Production and Evaluation of Ag85B:HspX:hFcγ1 Immunogenicity as an Fc Fusion Recombinant Multi-Stage Vaccine Candidate Against Mycobacterium tuberculosis.

An urgent need is to introduce an effective vaccine against Mycobacterium tuberculosis (M.tb) infection. In the present study, a multi-stage M.tb immunodominant Fcγ1 fusion protein (Ag85B:HspX:hFcγ1) was designed and produced, and the immunogenicity of purified protein was evaluated. This recombinant fusion protein was produced in the Pichia pastoris expression system. The HiTrap-rPA column affinity chromatography purified and confirmed the fusion protein using ELISA and Western blotting methods. The co-localisation assay was used to confirm its proper folding and function. IFN-γ, IL-12, IL-4, and TGF-ß expression in C57BL/6 mice then evaluated the immunogenicity of the construct in the presence and absence of BCG. After expression optimisation, medium-scale production and the Western blotting test confirmed suitable production of Ag85B:HspX:hFcγ1. The co-localisation results on antigen-presenting cells (APCs) showed that Ag85B:HspX:hFcγ1 properly folded and bound to hFcγRI. This strong co-localisation with its receptor can confirm inducing proper Th1 responses. The in vivo immunisation assay showed no difference in the expression of IL-4 but a substantial increase in the expression of IFN-γ and IL-12 (P ≤ 0.02) and a moderate increase in TGF-ß (P = 0.05). In vivo immunisation assay revealed that Th1-inducing pathways have been stimulated, as IFN-γ and IL-12 strongly, and TGF-ß expression moderately increased in Ag85B:HspX:hFcγ1 group and Ag85B:HspX:hFcγ1+BCG. Furthermore, the production of IFN-γ from splenocytes in the Ag85B:HspX:hFcγ1 group was enormously higher than in other treatments. Therefore, this Fc fusion protein can make a selective multi-stage delivery system for inducing appropriate Th1 responses and is used as a subunit vaccine alone or in combination with others.

Photoinactivation Effects of Curcumin, Nano-curcumin, and Erythrosine on Planktonic and Biofilm Cultures of Streptococcus mutans.

Introduction: This in vitro study was conducted to assess the phototoxic effects of curcumin, nano-curcumin, and erythrosine on the viability of Streptococcus mutans (S. mutans) in suspension and biofilm forms. Methods: Various concentrations of curcumin (1.5 g/L, 3 g/L), nano-curcumin (3 g/L), and erythrosine (100 µM/L, 250 µM/L) were examined for their impact on planktonic and biofilm cultures of S. mutans, either individually or in conjunction with light irradiation (photodynamic therapy or PDT). A blue light-emitting diode (LED) with a central wavelength of 450 nm served as the light source. The results were compared to 0.12% chlorhexidine digluconate (CHX) as the positive control, and a solution containing neither a photosensitizer (PS) nor a light source as the negative control group. The dependent variable was the number of viable microorganisms per experiment (CFU/mL). Results: Antimicrobial PDT caused a significant reduction in the viability of S. mutans in both planktonic and biofilm forms, compared to the negative control group (P<0.05). The highest cell killing was observed in PDT groups with curcumin 3 g/L or erythrosine 250 µmol/L, although the difference with PDT groups using curcumin 1.5 g/L or erythrosine 100 µmol/L was not significant (P>0.05). Antimicrobial treatments were more effective against planktonic S. mutans than the biofilm form. Conclusion: PDT with either curcumin 1.5 g/L or erythrosine 100 µmol/L may be suggested as an alternative to CHX to inactivate the bacteria in dental plaque or deep cavities. Nano-curcumin, at the selected concentration, exhibited lower efficacy in killing S. mutans compared to Curcumin or erythrosine.

Sustain-release lipid-liquid crystal formulations of pexiganan against Helicobacter pylori infection: in vitro evaluation in C57BL/6 mice.

INTRODUCTION: The Gram-negative bacterium Helicobacter pylori, H. pylori, is associated with significant digestive disorders. However, the effectiveness of bacterial eradication is declining due to drug resistance. A potent anti-H. pylori activity is shown by the natural antimicrobial peptide pexiganan. OBJECTIVE: The current study aimed to evaluate the effectiveness of pexiganan and its lipid-liquid crystals (LLCs) in inducing Helicobacter pylori in mice. METHODS: In this experimental study, H. pylori infection was first induced in C57BL/6 mice. Secondly, the antibacterial efficacy of pexiganan and its LLCs formulations was investigated to eliminate H. pylori infection. RESULTS: The H. pylori infection could not be completely eradicated by pexiganan peptide alone. However, incorporating pexiganan within the LLC formulation resulted in an increased elimination of H. pylori. Under the H&E strain, the pexiganan-LLCs formulation revealed minimal mucosal alterations and a lower amount of inflammatory cell infiltration in the stomach compared to the placebo. CONCLUSION: Clarithromycin was more effective than pexiganan at all tested concentrations. Furthermore, the pexiganan-loaded LLCs exhibited superior efficacy in curing H. pylori infection in a mouse model compared to pexiganan alone. This formulation can enhance H. pylori clearance while mitigating the adverse effects, typically associated with conventional drugs, leading to a viable alternative to current treatment options.

Antimicrobial photodynamic therapy against Lactobacillus casei using curcumin, nano-curcumin, or erythrosine and a dental LED curing device.

This study aimed to investigate the photodynamic effects of curcumin, nanomicelle curcumin, and erythrosine on Lactobacillus casei (L. casei). Various concentrations of curcumin (1.5 g/L, 3 g/L), nano-curcumin (3 g/L), and erythrosine (100 µM/L, 250 µM/L) were tested either alone or combined with light irradiation (PDT effect) against L. casei in planktonic and biofilm cultures. The light was emitted from a light-emitting diode (LED) with a central wavelength of 450 nm. A 0.12% chlorhexidine digluconate (CHX) solution served as the positive control, and a solution containing neither photosensitizer nor light was the negative control group. The number of viable microorganisms was determined using serial dilution. There was a significant difference in the viability of L. casei in both planktonic and biofilm forms (P < 0.05). In the planktonic culture, the antibacterial effects of CHX and PDT groups with curcumin 3 g/L and erythrosine 250 µM/L were significantly greater than the other groups (P < 0.05). For L. casei biofilms, the greatest toxic effects were observed in CHX and PDT groups with curcumin 3 g/L, erythrosine 250 µmol/L, erythrosine 100 µmol/L, and nanomicelle curcumin 3 g/L, with a significant difference to other groups (P < 0.05). The antibacterial effects of all photosensitizers (except erythrosine 250 µmol/L at planktonic culture) enhanced significantly when combined with light irradiation (P < 0.05). PDT with curcumin 3 g/L or erythrosine 250 µmol/L produced comparable results to CHX against L. casei at both planktonic and biofilm cultures. Alternatively, PDT with erythrosine 100 µmol/L or nanomicelle curcumin 3 g/L could be suggested to kill L. casei biofilms.

Mucosal and systemic immunization against tuberculosis by ISCOMATRIX nano adjuvant co-administered with alginate coated chitosan nanoparticles.

Objectives: BCG vaccine has no longer been appreciated to immunize against tuberculosis, worldwide, so novel appropriate adjuvants have been dedicated to improve immune responses. This study aimed to evaluate the immunomodulatory effects of ISCOMATRIX as an adjuvant to stimulate potent humoral and cellular immune responses of the PPE17 loaded alginate coated nanoparticles through subcutaneous and intranasal vaccination. Materials and Methods: Size, polydispersity index, and morphology of the resulting colloidal particles were explored by dynamic light scattering (DLS). The cellular and/or humoral immune stimulation properties of ISCOMATRIX adjuvant were measured by measuring the level of IFNγ, IL-4, IL-17, and TGFß in spleen cell cultures and IgG1 and IgG2a in serum and sIgA in nasal lavage of immunized mice, respectively. Results: The spherical cage-like particles of ISCOMATRIX adjuvant have optimal size of 59±6 nm appropriate for an immune adjuvant vaccine. ISCOMATRIX induced robust Th1 (IFN-γ) and IL-17 cytokine response also significant IgG2a and IgG1antibodies in both subcutaneous and intranasal routes and elicited mucosal sIgA response when administered intranasally. As a booster for BCG, ISCOMATRIX induced immune responses only in subcutaneous route. Conclusion: These findings indicate that ISCOMATRIX is a promising adjuvant with the potential for increasing cellular and humoral immunity both after subcutaneous and intranasal administration.

Anticancer activity of Pseudomonas aeruginosa derived peptide with iRGD in colon cancer therapy.

Objectives: Colon cancer is well-known as a life-threatening disease. Since the current treatment modalities for this type of cancer are powerful yet face some limitations, finding novel treatments is required to achieve better outcomes with fewer side effects. Here we investigated the therapeutic potential of Azurin-p28 alone or along with iRGD (Ac-CRGDKGPDC-amide) as a tumor-penetrating peptide and 5-fluorouracil (5-FU) for colon cancer. Materials and Methods: Inhibitory effect of p28 with or without iRGD/5-FU was studied in CT26 and HT29, as well as the xenograft animal model of cancer. The effect of p28 alone or along with iRGD/5-FU on cell migration, apoptotic activity, and cell cycle of the cell lines was assessed. Level of the BAX and BCL2 genes, tumor suppressor genes [(p53 and collagen type-Iα1 (COL1A1), collagen type-Iα2 (COL1A2)] were assessed by quantitative RT-PCR. Results: These findings show that using p28 with or without iRGD and 5-FU raised the level of p53 and BAX but decreased BCL2, compared with control and 5-FU groups in tissues of the tumor, which result in raising the apoptosis. Conclusion: It seems that p28 may be used as a new therapeutic approach in colon cancer therapy that can enhance the anti-tumor effect of 5-FU.

Novel approaches to overcome Colistin resistance in Acinetobacter baumannii: Exploring quorum quenching as a potential solution.

Acinetobacter baumannii is responsible for a variety of infections, such as nosocomial infections. In recent years, this pathogen has gained resistance to many antibiotics, and thus, carbapenems were used to treat infections with MDR A. baumannii strains in clinical settings. However, as carbapenem-resistant isolates are becoming increasingly prevalent, Colistin is now used as the last line of defense against resistant A. baumannii strains. Unfortunately, reports are increasing on the presence of Colistin-resistant phenotypes in infections caused by A. baumannii, creating an urgent need to find a substitute way to combat these resistant isolates. Quorum sensing inhibition, also known as quorum quenching, is an efficient alternative way of reversing resistance in different Gram-negative bacteria. Quorum sensing is a mechanism used by bacteria to communicate with each other by secreting signal molecules. When the population of bacteria increases and the concentration of signal molecules reaches a certain threshold, bacteria can implement mechanisms to adapt to a hostile environment, such as biofilm formation. Biofilms have many advantages for pathogens, such as antibiotic resistance. Different studies have revealed that disrupting the biofilm of A. baumannii makes it more susceptible to antibiotics. Although very few studies have been conducted on the biofilm disruption through quorum quenching in Colistin-resistant A. baumannii, these studies and similar studies bring hope in finding an alternative way of treating the Colistin-resistant isolates. In conclusion, quorum quenching has the potential to be used against Colistin-resistant A. baumannii.

Genotyping of Mycobacterium tuberculosis complex isolated from humans and animals in northeastern Iran.

The objective of this study was to genotype Mycobacterium tuberculosis complex isolated from humans and cattle in northern Iran. Over the course of one year, a total of 120 human and 21 cattle isolates were tested using region of difference (RD)-based polymerase chain reaction (PCR) and mycobacterial interspersed repetitive unites-variable number tandem repeats (MIRU-VNTR). In M. tuberculosis, out of 120 isolates investigated, the most common genotype detected was NEW-1 (53.3%), followed by CAS/ Delhi (24.1%), Haarlem (5%), Beijing (4.16%), Uganda I (4.16%), S (3.3%), Ural (0.83%), TUR (0.83%), Uganda II (0.83%), Lam (0.83%) and Cameroon (0.83%). The HGDI rate was 0.9981 and the clustering rate was 10.83. Of the isolates, QUB26 had the highest allele diversity (h: 0.76), while the loci Mtub29 and MIRU24 had the lowest (h: 0). In M. Bovis, out of 123 collected tissue samples, 21 (17%) grew on culture media. The HGDI rate was 0.71 and clustering rate was 85.7%. The locus ETRC had the highest allele diversity (h: 0.45). The findings of this study suggest that there is high genetic diversity among M. tuberculosis isolates in Khorasan Razavi Province, which is consistent with similar results from other studies in other provinces in Iran and neighboring countries. This indicates that the prevalent genotypes in this study are spreading in the Middle East region. Furthermore, considering that M. Bovis isolates were identified in two clusters, it seems that all of them have a common origin and are circulating among the livestock farms in the province.

Characterization of carbapenem-resistant Escherichia coli and Klebsiella: a role for AmpC-producing isolates.

Aim: This study aimed to investigate the role of AmpC enzymes in carbapenem resistance among AmpC/extended-spectrum ß-lactamase (ESBL)-producing clinical isolates of Escherichia coli and Klebsiella spp. Methods: Fifty-six bacterial strains that were AmpC producers were examined. The antibiotic susceptibility test was performed by the disk diffusion and E-test. The prevalence of the plasmid carbapenemase was determined using PCR. Results: The resistance to meropenem in the AmpC+/ESBL+ group was 64%, higher than that reported for the AmpC-/ESBL+ group. Ten isolates of the carbapenem-resistant AmpC producers were negative for carbapenemase-encoding genes. Conclusion: Carbapenem resistance among AmpC-producing isolates with negative results for carbapenemase-encoding genes potentially demonstrates the role of AmpC enzymes among these isolates.

Gene Expression Study of Host and Mycobacterium tuberculosis Interactions in the Manifestation of Acute Tuberculosis.

Mycobacterium tuberculosis (M.tb) could induce type IV hypersensitivity. The chemotaxis of the leukocytes toward the site of infection and producing matrix metalloproteinases (MMPs) are key factors in the immune pathogenesis of tuberculosis (TB). Mononuclear cells were isolated from bronchoalveolar lavage (BAL) specimens, and the target from genomic DNA was used for qPCR TB diagnosis and cDNA for specific RT-qPCR gene expression. The subjects were then classified into TB+ and TB- groups, and the expression levels of CFP-10, ESAT-6, CCR1, CCR12 and MMP3,9 were evaluated. The mean level of CCR1 expression in TB+ and TB- patients' BAL was 1.71 ± 0.78 and 0.5 ± 0.22, respectively, which was statistically different (p = 0.01). The CCR2 level, in TB+ (2.07 ± 1.4), was higher than in TB- patients (1.42 ± 0.89, p = 0.01). The MMP9 expression in TB+ was 2.56 ± 0.68, also higher than in TB- patients (1.13 ± 0.35), while MMP3 was lower in TB+ (0.22 ± 0.09) than in TB- (0.64 ± 0.230, p = 0.05). The CCR2/CCR1 and MMP3/MMP9 balance in TB+ were reduced, compared to the TB-. The CFP-10 and ESAT-6 were highly expressed in TB+ patients. The CFP-10 expression had a strong negative correlation with albumin (r = - 0.93, p = 0.001), and a negative correlation with neutrophil (r = - 0.444, p = 0.1 with 90% CI). The MMP-9 expression showed a positive correlation with WBC count (r = 0.61, p = 0.02), in TB+, and had a negative correlation with BMI (r = 0.59, p = 0.02) in TB-. The M.tb CFP-10 might be implicated in lowering CCR2 and MMP3 expression in favour of M.tb dissemination. Moreover, the balance of CCR2/CCR1 and MMP3/MMP9 can be used as prognostic factors in the severity of TB.

Effects of probiotic yogurt, casein phosphopeptide-amorphous calcium phosphate, and xylitol chewing gums on the salivary count of Streptococcus mutans: A single-blinded randomized controlled clinical trial.

Background: Dental caries is a preventable multifactorial disease, with Streptococcus mutans being suggested to be its primary pathogen. Our study aim was to compare the effects of three different low-cost and easy-to-use regimens with that of the gold standard (chlorhexidine [CHX] mouthwash) on the count of salivary S. mutans in dental students over 30 days. Materials and Methods: In this single-blinded parallel randomized controlled clinical trial, a total of 120 dental students were included and randomly allocated into four intervention groups: (1) CHX mouthwash (control(, (2) probiotic yogurt, (3) casein phosphopeptide-amorphous calcium phosphate chewing gum, and (4) xylitol chewing gum. Salivary S. mutans counts were evaluated at baseline, 15 days, and 30 days after initiation of the study and compared at different times and among different groups using the repeated measures analysis of variance design analysis and least significant difference test with SPSS software version 20. The level of significance was determined to be 0.05. Results: The microorganism count variable at baseline, first, and second follow-ups was significantly different for all groups except the probiotic yogurt group (P = 0.340). S. mutans count was significantly different when comparing the first follow-up and baseline values in the CHX and xylitol gum groups (P = 0.027, P = 0.037). When comparing the second follow-up with baseline values, a significant difference was observed in the xylitol gum group (P = 0.003). Conclusion: Xylitol chewing gum seems to be a viable alternative to the gold standard (CHX mouthwash) in reducing the salivary count of S. mutans.

Antimicrobial peptides: natural or synthetic defense peptides against HBV and HCV infections.

According to the literature, treatment of HCV and HBV infections faces challenges due to problems such as the emergence of drug-resistant mutants, the high cost of treatment, and the side effects of current antiviral therapy. Antimicrobial peptides (AMPs), a group of small peptides, are a part of the immune system and are considered as an alternative treatment for microbial infections. These peptides are water-soluble with amphiphilic (hydrophilic and hydrophobic surfaces) characteristics. AMPs are produced by a wide range of organisms including both prokaryotic and eukaryotic cells. The antiviral mechanisms of AMPs include inhibiting virus entry, inhibiting intracellular virus replication, inhibiting intracellular viral packaging, and inducing immune responses. In addition, AMPs are a new generation of antiviral biomolecules that have very low toxicity for human host cells, particularly liver cell lines. AMPs can be considered as one of the most important strategies for developing new adjuvant drugs in the treatment of HBV and HCV infections. In the present study, several groups of AMPs (with a net positive charge) such as Human cathelicidin, Claudin-1, Defensins, Hepcidin, Lactoferrin, Casein, Plectasin, Micrococcin P1, Scorpion venom, and Synthetic peptides were reviewed with antiviral properties against HBV and HCV.

Evaluation of Mutations Related to Streptomycin Resistance in Mycobacterium tuberculosis Clinical Isolates.

Drug resistance to streptomycin in the clinical isolates of Mycobacterium tuberculosis (MTB) needs special consideration. It can mostly be caused by mutations in four genes with the names rpsL, rrs, gidB, and whiB7. The main objective of this study was the evaluation of the type and frequency of mutations in these mentioned genes using the PCR-sequencing method. This study was performed on 15 streptomycin-resistant and five streptomycin-sensitive isolates. Among resistant isolates, 11 samples contained mutations in codon 43 of the rpsL gene, which caused the lysine to be converted to arginine. Additionally, all of the isolates had mutations in the gidB. Missense mutations in codons 92 and 20 of this gene result in the amino acids Glutamic acid or Arginine being changed to Aspartic acid or Proline, respectively. No mutations in the rrs or whiB7 were found in any of the samples. Simultaneous mutations of rpsL and gidB were found in 10 isolates, the majority of which were Beijing strain. The results showed that the mutations of rpsL and gidB genes are mostly responsible for the streptomycin resistance in the evaluated MTB isolates. Furthermore, the discovery of dual mutations in Beijing strains highlights the strain's considerable potential for developing Tuberculosis drug resistance.

Assessment of antibiotic resistance changes during the Covid-19 pandemic in northeast of Iran during 2020-2022: an epidemiological study.

BACKGROUND: The coronavirus disease 2019 seems to change antibiotic resistance pattern. Certain conditions in the Covid-19 era may be contributing to the rise of antimicrobial resistance (AMR). Due to the limited information on the impact of Covid-19 on antimicrobial resistance (AMR), the purpose of this research was to investigate the trend in antimicrobial resistance changes of E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii in Hasheminezhad hospital. This hospital was a Corona center in Mashhad at the onset of this epidemic. METHODS: 1672 clinical samples were collected between January 21, 2020 and January 30, 2022from patients hospitalized at Hasheminezhad Hospital in Mashhad, Conventional microbiological procedures for identifying gram-negative bacteria and antibiotic susceptibility testing were used, according to the clinical and laboratory standards institute (CLSI) 2021. The two years of the pandemic, from the initial stage of the outbreak until the 6th peak, (January 2020 to and January 2022) were divided into 9 periods according to the seasons. RESULTS: Highest resistance rates were seen in E. coli (615 samples), K. pneumoniae (351 samples), P. aeruginosa (362 samples) and A. baumannii (344 samples) to Ampicillin (89.6%), Ampicillin (98%), Imipenem (91.8%), and Ceftazidime (94.6%), respectively. The largest change in antibiotic resistance was seen between Summer 2020 and Summer 2021 for K. pneumoniae with about a 30% rise in antibiotic resistance to Ceftriaxone. CONCLUSIONS: All 4 species evaluated in this study, have shown rising AMR rates during the first year of the pandemic in the northeast of Iran. This study revealed that E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii strains in Northern Iran have a higher level of antibiotic resistance than what was measured in similar studies conducted before the pandemic. This will further restrict treatment choices and jeopardize global public health.

Effects of Magnesium Oxide Nanoparticles Incorporation on Shear Bond Strength and Antibacterial Activity of an Orthodontic Composite: An In Vitro Study.

This study aimed to evaluate the effects of magnesium oxide (MgO) nanoparticle (NP) incorporation on shear bond strength (SBS) and antibacterial property of orthodontic composites. A total of 100 mounted premolar teeth were randomly divided into five groups. In group 1 (control), the brackets were bonded to the teeth using the GC Ortho Connect orthodontic composite, while the brackets of groups 2 to 5 were bonded by the GC Ortho Connect orthodontic composite that contained 0.5%, 1%, 2%, and 4% weight percentages (w/w) of MgO NPs, respectively and then the SBS was measured. In the following, we evaluated the antibacterial properties of the MgO NP-containing composite on Streptococcus mutans (S. mutans) bacteria by the direct contact test method. According to results, there were no significant changes in the SBS as the MgO NP concentration was increased up to 1%, while the SBSs of the 2% and 4% MgO NPs were decreased when compared to the other three groups. The outcomes of the direct contact test indicated the case of 1% as being the minimum ratio of MgO NPs, which almost caused the entire annihilation of the S. mutans bacteria. In conclusion, the orthodontic composite containing 1% MgO NPs can display a significant antibacterial effect against S. mutans bacteria without inducing any negative effect on the SBS.

Bacterial Peptides and Bacteriocins as a Promising Therapy for Solid Tumor.

The conventional treatment is faced with limitations in treating solid tumors due to their specific pathophysiology. Several novel therapeutics have been introduced in recent decades to treat solid tumors. Among these new methods, tumor therapy using bacterial products like bacteriocins and peptides has been of great interest due to their unique characteristics and advantages of them in comparison to the conventional treatment, including that they can precisely target tumor cells, selective toxicity for tumor cells, low side effect on normal cells, toxicity activity for MDR cancer cells, used as the target delivery vehicles and enhancing drug delivery. Moreover, their small size and low molecular weight have made them easy to synthesize and modify. Furthermore, in recent years, genetic engineering has expanded the therapeutic ability of peptides to treat solid tumors, which results in overcoming the peptide drawbacks. The present review mainly focuses on the new advances in applying bacterial peptides and bacteriocins in treating human solid tumors.