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.

Background: Salmonellosis is one of the most important food-borne zoonotic disease affecting both animals and humans. The objective of the present study was to identify gastrointestinal (GI) lactic acid bacteria (LAB) of canineorigin from Salmonella-negative dogs’ faeces able to inhibit monophasic Salmonella Typhimurium previously isolated from dogs’ faeces, in order to be used as a potential probiotic in pet nutrition. Results: Accordingly, 37 LAB were isolated from Salmonella-negative dogs’ faeces and tested against monophasic S. Typhimurium using the spot on lawn method out of which 7 strains showed an inhibition halo higher than 2.5 cm. These 7 strains were also tested with the co-culture method and one showed the greatest inhibition value (p < 0.05). Subsequently, the isolate was identified through 16S rRNA sequencing and sequence homology and designated as Ligilactobacillus salivarius (L. salivarius). LAB from Salmonella-positive dogs were also identified and none was the selected strain. Finally, to identify the mechanism of inhibition of L. salivarius, the supernatant was analyzed, and a dose response effect was observed. Conclusions: It is concluded that the canine-origin L. salivarius, could possess some in vitro functional attributes of a candidate probiotic and could prevent monophasic S. Typhimurium colonization or inhibit its activity if the infection occurs.

Salmonella spp. is one of the most important zoonotic pathogens with economic impact in public health worldwide. The relevance of Salmonella increases with the appearance of resistant strains. The aim of this study was to determine the level of antimicrobial resistance in 332 Salmonella isolates selected from 3 different poultry productive orientations in Eastern Spain during 3 yr (2015−2017). Antimicrobial susceptibly was evaluated by broth microdilution method using 14 antibiotics. Epidemiological cut-off values (ECOFF) were used to evaluate the microbiological resistance to antibiotics. The rates of Salmonella resistance at least to one antibiotic were 96, 98, and 56% in broilers, turkeys, and layers, respectively. Regarding multidrug resistance, all productive orientations seems to present a decreasing trend along the study, being the mean rates 80% in turkeys followed by broilers (40%) and layers (6%). Throughout the study, the highest percentage of resistance was found to sulfamethoxazole in all productive orientations. Strains from broilers showed the highest resistance rates to sulfamethoxazole (73%), gentamicin (57%), ciprofloxacin (50%), nalidixic acid (29%), and tetracycline (24%). Relative to turkeys the highest resistance rates were to sulfamethoxazole (76%), ciprofloxacin (69%), tetracycline (75%), nalidixic acid (63%), and ampicillin (63%). Layers presented the most elevated resistance rates to sulfamethoxazole (39%) and tetracycline (13%). Regarding serovars the most MDR common serovars to the 3 productive orientations were S. Kentucky and S. Hadar. In the other hand, high MDR rates were found in other serovars like S. Infantis and S. Typhimurium in broilers and turkeys. Results shown in the present study suggest that the reduction in the use of antibiotics begins to be reflected in the reduction of the number of MDRs, especially in layers, with no MDR Salmonella strains in the last period. However, the level of resistances found in this study suggests the necessity of continuing working on the limitation of the use of antimicrobials in poultry to achieve (as in layers) the control of MDRs.

Stray cat populations can represent a significant threat of the transmission of zoonotic diseases such as salmonellosis. The objective of this study was to assess Salmonella carriage by free-living cats in Gran Canaria island and the Salmonella serovars involved, in order to inform to those responsible for the colonies about the possible risk factors. One hundred rectal swabs of feral cats were taken. Salmonella strains were serotyped in accordance with Kauffman-White-Le-Minor technique. Of a total of 100 animals under study, 19% were found to be positive to Salmonella spp. This is the first report that described the zoonotic serovars S. Nima, S. Bredeney, S. Grancanaria and S. Kottbus in cats. The present study demonstrates that feral cats may represent a source of risk for the spread of different Salmonella zoonotic serovars. It has been reported that there is a certain correlation between Salmonella isolates from pets and wild animals. Further studies are needed from other animal species and environmental sources to make this correlation.

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.

Different studies have reported the prevalence and antibiotic resistance of Salmonella in dromedaries’ camels and its role in camelid-associated salmonellosis in humans, but little is known about the epidemiology of Campylobacter in dromedaries. Here we investigate the prevalence, genetic diversity and antibiotic resistance of Campylobacter and Salmonella in dromedary camels (Camelus dromedarius). A total of 54 individuals were sampled from two unique dromedary farms located in Tenerife (Canary Islands, Spain). Whilst all the samples were Campylobacter-negative, Salmonella prevalence was 5.5% (3/54) and the only serovar isolated was S. Frintrop. The pulsed field gel electrophoresis analysis revealed a low genetic diversity, with all isolates showing a nearly identical pulsotype (similarity > 95%). Our results indicate that dromedaries’ camels could not be a risk factor for Campylobacter human infection, but seems to be a reservoir for Salmonella transmission. Since camel ride has become one of the main touristic attractions in several countries and its popularity has considerably risen in the last years, a mandatory control, especially for zoonotic pathogens, such as Campylobacter and Salmonella should be implemented.

Pork is considered a major source of Salmonella Typhimurium infection in humans in the EU, including monophasic strains. Widespread distribution of virulent serotypes such as monophasic variants of S. Typhimurium have emerged as a public health threat. Despite the current situation, within the EU there is no mandatory programme for the control of Salmonella at pork production level. In this context, the aims of this study were: to examine the presence of Salmonella in the swine production system from arrival at the slaughterhouse until the end of processing, and investigate the genetic relationship among the Salmonella serovars isolated. During the study, a total of 21 pig herds were intensively sampled during processing at the slaughterhouse. ERIC-PCR was performed among isolates recovered at the different steps in the slaughterhouse to assess the genetic relationship. Then, PFGE was done to study the pulsotypes among the different Salmonella serovars isolated. The results showed a high level of Salmonella pork batch contamination upon arrival at the slaughterhouse (71.4%) and at the end of the slaughtering process (66.7%), with mST the main serovar isolated from both origins (53.1% and 38.2%, respectively). The slaughter environment poses a potential risk for carcass contamination and it is considered an important source of Salmonella spp. Similarly, this study shows that 14.3% of the strains isolated from carcasses have the same Xbal-PFGE profile as those previously recovered in the slaughterhouse environment, but not in the live animals from that same batch. In conclusion, there is a high level of Salmonella swine batch contamination upon arrival at the slaughterhouse and at the end of the slaughtering process, mST being the most frequently isolated serovar. Moreover, a strong genetic relationship has been observed between Salmonella strains isolated from the batch on arrival at the slaughterhouse, the processing environment and pork carcass contamination. In this sense, it would be necessary to implement a control programme to reduce the bacterium from pork farms and raise the awareness of biosecurity measures.