Resistome analysis of Escherichia coli isolates from layers in Hungary.

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.

Investigation of the environmental presence of multidrug-resistant bacteria at small animal hospitals in Hungary.

Multidrug-resistant bacteria can cause severe nosocomial infections in both human and veterinary clinics. The aim of this study was to investigate the presence and antibiotic susceptibility of Enterococcus, Staphylococcus and Pseudomonas strains at four small animal clinics of Hungary in 2018, as these bacteria can reliably represent the level of antimicrobial resistance in the investigated environment. A total of 177 Staphylococcus colonies were found, including 22 Staphylococcus pseudintermedius and 13 Staphylococcus aureus. As regards enterococci, 9 Enterococcus faecium, 2 E. faecalis and further 286 Enterococcus strains were isolated. The number of Pseudomonas aeruginosa isolates (n = 34) was considered too low for relevant susceptibility testing. Among staphylococci, the highest resistance was found to sulphamethoxazole (82.9%), penicillin (65.7%) and erythromycin (54.3%), while in the case of enterococci, resistance to norfloxacin and rifampicin was the most common, with 25.5% of the strains being resistant to both antibiotics. Ten methicillin-resistant S. pseudintermedius (MRSP) and six vancomycin-resistant Enterococcus (VRE) strains could be identified. Only 5.7% of the Staphylococcus isolates were susceptible to all tested agents, while this ratio was 36.2% among enterococci. The results of this study have revealed a high prevalence of antibiotic-resistant bacteria in Hungarian small animal clinics, which highlights the importance of regular disinfection processes and stringent hygiene measures in veterinary clinics.

Antimicrobial Susceptibility Profiles of Pasteurella multocida Isolates from Clinical Cases of Waterfowl in Hungary between 2022 and 2023.

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.

Stability Studies of the Dilution Series of Different Antibiotic Stock Solutions in Culture Medium Incubated at 37 °C.

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.

Monitoring Changes in the Antimicrobial-Resistance Gene Set (ARG) of Raw Milk and Dairy Products in a Cattle Farm, from Production to Consumption.

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.

In Vitro Microevolution and Co-Selection Assessment of Amoxicillin and Cefotaxime Impact on Escherichia coli Resistance Development.

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.

Antiviral Drug Candidate Repositioning for Streptococcus suis Infection in Non-Tumorigenic Cell Models.

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.

Susceptibility of Actinobacillus pleuropneumoniae, Pasteurella multocida and Streptococcus suis Isolated from Pigs in Hungary between 2018 and 2021.

Porcine respiratory disease complex (PRDC) has been a major animal health, welfare, and economic problem in Hungary; therefore, great emphasis should be put on both the prevention and control of this complex disease. As antibacterial agents are effective tools for control, antibiotic susceptibility testing is indispensable for the proper implementation of antibacterial therapy and to prevent the spread of resistance. The best method for this is to determine the minimum inhibitory concentration (MIC) by the broth microdilution method. In our study, we measured the MIC values of 164 Actinobacillus pleuropneumoniae, 65 Pasteurella multocida, and 118 Streptococcus suis isolates isolated from clinical cases against the following antibacterial agents: amoxicillin, ceftiofur, cefquinome, oxytetracycline, doxycycline, tylosin, tilmicosin, tylvalosin, tulathromycin, lincomycin, tiamulin, florfenicol, colistin, enrofloxacin, and sulfamethoxazole-trimethoprim. Outstanding efficacy against A. pleuropneumoniae isolates was observed with ceftiofur (100%) and tulathromycin (100%), while high levels of resistance were observed against cefquinome (92.7%) and sulfamethoxazole-trimethoprim (90.8%). Ceftiofur (98.4%), enrofloxacin (100%), florfenicol (100%), and tulathromycin (100%) were found to be highly effective against P. multocida isolates, while 100% resistance was detected against the sulfamethoxazole-trimethoprim combination. For the S. suis isolates, only ceftiofur (100%) was not found to be resistant, while the highest rate of resistance was observed against the sulfamethoxazole-trimethoprim combination (94.3%). An increasing number of studies report multi-resistant strains of all three pathogens, making their monitoring a high priority for animal and public health.

Pharmacokinetics and Pharmacodynamics of Florfenicol in Plasma and Synovial Fluid of Pigs at a Dose of 30 mg/kgbw Following Intramuscular Administration.

A major problem of our time is the ever-increasing resistance to antimicrobial agents in bacterial populations. One of the most effective ways to prevent these problems is to target antibacterial therapies for specific diseases. In this study, we investigated the in vitro effectiveness of florfenicol against S. suis, which can cause severe arthritis and septicemia in swine herds. The pharmacokinetic and pharmacodynamic properties of florfenicol in porcine plasma and synovial fluid were determined. After a single intramuscular administration of florfenicol at 30 mg/kgbw, the AUC0-∞ was 164.45 ± 34.18 µg/mL × h and the maximum plasma concentration was 8.15 ± 3.11 µg/mL, which was reached in 1.40 ± 0.66 h, whereas, in the synovial fluid, these values were 64.57 ± 30.37 µg/mL × h, 4.51 ± 1.16 µg/mL and 1.75 ± 1.16 h, respectively. Based on the MIC values of the 73 S. suis isolates tested, the MIC50 and MIC90 values were 2 µg/mL and 8 µg/mL, respectively. We successfully implemented a killing-time curve in pig synovial fluid as a matrix. Based on our findings, the PK/PD breakpoints of the bacteriostatic (E = 0), bactericidal (E = -3) and eradication (E = -4) effects of florfenicol were determined and MIC thresholds were calculated, which are the guiding indicators for the treatment of these diseases. The AUC24h/MIC values for bacteriostatic, bactericidal and eradication effects were 22.22 h, 76.88 h and 141.74 h, respectively, in synovial fluid, and 22.42 h, 86.49 h and 161.76 h, respectively, in plasma. The critical MIC values of florfenicol against S. suis regarding bacteriostatic, bactericidal and eradication effects in pig synovial fluid were 2.91 ± 1.37 µg/mL, 0.84 ± 0.39 µg/mL and 0.46 ± 0.21 µg/mL, respectively. These values provide a basis for further studies on the use of florfenicol. Furthermore, our research highlights the importance of investigating the pharmacokinetic properties of antibacterial agents at the site of infection and the pharmacodynamic properties of these agents against different bacteria in different media.

Determining the In Vivo Efficacy of Plant-Based and Probiotic-Based Antibiotic Alternatives against Mixed Infection with Salmonella enterica and Escherichia coli in Domestic Chickens.

Restrictions on the use of antimicrobial compounds have led to a surge of interest in alternative solutions, such as natural, plant-based compounds. In our study, we investigated the efficacy of three feed supplements containing different additives, namely, probiotics (Lactobacillus spp., "Test substance A"), turmeric (Curcuma longa L., "Test substance B"), and fenugreek (Trigonella foenum graecum, "Test substance C"). In the experiment, we tested 180 birds of the Bábolna Tetra-SL laying hybrid breed that were infected with Salmonella enteritidis strains. The birds were randomly divided into six groups: three groups treated with the different additives, a negative control group, a positive control group, and an antibiotic-treated group using enrofloxacin. We examined the maturation and the time course of shedding of Salmonella; at the end of rearing, pathological and histopathological examinations were performed. When Salmonella was isolated from the cloacal swab samples, the enrofloxacin-treated group had a high number of animals shedding Salmonella by day 9, which was like the group treated with test material C. The greatest reduction in Salmonella shedding was observed in the groups treated with test materials A and B. In terms of pathological parameters, villus length and crypt depth were significantly better in the group treated with test material C compared to the positive and negative controls, and when comparing the body weight of the tested animals, the group treated with test material B had a significantly larger absorption surface area compared to the positive control group. Overall, the supplement with test material C proved to be the most effective. In the future, it is worthwhile to investigate the combination of the tested active substances for their possible synergistic effects and to perform a dose-response study to select the optimal dosage.

In Vitro Microevolution and Co-Selection Assessment of Florfenicol Impact on Escherichia coli Resistance Development.

The issue of antimicrobial resistance is becoming an increasingly serious challenge in both human and veterinary medicine. Prudent antimicrobial use in veterinary medicine is warranted and supported by international guidelines, with the Antimicrobial Advice Ad Hoc Expert Group (AMEG) placing particular emphasis on the critically important group B antimicrobials. These antimicrobials are commonly employed, especially in the poultry and swine industry. The impact of florfenicol, a veterinary antibiotic, was studied on the resistance development of Escherichia coli. The aim of the study was to investigate the effect of the use of florfenicol on the development of phenotypic and genomic resistances, not only to the drug itself but also to other drugs. The minimum inhibitory concentrations (MICs) of the antibiotics were investigated at 1×, 10×, 100× and 1000× concentrations using the adapted Microbial Evolution and Growth Arena (MEGA-plate) method. The results demonstrate that florfenicol can select for resistance to fluoroquinolone antibiotics (167× MIC value increase) and cephalosporins (67× MIC value increase). A total of 44 antimicrobial resistance genes were identified, the majority of which were consistent across the samples. Chromosomal point mutations, including alterations in resistance-associated and regulatory genes (acrB, acrR, emrR and robA), are thought to trigger multiple drug efflux pump activations, leading to phenotypically increased resistance. The study underscores the impact of florfenicol and its role in the development of antimicrobial resistance, particularly concerning fluoroquinolone antibiotics and cephalosporins. This study is the first to report florfenicol's dose-dependent enhancement of other antibiotics' MICs, linked to mutations in SOS-box genes (mdtABC-tolC, emrAB-tolC and acrAB-tolC) and increased multidrug efflux pump genes. Mutations in the regulatory genes acrR, emrR and rpbA support the possibility of increased gene expression. The results are crucial for understanding antimicrobial resistance and its development, highlighting the promising potential of in vitro evolutionary and coselection studies for future research.

Synovial and Systemic Pharmacokinetics of Florfenicol and PK/PD Integration against Streptococcus suis in Pigs.

Florfenicol is a member of the phenicol group, a broad-spectrum antibacterial agent. It has been used for a long time in veterinary medicine, but there are some factors regarding its pharmacokinetic characteristics that have yet to be elucidated. The aim of our study was to describe the pharmacokinetic profile of florfenicol in synovial fluid and plasma of swine after intramuscular (i.m.) administration. In addition, the dosage regimen of treatment of arthritis caused by S. suis was computed for florfenicol using pharmacokinetic/pharmacodynamic (PK/PD) indices. As the first part of our investigation, the pharmacokinetic (PK) parameters of florfenicol were determined in the plasma and synovial fluid of six pigs. Following drug administration (15 mg/kgbw, intramuscularly), blood was drawn at the following times: 10, 20, 30, 40, 50 and 60 min, 2, 3, 4, 5, 6, 7, 8, 12, 24, 48 and 72 h; synovial fluid samples were taken after 1, 2, 3, 4, 6, 8, 12, 24, 48 and 72 h. The concentration of florfenicol was determined by a validated liquid chromatography-mass spectrometry (LC-MS/MS) method via multiple reaction monitoring (MRM) modes. As the second part of our research, minimum inhibitory concentration (MIC) values of florfenicol were determined in 45 S. suis strains isolated from clinical samples collected in Hungary. Furthermore, a strain of S. suis serotype 2 (SS3) was selected, and killing-time curves of different florfenicol concentrations (0.5 µg/mL, 1 µg/mL and 2 µg/mL) were determined against this strain. Peak concentration of the florfenicol was 3.58 ± 1.51 µg/mL in plasma after 1.64 ± 1.74 h, while it was 2.73 ± 1.2 µg/mL in synovial fluid 3.4 ± 1.67 h after administration. The half-life in plasma was found to be 17.24 ± 9.35 h, while in synovial fluid it was 21.01 ± 13.19 h. The area under the curve (AUC24h) value was 54.66 ± 23.34 µg/mL·h for 24 h in plasma and 31.24 ± 6.82 µg/mL·h for 24 h in synovial fluid. The drug clearance scaled by bioavailability (Cl/F) in plasma and synovial fluid was 0.19 ± 0.08 L/h/kg and 0.29 ± 0.08 L/h/kg, respectively. The mean residence time (MRT) in plasma and synovial fluid was 24.0 ± 13.59 h and 27.39 ± 17.16 h, respectively. The steady-state volume of distribution (Vss) in plasma was calculated from Cl/F of 0.19 ± 0.08 L/h/kg, multiplied by MRT of 24.0 ± 13.59 h. For the PK/PD integration, average plasma and synovial fluid concentration of florfenicol was used in a steady-state condition. The obtained MIC50 value of the strains was 2.0 µg/mL, and MIC90 proved to be 16.0 µg/mL. PK/PD integration was performed considering AUC24h/MIC breakpoints that have already been described. This study is the first presentation of the pharmacokinetic behavior of florfenicol in swine synovia as well as a recommendation of extrapolated critical MICs of S. suis for therapeutic success in the treatment of S. suis arthritis in swine, but it should be noted that this requires a different dosage regimen to that used in authorized florfenicol formulations.

Photoreactive Coating Material as an Effective and Durable Antimicrobial Composite in Reducing Bacterial Load on Surfaces in Livestock.

Titanium dioxide (TiO2) is a well-known photocatalytic compound that can be used to effectively reduce the presence of pathogens in human and animal hospitals via ROS release. The aim of this study was to investigate the efficacy of a polymer-based composite layer containing TiO2 and zinc oxide (ZnO) against Escherichia coli (E. coli) of animal origin. We showed that the photocatalyst coating caused a significant (p < 0.001) reduction in pathogen numbers compared to the control with an average reduction of 94% over 30 min. We used six light sources of different wattages (4 W, 7 W, 9 W, 12 W, 18 W, 36 W) at six distances (35 cm, 100 cm, 150 cm, 200 cm, 250 cm, 300 cm). Samples (n = 2160) were taken in the 36 settings and showed no significant difference in efficacy between light intensity and distance. We also investigated the influence of organic contaminant that resulted in lower activity as well as the effect of a water jet and a high-pressure device on the antibacterial activity. We found that the latter completely removed the coating from the surface, which significantly (p < 0.0001) reduced its antibacterial potential. As a conclusion, light intensity and distance does not reduce the efficacy of the polymer, but the presence of organic contaminants does.

Molecular epidemiological study of Trichomonas gallinae focusing on central and southeastern Europe.

Trichomonas gallinae is a geographically widespread protozoan parasite of birds. In this study, oropharyngeal swab samples were collected in Hungary and Romania from 99 columbiform birds, including 76 feral pigeons (Columba livia domestica: 42 kept for racing, 32 with urban and two with rural habitat), four common wood pigeons (C. palumbus), 16 ring doves (Streptopelia risoria) and three Eurasian collared doves (S. decaocto). These samples were analyzed for the presence of T. gallinae using molecular methods. Racing feral pigeons had significantly higher prevalence of T. gallinae infection than urban feral pigeons. The rate of PCR-positivity was the highest among wood pigeons and ring doves. Based on 18S rRNA gene, T. gallinae was the most heterogenous among racing feral pigeons sampled in a trading-breeding place. Clinical signs were associated with only one 18S rRNA gene subtype. The most divergent 18S rRNA gene subtype, Trichomonas sp. Hu-TG37 clustered with T. canistomae and T. tenax and represents probably a new species. To our knowledge, this is the first report on the genetic diversity of T. gallinae in the southeastern European region. The results suggest that most detected T. gallinae 18S rRNA gene subtypes are not host-specific and do not cause clinical signs. The highest number of 18S rRNA gene subtypes was demonstrated among racing feral pigeons. Significantly more captive than free-living columbiform birds had T. gallinae infection. These data highlight the importance of epizootic monitoring of the genetic diversity and presence of T. gallinae in trading-breeding places of pigeons and doves.