BACKGROUND: The outer membrane vesicles (OMVs) produced by Gram-negative bacteria can modulate the immune system and have great potentials for bacterial vaccine development.
RESULTS: A highly active Acinetobacter baumannii phage lysin, LysP53, can stimulate the production of OMVs after interacting with A. baumannii, Escherichia coli, and Salmonella. The OMVs prepared by the lysin (LOMVs) from A. baumannii showed better homogeneity, higher protein yield, lower endotoxin content, and lower cytotoxicity compared to the naturally produced OMVs (nOMVs). The LOMVs contain a significantly higher number of cytoplasmic and cytoplasmic membrane proteins but a smaller number of periplasmic and extracellular proteins compared to nOMVs. Intramuscular immunization with either LOMVs or nOMVs three times provided robust protection against A. baumannii infections in both pneumonia and bacteremia mouse models. Intranasal immunization offered good protection in the pneumonia model but weaker protection (20-40%) in the bacteremia model. However, with a single immunization, LOMVs demonstrated better protection than the nOMVs in the pneumonia mouse model.
CONCLUSIONS: The novel lysin approach provides a superior choice compared to current methods for OMV production, especially for vaccine development.

Salmonella is a zoonotic pathogen that is commonly associated with foodborne disease outbreaks. This study found that a newly identified Gram-negative lysin LysP53 had good activity against a wide range of Salmonella, including Salmonella Newington, Salmonella Typhimurium, and Salmonella Dublin. Without the help of an outer membrane permeabilizer, 4 μM LysP53 could reduce 97.6% of planktonic Salmonella Enteritidis and 90% of the bacteria in biofilms. Moreover, LysP53 was highly thermostable because it maintained >90% activity even after exposure to temperatures up to 95 °C. Although high concentrations of salts could reduce the activity, LysP53 was found safe for oral gavage of mice without affecting body weights and cytokines in sera and able to reduce 90% of Salmonella Enteritidis loads on fresh romaine lettuce after 30 min of treatment. Because of its good activity against a wide range of bacteria, thermal stability, safe for oral administration, LysP53 could be used as a biocontrol agent for reducing bacterial loads in fresh vegetable food. KEY POINTS: • Lysin LysP53 has high bactericidal activity against Salmonella. • LysP53 is thermostable even at high temperature of up to 95 °C. • LysP53 can be used for topical decontamination of Salmonella on vegetables.

Klebsiella pneumoniae and Pseudomonas aeruginosa are two leading causes of burn and wound infections, pneumonia, urinary tract infections, and more severe invasive diseases, which are often multidrug resistant (MDR) or extensively drug resistant. Due to this, it is critical to discover alternative antimicrobials, such as bacteriophage lysins, against these pathogens. Unfortunately, most lysins that target Gram-negative bacteria require additional modifications or outer membrane permeabilizing agents to be bactericidal. We identified four putative lysins through bioinformatic analysis of Pseudomonas and Klebsiella phage genomes in the NCBI database and then expressed and tested their intrinsic lytic activity in vitro. The most active lysin, PlyKp104, exhibited >5-log killing against K. pneumoniae, P. aeruginosa, and other Gram-negative representatives of the multidrug-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, K. pneumonia, Acinetobacter baumannii, P. aeruginosa, and Enterobacter species) without further modification. PlyKp104 displayed rapid killing and high activity over a wide pH range and in high concentrations of salt and urea. Additionally, pulmonary surfactants and low concentrations of human serum did not inhibit PlyKp104 activity in vitro. PlyKp104 also significantly reduced drug-resistant K. pneumoniae >2 logs in a murine skin infection model after one treatment of the wound, suggesting that this lysin could be used as a topical antimicrobial against K. pneumoniae and other MDR Gram-negative infections.

In this study, we isolated a lytic Pseudomonas aeruginosa phage (vB_PaeP_ASP23) from the sewage of a mink farm, characterized its complete genome and analyzed the function of its putative lysin and holin. Morphological characterization and genome annotation showed that phage ASP23 belonged to the Krylovirinae family genus Phikmvvirus, and it had a latent period of 10 min and a burst size of 140 pfu/infected cell. In minks challenged with P. aeruginosa, phage ASP23 significantly reduced bacterial counts in the liver, lung, and blood. The whole-genome sequencing showed that its genome was a 42,735-bp linear and double-stranded DNA (dsDNA), with a G + C content of 62.15%. Its genome contained 54 predicted open reading frames (ORFs), 25 of which had known functions. The lysin of phage ASP23 (LysASP), in combination with EDTA, showed high lytic activity against P. aeruginosa L64. The holin of phage ASP23 was synthesized by M13 phage display technology, to produce recombinant phages (HolASP). Though HolASP exhibited a narrow lytic spectrum, it was effective against Staphylococcus aureus and Bacillus subtilis. However, these two bacteria were insensitive to LysASP. The findings highlight the potential of phage ASP23 to be used in the development of new antibacterial agents.

Wound infections are prone to attacks from infectious pathogens, including multidrug resistant bacteria that render conventional antimicrobials ineffective. Recently, lysins have been proposed as alternatives to conventional antimicrobials to tackle the menace of multidrug resistance pathogens. The coupling of lysins with a material that will cover the wound may prove beneficial in both protecting and treating wound infections. Hence, in this study, a Gram-negative lysin, LysP53, was coupled with a thermosensitive hydrogel, poloxamer P407, and its efficacy to treat wound infection was tested. In vitro, the addition of LysP53 to the poloxamer did not affect its thermosensitive characteristics, nor did it affect the hydrogel structure. Moreover, the lysin hydrogel could hydrolyze the peptidoglycan, demonstrating that it may have bactericidal activity. Up to 10.4% of LysP53 was released from the hydrogel gradually within 24 h, which led to a 4-log reduction of stationary phase Acinetobacter baumannii. Lastly, the lysin hydrogel was found safe with no cytotoxic effects observed in cells. Ex vivo, LysP53 hydrogel could inhibit bacterial growth on a pig skin decolonization model, with 3-log differences compared to non-treated groups. Overall, our results suggest that lysin-loaded hydrogels may provide a novel solution to treat wound infections caused by resistant bacteria.

The development of new antimicrobial agents is critically needed due to the alarming increase in antibiotic resistance in bacterial pathogens. Phages have been widely considered as effective alternatives to antibiotics. A novel phage vB_StaM_SA1 (hereinafter as SA1) that can infect multiple Staphylococcus strains was isolated from untreated sewage of a pig farm, which belonged to Myoviridae family. At MOI of 0.1, the latent period of phage SA1 was 55 min, and the final titer reached about 109 PFU/mL. The genome of phage SA1 was 260,727 bp, indicating that it can be classified as a jumbo phage. The genome of SA1 had 258 ORFs and a serine tRNA, while only 53 ORFs were annotated with functions. Phage SA1 contained a group of core genes that was characterized by multiple RNA polymerase subunits and also found in phiKZ-related jumbo phages. The phylogenetic tree showed that phage SA1 was a phiKZ-related phage and was closer to jumbo phages compared with Staphylococcus phages with small genome. Three proteins (lys4, lys210, and lys211) were predicted to be associated with lysins, and two proteins with lytic function were verified by recombinant expression and bacterial survival test. Both lys210 and lys211 possessed efficient bactericidal ability, and lys210 could lyse all test strains. The results show that phage SA1 and lys210/lys211 could be potentially used as antibiotic agents to treat Staphylococcus infection.

Staphylococcal-associated device-related infections (DRIs) represent a significant clinical challenge causing major medical and economic sequelae. Bacterial colonization, proliferation, and biofilm formation after adherence to surfaces of the indwelling device are probably the primary cause of DRIs. To address this issue, we incorporated constructs of silica-binding peptide (SiBP) with ClyF, an anti-staphylococcal lysin, into functionalized coatings to impart bactericidal activity against planktonic and sessile Staphylococcus aureus. An optimized construct, SiBP1-ClyF, exhibited improved thermostability and staphylolytic activity compared to its parental lysin ClyF. SiBP1-ClyF-functionalized coatings were efficient in killing MRSA strain N315 (>99.999% within 1 h) and preventing the growth of static and dynamic S. aureus biofilms on various surfaces, including siliconized glass, silicone-coated latex catheter, and silicone catheter. Additionally, SiBP1-ClyF-immobilized surfaces supported normal attachment and growth of mammalian cells. Although the recycling potential and long-term stability of lysin-immobilized surfaces are still affected by the fragility of biological protein molecules, the present study provides a generic strategy for efficient delivery of bactericidal lysin to solid surfaces, which serves as a new approach to prevent the growth of antibiotic-resistant microorganisms on surfaces in hospital settings and could be adapted for other target pathogens as well.

Antimicrobial resistance-related infections of Gram-negative pathogens pose a huge threat to global public health. Lysins, peptidoglycan hydrolases from bacteriophages, are expected as an alternative weapon against drug-resistant bacteria. In the present study, we report a new lysin LysP53 from Acinetobacter baumannii phage 53. Bioinformatic analysis revealed that LysP53 contains a positively charged N-terminal region and a putative peptidase catalytic domain. In vitro biochemical experiments showed that LysP53 is active against multiple antibiotic-resistant Gram-negative bacteria, including A. baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli, with a reduction of 5 logs in viable A. baumannii number after exposure to 100 μg/mL LysP53 for 1 h. Further studies showed that LysP53 contains a functional antimicrobial peptide, i.e., N-terminal 33 aa, with a comparable spectrum of activity to LysP53. In an A. baumannii-associated mouse model of burn infection, a single dose of 14 μg/mouse LysP53 (57.6 μM) showed higher decolonization efficacy than 4 μg/mouse minocycline- (874 μM; p < 0.05) and buffer-treated groups (p <0.001), leading to a bacterial reduction of 3 logs. Our findings collectively establish that LysP53 could be a promising candidate in the treatment of topical infections caused by multiple Gram-negative pathogens.

Pseudomonas aeruginosa is a Gram-negative pathogen that causes a variety of infections in humans and animals. Due to the inappropriate use of antibiotics, multi-drug resistant (MDR) P. aeruginosa strains have emerged and are prevailing. In recent years, cow mastitis caused by MDR P. aeruginosa has attracted attention. In this study, a microbial community analysis revealed that P. aeruginosa could be a cause of pathogen-induced cow mastitis. Five MDR P. aeruginosa strains were isolated from milk diagnosed as mastitis positive. To seek an alternative antibacterial agent against MDR, P. aeruginosa, a lytic phage, designated vB_PaeS_PAJD-1 (PAJD-1), was isolated from dairy farm sewage. PAJD-1 was morphologically classified as Siphoviridae and was estimated to be about 57.9 kb. Phage PAJD-1 showed broad host ranges and a strong lytic ability. A one-step growth curve analysis showed a relatively short latency period (20 min) and a relatively high burst size (223 PFU per infected cell). Phage PAJD-1 remained stable over wide temperature and pH ranges. Intramammary-administered PAJD-1 reduced bacterial concentrations and repaired mammary glands in mice with mastitis induced by MDR P. aeruginosa. Furthermore, the cell wall hydrolase (termed endolysin) from phage PAJD-1 exhibited a strong bacteriolytic and a wide antibacterial spectrum against MDR P. aeruginosa. These findings present phage PAJD-1 as a candidate for phagotherapy against MDR P. aeruginosa infection.

Worldwide, foods waste caused by putrefactive organisms and diseases caused by foodborne pathogens persist as public health problems even with a plethora of modern antimicrobials. Our over reliance on antimicrobials use in agriculture, medicine, and other fields will lead to a postantibiotic era where bacterial genotypic resistance, phenotypic adaptation, and other bacterial evolutionary strategies cause antimicrobial resistance (AMR). This AMR is evidenced by the emergence of multiple drug-resistant (MDR) bacteria and pan-resistant (PDR) bacteria, which produces cross-contamination in multiple fields and poses a more serious threat to food safety. A \"red queen premise\" surmises that the coevolution of phages and bacteria results in an evolutionary arms race that compels phages to adapt and survive bacterial antiphage strategies. Phages and their lysins are therefore useful toolkits in the design of novel antimicrobials in food protection and foodborne pathogens control, and the modality of using phages as a targeted vector against foodborne pathogens is gaining momentum based on many encouraging research outcomes. In this review, we discuss the rationale of using phages and their lysins as weapons against spoilage organisms and foodborne pathogens, and outline the targeted conquest or dodge mechanism of phages and the development of novel phage prospects. We also highlight the implementation of phages and their lysins to control foodborne pathogens in a farm-table-hospital domain in the postantibiotic era.