1. Investigación

Salmonella has been recognized as one of the most important zoonotic pathogens worldwide, being poultry derived products the main source of human infection. Among the most promising tools for Salmonella control at the field level in poultry production are included bacteriophages (or phages). However, little is known about the phage application impact on the rest of the gut microbiota. In this sense, new studies suggest that phage application may affects the gastrointestinal ecology homeostasis. Therefore, the general objective of this doctoral thesis was to apply bacteriophages for Salmonella control in broiler production, focusing on their effect on intestinal health, by means of genomic sequencing and metabolomic study. To achieve this goal, two different parts were performed. The first part studied the phage gastrointestinal dynamics in Salmonella-free broilers and its influence on microbiota and metabolome, meanwhile the second part studied the phage dynamics in Salmonella-infected broilers and its influence on microbiota and metabolome. The results aim to provide important insights into the use of phages as a preventative and biocontrol strategy against Salmonella infection from farm-to-fork.

Salmonellosis remains one of the main foodborne zoonoses in Europe, with poultry products as the main source of human infections. The slaughterhouse has been identified as a potential source for Salmonella contamination of poultry meat. Despite the mandatory programme of the EU, there are companies with persistent Salmonella that are unable to remove the bacteria from their processing environment, compromising the entire production line. In this context, an intensive sampling study was conducted to investigate a slaughterhouse with persistent Salmonella problems, establishing the genetic relationship among Salmonella strains isolated during the slaughter process. A total of 36 broiler flocks were sampled during processing at the slaughterhouse. Salmonella was identified based on ISO 6579-1:2017 (Annex D), serotyped by Kauffman-White-Le-Minor technique, and the genetic relationship was assessed with ERIC-PCR followed by PFGE. The outcomes showed that 69.4% of the batches sampled carried Salmonella upon arrival at the slaughterhouse and that 46.3% of the different samples from carcasses were contaminated with Salmonella. The two serovars isolated at the different steps in the slaughterhouse were Enteritidis (98.2%) and Kentucky (1.8%). Pulsed-field gel electrophoresis analysis revealed a low genetic diversity, with all S. Enteritidis isolates showing a nearly identical pulsotype (similarity >85%) and S. Kentucky strains showed the same XbaI PFGE profile (95.0% genetic similarity). The results of this study showed a high genetic relationship among isolates recovered from carcasses and environmental samples in the slaughterhouse from both Salmonella-positive and Salmonella- free flocks. Salmonella strains re-circulated across to poultry flocks and re-entered the slaughterhouse to survive on the processing line. Thus, it is necessary to implement molecular diagnosis methods in time at the field level to determine the Salmonella epidemiology of the flock, to make rapid decisions for the control of Salmonella and prevent entry into the slaughterhouse environment.

Bacteriophages selectively infect and kill their target bacterial host, being a promising approach to controlling zoonotic bacteria in poultry production. To ensure confidence in its use, fundamental questions of safety and toxicity monitoring of phage therapy should be raised. Due to its high specificity, a minimal impact on the gut ecology is expected; however, more in-depth research into key parameters that influence the success of phage interventions has been needed to reach a consensus on the impact of bacteriophage therapy in the gut. In this context, this study aimed to investigate the interaction of phages with animals; more specifically, we compared the caecum microbiome and metabolome after a Salmonella phage challenge in Salmonella-free broilers, evaluating the role of the phage administration route. To this end, we employed 45 caecum content samples from a previous study where Salmonella phages were administered via drinking water or feed for 24 h from 4, 5 to 6-weeks-old broilers. High-throughput 16S rRNA gene sequencing showed a high level of similarity (beta diversity) but revealed a significant change in alpha diversity between broilers with Salmonella-phage administered in the drinking water and control. Our results showed that the phages affected only a few genera of the microbiota’s structure, regardless of the administration route. Among these, we found a significant increase in Streptococcus and Sellimonas in the drinking water and Lactobacillus, Anaeroplasma and Clostridia_vadinBB60_group in the feed. Nevertheless, the LC-HRMS-based metabolomics analyses revealed that despite few genera were significantly affected, a substantial number of metabolites, especially in the phage administered in the drinking water were significantly altered (64 and 14 in the drinking water and feed groups, respectively). Overall, our study shows that preventive therapy with bacteriophages minimally alters the caecal microbiota but significantly impacts their metabolites, regardless of the route of administration.

Bacteriophage therapy is being considered as a promising tool to control Salmonella in poultry. Nevertheless, changes in gastrointestinal tract environmental conditions throughout the production cycle could compromise the efficacy of phages administered orally. The main objectives of this study were to assess the optimal timing of the phage administration over a 42-day production cycle and to compare microencapsulated and non-encapsulated phages and the spatial and temporal dynamics of the phage delivery along the gastrointestinal tract. Phage FGS011 was encapsulated in the pH-responsive polymer Eudragit® L100 using the process of spray drying. At different weeks of the chicken rearing period, 15 broilers were divided into three groups. Over a period of 24 h, group 1 received non-encapsulated phages (delivered through drinking water), group 2 received microencapsulated phages (incorporated in animal feed), and group 3 did not receive any phages. Microencapsulation was shown to enable efficient delivery of the bacteriophages to the animal gut and cecum throughout the animal rearing period. During the six weeks of application, the crop displayed the highest phage concentration for both phage delivery methods. The L100 based encapsulation offered significant protection to the phages from the harsh environmental conditions in the PV-Gizzard (not seen with phages administered in drinking water) which may help in the delivery of high phage doses to the cecum. Future Salmonella challenge studies are necessary to demonstrate the benefits of microencapsulation of phages using L100 formulation on phage therapy in field studies during the rearing period.