Stability study in selected conditions and biofilm-reducing activity of phages active against drug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is currently a serious threat to human health, especially to people with immunodeficiency as well as patients with prolonged hospital stays and those undergoing invasive medical procedures. The ever-increasing percentage of strains characterized by multidrug resistance to widely used antibiotics and their ability to form biofilms make it difficult to fight infections with traditional antibiotic therapy. In view of the above, phage therapy seems to be extremely attractive. Therefore, phages with good storage stability are recommended for therapeutic purposes. In this work, we present the results of studies on the stability of 12 phages specific for A. baumannii under different conditions (including temperature, different pH values, commercially available disinfectants, essential oils, and surfactants) and in the urine of patients with urinary tract infections (UTIs). Based on our long-term stability studies, the most optimal storage method for the A. baumannii phage turned out to be - 70 °C. In contrast, 60 °C caused a significant decrease in phage activity after 1 h of incubation. The tested phages were the most stable at a pH from 7.0 to 9.0, with the most inactivating pH being strongly acidic. Interestingly, ethanol-based disinfectants caused a significant decrease in phage titers even after 30 s of incubation. Moreover, copper and silver nanoparticle solutions also caused a decrease in phage titers (which was statistically significant, except for the Acba_3 phage incubated in silver solution), but to a much lesser extent than disinfectants. However, bacteriophages incubated for 24 h in essential oils (cinnamon and eucalyptus) can be considered stable.

Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses.

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs.

New Phage-Derived Antibacterial Enzyme PolaR Targeting Rothia spp.

Rothia is an opportunistic pathogen, particularly life-threatening for the immunocompromised. It is associated with pneumonia, endocarditis, peritonitis and many other serious infections, including septicemia. Of note, Rothia mucilaginousa produces metabolites that support and increase overgrowth of Pseudomonas aeruginosa, one of the ESKAPE bacteria. Endolysins are considered as antibacterial enzymes derived from bacteriophages that selectively and efficiently kill susceptible bacteria without harming human cells or the normal microbiome. Here, we applied a computational analysis of metagenomic sequencing data of the gastric mucosa phageome extracted from human patients' stomach biopsies. A selected candidate anti-Rothia sequence was produced in an expression system, purified and confirmed as a Rothia mucilaginosa- and Rothia dentocariosa-specific endolysin PolaR, able to destroy bacterial cells even when aggregated, as in a biofilm. PolaR had no cytotoxic or antiproliferative effects on mammalian cells. PolaR is the first described endolysin selectively targeting Rothia species, with a high potential to combat infections caused by Rothia mucilaginosa and Rothia dentocariosa, and possibly other bacterial groups. PolaR is the first antibacterial enzyme selected from the gastric mucosa phageome, which underlines the biological complexity and probably underestimated biological role of the phageome in the human gastric mucosa.

Biological Properties of 12 Newly Isolated Acinetobacter baumannii-Specific Bacteriophages.

Infections with the opportunistic Gram-negative bacterium Acinetobacter baumannii pose a serious threat today, which is aggravated by the growing problem of multi-drug resistance among bacteria, caused by the overuse of antibiotics. Treatment of infections caused by antibiotic-resistant A. baumannii strains with the use of phage therapy is not only a promising alternative, but sometimes the only option. Therefore, phages specific for clinical multi-drug resistant A. baumannii were searched for in environmental, municipal, and hospital wastewater samples collected from different locations in Poland. The conducted research allowed us to determine the biological properties and morphology of the tested phages. As a result of our research, 12 phages specific for A. baumannii, 11 of which turned out to be temperate and only one lytic, were isolated. Their lytic spectra ranged from 11 to 75%. The plaques formed by most phages were small and transparent, while one of them formed relatively large plaques with a clearly marked 'halo' effect. Based on Transmission Electron Microscopy (TEM), most of our phages have been classified as siphoviruses (only one phage was classified as a podovirus). All phages have icosahedral capsid symmetry, and 11 of them have a long tail. Optimal multiplicity of infections (MOIs) and the adsorption rate were also determined. MOI values varied depending on the phage-from 0.001 to 10. Based on similarities to known bacteriophages, our A. baumannii-specific phages have been proposed to belong to the Beijerinckvirinae and Junivirinae subfamilies. This study provides an additional tool in the fight against this important pathogen and may boost the interest in phage therapy as an alternative and supplement to the current antibiotics.

Experimental Identification of Cross-Reacting IgG Hotspots to Predict Existing Immunity Evasion of SARS-CoV-2 Variants by a New Biotechnological Application of Phage Display.

Multiple pathogens are competing against the human immune response, leading to outbreaks that are increasingly difficult to control. For example, the SARS-CoV-2 virus continually evolves, giving rise to new variants. The ability to evade the immune system is a crucial factor contributing to the spread of these variants within the human population. With the continuous emergence of new variants, it is challenging to comprehend all the possible combinations of previous infections, various vaccination types, and potential exposure to new variants in an individual patient. Rather than conducting variant-to-variant comparisons, an efficient approach could involve identifying key protein regions associated with the immune evasion of existing immunity against the virus. In this study, we propose a new biotechnological application of bacteriophages, the phage display platform for experimental identification of regions (linear epitopes) that may function as cross-reacting IgG hotspots in SARS-CoV-2 structural proteins. A total of 34,949 epitopes derived from genomes of all SARS-CoV-2 variants deposited prior to our library design were tested in a single assay. Cross-reacting IgG hotspots are protein regions frequently recognized by cross-reacting antibodies in many variants. The assay facilitated the one-step identification of immunogenic regions of proteins that effectively induced specific IgG in SARS-CoV-2-infected patients. We identified four regions demonstrating both significant immunogenicity and the activity of a cross-reacting IgG hotspot in protein S (located at NTD, RBD, HR1, and HR2/TM domains) and two such regions in protein N (at 197-280 and 358-419 aa positions). This novel method for identifying cross-reacting IgG hotspots holds promise for informing vaccine design and serological diagnostics for COVID-19 and other infectious diseases.

Immunogenicity of Endolysin PlyC.

Endolysins are bacteriolytic enzymes derived from bacteriophages. They represent an alternative to antibiotics, since they are not susceptible to conventional antimicrobial resistance mechanisms. Since non-human proteins are efficient inducers of specific immune responses, including the IgG response or the development of an allergic response mediated by IgE, we evaluated the general immunogenicity of the highly active antibacterial enzyme, PlyC, in a human population and in a mouse model. The study includes the identification of molecular epitopes of PlyC. The overall assessment of potential hypersensitivity to this protein and PlyC-specific IgE testing was also conducted in mice. PlyC induced efficient IgG production in mice, and the molecular analysis revealed that PlyC-specific IgG interacted with four immunogenic regions identified within the PlyCA subunit. In humans, approximately 10% of the population demonstrated IgG reactivity to the PlyCB subunit only, which is attributed to cross-reactions since this was a naïve serum. Of note, in spite of being immunogenic, PlyC induced a normal immune response, without hypersensitivity, since both the animals challenged with PlyC and in the human population PlyC-specific IgE was not detected.