Search

Introduction: One of the greatest challenges of our time is antimicrobial resistance, which could become the leading cause of death globally within a few decades. In the context of One Health, it is in the common interest to mitigate the global spread of antimicrobial resistance by seeking alternative solutions, alongside appropriate drug selection and responsible use. Probiotics offer a potential avenue to reduce antibiotic usage; however, there is a scarcity of research that examines commercial products in terms of carrying antimicrobial resistance genes (ARGs) involved in resistance development through microbial vectors., Methods: Our study investigated 10 commercially available probiotic products for cats and dogs. Initially, we conducted phenotypic testing through determination of minimum inhibitory concentration (MIC) for antibiotics important in animal and public health. Subsequently, we performed next-generation sequencing (NGS) of the products to elucidate the genetic background behind the decrease in phenotypic sensitivity., Results: In total, 19 types of ARGs were identified, with 57.9% being found on plasmids, and in two cases, carriage as mobile genetic elements were found. One of the genes identified was the APH(3 ' )-Ia gene, capable of inactivating aminoglycoside antibiotics through phosphotransferase enzyme production regulation, while the other was the tetS gene, capable of conferring reduced sensitivity to tetracycline antibiotics through target protection., Discussion: Our findings underscore the importance of approaching antimicrobial resistance investigations from a broader perspective. We suggest that further studies in this area are justified and raise questions regarding the need to extend legally required studies on probiotic products from their use in economic livestock to their use in companion animals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kerek, Szabó, Szabó, Papp, Bányai, Kardos, Kaszab, Bali and Jerzsele.)

Drug innovation traditionally follows a de novo approach with new molecules through a complex preclinical and clinical pathway. In addition to this strategy, drug repositioning has also become an important complementary approach, which can be shorter, cheaper, and less risky. This review provides an overview of drug innovation in both human and veterinary medicine, with a focus on drug repositioning. The evolution of drug repositioning and the effectiveness of this approach are presented, including the growing role of data science and computational modeling methods in identifying drugs with potential for repositioning. Certain business aspects of drug innovation, especially the relevant factors of market exclusivity, are also discussed. Despite the promising potential of drug repositioning for innovation, it remains underutilized, especially in veterinary applications. To change this landscape for mutual benefits of human and veterinary drug innovation, further exploitation of the potency of drug repositioning is necessary through closer cooperation between all stakeholders, academia, industry, pharmaceutical authorities, and innovation policy makers, and the integration of human and veterinary repositioning into a unified innovation space. For this purpose, the establishment of the conceptually new "One Health Drug Repositioning Platform" is proposed. Oncology is one of the disease areas where this platform can significantly support the development of new drugs for human and dog (or other companion animals) anticancer therapies. As an example of the utilization of human and veterinary drugs for veterinary repositioning, the use of COX inhibitors to treat dog cancers is reviewed., Competing Interests: This study was sponsored by the National Research, Development and Innovation Office (Hungary) and the E-GROUP ICT SOFTWARE Information Technology Private Limited Company. There is no conflicts of interest between the authors and the sponsors.

Inhibitors of the serine protease furin have been widely studied as antimicrobial agents due to their ability to block the cleavage and activation of certain viral surface proteins and bacterial toxins. In this study, the antipseudomonal effects and safety profiles of the furin inhibitors MI-1851 and MI-2415 were assessed. Fluorescence quenching studies suggested no relevant binding of the compounds to human serum albumin and α 1 -acid glycoprotein. Both inhibitors demonstrated significant antipseudomonal activity in Madin-Darby canine kidney cells, especially compound MI-1851 at very low concentrations (0.5 µM). Using non-tumorigenic porcine IPEC-J2 cells, neither of the two furin inhibitors induced cytotoxicity (CCK-8 assay) or altered significantly the intracellular (Amplex Red assay) or extracellular (DCFH-DA assay) redox status even at a concentration of 100 µM. The same assays with MI-2415 conducted on primary human hepatocytes also resulted in no changes in cell viability and oxidative stress at up to 100 µM. Microsomal and hepatocyte-based CYP3A4 activity assays showed that both inhibitors exhibited a concentration-dependent inhibition of the isoenzyme at high concentrations. In conclusion, this study indicates a good safety profile of the furin inhibitors MI-1851 and MI-2415, suggesting their applicability as antimicrobials for further in vivo investigations, despite some inhibitory effects on CYP3A4.

In dogs, chronic enteropathies, and impaired gut integrity, as well as microbiome imbalances, are a major problem. These conditions may represent a continuous low endotoxin load, which may result in the development of diseases that are attributable to chronic inflammation. Flavonoids are polyphenolic plant compounds with numerous beneficial properties such as antioxidant, anti-inflammatory and antimicrobial effects. For our experiments, we isolated primary white blood cells (peripheral blood mononuclear cells and polymorphonuclear leukocytes) from healthy dogs and induced inflammation and oxidative stress with Escherichia coli and Salmonella enterica serovar Enteritidis lipopolysaccharide (LPS). In parallel, we treated the cell cultures with various flavonoids luteolin, quercetin and grape seed extract oligomeric proanthocyanidins (GSOP) alone and also in combination with LPS treatments. Then, changes in viability, reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α) levels were measured in response to treatment with quercetin, luteolin and GSOP at 25 and 50 μg/mL concentrations. We found that ROS levels were significantly lower in groups which were treated by flavonoid and LPS at the same time compared to LPS-treated groups, whereas TNF-α levels were significantly reduced only by luteolin and quercetin treatment. In contrast, treatment with lower concentrations of GSOP caused an increase in TNF-α levels, while higher concentrations caused a significant decrease. These results suggest that the use of quercetin, luteolin and GSOP may be helpful in the management of chronic intestinal diseases in dogs with reduced intestinal barrier integrity or altered microbiome composition, or in the mitigation of chronic inflammatory processes maintained by endotoxemia. Further in vitro and in vivo studies are needed before clinical use., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Móritz, Kovács, Jerzsele, Psáder and Farkas.)

Intestinal infections caused by Escherichia coli and Salmonella enterica pose a huge economic burden on the swine industry that is exacerbated by the development of antimicrobial resistance in these pathogens, thus raising the need for alternative prevention and treatment methods. Our aim was to test the beneficial effects of the flavonoid luteolin in an in vitro model of porcine intestinal infections. We infected the porcine intestinal epithelial cell line IPEC-J2 with E. coli and S . enterica subsp. enterica serovar Typhimurium (10 6 CFU/mL) with or without previous, concurrent, or subsequent treatment with luteolin (25 or 50 µg/mL), and measured the changes in the reactive oxygen species and interleukin-6 and -8 levels of cells. We also tested the ability of luteolin to inhibit the adhesion of bacteria to the cell layer, and to counteract the barrier integrity damage caused by the pathogens. Luteolin was able to alleviate oxidative stress, inflammation, and barrier integrity damage, but it could not inhibit the adhesion of bacteria to IPEC-J2 cells. Luteolin is a promising candidate to be used in intestinal infections of pigs, however, further studies are needed to confirm its efficacy. The use of luteolin in the future could ultimately lead to a reduced need for antibiotics in pig production.

Probiotics are widely used in broiler chickens to support the gut microbiome, gut health, and to reduce the amount of antibiotics used. Despite their benefits, there is concern over their ability to carry and spread antimicrobial resistance genes (ARGs), posing a significant public health risk. This study utilized next-generation sequencing to investigate ARGs in probiotics approved for poultry, focusing on their potential to be transferred via mobile genetic elements such as plasmids and phages. We examined the gut microbiome and resistome changes in 60 broiler chickens over their rearing period, correlating these changes with different probiotic treatments. Specific resistance mechanisms against critically important antibiotics were identified, including genes related to fluoroquinolone resistance and peptide antibiotic resistance. We also found genes with significant relevance to public health ( aadK , AAC(6')-Ii ) and multiple drug-resistance genes ( vmlR , ykkC , ykkD , msrC , clbA , eatAv ). Only one phage-encoded gene ( dfrA43 ) was detected, with no evidence of plasmid or mobile genetic element transmission. Additionally, metagenomic analysis of fecal samples showed no significant changes corresponding to time or diet across groups. Our findings highlight the potential risks associated with the use of probiotics in poultry, particularly regarding the carriage of ARGs. It is crucial to conduct further research into the molecular genetics of probiotics to develop strategies that mitigate the risk of resistance gene transfer in agriculture, ensuring the safe and effective use of probiotics in animal husbandry.

The long-term stability of antibiotics in culture media remains underexplored in scientific literature. This study evaluated the stability of eight distinct antibiotic stock solutions-amoxicillin, cefotaxime, neomycin, oxytetracycline, florfenicol, enrofloxacin, colistin, and potentiated sulfonamide-and their 10-fold dilution series in tryptone soy broth (TSB) at 37 °C, over 12 days. Samples were collected immediately after preparation and on days 1, 2, 5, 7, 9, and 12, with active substance concentrations measured using ultra-high-performance liquid chromatography (UHPLC) coupled with mass spectrometry. The results indicated that among the ultrapure water stock solutions, neomycin, florfenicol, and potentiated sulfonamide maintained stability (>95%). Within the culture medium, florfenicol showed consistent stability (100%) throughout the study, potentiated sulfonamide experienced minor degradation (>85%), and neomycin underwent significant degradation. Amoxicillin, oxytetracycline, and colistin displayed considerable degradation in both solution types but were more stable in ultrapure water solutions. The stability of cefotaxime and enrofloxacin in ultrapure water solutions and in the medium was very similar when compared; however, 3.6% of the former and 88.7% of the latter remained detectable by day 12. These findings are crucial for minimum inhibitory concentration (MIC) assessments, especially in minimum bactericidal concentration (MBC) studies, and in experiments concerning long-term evolution and co-selection. This study underscores the necessity of stability assessments in culture media to validate future experimental outcomes.

Raw milk and dairy products can serve as potential vectors for transmissible bacterial, viral and protozoal diseases, alongside harboring antimicrobial-resistance genes. This study monitors the changes in the antimicrobial-resistance gene pool in raw milk and cheese, from farm to consumer, utilizing next-generation sequencing. Five parallel sampling runs were conducted to assess the resistance gene pool, as well as phage or plasmid carriage and potential mobility. In terms of taxonomic composition, in raw milk the Firmicutes phylum made up 41%, while the Proteobacteria phylum accounted for 58%. In fresh cheese, this ratio shifted to 93% Firmicutes and 7% Proteobacteria. In matured cheese, the composition was 79% Firmicutes and 21% Proteobacteria. In total, 112 antimicrobial-resistance genes were identified. While a notable reduction in the resistance gene pool was observed in the freshly made raw cheese compared to the raw milk samples, a significant growth in the resistance gene pool occurred after one month of maturation, surpassing the initial gene frequency. Notably, the presence of extended-spectrum beta-lactamase (ESBL) genes, such as OXA-662 (100% coverage, 99.3% identity) and OXA-309 (97.1% coverage, 96.2% identity), raised concerns; these genes have a major public health relevance. In total, nineteen such genes belonging to nine gene families ( ACT , CMY , EC , ORN , OXA , OXY , PLA , RAHN , TER ) have been identified. The largest number of resistance genes were identified against fluoroquinolone drugs, which determined efflux pumps predominantly. Our findings underscore the importance of monitoring gene pool variations throughout the product pathway and the potential for horizontal gene transfer in raw products. We advocate the adoption of a new approach to food safety investigations, incorporating next-generation sequencing techniques.

The waterfowl industry represents a narrow, yet economically significant, sector within the poultry industry. Although less prominent, the waterfowl sector is nonetheless of equal importance to any other livestock sector in terms of antimicrobial resistance and animal health issues. This study assesses the antimicrobial resistance profile of Pasteurella multocida bacterial strains isolated from clinical cases in Hungary's duck and goose populations, determining the minimal inhibitory concentration (MIC) of 27 samples collected from 15 different locations. The results indicate that the isolated strains were susceptible to most antibiotics, except for notable resistance to enrofloxacin. These findings support that Pasteurella multocida largely retained its susceptibility. However, the observed resistance to enrofloxacin suggests overuse of fluoroquinolones, which indicates the potential need for stricter regulation of their use in the poultry industry.

The authors aimed to investigate eight strains of Escherichia coli (E. coli) strains from Hungarian layer flocks for antimicrobial resistance genes (ARG), using metagenomic methods. The strains were isolated from cloacal swabs of healthy adult layers. This study employed shotgun sequencing-based genetic and bioinformatic analysis along with determining phenotypic minimum inhibitory concentrations. A total of 59 ARGs were identified in the eight E. coli isolates, carrying ARGs against 15 groups of antibiotics. Among these, 28 ARGs were identified as transferable. Specifically, four ARGs were plasmid-derived, 18 ARGs were phage-derived and an additional six ARGs were predicted to be mobile, contributing to their mobility and potential spread between bacteria.

The increasing prevalence of antimicrobial resistance against zoonotic bacteria, including Streptococcus (S.) suis , highlights the need for new therapeutical strategies, including the repositioning of drugs. In this study, susceptibilities of bacterial isolates were tested toward ten different 3-amidinophenyalanine (Phe(3-Am)) derivatives via determination of minimum inhibitory concentration (MIC) values. Some of these protease inhibitors, like compounds MI-432, MI-471, and MI-476, showed excellent antibacterial effects against S. suis . Their drug interaction potential was investigated using human liver microsomal cytochrome P450 (CYP450) measurements. In our work, non-tumorigenic IPEC-J2 cells and primary porcine hepatocytes were infected with S. suis , and the putative beneficial impact of these inhibitors was investigated on cell viability (Neutral red assay), on interleukin (IL)-6 levels (ELISA technique), and on redox balance (Amplex red method). The antibacterial inhibitors prevented S. suis -induced cell death (except MI-432) and decreased proinflammatory IL-6 levels. It was also found that MI-432 and MI-476 had antioxidant effects in an intestinal cell model upon S. suis infection. Concentration-dependent suppression of CYP3A4 function was found via application of all three inhibitors. In conclusion, our study suggests that the potential antiviral Phe(3-Am) derivatives with 2',4' dichloro-biphenyl moieties can be considered as effective drug candidates against S. suis infection due to their antibacterial effects.

The global spread of antimicrobial resistance has become a prominent issue in both veterinary and public health in the 21st century. The extensive use of amoxicillin, a beta-lactam antibiotic, and consequent resistance development are particularly alarming in food-producing animals, with a focus on the swine and poultry sectors. Another beta-lactam, cefotaxime, is widely utilized in human medicine, where the escalating resistance to third- and fourth-generation cephalosporins is a major concern. The aim of this study was to simulate the development of phenotypic and genotypic resistance to beta-lactam antibiotics, focusing on amoxicillin and cefotaxime. The investigation of the minimal inhibitory concentrations (MIC) of antibiotics was performed at 1×, 10×, 100×, and 1000× concentrations using the modified microbial evolution and growth arena (MEGA-plate) method. Our results indicate that amoxicillin significantly increased the MIC values of several tested antibiotics, except for oxytetracycline and florfenicol. In the case of cefotaxime, this increase was observed in all classes. A total of 44 antimicrobial resistance genes were identified in all samples. Chromosomal point mutations, particularly concerning cefotaxime, revealed numerous complex mutations, deletions, insertions, and single nucleotide polymorphisms (SNPs) that were not experienced in the case of amoxicillin. The findings suggest that, regarding amoxicillin, the point mutation of the acrB gene could explain the observed MIC value increases due to the heightened activity of the acrAB-tolC efflux pump system. However, under the influence of cefotaxime, more intricate processes occurred, including complex amino acid substitutions in the ampC gene promoter region, increased enzyme production induced by amino acid substitutions and SNPs, as well as mutations in the acrR and robA repressor genes that heightened the activity of the acrAB-tolC efflux pump system. These changes may contribute to the significant MIC increases observed for all tested antibiotics. The results underscore the importance of understanding cross-resistance development between individual drugs when choosing clinical alternative drugs. The point mutations in the mdtB and emrR genes may also contribute to the increased activity of the mdtABC-tolC and emrAB-tolC pump systems against all tested antibiotics. The exceptionally high mutation rate induced by cephalosporins justifies further investigations to clarify the exact mechanism behind.