Are bacteriophages replacing antibiotics? In pursuit of an engineered phage alternative for the treatment of UTIs
produce many different phages. This makes phage therapy a highly personalised treatment option to select patients. Furthermore, the regulatory environment surrounding phage therapy is a complicated issue, as phages are classified as biological products and their use as therapeutic agents is not well-regulated in many countries. This lack of regulation is hindering the ability to conduct clinical studies and administer phage therapy to patients, slowing the development of this option and the creation of standardised treatment protocols. While there have been several case reports, case series, and a few randomised controlled trials (RCT) that did not reveal safety concerns, there is still a lack of high-quality efficacy data. More research is needed to get phage therapy widely adopted as a mainstream treatment option.
phages with enhanced homologous and heterologous therapeutic effects (Figure 3).
Dr. Lorenz Leitner Department of Neuro-Urology, Balgrist University Hospital, University of Zürich (CH)
lorenz.leitner@gmail. com
Bacteriophages, also known as phages, are viruses that infect, replicate within, and lyse bacteria. They were discovered in 1917 by the French-Canadian microbiologist Dr. Félix d'Hérelle, who observed their ability to kill bacterial pathogens in a laboratory setting. Since then, phage therapy has been explored as a treatment for bacterial infections, however, with the discovery of antibiotics, there was a decline in interest. The use of phages in medicine has also been hindered by a lack of understanding and regulation, as well as the difficulty of mass producing and standardising them. Despite these challenges, recent advances in the understanding of phages and their potential applications have renewed interest in phage therapy as a viable alternative to antibiotics. History of phage therapy and biology Phage therapy is a form of treatment for bacterial infections that utilises phages to target and kill specific pathogens (Figure 1). [1] In 1917, Dr. d'Hérelle observed an agent able to kill bacteria in a petri dish and he later named it "bacteriophage," derived from the Greek words βακτήριoν (baktérion) and ϕ αγε ῖ ν (phageín), meaning “to devour rods” or “bacteria eater”. He hypothesised that phages specifically targeted and infected bacteria and then applied this principle to develop a treatment for bacterial infections. In 1919, Dr. d'Hérelle used phage for the first time to successfully treat a patient with dysentery. The discovery of phages came at a crucial time in medical history when bacterial infections were a significant cause of morbidity and mortality. Phage therapy gained popularity as a treatment option in the 1920s and 1930s, particularly in Eastern Europe to treat a variety of bacterial infections. It is still used today in some parts of the former Soviet Union as an integral part of their healthcare system. However, the development of antibiotics in the 1940s led to a decline in phage therapy research and usage. Phages are the most abundant replicating units on earth, they coexist and coevolve with bacteria, and they modify and shape bacterial communities and evolution. They can be classified into two main types based on their life cycle: lytic and temperate phages (Figure 2). Lytic phages enter a bacterial cell and immediately begin replicating, eventually causing the host cell to burst and release new phages. Temperate phages, on the other hand, integrate their genetic material into the host cell's genome and remain dormant until triggered to enter the lytic cycle. As temperate phages can serve as vectors for horizontal gene exchange between
Figure 3: Illustrates a strategy for diagnosing and treating urinary tract infections (UTIs) using engineered phages. 3a) Urine samples are collected from patients with a UTI and incubated with reporter phages. If bioluminescence is detected within 5 hours, treatment with a specific phage can be started. 3b) The genome of a wild-type phage (shown in blue) is equipped with a gene for bioluminescence (shown in yellow). If the engineered reporter phage (shown in yellow) infects the bacteria, bioluminescence can be detected after the engineered protein is expressed . 3c) The same wild-type phage (shown in blue) can be equipped with both homologous and heterologous antimicrobial effector genes (shown in red). These payload proteins allow for enhanced
Phage therapy in the context of urology Urinary tract infections (UTIs) represent an
important model disease to optimise the evidence base for phage therapy. As a closed compartment, the urinary bladder allows for a longer interaction with existing bacteria after instillation of a phage preparation. Since many patients, especially those with neurogenic lower urinary tract dysfunction, rely on intermittent catheterisation or an indwelling catheter in their daily lives, phage therapy could become an important treatment option for acute or recurrent UTIs in this population. Several successful urological applications of phages have already been published. In vitro coating of permanent catheters with specific phages against Pseudomonas aeruginosa, Escherichia coli , and Proteus mirabilis resulted in a reduction of biofilm formation. Khawaldeh et al. succeeded in eradicating P. aeruginosa and achieving clinical improvement in a patient with recurrent foreign- body-associated (double-J-stent) UTIs, using a combination of phages and antibiotics after repeated antibiotic therapy failed. In 2018, our research group was able to show a reduction in the bacterial load in 67% of all patients with asymptomatic bacteriuria in a clinical application of a phage cocktail licensed in Georgia. The world's first RCT on the use of phages for UTIs found a comparable success rate for phages and antibiotics, but no superiority of phages over placebo in patients with UTIs undergoing transurethral resection of the prostate. [4] Important note, the overall success rate in all three groups in this trial was unusually low. Locus Biosciences, a clinical- stage biotechnology company, posted preliminary results in 2021 from their multi-centre phase 1b RCT (LBx-1001 Study, CT.gov NCT04191148.) using engineered phages, with the main finding of a 2-3 log reduction of urine E. coli concentration (colonies forming units (CFU)/mL) compared to placebo across the duration of the treatment. Overcoming limitations with engineered phages Genetic engineering can be a useful strategy to increase the speed of testing the sensitivity of pathogens to phages, and to increase the host range or antimicrobial activity. This would allow the use of phages to be expanded beyond highly personalised treatment and applied to a broader range of patients. With natural phages, the use of multiple phage "cocktail" that can target many strains of bacteria at once, is necessary to be active against different strains. However, it remains challenging to determine the optimal number and type of phages in such a "cocktail", and to evaluate in each case, how many phages would have therapeutic value for a specific infection. Additionally, combining phages in “cocktail” solutions can result in stability issues that cause low concentrations of individual phages and can compromise their therapeutic value. Research projects In collaboration with a national and international network of leading phage experts, as well as basic and clinical scientists, we are pursuing an engineered phage alternative strategic approach for the treatment of UTIs. To achieve this, two important research projects are ongoing - CAUTIphage: Engineered bacteriophages as antibiotic alternatives for treating catheter associated urinary tract infections (http://p3.snf.ch/project-189957); and ImmnoPhage: (https://www.hochschulmedizin.uzh. ch/de/projekte/immunophage.html). After identifying phages with a broad host spectrum, these phages are genetically modified and engineered into so-called reporter phages and
killing of the uropathogen and additional killing of polymicrobial communities after bacterial lysis.
bacteria, including antibiotic resistance genes, mainly lytic phages are selected for therapeutic purposes. [1] Phages vs. common antibiotic therapy The main advantages and disadvantages of phage therapy, as well as the distinctions from conventional antibiotics, are closely connected to and stem from their biology. Phages are not only specific for certain bacterial species but even possess strain level specificity. The ability to target definite bacterial strains makes them a useful tool to treat infection- causing pathogens without the risk of collateral damage to the patient's beneficial microbiome, which is a common side effect of antibiotics. At the same time, this high level of specificity requires sensitivity testing of the patient’s bacterial isolate prior to starting treatment, similar to what is done for antibiotic sensitivity testing. A concern for any antibacterial therapy – old or new – is the development of resistance. The close coexistence and coevolution of phages with their host bacteria has led to the development of several bacterial defence strategies against phage that can allow bacteria to become resistant. However, the high abundance of phages in nature usually allows for the identification of new candidate phages for therapy, even in cases where the target bacteria are resistant or develop resistance. This contrasts to the difficulty of finding effective antibiotics in cases of multidrug-resistant bacteria. Interestingly, resistance to phage may result in changes to the host bacteria that make it less pathogenic or even revert antibiotic sensitivity due to survival pressure. [3] Phages that use different mechanisms of bacterial infection can be combined together or combined with antibiotics to result in highly synergistic effects for the inactivation of bacteria and reduction of antibiotic resistance. The logical trade-off of an antimicrobial strategy with such high specificity is the requirement to have rather large phage banks at one’s disposition and to perform extensive sensitivity testing against the infection-causing pathogens. Furthermore, subsequent production of the right phages for a target infection can be difficult, expensive, labour intensive and time-consuming. The production process for phages is complex, making it difficult to Figure 2: Illustrates the two main life cycles of phages: lytic and temperate. When a phage infects a susceptible bacteria, it injects its genetic material into the host. In the lytic pathway (left), the bacterial genome breaks down and the phage genome replicates, resulting in mature new phage clones that burst the host bacteria, which then diffuse through the surrounding environment and can infect new susceptible bacteria. In contrast, temperate phages (right) have a different strategy. After injection of the genome, it can be integrated into a specific section of the bacterial genome and will be passively replicated every time the bacterial cell divides. The phage genome that is integrated in the bacterial genome or existing as an extrachromosomal plasmid is called a prophage. Environmental factors (such as starvation or other unfavourable growth conditions) can induce a temperate phage to enter the lytic cycle (left). Phage therapy mainly uses lytic phages to treat pathogenic bacterial infections. [1]
By engineering a nanoluciferase reporter gene into the phage genome, we developed a rapid phage- based diagnostic assay to detect the most prevalent pathogens causing UTIs. Upon host infection and expression of the nanoluciferase protein, bioluminescence can be detected. Overall, E. coli, Klebsiella spp., and Enterococcus spp. were each detected with high sensitivity (68%, 78%, 85%) and specificity (99%, 99%, 99%) at a resolution of 10^3 CFU/mL within 5 hours directly in patient urine. [5] This technique can be used to identify phages that should be effective for therapy. The very same phages were additionally engineered for target- specific effector gene delivery and host-dependent production of colicin-like bacteriocins and cell wall hydrolases. Testing these engineered phages ex vivo in patient urine, superior dual phage- and effector- mediated enhanced killing and suppression of resistance of uropathogen growth could be demonstrated compared to wild-type phages. [6] Conclusions Phage therapy has the potential to be an effective alternative to traditional antibiotic treatment for bacterial infections. However, there are still several challenges that need to be overcome before it can be widely adopted. These include the lack of standardised protocols and regulation, the difficulty of producing phages on a large scale, and the need for more research to identify the best suited patients for phage therapy. Despite these challenges, the potential of engineered phages, together with the growing need for new treatments for antibiotic- resistant bacterial infections, make phage therapy an area of research worth exploring further. References 1 Salmond, G. P. & Fineran, P. C. A century of the phage: past, present and future. Nat Rev Microbiol 13, 777-786, doi:10.1038/nrmicro3564 (2015). 2 Leitner, L., Kessler, T. M. & Klumpp, J. Bacteriophages: a Panacea in Neuro-Urology? Eur Urol Focus 6, 518-521, doi:10.1016/j.euf.2019.10.018 (2020). 3 Valerio, N. et al. Effects of single and combined use of bacteriophages and antibiotics to inactivate Escherichia coli. Virus Res 240, 8-17, doi:10.1016/j. virusres.2017.07.015 (2017). 4 Leitner, L. et al. Intravesical bacteriophages for treating urinary tract infections in patients undergoing transurethral resection of the prostate: a randomised, placebo-controlled, double-blind clinical trial. Lancet Infect Dis 21, 427-436, doi:10.1016/S1473- 3099(20)30330-3 (2021). 5 Meile, S. et al. Engineered reporter phages for rapid detection of Escherichia coli, Klebsiella spp., and Enterococcus spp. in urine. b ioRxiv, 2022.2011.2023.517494, doi:10.1101/2022.11.23.517494 (2022). 6 Du, J. et al . Enhancing bacteriophage therapeutics through in situ production and release of heterologous antimicrobial effectors. bioRxiv , 2022.2003.2009.483629, doi:10.1101/2022.03.09.483629 (2022).
Figure 1: Illustrates the morphology and action of phages. A free-floating phage seeks out a susceptible bacterial host and attaches to the cell wall using its tail fibres to bind with surface receptors. Once attached, the phage infects the bacteria by injecting its genetic material through a central tail tube. It's worth noting that phage tail fibres are usually highly specific for the surface receptors on a bacterium, which means that a particular phage can only infect a narrow range of bacteria. [2]
Sunday 12 March, 09:13 - 09:21 Plenary Session: Challenges in urogenital infections Yellow Area, eURO Auditorium 2
European Urology Today
20
February/March 2023
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