Molecular Markers and Antimicrobial Resistance Patterns of Extraintestinal Pathogenic Escherichia coli from Camel Calves Including Colistin-Resistant and Hypermucoviscuous Strains.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are capable of causing various systemic infections in both humans and animals. In this study, we isolated and characterized 30 E. coli strains from the parenchymatic organs and brains of young (<3 months of age) camel calves which died in septicemia. Six of the strains showed hypermucoviscous phenotype. Based on minimum inhibitory concentration (MIC) values, seven of the strains were potentially multidrug resistant, with two additional showing colistin resistance. Four strains showed mixed pathotypes, as they carried characteristic virulence genes for intestinal pathotypes of E. coli: three strains carried cnf1, encoding cytotoxic necrotizing factor type 1, the key virulence gene of necrotoxigenic E. coli (NTEC), and one carried eae encoding intimin, the key virulence gene of enteropathogenic E. coli (EPEC). An investigation of the integration sites of pathogenicity islands (PAIs) and the presence of prophage-related sequences showed that the strains carry diverse arrays of mobile genetic elements, which may contribute to their antimicrobial resistance and virulence patterns. Our work is the first to describe ExPEC strains from camels, and points to their veterinary pathogenic as well as zoonotic potential in this important domestic animal.

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.

Half-life of serum anti-Müllerian hormone and changes after gonadectomy in adult female and male dogs with normal and abnormal gonads.

Anti-Müllerian hormone (AMH) is a biomarker for the presence of gonadal tissue. Changes in serum AMH after gonadectomy are not well established, and its serum half-life is unknown in dogs. We measured serum AMH with a validated electro-chemiluminescent immunoassay in adult female (n = 12) and male (n = 7) dogs with normal gonads, as well as in dogs with gonadal pathology (ovarian remnant syndrome, ORS n = 3, testicular tumor [Leydig cell, Sertoli cell, seminoma] n = 3, unilateral abdominal cryptorchid n = 4) on the day of gonadectomy (D0), and on D3, D7, D14 (females and males), and D21, D28 (males only). Males had higher AMH concentrations than females independent of gonadal status (P < 0.001). Dogs with ORS had lower initial AMH (0.45 ± 0.43 ng/ml) than bitches with normal gonads (1.16 ± 0.44 ng/ml; P = 0.027). Cryptorchid dogs had higher initial concentrations (80.57 ± 52.81 ng/ml) than males with normal gonads (7.92 ± 2.45 ng/ml; P = 0.004), and those with testicular tumors (18.63 ± 5.04 ng/ml) were intermediate (P ≥ 0.250). AMH decreased over time (P ≤ 0.012) and was 0.01-0.04 ng/ml by D14 in females and 0.02-0.12 ng/ml by D28 in males. Serum half-life in the whole study population was 2.85 ± 0.51 days and did not differ between groups. In conclusion, serum AMH can differentiate between intact and gonadectomized status of adult dogs by 14 days after ovario(hyster)ectomy in females and by 28 days after surgical castration in males.

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.

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.

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.

Antimicrobial susceptibility profiles of Mycoplasma hyorhinis strains isolated from five European countries between 2019 and 2021.

Mycoplasma hyorhinis is an emerging swine pathogen bacterium causing polyserositis and polyarthritis in weaners and finishers. The pathogen is distributed world-wide, generating significant economic losses. No commercially available vaccine is available in Europe. Therefore, besides improving the housing conditions for prevention, antimicrobial therapy of the diseased animals is the only option to control the infection. Our aim was to determine the minimal inhibitory concentrations (MIC) of ten antimicrobials potentially used against M. hyorhinis infection. The antibiotic susceptibility of 76 M. hyorhinis isolates from Belgium, Germany, Hungary, Italy and Poland collected between 2019 and 2021 was determined by broth micro-dilution method and mismatch amplification mutation assay (MAMA). Low concentrations of tiamulin (MIC90 0.312 µg/ml), doxycycline (MIC90 0.078 µg/ml), oxytetracycline (MIC90 0.25 µg/ml), florfenicol (MIC90 2 µg/ml) and moderate concentrations of enrofloxacin (MIC90 1.25 µg/ml) inhibited the growth of the isolates. For the tested macrolides and lincomycin, a bimodal MIC pattern was observed (MIC90 >64 µg/ml for lincomycin, tulathromycin, tylosin and tilmicosin and 5 µg/ml for tylvalosin). The results of the MAMA assay were in line with the conventional method with three exceptions. Based on our statistical analyses, significant differences in MIC values of tiamulin and doxycycline were observed between certain countries. Our results show various levels of antimicrobial susceptibility among M. hyorhinis isolates to the tested antibiotics. The data underline the importance of susceptibility monitoring on pan-European level and provides essential information for proper antibiotic choice in therapy.

Characterization and ex vivo evaluation of excised skin samples as substitutes for human dermal barrier in pharmaceutical and dermatological studies.

BACKGROUND: Excised animal and human skins are frequently used in permeability testing in pharmaceutical research. Several factors exist that may have influence on the results. In the current study some of the skin parameters that may affect drug permeability were analysed for human, mouse, rat and pig skin. MATERIALS AND METHODS: Classic biophysical skin parameters were measured (e.g. pH, hydration, permittivity, transepidermal water loss). Physiological characteristics of the skins were also analysed by confocal Raman spectroscopy, scanning electron microscopy and two-photon microscopy. RESULTS: Based on biophysical testing, skin barrier function was damaged in psoriatic mouse skin and in marketed pig skin. Hydration and pH values were similar among the species, but freezing and thawing reduced the water content of the skins and shifted the surface pH to acidic. Aging reduced hydration and permittivity, resulting in impaired barrier function. Mechanical sensitization used in permeability studies resulted in proportional thinning of dead epidermis. DISCUSSION: Results indicate that depending on the scientific question it should be considered whether fresh or frozen tissue is used, and for certain purposes rodent skins are well usable. The structure of the skin tissue (ceramide, cholesterol, keratin, natural moisturizing factor or urea) is similar in rats and mice, but due to the higher skin thickness the lipid distribution is different in porcine skin. Psoriasis led to irregular chemical composition of the skin. CONCLUSION: A comprehensive evaluation of skin samples of four species was performed. The biophysical and microscopic observations should be considered when selecting drug penetration models and experimental conditions.

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.

Motorway Measurement Campaign to Support R&D Activities in the Field of Automated Driving Technologies.

A spectacular measurement campaign was carried out on a real-world motorway stretch of Hungary with the participation of international industrial and academic partners. The measurement resulted in vehicle based and infrastructure based sensor data that will be extremely useful for future automotive R&D activities due to the available ground truth for static and dynamic content. The aim of the measurement campaign was twofold. On the one hand, road geometry was mapped with high precision in order to build Ultra High Definition (UHD) map of the test road. On the other hand, the vehicles-equipped with differential Global Navigation Satellite Systems (GNSS) for ground truth localization-carried out special test scenarios while collecting detailed data using different sensors. All of the test runs were recorded by both vehicles and infrastructure. The paper also showcases application examples to demonstrate the viability of the collected data having access to the ground truth labeling. This data set may support a large variety of solutions, for the test and validation of different kinds of approaches and techniques. As a complementary task, the available 5G network was monitored and tested under different radio conditions to investigate the latency results for different measurement scenarios. A part of the measured data has been shared openly, such that interested automotive and academic parties may use it for their own purposes.

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.

Alternative treatment of serious and mild Pasteurella multocida infection in New Zealand White rabbits.

BACKGROUND: Pasteurella multocida causes numerous economically relevant diseases in livestock including rabbits. Immunisation is only variably effective. Prophylactic antibiotics are used in some species but are contra-indicated in rabbits, due to their adverse effects on the rabbit microbiota. There is therefore a substantial need for alternative forms of infection control in rabbits; we investigated the effect of oral ß-glucan on P. multocida infection in this species. RESULTS: Thirthy-five New Zealand White rabbits were randomly divided into five groups of seven animals. Three groups were inoculated with Pasteurella multocida intranasally (in.), a physiologically appropriate challenge which reproduces naturally acquired infection, and received either (1-3), (1-6) ß-glucans or placebo. Four other groups were inoculated both in. and intramuscularly (im.), representing a supra-physiological challenge, and received either (1-3), (1-6) ß-glucans, antibiotic or placebo. ß-glucans given prophylactically were highly effective in protecting against physiological (in.) bacterial challenge. They were less effective in protecting against supra-physiological bacterial challenge (in. and im.), although they extended survival times. This latter finding has practical relevance to breeders as it extends the window in which heavily infected and symptomatic animals can be salvaged with antibiotics. CONCLUSIONS: In our study, (1-3), (1-6) ß-glucans were highly effective in protecting against a model of naturally acquired P. multocida infection and extended survival times in the supra-physiological model. Enrofloxacin was effective in protecting against supra-physiological infection. We are currently reviewing the use of combined prophylaxis.

Toxicity of selenate and selenite to the potworm Enchytraeus albidus (Annelida: Enchytraeidae): a laboratory test.

Little information is available about the toxicity of inorganic selenium forms in soil animals. Therefore, the effects of selenate and selenite on the mortality and reproduction of Enchytraeus albidus were examined in standard laboratory tests with chronic exposure. Total and available amount of selenate and selenite were tested in a calcareous loamy chernozem soil. The LC(50) of selenate was 5.69 (2.7-8.12) mg kg(-1) dry wt. for total Se and 4.74 (2.14-6.98) mg kg(-1) dry wt. for available Se. Selenite LC(50) was as high as 22.5 (19.6-25.7) mg kg(-1) dry wt. for total Se and 8.10 (6.8-9.6) mg kg(-1) dry wt. for available Se. The EC(50) of selenate was 0.41 (0.35-0.48) mg kg(-1) dry wt. for total Se and 0.28 (0.24-0.34) mg kg(-1) dry wt. for available Se. Selenite EC(50) was as high as 7.3 (6.2-8.5) mg kg(-1) dry wt. for total Se and 2.46 (2.05-2.91) mg kg(-1) dry wt. for available Se. The response in reproduction was more sensitive to Se toxicity than the response in mortality. Selenate proved to be more toxic than selenite. Available data show that E. albidus may function as a biological indicator for some inorganic selenium forms in the soil.