Bacterial biofilms formed by pathogens are known to be hundreds of times more resistant to antimicrobial agents than planktonic cells, making it extremely difficult to cure biofilm-based infections despite the use of antibiotics, which poses a serious threat to human health. Therefore, there is an urgent need to develop promising alternative antimicrobial therapies to reduce the burden of drug-resistant bacterial infections caused by biofilms. As natural enemies of bacteria, bacteriophages (phages) have the advantages of high specificity, safety and non-toxicity, and possess great potential in the defense and removal of pathogenic bacterial biofilms, which are considered to be alternatives to treat bacterial diseases. This work mainly reviews the composition, structure and formation process of bacterial biofilms, briefly discusses the interaction between phages and biofilms, and summarizes several strategies based on phages and their derivatives against biofilms and drug-resistant bacterial infections caused by biofilms, serving the purpose of developing novel, safe and effective treatment methods against biofilm-based infections and promoting the application of phages in maintaining human health.

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Phages against Pathogenic Bacterial Biofilms and Biofilm-Based Infections: A Review

Endolysin-based therapeutics are promising antibacterial agents and can successfully supplement the existing antibacterial drugs array. It is specifically important in the case of Gram-negative pathogens, e.g., ESKAPE group bacteria, which includes Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, and are highly inclined to gain multiple antibiotic resistance. Despite numerous works devoted to the screening of new lytic enzymes and investigations of their biochemical properties, there are significant breaches in some aspects of their operating characteristics, including safety issues of endolysin use. Here, we provide a comprehensive study of the antimicrobial efficacy aspects of four Gram-negative bacteria-targeting endolysins LysAm24, LysAp22, LysECD7, and LysSi3, their in vitro and in vivo activity, and their biological safety. These endolysins possess a wide spectrum of action, are active against planktonic bacteria and bacterial biofilms, and are effective in wound and burn skin infection animal models. In terms of safety, these enzymes do not contribute to the development of short-term resistance, are not cytotoxic, and do not significantly affect the normal intestinal microflora in vivo. Our results provide a confident base for the development of effective and safe candidate dosage forms for the treatment of local and systemic infections caused by Gram-negative bacterial species.

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Discovering the Potentials of Four Phage Endolysins to Combat Gram-Negative Infections.

Bacteriophages (phages) are selective viral predators of bacteria. Abundant and ubiquitous in nature, phages can be used to treat bacterial infections (phage therapy), including refractory infections and those resistant to antibiotics. However, despite an abundance of anecdotal evidence of efficacy, significant hurdles remain before routine implementation of phage therapy into medical practice, including a dearth of robust clinical trial data. Phage–bacterium interactions are complex and diverse, characterized by co‐evolution trajectories that are significantly influenced by the environments in which they occur (mammalian body sites, water, soil, etc.). An understanding of the molecular mechanisms underpinning these dynamics is essential for successful clinical translation. This review aims to cover key aspects of bacterium–phage interactions that affect bacterial killing by describing the most relevant published literature and detailing the current knowledge gaps most likely to influence therapeutic success.

Biological foundations of successful bacteriophage therapy

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. 

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

Bacteriophage-encoded depolymerases are responsible for degrading capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) of the host bacteria during phage invasion. They have been considered as promising antivirulence agents in controlling bacterial infections, including those caused by multidrug-resistant (MDR) bacteria. This feature inspires hope of utilizing these enzymes to disarm the polysaccharide capsules of the bacterial cells, which then strengthens the action of antibiotics. Here we have identified, cloned, and expressed a depolymerase Dpo71 from a bacteriophage specific for the gram-negative bacterium Acinetobacter baumannii in a heterologous host Escherichia coli. Dpo71 sensitizes the MDR A. baumannii to the host immune attack, and also acts as an adjuvant to assist or boost the action of antibiotics, for example colistin. Specifically, Dpo71 at 10 μg/ml enables a complete bacterial eradication by human serum at 50% volume ratio. A mechanistic study shows that the enhanced bactericidal effect of colistin is attributed to the improved outer membrane destabilization capacity and binding rate to bacteria after stripping off the bacterial capsule by Dpo71. Dpo71 inhibits biofilm formation and disrupts the pre-formed biofilm. Combination of Dpo71 could significantly enhance the antibiofilm activity of colistin and improve the survival rate of A. baumannii infected Galleria mellonella. Dpo71 retains the strain-specificity of the parent phage from which Dpo71 is derived: the phage-sensitive A. baumannii strains respond to Dpo71 treatment, whereas the phage-insensitive strains do not. In summary, our work demonstrates the feasibility of using recombinant depolymerases as an antibiotic adjuvant to supplement the development of new antibacterials and to battle against MDR pathogens. 

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Phage-Derived Depolymerase as an Antibiotic Adjuvant Against Multidrug-Resistant Acinetobacter baumannii

Bacteriophage endolysins (lysins, or murein hydrolases) are enzymes that bacteriophages utilize to degrade the cell wall peptidoglycans (PG) and subsequently disintegrate bacterial cells from within. Due to their muralytic activity, lysins are considered as potential candidates to battle against antibiotic resistance. However, most lysins in their native form lack the capability of trespassing the outer membrane (OM) of Gram-negative (G-ve) bacteria. To turn the bacteriophage enzymes into antibacterial weapons against G-ve bacteria, endowing these enzymes the capability of accessing the PG substrate underneath the OM is critical. Here we show that fusing a membrane-permeabilizing peptide CeA at the C-terminus of a muralytic enzyme LysAB2 renders a two-step mechanism of bacterial killing and increases the activity of LysAB2 against the multidrug resistant Acinetobacter baumannii by up to 100 000-folds. The engineered LysAB2, termed LysAB2-KWK here, also shows remarkable activity against A. baumannii at the stationary phase and a prominent capability to disrupt biofilm formation. In addition, the enzyme shows a broad antibacterial spectrum against G-ve bacteria, a decent tolerance to serum, and a prolonged storage life. LysAB2-KWK rescues the larva of the greater wax moth Galleria mellonella from A. baumannii infection through systemic administration. Altogether, our work equips a globular lysin with OM permeabilization activity to enable effective killing of G-ve bacteria, reveals the critical role of the C-terminus of a globular lysin in the antibacterial activity, and points toward a viable route to engineer globular lysins as antibacterial enzymes for potential clinical use against multidrug resistant G-ve bacteria.

Membrane-Permeable Antibacterial Enzyme against Multidrug-Resistant Acinetobacter baumannii.

Facing the increasing threat of multi-drug antimicrobial resistance (AMR), humans strive to search for antibiotic drug candidates and antibacterial alternatives from all possible places, from soils...

Bacteriophage endolysins against gram-positive bacteria, an overview on the clinical development and recent advances on the delivery and formulation strategies

Pengfei Zhang

Papers

Membrane-Permeable Antibacterial Enzyme against Multidrug-Resistant Acinetobacter baumannii.

Bacteriophage endolysins against gram-positive bacteria, an overview on the clinical development and recent advances on the delivery and formulation strategies