Emerging applications of phage therapy and fecal virome transplantation for treatment of Clostridioides difficile infection: challenges and perspectives.

Clostridioides difficile, which causes life-threatening diarrheal disease, is considered an urgent threat to healthcare setting worldwide. The current standards of care solely rely on conventional antibiotic treatment, however, there is a risk of promoting recurrent C. difficile infection (rCDI) because of the emergence of antibiotic-resistant strains. Globally, the alarming spread of antibiotic-resistant strains of C. difficile has resulted in a quest for alternative therapeutics. The use of fecal microbiota transplantation (FMT), which involves direct infusion of fecal suspension from a healthy donor into a diseased recipient, has been approved as a highly efficient therapeutic option for patients with rCDI. Bacteriophages or phages are a group of viruses that can infect and destroy bacterial hosts, and are recognized as the dominant viral component of the human gut microbiome. Accumulating data has demonstrated that phages play a vital role in microbial balance of the human gut microbiome. Recently, phage therapy and fecal virome transplantation (FVT) have been introduced as promising alternatives for the treatment of C. difficile -related infections, in particular drug-resistant CDI. Herein, we review the latest updates on C. difficile- specific phages, and phage-mediated treatments, and highlight the current and future prospects of phage therapy in the management of CDI.

Rapid SARS-CoV-2 testing with duplexed recombinase polymerase amplification and a bacteriophage internal control

OBJECTIVES/GOALS: Current COVID-19 rapid molecular tests require cartridge-reader detection, expensive circuitry, and complex microfluidics making the most accurate tests unavailable to the masses. Here we present a rapid molecular diagnostic leveraging isothermal amplification and paper-based microfluidics for a low-cost ultra-sensitive COVID-19 assay. METHODS/STUDY POPULATION: We designed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of SARS-CoV-2 and bacteriophage MS2 RNA. RT-RPA is a sequence specific, ultrasensitive, rapid isothermal DNA amplification technique that is well suited to home based testing due to its rapid assay time, robustness, ease of use, and readout options. RT-RPA reagents are added to a tube and incubated at 39°C in a fluorometer. Realtime fluorometer data gives results in under 15 minutes. This assay also provides visual detection via lateral flow readout with results in 23 minutes. RESULTS/ANTICIPATED RESULTS: We have developed a rapid multiplexed nucleic acid amplification assay with an internal process control for SARS-CoV-2 using single-pot RT-RPA. We screened 21 primer combinations to select primers that demonstrated excellent performance and target specificity against common respiratory viruses. We demonstrate the ability to multiplex SARS-CoV-2 and MS2 detection, utilizing MS2 as an internal process control for lysis, reverse transcription, amplification, and readout. We show duplexed detection using both fluorescence readout and visual readout using lateral flow strips. Duplexed fluorescence detection shows a limit of detection of 25 copies per reaction. Duplexed lateral flow readout shows a limit of detection of 50 copies per reaction DISCUSSION/SIGNIFICANCE: We developed a duplexed RT-RPA assay for SARS-CoV-2 with fluorescence or lateral flow readout. Our assay does not re-quire expensive reader, circuity, or fluid handling. The low material cost, temperature, and robustness make it ideal for a more accurate home-based COVID-19 diagnostic.

An Explorative Review on Advanced Approaches to Overcome Bacterial Resistance by Curbing Bacterial Biofilm Formation.

The continuous emergence of multidrug-resistant pathogens evoked the development of innovative approaches targeting virulence factors unique to their pathogenic cascade. These approaches aimed to explore anti-virulence or anti-infective therapies. There are evident concerns regarding the bacterial ability to create a superstructure, the biofilm. Biofilm formation is a crucial virulence factor causing difficult-to-treat, localized, and systemic infections. The microenvironments of bacterial biofilm reduce the efficacy of antibiotics and evade the host's immunity. Producing a biofilm is not limited to a specific group of bacteria; however, Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus biofilms are exemplary models. This review discusses biofilm formation as a virulence factor and the link to antimicrobial resistance. In addition, it explores insights into innovative multi-targeted approaches and their physiological mechanisms to combat biofilms, including natural compounds, phages, antimicrobial photodynamic therapy (aPDT), CRISPR-Cas gene editing, and nano-mediated techniques.

Phages and SARS-CoV-2

The emerging human pathogenic viruses, including the recently emerged severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), have markedly affected the human health and have become a challenge for researchers worldwide. Antibiotic therapy and existing vaccines have reduced the pandemic burden to some extent. However, there is still need for efficient treatment, vaccination, and antiviral agents to control the pandemic. This chapter illustrates the role of bacteriophage in bacterial infections, SARS-COV-2 infected patients, biological activities of phages, phage display method, phages as potential inducers of antiviral immunity, phage-based vaccines, CRISPR and phage-based SARS-CoV-2 vaccines, and possible advantages of phage-based vaccines. It is concluded that phages have considerable breadth in the SARS-CoV-2 pandemic and offer many substantial advantages, such as clearing respiratory bacterial infections, which significantly reduce the burden of mortalities. Phage plays a vital role in triggering antiviral immunity by inducing cytokines such as IFN-α and IL-12. It suggests the role in driving antiviral immunity, triggering TLR3-dependent pattern recognition receptors, inhibiting TNF-driving type I IFN, inducing antiviral immunity through upregulation of the expression of defensin in IL-2, and encouraging a marked upregulation of gene hBD2 that induces virucidal effects, thus playing a key role in anti-SARS-COV-2 immunity. Moreover, phages have been presented as an alternative universal adjuvant-free nano-vaccine platform in which single-phage scaffolds are used to incorporate multiple targets. © 2023 Elsevier Inc. All rights reserved.

The Breadth of Bacteriophages Contributing to the Development of the Phage-Based Vaccines for COVID-19: An Ideal Platform to Design the Multiplex Vaccine.

Phages are highly ubiquitous biological agents, which means they are ideal tools for molecular biology and recombinant DNA technology. The development of a phage display technology was a turning point in the design of phage-based vaccines. Phages are now recognized as universal adjuvant-free nanovaccine platforms. Phages are well-suited for vaccine design owing to their high stability in harsh conditions and simple and inexpensive large-scale production. The aim of this review is to summarize the overall breadth of the antiviral therapeutic perspective of phages contributing to the development of phage-based vaccines for COVID-19. We show that phage vaccines induce a strong and specific humoral response by targeted phage particles carrying the epitopes of SARS-CoV-2. Further, the engineering of the T4 bacteriophage by CRISPR (clustered regularly interspaced short palindromic repeats) presents phage vaccines as a valuable platform with potential capabilities of genetic plasticity, intrinsic immunogenicity, and stability.

Removal of virus aerosols by the combination of filtration and UV-C irradiation

The COVID-19 pandemic remains ever prevalent and afflicting—partially because one of its transmission pathways is aerosol. With the widely used central air conditioning systems worldwide, indoor virus aerosols can rapidly migrate, thus resulting in rapid infection transmission. It is therefore important to install microbial aerosol treatment units in the air conditioning systems, and we herein investigated the possibility of combining such filtration with UV irradiation to address virus aerosols. Results showed that the removal efficiency of filtration towards f2 and MS2 phages depended on the type of commercial filter material and the filtration speed, with an optimal velocity of 5 cm/s for virus removal. Additionally, it was found that UV irradiation had a significant effect on inactivating viruses enriched on the surfaces of filter materials;MS2 phages had greater resistance to UV-C irradiation than f2 phages. The optimal inactivation time for UV-C irradiation was 30 min, with higher irradiation times presenting no substantial increase in inactivation rate. Moreover, excessive virus enrichment on the filters decreased the inactivation effect. Timely inactivation is therefore recommended. In general, the combined system involving filtration with UV-C irradiation demonstrated a significant removal effect on virus aerosols. Moreover, the system is simple and economical, making it convenient for widespread implementation in air-conditioning systems.

Support for Recommendations of Precleaning Before Disinfection: Limited Efficacy of Soapy Water as a Disinfectant against SARS-CoV-2

The World Health Organization (WHO) and US Centers for Disease Control and Prevention (CDC) recommend cleaning soiled surfaces with soap and water, followed by use of approved disinfectant. However, data are lacking on the potential efficacy of soapy water alone as a disinfectant for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is relevant to low-resource settings where soapy water is prevalent for handwashing. To our knowledge, no appropriate biosafety level 1 (BSL-1) surrogate has been identified and confirmed for use in studies with soapy water and the highly infectious SARS-CoV-2. Therefore, our objectives were to determine the efficacy of soapy water alone as a disinfectant against SARS-CoV-2 and if commonly used BSL-1 bacteriophage surrogates could serve as a surrogate model for testing soapy water as a disinfectant. Overall, results indicate that SARS-CoV-2 can be reduced >4 log10 in suspension but only 0.50 log10 on a nonporous surface with 10 min of exposure to 0.5% soapy water. This difference is potentially attributed to less area of exposure on surfaces than in suspension. Phi6 (a verified SARS-CoV-2 surrogate for other disinfectants) was not appropriate for SARS-CoV-2 disinfection with soapy water. Further research is needed to determine an appropriate surrogate for SARS-CoV-2 disinfection with soapy water as disinfection of MS2 was similar to SARS-CoV-2 on surfaces only. Our work highlights the importance of confirming surrogates for each disinfectant used. Based on our results, we do not recommend a change to the current WHO and CDC surface disinfection protocols that recommend using soapy water to preclean a surface before applying disinfectant.

Research in brief

The authors reported no adverse reactions to phage therapy, regardless of type of bacterial infection, type of phages used, or method of treatment. 11 patients displayed some measure of symptom improvement or reduced bacterial presence;four exhibited no response to treatment. T-helper cells key to malaria vaccine Scientists studying why immunity against Plasmodium falciparum lasts only a short time after immunisation found that T-helper cells reacted exclusively to the protein sequence of the vaccine strain and showed hardly any cross-reactivity with naturally occurring variants. For more on cancer drug and SARS-CoV-2 see ACS Infect Dis 2022;published online June 29. https://doi.org/10.1021/acsinfecdis.2c00008 For more on COVID-19 in pregnancy in sub-Saharan Africa see Clin Infect Dis 2022;published online June 8. https://doi.org/10.1093/cid/ciac294 For more on the spread of enteric viruses through saliva see Nature 2022;published online June 29. https://doi.org/10.1038/s41586-022-04895-8 For more on influenza vaccination and Alzheimer's disease see J Alzheimers Dis 2022;published online June 13. https://doi.org/10.3233/jad-220361 For more on extensively drug-resistant Neisseria gonorrhoeae see Euro Surveill 2022;27: 2200455 For more on bacteriophage therapy case series see Clin Infect Dis 2022;published online June 9. https://doi.org/10.1093/cid/ciac453 For more on T-helper cells and malaria vaccine see Sci Immunol 2022;7: eabm9644

Detailed analysis of antibody responses to SARS-CoV-2 vaccination and infection in macaques

Macaques are a commonly used model for studying immunity to human viruses, including for studies of SARS-CoV-2 infection and vaccination. However, it is unknown whether macaque antibody responses resemble the response in humans. To answer this question, we employed a phage-based deep mutational scanning approach (Phage-DMS) to compare which linear epitopes are targeted on the SARS-CoV-2 Spike protein in convalescent humans, convalescent (re-infected) rhesus macaques, mRNA-vaccinated humans, and repRNA-vaccinated pigtail macaques. We also used Phage-DMS to determine antibody escape pathways within each epitope, enabling a granular comparison of antibody binding specificities at the locus level. Overall, we identified some common epitope targets in both macaques and humans, including in the fusion peptide (FP) and stem helix-heptad repeat 2 (SH-H) regions. Differences between groups included a response to epitopes in the N-terminal domain (NTD) and C-terminal domain (CTD) in vaccinated humans but not vaccinated macaques, as well as recognition of a CTD epitope and epitopes flanking the FP in convalescent macaques but not convalescent humans. There was also considerable variability in the escape pathways among individuals within each group. Sera from convalescent macaques showed the least variability in escape overall and converged on a common response with vaccinated humans in the SH-H epitope region, suggesting highly similar antibodies were elicited. Collectively, these findings suggest that the antibody response to SARS-CoV-2 in macaques shares many features with humans, but with substantial differences in the recognition of certain epitopes and considerable individual variability in antibody escape profiles, suggesting a diverse repertoire of antibodies that can respond to major epitopes in both humans and macaques. Differences in macaque species and exposure type may also contribute to these findings.

Specific anti-SARS-CoV-2 S1 IgY-scFv is a promising tool for recognition of the virus

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread globally, a series of vaccines, antibodies and drugs have been developed to combat coronavirus disease 2019 (COVID-19). High specific antibodies are powerful tool for the development of immunoassay and providing passive immunotherapy against SARS-CoV-2 and expected with large scale production. SARS-CoV-2 S1 protein was expressed in E. coli BL21 and purified by immobilized metal affinity chromatography, as antigen used to immunize hens, the specific IgY antibodies were extracted form egg yolk by PEG-6000 precipitation, and the titer of anti-S1 IgY antibody reached 1:10,000. IgY single chain variable fragment antibody (IgY-scFv) was generated by using phage display technology and the IgY-scFv showed high binding sensitivity and capacity to S1 protein of SARS-CoV-2, and the minimum detectable antigen S1 protein concentration was 6 ng/µL. The docking study showed that the multiple epitopes on the IgY-scFv interacted with multiple residues on SARS-CoV-2 S1 RBD to form hydrogen bonds. This preliminary study suggests that IgY and IgY-scFv are suitable candidates for the development of immunoassay and passive immunotherapy for COVID-19 to humans and animals.

Disinfection of an ambulance using a compact atmospheric plasma device.

AIMS: The worldwide spread of the coronavirus SARS-CoV-2 has highlighted the need for fast and simple disinfection processes, amongst others for ambulance cars on site. To overcome current drawbacks regarding room disinfection, the use of cold atmospheric plasma in remote operation represents a promising alternative for the disinfection of larger volumes. In this study, a compact plasma system was evaluated regarding its disinfection efficiency inside an ambulance car. METHODS AND RESULTS: The developed plasma device is based on a dielectric barrier discharge (DBD) and operates with ambient air as process gas. The humidified afterglow from the plasma nozzle was introduced into an ambulance car with a volume of approximately 10 m3 while Bacillus atrophaeus endospores, Staphylococcus aureus or Phi 6 bacteriophages dried on different surfaces (PET-films, glass slides or aluminum foil) were exposed to the reactive gas inside the ambulance vehicle at eight different positions. Reductions of spores by more than 4 orders of magnitude were found on all surfaces and positions within 2 h. Due to their higher susceptibility, Phi 6 bacteriophages and S. aureus counts were reduced by at least 4 orders of magnitude within 30 min on all surfaces. CONCLUSION: The results show that different microorganisms dried on variable surfaces can be inactivated by several orders of magnitude inside an ambulance by plasma gas from of a compact DBD plasma nozzle. SIGNIFICANCE AND IMPACT OF THE STUDY: Plasma gas generated on site by a DBD plasma nozzle proved to be highly efficient for the disinfection of the interior of an ambulance car. Compact plasma systems could be a viable alternative for the disinfection of vehicles or rooms.

The International Virus Bioinformatics Meeting 2022.

The International Virus Bioinformatics Meeting 2022 took place online, on 23-25 March 2022, and has attracted about 380 participants from all over the world. The goal of the meeting was to provide a meaningful and interactive scientific environment to promote discussion and collaboration and to inspire and suggest new research directions and questions. The participants created a highly interactive scientific environment even without physical face-to-face interactions. This meeting is a focal point to gain an insight into the state-of-the-art of the virus bioinformatics research landscape and to interact with researchers in the forefront as well as aspiring young scientists. The meeting featured eight invited and 18 contributed talks in eight sessions on three days, as well as 52 posters, which were presented during three virtual poster sessions. The main topics were: SARS-CoV-2, viral emergence and surveillance, virus-host interactions, viral sequence analysis, virus identification and annotation, phages, and viral diversity. This report summarizes the main research findings and highlights presented at the meeting.

Demonstrated SARS-CoV-2 surface disinfection using ozone

SARS-CoV-2 has resulted in a global pandemic resulting in the infections of many millions and deaths of well over a million people. The ease of SARS-CoV-2 spread and the infectious nature of the virus in humans has resulted in an urgent need for effective disinfection strategies. Ozone (O3) is a promising disinfectant for SARS-CoV-2 due to its ability to overcome limitations with topical disinfection or sanitation products. In this work, we utilized RT-qPCR to measure RNA from a recombinant mammalian cell product, bacteriophage MS2, and SARS-CoV-2 sourced from municipal wastewater, after exposure to 4.5 and 9 ppmv O3.