Phage Hunt Targets Stubborn Bone Infections
Scientists Isolate Bacteriophage to Combat Drug-Resistant Bacteria
Researchers are intensifying the search for alternative treatments against persistent bacterial infections, with a recent study focusing on bacteriophages—viruses that specifically target bacteria. This investigation, aimed at combating a particularly resilient strain of *Pseudomonas aeruginosa* found in chronic bone infections, highlights a promising avenue in the fight against antimicrobial resistance.
Unearthing Potential Phage Warriors
The study began with the isolation of *P. aeruginosa* strains from a patient suffering from chronic osteomyelitis at Peking University Third Hospital. These bacterial samples were then identified using advanced genome sequencing. Simultaneously, wastewater from the same hospital was collected, forming the source for isolating and enriching potent bacteriophages.
From Lab Bench to Phage Purification
Wastewater samples underwent centrifugation to concentrate potential phage candidates. These, along with host bacteria cultures, were introduced into a nutrient-rich liquid medium for overnight enrichment. Following further centrifugation and sterile filtration through a 0.22-µm filter, the refined phage concentrate was ready for purification. This involved serial dilution and plating with molten semi-solid agar to identify bacterial plaque formation—clear zones indicating phage activity.
Phage purification involved multiple rounds to ensure the isolation of potent viral agents. The purified phages were then stored in SM buffer, a solution designed to maintain their stability. Phage concentration, or titer, was meticulously determined using a double-layer agar (DLA) plaque assay, a standard method for quantifying infectious viral particles.
Characterizing Phage Strength and Structure
The physical structure of the isolated phages was visualized using transmission electron microscopy (TEM). Samples were prepared by depositing them onto electron microscope mesh, followed by staining with uranyl acetate. Images captured at 80 kV revealed the distinct morphology of these bacterial predators.
To ascertain their effectiveness, phages were serially diluted and mixed with bacterial cultures in their active growth phase. The double-layer agar method was again employed. After overnight incubation, the number of plaques was counted to determine the phage titer, a crucial measure of their potency.
Optimizing Phage-Bacterial Interaction
The study also focused on determining the optimal multiplicity of infection (MOI)—the ratio of phages to bacteria. Host bacterial solutions were cultured to the logarithmic growth phase. Various MOI values were tested by mixing phage dilutions with the bacterial suspension. After incubation, phage titers in the resulting supernatant were measured. The MOI that yielded the highest phage concentration was identified as the most effective for subsequent experiments.
Assessing Phage Resilience: Temperature and pH Tolerance
The stability of the phages under different environmental conditions was rigorously tested. For temperature tolerance, phage solutions were incubated for one hour at temperatures ranging from 37°C to 70°C, with their potency subsequently measured. Similarly, phage suspensions were exposed to a spectrum of pH values, from highly acidic (pH 1.0) to highly alkaline (pH 12.0), for one hour at 37°C.
The phage titers obtained at 37°C and pH 7.0 served as baseline references for comparison. Control titers were established under standard conditions to ensure the accuracy of subsequent measurements.
Mapping Phage Attachment and Growth Dynamics
An adsorption curve was generated to understand how quickly the phages bind to their bacterial hosts. Phages and bacterial suspensions were incubated together at room temperature. Samples were collected at regular intervals, centrifuged, and the phage titer in the supernatant was measured to track the rate of bacterial infection.
Furthermore, a one-step growth curve experiment was conducted to map the phage replication cycle. Bacteria were infected with phages at a specific MOI (10) and incubated. Samples were collected periodically. After washing to remove unbound phages, the phage yield was measured over time, illustrating the burst size and replication rate of the phages.
The ongoing research into bacteriophage therapy is gaining momentum. For instance, the U.S. Food and Drug Administration (FDA) has been increasingly authorizing expanded access protocols for phage therapy, allowing critically ill patients access to these viral agents when conventional antibiotics fail. As of early 2024, over 150 clinical trials involving bacteriophages were reportedly underway globally, targeting a range of bacterial infections, including those affecting bone and implants (Nature Reviews Microbiology, 2023).
