## 4 Challenges Faced in the Application of _E. coli_ O157:H7 Phages  
There are a number of difficulties and factors to take into account when using phages as a biocontrol for _E. coli_ O157:H7 either on food products or as a medication in humans and animals. Phages are very specific to individual bacterial strains [144]. Since _E. coli_ O157:H7 strains can vary genetically and develop resistance to phages over time, it can be difficult to find phages that effectively target these strains. As a result, a combination of different phages in the form of a cocktail may be necessary for effective biocontrol of this pathogen. _E. coli_ O157:H7 has a low infectious dosage (10 to 100 CFU/mL) and can survive in low-pH conditions, such as the acidic environment of the stomach and acidic food, which is one of its stress resistance mechanisms [145]. It can be therefore difficult to deliver phages to the site of infection because they may need to endure stomach acid and other digestive processes in order to reach the intestinal _E. coli_ O157:H7 in the gut. For phages to be considered effective at inhibiting _E. coli_ O157:H7, they must possess acid resistance in order to survive in the acidic environment of the stomach. Furthermore, phage products can be expensive to create, produce, and distribute due to their sensitivity to environmental factors like pH and temperature [146]; their quality and effectiveness can be compromised if they are transported and stored in unsuitable environmental conditions. The issue of storage and transportation has made it difficult for patients situated in low-resource environments to have access to them. Additionally, research has shown that phage therapy may be more effective when combined with other treatments, such as antibiotics [147; 148]. Since its usage in humans could raise ethical concerns, it is crucial to carefully consider the best sequence and combination of therapies that will not cause further complications in infected patients. Inadequate clinical data also pose a challenge in the application of _E. coli_ O157:H7 phages; while there have been some case reports and small clinical studies on phage therapy [149], more large-scale, randomised controlled trials are required to establish the safety and efficacy of phages as a biocontrol agent in treating _E. coli_ O157:H7 infections. Figure 3: Phage-based products that have already received approval around the world. The manufacturer, the target microorganisms, and the food matrix where the product is intended to be applied are all listed in the product description. The FDA, FSANZ, and the companies’ news websites were used to create the timeline. Adapted with permission from: [PERSON] et al. [80], 2023, Taylor & Francis Ltd., [[http://www.tandfonline.com](http://www.tandfonline.com)]([http://www.tandfonline.com](http://www.tandfonline.com)), 1 September 2023. _Phage Resistance by E. coli O157:H7_  
The evolution of phage resistance in _E. coli_ O157:H7 is a continuous and natural process. As a result of repeated use of phages against _E. coli_ O157:H7, in vitro experiments have shown that _E. coli_ O157:H7 can develop phage resistance [150]. To avoid phage infection, they employ a variety of processes that involve the following techniques. Phage inability to attach: _E. coli_ O157:H7 may alter the architecture of its cell surfaces to restrict phages from attaching to its surface [151]. To attach to particular receptors on the bacterial cell wall, phages often use receptor-binding proteins [152]. Phage attachment may be prevented by changes to these receptors. _E. coli_ O157:H7 can change its surface characteristics, such as the lipopolysaccharideacharide (LPS) layer, thereby making it challenging for phages to adhere to the bacterial cell [153]. CRISPR-Cas Systems: Some _E. coli_ O157:H7 strains have Clustered Regularly Inter-spaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) systems, which are a form of adaptive immunity against phages.