Sandra Sevilla-Navarro

Introduction: Enterococcus faecalis is an opportunistic pathogen of One Health relevance and an important reservoir of antimicrobial resistance in poultry production systems. In this study, we generated a large-scale quantitative phage-host interaction dataset to support the rational design of phage cocktails targeting multidrug-resistant poultry-associated E. faecalis . Methods: Thirty-eight lytic phages were isolated from poultry farm samples, genomically characterized, and evaluated against 51 clinical poultry isolates using efficiency of plating as a quantitative measure of productive infectivity. Results: The complete phage collection achieved 94.12% cumulative strain coverage, while genomic relatedness showed only a moderate association with infectivity profiles, highlighting the need for functional screening. Computational optimization identified a six-phage cocktail, CKT6, which displayed broad in vitro activity against diverse strain backgrounds, reaching 92.16% coverage at MOI 1. Importantly, cocktail performance was not fully predictable from individual phage activity, as host-dependent synergistic and antagonistic effects were observed. Discussion: These findings support quantitative phage-host interaction profiling as a robust framework for reproducible and adaptive phage-cocktail design. Further validation under biofilm-associated, formulation-relevant and field-like poultry-production conditions will be required before practical application.

Salmonella is responsible for millions of foodborne illnesses worldwide. The emergence of antibiotic-resistant Salmonella strains necessitates the development of alternatives for controlling this microorganism in the food supply chain. In Ecuador, Salmonella Infantis (S. Infantis) is the most frequently isolated serovar in poultry farms, poultry food products, and human infections. The objective of this study was to isolate and characterize lytic bacteriophages against a S. Infantis strain from poultry products in Ecuador to evaluate their potential for biocontrol. Three bacteriophages, GS71, GS156, and GS166, were isolated from chicken feces samples and showed short latent times (5–10 min), burst sizes of 205–231 PFU/cell, and stability up to 50 °C and pH = 10. Despite being isolated at different times and locations, they exhibited high genomic similarity (91.9–98.7%), reflecting the low diversity of Ecuadorian S. Infantis strains. VIRIDIC and phylogenetic analyses placed them within the Tlsvirus genus, showing conserved gene modules for replication, morphogenesis, and lysis. Putative endolysin and depolymerase genes were identified, supporting their anti-biofilm activity against biofilm-forming bacteria. Host range assays showed GS71 and GS166 lysed most S. Infantis field strains, whereas GS156 had a narrower spectrum linked to a unique polynucleotide kinase insertion. TEM confirmed Siphovirus-like morphology with icosahedral capsids (~55 nm) and long non-contractile tails. No genes associated with lysogeny, virulence, or antibiotic resistance were found. These findings support GS71, GS156, and GS166 as safe and effective candidates for bacteriophage cocktails targeting multidrug-resistant S. Infantis in poultry production.

Background/Objectives: As bacteriophage-based strategies to control bacterial pathogens continue to gain momentum, phage therapy is increasingly being explored across various fields. In the poultry industry, efforts to minimize the public health impact of Salmonella have spurred growing interest in phage applications, particularly as prophylactic and disinfecting agents. Although the disinfecting potential of bacteriophages has been recognized, in-depth studies examining their efficacy under varying environmental conditions remain limited. This study focused on evaluating the effectiveness of bacteriophages as disinfecting agents against biofilm-forming Salmonella Infantis under different environments. Methods: A comprehensive screening of biofilm-producing strains was conducted using Congo Red Agar and 96-well plate assays. Two strains with distinct biofilm-forming capacities were selected for further analysis under different environmental conditions: aerobic and microaerobic atmospheres at both 25 °C and 37 °C. The resulting biofilms were then treated with four phage preparations: three individual phages and one phage cocktail. Biofilm reduction was assessed by measuring optical density and CFU/well. Additionally, scanning electron microscopy was used to visualize both untreated and phage-treated biofilms. Results: The results demonstrated that all S. Infantis strains were capable of forming biofilms (21/21). All three phage candidates exhibited biofilm-disrupting activity and were able to lyse biofilm-embedded Salmonella cells. Notably, the lytic efficacy of the phages varied depending on environmental conditions, highlighting the importance of thorough phage characterization prior to application. Conclusions: These findings underscore that the effectiveness of bacteriophages as surface disinfectants can be significantly compromised if inappropriate phages are used, especially in the presence of biofilms.

Despite meticulous monitoring of Salmonella spp. throughout the food chain to ensure safer animal food products for consumers, the number of salmonellosis cases in humans continues to rise annually in Europe. Phage therapy emerges as a promising tool for controlling and eradicating Salmonella in primary production. This study aimed to fully characterize new phage therapy candidates isolated from animal sources. To achieve this, a phenotypic and genetic characterization of five phage isolates was conducted. The five phages demonstrated physical stability across a wide range of temperatures and pH levels, effectively lysing 12 different Salmonella serovars, including the most prevalent ones in the European Union in recent years, as well as multidrug-resistant strains isolated from the field. Additionally, four of the phages exhibited depolymerase production in the host range, with genomic analysis confirming that all five possessed sequences encoding for this activity, suggesting their potential as surface-disinfecting agents. Genetic analysis further revealed that the phages belong to distinct genera: Felixounavirus, Cornellvirus, Skatevirus, Agtevirus and Berlinvirus. Notably, none of the phages contained harmful sequences that could compromise their future application, such as virulence factors, antibiotic resistance genes or temperate markers. Overall, these five phages show promise as suitable candidates for phage therapy applications or phage-based Salmonella eradication strategies, where their integration in the existing biocontrol measures may enhance both food safety and public health.

Background/Objectives: Antimicrobial resistance (AMR) in Enterococcus species from poultry production represents a significant public health threat due to the potential transmission of AMR through the food chain. This study aimed to examine the relative prevalence, resistance patterns, and mannitol fermentation capacity of Enterococcus isolates from various poultry production systems in Spain over a seven-year period (2017–2023). Methods: A total of 215 Enterococcus isolates were analyzed. Phenotypic assessments were conducted to determine resistance rates and metabolic capacities, while genotypic characterization focused on detecting vancomycin-resistance genes (vanA, vanB, vanC, and vanD). Results: Enterococcus faecalis (62.3%) and Enterococcus faecium (29.77%) were the predominant species, primarily isolated from broilers (74.88%), with the highest frequency observed in one-week-old chicks (31.16%). High resistance rates to tetracyclines and streptogramins were identified, while resistance to vancomycin (0.47%) and tigecycline (3.03%) was low. Interestingly, a significant reduction in tetracyclines resistance was shown in this period for Enterococcus faecalis (from 100% to 70% (2017–2023) and Enterococcus faecium (from 100% to 40% (2018–2023)). Multidrug resistance (MDR) was detected in 26.98% of isolates. Mannitol fermentation tests revealed high metabolic capacity in Enterococcus faecalis (99.25%) and Enterococcus faecium (96.88%), associated with adaptability and virulence potential. Genotypic analysis showed a very low prevalence of vanB and vanC genes. Conclusions: These findings highlight the critical need for targeted surveillance and intervention strategies in poultry production to mitigate the risks posed by MDR Enterococcus to public health.

Salmonella enterica serovar Infantis (S. Infantis) poses a growing issue in the poultry sector, with phage-based products emerging as a safe and effective control measure. This study investigated the emergence of reduced-phage-susceptibility variants (RPSV) of S. Infantis in PhagoVet-treated broilers, given that RPSV could undermine phage treatment efficacy. The bacteriophages in the PhagoVet product were characterized using transmission electron microscopy (TEM), genome sequencing, and infection profiling. Furthermore, two broiler trials were conducted: a challenge group (T1) and a challenge-and-treated group (T2). The S. Infantis infective dose was set at 10⁴ and 10⁶ colony-forming units (CFUs) per animal, with PhagoVet administration at 10⁶ and 10⁸ plaque-forming units (PFUs) per animal, in Trials 1 and 2, respectively. The results revealed that the four PhagoVet bacteriophages belonged to different genera. PhagoVet evidenced broad-spectrum efficacy against 271 strains representing 18 Salmonella serovars. In Trial 1, PhagoVet reduced bacterial counts in feces to nearly undetectable levels by day 42, with no RPSV detected. However, in Trial 2, three and five RPSVs were detected in feces and ceca, respectively. Consequently, PhagoVet demonstrated efficacy against S. Infantis in broilers, and the potential impact of RPSV is deemed unlikely to compromise its efficacy.

Introduction: Salmonella is a bacterium that can cause food-borne infections and is responsible for the most common gastrointestinal illnesses. The emergence of multi-drug resistant (MDR) strains worldwide is a major threat, representing a major challenge in public health. To reduce its incidence, the One Health approach is required, and the development of new biocontrol protocols will help prevent or eliminate the spread of Salmonella. Prevention measures, such as on-farm cleaning and disinfection protocols, are a crucial step in reducing infection to new flocks and eliminating bacteria that remain in the facilities. However, MDR Salmonella species, such as S. Infantis, are highly resistant to conventional cleaning and disinfection protocols, with an increased ability to persist in the broiler farm environment. The need for alternative biocontrol methods has led to the use of bacteriophages or phages, viruses that target bacteria, as promising tools. Thus, the aim of this study was to evaluate the efficacy of phages as a biocide against S. Infantis isolates in combination with cleaning and disinfection protocols in 10 commercial poultry farms.Methods: All commercial farms selected in this study had persistent Salmonella, even after the routinely used cleaning and disinfection procedures. In addition, Salmonella isolated before treatment were phenotypically characterized by antimicrobial resistance patterns.Results: The results showed that 100% of S. Infantis were resistant to at least one antibiotic, and > 70% were MDR. Phages were then isolated against the in-farm bacteria, purified, and multiplied for each poultry farm. The cleaning and disinfection protocols included the application of the lytic phages (vB_Si_CECAV_FGS009; vB_Si_CECAV_FGS017; vB_Si_CECAV_FGS029 and vB_Si_CECAV _FGS030) twice at 24-h intervals between cleaning and disinfection. Following the cleaning and disinfection procedures, Salmonella detection was reduced from 100% after cleaning to 36% after applying the phages and dropped to 0% after the final step of disinfection, thus eliminating Salmonella from the farm facilities.Discussion: This study demonstrates that bacteriophage application after cleaning and before disinfection enhances the removal of MDR Salmonella Infantis in commercial broiler farms, suggesting their use as biocontrol agents to reduce Salmonella, a major public health concern.