**5. Phage/antibiotic synergy (PAS)**

The efficacy of combinatorial medicinal treatments is well-documented and have proven successful in treating a range of human diseases such as cancer, HIV, and malaria [59]. Similarly, the use of phages and antibiotics synergistically (PAS) has been explored and experimental studies have shown using phages and antibiotics in combination could enhance bacterial suppression and lower emergence of bacterial resistance. Furthermore, a combined approach can lead to re-establishment of antibiotic sensitivity, for example in cases where phages bind to bacterial drug efflux pumps [60]. *In vitro* studies have investigated PAS activity for the control of *S*. Typhimurium with the well-studied phage P22 and antibiotics ceftriaxone and ciprofloxacin. The study found pre-treatment of *S*. Typhimurium with phage P22 prior to antibiotic addition was the most effective approach in comparison to treating with phages 6 hours after antibiotic treatment [61]. The timing and order of phage and antibiotics needs to be considered as it can influence PAS activity. It was also reported that the presence of antibiotics did not negatively influence phage binding to *Salmonella* cells, and a significant increase in phage lytic activity was observed [62].

To date, no *in vivo* PAS studies have been conducted in *Salmonella* challenged chickens and pigs. Therefore, further *in vivo* work is required for the underlying dynamics of PAS to be understood and developed into useful combinatorial therapies. Within the context of phage therapeutics in agricultural settings (and potentially in the clinic) PAS may well provide an exciting route of research for development into a parallel treatment with antibiotics. The emergence of resistance from the target bacterium to both antibiotics and phage treatment, choice of antibiotics and phage combinations, and potential efficacy-improving interactions with immunological responses will be important factors for consideration [59].

#### **6. Potential challenges of using phages in poultry and pigs**

The use of phages against *Salmonella* in farming, either pre or post-slaughter, have some challenges. Some of those difficulties are common to phage therapy in general and fall in to four categories initial phage selection; phage delivery; resistance development; and regulatory approval.

#### **6.1 Phage selection**

On the initial phage selection, potential phage candidates need to be virulent and propagate via the lytic cycle as opposed to temperate (can propagate via lysogenic or lytic cycle), which need to be confirmed by sequencing. This is to ensure the phage will not integrate on host genome avoiding transduction and horizontal gene transfer [21, 63, 64].

#### **6.2 Phage delivery**

The topic of phage therapy pharmacodynamics and pharmacokinetics is complex and more specific reviews have recently been published on this topic [65, 66]. In brief, phages need to reach the site of bacterial colonisation, and in poultry and pigs, *Salmonella* initially colonises the gut. Many studies have been designed to establish if phages can be delivered to the gastrointestinal tract and beyond via oral administration, either in feed or drinking water. For post-slaughter application, phage preparations can be applied by directly applying to carcasses, meat, skin,

packaging materials as well as surfaces in the abattoir or meat processing facilities. Both pre- and post-slaughter applications present challenges to phage delivery [36].

A particular challenge to phages delivered orally to control *Salmonella* is to ensure they will be active in the gut pH, despite the fact that they are sensitive biological entities and will encounter changes in pH (**Figure 3**) and temperature. Phages are typically stable between pH 4 and pH 10 [67]. However, the studies discussed in Section 3 highlight natural phages retain lytic activity through the passage of the gut and do reach the focal point of infection.

Alternative solutions have been developed to protect phages from the acidic conditions by using dry or liquid formulation solutions. For example, it was shown that Felix O1 microencapsulation in chitosan-alginate microspheres could fully preserve phage viability upon 1 hour exposure to simulated gastric fluid (pH = 2.4 with 3.2 mg ml−1 pepsin) and 3 hour exposure to 2% (wt/vol) porcine bile extract [68]. Other studies have shown that liposome-encapsulated phages (UAB\_Phi20, UAB\_Phi78, and UAB\_Phi87) were significantly more stable in simulated gastric fluid (pH = 2.8 with 3.0 mg ml − 1 pepsin) when compared to free phages *in vitro* while the preparation was stable at 4°C for at least 3 months [69]. These data show that the challenge of gut pH range that the phage has to endure, when administered orally, can be overcome by selecting phages that remain viable and withstand wide pH variations or, in addition or as an alternative, shield the phages by means of pH resistant pharmaceutical formulation development.

#### **6.3 Overcoming phage resistance**

Phages are no different from other antimicrobials that are used to kill bacteria, and can become resistant to them following exposure. Often, the use of phage cocktails and rotation schedules is used to limit or avoid the development of resistant mutants. When phages are used post-slaughter as disinfectants, they can be deployed at a high titer, to reduce the build up of phage-resistant bacteria [70]. In order to reduce the accumulation of phages on surfaces after their intended use in

**Figure 3.** *The gastrointestinal pH changes in the gut of pigs and chickens.*

*Potential Roles for Bacteriophages in Reducing* Salmonella *from Poultry and Swine DOI: http://dx.doi.org/10.5772/intechopen.96984*

the food industry, some disinfectants were tested and proved to be successful at neutralizing phages, such as peracetic acid [71].

When using phages within farm settings the challenge of phage persistence, spread and resistance development is more significant because successive animals will be housed in the same facilities and disinfection must be thorough. A recent study using a patented six phage cocktail against *Salmonella* (SalmoFREE) showed that after the first trial, SalmoFREE phages were detected from the beginning of the second trial in treated and control groups houses, showing that even after the cleaning and disinfection process, phages persisted in the environment and survived between trials. As a consequence, birds in the second trial (control and treatment group) showed unexpected reduction of *Salmonella* counts even before treatment/placebo administration at day 17 [46].

Effective disinfection practices and phage cocktail rotation may be the solution to the phage persistence challenge, however the effect of disinfectants should be tested on a case-by-case basis to determine their efficacy at neutralizing the respective phage(s) cocktail [72].

#### **6.4 Regulating phage products**

As discussed above, phages may be used as a feed additive to prevent or treat infection, as a medicine to treat infection or as a post product treatment for carcasses or meat. Phages could also be used to decontaminate either the environment that the animals are living in, or facilities regarding production of the final product. Phages go through specific regulatory pathways depending on which of these intervention points that they are used in, and on the level of claims associated with their use. To take a product to market requires the developer to know which regulatory route they will take, in order to gather appropriate data on safety and efficacy [73].

In recent years there has been a significant amount of engagement from regulating bodies, who are also acutely aware of the need to find novel antimicrobials. They are also aware that this is often seen as a major hurdle to developing the technology and are keen to help. It is important to state that by working with regulators there is an opportunity to impose a regulatory system that will allow the exploration of this technology whilst hopefully mitigating against many of the mistakes that we have previously made in terms of overusing antibiotics from the outset. Antibiotic stewardship was largely implemented after extensive bacterial resistance to antibiotics had already been achieved however sensible regulation could work hand in hand with a stewardship program to maintain effective phage use for future generations [74].

Establishing how phages fit into traditional drug/veterinary medical product regulatory systems is not always trivial. In the USA, phages are regulated by the Food and Drug Administration (FDA), regardless as to whether they are to be used in humans or animals, although they go through different parts of this depending on exactly how they are being used. Interestingly the FDA regulates phages in the same way regardless of whether they are 'natural' or engineered [73].

In Europe phages are currently regulated by the European Commission through the European Medicines Agency. Unlike the system in the USA, if phages are genetically altered, they are regulated differently. In the UK if phages are to be used within animals, they are regulated by the Veterinary Medical Directorate but if their end use is in humans they are regulated by the Medicines and Healthcare products Regulatory Agency and in food they are regulated by the Food Standards Agency. The different regulatory authorities do communicate with each other to identify

commonalities and routes forward. Clearly there are parallels with other biologicals such as monoclonal antibodies, which will inform how phages are effectively regulated [73].
