A novel temperate phage vB_KpnP_ZX1 was isolated from hospital sewage samples using the clinically derived K57-type Klebsiella pneumoniae as a host. Phage vB_KpnP_ZX1, encoding three lysogen genes, the repressor, anti-repressor, and integrase, is the fourth phage of the genus Uetakevirus, family Podoviridae, ever discovered. Phage vB_KpnP_ZX1 did not show ideal bactericidal effect on K. pneumoniae 111-2, but TEM showed that the depolymerase Dep_ZX1 encoded on the short tail fiber protein has efficient capsule degradation activity. In vitro antibacterial results show that purified recombinant Dep_ZX1 can significantly prevent the formation of biofilm, degrade the formed biofilm, and improve the sensitivity of the bacteria in the biofilm to the antibiotics kanamycin, gentamicin, and streptomycin. Furthermore, the results of animal experiments show that 50 µg Dep_ZX1 can protect all K. pneumoniae 111-2-infected mice from death, whereas the control mice infected with the same dose of K. pneumoniae 111-2 all died. The degradation activity of Dep_ZX1 on capsular polysaccharide makes the bacteria weaken their resistance to immune cells, such as complement-mediated serum killing and phagocytosis, which are the key factors for its therapeutic action. In conclusion, Dep_ZX1 is a promising anti-virulence agent for the K57-type K. pneumoniae infection or biofilm diseases.

Phages demonstrate remarkable promise as antimicrobial agents against antibiotic-resistant bacteria. However, the emergence of phage-resistant strains poses challenges to their effective application. In this paper, we presented the isolation of a phage adaptive mutant that demonstrated enhanced and sustained antibacterial efficacy through the co-evolution of Klebsiella pneumoniae (K. pneumoniae) 111-2 and phage ZX1Δint in vitro. Our experiments revealed that phage ZX1Δint successfully completed the adsorption phase by binding to the host surface, specifically targeting the capsular polysaccharide (CPS) receptor via the primary receptor-binding protein (RBP) ORF60 and the auxiliary RBP ORF59. Upon exposure to phage predation, mutations in genes wbaP, wbaZ or wzc, which encode the synthesis of the CPS, conferred resistance by reducing phage adsorption. In response to these host defense mechanisms, the adaptive mutant phages have evolved to utilize an alternative binding site located on an O-antigen site of lipopolysaccharide (LPS) through a mutation in the backup RBP ORF59. This evolutionary change enabled the phages to reinfect previously phage-resistant strains. Notably, the adaptive mutant phage PR2 carrying the ORF59 mutation Q777R, demonstrated the capacity to infect both wild-type and resistant strains, exhibiting prolonged antimicrobial activity against the wild strains. In conclusion, our findings elucidated a complex phage-host adsorption-antagonism mechanism characterized by mutation-driven alterations in phage receptor recognition. This work contributes to a deeper understanding of phage adaptability and highlights the potential for phages to combat phage-resistant bacteria through an in vitro evolutionary approach.

The rise of antibiotic-resistant bacteria, particularly multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa), represents a critical challenge to global health security. Bacteriophages, with their ability to target and lyse specific bacterial pathogens, present a promising alternative through phage therapy. However, their narrow host range limits therapeutic applications. In this study, we developed an evolutionary system to expand the host range of P. aeruginosa phage pap17. Through one-host and dual-host experimental evolution, pap17 was co-cultured with permissive and non-permissive bacterial hosts. Post-evolution, pap17 gained the ability to infect previously non-permissive strains while retaining infectivity against original hosts. Genomic analysis identified key mutations in the tail fiber protein, critical for host range expansion. Evolved phages exhibited enhanced stability and lytic activity under varying environmental conditions. These findings demonstrate that evolved phages can be trained to combat a broader range of antibiotic-resistant strains, offering a potential solution to multidrug-resistant P. aeruginosa infections.

The prevalence of multidrug-resistant highly virulent Klebsiella pneumoniae (MDR-hvKP) requires the development of new therapeutic agents. Herein, a novel lytic phage vB_KpnS_ZX4 against MDR-hvKP was discovered in hospital sewage. Phage vB_KpnS_ZX4 had a short latent period (5 min) and a large burst size (230 PFU/cell). It can rapidly reduce the number of bacteria in vitro and improve survival rates of bacteremic mice in vivo from 0 to 80 % with a single injection of 10⁸ PFU. LysZX4, an endolysin derived from vB_KpnS_ZX4, exhibits potent antimicrobial activity in vitro in combination with ethylenediaminetetraacetic acid (EDTA). The antimicrobial activity of LysZX4 was further enhanced by the fusion of KWKLFKI residues from cecropin A (LysZX4-NCA). In vitro antibacterial experiments showed that LysZX4-NCA exerts broad-spectrum antibacterial activity against clinical Gram-negative bacteria, including MDR-hvKP. Moreover, in the mouse model of MDR-hvKP skin infection, treatment with LysZX4-NCA resulted in a three-log reduction in bacterial burden on the skin compared to the control group. Therefore, the novel phages vB_KpnS_ZX4 and LysZX4-NCA are effective reagents for the treatment of systemic and local MDR-hvKP infections.

The study of the interaction between temperate phages and bacteria is vital to understand their role in the development of human diseases. In this study, a novel temperate Escherichia coli phage, vB_EcoP_ZX5, with a genome size of 39,565 bp, was isolated from human fecal samples. It has a short tail and belongs to the genus Uetakevirus and the family Podoviridae. Phage vB_EcoP_ZX5 encodes three lysogeny-related proteins (ORF12, ORF21, and ORF4) and can be integrated into the 3′-end of guaA of its host E. coli YO1 for stable transmission to offspring bacteria. Phage vB_EcoP_ZX5 in lysogenized E. coli YO1+ was induced spontaneously, with a free phage titer of 10⁷ PFU/mL. The integration of vB_EcoP_ZX5 had no significant effect on growth, biofilm, environmental stress response, antibiotic sensitivity, adherence to HeLa cells, and virulence of E. coli YO1. The ORF4 anti-repressor, ORF12 integrase, and ORF21 repressors that affect the lytic–lysogenic cycle of vB_EcoP_ZX5 were verified by protein overexpression. We could tell from changes of the number of total phages and the transcription level of phage genes that repressor protein is the key determinant of lytic-to-lysogenic conversion, and anti-repressor protein promotes the conversion from lysogenic cycle to lytic cycle.

Cancer incidence is increasing globally, presenting significant health challenges due to its severe impact on morbidity and mortality. As a disease closely linked to aging, the prevalence of cancer is expected to increase with increasing age, underscoring the need for comprehensive research into its mechanisms and treatments. Macrophages, which are central to the immune system, play a paradoxical dual role in cancer progression. While they can suppress tumor growth, tumor-associated macrophages (TAMs) frequently facilitate tumor development and metastasis, a complexity that is further intricate by the aging process. As macrophages transition into senescent cells, they undergo changes, including shifts in cytokine profiles, reduced phagocytic activity, and altered metabolism. These senescent macrophages contribute to cancer progression by creating an immunosuppressive environment, promoting angiogenesis, and supporting tumor invasion. This review explores the intricate functions of senescent macrophages in cancer, highlighting their implications for tumor biology and their potential as therapeutic targets. We discuss strategies to manipulate senescent macrophages to enhance current cancer therapies, emphasizing the importance of understanding their mechanisms to advance cancer treatment.