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Abstract Phage therapy holds promise as an alternative to antibiotics for combating multidrug-resistant bacteria. However, host bacteria can quickly produce progeny that are resistant to phage infection. In this study, we investigated the mechanisms of bacterial resistance to phage infection. We found that Rsm1, a mutant strain of Salmonella enteritidis (S. enteritidis) sm140, exhibited resistance to phage Psm140, which was originally capable of lysing its host at sm140. Whole genome sequencing analysis revealed a single nucleotide mutation at position 520 (C → T) in the rfbD gene of Rsm1, resulting in broken lipopolysaccharides (LPS), which is caused by the replacement of CAG coding glutamine with a stop codon TAG. The knockout of rfbD in the sm140ΔrfbD strain caused a subsequent loss of sensitivity toward phages. Furthermore, the reintroduction of rfbD in Rsm1 restored phage sensitivity. Moreover, polymerase chain reaction (PCR) amplification of rfbD in 25 resistant strains revealed a high percentage mutation rate of 64% within the rfbD locus. We assessed the fitness of four bacteria strains and found that the acquisition of phage resistance resulted in slower bacterial growth, faster sedimentation velocity, and increased environmental sensitivity (pH, temperature, and antibiotic sensitivity). In short, bacteria mutants lose some of their abilities while gaining resistance to phage infection, which may be a general survival strategy of bacteria against phages. This study is the first to report phage resistance caused by rfbD mutation, providing a new perspective for the research on phage therapy and drug-resistant mechanisms.

ABSTRACT: In the face of the increasingly severe problem of antibiotic resistance, phage therapy is regarded as a highly potential alternative. Compared with traditional antimicrobial agents, a key research area of phage therapy is the study of phage-resistant mutant bacteria. To effectively monitor and prevent this resistance, it is crucial to conduct in-depth exploration of the mechanism behind phage resistance. In this study, a strain of Salmonella enteritidis (sm140) and the corresponding phage (Psm140) were isolated from chicken liver and sewage, respectively. Using the double-layer plate method, successfully screened out phage-resistant mutant strains. Whole-genome resequencing of 3 resistant strains found that the wbaP gene of all 3 strains had mutations at a specific position (1,118), with the base changing from G to A. This mutation causes the gene-encoded glycine to be replaced by aspartic acid. Subsequent studies found that the frequency of this gene mutation is extremely high, reaching 84%, and all mutations occur at the same position. To further explore the relationship between the wbaP gene and phage resistance, knockout strains and complement strains of the wbaP gene were constructed. The experimental results confirmed the association between the wbaP gene and phage resistance. At the same time, biological characteristics and virulence were evaluated for wild strains, resistant strains, knockout strains, and complement strains. It was found that mutations or deletions of the wbaP gene lead to a decrease in bacterial environmental adaptability and virulence. Through systematic research on the mechanism and biological characteristics of phage resistance, this study provides important references and guidance for the development of new phage therapies, promoting progress in the field of antimicrobial treatment. At the same time, the emergence of phage resistance due to wbaP gene mutations is reported for the first time in salmonella, providing a new perspective and ideas for further studying phage resistance mechanisms.

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.