Rapid Antibacterial Activity Assessment of Chimeric Lysins.

Various chimeric lysins have been developed as efficacious antibiotics against multidrug-resistant bacteria, but direct comparisons of their antibacterial activities have been difficult due to the preparation of multiple recombinant chimeric lysins. Previously, we reported an Escherichia coli cell-free expression method to better screen chimeric lysins against Staphylococcus aureus, but we still needed to increase the amounts of expressed proteins enough to be able to detect them non-isotopically for quantity comparisons. In this study, we improved the previous cell-free expression system by adding a previously reported artificial T7 terminator and reversing the different nucleotides between the T7 promoter and start codon to those of the T7 phage. The new method increased the expressed amount of chimeric lysins enough for us to detect them using Western blotting. Therefore, the qualitative comparison of activity between different chimeric lysins has become possible via the adjustment of the number of variables between samples without protein purification. We applied this method to select more active chimeric lysins derived from our previously reported chimeric lysin (ALS2). Finally, we compared the antibacterial activities of our selected chimeric lysins with reported chimeric lysins (ClyC and ClyO) and lysostaphin and determined the rank orders of antibacterial activities on different Staphylococcus aureus strains in our experimental conditions.

Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation.

We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin-an enzyme that degrades the cell wall of Staphylococcus aureus-while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para-azidophenylalanine. The incorporation of this "reactive handle" enabled the orthogonal site-specific modification of the enzyme variants with polyethylene glycol (PEG) using copper-free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site-specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.

The choice of chromatographic resin for the purification of recombinant lysostaphin affects its activity.

Lysostaphin is a zinc-dependent endopeptidase that is effective against both antibiotic-sensitive and antibiotic-resistant strains of Staphylococcus aureus. Lysostaphin is typically purified on cation-exchange or metal-chelate affinity resins, and there are data indicating potential influence of the chromatographic resin on the lysostaphin activity. In this study, we systematically investigated the impact of the resin used to purify the recombinant lysostaphin on its activity. To this end, recombinant lysostaphin with an additional histidine tag at the C-terminus was purified using a cation-exchange resin, three types of nickel-chelate resins with different strength of metal ion binding, or a zinc-chelate resin. Lysostaphin samples purified on the cation-exchange resin (WorkBeads 40S), the nickel-chelate resin with a strong nickel ion binding (WorkBeads NiMAC), and the zinc-chelate resin (WorkBeads NTA with immobilized zinc ions) had equal activity. On the contrary, the activity of lysostaphin preparations purified on nickel-chelate resins with medium (WorkBeads Ni-NTA) and relatively weak (WorkBeads Ni-IDA) nickel ion binding was significantly reduced. The decrease in activity can be explained by the interaction of lysostaphin with the nickel ions leached from the resin and is caused by either the exchange of the zinc ion in the lysostaphin active center with a nickel ion from the resin, or binding of an additional ion that inhibits the enzymatic activity. Removal of the metal ions from the active site of lysostaphin and subsequent incorporation of the native zinc ions lead to complete restoration of the activity of the enzyme.

Characterization of the LysP2110-HolP2110 Lysis System in Ralstonia solanacearum Phage P2110.

Ralstonia solanacearum, a pathogen causing widespread bacterial wilt disease in numerous crops, currently lacks an optimal control agent. Given the limitations of traditional chemical control methods, including the risk of engendering drug-resistant strains and environmental harm, there is a dire need for sustainable alternatives. One alternative is lysin proteins that selectively lyse bacteria without contributing to resistance development. This work explored the biocontrol potential of the LysP2110-HolP2110 system of Ralstonia solanacearum phage P2110. Bioinformatics analyses pinpointed this system as the primary phage-mediated host cell lysis mechanism. Our data suggest that LysP2110, a member of the Muraidase superfamily, requires HolP2110 for efficient bacterial lysis, presumably via translocation across the bacterial membrane. LysP2110 also exhibits broad-spectrum antibacterial activity in the presence of the outer membrane permeabilizer EDTA. Additionally, we identified HolP2110 as a distinct holin structure unique to the Ralstonia phages, underscoring its crucial role in controlling bacterial lysis through its effect on bacterial ATP levels. These findings provide valuable insights into the function of the LysP2110-HolP2110 lysis system and establish LysP2110 as a promising antimicrobial agent for biocontrol applications. This study underpins the potential of these findings in developing effective and environment-friendly biocontrol strategies against bacterial wilt and other crop diseases.

Comparative study of antibacterial activity and stability of D-enantiomeric and L-enantiomeric bovine NK-lysin peptide NK2A.

L-enantiomers of antimicrobial peptides (AMPs) are sensitive to proteolytic degradation; however, D-enantiomers of AMPs are expected to provide improved proteolytic resistance. The present study aimed to comparatively investigate the in vitro antibacterial activity, trypsin and serum stability, toxicity, and in vivo antibacterial activity of L-enantiomeric bovine NK2A (L-NK2A) and its D-enantiomeric NK2A (D-NK2A). Circular dichroism spectroscopy of D-NK2A and L-NK2A in anionic liposomes showed α-helical structures and the α-helical conformation of D-NK2A was a mirror image of L-NK2A. Both D-NK2A and L-NK2A displayed minimal in vitro and in vivo toxicities. RP-HPLC and mass spectrometry analyses revealed that D-NK2A, but not L-NK2A, was resistant to trypsin digestion. D-NK2A and L-NK2A showed similar in vitro bacterial killing activities against Histophilus somni. Slightly reduced antibacterial activity was observed when D-NK2A and L-NK2A were pre-incubated with serum. Confocal and transmission electron microscopic findings confirmed that both peptides induced disruption of bacterial inner- and outer-membranes. Improved survivals with D-NK2A treatment were observed when compared to L-NK2A in a murine model of acute H. somni septicemia. We conclude that antibacterial activity and mode of action of NK2A are not chiral specific. With further optimization, D-NK2A may be a viable AMP candidate to combat bacterial infections.

NK-lysin antimicrobial peptide-functionalized nanoporous alumina membranes as biosensors for label-free bacterial endotoxin detection.

We report an NK-lysin peptide-functionalized nanoporous anodized aluminum oxide (NAAO) based biosensor to detect bacterial endotoxin. Bovine NK-lysin-derived peptides show antimicrobial activity against bacterial pathogens, and bactericidal activity is primarily due to the membranolysis activity. Antimicrobial activity of NK-lysin NK2A was confirmed against a Gram-negative Mannheimia haemolytica and a Gram-positive Staphylococcus aureus. Electron microscopic examination showed the localization of NK2A conjugated silver nanoparticles, but not unconjugated silver nanoparticles used as control, to the bacterial outer membrane and cell wall. NK2A functionalized NAAO membranes were used in a previously developed four-electrode electrochemical configuration to detect the presence of Gram-negative bacterial lipopolysaccharides (LPS) and Gram-positive bacterial lipoteichoic acid (LTA) molecules. NK2A-functionalized NAAO biosensor could detect LPS with a detection limit of 10 ng/mL within an appreciable signal/noise ratio. Biosensors functionalized with a scrambled amino acid version of NK2A (Sc-NK2A) that lacks antimicrobial activity could not detect the presence of LPS. However, both NK2A and Sc-NK2A functionalized biosensors showed sensing signals with Gram-positive bacterial lipoteichoic acids. These results suggest that the specific binding of NK2A-LPS on the NAAO membrane surface is responsible for the observed biosensor signals. These findings suggest that NK2A-functionalized biosensors can be used for rapid and sensitive label-free LPS detection.

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

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 in remote areas to deep in the sea. In this "gold rush for antibacterials," researchers turn to the natural enemy of bacterial cells, bacteriophage (phages), and find them a rich source of weapons for AMR bacteria. Endolysins (lysins), the enzymes phages use to break the bacterial cells from within, have been shown to be highly selective and efficient in killing their target bacteria from outside while maintaining a low occurrence of bacterial resistance. In this review, we start with the structures and mechanisms of action of lysins against Gram-positive (GM+) bacteria. The developmental history of lysins is also outlined. Then, we detail the latest preclinical and clinical research on their safety and efficacy against GM+ bacteria, focusing on the formulation strategies of these enzymes. Finally, the challenges and potential hurdles are discussed. Notwithstanding these limitations, the trends in development indicate that the first, approved lysin drugs will be available soon in the near future. Overall, this review presents a timely summary of the current progress on lysins as antibacterial enzymes for AMR GM+ bacteria, and provides a guidebook for biomaterial researchers who are dedicating themselves to the battle against bacterial infections.

Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium that presents resistance to several antibiotics, thus, representing a major threat to human and animal health. Phage-derived products, namely lysins, or peptidoglycan-hydrolyzing enzymes, can be an effective weapon against antibiotic-resistant bacteria. Whereas in Gram-positive bacteria, lysis from without is facilitated by the exposed peptidoglycan layer, this is not possible in the outer membrane-protected peptidoglycan of Gram-negative bacteria. Here, we suggest the encapsulation of lysins in liposomes as a delivery system against Gram-negative bacteria, using the model of P. aeruginosa. Bioinformatic analysis allowed for the identification of 38 distinct complete prophages within 66 P. aeruginosa genomes (16 of which newly sequenced) and led to the identification of 19 lysins of diverse sequence and function, 5 of which proceeded to wet lab analysis. The four purifiable lysins showed hydrolytic activity against Gram-positive bacterial lawns and, on zymogram assays, constituted of autoclaved P. aeruginosa cells. Additionally, lysins Pa7 and Pa119 combined with an outer membrane permeabilizer showed activity against P. aeruginosa cells. These two lysins were successfully encapsulated in DPPC:DOPE:CHEMS (molar ratio 4:4:2) liposomes with an average encapsulation efficiency of 33.33% and 32.30%, respectively. The application of the encapsulated lysins to the model P. aeruginosa led to a reduction in cell viability and resulted in cell lysis as observed in MTT cell viability assays and electron microscopy. In sum, we report here that prophages may be important sources of new enzybiotics, with prophage lysins showing high diversity and activity. In addition, these enzybiotics following their incorporation in liposomes were able to potentiate their antibacterial effect against the Gram-negative bacteria P. aeruginosa, used as the model.

Cloning and Characterization of a Thermostable Endolysin of Bacteriophage TP-84 as a Potential Disinfectant and Biofilm-Removing Biological Agent.

The obligatory step in the life cycle of a lytic bacteriophage is the release of its progeny particles from infected bacterial cells. The main barrier to overcome is the cell wall, composed of crosslinked peptidoglycan, which counteracts the pressure prevailing in the cytoplasm and protects the cell against osmotic lysis and mechanical damage. Bacteriophages have developed two strategies leading to the release of progeny particles: the inhibition of peptidoglycan synthesis and enzymatic cleavage by a bacteriophage-coded endolysin. In this study, we cloned and investigated the TP84_28 endolysin of the bacteriophage TP-84, which infects thermophilic Geobacillus stearothermophilus, determined the enzymatic characteristics, and initially evaluated the endolysin application as a non-invasive agent for disinfecting surfaces, including those exposed to high temperatures. Both the native and recombinant TP84_28 endolysins, obtained through the Escherichia coli T7-lac expression system, are highly thermostable and retain trace activity after incubation at 100 °C for 30 min. The proteins exhibit strong bacterial wall digestion activity up to 77.6 °C, decreasing to marginal activity at ambient temperatures. We assayed the lysis of various types of bacteria using TP84_28 endolysins: Gram-positive, Gram-negative, encapsulated, and pathogenic. Significant lytic activity was observed on the thermophilic and mesophilic Gram-positive bacteria and, to a lesser extent, on the thermophilic and mesophilic Gram-negative bacteria. The thermostable TP84_28 endolysin seems to be a promising mild agent for disinfecting surfaces exposed to high temperatures.

Exploration of Synergistic Action of Cell Wall-Degrading Enzymes against Mycobacterium tuberculosis.

The major global health threat tuberculosis is caused by Mycobacterium tuberculosis. M. tuberculosis has a complex cell envelope-a partially covalently linked composite of polysaccharides, peptidoglycan, and lipids, including a mycolic acid layer-which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating Gram-positive and -negative infections with cell wall-degrading enzymes, we investigated such an approach for M. tuberculosis. In this study, we aimed to (i) develop an M. tuberculosis microtiter growth inhibition assay that allows undisturbed cell envelope formation to overcome the invalidation of results by typical clumped M. tuberculosis growth in surfactant-free assays, (ii) explore anti-M. tuberculosis potency of cell wall layer-degrading enzymes, and (iii) investigate the concerted action of several such enzymes. We inserted a bacterial luciferase operon in an auxotrophic M. tuberculosis strain to develop a microtiter assay that allows proper evaluation of cell wall-degrading anti-M. tuberculosis enzymes. We assessed growth inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase [LysB], fungal α-amylase, and human and chicken egg white lysozymes) and combinations thereof in the presence or absence of biopharmaceutically acceptable surfactant. Our biosafety level 2 assay identified both LysB and lysozymes as potent M. tuberculosis inhibitors but only in the presence of surfactant. Moreover, the most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, α-amylase, and polysorbate 80. Synergistically acting cell wall-degrading enzymes are potently inhibiting M. tuberculosis, which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

Structural and biochemical analyses of the tetrameric cell binding domain of Lys170 from enterococcal phage F170/08.

Lysins are a class of hydrolytic enzymes used by bacteriophages to target and cleave the peptidoglycan of bacterial cell walls during their lytic cycle. The lysins from bacteriophages that infect Gram-positive bacteria are typically monomeric and consist of one or two catalytic domains (CD) and a cell binding domain (CBD). However, multimeric lysins encoded by a single gene have also been reported, among which Lys170 from enterococcal phage F170/08 was one of the first identified. Here, we determined the crystal structure of Lys170 CBD at 1.40 Å resolution. The structure reveals that Lys170 CBDs assemble into a tetrameric functional unit and that each monomer folds into a three-stranded ß-sheet core capped on each side by an α-helix. In addition, we identified key residues of Lys170 CBD involved in host cell binding. Our work provides a basis for designing highly efficient lysins targeting Enterococcus faecalis.

Bovine NK-lysin-derived peptides have bactericidal effects against Mycobacterium avium subspecies paratuberculosis.

Infection with Mycobacterium avium subspecies paratuberculosis (MAP) is complex, but little is known about the role that natural killer (NK) cells play. In the present study, four bovine NK-lysin peptides were synthesized to evaluate their bactericidal activity against MAP. The results demonstrated that bNK-lysin peptides were directly bactericidal against MAP, with bNK1 and bNK2A being more potent than bNK2B and bNK2C. Mechanistically, transmission electron microscopy revealed that the incubation of MAP with bNK2A resulted in extensive damage to cell membranes and cytosolic content leakage. Furthermore, the addition of bNK2A linked with a cell-penetrating peptide resulted in increased MAP killing in a macrophage model.

Chemotherapy with Phage Lysins Reduces Pneumococcal Colonization of the Respiratory Tract.

Bacteriophage-borne lytic enzymes, also named lysins or enzybiotics, are efficient agents for the killing of bacterial pathogens. The colonization of the respiratory tract by Streptococcus pneumoniae is a prerequisite for the establishment of the infection process. Hence, we have evaluated the antibacterial activities of three different lysins against pneumococcal colonization using human nasopharyngeal and lung epithelial cells as well as a mouse model of nasopharyngeal colonization. The lysins tested were the wild-type Cpl-1, the engineered Cpl-7S, and the chimera Cpl-711. Moreover, we included amoxicillin as a comparator antibiotic. Human epithelial cells were infected with three different multidrug-resistant clinical isolates of S. pneumoniae followed by a single dose of the corresponding lysin. The antimicrobial activities of these lysins were also evaluated using a mouse nasopharyngeal carriage model. The exposure of the infected epithelial cells to Cpl-7S did not result in the killing of any of the pneumococcal strains investigated. However, the treatment with Cpl-1 or Cpl-711 increased the killing of S. pneumoniae organisms adhered to both types of human epithelial cells, with Cpl-711 being more effective than Cpl-1, at subinhibitory concentrations. In addition, a treatment with amoxicillin had no effect on reducing the carrier state, whereas mice treated by the intranasal route with Cpl-711 showed significantly reduced nasopharyngeal colonization, with no detection of bacterial load in 20 to 40% of the mice. This study indicates that Cpl-1 and Cpl-711 lysins might be promising antimicrobial candidates for therapy against pneumococcal colonization.

Efficacy of Novel Antistaphylococcal Ectolysin P128 in a Rat Model of Methicillin-Resistant Staphylococcus aureus Bacteremia.

Staphylococcus aureus causes systemic infections with high morbidity and mortality, and the emergence of drug-resistant strains is a rapidly growing clinical concern. Novel therapeutic agents are required to tackle S. aureus infections. P128 is a bacteriophage-derived chimeric ectolysin with potent and rapid bactericidal activity against S. aureus In the present study, the efficacy of P128 was evaluated in a newly developed rat model of S. aureus bacteremia. Prior to in vivo testing, P128 was shown to be stable in whole blood by incubation in rat blood for up to 6 h and testing its bactericidal activity against the methicillin-resistant S. aureus isolate USA300. Rats succumbed to intravenous challenge with 109 CFU of S. aureus USA300, resulting in 80 to 100% mortality by day 14. Evaluation of the bacterial load in various organs at 96 h postinfection revealed high bacterial counts in the kidney, and this correlated with the presence of renal abscesses. Treatment of infected animals with P128 either by intravenous bolus administration via tail vein or by 1-h infusion via the jugular vein at 2 h postinfection resulted in the dose-dependent survival of rats. P128 treatment also resulted in very few or no abscesses in the kidneys. These data show that P128 is stable in the physiological milieu and that intravenous treatment with P128 is highly effective in rescuing rats from S. aureus bacteremia. P128 can be a novel therapeutic option for treatment of S. aureus systemic infections.

Applications of bacteriophages versus phage enzymes to combat and cure bacterial infections: an ambitious and also a realistic application?

Bacteriophages (phages) are viruses that infect bacteria. The "predator-prey" interactions are recognized as a potentially effective way to treat infections. Phages, as well as phage-derived proteins, especially enzymes, are intensively studied to become future alternative or supportive antibacterials used alone or in combination with standard antibiotic regimens treatment. There are many publications presenting phage therapy aspects, and some papers focused separately on the application of phage-derived enzymes. In this review, we discuss advantages and limitations of both agents concerning their specificity, mode of action, structural issues, resistance development, pharmacokinetics, product preparation, and interactions with the immune system. Finally, we describe the current regulations for phage-based product application.

Bacteriophages and their derivatives for the treatment and control of food-producing animal infections.

Nowadays, the world is facing an increasing emergence of antibiotic resistant bacteria. Simultaneously, the banning of some existing antibiotics and the lack of development of new antimicrobials have created an urgent need to find new alternatives against animal infections. Bacteriophages (phages) are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and harmless to animals. For these reasons, phages and their derivatives are being considered valuable antimicrobial alternatives and an opportunity to reduce the current use of antibiotics in agri-food production, increasing animal productivity and providing environmental protection. Furthermore, the possibility of combining phage genetic material with foreign genes encoding peptides of interest has enabled their use as vaccine delivery tools. In this case, besides bacterial infections, they might be used to prevent viral infections. This review explores current data regarding advances on the use of phages and phage-encoded proteins, such as endolysins, exolysins and depolymerases, either for therapeutic or prophylactic applications, in animal husbandry. The use of recombinant phage-derived particles or genetically modified phages, including phage vaccines, will also be reviewed.

In vitro gene expression-coupled bacterial cell chip for screening species-specific antimicrobial enzymes.

Targeting infectious bacterial pathogens is important for reducing the evolution of antibiotic-resistant bacteria and preserving the endogenous human microbiome. Cell lytic enzymes including bacteriophage endolysins, bacterial autolysins, and other bacteriolysins are useful antibiotic alternatives due to their exceptional target selectivity, which may be used to lysins rapidly kill target bacteria and their high specificity permit the normal commensal microflora to be left undisturbed. Genetic information of numerous lysins is currently available, but the identification of their antimicrobial function and specificity has been limited because most lysins are often poorly expressed and exhibit low solubilities. Here, we report the development of bacterial cell chip for rapidly accessing the function of diverse genes that are suggestive of encoding lysins. This approach can be used to evaluate rapidly the species-specific antimicrobial activity of diverse lysins synthesized from in vitro transcription and translation (TNT) of plasmid DNA. In addition, new potent lysins can be assessed that are not expressed in hosts and display low solubility. As a result of evaluating the species-specific antimicrobial function of 11 (un)known lysins with an in vitro TNT-coupled bacterial cell chip, a potent recombinant lysin against Staphylococcus strains, SA1, was identified. The SA1 was highly potent against not only S. aureus, but also both lysostaphin-resistant S. simulans and S. epidermidis cells. To this end, the SA1 may be applicable to treat both methicillin-resistant S. aureus (MRSA) and lysostaphin-resistant MRSA mutants. Biotechnol. Bioeng. 2017;114: 1648-1657. © 2017 Wiley Periodicals, Inc.

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process.

Bacteriophages are bacterial viruses that infect the host after successful receptor recognition and adsorption to the cell surface. The irreversible adherence followed by genome material ejection into host cell cytoplasm must be preceded by the passage of diverse carbohydrate barriers such as capsule polysaccharides (CPSs), O-polysaccharide chains of lipopolysaccharide (LPS) molecules, extracellular polysaccharides (EPSs) forming biofilm matrix, and peptidoglycan (PG) layers. For that purpose, bacteriophages are equipped with various virion-associated carbohydrate active enzymes, termed polysaccharide depolymerases and lysins, that recognize, bind, and degrade the polysaccharide compounds. We discuss the existing diversity in structural locations, variable architectures, enzymatic specificities, and evolutionary aspects of polysaccharide depolymerases and virion-associated lysins (VALs) and illustrate how these aspects can correlate with the host spectrum. In addition, we present methods that can be used for activity determination and the application potential of these enzymes as antibacterials, antivirulence agents, and diagnostic tools.

Enhancement of the direct antimicrobial activity of Lysep3 against Escherichia coli by inserting cationic peptides into its C terminus.

Phage lysins are considered promising antimicrobials against resistant bacterial infections. Some lysins have been reported for the prevention and treatment of Gram-positive bacterial infection. Gram-negative bacterial phage lysins, however, can only destroy the bacterial cell wall from inside because of the obstruction of the bacterial outer membrane that prevents direct hydrolysis of the bacterial wall peptidoglycan from the outside, severely restricting the development of lysins against Gram-negative bacteria. In this study, genetic engineering techniques were used to fuse a 5 cationic amino acid polypeptide (KRKRK), a 10 cationic amino acid polypeptide (KRKRKRKRKR), a 15 cationic amino acid polypeptide (KRKRKRKRKRKRKRK), and a polypeptide including both cationic and hydrophobic amino acids (KRKRKFFVAIIP) to the C-terminus of the Escherichia coli phage lysin Lysep3 to obtain four fusion lysins (5aa, 10aa, 15aa, Mix). The bactericidal effects of those four lysins on E. coli were then compared in vitro. Our results showed that the fusion of hydrophobic and positively charged amino acids, Mix, can kill E. coli effectively; the fusion of positively charged amino acids alone at the C-terminus (5aa, 10aa, 15aa) also showed bactericidal activity against E. coli from the outside, with the bactericidal activity gradually increasing with the positive charge at the C-terminus of the lysin. Collectively, improving the positive charge at the C-terminus of E. coli bacteriophage lysin Lysep3 increases its bactericidal ability from outside E. coli, providing a new practical method for the development of anti-Gram-negative bacterial lysins.

Characterization and genome sequencing of a Citrobacter freundii phage CfP1 harboring a lysin active against multidrug-resistant isolates.

Citrobacter spp., although frequently ignored, is emerging as an important nosocomial bacterium able to cause various superficial and systemic life-threatening infections. Considered to be hard-to-treat bacterium due to its pattern of high antibiotic resistance, it is important to develop effective measures for early and efficient therapy. In this study, the first myovirus (vB_CfrM_CfP1) lytic for Citrobacter freundii was microbiologically and genomically characterized. Its morphology, activity spectrum, burst size, and biophysical stability spectrum were determined. CfP1 specifically infects C. freundii, has broad host range (>85 %; 21 strains tested), a burst size of 45 PFU/cell, and is very stable under different temperatures (-20 to 50 °C) and pH (3 to 11) values. CfP1 demonstrated to be highly virulent against multidrug-resistant clinical isolates up to 12 antibiotics, including penicillins, cephalosporins, carbapenems, and fluroquinoles. Genomically, CfP1 has a dsDNA molecule with 180,219 bp with average GC content of 43.1 % and codes for 273 CDSs. The genome architecture is organized into function-specific gene clusters typical for tailed phages, sharing 46 to 94 % nucleotide identity to other Citrobacter phages. The lysin gene encoding a predicted D-Ala-D-Ala carboxypeptidase was also cloned and expressed in Escherichia coli and its activity evaluated in terms of pH, ionic strength, and temperature. The lysine optimum activity was reached at 20 mM HEPES, pH 7 at 37 °C, and was able to significantly reduce all C. freundii (>2 logs) as well as Citrobacter koseri (>4 logs) strains tested. Interestingly, the antimicrobial activity of this enzyme was performed without the need of pretreatment with outer membrane-destabilizing agents. These results indicate that CfP1 lysin is a good candidate to control problematic Citrobacter infections, for which current antibiotics are no longer effective.