Comparative in vitro activity of bacteriophage endolysin HY-133 against Staphylococcus aureus attached to vascular graft surface.

Novel strategies are needed for combating Staphylococcus aureus biofilm in vascular graft infections. We investigated the in vitro activity of bacteriophage endolysin HY-133, daptomycin and rifampin against S. aureus attached to vascular graft surface. Daptomycin showed rapid bactericidal effect on surface-associated S. aureus, while the activity of HY-133 on graft surface-adherent cells was moderate and rifampin did not achieve bactericidal effect. Even in the highest concentrations, all antimicrobials used failed in a complete eradication of the surface-adherent bacteria.

Expression and characterization of novel chimeric endolysin CHAPk-SH3bk against biofilm-forming methicillin-resistant Staphylococcus aureus.

The continuous evolution of antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) due to the misuse of antibiotics lays out the need for the development of new antimicrobials with higher activity and lower resistance. In this study, we have expressed novel chimeric endolysin CHAPk-SH3bk derived from LysK to investigate its antibacterial activity against planktonic and biofilm-forming MRSA. The molecular docking and MD simulation results identified critical amino acids (ASP47, ASP56, ARG71, and Gly74) of CHAPk domain responsible for its catalytic activity. Chimeric endolysin CHAPk-SH3bk showed an effective binding to peptidoglycan fragment using 14 hydrogen bonds. The in-vitro antibacterial assays displayed higher activity of CHAPk against planktonic MRSA with 2-log10 reduction in 2 h. Both CHAPk and CHAPk-SH3bk displayed bactericidal activity against MRSA with ∼4log10 and ∼3.5log10 reduction in 24 h. Biofilm reduction activity displayed CHAPk-SH3bk reduced 33 % and 60 % of hospital-associated ATCC®BAA-44™ and bovine origin SA1 respectively. The CHAPk treatment reduced 47 % of the preformed biofilm formed by bovine-origin MRSA SA1. This study indicates an effective reduction of preformed MRSA biofilms of human and animal origin using novel chimeric construct CHAPk-SH3bk. Stating that the combination and shuffling of different domains of phage endolysin potentially increase its bacteriolytic effectiveness against MRSA.

Integration of Lysin into Chitosan Nanoparticles for Improving Bacterial Biofilm Inhibition.

Bacterial biofilms (BBFs) exhibit high drug resistance, antiphagocytosis, and extremely strong adhesion, and therefore can cause various diseases. They are also one of the important causes of bacterial infections. Thus, the effective removal of BBFs has attracted considerable research interest. Endolysins, which are efficient antibacterial bioactive macromolecules, have recently been receiving increasing attention. In this study, we overcame the deficiencies of endolysins via immobilization on chitosan nanoparticles (CS-NPs) by preparing LysST-3-CS-NPs using the ionic cross-linking reaction between CS-NPs and LysST-3, an endolysin purified using phage ST-3 expression. The obtained LysST-3-CS-NPs were verified and thoroughly characterized, their antimicrobial activity was investigated using microscopy, and their antibacterial efficacy on polystyrene surfaces was studied. The results obtained suggested that LysST-3-CS-NPs exhibit enhanced bactericidal properties and increased stability and can serve as reliable biocontrol agents for the prevention and treatment of Salmonella biofilm infections.

Bacteriophage endolysin powders for inhaled delivery against pulmonary infections.

Endolysins are bacteriophage-encoded enzymatic proteins that have great potential to treat multidrug-resistant bacterial infections. Bacteriophage endolysins Cpl-1 and ClyJ-3 have shown promising antimicrobial activity against Streptococcus pneumoniae, which causes pneumonia in humans. This is the first study to investigate the feasibility of spray-dried endolysins Cpl-1 and ClyJ-3 with excipients to produce inhalable powders. The two endolysins were individually tested with leucine and sugar (lactose or trehalose) for spray drying method followed by characterization of biological and physico-chemical properties. A complete loss of ClyJ-3 bioactivity was observed after atomization of the liquid feed solution（before the drying process）, while Cpl-1 maintained its bioactivity in the spray-dried powders. Cpl-1 formulations containing leucine with lactose or trehalose showed promising physico-chemical properties (particle size, crystallinity, hygroscopicity, etc.) and aerosol performances (fine particle fraction values above 65%). The results indicated that endolysin Cpl-1 can be formulated as spray dried powders suitable for inhaled delivery to the lungs for the potential treatment of pulmonary infections.

Development of a Magnetic Bead-Based Method for Specific Detection of Enterococcus faecalis Using C-Terminal Domain of ECP3 Phage Endolysin.

Bacteriophage endolysins are peptidoglycan hydrolases composed of cell binding domain (CBD) and an enzymatically active domain. A phage endolysin CBD can be used for detecting bacteria owing to its high specificity and sensitivity toward the bacterial cell wall. We aimed to develop a method for detection of Enterococcus faecalis using an endolysin CBD. The gene encoding the CBD of ECP3 phage endolysin was cloned into the Escherichia coli expression vector pET21a. A recombinant protein with a C-terminal 6-His-tag (CBD) was expressed and purified using a His-trap column. CBD was adsorbed onto epoxy magnetic beads (eMBs). The bacterial species specificity and sensitivity of bacterial binding to CBD-eMB complexes were determined using the bacterial colony counting from the magnetic separations after the binding reaction between bacteria and CBD-eMB complexes. E. faecalis could bind to CBD-eMB complexes, but other bacteria (such as Enterococcus faecium, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Streptococcus mutans, and Porphyromonas gingivalis) could not. E. faecalis cells were fixed onto CBD-eMB complexes within 1 h, and >78% of viable E. faecalis cells were recovered. The E. faecalis recovery ratio was not affected by the other bacterial species. The detection limit of the CBD-eMB complex for E. faecalis was >17 CFU/ml. We developed a simple method for the specific detection of E. faecalis using bacteriophage endolysin CBD and MBs. This is the first study to determine that the C-terminal region of ECP3 phage endolysin is a highly specific binding site for E. faecalis among other bacterial species.

Bacteriophage endolysin Ply113 as a potent antibacterial agent against polymicrobial biofilms formed by enterococci and Staphylococcus aureus.

Antibiotic resistance in Enterococcus faecium, Enterococcus faecalis, and Staphylococcus aureus remains a major public health concern worldwide. Furthermore, these microbes frequently co-exist in biofilm-associated infections, largely nullifying antibiotic-based therapy. Therefore, it is imperative to develop an efficient therapeutic strategy for combating infections caused by polymicrobial biofilms. In this study, we investigated the antibacterial and antibiofilm activity of the bacteriophage endolysin Ply113 in vitro. Ply113 exhibited high and rapid lytic activity against E. faecium, E. faecalis, and S. aureus, including vancomycin-resistant Enterococcus and methicillin-resistant S. aureus isolates. Transmission electron microscopy revealed that Ply113 treatment led to the detachment of bacterial cell walls and considerable cell lysis. Ply113 maintained stable lytic activity over a temperature range of 4-45°C, over a pH range of 5.0-8.0, and in the presence of 0-400 mM NaCl. Ply113 treatment effectively eliminated the mono-species biofilms formed by E. faecium, E. faecalis, and S. aureus in a dose-dependent manner. Ply113 was also able to eliminate the dual-species biofilms of E. faecium-S. aureus and E. faecalis-S. aureus. Additionally, Ply113 exerted potent antibacterial efficacy in vivo, distinctly decreasing the bacterial loads in a murine peritoneal septicemia model. Our findings suggest that the bacteriophage endolysin Ply113 is a promising antimicrobial agent for the treatment of polymicrobial infections.

E. coli Cell Lysis Induced by Lys394 Enzyme Assisted by Magnetic Nanoparticles Exposed to Non-Heating Low-Frequency Magnetic Field.

The spreading of microbial pathogens with more and more resistance to traditional low-molecular antibiotic agents demands new approaches to antibacterial therapy. The employment of bacteriophage enzymes capable of breaking bacterial cell walls has attracted much interest within this context. The specific features of the morphology of Gram-negative bacteria prevent the effective direct usage of lytic enzymes and require assistance from additional helpers to facilitate cell lysis. The current work is devoted to the study of boosting the lysis of Escherichia coli (E. coli) JM 109 and MH 1 strains induced by Lys394 bacteriophage endolysin by means of rod-like (56 × 13 nm) magnetic nanoparticles (MNPs) activated by a non-heating low-frequency magnetic field (LF MF) with a frequency of 50 Hz and a flux density of 68.5 mT in a pulse-pause mode (1 s on and 0.3 s off). According to theoretical assumptions, the mechanism of MNP assistance is presumably based upon the disordering of the outer membrane that facilitates enzyme permeation into peptidoglycans to its substrate. It is found that the effect of the LF MF reaches an almost a twofold acceleration of the enzyme reaction, resulting in almost 80 and 70%, respectively, of lysed E. coli JM 109 and MH 1 cells in 21 min. An increase in the membrane permeability was proven by two independent experiments employing ß-lactamase periplasmic enzyme leakage and Nile Red (NR) hydrophobic dye fluorescence. It is shown that the outer membrane disordering of E. coli caused by exposure to LF MF nanoparticle movement leads to almost complete (more than 80%) ß-lactamase release out of the cells' periplasm to the buffer suspension. Experiments with NR (displaying fluorescence in a non-polar medium only) reveal a drastic reduction in NR fluorescence intensity, reaching a change of an order of magnitude when exposed to LF MF. The data obtained provide evidence of changes in the bacterial cell wall structure. The result shown open up the prospects of non-heating LF MF application in enhancing enzyme activity against Gram-negative pathogens.

Yeast Surface Display System for Facilitated Production and Application of Phage Endolysin.

Bacteriophage endolysin is one of the potential alternatives of conventional antibiotics, but the intrinsic limitations of the bacterial expression system may undermine the comprehensive application of this therapeutic protein. To circumvent such limitations, we adopted a yeast surface display system as a novel expression platform for endolysin. Endolysin LysSA11 from staphylococcal phage SA11 was expressed and surface-displayed in Saccharomyces cerevisiae to exhibit sufficient antimicrobial activity against Staphylococcus aureus. Without any protein isolation or purification procedures, we showed that direct treatment of LysSA11-displaying yeast cells could accomplish a 5-log reduction of viable Staphylococcus aureus within 3 h. Furthermore, the surface-displayed LysSA11 exhibited superior stability over the soluble form of purified LysSA11 during 14 days of storage in a refrigerated environment. We suggest that the yeast surface display system is an efficient, stable, and straightforward platform for the production and antibacterial applications of endolysin.

External lysis of Escherichia coli by a bacteriophage endolysin modified with hydrophobic amino acids.

Drug-resistant bacteria are a serious threat to global public health. Gram-positive bacterial endolysin preparations have been successfully used to fight Gram-positive bacteria as a novel antimicrobial replacement strategy. However, Gram-negative bacterial phage endolysins cannot be applied directly to destroy Gram-negative strains due to the externally inaccessible peptidoglycan layer of the cell wall; this has seriously hampered the development of endolysin-like antibiotics against Gram-negative bacteria. In this study, 3-12 hydrophobic amino acids were successively added to the C-terminus of Escherichia coli phage endolysin Lysep3 to create five different hydrophobic-modified endolysins. Compared with endogenous Lysep3, endolysins modified with hydrophobic amino acids surprisingly could kill E. coli from outside of the cell at the appropriate pH and endolysin concentration. The lysis ability of modified endolysins were enhanced with increasing numbers of hydrophobic amino acids at the C-terminus of endolysin. Thus, these findings demonstrate that the enhancement of hydrophobicity at the C-terminus enables the endolysin to act upon E. coli from the outside, representing a novel method of lysing Gram-negative antibiotic-resistant bacteria.

Kinetics of inactivation of staphylolytic enzymes: Qualitative and quantitative description.

Lysin 2638aR and chimeric Ply187AN-KSH3b fusion protein are capable of lysing antibiotic-resistant strains of Staphylococcus aureus and are promising alternatives to antibiotics. Studies on the stability and structure of lysins 2638aR and Ply187AN-KSH3b are important for assessing the feasibility of their practical use. Both lysins are highly active at physiological pH (7.5) and at low salt content (the concentration of NaCl in the reaction medium is not more than 250 mM). Lysins are inactivated by a monomolecular mechanism and have high stability at 4 °C (storage temperature). The maximum value of the half-inactivation time for lysin 2638aR is 190-200 days (500-1000 mM NaCl, pH 6.0-7.5), for lysin Ply187AN-KSH3b is 320-340 days (10-1000 mM NaCl, pH 6.0). The lysins are pretty stable in human blood serum (the half-inactivation time is 0.5-2 h) at 37 °C. The lysins undergo denaturation in large part due to the destruction of the α-helices at temperatures above 40 °C.

Bactericidal activity of bacteriophage endolysin HY-133 against Staphylococcus aureus in comparison to other antibiotics as determined by minimum bactericidal concentrations and time-kill analysis.

Methicillin-resistant Staphylococcus aureus (MRSA) decolonization is expensive and time consuming, and new agents are necessary due to increasing resistance rates. The administration of bacteriophages or particularly their endolysins may offer an alternative treatment strategy and could provide a solution to overcome the selection pressure due to classical antibiotics. Here, the bactericidal activity was characterized for the recombinant chimeric bacteriophage endolysin HY-133 in comparison to other antimicrobials. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for 2 reference strains, 24 clinical MRSA and methicillin-susceptible S. aureus (MSSA) isolates, as well as 6 isolates with high-level mupirocin resistance. Additionally, HY-133 activity against bacteria in stationary or exponential growth phase was compared in 12 isolates. Time-kill curves were performed with 2 representative isolates to investigate the pharmacodynamics until 48-h incubation time. All experiments were performed in comparison to daptomycin and mupirocin. The MIC50/90 and MBC50/90 values were in the range 0.12-0.5 mg/L for all 3 growth conditions comparable to daptomycin with 0.5/0.5 mg/L, respectively. The MBC was almost always equal the MIC and without considerable differences between MSSA and MRSA. Time-kill curves revealed a rapid bactericidal effect of HY-133 within the first 2 h, similar to daptomycin. Even with low concentrations, the recombinant endolysin HY-133 was highly active against all tested MSSA and MRSA isolates including mupirocin-resistant isolates. The application of this alternative agent may offer a future strategy for MRSA/MSSA decolonization and, potentially, for treatment purposes.

Thermally triggered release of the bacteriophage endolysin CHAPK and the bacteriocin lysostaphin for the control of methicillin resistant Staphylococcus aureus (MRSA).

Staphylococcus aureus infections of the skin and soft tissue pose a major concern to public health, largely owing to the steadily increasing prevalence of drug resistant isolates. As an alternative mode of treatment both bacteriophage endolysins and bacteriocins have been shown to possess antimicrobial efficacy against multiple species of bacteria including otherwise drug resistant strains. Despite this, the administration and exposure of such antimicrobials should be restricted until required in order to discourage the continued evolution of bacterial resistance, whilst maintaining the activity and stability of such proteinaceous structures. Utilising the increase in skin temperature during infection, the truncated bacteriophage endolysin CHAPK and the staphylococcal bacteriocin lysostaphin have been co-administered in a thermally triggered manner from Poly(N-isopropylacrylamide) (PNIPAM) nanoparticles. The thermoresponsive nature of the PNIPAM polymer has been employed in order to achieve the controlled expulsion of a synergistic enzybiotic cocktail consisting of CHAPK and lysostaphin. The point at which this occurs is modifiable, in this case corresponding to the threshold temperature associated with an infected wound. Consequently, bacterial lysis was observed at 37°C, whilst growth was maintained at the uninfected skin temperature of 32°C.

Lytic activity of the staphylolytic Twort phage endolysin CHAP domain is enhanced by the SH3b cell wall binding domain.

Increases in the prevalence of antibiotic-resistant strains of Staphylococcus aureus have elicited efforts to develop novel antimicrobials to treat these drug-resistant pathogens. One potential treatment repurposes the lytic enzymes produced by bacteriophages as antimicrobials. The phage Twort endolysin (PlyTW) harbors three domains, a cysteine, histidine-dependent amidohydrolases/peptidase domain (CHAP), an amidase-2 domain and a SH3b-5 cell wall binding domain (CBD). Our results indicate that the CHAP domain alone is necessary and sufficient for lysis of live S. aureus, while the amidase-2 domain is insufficient for cell lysis when provided alone. Loss of the CBD results in ∼10X reduction of enzymatic activity in both turbidity reduction and plate lysis assays compared to the full length protein. Deletion of the amidase-2 domain resulted in a protein (PlyTW Δ172-373) with lytic activity that exceeded the activity of the full length construct in both the turbidity reduction and plate lysis assays. Addition of Ca(2+) enhanced the turbidity reduction activity of both the full length protein and truncation constructs harboring the CHAP domain. Chelation by addition of EDTA or the addition of zinc inhibited the activity of all PlyTW constructs.

Peptidoglycan degrading activity of the broad-range Salmonella bacteriophage S-394 recombinant endolysin.

The use of bacteriophage endolysins as specific antibacterial agents is a prospective strategy to treat bacterial infections caused by antibiotic-resistant pathogens. In case of Gram-negative species this strategy has limited applications since outer membrane shields the enzyme target and prevents bacteria lysis. We aimed to obtain and characterize the endolysin of the newly discovered anti-Salmonella bacteriophage S-394 (Lys394) and to choose an appropriate permeabilizing agent to disrupt Escherichia coli cells suspended in buffer solution and grown on agar surface. Lys394 synthesized in E. coli C41(DE3) was obtained as an electrophoretically homogenous protein. The protein of 18 kDa molecular weight shows high muralytic activity against various genera of chloroform treated Gram-negatives. Maximum of enzyme activity was observed at pH 8.5 and low ionic strength. In silico analysis of amino acid sequence identified Lys394 as an endopeptidase. Various outer membrane permeabilizers were analyzed in combination with Lys394 to degrade laboratory strain of E. coli CR63. Permeabilizing activity was evaluated using a periplasmic ß-lactamase leakage test with untreated E. coli cells as a substrate. The highest rate of planktonic E. coli lysis was reached for Lys394 applied together with 25 µg/ml of poly-l-arginine with molecular weight distribution from 5 to 15 kDa or 20 µg/ml PGLa peptide. Lawn E. coli colony forming ability was decreased by 4 orders of magnitude after 30 min treatment with 25 µg of Lys394, 1 mM EDTA and 50 µg/ml of PGLa peptide at a room temperature.