The emergence of phage-resistant bacterial strains is one of the biggest challenges for phage therapy. However, the emerging phage-resistant bacteria are often accompanied by adaptive trade-offs, which supports a therapeutic strategy called “phage steering”. The key to phage steering is to guide the bacterial population toward an evolutionary direction that is favorable for treatment. Thus, it is important to systematically investigate the impacts of phages targeting different bacterial receptors on the fitness of the bacterial population. Herein, we employed 20 different phages to impose strong evolutionary pressure on the host Pseudomonas aeruginosa PAO1 and examined the genetic and phenotypic responses of their phage-resistant mutants. Among these strains with impaired adsorptions, four types of mutations associated with bacterial receptors were identified, namely, lipopolysaccharides (LPSs), type IV pili (T4Ps), outer membrane proteins (OMPs), and exopolysaccharides (EPSs). PAO1, responding to LPS- and EPS-dependent phage infections, mostly showed significant growth impairment and virulence attenuation. Most mutants with T4P-related mutations exhibited a significant decrease in motility and biofilm formation ability, while the mutants with OMP-related mutations required the lowest fitness cost out of the bacterial populations. Apart from fitness costs, PAO1 strains might lose their resistance to antibiotics when counteracting with phages, such as the presence of large-fragment mutants in this study, which may inspire the usage of phage–antibiotic combination strategies. This work provides methods that leverage the merits of phage resistance relative to obtaining therapeutically beneficial outcomes with respect to phage-steering strategies.

Stenotrophomonas maltophilia (S. maltophilia) is widely distributed in nature and frequently causes nosocomial infections. In this work, the biological characteristics and genome of a new S. maltophilia phage BUCT609 isolated from hospital sewage with S. maltophilia strain No. 3015 as host was analyzed and its therapeutic effect in vivo was explored. It was observed by TEM that phage BUCT609 belongs to the Podoviridae with a 10 nm tail structure and a capsid with a diameter of about 50 nm. It has a short latent period (about 10 min) and its burst size is 382 PFU /cell when multiplicity of infection (MOI) is 0.01. Furthermore, it has a high survival rate in the environment with a pH range from 3 to 10 and temperature range from 4°C to 55°C. The complete genome of phage BUCT609 is linear double-stranded DNA of 43,145 bp in length, and the GC content is 58%. The genome sequence of phage BUCT609 shares <45% homology with other phages. No virulence genes and antibiotic resistance genes were found in bacteriophage BUCT609. In vivo animal experiments showed that the survival rate of mice infected with S. maltophilia was significantly improved after the intranasal injection of phage BUCT609. Therefore, our study supports that phage BUCT609 could be used as a promising antimicrobial candidate for treating S. maltophilia infections.

Quorum sensing (QS) can regulate diverse critical phenotypic responses in Pseudomonas. aeruginosa ( P. aeruginosa ), enabling bacterial adaptation to external environmental fluctuations and optimizing population advantages. While there is emerging evidence of QS’s involvement in influencing phage infections, our current understanding remains limited, necessitating further investigation. In this study, we isolated and characterized a novel phage designated as BUCT640 that infected P. aeruginosa PAO1. This phage belonged to class Caudoviricetes , genus Bruynoghevirus , with a podovirus morphology, and its adsorption was dependent on Psl polysaccharides, a repeating pentamer used to support biofilm structure. Leveraging phage BUCT640 as a model, we analyzed the role of both rhl QS and las QS in bacteria–phage interactions. Based on its distinctive plaque formation performances on different QS-related mutants, we investigated the variations of phage sensitivity to these strains and ultimately elucidated the mechanism underlying how QS inhibited phage infection to PAO1. Specifically, we unveiled that the las QS could inhibit phage adsorption, which is related to the thickness change caused by biofilm differentiation. Our findings suggest that the inhibition of QS may enhance phage infectivity, potentially facilitating advanced phage therapy combined with QS interference. IMPORTANCE: Phage therapy is a powerful solution to combat drug-resistant pathogenic bacterial infections and has earned remarkable success in clinical treatment. However, recent insights underscore the potential impact of bacterial QS on phage infection dynamics. Here, we reported a unique phenomenon wherein QS, particularly in the las QS pathway, showed distinctive plaque formation behaviors by enlarging halos around plaques in mutant strains. In addition to this, we first elucidated the correlation between biofilm formation and phage infection. Notably, the las QS could inhibit phage adsorption, an effect closely related to biofilm thickness. Such research could be the evidence to steer bacterial QS toward favorable therapeutical outcomes. Therefore, our work can extend the comprehension of the interactions between bacteria and phages influenced by QS, thereby providing new perspectives on leveraging QS interference to enhance the efficacy of phage therapy for clinical applications.

The spread of multidrug-resistant Klebsiella pneumoniae (MDR-KP) has become an emerging threat as a result of the overuse of antibiotics. Bacteriophage (phage) therapy is considered to be a promising alternative treatment for MDR-KP infection compared with antibiotic therapy. In this research, a lytic phage BUCT610 was isolated from hospital sewage. The assembled genome of BUCT610 was 46,774 bp in length, with a GC content of 48%. A total of 83 open reading frames (ORFs) and no virulence or antimicrobial resistance genes were annotated in the BUCT610 genome. Comparative genomics and phylogenetic analyses showed that BUCT610 was most closely linked with the Vibrio phage pYD38-A and shared 69% homology. In addition, bacteriophage BUCT610 exhibited excellent thermal stability (4–75 °C) and broad pH tolerance (pH 3–12) in the stability test. In vivo investigation results showed that BUCT610 significantly increased the survival rate of Klebsiella pneumonia-infected Galleria mellonella larvae from 13.33% to 83.33% within 72 h. In conclusion, these findings indicate that phage BUCT610 holds great promise as an alternative agent with excellent stability for the treatment of MDR-KP infection.