A novel biosensing strategy for identification of three important bacteria causing meningitis.

Bacterial meningitis is an acute infection which requires rapid diagnosis and treatment due to the high mortality and serious consequences of the disease. The purpose of this study was to design a homemade multiplex PCR and a novel fluorescence biosensor on chip (FBC) to detect three important agents of meningitis including Streptococcus pneumoniae (S. pneumoniae), Neisseria meningitidis (N. meningitidis), and Haemophilus influenzae (H. influenzae). The homemade multiplex PCR can diagnose three bacterial species simultaneously. Fabrication of FBC was carried out based on the deposition of lead nanoparticles on a quartz slide using the thermal evaporation method. Then, the SH-Cap Probe/Target ssDNA /FAM-Rep probe was loaded on lead film. The evaluation of the fluorescence reaction when the probes bind to the target ssDNA was assessed by a Cytation 5 Cell Imaging Multimode Reader Bio-Tek. The limit of detections (LOD) in homemade PCR and FBC to identify S. pneumoniae were 119 × 102 CFU/mL (0.27 ng/µL) and 380 CFU/mL (9 pg/µL), respectively. The LODs of homemade PCR and FBC for detection of N. meningitidis were 4.49 CFU/mL (1.1 pg/µL) and 13 × 103 CFU/mL (30 pg/µL), respectively. Our results confirmed the LODs of homemade PCR and FBC in detection of H. influenzae were 15.1 CFU/mL (30 fg/µL) and 41 × 102 CFU/mL (90 pg/ µL), respectively. Both techniques had appropriate sensitivity and specificity in detection of S. pneumoniae, N. meningitidis and H. influenzae.

Reduction of UreB and CagA expression level by siRNA construct in Helicobacter pylori strain SS1.

BACKGROUND: Two important virulence factors, urease and cagA, play an important role in Helicobacter pylori (H. pylori) gastric cancer. Aim of this study was to investigate the expression level and function of ureB and cagA using small interfering RNAs (siRNA). METHODS: SS1 strain of H. pylori was considered as host for natural transformation. siRNA designed for ureB and cagA genes were inserted in pGPU6/GFP/Neo siRNA plasmid vector to evaluate using phenotypic and genotypic approaches. Then, qPCR was performed for determining inhibition rate of ureB and cagA gene expression. RESULTS: The expression levels of siRNA-ureB and siRNA-cagA in the recombinant strain SS1 were reduced by about 5000 and 1000 fold, respectively, compared to the native H. pylori strain SS1. Also, preliminary evaluation of siRNA-ureB in vitro showed inhibition of urea enzyme activity. These data suggest that siRNA may be a powerful new tool for gene silencing in vitro, and for the development of RNAi-based anti-H. pylori therapies. CONCLUSION: Our results show that targeting ureB and cagA genes with siRNA seems to be a new strategy to inhibit urease enzyme activity, reduce inflammation and colonization rate.

In silico designing and immunoinformatics analysis of a novel peptide vaccine against metallo-beta-lactamase (VIM and IMP) variants.

The rapid spread of acquired metallo-beta-lactamases (MBLs) among gram negative pathogens is becoming a global concern. Improper use of broad-spectrum antibiotics can trigger the colonization and spread of resistant strains which lead to increased mortality and significant economic loss. In the present study, diverse immunoinformatic approaches are applied to design a potential epitope-based vaccine against VIM and IMP MBLs. The amino acid sequences of VIM and IMP variants were retrieved from the GenBank database. ABCpred and BCPred online Web servers were used to analyze linear B cell epitopes, while IEDB was used to determine the dominant T cell epitopes. Sequence validation, allergenicity, toxicity and physiochemical analysis were performed using web servers. Seven sequences were identified for linear B cell dominant epitopes and 4 sequences were considered as dominant CD4+ T cell epitopes, and the predicted epitopes were joined by KK and GPGPG linkers. Stabilized multi-epitope protein structure was obtained using molecular dynamics simulation. Molecular docking showed that the designed vaccine exhibited sustainable and strong binding interactions with Toll-like receptor 4 (TLR4). Finally, codon adaptation and in silico cloning studies were performed to design an effective vaccine production strategy. Immune simulation significantly provided high levels of immunoglobulins, T helper cells, T-cytotoxic cells and INF-γ. Even though the introduced vaccine candidate demonstrates a very potent immunogenic potential, but wet-lab validation is required to further assessment of the effectiveness of this proposed vaccine candidate.

Designing multi-epitope vaccine against important colorectal cancer (CRC) associated pathogens based on immunoinformatics approach.

BACKGROUND: It seems that several members of intestinal gut microbiota like Streptococcus bovis, Bacteroides fragilis, Helicobacter pylori, Fusobacterium nucleatum, Enterococcus faecalis, Escherichia coli, Peptostreptococcus anaerobius may be considered as the causative agents of Colorectal Cancer (CRC). The present study used bioinformatics and immunoinformatics approaches to design a potential epitope-based multi-epitope vaccine to prevent CRC with optimal population coverage. METHODS: In this study, ten amino acid sequences of CRC-related pathogens were retrieved from the NCBI database. Three ABCpred, BCPREDS and LBtope online servers were considered for B cells prediction and the IEDB server for T cells (CD4+ and CD8+) prediction. Then, validation, allergenicity, toxicity and physicochemical analysis of all sequences were performed using web servers. A total of three linkers, AAY, GPGPG, and KK were used to bind CTL, HTL and BCL epitopes, respectively. In addition, the final construct was subjected to disulfide engineering, molecular docking, immune simulation and codon adaptation to design an effective vaccine production strategy. RESULTS: A total of 19 sequences of different lengths for linear B-cell epitopes, 19 and 18 sequences were considered as epitopes of CD4+ T and CD8+ cells, respectively. The predicted epitopes were joined by appropriate linkers because they play an important role in producing an extended conformation and protein folding. The final multi-epitope construct and Toll-like receptor 4 (TLR4) were evaluated by molecular docking, which revealed stable and strong binding interactions. Immunity simulation of the vaccine showed significantly high levels of immunoglobulins, helper T cells, cytotoxic T cells and INF-γ. CONCLUSION: Finally, the results showed that the designed multi-epitope vaccine could serve as an excellent prophylactic candidate against CRC-associated pathogens, but in vitro and animal studies are needed to justify our findings for its use as a possible preventive measure.

Bacterial variation in the oral microbiota in multiple sclerosis patients.

BACKGROUND: Microorganisms in oral cavity are called oral microbiota, while microbiome consists of total genome content of microorganisms in a host. Interaction between host and microorganisms is important in nervous system development and nervous diseases such as Autism, Alzheimer, Parkinson and Multiple Sclerosis (MS). Bacterial infections, as an environmental factor in MS pathogenesis play role in T helper 17(Th17) increase and it enhancing the production of pro-inflammatory cytokines such as Interlukin-21(IL-21), IL-17 and IL -22. Oral microbiota consists diverse populations of cultivable and uncultivable bacterial species. Denaturing gradient gel electrophoresis (DGGE) is an acceptable method for identification of uncultivable bacteria. In this study, we compared the bacterial population diversity in the oral cavity between MS and healthy people. METHODS: From October to March 2019, samples were taken at Kermanshah University of Medical Sciences' MS patients center. A total of 30 samples were taken from MS patients and another 30 samples were taken from healthy people. Phenotypic tests were used to identify bacteria after pure cultures were obtained. DNA was extracted from 1 mL of saliva, and PCR products produced with primers were electrophoresed on polyacrylamide gels. RESULTS: The genera Staphylococcus, Actinomyces, Fusobacterium, Bacteroides, Porphyromonas, Prevotella, Veillonella, Propionibacterium and uncultivable bacteria with accession number MW880919-25, JQ477416.1, KF074888.1 and several other un-culturable strains were significantly more abundant in the MS group while Lactobacillus and Peptostreptococcus were more prevalent in the normal healthy group according to logistic regression method. CONCLUSION: Oral micro-organisms may alleviate or exacerbate inflammatory condition which impact MS disease pathogenesis. It may be assumed that controlling oral infections may result in reduction of MS disease progression.

An update review of globally reported SARS-CoV-2 vaccines in preclinical and clinical stages.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the rapidly spreading pandemic COVID-19 in the world. As an effective therapeutic strategy is not introduced yet and the rapid genetic variations in the virus, there is an emerging necessity to design, evaluate and apply effective new vaccines. An acceptable vaccine must elicit both humoral and cellular immune responses, must have the least side effects and the storage and transport systems should be available and affordable for all countries. These vaccines can be classified into different types: inactivated vaccines, live-attenuated virus vaccines, subunit vaccines, virus-like particles (VLPs), nucleic acid-based vaccines (DNA and RNA) and recombinant vector-based vaccines (replicating and non-replicating viral vector). According to the latest update of the WHO report on April 2nd, 2021, at least 85 vaccine candidates were being studied in clinical trial phases and 184 candidate vaccines were being evaluated in pre-clinical stages. In addition, studies have shown that other vaccines, including the Bacillus Calmette-Guérin (BCG) vaccine and the Plant-derived vaccine, may play a role in controlling pandemic COVID-19. Herein, we reviewed the different types of COVID-19 candidate vaccines that are currently being evaluated in preclinical and clinical trial phases along with advantages, disadvantages or adverse reactions, if any.

Assessment of biofilm formation among clinical isolates of Acinetobacter baumannii in burn wounds in the west of Iran.

Burn wound infection by A. baumannii is one of the predominant cause of mortality worldwide. The present investigation aimed at determination of antimicrobial resistance profile and expression of the biofilm-related genes in A. baumannii isolated from hospitalized patients with burn wound infection in Kermanshah hospitals. Sixty four isolates of A. baumannii were recovered from burn wound of hospitalized patients at hospitals in Kermanshah. The antimicrobial susceptibility testing (AST) was performed. Biofilm formation was measured and antibiotic resistance was compared between before and after of biofilm formation. The polymerase chain reaction (PCR) and Real-Time PCR were performed to detect of abaI and pgaD genes. The biofilm producer isolates and the most resistant isolates were exposed to ozone gas .More than 70% strains were resistance to Erythromycin, Ofloxacin, Ceftazidime, Ceftriaxone, and Ticarcillin-clavulanic acid and 50% isolates were resistant to Imipenem. Thirty one (48.4%) isolates were biofilm producer. The pgaD and abaI genes were positive in 29 (45.3%) and 9 (14%) isolates, respectively. Real time PCR demonstrated that the copy numbers of the pgaD and abaI genes after biofilm formation were increased. After exposure to ozone, biofilm formation reduced in all very strong biofilm producing isolates. Our results showed that after biofilm formation, an increased resistance was observed in most isolates. Also rising expression of abaI gene was associated with biofilm formation and an increase of antibiotic resistance. In the current study, both biofilm formation and antibiotic resistance were reduced after O3 exposure.

Colorimetric detection of Helicobacter pylori DNA using isothermal helicase-dependent amplification and gold nanoparticle probes.

This study describes a nanodiagnostic method using thermophilic helicase-dependent isothermal amplification (tHDA) and gold nanoparticle probes for colorimetric detection of Helicobacter pylori DNA. The primers targeting ureC gene were used for the amplification of bacterial DNA by the isothermal tHDA reaction, resulting in the accumulation of DNA amplicons. The amplicons were hybridized with specific gold nanoparticle probes. The hybrids were colorimetrically detected by the assembly of gold nanoparticles. Using this method, we detected as little as 10 CFU mL(-1) of H. pylori within less than 1 h. Results obtained from the gastric biopsy samples showed 92.5% and 95.4% of sensitivity and specificity, respectively, in comparison with culture results, and 100% and 98.8% of sensitivity and specificity, respectively, in comparison with those of the histologic studies. Owing to its ease of operation, this assay significantly reduces the time and cost needed for the molecular diagnosis of H. pylori and has the potential to facilitate early detection of this pathogen.