Prophages can alter their bacterial hosts to prevent other phages from infecting the same cell, a mechanism known as superinfection exclusion (SIE). Such alterations are facilitated by phage interactions with critical bacterial components involved in motility, adhesion, biofilm production, conjugation, antimicrobial resistance, and immune evasion. Therefore, the impact of SIE extends beyond the immediate defense against superinfection, influencing the overall fitness and virulence of the bacteria. Evaluating the interactions between phages and their bacterial targets is critical for leading phage therapy candidates like Pseudomonas aeruginosa, a Gram-negative bacterium responsible for persistent and antibiotic-resistant opportunistic infections. However, comprehensive literature on the mechanisms underlying SIE remains scarce. Here, we provide a compilation of well-characterized and potential mechanisms employed by Pseudomonas phages to establish SIE. We hypothesize that the fitness costs imposed by SIE affect bacterial virulence, highlighting the potential role of this mechanism in the management of bacterial infections.

Background/Objectives: Antimicrobial resistance (AMR) is a growing threat that undermines the effectiveness of global healthcare. The Centers for Disease Control and Prevention and the World Health Organization have identified numerous microbial organisms, particularly members of the ESKAPEE pathogens, as critical threats to global health and economic security. Many clinical isolates of these pathogens have become completely resistant to current antibiotics, making treatment nearly impossible. Herbal remedies, such as those found in Traditional Chinese Medicine (TCM), have been practiced for thousands of years and successfully used to treat a wide range of ailments, including infectious diseases. Surprisingly, despite this extensive knowledge of folk medicine, no plant-derived antibacterial drugs are currently approved for clinical use. As such, the objective of this study is to evaluate the antimicrobial properties of extracts derived from TCM plants. Methods: This study explores a comprehensive library comprising 664 extracts from 132 distinct TCM plant species for antimicrobial properties against gram-negative (Escherichia coli) and gram-positive (Micrococcus luteus) bacteria using liquid and solid in vitro assays. Results: Intriguingly, our results reveal 17 plant species with potent antimicrobial properties effective primarily against gram-positive organisms, including Streptococcus aureus and epidermidis. A literature search revealed that nearly 100 purified compounds from the identified TCM plants were previously isolated and confirmed for their antimicrobial properties, collectively inhibiting 45 different bacterial species. Conclusions: Our results indicate that phytobiotics from the identified plants could serve as potential candidates for novel antimicrobials.

Bacteria and other microbes can naturally produce antibiotics within their native soil environment, but often at sub-inhibitory concentrations; consequently, the exact role of antibiotics within bacterial native communities remains unknown. We have shown that subtherapeutic quantities of naturally occurring antibiotics can induce the Pseudomonas prophage Pf4, and superinfection of Pseudomonas aeruginosa cells by this phage leads to their reduced virulence, as demonstrated by impaired twitching motility, compromised macrophage evasion, and increased killing by macrophages in vitro . Thus, the production of subtherapeutic concentrations of antibiotics by environmental microbes may provide the producers an evolutionary advantage associated with reduced fitness induced by prophages in the competing bacteria. Collectively, these results reveal the role of naturally occurring antibiotics in altering fitness by phage-mediated superinfection exclusion and provide potential clinical implications in the application of phage therapy. Significance Statement: This study provides insights into the mechanisms by which sub-inhibitory concentrations of environmentally-produced antibiotics induce Pseudomonas aeruginosa prophages, revealing a potential evolutionary strategy for competitive advantage among bacteria. By activating prophages, antibiotics can induce fitness defects in neighboring bacteria, impacting their motility, phagocytosis, and survival within macrophages. Such previously unrecognized role for environmental antibiotics in bacterial ecosystems may offer insights into enhancing phage therapy by exploiting phage-antibiotic synergies. Understanding phage-host interactions can enhance therapeutic strategies to mitigate bacterial infections and antimicrobial resistance.

Phage therapy is an exciting strategy against antimicrobial-resistant bacterial infections, but critical knowledge gaps regarding its clinical application persist. Studying a patient with a life-threatening, chronic bacterial infection who failed phage therapy, we uncovered important biological concepts with direct translational impact. Using longitudinal clinical samples, we found that patients can harbour pre-existing antibodies against active prophages induced from the genome of the causative pathogen. Notably, these antibodies can contribute to clinical failure by cross-reacting with and effectively neutralising therapeutic phage. We also uncovered bacterial heteroresistance, characterised by bacterial subpopulations from the initial infection with reduced phage susceptibility, as a further contributor to treatment failure. These findings highlight the intricate interplay between host immunology, bacterial genetic diversity and phage biology, bearing broad significance for clinical phage therapy. Future phage therapy patients, especially those with chronic infections, should be screened for antiphage immunity and bacterial heteroresistance prior to phage treatment.

Antimicrobial resistance (AMR) is a growing threat that undermines the effectiveness of global healthcare. The Centers for Disease Control and Prevention and the World Health Organization have identified numerous microbial organisms, particularly members of the ESKAPEE pathogens, as critical threats to global health and economic security. Many clinical isolates of these pathogens have become completely resistant to current antibiotics, making treatment nearly impossible. Herbal remedies, such as those found in Traditional Chinese Medicine (TCM), have been practiced for thousands of years and successfully used to treat a wide range of ailments, including infectious diseases. Surprisingly, despite this extensive knowledge of folk medicine, no plant-derived antibacterial drugs are currently approved for clinical use. This study explores a comprehensive library comprising 664 extracts from 132 distinct TCM plant species for antimicrobial properties against gram-negative (Escherichia coli) and gram-positive (Micrococcus luteus) bacteria using liquid and solid in vitro assays. Intriguingly, our results reveal 17 plant species with potent antimicrobial properties effective primarily against gram-positive organisms, including Streptococcus aureus and epidermidis. Our results indicate that phytobiotics from the identified plants may be potential candidates for novel antimicrobials.