Distinct mode of action of a highly stable, engineered phage lysin killing Gram-negative bacteria.

IMPORTANCE: Engineered lysins are considered as highly promising alternatives for antibiotics. Our previous screening study using VersaTile technology identified 1D10 as a possible lead compound with activity against Acinetobacter baumannii strains under elevated human serum concentrations. In this manuscript, we reveal an unexpected mode of action and exceptional thermoresistance for lysin 1D10. Our findings shed new light on the development of engineered lysins, providing valuable insights for future research in this field.

Characterization and genomic analysis of novel bacteriophage NK20 to revert colistin resistance and combat pandrug-resistant Klebsiella pneumoniae in a rat respiratory infection model.

AIM: We investigated the therapeutic capacity of the isolated Klebsiella bacteriophage NK20 against pandrug-resistant strains. Moreover, we assessed the impact of resistance development on the overall therapeutic outcome both in vitro and in vivo. MAIN METHODS: The pandrug-resistant K. pneumoniae Kp20 is used as a host strain for the isolation of bacteriophages using sewage samples. Spot assay was then used to compare the spectra of the isolated phages, while kinetic and genomic analysis of the phage with the broadest spectrum was assessed. Antibacterial potential of the phage was assessed using turbidimetric assay and MIC with and without colistin. Finally, the therapeutic efficacy was evaluated in vivo using a rat respiratory infection model. KEY FINDINGS: The isolated lytic bacteriophage (NK20) showed a relatively broad spectrum and an acceptable genomic profile. In vitro antibacterial assay revealed bacterial resistance development after 12 h. Colistin inhibited bacterial regrowth and reduced pandrug-resistant strains' colistin MICs. Despite the isolation of resistant clones, intranasal administration of NK20 significantly (p < 0.05) reduced the bacterial load in both the pulmonary and blood compartments and rescued 100 % of challenged rats. Histological and immunological analysis of treated animals' lung tissue revealed less inflammation and lower TNF-α and caspase-3 expression. SIGNIFICANCE: NK20 is a promising candidate that rescued rats from untreatable, pan-drug-resistant K. pneumoniae Kp20. Moreover, it steers the evolution of resistant mutants with higher sensitivity to colistin and less virulence, opening the door for using phages as sensitizing and anti-virulence entities rather than direct killer.

Cecropin A Improves the Antibacterial Activity of Hen Egg White Lysozyme against Challenging Salmonella enterica Serovars.

The prevalence of multidrug-resistant Salmonella enterica among animal- and plant-derived food products threatens global healthcare and economic sectors. Hen egg white lysozyme is widely exploited as a food preservative against Gram-positive pathogens. Nevertheless, its limited penetration of the outer membrane renders it ineffective against Gram-negative bacteria. Herein, we present a safe and effective approach to facilitate HEWL access to peptidoglycan layers using cecropin A. In silico analysis of cecropin A peptide revealed an amphipathic α-helical peptide with potential outer membrane permeabilizing activity through its interaction with both hydrophobic and ionic stabilizing forces. Evaluation of HEWL/cecropin A combination showed a cecropin A dose-dependent bacterial count reduction up to 4.16 and 3.18 ± 0.26 log units against Salmonella enterica ATCC 35664 at the logarithmic and stationary growth phases, respectively. Moreover, the combination displayed antibacterial activity of 2.1 ± 0.31 and ~1 log-unit reductions against Salmonella enterica serovars Kentucky, Typhimurium, and Enteritidis, respectively, whereas Hato and Shangani were found irresponsive. The cytotoxicity assay revealed compatibility of cecropin A with oral epithelial cells. These observations suggest HEWL/cecropin A combination as an effective and safe alternative to lysozyme against Salmonella enterica.

Mechanistic study of the antibacterial potential of the prenylated flavonoid auriculasin against Escherichia coli.

Bacterial resistance is spreading in an alarming manner, outpacing the rate of development of new antibacterial agents and surging the need for effective alternatives. Prenylated flavonoids are a promising class of natural antibiotics with reported activity against a wide range of resistant pathogens. Here, a large library of natural flavonoids (1718 structures) was virtually screened for potential candidates inhibiting the B-subunit of gyrase (Gyr-B). Twenty-eight candidates, predominated by prenylated flavonoids, appeared as promising hits. Six of them were selected for further in vitro antibacterial and Gyr-B enzyme inhibitory activities. Auriculasin is presented as the most potent antibacterial candidate, with a MIC ranging from 2 to 4 µg/ml against two clinically isolated multidrug-resistant Escherichia coli strains. Mechanistic antibacterial analysis revealed auriculasin inhibitory activity towards the Gyr-B enzyme on the micromolar scale (IC50 = 0.38 ± 0.15 µM). Gyr-B interaction was further detailed by conducting an isothermal titration calorimetric experiment, which revealed a competitive inhibition with a high affinity for the Gyr-B active site, achieved mostly through enthalpic interactions (ΔGbinding = -10.69 kcal/mol). Molecular modeling and physics-based simulations demonstrated the molecule's manner of fitting inside the Gyr-B active site, indicating a very potential nucleus for the future generation of more potent derivatives. To conclude, prenylated flavonoids are interesting antibacterial candidates with anti-Gyr-B mechanism of action that can be obtained from a plant-derived flavonoid.

The Specific Capsule Depolymerase of Phage PMK34 Sensitizes Acinetobacter baumannii to Serum Killing.

The rising antimicrobial resistance is particularly alarming for Acinetobacter baumannii, calling for the discovery and evaluation of alternatives to treat A. baumannii infections. Some bacteriophages produce a structural protein that depolymerizes capsular exopolysaccharide. Such purified depolymerases are considered as novel antivirulence compounds. We identified and characterized a depolymerase (DpoMK34) from Acinetobacter phage vB_AbaP_PMK34 active against the clinical isolate A. baumannii MK34. In silico analysis reveals a modular protein displaying a conserved N-terminal domain for anchoring to the phage tail, and variable central and C-terminal domains for enzymatic activity and specificity. AlphaFold-Multimer predicts a trimeric protein adopting an elongated structure due to a long α-helix, an enzymatic ß-helix domain and a hypervariable 4 amino acid hotspot in the most ultimate loop of the C-terminal domain. In contrast to the tail fiber of phage T3, this hypervariable hotspot appears unrelated with the primary receptor. The functional characterization of DpoMK34 revealed a mesophilic enzyme active up to 50 °C across a wide pH range (4 to 11) and specific for the capsule of A. baumannii MK34. Enzymatic degradation of the A. baumannii MK34 capsule causes a significant drop in phage adsorption from 95% to 9% after 5 min. Although lacking intrinsic antibacterial activity, DpoMK34 renders A. baumannii MK34 fully susceptible to serum killing in a serum concentration dependent manner. Unlike phage PMK34, DpoMK34 does not easily select for resistant mutants either against PMK34 or itself. In sum, DpoMK34 is a potential antivirulence compound that can be included in a depolymerase cocktail to control difficult to treat A. baumannii infections.

Engineering a Lysin with Intrinsic Antibacterial Activity (LysMK34) by Cecropin A Fusion Enhances Its Antibacterial Properties against Acinetobacter baumannii.

Bacteriophage-encoded lysins are increasingly reported as alternatives to combat Acinetobacter baumannii infections, for which limited therapeutic options are available. Some lysins, such as LysMK34, have a C-terminal amphipathic helix allowing them to penetrate the otherwise-impermeable outer membrane barrier. Another approach to kill Gram-negative pathogens with lysins relies on fusion of a peptide with outer membrane-permeabilizing properties to the lysin. In this work, we aimed to leverage the intrinsic antibacterial activity of LysMK34 by fusing the peptide cecropin A to its N terminus via a linker of three Ala-Gly repeats, resulting in engineered LysMK34 (eLysMK34). The engineered lysin has an improved antibacterial activity compared to that of the parental lysin, LysMK34, in terms of MICs (0.45 to 1.2 µM), killing rate, and killing extent. eLysMK34 has a ≥2-fold-increased activity against stationary-phase cells, and the bactericidal effect becomes less dependent on the intracellular osmotic pressure. In particular, colistin-resistant strains become highly susceptible to eLysMK34, and enhanced antibacterial activity is observed in complement-deactivated human serum. These observations demonstrate that fusion of a lysin with intrinsic antibacterial activity with a selected outer membrane-permeabilizing peptide is a useful strategy to further improve the in vitro antibacterial properties of such lysins. IMPORTANCE Phage lysins are a new class of enzyme-based antibiotics that increasingly gain interest. Lysins kill cells through rapid degradation of the peptidoglycan layer, resulting in sudden osmotic lysis. Whereas Gram-positive bacteria are readily susceptible to the actions of lysins, Gram-negative bacteria are naturally resistant, as the outer membrane protects their peptidoglycan layer. This work reveals that fusing an outer membrane-permeabilizing peptide to a lysin with intrinsic antibacterial activity results in a superior lysin that shows improved robustness in its antibacterial activity, including against the most worrisome colistin-resistant A. baumannii strains.

Lysin LysMK34 of Acinetobacter baumannii Bacteriophage PMK34 Has a Turgor Pressure-Dependent Intrinsic Antibacterial Activity and Reverts Colistin Resistance.

The prevalence of extensively and pandrug-resistant strains of Acinetobacter baumannii leaves little or no therapeutic options for treatment for this bacterial pathogen. Bacteriophages and their lysins represent attractive alternative antibacterial strategies in this regard. We used the extensively drug-resistant A. baumannii strain MK34 to isolate the bacteriophage PMK34 (vB_AbaP_PMK34). This phage shows fast adsorption and lacks virulence genes; nonetheless, its narrow host spectrum based on capsule recognition limits broad application. PMK34 is a Fri1virus member of the Autographiviridae and has a 41.8-kb genome (50 open reading frames), encoding an endolysin (LysMK34) with potent muralytic activity (1,499.9 ± 131 U/µM), a typical mesophilic thermal stability up to 55°C, and a broad pH activity range (4 to 10). LysMK34 has an intrinsic antibacterial activity up to 4.8 and 2.4 log units for A. baumannii and Pseudomonas aeruginosa strains, respectively, but only when a high turgor pressure is present. The addition of 0.5 mM EDTA or application of an osmotic shock after treatment can compensate for the lack of a high turgor pressure. The combination of LysMK34 and colistin results in up to 32-fold reduction of the MIC of colistin, and colistin-resistant strains are resensitized in both Mueller-Hinton broth and 50% human serum. As such, LysMK34 may be used to safeguard the applicability of colistin as a last-resort antibiotic.IMPORTANCEA. baumannii is one of the most challenging pathogens for which development of new and effective antimicrobials is urgently needed. Colistin is a last-resort antibiotic, and even colistin-resistant A. baumannii strains exist. Here, we present a lysin that sensitizes A. baumannii for colistin and can revert colistin resistance to colistin susceptibility. The lysin also shows a strong, turgor pressure-dependent intrinsic antibacterial activity, providing new insights in the mode of action of lysins with intrinsic activity against Gram-negative bacteria.

The Preclinical and Clinical Progress of Bacteriophages and Their Lytic Enzymes: The Parts are Easier than the Whole.

The therapeutic potential of phages has been considered since their first identification more than a century ago. The evident concept of using a natural predator to treat bacterial infections has, however, since then been challenged considerably. Initially, the vast success of antibiotics almost eliminated the study of phages for therapy. Upon the renaissance of phage therapy research, the most provocative and unique properties of phages such as high specificity, self-replication and co-evolution prohibited a rapid preclinical and clinical development. On the one hand, the typical trajectory followed by small molecule antibiotics could not be simply translated into the preclinical analysis of phages, exemplified by the need for complex broad spectrum or personalized phage cocktails of high purity and the more complex pharmacokinetics. On the other hand, there was no fitting regulatory framework to deal with flexible and sustainable phage therapy approaches, including the setup and approval of adequate clinical trials. While significant advances are incrementally made to eliminate these hurdles, phage-inspired antibacterials have progressed in the slipstream of phage therapy, benefiting from the lack of hurdles that are typically associated with phage therapy. Most advanced are phage lytic enzymes that kill bacteria through peptidoglycan degradation and osmotic lysis. Both phages and their lytic enzymes are now widely considered as safe and have now progressed to clinical phase II to show clinical efficacy as pharmaceutical. Yet, more initiatives are needed to fill the clinical pipeline to beat the typical attrition rates of clinical evaluation and to come to a true evaluation of phages and phage lytic enzymes in the clinic.