Bacteriophages and bacterial extracellular vesicles, threat or opportunity?

Emergence of antimicrobial-resistant bacteria (AMR) is one of the health major problems worldwide. The scientists are looking for a novel method to treat infectious diseases. Phage therapy is considered a suitable approach for treating infectious diseases. However, there are different challenges in this way. Some biological aspects can probably influence on therapeutic results and further investigations are necessary to reach a successful phage therapy. Bacteriophage activity can influence by bacterial defense system. Bacterial extracellular vesicles (BEVs) are one of the bacterial defense mechanisms which can modify the results of bacteriophage activity. BEVs have the significant roles in the gene transferring, invasion, escape, and spreading of bacteriophages. In this review, the defense mechanisms of bacteria against bacteriophages, especially BEVs secretion, the hidden linkage of BEVs and bacteriophages, and its possible consequences on the bacteriophage activity as well phage therapy will be discussed.

A simplified method of bacteriophage preparation for transmission electron microscope.

Bacteriophages are viruses that infect bacteria. Researchers use different methods to study the characteristics of bacteriophages. Transmission electron microscope (TEM) is considered the best method to analyze these characteristics. However, the quality of TEM micrographs is significantly influenced by the preparation methods used to prepare the bacteriophages sample. In this study, researchers compared two different methods for preparing the bacteriophage samples. In one method was used SM buffer, while in the other used deionized water. The results were analyzed by TEM and compared with each other. Additionally, the viability of bacteriophage in deionized water and SM buffer at 4°C was determined through plaque assay within 72 hours. TEM micrographs showed that the quality of bacteriophage sample prepared with deionized water is superior to those prepared with SM buffer. Furthermore, the titer of the bacteriophages did not show a significant reduction during 72 hours in both SM and deionized water. In conclusion, the results suggested that preparation method can significantly impact the quality of TEM micrographs. Using sterile deionized water for the preparation of bacteriophages is a simple way to improve the quality of TEM micrographs and it is advisable to send the samples to the laboratory within 72 hours.

A novel Enterococcus faecium phage EF-M80: unveiling the effects of hydrogel-encapsulated phage on wound infection healing.

Background: Enterococcus faecium is one of the members of ESKAPE pathogens. Due to its resistance to antimicrobial agents, treating this bacterium has become challenging. The development of innovative approaches to combat antibiotic resistance is necessary. Phage therapy has emerged as a promising method for curing antibiotic-resistant bacteria. Methods: In this study, E. faecium phages were isolated from wastewater. Phage properties were characterized through in vitro assays (e.g. morphological studies, and physicochemical properties). In addition, whole genome sequencing was performed. A hydrogel-based encapsulated phage was obtained and its structure characteristics were evaluated. Wound healing activity of the hydrogel-based phage was assessed in a wound mice model. Results: The purified phage showed remarkable properties including broad host range, tolerance to high temperature and pH and biofilm degradation feature as a stable and reliable therapeutic agent. Whole genome sequencing revealed that the genome of the EF-M80 phage had a length of 40,434 bp and harbored 65 open reading frames (ORFs) with a GC content of 34.9% (GenBank accession number is OR767211). Hydrogel-based encapsulated phage represented an optimized structure. Phage-loaded hydrogel-treated mice showed that the counting of neutrophils, fibroblasts, blood vessels, hair follicles and percentage of collagen growth were in favor of the wound healing process in the mice model. Conclusion: These findings collectively suggest the promising capability of this phage-based therapeutic strategy for the treatment of infections associated with the antibiotic-resistant E. faecium. In the near future, we hope to expect the presence of bacteriophages in the list of antibacterial compounds used in the clinical settings.

Multicenter Study of Rotavirus Infection, Diversity of Circulating Genotypes and Clinical Outcomes in Children ≤5 Years Old in Iran.

BACKGROUND: To determine the epidemiology of rotavirus group A (RVA) infection in symptomatic children, and analyze genotype diversity in association with clinical characteristics, geographical and seasonal changes. METHODS: The stool samples of symptomatic children 5≥ years old were collected from 5 different hospitals during December 2020 and March 2022. Rotavirus stool antigen test was done and G and P genotypes of the positive samples were determined. Associations of the infection and genotype diversity with demographical and clinical data were assessed by statistical methods. RESULTS: RVA infection was detected in 32.1% (300/934) of the patients (Ranges between 28.4% and 47.4%). An inverse association with age was detected, where the highest frequency was measured in children ≤12 months of age (175/482, 36.3%). The infection was more frequent during winter (124/284, 43.7%) and spring (64/187, 34.2%). Children who were exclusively fed with breast milk showed a lower rate of infection (72/251, 28.6%). Among the 46 characterized genotypes (17 single- and 29 mixed-genotype infections), G1P[8] and G9P[4] were more frequently detected in children <36 (67/234, 28.63%) and 36-60 (7/24, 29.16%) months of age children, respectively. A seasonal diversity in the circulating genotypes was detected in different cities. Children with G1P[8], G1P[6], and mixed-genotype infection experienced a shorter duration of hospitalization, and a higher frequency of nausea and severe diarrhea, respectively. CONCLUSIONS: In this study high frequency of RVA infection was detected in symptomatic children in Iran. Moreover, genotype diversity according to geographic area, seasons, age groups, and clinical features of disease was detected.

Antiviral activity of biosynthesized copper nanoparticle by Juglans regia green husk aqueous extract and Iron nanoparticle: molecular docking and in-vitro studies.

Background and Objectives: The interaction between nanoparticles (NPs) and viruses is attracting interest because of the antiviral potential of NPs. This study aims to investigate the antiviral potential of NPs against Herpes simplex virus types 1 (HSV-1). Materials and Methods: Molecular docking studies were conducted by Molegro virtual docker software. An extract of Juglans regia green husk was utilized to biosynthesize copper-oxide nanoparticles (CuNPs). The cytotoxicity of NPs was evaluated by MTT assay. Different treatment assays were conducted. Another assay was designed to employ the concentration of 300 µg/ml of CuNPs, which is the highest concentration that did not precipitate. Finally, chemically synthesized Iron oxide nanoparticles (FeNPs) were utilized to adsorb CuNPs. The antiviral effect of FeNPs was investigated, separately. Results: Docking results confirmed that NPs could interact with the HSV-1 glycoproteins and prevent viral entry. MTT assay results illustrated that the minimum non-toxic concentration (MNTD) of CuNPs is 100 µg/ml which did not exhibit antiviral properties. Employing a noncytotoxic concentration of FeNPs (300 mg/ml) in combination with cytotoxic concentration of CuNPs (300 µg / ml), eliminated the cytotoxicity effects of CuNPs. Exposure of the virus with the combination of CuNPs and FeNPs resulted in 4.5 log10 TCID50 reductions in HSV-1. While treating HSV-1 with only FeNPs reduced the titer of virus by 3.25 log10 TCID50. Conclusion: The results highlight that combination of CuNPs and FeNPs have antiviral activity against HSV-1. Moreover, FeNPs demonstrated antiviral properties against HSV-1 separately.

Seroepidemiological and Molecular Survey for the Detection of SARS-CoV-2 Infection among Children in Iran, September 2020 to June 2021: 1-Year Cross-Sectional Study.

A population-based seroepidemiological and molecular survey on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was performed to detect induced antibodies to prior exposure and active infection of children aged 14 years or less in Tehran between 19 September 2020 and 21 June 2021. Moreover, correlations between the children's demographic data and coronavirus disease 2019 (COVID-19) symptoms with the infection status were investigated. Out of 1517 participants, cardinal symptoms of COVID-19 (fever > 38 °C and/or cough and/or diarrhea) were detected in 18%, and serological history of SARS-CoV-2 infection and polymerase chain reaction (PCR) positivity were confirmed in 33.2% and 10.7% of the weighted population, respectively. The prevalence of SARS-CoV-2 infection was significantly higher among 10-14-year-old children. Active infection was significantly higher in symptomatic children and during autumn 2020 and spring 2021. The quantitative reverse transcription real-time PCR (RT-qPCR) positivity was significantly higher among families with a lower socioeconomic status, whereas no association between RT-qPCR or seropositivity was determined with household size, underlying diseases, or gender. In conclusion, high SARS-CoV-2 infection prevalence and seroprevalence were detected in children in Tehran in different seasons. Infection prevalence was significantly higher in older children and in those with a positive history of close contact with infected cases and/or lower socioeconomic status.

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.

Evaluation of miR-222 Expression in HBV Infected Patients in Comparison with HDV and HBV Co-infected Patients.

BACKGROUND: Liver disease is more severe in HDV+HBV co-infected patients than HBV infected patients which seems to be related to differences in the expression of genes and other factors such as MicroRNAs (miRNAs). The aim of this study was to investigate miR-222 expression in HBV infected patients in comparison with HDV+HBV co-infected patients. METHODS: First, total RNA was extracted from the serum samples and then, complementary DNA (cDNA) was produced using cDNA synthesis kit. Finally, miR-222 gene expression was measured using U6 as the internal control by quantitative PCR (qPCR). RESULTS: The level of miR-222 expression in HDV+HBV co-infected samples was significantly up regulated. The fold change of the miR-222 expression between two groups was 3.3 (95% CI; 0.011-17.63) with p<0.001. CONCLUSION: The expression of miR-222 was higher in HBV+HDV co-infected patients than HBV infected patients. Further studies should be conducted to confirm whether miR-222 can be a biomarker for prognosis of severe liver diseases.

Inactivation of model viruses and bacteria in human fresh frozen plasma using riboflavin and long wave ultraviolet rays.

BACKGROUND AND OBJECTIVES: Pathogen reduction technologies are among methods to eliminate transfusion transmitted infections. Mirasol method using riboflavin in combination with ultraviolet rays is one of them. The aims of this study were to investigate the effectiveness of Mirasol method to inactivate some model pathogens as well as examination of the sensitivity of plasma proteins after treatment. MATERIALS AND METHODS: Riboflavin in 50µM concentration and ultraviolet (365 nm) in three different energy doses (3.6, 7.2, and 10.8 j/cm2) were employed to inactivate model pathogens. Four standard viruses were used in this study including Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus1 (HSV-1), Bovine Viral Diarrhea Virus (BVDV) and Polio Virus. 50% Tissue Culture Infectious Dose (TCID50) and Reed-Muench Methods were used to estimate viruses' titers. E. coli and Staphylococcus aureus were used as bacterial models. Four plasma proteins including factor V, VIII, fibrinogen and antithromin were used to determine their sensitivity to pathogen inactivation treatment. RESULTS: The most pathogen reduction titre was determined for 15 minutes irradiation period equal to 10.8 J/cm2 that is corresponding to Log 6.10 for BVDV, Log 6.09 for HSV-1, Log 6.62 for VSV and Log 3.36 for Polio. Bacterial reduction titer was Log 6.94 for E. coli and Log 7.00 for S. aureus. Indicator proteins for plasma activity were determined to be 75% for factor V, 88% for factor VIII, 52% for fibrinogen and 94% for antithrombin. CONCLUSION: Results showed that the employed method can inactivate most of the pathogens in fresh frozen plasma. The acceptable activities of selected plasma proteins remained after treatment.

Inactivation of model viruses suspended in fresh frozen plasma using novel methylene blue based device.

BACKGROUND AND OBJECTIVE: There is a concern on safety of human Fresh Frozen Plasma (FFP) as it is a source of some medicinal products. The possibility of transmission of blood-borne are reported often due to emerging viruses. There are some Pathogen Reduction Technologies (PRT) to inactivate viruses. Methylene Blue (MB) based method is one of them. The aim of this study was to examine new designated device to inactivate model viruses. MATERIALS AND METHODS: Four model viruses were used in this study:Vesicular stomatitis virus (VSV), Herpes Simplex Virus I (HSV-1), Bovine Viral DiarrheaVirus(BVDV) and Polio Virus.50% Tissue Culture Infective Dose (TCID 50) and Reed-Muench Methods were used to titer the viruses. MB in two final concentration of 0.1 µM and 1 µM and illumination in about 627nm with red LED (Lamp Emitting Diode) for 15, 30, 45 and 60 minutes were used. Three replicates employed for each experiments. RESULTS: 1µMconcentration of MB showed more effective than 0.1µMin all designed illumination period for inactivation of HSV, VSV and BVDV. This method also demonstrated best results for enveloped model viruses. The most Log reduction for HSV, VSV and BVDV were6.28, 5.54 and 6.22, respectively. For HSV and BVDV inactivation, the best illumination period was 45 minutes. CONCLUSION: Model viruses showed sensitivity combination of MB and illumination using red LEDs. As results show this device could inactivate model viruses and reduce their titer very close to approved commercial devices, in compare.