Plaque-forming Units Research Articles

Mayaro virus pathogenesis and immunity in rhesus macaques.

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that causes debilitating and persistent arthritogenic disease. While MAYV was previously reported to infect non-human primates (NHP), characterization of MAYV pathogenesis is currently lacking. Therefore, in this study we characterized MAYV infection and immunity in rhesus macaques. To inform the selection of a viral strain for NHP experiments, we evaluated five MAYV strains in C57BL/6 mice and showed that MAYV strain BeAr505411 induced robust tissue dissemination and disease. Three male rhesus macaques were subcutaneously challenged with 105 plaque-forming units of this strain into the arms. Peak plasma viremia occurred at 2 days post-infection (dpi). NHPs were taken to necropsy at 10 dpi to assess viral dissemination, which included the muscles and joints, lymphoid tissues, major organs, male reproductive tissues, as well as peripheral and central nervous system tissues. Histological examination demonstrated that MAYV infection was associated with appendicular joint and muscle inflammation as well as presence of perivascular inflammation in a wide variety of tissues. One animal developed a maculopapular rash and two NHP had viral RNA detected in upper torso skin samples, which was associated with the presence of perivascular and perifollicular lymphocytic aggregation. Analysis of longitudinal peripheral blood samples indicated a robust innate and adaptive immune activation, including the presence of anti-MAYV neutralizing antibodies with activity against related Una virus and chikungunya virus. Inflammatory cytokines and monocyte activation also peaked coincident with viremia, which was well supported by our transcriptomic analysis highlighting enrichment of interferon signaling and other antiviral processes at 2 days post MAYV infection. The rhesus macaque model of MAYV infection recapitulates many of the aspects of human infection and is poised to facilitate the evaluation of novel therapies and vaccines targeting this re-emerging virus.

Identification of 2,4-Diaminoquinazoline Derivative as a Potential Small-Molecule Inhibitor against Chikungunya and Ross River Viruses.

Alphaviruses are serious zoonotic threats responsible for significant morbidity, causing arthritis or encephalitis. So far, no licensed drugs or vaccines are available to combat alphaviral infections. About 300,000 chikungunya virus (CHIKV) infections have been reported in 2023, with more than 300 deaths, including reports of a few cases in the USA as well. The discovery and development of small-molecule drugs have been revolutionized over the last decade. Here, we employed a cell-based screening approach using a series of in-house small-molecule libraries to test for their ability to inhibit CHIKV replication. DCR 137, a quinazoline derivative, was found to be the most potent inhibitor of CHIKV replication in our screening assay. Both, the cytopathic effect, and immunofluorescence of infected cells were reduced in a dose-dependent manner with DCR 137 post-treatment. Most importantly, DCR 137 was more protective than the traditional ribavirin drug and reduced CHIKV plaque-forming units by several log units. CHIKV-E2 protein levels were also reduced in a dose-dependent manner. Further, DCR 137 was probed for its antiviral activity against another alphavirus, the Ross River virus, which revealed effective inhibition of viral replication. These results led to the identification of a potential quinazoline candidate for future optimization that might act as a pan-alphavirus inhibitor.

Control of Escherichia coli in Fresh-Cut Mixed Vegetables Using a Combination of Bacteriophage and Carvacrol.

The continual emergence of antibiotic-resistant bacteria and the slow development of new antibiotics has driven the resurgent interest in the potential application of bacteriophages as antimicrobial agents in different medical and industrial sectors. In the present study, the potential of combining phage biocontrol and a natural plant compound (carvacrol) in controlling Escherichia coli on fresh-cut mixed vegetable was evaluated. Four coliphages, designated Escherichia phage SUT_E420, Escherichia phage SUT_E520, Escherichia phage SUT_E1520 and Escherichia phage SUT_E1620, were isolated from raw sewage. Biological characterization revealed that all four phages had a latent period of 20-30 min and a burst size ranging from 116 plaque-forming units (PFU)/colony forming units (CFU) to 441 PFU/CFU. The phages effectively inhibited the growth of respective host bacteria in vitro, especially when used at a high multiplicity of infection (MOI). Based on transmission electron microscopy analysis, all phages were classified as tailed phages in the class of Caudoviricetes. Additionally, next generation sequencing indicated that none of the selected coliphages contained genes encoding virulence or antimicrobial resistance factors, highlighting the suitability of isolated phages as biocontrol agents. When a phage cocktail (~109 PFU/mL) was applied alone onto fresh-cut mixed vegetables artificially contaminated with E. coli, no bacteria were recovered from treated samples on Day 0, followed by a gradual increase in the E. coli population after 24 h of incubation at 8 °C. On the other hand, no significant differences (p < 0.05) were observed between treated and non-treated samples in terms of E. coli viable counts when carvacrol at the minimum inhibitory concentration (MIC) of 6.25 μL/mL was applied alone. When a phage cocktail at an MOI of ~1000 and MIC carvacrol were applied in combination, no E. coli were recovered from treated samples on Day 0 and 1, followed by a slight increase in the E. coli population to approximately 1.2-1.3 log CFU/mL after 48 h of incubation at 8 °C. However, total elimination of E. coli was observed in samples treated with a phage cocktail at a higher MOI of ~2000 and carvacrol at MIC, with a reduction of approximately 4 log CFU/mL observed at the end of Day 3. The results obtained in this study highlight the potential of combined treatment involving phage biocontrol and carvacrol as a new alternative method to reduce E. coli contamination in minimally processed ready-to-eat foods.

Optimization of Virus Yield as a Strategy to Increase the Efficiency of Rabies Vaccine by BHK-21 Cells in a Bioreactor

Background: Rabies is a usually fatal viral zoonotic and preventable disease. The efficacy and safety of animal rabies vaccination made in permanent BHK-21 cell culture have been proven over a long period of use. By increasing the yield of cells and viruses, the efficacy of the vaccine can be increased. Objectives: The objective of this study was to optimize and maximize the output of a rabies vaccine made on BHK cells in a bioreactor. Methods: This study examined the impacts of independent parameters, such as pH, temperature, cell density, and dissolved oxygen (DO), on rabies virus strain PV-PARIS yield for a central composite design. To achieve high viral production, this study used the central composite approach to optimize cell development. Results: The findings showed that BHK-21 cells were grown under the ideal conditions of pH 7.21, temperature 35.05ºC, 68.75% for DO, and 2.30 × 106 cell/mL of cell density to produce high titers of rabies virus (4.7 × 107 plaque-forming unit [PFU]/mL). High correlation coefficients (0.927) validated that the predicted model was well-fitted with the data, and the statistical analysis of the collected data indicated that the experimental data and predicted model were well-matched. The accuracy of this model’s predictions was correlated with values of adjusted R-squared (R2Adj) and predicted R-squared (R2Pred). Conclusions: These upgrades lead to a more reliable and economical procedure that makes industrialization and commercialization easier.

Household dishwashing detergents efficiently inactivate HSV-1, but not the non-enveloped viruses HAV and MNV, while all viruses were removed from glass by manual scrubbing

This study investigated the stability of herpes simplex virus 1 (HSV-1), hepatitis A virus (HAV) and murine norovirus (MNV) on glass, their inactivation by three household dishwashing detergents and during a glass rinsing process. After drying on glass and storage in darkness, virus titers decreased by 4.7, 5.5 and 4.5 log10 plaque forming units (PFU) in 21, 28 and 14 days for HSV-1, HAV and MNV, respectively. Daylight storage resulted in shorter survival times. While HAV and MNV titers were not significantly affected by 60 s incubation in dishwashing detergents at up to 43 °C, HSV-1 titer decreased significantly by > 4 log10 PFU/mL at the same conditions with each detergent used. After cleaning artificially contaminated drinking glasses with a manual glass scrubbing device, HSV-1, HAV and MNV titers decreased significantly by 4.4 ± 0.1 to 5.3 ± 0.0 log10 PFU. Our study shows, that all tested viruses can remain infectious after drying on glass up to several weeks. Household dishwashing detergents efficiently inactivate the enveloped virus HSV-1, but not the non-enveloped viruses HAV and MNV. The applied manual glass rinsing procedure efficiently removed all three viruses from drinking glasses, probably due to the combination of chemical and mechanical treatments.

Rapid detection of vaccinia virus using biofunctionalized fiber-optic ball-tip biosensors

In this work, we present the development and biofunctionalization of a fiber-optic ball-resonator biosensor for the real-time detection of vaccinia poxvirus. We fabricated several ball-tip resonators, functionalized through a silanization process to immobilize two bioreceptors: the monoclonal anti-L1R antibody targeting the L1R protein, and the polyclonal rabbit serum antibodies targeting the whole vaccinia virus (VV) pathogen. Experimental measurements were carried out to detect VV in concentrations from 103 to 108 plaque-forming units (PFU), with a limit of detection of around 1.7–4.3 × 103 PFU and a log-quadratic pattern, with a response up to 5 × 10−4 RIU (refractive index units). The specificity was assessed against herpes simplex virus, used as a non-specific control, with the best results obtained with anti-L1R monoclonal antibodies, and through the detection of vaccinia virus/herpes simplex-1 combination. The obtained results provide a real-time viral recognition with a label-free sensing platform, having rapid response and ease of manufacturing, and paving the road to the seamless detection of poxviruses affecting different human and animal species using optical fibers.

Stability of magistral phage preparations before therapeutic application in patients with chronic rhinosinusitis, sepsis, pulmonary, and musculoskeletal infections.

Bacteriophages have (re)gained popularity as an adjunct antibacterial therapy. On top of the self-amplifying character of phages, their titer can be influenced by several factors which could lead to reduced titers after preparation and storage. The stability of phage preparations, used for the treatment of chronic rhinosinusitis (CRS), sepsis, pulmonary, and musculoskeletal infections, was investigated. Staphylococcus aureus phage ISP and Pseudomonas aeruginosa phages PNM, 14-1 and PT07 were evaluated. These phages were magistrally prepared by diluting active pharmaceutical products and storing them in different polypropylene/polycarbonate syringes at 4°C. Titers were monitored for 7-21 days using the double-agar overlay method. For 7 days, titers remained stable. Afterwards, the phage titer reduced for all phages with an average reduction of 0.78 log (P < 0.0001) plaque-forming units (PFUs)/mL after 14 days and 1 log PFU/mL after 21 days (P < 0.0001). PT07 titers dropped below the predefined threshold after 2 days in polycarbonate syringes. In addition, the stability after irrigation using a nasal douche (CRS) and infusion through a catheter (sepsis) was assessed. No changes were observed after using these devices with a mean change in titer of -0.07 log PFU/mL (P = 0.9062) and -0.03 log PFU/mL (P = 0.7350), respectively. In conclusion, a decreased titer after storage of a phage solution can lead to administration of subtherapeutic concentrations. In our experiments, we demonstrated that liquid phage solutions can be stored in syringes at 4°C for 5-7 days. PT07 appeared to be less stable, especially in polycarbonate syringes. Irrigation using a nasal douche or infusion through a catheter does not impact phage titer. IMPORTANCE As antimicrobial resistance becomes more prevalent, the application of (bacterio)phage therapy as an alternative treatment for difficult-to-treat infections is (re)gaining popularity. Over the past decade, numerous promising case reports and series have been published demonstrating the therapeutic potential of phage therapy. However, important questions remain regarding the optimal treatment protocol and, unlike for medicinal products, there are currently no predefined quality standards for the stability of phage preparations. Phage titers can be influenced by several factors which could lead to reduced titers after preparation and storage and, ultimately, subtherapeutic applications. Determining the stability of different phages in different recipients according to the route of administration is therefore one of the first important steps in establishing a standardized protocol for phage therapy.

Immunity induced by vaccination with recombinant influenza B virus neuraminidase protein breaks viral transmission chains in guinea pigs in an exposure intensity-dependent manner.

Vaccines that can slow respiratory virus transmission in the population are urgently needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus. Here, we describe how a recombinant neuraminidase-based influenza virus vaccine reduces transmission in vaccinated guinea pigs in an exposure intensity-based manner.

Simple Point-of-Care Nucleic Acid Amplification Test for Rapid SARS-CoV-2 Infection Diagnosis.

After three years of the SARS-CoV-2 pandemic, the demand for developing field-deployable point-of-care (PoC) molecular diagnostic tests has increased. Although RT-qPCR is the molecular diagnostic gold standard and is accurate, it is not readily applied to point-of-care testing (POCT). Meanwhile, rapid diagnostic kits have the disadvantage of low sensitivity. Recently, rapid isothermal nucleic acid amplification technology has emerged as an alternative for rapid diagnosis. Here, we developed a rapid SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP)-lateral flow assay (LFA) kit. This kit includes a Chelex-100/boiling nucleic acid extraction device and a one-step amplification detection apparatus capable of performing the entire process, from RNA extraction to detection, and diagnosing SARS-CoV-2 infection within 40 min without contamination. The detection limits of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 100 plaque-forming units (PFUs) mL-1 and 10-1 PFU mL-1 for RNA samples extracted using the Chelex-100/boiling nucleic acid extraction device and commercial AdvansureTM E3 system, respectively. The sensitivity and specificity of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 97.8% and 100%, respectively. Our SARS-CoV-2 RT-LAMP-LFA kit exhibited high sensitivity and specificity within 40 min without requiring laboratory instruments, suggesting that the kit could be used as a rapid POC molecular diagnostic test for SARS-CoV-2.

A lytic phage to control multidrug-resistant avian pathogenic Escherichia coli (APEC) infection.

The inappropriate use of antibiotics has led to the emergence of multidrug-resistant strains. Bacteriophages (phages) have gained renewed attention as promising alternatives or supplements to antibiotics. In this study, a lytic avian pathogenic Escherichia coli (APEC) phage designated as PEC9 was isolated and purified from chicken farm feces samples. The morphology, genomic information, optimal multiplicity of infection (MOI), one-step growth curve, thermal stability, pH stability, in vitro antibacterial ability and biofilm formation inhibition ability of the phage were determined. Subsequently, the therapeutic effects of the phages were investigated in the mice model. The results showed that PEC9 was a member of the siphovirus-like by electron microscopy observation. Biological characterization revealed that it could lyse two serotypes of E. coli, including O1 (9/20) and O2 (6/20). The optimal multiplicity of infection (MOI) of phage PEC9 was 0.1. Phage PEC9 had a latent period of 20 min and a burst period of 40 min, with an average burst size of 68 plaque-forming units (PFUs)/cell. It maintained good lytic activity at pH 3-11 and 4-50°C and could efficiently inhibit the bacterial planktonic cell growth and biofilm formation, and reduce bacterial counts within the biofilm, when the MOI was 0.01, 0.1, and 1, respectively. Whole-genome sequencing showed that PEC9 was a dsDNA virus with a genome of 44379 bp and GC content of 54.39%. The genome contains 56 putative ORFs and no toxin, virulence, or resistance-related genes were detected. Phylogenetic tree analysis showed that PEC9 is closely related to E. coli phages vB_EcoS_Zar3M, vB_EcoS_PTXU06, SECphi18, ZCEC10, and ZCEC11, but most of these phages exhibit different gene arrangement. The phage PEC9 could successfully protect mice against APEC infection, including improved survival rate, reduced bacterial loads, and organ lesions. To conclude, our results suggest that phage PEC9 may be a promising candidate that can be used as an alternative to antibiotics in the control of APEC infection.

Refined semi-lethal aerosol H5N1 influenza model in cynomolgus macaques for evaluation of medical countermeasures

Highly pathogenic avian influenza A H5N1 viruses cause high mortality in humans and have pandemic potential. Effective vaccines and treatments against this threat are urgently needed. Here, we have refined our previously established model of lethal H5N1 infection in cynomolgus macaques. An inhaled aerosol virus dose of 5.1 log10 plaque-forming unit (pfu) induced a strong febrile response and acute respiratory disease, with four out of six macaques succumbing after challenge. Vaccination with three doses of adjuvanted seasonal quadrivalent influenza vaccine elicited low but detectable neutralizing antibody to H5N1. All six vaccinated macaques survived four times the 50% lethal dose of aerosolized H5N1, while four of six unvaccinated controls succumbed to disease. Although vaccination did not protect against severe influenza, vaccinees had reduced respiratory dysfunction and lower viral load in airways compared to controls. We anticipate that our macaque model will play a vital role in evaluating vaccines and antivirals against influenza pandemics.

Antibody responses to recombinant vesicular stomatitis virus-Zaire Ebolavirus vaccination for Ebola virus disease across doses and continents: 5-year durability

ObjectivesTo report 5-year persistence and avidity of antibodies produced by the live-attenuated recombinant vesicular stomatitis virus (rVSV) expressing the Zaire Ebolavirus (ZEBOV) glycoprotein (GP), known as rVSV-ZEBOV (Ervebo®). MethodsHealthy adults vaccinated with 300,000 or 10–50 million plaque-forming units of rVSV-ZEBOV in the WHO-coordinated trials of 2014–2015 were followed for up to 4 (Lambaréné, Gabon) and 5 (Geneva, Switzerland) years. We report seropositivity rates, geometric mean titres (GMTs), and population distribution of ZEBOV-GP ELISA IgG antibodies, neutralizing antibodies (pseudovirus and live-virus neutralization) and antibody avidity; the primary outcome was ZEBOV-GP ELISA IgG GMTs at 4 or 5 years compared with 1 year (Y1) after immunization. ResultsAmong the 168 eligible vaccinees (Geneva: 97 and Lambaréné: 71) enrolled 1 year post-immunization, 146 (87%) remained enrolled at 4 years (Geneva: n = 88, Lambaréné: n = 58), and 84 (87%, Geneva) at 5 years post-vaccination. ZEBOV-GP ELISA IgG GMTs plateaued, with no declining trend from 1 year through the last time point assessed (1147.8 [95% CI 874.3–1507.0] at Y1 versus 1548.1 [95% CI 1136.6–2108.5] at Y5 in Geneva volunteers receiving ≥10 million plaque-forming units of rVSV-ZEBOV), their avidity matching that of ZEBOV convalescents. Live-virus neutralizing antibodies were detected for shorter periods and in fewer vaccinees (53/95 [56%] at Y1 versus 35/84 [42%] at Y5 in Geneva volunteers, all dose levels). DiscussionTitres at Y1 emerged as a correlate of antibody persistence at Y5. The findings of persistent ZEBOV-GP ELISA IgG titres yet shorter-lasting, lower titres of live-virus neutralizing antibodies suggest the contribution of antibody-mediated protective mechanisms other than neutralization. Long-term clinical efficacy of rVSV-ZEBOV, however, requires further study.

The elucidation of plasma lipidome profiles during severe influenza in a mouse model

Although influenza virus infection has been shown to affect lipid metabolism, details remain unknown. Therefore, we elucidated the kinetic lipid profiles of mice infected with different doses of influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) by measuring multiple lipid molecular species using untargeted lipidomic analysis. C57BL/6 male mice were intranasally infected with PR8 virus at 50 or 500 plaque-forming units to cause sublethal or lethal influenza, respectively. Plasma and tissue samples were collected at 1, 3, and 6 days post-infection (dpi), and comprehensive lipidomic analysis was performed using high-performance liquid chromatography–linear trap quadrupole–Orbitrap mass spectrometry, as well as gene expression analyses. The most prominent feature of the lipid profile in lethally infected mice was the elevated plasma concentrations of phosphatidylethanolamines (PEs) containing polyunsaturated fatty acid (PUFA) at 3 dpi. Furthermore, the facilitation of PUFA-containing phospholipid production in the lungs, but not in the liver, was suggested by gene expression and lipidomic analysis of tissue samples. Given the increased plasma or serum levels of PUFA-containing PEs in patients with other viral infections, especially in severe cases, the elevation of these phospholipids in circulation could be a biomarker of infection and the severity of infectious diseases.

Isolation, biological and whole genome characteristics of a Proteus mirabilis bacteriophage strain

Proteus mirabilis, a naturally resistant zoonotic bacterium belonging to the Enterobacteriaceae family, has exhibited an alarming increase in drug resistance. Consequently, there is an urgent need to explore alternative antimicrobial agents. Bacteriophages, viruses that selectively target bacteria, are abundant in the natural environment and have demonstrated potential as a promising alternative to antibiotics. In this study, we successfully isolated four strains of Proteus mirabilis phages from sewage obtained from a chicken farm in Sichuan, China. Subsequently, we characterized one of the most potent lytic phages, Q29, by examining its biological and genomic features. Comparative genomic analysis revealed the functional genes and phylogenetic evolution of Q29 phages. Our findings revealed that Proteus mirabilis bacteriophage Q29 possesses an icosahedral symmetrical head with a diameter of 95 nm and a tail length of 240 nm. Moreover, phage Q29 exhibited stability within a temperature range of 37 ℃ to 55 ℃ and under pH conditions ranging from 4 to 9. The optimal multiplicity of infection (MOI) for this phage was determined to be 0.001. Furthermore, the one-step growth curve results indicated an incubation period of approximately 15 min, an outbreak period of approximately 35 min, and an average cleavage quantity of approximately 60 plaque-forming units (PFU) per cell. The genome of phage Q29 was found to have a total length of 58,664 base pairs and encoded 335 open reading frames (ORFs) without carrying any antibiotic resistance genes. Additionally, genetic evolutionary analysis classified phage Q29 within the family Caudalidae and the genus Myotail. This study provides valuable research material for further development of Proteus mirabilis bacteriophage biologics as promising alternatives to antibiotics, particularly in light of the growing challenge of antibiotic resistance posed by this bacterium.

A stability study of transparent conducting WO3/Cu/WO3 coatings with antimicrobial properties

WO3/Cu/WO3 coatings are transparent electrodes, but conductivity and transmittance have been observed to decrease with time. This paper reports the improved stability of WO3/Cu/WO3 coatings deposited by magnetron sputtering on glass and polyethylene terephthalate substrates. The stability issues due to Cu oxidation and migration can be addressed by adjusting the deposition parameters. Lowering the sputtering pressure results in denser WO3 films, confirmed by spectroscopic ellipsometry, and thus more stable coatings. The coatings retain their properties in an inert atmosphere, indicating that Cu oxidation is the main reason for the decrease in conductivity, rather than its migration observed by X-ray photoelectron spectroscopy. Optical property modeling is used to optimize the thickness of the three-layer coatings to obtain the highest figure-of-merit for a transparent electrode. A structure of glass/WO3 (70 nm)/Cu (10 nm)/WO3 (45 nm) gives a sheet resistance of 14 Ω/sq. and a light transmittance of 65% at 600 nm. In addition, the antimicrobial properties of these coatings are revealed. A decrease up to 105 of the gram-negative Escherichia coli and gram-positive Staphylococcus aureus bacterial colony formation units is found for several WO3/Cu/WO3-based coatings. In the case of the MS2 (Emesvirus zinderi) bacteriophage, a decrease in infectious particles for up to 104 plaque-forming units is obtained. The results indicate that more stable samples also had higher antimicrobial activity.

Efficacy of a far-ultraviolet-C light technology for continuous decontamination of air and surfaces.

A wall-mounted, far-ultraviolet-C light technology reduced aerosolized bacteriophage MS2 by >3 log10 plaque-forming units within 30 minutes. Vegetative bacterial pathogens on steel disk carriers in the center of the room were reduced by >3 log10 after 45 minutes of exposure, but Candida auris and Clostridioides difficile spores were not.

Compounding Achromobacter Phages for Therapeutic Applications.

Achromobacter species colonization of Cystic Fibrosis respiratory airways is an increasing concern. Two adult patients with Cystic Fibrosis colonized by Achromobacter xylosoxidans CF418 or Achromobacter ruhlandii CF116 experienced fatal exacerbations. Achromobacter spp. are naturally resistant to several antibiotics. Therefore, phages could be valuable as therapeutics for the control of Achromobacter. In this study, thirteen lytic phages were isolated and characterized at the morphological and genomic levels for potential future use in phage therapy. They are presented here as the Achromobacter Kumeyaay phage collection. Six distinct Achromobacter phage genome clusters were identified based on a comprehensive phylogenetic analysis of the Kumeyaay collection as well as the publicly available Achromobacter phages. The infectivity of all phages in the Kumeyaay collection was tested in 23 Achromobacter clinical isolates; 78% of these isolates were lysed by at least one phage. A cryptic prophage was induced in Achromobacter xylosoxidans CF418 when infected with some of the lytic phages. This prophage genome was characterized and is presented as Achromobacter phage CF418-P1. Prophage induction during lytic phage preparation for therapy interventions require further exploration. Large-scale production of phages and removal of endotoxins using an octanol-based procedure resulted in a phage concentrate of 1 × 109 plaque-forming units per milliliter with an endotoxin concentration of 65 endotoxin units per milliliter, which is below the Food and Drugs Administration recommended maximum threshold for human administration. This study provides a comprehensive framework for the isolation, bioinformatic characterization, and safe production of phages to kill Achromobacter spp. in order to potentially manage Cystic Fibrosis (CF) pulmonary infections.

Characterization of Salmonella Thompson-specific bacteriophages and their preventive application against Salmonella Thompson biofilm on eggshell as a promising antimicrobial agent in the food industry

Salmonella Thompson is a foodborne pathogen that has attracted attention in Korea since it was identified as the cause of a large-scale domestic food poisoning outbreak in 2018. The present study aims to isolate and characterize bacteriophages specific to Salmonella Thompson and evaluate their efficacy in reducing Salmonella Thompson biofilm on eggshell. In addition, advanced microscopic analysis using field-emission scanning electron microscopy and confocal laser scanning microscopy was conducted to confirm the anti-biofilm activity. The two isolated bacteriophages CAU-STP-1 and CAU-STP-2 showed very high specificity by targeting only Salmonella Thompson. They were determined to be stable at 60–65 °C and pH 4–11. Bacteriophage particles quickly adsorbed to the host cells, with a latent period and burst size of 30–40 min and 19–100 plaque-forming units per cell, respectively. When comparing the two bacteriophages individually, CAU-STP-1 exhibited the highest in vitro inhibition ability, but treatment with both bacteriophages together in a cocktail was significantly more effective than with CAU-STP-1 alone. This bacteriophage cocktail was consequently employed to treat Salmonella Thompson biofilm on eggshell at different storage temperatures. There was a significant reduction in the biofilm on the eggshell with bacteriophage treatment at 10 and 20 °C after 7 days. For example, After 7 days at 10 °C, the biofilm on eggshell was reduced by 0.26, 0.96, and 2.50 log CFU/cm2 for MOI 103, MOI 104, and MOI 105, respectively. On the other hand, at 20 °C, the biofilm on eggshells was reduced by 0.10, 0.24, and 0.33 log CFU/cm2 for MOI 103, MOI 104, and MOI 105, respectively This study thus provides promising information for the potential development of an effective biocontrol agent against Salmonella Thomson within the food industry.

Photocatalytic paint for phage inactivation in dairy industry: inactivation constants and efficiencies

A photocatalytic paint, formulated with commercial carbon doped anatase TiO2, was tested for the inactivation of sixteen bacteriophages infective to lactic acid bacteria. Phages were deposited on borosilicate glass plates coated with paint and subjected under radiation typically used for indoor illumination (λ = 360–720 nm). Inactivation assays for a total time of 20 h in a laboratory photoreactor at constant temperature and relative humidity were performed. Plaque Forming Units (PFU) as function of time were followed demonstrating that eight phages were completely inactivated in a short time (1.5–5 h, phage reductions higher than 2.79 log orders) whereas the eight remaining phages partially reduced their infectivity with varied log reductions and assay times (reductions of 2.33–5.88 log orders within 4–20 h). Apparent inactivation kinetic constant (kT) ranged from 0.30 to 7.52 1/h. The highest and lowest photonic inactivation efficiency were of 4.06 × 1012 PFU/Einstein and 3.00 × 109 PFU/Einstein, respectively; whereas the quantum inactivation efficiencies varied between 9.17 × 1012 PFU/Einstein and 6.00 × 109 PFU/Einstein. Results demonstrated the efficiency of photocatalytic paint to inactive phages in short times. Application of this paint on diverse surfaces of dairies using the own illumination system would contribute to the diminution of phage concentration in the environment, thus reducing the risk of phage attacks to bacterial strains belonged to starter cultures.

Catalpol Prevents Glomerular Angiogenesis Induced by Advanced Glycation End Products via Inhibiting Galectin-3.

The main characteristics of diabetic nephropathy (DN) at the early stage are abnormal angiogenesis of glomerular endothelial cells (GECs) and macrophage infiltration. Galectin-3 plays a pivotal role in the pathogenesis of DN via binding with its ligand, advanced glycation end products (AGEs). Catalpol, an iridoid glucoside extracted from Rehmannia glutinosa, has been found to ameliorate vascular inflammation, reduce endothelial permeability, and protect against endothelial damage in diabetic milieu. However, little is known about whether catalpol could exert an anti-angiogenesis and anti-inflammation effect induced by AGEs. Mouse GECs (mGECs) and RAW 264.7 macrophages were treated with different concentrations of AGEs (0, 50, 100, 200 and 400 µg/mL) for different time (0, 6, 12, 24 and 48 h) to determine the optimal concentration of AGEs and treatment time. Cells were treated with catalpol (10 µmol/L), GB1107 (1 µmol/L, galectin-3 inhibitor), PX-478 (50 µmol/L, HIF-1α inhibitor), adenovirus-green fluorescent protein (Ad-GFP) [3×107 plaque-forming unit (PFU)/mL] or Ad-galectin-3-GFP (2×108 PFU/mL), which was followed by incubation with 50 µg/mL AGEs. The levels of galectin-3, vascular endothelial growth factor A (VEGFA) and pro-angiogenic factors angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), tunica interna endothelial cell kinase-2 (Tie-2) were detected by enzymelinked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8) assay was used to evaluate the proliferation of these cells. The expression levels of galectin-3, vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and hypoxia-inducible factor-1α (HIF-1α) in mGECs and those of galectin-3 and HIF-1α in RAW 264.7 macrophages were detected by Western blotting and immunofluorescence (IF) staining. The rat DN model was established. Catalpol (100 mg/kg) or GB1107 (10 mg/kg) was administered intragastrically once a day for 12 weeks. Ad-galectin-3-GFP (6×107 PFU/mL, 0.5 mL) or Ad-GFP (6×106 PFU/mL, 0.5 mL) was injected into the tail vein of rats 48 h before the sacrifice of the animals. The expression of galectin-3, VEGFR1, VEGFR2, and HIF-1α in renal cortices was analyzed by Western blotting. The expression of galectin-3, F4/80 (a macrophage biomarker), and CD34 (an endothelium biomarker) in renal cortices was detected by IF staining, and collagen accumulation by Masson staining. The expression levels of galectin-3 and VEGFA were significantly higher in mGECs and RAW 264.7 macrophages treated with 50 µg/mL AGEs for 48 h than those in untreated cells. Catalpol and GB1107 could block the AGEs-induced proliferation of mGECs and RAW 264.7 macrophages. Over-expression of galectin-3 was found to reduce the inhibitory effect of catalpol on the proliferation of cells. Catalpol could significantly decrease the levels of Ang-1, Ang-2 and Tie-2 released by AGEs-treated mGECs, which could be reversed by over-expression of galectin-3. Catalpol could significantly inhibit AGEs-induced expression of galectin-3, HIF-1α, VEGFR1, and VEGFR2 in mGECs. The inhibitory effect of catalpol on galectin-3 in AGEs-treated mGECs was impaired by PX-478. Moreover, catalpol attenuated the AGEs-activated HIF-1α/galectin-3 pathway in RAW 264.7 macrophages, which was weakened by PX-478. Additionally, catalpol significantly inhibited the expression of galectin-3, macrophage infiltration, collagen accumulation, and angiogenesis in the kidney of diabetic rats. Over-expression of galectin-3 could antagonize these inhibitory effects of catalpol. Catalpol prevented the angiogenesis of mGECs and macrophage proliferation via inhibiting galectin-3. It could prevent the progression of diabetes-induced renal damage.