers. The niosomes' size, zeta potential, shape, stability, drug entrapment efficacy, drug release pattern and antibacterial activity were assessed. Several clinical samples were isolated from the lungs of patients hospitalized at Loghman hospital, Tehran, Iran. The biofilm formation of most lethal clinical isolates of A. baumannii was analyzed. The pneumonia model was generated by intranasally administering A. baumannii suspension to anesthetized mice whose immune systems was compromised twice by cyclophosphamide. Lung infection of the mouse with A. baumannii was confirmed using PCR. After treatment, the lungs were excised under sterile conditions and stained with hematoxylin and eosin (H&E) to determine histological symptoms, inflammation and intercellular secretions. The niosomes contained minocycline and GaN had an average size of 230 nm and a zeta potential of -40 mV, respectively. The percentage of drug entrapment and delayed drug release was both high in niosomal formulations. Niosomes containing minocycline and GaN dispersed 1, 3 and 5 day old biofilms. The mice given the combination of two compounds required less time to be treated than the animals given the single medication (minocycline). The minocycline& GaN-loaded niosomes could be considered as promising candidates to treat the infections caused by A. baumannii biofilm.

Immunization with recombinant DcaP-like protein and AbOmpA revealed protections against sepsis infection of multi-drug resistant Acinetobacter baumannii ST2Pas in a C57BL/6 mouse model.

BACKGROUNDS: The prevalence of infections associated with multi-drug resistant (MDR) Acinetobacter baumannii is increasing worldwide. Therefore, the introduction of effective vaccines against this bacterium seems necessary. METHODS: AbOmpA and DcaP-like protein were selected as promising and putative immunogenic candidates based on previous in silico studies. Three formulations including AbOmpA, DcaP-like protein, and AbOmpA + DcaP-like protein were injected into C57BL/6 mice three times with Alum adjuvant. The specific production of IgG antibodies (e.g. total IgG, IgG1 and IgG2c) and cytokines (e.g. IL-4, IL-6, and IL-17A), were evaluated. LD50% of MDR A. baumannii ST2Pas was measured using Probit's method. After the challenge with bacteria, a decrease in bacterial loads (DLs) in the lung and spleen of mice was measured. Then serum bactericidal assay was performed to determine the function of antibodies on day 42. In addition, histopathological examinations of the spleen and lung, the number of macrophage and neutrophil, as well as the rate of lymphocyte infiltration were assessed. RESULTS: The highest level of total IgG was reported in the group immunized with DcaP-like protein on day 42. The survival rate of mice was 80% in the AbOmpA immunized group and 100% for the rest of two groups. DLs in the spleen of mice immunized with AbOmpA, DcaP-like protein, and combination form were 3.5, 3, and 3.4 Log10 (CFU/g), respectively. While in the lung, the DLs were 7.5 Log10 (CFU/g) for the AbOmpA group and 5 for the rest of two groups. The levels of IL-6, IL-4, and IL-17A were significantly decreased in all immunized groups after the bacterial challenge (except for IL-17A in the group of AbOmpA). The bactericidal effect of antibodies against DcaP-like protein was more effective. No histopathological damage was observed in the combination immunized group. The DcaP-like protein was more effective in neutrophil and macrophage deployment and decreased lymphocyte infiltration. CONCLUSION: The results of immunization with AbOmpA + DcaP-like protein induced a protective reaction against the sepsis infection of MDR A. baumannii. It seems that in the future, these proteins can be considered as promising components in the development of the A. baumannii vaccine.

Isolation, characterization, and antibacterial activity of lytic bacteriophage against methicillin-resistant Staphylococcus aureus causing bedsore and diabetic wounds.

Background and Objectives: Phage therapy has gained interest as an alternative treatment for methicillin-resistant Staphylococcus aureus (MRSA) infections. The purpose of this study was to isolate and characterize an effective bacteriophage against isolates of MRSA. Materials and Methods: Bacteriophage was isolated from hospital sewage. Lytic activity and the titers of phage lysates were measured using spot test and double-layer plaque assay. The phage characterization was determined through transmission electron microscopy. Adsorption rate, host range and stability tests were investigated. The latent period and burst size were estimated from a one-step growth curve. The effect of bacteriophage against MRSA biofilms was determined and Real-time PCR was used to assess the effects of the bacteriophage on the expression of the biofilm-associated genes. Results: TEM results showed that the phage resembled the Cystoviridae family. Its latent period was 30 min, corresponding to about 71/43 phage particles per infected cell. The phage had a broad host range and it was most stable at 37°C and pH 7. It was sensitive to NaCl concentrations. The expressions of the biofilm-associated genes were significantly reduced in the presence of the phage. Conclusion: The isolated phage was effective against MRSA strains and it can be an optional strategy of controlling biofilm development.

Electrosprayed cefazolin-loaded niosomes onto electrospun chitosan nanofibrous membrane for wound healing applications.

Chronic wounds are among the most therapeutically challenging conditions, which are commonly followed by bacterial infection. The ideal approach to treat such injuries are synergistic infection therapy and skin tissue regeneration. In the recent decades, nanotechnology has played a critical role in eradicating bacterial infections by introducing several carriers developed for drug delivery. Moreover, advances in tissue engineering have resulted in new drug delivery systems that can improve the skin regeneration rate and quality. In this study, cefazolin-loaded niosomes were electrosprayed onto chitosan membrane for wound healing applications. For this purpose, niosomes were obtained by the thin-film hydration method; electrospinning was then conducted to fabricate nanofibrous mats. In vitro characterization of the scaffold was performed to evaluate the physicochemical and biological properties. Finally, in vivo studies were carried out to evaluate the potential use of the membrane for skin regeneration. In vitro results indicated the antibacterial properties of the membrane against Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) due to the gradual release of cefazolin from niosomes. The scaffolds also showed no cell toxicity. In vivo studies also confirmed the ability of the membrane to enhance skin regeneration by improving re-epithelialization, tissue remodeling, and angiogenesis. The current study could well show the promising role of the prepared scaffold for skin regeneration and bacterial infection elimination.

Effects of cefazolin-containing niosome nanoparticles against methicillin-resistant Staphylococcus aureus biofilm formed on chronic wounds.

The ability of biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA) causes significant mortality and morbidity in wound infections. Nanoparticles because of the drug concentration increment at the point of contact of nanoparticles and bacteria, and slower release of the drug at the desired location are considered as proper tools to overcome the therapeutic problem of antimicrobial-resistant infections. This study was aimed to evaluate the anti-biofilm activity of cefazolin-loaded nanoparticles against MRSA isolates. The 27 clinical isolates of MRSA were collected from patients with pressure sores and diabetic ulcers referred to Loghman Hospital in Tehran-Iran. MRSA isolates were detected by polymerase chain reaction (PCR) and biochemical tests. Cefazolin-loaded niosome was synthesized using the thin-film hydration method and were characterized by zeta potential measurement and transmission electron microscopy (TEM). The round-shaped cefazolin-loaded niosomes had a diameter of 100 nm and a -63 mV zeta potential. The cefazolin-containing niosomes removed 1, 3, and 5 d old biofilms at the concentration of 128 µg ml-1, 128 µg ml-1, and 256 µg ml-1, respectively. Histological results indicated that BALB/c mice receiving cefazolin-loaded niosomes were treated effectively faster than those treated by cefazolin or untreated group. In conclusion, the cefazolin-loaded niosome could be considered as a promising candidate for the treatment of biofilm-mediated infections of MRSA.