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This review delves into the increasing interest in probiotics and paraprobiotics as a viable alternative to antibiotics in aquaculture, highlighting their potential to enhance fish health and prevent diseases. As the aquaculture industry continues its global expansion, addressing the challenges associated with disease outbreaks in high-density fish populations becomes imperative. The review underscores the promising role of probiotics and paraprobiotics as a sustainable strategy to mitigate these challenges. The diverse positive impacts of various probiotic strains such as Arthrobacter , Bacillus , Lactobacillus , Bifidobacterium , Clostridium , and others emphasize their roles in enhancing growth, resistance to diseases (including bacterial, viral, and parasitic infections), stress reduction, water quality management, and environmental sustainability. Challenges such as stability, host specificity, and regulatory considerations must be addressed to optimize the use of probiotics in aquaculture. Additionally, paraprobiotics, or non-viable microbial cells, present a safer alternative to the criticized antibiotics and even live probiotics in environments where microbial viability poses a risk. These inactivated cells retain the ability to modulate the immune system and improve gut health, offering a promising complementary approach to fish disease prevention. The review advocates for a systematic approach combining research, innovation, and collaboration to effectively integrate probiotics and paraprobiotics into fish farming practices. Furthermore, the mechanisms by which probiotics and paraprobiotics modulate gut microbiota, produce antimicrobial compounds, and strengthen fish’s immune system have been elucidated. Moreover, the practical applications of probiotics in fish farming, including optimal administration methods and the challenges and limitations faced by the industry, have been discussed. Emphasis on the importance of continued research to explore new probiotic and paraprobiotic strains and develop innovative delivery systems to ensure the sustainability of aquaculture has been discussed. By enhancing fish health, reducing the need for antibiotics, and improving water quality, probiotics, and paraprobiotics contribute to more sustainable and environmentally responsible aquaculture operations.

This review provides a comprehensive overview of the current state of probiotic research, covering a wide range of topics, including strain identification, functional characterization, preclinical and clinical evaluations, mechanisms of action, therapeutic applications, manufacturing considerations, and future directions. The screening process for potential probiotics involves phenotypic and genomic analysis to identify strains with health-promoting properties while excluding those with any factor that could be harmful to the host. In vitro assays for evaluating probiotic traits such as acid tolerance, bile metabolism, adhesion properties, and antimicrobial effects are described. The review highlights promising findings from in vivo studies on probiotic mitigation of inflammatory bowel diseases, chemotherapy-induced mucositis, dysbiosis, obesity, diabetes, and bone health, primarily through immunomodulation and modulation of the local microbiota in human and animal models. Clinical studies demonstrating beneficial modulation of metabolic diseases and human central nervous system function are also presented. Manufacturing processes significantly impact the growth, viability, and properties of probiotics, and the composition of the product matrix and supplementation with prebiotics or other strains can modify their effects. The lack of regulatory oversight raises concerns about the quality, safety, and labeling accuracy of commercial probiotics, particularly for vulnerable populations. Advancements in multi-omics approaches, especially probiogenomics, will provide a deeper understanding of the mechanisms behind probiotic functionality, allowing for personalized and targeted probiotic therapies. However, it is crucial to simultaneously focus on improving manufacturing practices, implementing quality control standards, and establishing regulatory oversight to ensure the safety and efficacy of probiotic products in the face of increasing therapeutic applications.

Pursuing new probiotic targets has surged, driven by next-generation sequencing, facilitating a thorough exploration of bacterial traits. The genus Geobacillus stands out as a promising candidate for probiotics. The study explored the genetic attributes of the genus Geobacillus for their resilience to gastrointestinal conditions, nutrient production, and immunomodulatory compound creation, revealing potential probiotic traits. Predictive analyses of genomic elements like prophages, CRISPR-Cas systems, insertion sequences, genomic islands, antibiotic resistance genes, and CAZymes were conducted to assess safety. Comparative genomic analysis was performed using 18 published Geobacillus genomes and a few Lactobacillus and Bifidobacterium genomes as controls. Genes associated with probiotic traits, such as adhesion, stress tolerance (acid/bile, osmotic, oxidative), immune modulation, and molecular chaperones, were uniformly detected in Geobacillus . Mobile genetic elements (such as plasmids, prophages, and insertion sequences), virulence factors, toxins, and antibiotic resistance genes were absent, while CRISPR-Cas systems and CAZymes were present. The pan-genome comprised 25,284 protein-coding genes. Comparative genomic analysis revealed an open pan-genome for Geobacillus . Pan-genome exhibited variability, particularly in genes linked to environmental interaction and secondary metabolite synthesis. Geobacillus appears potentially safe and well-suited for the gut habitat. However, further in vitro studies are essential to confirm its probiotic potential.

Background Probiotics have gained attention for their potential maintaining gut and immune homeostasis. They have been found to confer protection against pathogen colonization, possess immunomodulatory effects, enhance gut barrier functionality, and mitigate inflammation. However, a thorough understanding of the unique mechanisms of effects triggered by individual strains is necessary to optimize their therapeutic efficacy. Probiogenomics, involving high-throughput techniques, can help identify uncharacterized strains and aid in the rational selection of new probiotics. This study evaluates the potential of the Escherichia coli CEC15 strain as a probiotic through in silico , in vitro , and in vivo analyses, comparing it to the well-known probiotic reference E. coli Nissle 1917. Genomic analysis was conducted to identify traits with potential beneficial activity and to assess the safety of each strain (genomic islands, bacteriocin production, antibiotic resistance, production of proteins involved in host homeostasis, and proteins with adhesive properties). In vitro studies assessed survival in gastrointestinal simulated conditions and adhesion to cultured human intestinal cells. Safety was evaluated in BALB/c mice, monitoring the impact of E. coli consumption on clinical signs, intestinal architecture, intestinal permeability, and fecal microbiota. Additionally, the protective effects of both strains were assessed in a murine model of 5-FU-induced mucositis. Results CEC15 mitigates inflammation, reinforces intestinal barrier, and modulates intestinal microbiota. In silico analysis revealed fewer pathogenicity-related traits in CEC15, when compared to Nissle 1917, with fewer toxin-associated genes and no gene suggesting the production of colibactin (a genotoxic agent). Most predicted antibiotic-resistance genes were neither associated with actual resistance, nor with transposable elements. The genome of CEC15 strain encodes proteins related to stress tolerance and to adhesion, in line with its better survival during digestion and higher adhesion to intestinal cells, when compared to Nissle 1917. Moreover, CEC15 exhibited beneficial effects on mice and their intestinal microbiota, both in healthy animals and against 5FU-induced intestinal mucositis. Conclusions These findings suggest that the CEC15 strain holds promise as a probiotic, as it could modulate the intestinal microbiota, providing immunomodulatory and anti-inflammatory effects, and reinforcing the intestinal barrier. These findings may have implications for the treatment of gastrointestinal disorders, particularly some forms of diarrhea.

Probiogenomics can provide important insights bout probiotic candidate bacteria. This study aimed to perform an in-depth genomic characterization of the probiotic candidate Lactiplantibacillus plantarum CNPC024 to investigate its probiosis mechanisms, identify metabolic pathways that might benefit the host, and improve the safety assessment for this strain to be effectively used as a probiotic. After whole-genome sequencing in Illumina MiSeq platform, the de novo genome assembly resulted in a 3.2 Mb draft genome. According to the Average Nucleotide Identity (ANI) analysis with 46 randomly validated probiotic LAB belonging to the Lactobacillaceae family, the strain showed a 99% nucleotide identity with other L. plantarum probiotic species. We identified a set of determinants conferring tolerance to bile salts and low pH conditions, as well as temperature, oxidative and osmotic stressors via the glutathione-glutaredoxin system (Grxs). As a β‑galactosidase‑producing strain, it has the potential to be used in fermented dairy products for lactose-intolerant individuals. There were no significant hits for transferable antibiotic-resistance genes. We also identified gene clusters associated with production of bacteriocins (plantaricins E, F and K). Lastly, we detected metabolic pathways associated with the production of tryptophan-derived metabolites that could potentially modulate the host’s immune responses.

This study aimed to explore the probiogenomic characteristics of artisanal bacteriocin-producing Enterococcus faecium BGZLM1-5 and its potential application in reducing Listeria monocytogenes in a milk model. The BGZLM1-5 strain was isolated from raw cow’s milk from households in the Zlatar Mountain region. The whole genome sequencing approach and bioinformatics analyses reveal that the strain BGZLM1-5 is non-pathogenic to humans. Bacteriocin-containing supernatant was thermally stable and antimicrobial activity retained 75% of the initial activity compared with that of the control after treatment at 90 °C for 30 min. Antimicrobial activity maintained relative stability at pH 3–11 and retained 62.5% of the initial activity compared with that of the control after treatment at pH 1, 2, and 12. The highest activity of the partially purified bacteriocin was obtained after precipitation at 40% saturation with ammonium sulfate and further purification by mixing with chloroform. Applying 3% and 5% (v/v) of the bacteriocin-containing supernatant and 0.5% (v/v) of the partially purified bacteriocin decreased the viable number of L. monocytogenes ATCC19111 after three days of milk storage by 23.5%, 63.5%, and 58.9%, respectively.

This study aimed to investigate the probiogenomic features of artisanal bacteriocin-producing Enterococcus faecium BGPAS1-3 and the use of the improved pMALc5HisEk expression vector for overexpressing class II bacteriocins and the application of purified bacteriocin 31 in a milk model as a preservative against L. monocytogenes. The BGPAS1-3 strain was isolated from traditional fresh soft cheese manufactured in households on a small scale in rural locations surrounding Pale Mountain City in Bosnia and Herzegovina. The whole-genome sequencing approach and bioinformatics analyses revealed that the strain BGPAS1-3 was non-pathogenic to humans. The presence of bacteriocin operons suggested the ability of the isolate to suppress the growth of pathogens. Coding regions for three maturated bacteriocins (bacteriocin 31, bacteriocin 32, and enterocin P) produced by BGPAS1-3 were amplified and expressed in Escherichia coli ER2523 using the pMALc5HisEk system. All three bacteriocins were successfully overexpressed and purified after enterokinase cleavage but showed different antimicrobial activity. Bacteriocin 31 showed significantly stronger antimicrobial activity compared with bacteriocin 32. It was the only one that proved to be suitable for use as a food preservative against L. monocytogenes in a milk model.

Members of the genus Lactobacillus represent the most common colonizers of the human vagina and are well-known for preserving vaginal health and contrasting the colonization of opportunistic pathogens. Remarkably, high abundance of Lactobacillus crispatus in the vaginal environment has been linked to vaginal health, leading to the widespread use of many L. crispatus strains as probiotics. Nevertheless, despite the scientific and industrial relevance of this species, a comprehensive investigation of the genomics of L. crispatus taxon is still missing. For this reason, we have performed a comparative genomics analysis of 97 L. crispatus strains, encompassing 16 strains sequenced in the framework of this study alongside 81 additional publicly available genome sequences. Thus, allowing the dissection of the L.crispatus pan-genome and core-genome followed by a comprehensive phylogenetic analysis based on the predicted core genes that revealed clustering based on ecological origin. Subsequently, a genomics-targeted approach, i.e., probiogenomics analysis, was applied for in-depth analysis of the eight L. crispatus strains of human origin sequenced in this study. In detail their genetic repertoire was screened for strain-specific genes responsible for phenotypic features that may guide the identification of optimal candidates for next-generation probiotics. The latter includes bacteriocin production, carbohydrates transport and metabolism, as well as a range of features that may be responsible for improved ecological fitness. In silico results regarding the genetic repertoire involved in carbohydrate metabolism were also validated by growth assays on a range of sugars, leading to the selection of putative novel probiotic strains.

Lactiplantibacillus sp. LM14-2, isolated from Thai-fermented mussel ( Hoi-dong ), showed attractive probiotic properties. This strain was identified as Lactiplantibacillus plantarum based on its phenotypic, chemotaxonomic, and genetic characteristics including whole-genome sequencing (WGS). The draft genome sequence was analyzed and annotated for the molecular mechanisms involved in the safety assessment, the adaptation and adhesion of L. plantarum LM14-2 to the gastrointestinal tract (GIT), and the beneficial genes involved in bacteria–host interactions. The L. plantarum LM14-2 exhibited bile salt hydrolase (BSH) activity, assimilated cholesterol at 86.07 ± 5.03%, stimulated the secretion of interleukin-12, interferon-gamma, and human beta defensin-2, and induced nitric oxide production. In addition, L. plantarum LM14-2 showed excellent gastrointestinal tolerance and adhesion ability to Caco-2 cells. Furthermore, the in silico analysis showed that L. plantarum LM14-2 was a non-human pathogen and did not contain antibiotic resistance genes or plasmids. L. plantarum LM14-2 also contained potential genes associated with various probiotic characteristics and health-promoting effects. Consequently, this study suggested that L. plantarum LM14-2 could be considered safe, with potential probiotic properties and health-promoting impacts, which could facilitate its probiotic application.

A Lactobacillus delbrueckii ssp. lactis strain named A4, isolated from the gut of an Armenian honeybee, was subjected to a probiogenomic characterization because of its unusual origin. A whole-genome sequencing was performed, and the bioinformatic analysis of its genome revealed a reduction in the genome size and the number of the genes—a process typical for the adaptation to endosymbiotic conditions. Further analysis of the genome revealed that Lactobacillus delbrueckii ssp. lactis strain named A4 could play the role of a probiotic endosymbiont because of the presence of intact genetic sequences determining antioxidant properties, exopolysaccharides synthesis, adhesion properties, and biofilm formation, as well as an antagonistic activity against some pathogens which is not due to pH or bacteriocins production. Additionally, the genomic analysis revealed significant potential for stress tolerance, such as extreme pH, osmotic stress, and high temperature. To our knowledge, this is the first report of a potentially endosymbiotic Lactobacillus delbrueckii ssp. lactis strain adapted to and playing beneficial roles for its host.