Designing a Multiple-Epitope Vaccine Candidate against Leishmania major and Leishmania infantum for Monocyte-Derived Exosome Preparation.

Background: Leishmania is a vector-borne protozoon, which causes visceral, cutaneous and mucocutaneous leishmaniosis in human and animals. Monocyte-derived exosome vaccines can be used as prophylaxis and immunotherapy strategies. The aim of this study was to design a multiple-epitope candidate vaccine using leishmaniolysin (GP63) and rK39 proteins against Leishmania major and L. infantum for monocyte-derived exosome preparation. Methods: This study was carried out in Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran, 2023-2024. Effective immunodominant epitopes were selected from two antigenic proteins of GP63 and rK39 using various immunoinformatics and bioinformatics approaches. Vibrio cholerae ß-subunit was used as an adjuvant to stimulate immune responses. Then, appropriate linkers were selected for the fusion of epitopes. The 3D model of candidate vaccine was predicted and validated. Results: This designed candidate vaccine could effectively be used as a prophylaxis strategy against leishmaniosis. Conclusion: A candidate vaccine was designed using bioinformatic and immunoinformatic studies with virtual acceptable quality; however, effectiveness of this vaccine should be verified through further in-vitro and in-vivo studies.

Comparative Analysis of 198 SARS-CoV-2 Genomes from Iran and West Asia, February 2020 to December 2021.

Background & Objective: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in a worldwide pandemic. The first case of COVID-19 was reported from Wuhan in the Hubei Province of China in December 2019; however, the disease's origin is still mysterious. Whole-genome sequence analysis is essential for monitoring the spread of infectious diseases as well as studying the pathogenesis and evolution of viruses. In this study, analysis of 198 fully sequenced genomes from Iran and West Asia was carried out to study mutations, phylogeny, amino acid changes, clades, and lineages of these genomes as well as comparison of these sequences with those of reference Wuhan genome of NC_045512.2. Methods: In total, 198 completely sequenced genome data from Iran and West Asia were collected from GenBank. Mutation detection was carried out using a trial version of CLC Genomics Workbench v.21.0 (QIAGEN, Germany). Online tools such as GISAID Mutations App and Pangolin were used for further analysis of the results. Results: In this study, several unique mutation sites were identified in the Iranian genomes (n = 8); positions 1397 G>A and 29742 G>T were the most frequent changes in more than 85% of the Iranian genomes. Mutation rate, mutation per sequence, and transition versus transversion for the Iranian genomes included 4.73, 14.14, and 1.6, respectively. Generally, C>T alteration was the most common substitution in all the sequences. Conclusion: The ORF1ab, N, and S were the genes with the most changes. The current data can help researchers predict future epidemics and establish better strategies to control viral pandemics.

Lytic potential of a filamentous bacteriophage isolated from sewage water in Tehran on clinical carbapenem-resistant strains.

Background and Objectives: Acinetobacter baumannii (A. baumannii), an opportunistic pathogen, has been isolated from sewage, soil, and hospital wards. The prevalence of multidrug-resistance A. baumannii has seriously caused a health crisis in hospital settings. Bacteriophages have been used as an alternative therapy for control carbapenem-resistant bacteria-caused infections. We aimed to assay lytic effect of a filamentous phage on clinical bacterial strains. Materials and Methods: 500-ml water samples was collected from sewage in Tehran. Sewage samples were precipitated at 6000 rpm for 10 minutes and filtered using 0.45-µm syringe filters. Bacteriophage was isolated using double-layer agar assay and evaluated its stability at various pH and temperature ranges. In addition, the stability of the phage was assayed at chloroform 0.1%. Results: Transmission electron microscopy imagine showed phage is filamentous called vB-AbaI-TMU2. The phage affected on its own host so that could not effect on any Pseudomonas aeruginosa and Klebsiella pneumoniae strains. vB-AbaITMU2 phage was stable at pH 5 and 7 and also temperatures 25 and 37°C. vB-AbaI-TMU2 was stable at chloroform 0.1%. vB-AbaI-TMU2 phage infected carbapenem-resistant A. baumannii strains while other bacterial strains were resistant to. Conclusion: The present study indicated the isolated phage had a narrow host range and is susceptible to various pH values and temperatures.

Effects of Poly-N-isopropylacrylamide Microgels Containing Antibiofilm Substances on Pseudomonas aeruginosa Isolated from Chronic Wounds.

Background: Biofilm formation helps Pseudomonas aeruginosa (P. aeruginosa) survive in various environments. Microgels can be effective in treatment of bacterial infections. The major aim of this study was to investigate effects of poly-N-isopropylacrylamide microgels (PNIPAM) on P. aeruginosa. Methods: Totally, 100 P. aeruginosa strains were isolated from chronic wound infections. Quantitative assessments of biofilm formation and antibiotic susceptibility were carried out. Furthermore, algD, lasR, and PA2714 genes were amplified to investigate gene frequencies and expression rates. Results: Significant decreases were seen in lasR expression in EDTA-treated samples. Significant decreases were observed in expression of algD and lasR treated with xylitol. Decreased expression of PA2714 was seen in samples treated with xylitol with no significance. Conclusion: The PNIPAM containing xylitol or EDTA could penetrate biofilms of P. aeruginosa and significantly decrease expression of lasR and algD. This can be a novel strategy in the management of chronic wounds.

Genome Analysis of an Enterococcal Prophage, Entfac.MY.

Background: Bacteriophages are bacterial parasites. Unlike lytic bacteriophages, lysogenic bacteriophages do not multiply immediately after entering the host cells and may integrate their genomes into the bacterial genomes as prophages. Prophages can include various phenotypic and genotypic effects on the host bacteria. Enterococcus spp. are Gram-positive bacteria that cause infections in humans and animals. In recent decades, these bacteria have become resistant to various antimicrobials, including vancomycin. The aim of this study was to analyze genome of an enterococcal prophage. Methods: In this study, Enterococcus faecium EntfacYE was isolated from biological samples and its genome was analyzed using next-generation sequencing method. Results: Overall, 254 prophage genes were identified in the bacterial genome. The prophage included 39 housekeeping, 41 replication and regulation, 80 structural and packaging, and 48 lysis genes. Moreover, 46 genes with unknown functions were identified. All genes were annotated in DNA Data Bank of Japan. Conclusion: In general, most prophage genes were linked to packaging and structure (31.5%) gene group. However, genes with unknown functions included a high proportion (18.11%), which indicated necessity of further analyses. Genomic analysis of the prophages can be effective in better understanding of their roles in development of bacterial resistance to antibiotics. Moreover, identification and study of prophages can help researchers develop genetic engineering tools and novel infection therapies.

Genome Analysis of the Enterococcus faecium Entfac.YE Prophage.

Background: Bacteriophages are viruses that infect bacteria. Bacteriophages are widely distributed in various environments. The prevalence of bacteriophages in water sources, especially wastewaters, is naturally high. These viruses affect evolution of most bacterial species. Bacteriophages are able to integrate their genomes into the chromosomes of their hosts as prophages and hence transfer resistance genes to the bacterial genomes. Enterococci are commensal bacteria that show high resistance to common antibiotics. For example, prevalence of vancomycin-resistant enterococci has increased within the last decades. Methods: Enterococcal isolates were isolated from clinical samples and morphological, phenotypical, biochemical, and molecular methods were used to identify and confirm their identity. Bacteriophages extracted from water sources were then applied to isolated Enterococcus faecium (E. faecium). In the next step, the bacterial genome was completely sequenced and the existing prophage genome in the bacterial genome was analyzed. Results: In this study, E. faecium EntfacYE was isolated from a clinical sample. The EntfacYE genome was analyzed and 88 prophage genes were identified. The prophage content included four housekeeping genes, 29 genes in the group of genes related to replication and regulation, 25 genes in the group of genes related to structure and packaging, and four genes belonging to the group of genes associated with lysis. Moreover, 26 genes were identified with unknown functions. Conclusion: In conclusion, genome analysis of prophages can lead to a better understanding of their roles in the rapid evolution of bacteria.

Comparative Effects of Carum copticum Essential Oil on Bacterial Growth and Shiga-Toxin Gene Expression of Escherichia coli O157:H7 at Abused Refrigerated Temperatures.

Abused refrigerated temperatures are described as unacceptable deviations from the optimal temperature, occurring frequently during transportation of food products. Escherichia coli O157:H7 is a serious contaminant of meats and meat products due to its ability to grow at abused temperatures (> 10 °C). The aim of this study was to evaluate the antibacterial activity of Carum copticum essential oil for the control of Escherichia coli O157:H7 using laboratory media and minced beef at severe abused refrigerated temperature (15 °C). A comparative quantitative reverse transcription real-time PCR was used to assess effects of temperature and Carum copticum essential oil at sub-minimum inhibitory concentrations on bacterial growth and Shiga-toxin gene (stx1A and stx2A) expression. Results indicated that Carum copticum essential oil inhibited growth of E. coli O157:H7 in tryptone soy broth (TSB) media at all sub-MIC values until Hour 48. However, bacterial population increased progressively until Hour 72 at essential oil concentration of 0.75% (ml g-1) and reached 8.6 log CFU g-1 in minced beef. The essential oil at concentration of 0.005% (ml g-1) increased stx gene expression at all times, but increased stx gene expression (0.015%) at Hour 24 in TSB media. The expression rate of stx1A in minced beef decreased progressively (10.39 and 7.67 folds for 0.5 and 0.75%, respectively) and expression of stx2A was variable in minced beef during storage. In conclusion, results from this study have shown that effects of Carum copticum essential oil on growth and virulence gene expression are not necessarily correlated and temperature, essential oil concentration, investigated gene type, and bacterial growth environment (in vivo or in vitro) are effective as well.

Effects of vitamin D supplements on frequency of CD4+ T-cell subsets in women with Hashimoto's thyroiditis: a double-blind placebo-controlled study.

BACKGROUND: Vitamin D is a modulator of immune functions. Investigations on the mechanisms of vitamin D action and pathogenesis of Hashimoto's thyroiditis (HT) have revealed that vitamin D can reduce damages to thyroid cells caused by autoreactive immune cells. METHODS: Totally, 48 female patients with HT disease were introduced to the study by endocrinologists. Patients were divided into two major groups of 24 individuals and treated weekly with 50,000 IU of cholecalciferol (vitamin D group) or placebo (placebo group) using oral administration for 3 months. Eventually, 17 of the 24 patients in each group finished the study. Before and after supplementation, frequencies of Th1, Th17, Th2 and Tr1 cells and mean fluorescent intensity (MFI) of the associated cytokines, including IFN-γ, IL-17, IL-4 and IL-10, were assessed using flow cytometry. Furthermore, gene expression of IL-10 was assessed using real-time PCR. RESULTS: Results of this study showed that cholecalciferol supplementation caused a significant decrease in Th17/Tr1 ratio. The proportion and MFI of Th1, Th2, Tr1 and Th17 cells included no significant changes in vitamin D group, compared to those in placebo group. Expression rate and MFI of IL-10 increased in both groups. This increase was higher in vitamin D group than placebo group with no significance. CONCLUSIONS: In this novel preliminary clinical trial study, supplementation with cholecalciferol in HT patients for 3 months changed the balance of CD4+ T-cell subsets to improve the disease control. However, further studies are necessary to investigate effects of vitamin D on immune functions in HT patients.

Effects of non-digestive polymers used in iron encapsulation on calcium and iron apparent absorption in rats fed by infant formula.

Iron deficiency anemia is a common problem of all ages in developed and developing countries. Various strategies are used by governments and industries to solve this problem. One of these strategies is iron fortification. In the present study, novel iron microcapsules were designed without any changes in their effects on other ingredients in infant milk formulas. Resistant starch-pectin-iron and pectin-iron microparticles were added to infant powdered milk models. Furthermore, animal studies were carried out. Fecal iron and calcium were assessed using flame atomic absorption spectroscopy and inductively coupled plasma optical emission spectroscopy, respectively. Then, apparent iron and calcium absorptions were calculated. Sensory evaluation was carried out on reconstituted powdered milks. Results showed that iron absorption in rats treated by pectin-coated particles was significantly higher than that in controls with no significant effects on calcium absorption. No significant differences were observed in sensory evaluation.

Weight Changes and Metabolic Outcomes in Calorie-Restricted Obese Mice Fed High-Fat Diets Containing Corn or Flaxseed Oil: Physiological Role of Sugar Replacement with Polyphenol-Rich Grape.

OBJECTIVE: Because diet components are important during dieting in obesity treatment, we examined possible beneficial effects of substituting corn oil and sugar with flaxseed oil and grape in calorie-restricted high-fat diets on weight changes as well as improvement in some metabolic markers and related gene expression. METHODS: Seventy-five C57BL/6J male mice were given free access to a high-fat (36% of energy from fat) diet containing corn oil plus sugar (CO + S). After 11 weeks, 15 mice were sacrificed and another 60 were divided among 4 high-fat diet groups with 30% calorie restriction (CR) for the next 12 weeks. The diets contained corn oil (CO) or flaxseed oil (FO) with sugar (S) or grape (G). RESULTS: Despite CR, a weight loss trend was observed only during the first 4 weeks in all groups. CR did not significantly increase SIRT1 gene expression. Higher liver weight was observed in mice consuming FO (p < 0.05). Proliferator-activated receptor gamma (PPARγ) expression decreased in FO + G-CR significantly and even with a reduction of adiposity and higher adiponectin levels, fasting blood sugar (FBS) was significantly higher than in CO + G-CR. Grape intake increased Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) expression and decreased insulin resistance in CO + G-CR. CONCLUSIONS: Sugar replacement with polyphenol-rich grape along with CR improved glucose homeostasis, and substituting corn oil with flaxseed oil in obese mice reduced fat mass, but even with no change in adiponectin levels it could not decrease insulin resistance. However, none of the food item combinations facilitated weight reduction in the long-term CR. Therefore, regardless of the total calorie intake, different diet components and fat contents may have unexpected effects on metabolic regulation.

Invasion of HEp-2 cells by Shigella spp. isolated from acute pediatric diarrhea.

Aim: Shigella infection is an important global health problem in developing countries where hygiene is poor and hence shigellosis is a main cause of diarrhoea-associated mortality and morbidity, particularly in children under the age of five. The bacterial entry into colon and rectal epithelial cells has been named 'bacterium-directed phagocytosis'. This term highlights that the bacteria actively stimulate their own uptake into non-professional phagocytes. The aim of this study was to demonstrate the invasion of HEp-2 cells by Shigella spp. isolated from acute pediatric diarrhea in Tehran, Iran. Methods: Three-hundred and ten non-duplicative diarrheal stool samples were collected from the children admitted to Children's Medical Center in Tehran, Iran. Samples were cultured and suspected colonies were identified by routine microbiological and biochemical tests. The invasion of the two isolated Shigella spp. to HEp-2 cells was studied. Results: Of 310 stool samples, 16 (5.2%) Shigella spp. were isolated, including seven (43.7%) S. sonnei and nine (56.3%) S. flexneri. Four (44.4%) S. sonnei and seven (42.8%) S. flexneri showed invasive phenotype to HEp-2. Conclusion: Shigella sonnei and S. flexneri are reported as the most prevalent Shigella spp. in nature which infect humans. Invasion of various cell lines gives the chance of survival to Shigella spp. This ability causes more virulent infections in the host. Despite costly and time consuming cell culture techniques, the current method described in this paper is reliable for detecting invasive behavior of Shigella spp. Results have also shown that not all the Shigella spp. are able to invade intestinal epithelial cells.

Prevalence, virulence and antimicrobial resistance patterns of Aeromonas spp. isolated from children with diarrhea.

BACKGROUND: Aeromonas spp. cause various intestinal and extraintestinal diseases. These bacteria are usually isolated from fecal samples, especially in children under five years old. The aim of this study was to assess the prevalence of Aeromonas spp. and their antimicrobial resistance profile in children with diarrhea referred to the Children Medical Center in Tehran, between 2013 and 2014. METHODS: A total number of 391 stool samples were collected from children with ages between 1 day and 14 years old, with diarrhea (acute or chronic), referred to the Children Hospital, Tehran, Iran, between 2013 and 2014. Samples were enriched in alkaline peptone water broth for 24 hours at 37 °C and then cultured. Suspicious colonies were analyzed through biochemical tests. Furthermore, antimicrobial susceptibility tests were carried out for the isolates. Isolates were further studied for act, ast, alt, aerA and hlyA virulence genes using polymerase chain reaction. RESULTS: In total, 12 isolates (3.1%) were identified as Aeromonas spp.; all were confirmed using the API-20E test. Of these isolates, five A. caviae (42%), four A. veronii (33%) and three A. hydrophila (25%) were identified in cases with gastroenteritis. Second to ampicillin (which was included in the growth medium used), the highest rate of antimicrobial resistance was seen against nalidixic acid and trimethoprim-sulfamethoxazole (5 isolates each, 41.6%) and the lowest rate of antimicrobial resistance was seen against gentamicin, amikacin and cefepime (none of the isolates). Results included 76.4% act, 64.7% ast, 71.5% alt, 83.3% aerA and 11.7% hlyA genes. CONCLUSION: Aeromonas spp. are important due to their role in diarrhea in children; therefore, isolation and identification of these fecal pathogens should seriously be considered in medical laboratories. Since virulence genes play a significant role in gastroenteritis symptoms caused by these bacteria, Aeromonas species that include virulence genes are potentially suspected to cause severe infections. Moreover, bacterial antimicrobial resistance is increasing, especially against trimethoprim-sulfamethoxazole and nalidixic acid.

A Novel Combination of Docosahexaenoic Acid, All-Trans Retinoic Acid, and 1, 25-Dihydroxyvitamin D3 Reduces T-Bet Gene Expression, Serum Interferon Gamma, and Clinical Scores but Promotes PPARγ Gene Expression in Experimental Autoimmune Encephalomyelitis.

Vitamins are immunologically interesting due to their significant immunomodulatory activities. Experimental autoimmune encephalomyelitis (EAE) is one of the most commonly used experimental models for studying autoimmune disorder in multiple sclerosis (MS). The aim of this study was to evaluate the protective and ameliorative effects of novel combination of all-trans retinoic acid (ATRA), 1,25-dihydroxyvitamin D3 (D3), and docosahexaenoic acid (DHA) on EAE-specific determinants and target gene expressions. Mice were randomly categorized into three groups before EAE induction [non-treated EAE (Group E), treated EAE (Group T), and healthy mice (Group H)]. Encephalomyelitis was induced in female C57BL/6 mice by subcutaneous immunization using commercial kits. Preceding day of EAE induction, combination of ATRA, D3, and DHA was administered with a single IP injection every 48 h and continued until day 26. Findings of present study showed that administration of vitamins A, D, and DHA significantly decreased average clinical scores, cumulative EAE score, and EAE incidence in Group T, compared to Group E (p values <0.001). Interferon γ secretion in serum and T-bet mRNA expression in splenocytes were significantly reduced (p = 0.004, p = 0.029, respectively) while PPARγ mRNA expression was significantly increased in Group T compared to Group E (p = 0.021). These findings highlighted that ATRA, D3, and DHA combination modulated PPARγ and T-bet gene expression and resulted in decrease in Th1 response and lymphocyte invasion into the central nervous system (CNS) and resultant inflammation. In conclusion, the results of this study suggested the potential use of this intervention in treatment and/or prevention of EAE/MS and probably other Th1 cell-mediated autoimmune diseases.

Molecular identification and phylogenic analysis of Bartonella henselae isolated from Iranian cats based on gltA gene.

One of the most important species of the Bartonella genus is B. henselae that causes a zoonotic infection, cat scratch disease (CSD). The main source of the bacteria is cat and the carrier is Ctenocephalides felis flea. One hundred and forty nail and saliva samples were collected from 70 domestic cats. Positive samples for B. henselae were characterized by polymerase chain reaction (PCR) and sequencing. Sequences of gltA gene were trimmed using BioEdit software and then compared with the sequences of the same gene from B. henselae isolated from cats and humans in GenBank database. Phylogenic tree was constructed using CLC Sequence Viewer software and unweighted pair group method with arithmetic mean (UPGMA) method. Molecular assessments showed that five samples out of 70 nail samples (7.14%) and one sample out of 70 saliva samples (1.42%) were genetically positive for B. henselae. At least an 87.00% similarity was seen between the gene sequences from the current study and the reference sequences from the GenBank database. Phylogenic analysis has shown that strains isolated in this study were grouped in a different haplo group, compared to other strains. Among the Asian countries, the prevalence of the bacteria in Iran was close to that in Japan and Turkey. In conclusion, findings of this study showed the prevalence of B. henselae in Iranian cats which is important due to its public health issues, especially for the immunocompromised pet owners.

Novel Bacteriophages in Enterococcus spp.

Most of the bacteriophages (phages) currently reported in Enterococcus spp. belong to tailed families of bacteriophages Podoviridae, Siphoviridae, and Myoviridae. There is a little information on non-tailed bacteriophages isolated from enterococci. Samples of sewage and piggery effluents were tested on pig and chicken isolates of Enterococcus faecalis, E. faecium and E. gallinarum for lytic phages. In addition, isolates were exposed to mitomycin C to induce lysogenic phages. Bacteriophages that were detected were visualized by electron microscopy. Ten bacteriophages were of isometric shape with long flexible or non-flexible tails, while one had a long head with a long flexible tail; all contained double-stranded DNA molecules. Seven Polyhedral, filamentous, and pleomorphic-shaped phages containing DNA or RNA were also observed. The pleomorphic phages were droplet- or lemon-shaped in morphology. This study is the first report on polyhedral phages in Enterococcus spp. of animal origin and also the first report of filamentous and pleomorphic phages in enterococci.