Javier Feito

Background/Objectives: Antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional antibiotics in livestock, offering a sustainable strategy for controlling bacterial pathogens in food production systems. In addition to their direct antimicrobial effects, AMPs play a key role in modulating host-associated microbiomes, influencing both microbial composition and function. Advances in metagenomic sequencing and bioinformatic tools now enable comprehensive exploration of AMP diversity and activity within complex microbial ecosystems. Methods: In this study, we employed Illumina-based next-generation sequencing (NGS) to analyze intestinal contents from six gut sections of broiler chickens obtained from a Spanish slaughterhouse. Results: Through de novo assembly and bioinformatic annotation, we identified biosynthetic gene clusters (BGCs) encoding ribosomally synthesized and post-translationally modified peptides (RiPPs), other specialized bioactive secondary metabolites, antimicrobial resistance genes (ARGs), virulence factor genes (VFGs), and a diverse microbial community. Among all gut sections, the cecum exhibited the highest genetic richness, characterized by a high diversity of RiPP-like clusters and antimicrobial resistance determinants. Conclusions: These findings highlight the poultry gut, particularly the cecum, as a significant reservoir of antimicrobial peptides (AMPs) with potential implications in antibiotic-free poultry production and enhanced food safety.

Background/Objectives: The overuse and misuse of antibiotics has contributed significatively to the growing problem of the emergence and spread of antibiotic resistance genes among bacteria, posing a serious global challenge to the treatment of bacterial infectious diseases. For these reasons, there is a current and growing interest in the development of effective alternative or complementary strategies to antibiotic therapy for the prevention of fish diseases, which are mainly based on the use of probiotics—in particular, those belonging to the Lactic Acid Bacteria (LAB) group. In this context, the aim of the present study was to characterise, evaluate the genetic diversity and assess the safety of candidate probiotic LAB strains for aquaculture isolated from faeces and intestines of European hakes (Merluccius merluccius, L.) caught in the Northeast Atlantic Ocean (Ireland). Methods: The direct antimicrobial activity of the LAB isolates was tested by the Stab-On-Agar method against key ichthyopathogens. Subsequently, their taxonomic classification and genetic diversity were determined by 16SrDNA sequencing and Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), respectively. To ensure the in vitro safety of the LAB isolates, their biofilm-forming ability was assessed by a microtiter plate assay; their sensitivity to major antibiotics used in aquaculture, human and veterinary medicine by a broth microdilution method and their haemolytic and gelatinase activity by microbiological assays. Results: All LAB isolates were biofilm producers and susceptible to chloramphenicol, oxytetracycline, flumequine and amoxicillin. A total of 30 isolates (85.7%) were resistant to at least one of the tested antibiotics. None of the 35 LAB isolates showed haemolytic or proteolytic activity. Conclusions: Among the isolated strains, five LAB strains exhibiting the highest antimicrobial activity against aquaculture-relevant ichthyopathogens, taxonomically identified as Streptococcus salivarius, Enterococcus avium and Latilactobacillus sakei, were selected for further characterisation as potential probiotic candidates to promote sustainable aquaculture. To our knowledge, this is the first study to report that hake intestines and faeces represent viable ecological niches for the isolation of LAB strains with antimicrobial activity.

Background/Objectives: Antimicrobial-resistant (AMR) pathogens represent a serious threat to public health, particularly in food production systems where antibiotic use remains widespread. As a result, alternative antimicrobial treatments to antibiotics are essential for effectively managing bacterial infections. This study aimed to identify and characterize novel antimicrobial peptides produced by bacteria, known as bacteriocins, as well as to recognize safe bacteriocin-producing strains, sourced from poultry slaughterhouse effluents. Methods: A total of 864 bacterial isolates were collected across eight stages of a poultry slaughter line and screened for antimicrobial activity against Gram-positive and Gram-negative indicator strains. Whole-genome sequencing (WGS) was performed on 12 selected strains, including Enterococcus faecium (6 isolates), Lactococcus lactis (1 isolate), Lactococcus garvieae (1 isolate) and Escherichia coli (4 isolates). The presence of bacteriocin gene clusters (BGC), antibiotic resistance genes (ARG), and virulence factors (VF) was analyzed. The antimicrobial activity of a novel bacteriocin was further evaluated using in vitro cell-free protein synthesis (IV-CFPS). Results: WGS revealed multiple BGCs, including a novel class IId bacteriocin, lactococcin P1A (LcnP1A), in L. lactis SWD9. LcnP1A showed antimicrobial activity against various indicator strains, including Listeria monocytogenes. While most bacteriocin-encoding strains harbored ARGs and VFs, E. faecium SWG6 was notable for its absence of ARGs and minimal VFs, highlighting its potential as a probiotic. Conclusions: These findings underscore the importance of discovering novel bacteriocins and safer bacteriocin producing strains to address antimicrobial resistance in the food chain. Further research would validate the efficacy of both the novel lactococcin P1A bacteriocin and the E. faecium SWG6 isolate for application in processed food and animal production systems.

Frequently, diseases in aquaculture have been fought indiscriminately with the use of antibiotics, which has led to the development and dissemination of (multiple) antibiotic resistances in bacteria. Consequently, it is necessary to look for alternative and complementary approaches to chemotheraphy that are safe for humans, animals, and the environment, such as the use of probiotics in fish farming. The objective of this work was the Whole-Genome Sequencing (WGS) and bioinformatic and functional analyses of S. salivarius MDI13 and L. sakei MEI5, two LAB strains isolated from the gut of commercial European hakes (M. merluccius, L.) caught in the Northeast Atlantic Ocean. The WGS and bioinformatic and functional analyses confirmed the lack of transferable antibiotic resistance genes, the lack of virulence and pathogenicity issues, and their potentially probiotic characteristics. Specifically, genes involved in adhesion and aggregation, vitamin biosynthesis, and amino acid metabolism were detected in both strains. In addition, genes related to lactic acid production, active metabolism, and/or adaptation to stress and adverse conditions in the host gastrointestinal tract were detected in L. sakei MEI5. Moreover, a gene cluster encoding three bacteriocins (SlvV, BlpK, and BlpE) was identified in the genome of S. salivarius MDI13. The in vitro-synthesized bacteriocin BlpK showed antimicrobial activity against the ichthyopathogens Lc. garvieae and S. parauberis. Altogether, our results suggest that S. salivarius MDI13 and L. sakei MEI5 have a strong potential as probiotics to prevent fish diseases in aquaculture as an appropriate alternative/complementary strategy to the use of antibiotics.

Aquaculture is a rapidly expanding agri-food industry that faces substantial economic losses due to infectious disease outbreaks, such as bacterial infections. These outbreaks cause disruptions and high mortalities at various stages of the rearing process, especially in the larval stages. Probiotic bacteria are emerging as promising and sustainable alternative or complementary strategies to vaccination and the use of antibiotics in aquaculture. In this study, potential probiotic candidates for larviculture were isolated from a rotifer-rearing tank used as the first live feed for turbot larvae. Two Lacticaseibacillus paracasei and two Lactiplantibacillus plantarum isolates were selected for further characterization due to their wide and strong antimicrobial activity against several ichthyopathogens, both Gram-positive and Gram-negative. An extensive in vitro safety assessment of these four isolates revealed the absence of harmful traits, such as acquired antimicrobial resistance and other virulence factors (i.e., hemolytic and gelatinase activities, bile salt deconjugation, and mucin degradation, as well as PCR detection of biogenic amine production). Moreover, Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) analyses unveiled their genetic relatedness, revealing two divergent clusters within each species. To our knowledge, this work reports for the first time the isolation and characterization of Lactic Acid Bacteria (LAB) with potential use as probiotics in aquaculture from rotifer-rearing tanks, which have the potential to optimize turbot larviculture and to introduce novel microbial management approaches for a sustainable aquaculture.

Aquaculture plays a crucial role in meeting the increasing global demand for food and protein sources. However, its expansion is followed by increasing challenges, such as infectious disease outbreaks and antibiotic misuse. The present study focuses on the genetic and functional analyses of two Lacticaseibacillus paracasei (BF3 and RT4) and two Lactiplantibacillus plantarum (BF12 and WT12) strains isolated from a rotifer cultivation tank used for turbot larviculture. Whole-genome sequencing (WGS) and bioinformatics analyses confirmed their probiotic potential, the absence of transferable antibiotic resistance genes, and the absence of virulence and pathogenicity factors. Bacteriocin mining identified a gene cluster encoding six plantaricins, suggesting their role in the antimicrobial activity exerted by these strains. In vitro cell-free protein synthesis (IV-CFPS) analyses was used to evaluate the expression of the plantaricin genes. The in vitro-synthesized class IIb (two-peptide bacteriocins) plantaricin E/F (PlnE/F) exerted antimicrobial activity against three indicator microorganisms, including the well-known ichthyopathogen Lactococcus garvieae. Furthermore, MALDI-TOF MS on colonies detected the presence of a major peptide that matches the dimeric form of plantaricins E (PlnE) and F (PlnF). This study emphasizes the importance of genome sequencing and bioinformatic analysis for evaluating aquaculture probiotic candidates. Moreover, it provides valuable insights into their genetic features and antimicrobial mechanisms, paving the way for their application as probiotics in larviculture, which is a major bottleneck in aquaculture.

The indiscriminate use of antibiotics has contributed to the dissemination of multiresistant bacteria, which represents a public health concern. The aim of this work was to characterize 27 coagulase-negative staphylococci (CoNS) isolated from eight wild Northeast Atlantic hakes (Merluccius merluccius, L.) and taxonomically identified as Staphylococcus epidermidis (n = 16), Staphylococcus saprophyticus (n = 4), Staphylococcus hominis (n = 3), Staphylococcus pasteuri (n = 2), Staphylococcus edaphicus (n = 1), and Staphylococcus capitis (n = 1). Biofilm formation was evaluated with a microtiter assay, antibiotic susceptibility testing was performed using the disk diffusion method, and antibiotic resistance and virulence determinants were detected by PCR. Our results showed that all staphylococci produced biofilms and that 92.6% of the isolates were resistant to at least one antibiotic, mainly penicillin (88.8%), fusidic acid (40.7%), and erythromycin (37%). The penicillin resistance gene (blaZ) was detected in 66.6% (18) of the isolates, of which 10 also carried resistance genes to macrolides and lincosamides (mphC, msr(A/B), lnuA, or vgaA), 4 to fusidic acid (fusB), and 3 to trimethoprim-sulfamethoxazole (dfrA). At least one virulence gene (scn, hla, SCCmecIII, and/or SCCmecV) was detected in 48% of the isolates. This study suggests that wild European hake destined for human consumption could act as a vector of CoNS carrying antibiotic resistance genes and/or virulence factors.

Lactococcus garvieae is a main ichthyopathogen in rainbow trout (Oncorhynchus mykiss, Walbaum) farming, although bacteriocinogenic L. garvieae with antimicrobial activity against virulent strains of this species have also been identified. Some of the bacteriocins characterized, such as garvicin A (GarA) and garvicin Q (GarQ), may show potential for the control of the virulent L. garvieae in food, feed and other biotechnological applications. In this study, we report on the design of Lactococcus lactis strains that produce the bacteriocins GarA and/or GarQ, either alone or together with nisin A (NisA) or nisin Z (NisZ). Synthetic genes encoding the signal peptide of the lactococcal protein Usp45 (SP_usp45), fused to mature GarA (lgnA) and/or mature GarQ (garQ) and their associated immunity genes (lgnI and garI, respectively), were cloned into the protein expression vectors pMG36c, which contains the P₃₂ constitutive promoter, and pNZ8048c, which contains the inducible P_nisA promoter. The transformation of recombinant vectors into lactococcal cells allowed for the production of GarA and/or GarQ by L. lactis subsp. cremoris NZ9000 and their co-production with NisA by Lactococcus lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24. The strains L. lactis subsp. cremoris WA2-67 (pJFQI), a producer of GarQ and NisZ, and L. lactis subsp. cremoris WA2-67 (pJFQIAI), a producer of GarA, GarQ and NisZ, demonstrated the highest antimicrobial activity (5.1- to 10.7-fold and 17.3- to 68.2-fold, respectively) against virulent L. garvieae strains.

Probiotics are a viable alternative to traditional chemotherapy agents to control infectious diseases in aquaculture. In this regard, Lactococcus lactis subsp. cremoris WA2-67 has previously demonstrated several probiotic features, such as a strong antimicrobial activity against ichthyopathogens, survival in freshwater, resistance to fish bile and low pH, and hydrophobicity. The aim of this manuscript is an in silico analysis of the whole-genome sequence (WGS) of this strain to gain deeper insights into its probiotic properties and their genetic basis. Genomic DNA was purified, and libraries prepared for Illumina sequencing. After trimming and assembly, resulting contigs were subjected to bioinformatic analyses. The draft genome of L. cremoris WA2-67 consists of 30 contigs (2,573,139 bp), and a total number of 2493 coding DNA sequences (CDSs). Via in silico analysis, the bacteriocinogenic genetic clusters encoding the lantibiotic nisin Z (NisZ) and two new bacteriocins were identified, in addition to several probiotic traits, such as the production of vitamins, amino acids, adhesion/aggregation, and stress resistance factors, as well as the absence of transferable antibiotic resistance determinants and genes encoding detrimental enzymatic activities and virulence factors. These results unveil diverse beneficial properties that support the use of L. cremoris WA2-67 as a probiotic for aquaculture.

Weissella cibaria P71 is a lactic acid bacterium that was isolated from common octopus ( Octopus vulgaris ) and previously showed interesting probiotic properties for turbot ( Scophthalmus maximus L.) farming. The draft genome sequence of this strain provides further data to support its potential as a probiotic for aquaculture.