Potential Roles for Bacteriophages in Reducing *Salmonella* from Poultry and Swine

*Anisha M. Thanki, Steve Hooton, Adriano M. Gigante, Robert J. Atterbury and Martha R.J. Clokie*

## **Abstract**

This chapter discusses application of natural parasites of bacteria, bacteriophages (phages), as a promising biological control for *Salmonella* in poultry and swine. Many studies have shown phages can be applied at different points from farm-tofork, from pre to post slaughter, to control the spread of *Salmonella* in the food chain. Pre-slaughter applications include administering phages via oral gavage, in drinking water and in feed. Post slaughter applications include adding phages to carcasses and during packaging of meat products. The research discussed in this chapter demonstrate a set of promising data that relate to the ability of phages to reduce *Salmonella* colonisation and abundance. Collectively the studies support the viability of phage as antimicrobial prophylactics and therapeutics to prevent and control *Salmonella* in the food chain.

**Keywords:** Bacteriophages, phages, swine, poultry, delivery

### **1. Introduction**

The global problem of antimicrobial resistance (AMR) is driving the search for novel treatments to control multidrug-resistant (MDR) pathogenic bacteria. Infections caused by MDR pathogens impose a significant burden on healthcare systems and economic productivity and are a major cause of mortality. Globally, AMR is associated with 700,000 deaths annually, with the prospect of this reaching 10,000,000 by 2050 if no resolution is found [1].

A One Health approach, that considers the intrinsic associations between antibiotic use in livestock and agriculture, the emergence of MDR pathogens, and the societal impact of AMR in developed and developing nations is required [2, 3]. However, integrating these approaches is challenging as antibiotic use in agriculture is generally widespread [4, 5]. For example, prophylactic administration of antibiotics to pigs during the weaning process is a standard technique employed in many countries [6]. Over recent years, efforts to limit antibiotic use other than specifically to control active bacterial infections have been implemented. Consequently, the use of antibiotics as growth promoters in food

production animals was banned in the European Union (EU) in 2006 and in the United States of America (USA) in 2017 [7].

Gram negative *Enterobacteriaceae* are an important component of human, animal, and environmental microbiomes and can be associated with both health and disease. While the family contains several notorious pathogens (e.g. certain *E. coli*, *Klebsiella* spp., *Shigella* spp. *etc*.), the genus *Salmonella* presents a problem for AMR due to its ubiquitous distribution in food production environments and MDR phenotypes [8]. Worryingly, clinically important antibiotics are becoming ineffective, including colistin, which is a human critical antibiotic [9]. As such, alternative strategies to control/eliminate MDR *Salmonella* that may replace or complement antibiotics are needed.

Globally, dominant *Salmonella* serovars display a distribution pattern in pigs and poultry reflective of each industry. In pigs, *S*. Typhimurium (e.g. U288, U302, DT193, DT104), monophasic 4,[5],12:i:- and other variants such as 4,12:i:- are the dominant strains at both farm and slaughterhouse facilities in the UK and EU [10–13]. Other serovars such as *S*. Derby, *S*. Enteritidis, *S*. Bovismorbificans, *S*. Kedougou, *S*. Rissen, and *S*. Brandenburg are also reported [13, 14]. In the USA and China the dominant *Salmonella* serovars include *S*. Typhimurium, monophasic 4,[5],12:i:- *S*. Infantis, and *S*. Brandenburg [15].

For poultry, and in parallel with the global emergence of strains such as 4, [5],12:i:- the most prevalent serovar in UK production facilities is an *S*. Typhimurium derivative 13,23:i:- that accounted for almost a quarter of all isolations in 2019 [13]. Across the EU, the USA and China monophasic strains continue to expand throughout poultry production facilities. Other serovars such as *S*. Enteritidis, *S*. Berta, *S*. Typhimurium, *S*. Infantis, *S*. Hadar, *S*. Kentucky, and *S*. Heidelberg have all been isolated and/or linked to outbreaks [16–18]. The global diversity of *Salmonella* spp. within pig and poultry production constitutes a significant source of disease for humans and animals alike.

Controlling *Salmonella* requires intervention strategies capable of implementation at the national/international level. One such strategy is the targeted application of natural bacterial predators, bacteriophages (phages). Over the last decade, a robust body of evidence has demonstrated that phages can be applied at various points from farm-to-fork for pathogen control [19, 20]. Phage application could be implemented at the stage of rearing [21, 22], slaughter and processing [23], or at pre-retail/packaging [24, 25].

#### **2. Phages**

Phages are viruses that specifically infect and kill bacteria and with few reported side-effects in humans and animals. Phages are the most abundant biological entity on Earth, with estimated numbers ten times greater than bacterial cells [26]. Phages were independently discovered by Frederick Twort and Felix d'Herelle in 1915 and 1917 respectively. D'Herelle was the first to test phage efficacy in animals and showed phage treatment increased the survival of chickens suffering from fowl typhoid by 95–100% compared with 0–25% in untreated birds [27]. Despite this, phage therapy research slowed markedly following the discovery of antibiotics. However, research into phage therapy has been renewed since the emergence of AMR as it offers a promising alternative to antibiotics. Studies have shown phages are able to lyse MDR strains [28, 29] and there are multiple examples of successful phage therapy in humans [30] and animals [31]. Furthermore, phages can be applied to food to reduce bacterial loads and globally are being used commercially to improve food safety [32].

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

#### **2.1 Phage morphology and infection cycle**

Phages are characterised based on their virion morphology, genome type and sequence, and the infection cycle they follow. Phages are approximately a hundred times smaller than bacterial cells by volume, and generally only infect a subset of strains within a host species. Over 5,000 phages have been viewed under the transmission electron microscope (TEM) [33] and over 96% of phages studied are tailed phages and belong to the order *Caudovirales*. Siphoviruses, myoviruses and podoviruses are the most common phage types and constitute 61, 25 and 14% of all isolated tailed phages respectively (**Figure 1**) [34].

Phages are obligate parasites of bacteria as they lack the capacity to replicate independently. Phage replication occurs through either a lytic or lysogenic cycle (**Figure 2**). Phages following the lytic cycle attach to receptor(s) on the host cell surface using tail fibres, after which they inject their DNA and sequester the host's metabolic processes to produce more phage, eventually leading to cell lysis and release of the virions for further cycles of infection [35]. In comparison, during the lysogenic cycle phage DNA is incorporated into the bacterial cell and is replicated along with the host. Under certain conditions, e.g. stress and DNA damage, the phage can enter a lytic cycle as above. The lifestyle of the phage is determined via sequencing where the absence of recognisable integrases and other genes involved in the process of integration can be taken as indicative of a strictly lytic life cycle [36]. As lytic phages kill their target cells directly, they are preferred for therapeutic applications.

**Figure 1.** *Morphology of tailed phages viewed under TEM. The images show the typical structure of a (a) siphovirus, (b) myovirus and (c) podovirus. TEM images were taken by the Electron Microscopy Facility at the University of Leicester.*

#### **Figure 2.**

*Phage lytic and lysogenic infection cycle. (a) phages attach to a receptor on the bacterial cell, after which (b) they inject their DNA (red line) into the cytoplasm of the cell. Phages can then go on to follow the lytic cycle (c-d) or the lysogenic cycle (f-g). In the lytic cycle (c) phages take over the host cells machinery to replicate their nucleic acids and proteins (d) to form new phage progeny. This (e) leads to lysis of the bacterial cell to release the phage progeny and the phages go on to infect more target bacterial cells. In the lysogenic cycle (f) phage DNA is integrated into the bacterial genome and (e) as the bacterial cells are replicated the prophage is replicated simultaneously.*

#### **2.2 Phage isolation, host range and resistance**

Phages can be isolated from any environment their hosts inhabit. *Salmonella*specific phages have been isolated from faecal material obtained from pig and chicken farms, food processing plants, wild boar reserve [29], slurry lagoons [37], and sewage [22, 23]. Consequently, as phages are found in nature, humans and animals are continuously exposed to them, which is a major advantage in using them, as new entities would not be introduced into biological systems when phages are applied therapeutically [38].

The lytic spectrum (host range) of a phage is determined by screening against multiple strains of the target pathogen. Both narrow and broad host range phages have potential uses as therapeutics [39], for example a highly-specific, narrow host range phage can be applied with minimal perturbation to other residual microbial populations. Broad host-range phages provide a better scope of lysis and are therefore the desired components of most phage therapeutic applications. Multiple phages can be combined as a cocktail to improve phage coverage of the target species [40].

Emergence of resistance against therapeutic phages is a possibility as both phages and bacteria are in a continuous arms race. The mechanisms of phage resistance include altering the phage receptor, blocking phage DNA injection or inhibiting phage replication. This resistance can be countered by using cocktails of phage which bind to different receptors, as its unlikely resistance to all phage in the cocktail will emerge concurrently. Moreover, phage resistance can lead to a fitness cost for the bacterial cells [41]. Different multiplicities of infections (MOI's), which is ratio of phages to bacterial cells can also be trialed to limit resistance [42].

## **3. Experimental phage studies in chickens and pigs**

In this section, the application of phages pre- and post-slaughter to reduce *Salmonella* numbers in chickens and pigs is discussed. Studies have varying levels of success in reducing *Salmonella* in challenge models, but with each study, valuable information is gained on phage dose, route of administration and resistance.

#### **3.1 Experimental phage studies in chickens**

#### *3.1.1* In vivo *phage studies in chickens at farm level*

One of the first studies that investigated phage therapy against *Salmonella* challenged chickens dates back to 1991 [43]. The authors orally challenged one day old Rhode Island Red chickens with *S*. Typhimurium (108 Colony Forming Units (CFU)) and 10 minutes later administered a single phage orally at dose 1012 Plaque Forming Units (PFU)/mL. The mortality of untreated chickens was 56% 21 days post-challenge but in chickens treated with phage mortality was reduced to 20%. The authors demonstrated phage transition and replication in the gut at sites of *Salmonella* colonization such as the crop, intestine and caecum. Similarly, Atterbury et al. [21] showed in two different broiler chicken studies phage treatment (1011 PFU/mL) administered two days after challenge (108 CFU/mL), reduced ceacal colonisation by 4.2 and 2.2 log10 CFU/mL in birds challenged with Enteritidis P125109 or Typhimurium 4/74 respectively after 48 hours.

Goncalves and colleagues [44] compared the efficacy of three different phage cocktails in 45-day-old broiler chickens. The phages were administered at a dose of 10<sup>9</sup> PFU/mL via oral gavage, 1 hour post challenge with *S.* Enteritidis at

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

107 CFU/mL. Two of the three phage cocktails reduced caecal *Salmonella* counts by ~2 log10 CFU/mL in 12 hours, and *Salmonella* counts were below the detectable limit in the crop.

Toro et al. [45] designed a cocktail of three phages which could infect the top seven serotypes commonly associated with chickens. This cocktail was administered orally on days 4, 5, 6, 18, 19 and 20 at a dose of 5.4 × 106 PFU/bird and birds were challenged on day 7 with *S*. Typhimurium (105 CFU/mL). The phage treatment reduced *Salmonella* colonisation in the caeca by ten-fold, 4 days post-challenge, and 48 hours after treatment phages were isolated in the birds' faeces. Interestingly, the authors found phage treatment had a beneficial effect and chickens given the treatment gained more weight in comparison to challenged birds.

Delivering phages to chickens individually, via oral gavage, would be impractical commercially, however they could be administered easily through drinking water. Clavijo and colleagues [46] added a six-phage cocktail (named SalmoFREE®) at dose of 108 PFU/mL to drinking water on days 18, 26 and 34 (chickens were slaughtered on day 35), which was sufficient to reduce ceacal *Salmonella* counts to below the detectable limit (below 100 CFU/mL). The trial was conducted at a commercial farm where there was a record of *Salmonella* outbreaks and included 34,680 broiler chickens. This is the biggest and the only trial to date evaluating phage efficacy against *Salmonella* in a commercial setting. There was no difference in mortality or productivity measurements between untreated control birds and those treated with phage only, suggesting the cocktail was safe. Furthermore, the authors conducted a microbiome study and showed phage treatment had no detrimental effect on the chicken's microbiota [47]. Their studies provide further valuable evidence into the effectiveness and safety of phage treatment.

Delivering phages as feed additives has been investigated. Sklar and Joerger. [48] added a single phage dose (A) and a three-phage cocktail (B) to starter broiler feed at a dose of 107 PFU/g. The treated feed was available throughout the trial and chickens were challenged with *S.* Enteritidis at 104 CFU on day 1. After 14 days phage A reduced caecal colonisation by 1.9 log10 CFU/g and cocktail B by 0.6 log10 CFU/g. The authors found that the process of mixing phage with feed and storing feed in bird rearing conditions over 14 days caused a 2 log10 PFU/g reduction in phage numbers. Phage stability in feed could be a limitation and further research is needed to determine the impact storage conditions have on phage stability, such as factors as humidity and temperature.

#### *3.1.2 Experimental post-slaughter phage studies in chickens*

Following processing and packaging, meat is refrigerated to avoid bacterial growth, but *Salmonella* can survive under these conditions and phages could be used to reduce surface contamination of *Salmonella*. Goode et al. [38] applied a single phage to chicken skin artificially contaminated with *S*. Enteritidis at 103 CFU/cm3 . Phage applied at doses above 105 PFU/mL reduced bacterial numbers by over 98% and phages amplified on the surface of the infected skin by three-fold over 48 hours. In comparison, in the uninfected samples the phage titre reduced by 1 log10 PFU/cm3 , which suggests phages don't linger in absence of their target pathogen.

Atterbury et al. [49] showed phage treatment at dose 109 PFU/mL reduced levels of *S*. Enteritidis and *S*. Typhimurium by 72.2% and 38.9% respectively on spiked chicken skin samples (106 CFU/ml). The authors confirmed phage infection was occurring on the surface of the chicken skin by spreading a bioluminescent *S*. Typhimurium strain on the surface of chicken skin and then monitored its growth using photon counting. Further studies have shown the efficacy of phage treatment to reduce *Salmonella*

numbers on chicken skins are comparable to the typical chemical agents used by the food industry [50]. In addition, combining phage and chemical treatment was able to further decrease *Salmonella* counts to below detection levels [51].

To date only one study has investigated phage application on whole carcasses. Higgins et al. [52] spiked chicken carcasses with *S*. Enteritidis at 20 CFU, after which carcasses were sprayed with phage at different doses. The authors found only high phage doses of 108 and 1010 PFU/ml were effective and after 24 hours, *Salmonella* was only isolated from one out of fifteen carcasses. The phage counts were not monitored in the study, therefore it's unclear if there was phage amplification.

Phage treatment of raw meat samples has been shown to be effective at reducing bacterial load and consequently reducing its presence in the final consumer product. Duc et al. [53] tested the lytic activity of a five-phage cocktail at dose 109 PFU on chicken breasts inoculated with either *S*. Enteritidis or *S*. Typhimurium at 105 CFU. The phage cocktail reduced counts of both strains by ~1.6 log10 CFU/piece of chicken breast, when stored at 8°C, over 24 hours. However, when the meat was stored at 25°C phage treatment was more effective and reduced *S*. Enteritidis or *S*. Typhimurium by 3.1 and 2.2 log10 CFU/piece respectively over 24 hours. This could suggest phage activity is temperature dependent. However, another study showed phage activity was unaltered when spiked chicken breasts (105 CFU/ml) were treated with phage at doses 106 and 107 PFU/mL and stored at 4°C and 25°C. Under both conditions, phage treatment reduced bacterial counts to undetectable levels after just 12 hours [54]. The studies suggest phage temperature stability can vary between phages and its stability needs to be tested to determine which are more effective at food storage temperatures.

#### **3.2 Experimental phage studies in pigs**

#### *3.2.1 Phage therapy in pre-market and market-weight pigs*

Very few studies have examined the efficacy of phage treatments to control *Salmonella* in live pigs and this is largely due to the inherent difficulties of performing longitudinal studies from piglets to finished pigs. One pioneering study did exactly that and the efficacy of a fifteen-phage cocktail were tested in challenged piglets and market-weight pigs [22]. In the first study, the phage cocktail (109 PFU/mL) and challenge strain *S*. Typhimurium γ4232 (5 × 108 CFU/pig) were co-administered via oral gavage to piglets. Piglets were euthanised 6 hours postinoculation in order to mimic the amount of time spent in a holding pen. Overall, the activity of the phage cocktail was sufficient to achieve 2–3 log10 CFU (~99%) reductions in the ileum, tonsils and caecum. In collected ileum and caecal samples, in five out of six phage-treated pigs *S*. Typhimurium counts were reduced to below the limits of detection (~100 CFU/mL).

The authors next assessed the efficacy of the phage cocktail in marketweight pigs. Four pigs (in three replicates) were inoculated via oral gavage with 5 × 10<sup>9</sup> CFU *S*. Typhimurium and allowed to contaminate a holding pen for a period of 48 hours. Following this, sixteen naïve pigs (non-*Salmonella* infected – eight phage-treated/eight mock treatments controls) were introduced to the holding pens and allowed to co-mingle with the seeder pigs for 6 hours. Phage cocktail administration involved an initial oral gavage of 10<sup>9</sup> PFU/mL followed by further identical doses every 2 hours for a total of 6 hours. After 6 hours of co-mingling between *S*. Typhimurium γ4232-infected, phage cocktail-treated, and mock control-treated pigs, each cohort was euthanised. In phage treated pigs there was 1 to 1.5 log10 CFU/mL reductions in *Salmonella* colonisation in ceacal and ileal samples. The role phages can play in controlling *Salmonella*

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

infection in pigs at a critical stage of the production process is evident from the work performed by Wall et al. [22].

A similar degree of efficacy was observed when applying a microencapsulated phage cocktail treatment to control shedding of *S*. Typhimurium during a holding period of 6 hours [55]. Saez et al. found that shedding of *Salmonella* from pigs in the phage-treated group (PT) was less common than non-phage treated pigs (nPT) at 2 hours (% pigs shedding PT-38.1%, nPT-71.4%) and 4 hours (PT-42,9% - nPT-81.1%). Sampling of caecal and ileal contents 6 hours postinfection showed that phage-treated pigs had significantly less *S*. Typhimurium levels at both anatomical sites by 1 log10 CFU/mL. Another study produced some promising results by showing how dietary supplementation with probiotics (*Saccharomyces cerevisiae*, *Lactobacillus acidophilus*, and *Bacillus subtilis*) and phages can positively influence growth performance of pigs. A phage cocktail (~10<sup>9</sup> PFU/g) designed to target a diverse selection of bacteria (*S*. Typhimurium, *S*. Enteritidis, *S*. Choleraesuis, *S*. Derby, *Staphylococcus aureus*, *Escherichia coli*, and *Clostridium perfringens* types A and C) was administered as part of a feed supplement. Interestingly, the addition of phage was found to be more effective than probiotics. Phages may therefore offer an attractive alternative to replace the use of antibiotics as growth promoters in pigs [56].

#### *3.2.2 Phage decontamination of pigskin*

Post-slaughter application of phages has the potential to reduce risks associated with pork contaminated with *Salmonella* prior to general retail. An investigation into the stability of phages at retail temperatures (fresh 4°C and frozen −20°C) and also their ability to control the endemic UK pig pathogen *S*. Typhimurium U288 was examined [23]. Hooton et al. tested killing activity of *Salmonella*-specific phages against a diverse panel of *Salmonella* serovars prior to formulation as a four phage cocktail (PC1). PC1 consisted of three novel *Salmonella* phage isolates (ΦSH17, ΦSH18, and ΦSH19) combined with the broad-host range *Salmonella* phage Felix 01 in equal volumes/titres for a final concentration of 108 PFU/mL. Initially it was shown that both *S*. Typhimurium U288 and the phage components of PC1 are both stable on experimentally-contaminated pigskin pieces stored at temperatures reflective of those at retail. The efficacy of PC1 was subsequently tested on spiked pigskin over a five-day trial under fresh conditions (4°C). A 3 × 3 matrix of CFU (106 , 104 , and 103 ) versus PC1 PFU (107 , 105 , and 104 ) was used to examine a range of MOIs (0.01–10,000) to determine the most effective combination.

The phage cocktail applied at MOI's of 1000 (10<sup>7</sup> PC1 V 104 U288) and 10 (105 PC1 v 104 U288) reduced *S*. Typhimurium U288 levels by ~92% after 1 hour post challenge. After 48 hours *Salmonella* counts were significantly reduced by ~1.4 log10 CFU/4 cm2 . The first reductions of *S*. Typhimurium U288 below the limits of detection were also reported at the 48 hour timepoint, specifically when an MOI of 10 was employed against low level contamination. At 96 hours post-inoculation it was evident that MOIs in excess of the target bacterium could reduce low-level bacterial contamination to below the limits of detection. The results reported here indicate that phages may provide useful tools for the post-harvest reduction of *S*. Typhimurium U288 on pork products [23].

#### **4. Commercial phage products**

A handful of phage products that target *Salmonella* in pre- and post- slaughter stages of the food chain are commercially available and summarized in **Table 1**.


#### **Table 1.**

*Patented and approved* Salmonella *phage products.*

Some products (SalmoFresh® and SalmoPro®) have already obtained clearance from specific regulatory agencies, such as FDA, and are available to purchase, while others are patented but not approved by any regulatory authority, at the time of writing. However relevant scientific data about the product has been published, such as for SalmoFree® [20].

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