Generation and characterization of two acid-resistant macrocin O-methyltransferase variants with a higher enzyme activity at 30 °C from Streptomyces fradiae.

Tylosin is an important macrolide antibiotic produced by Streptomyces fradiae. In the biosynthesis of tylosin, macrocin O-methyltransferase TylF catalyzes the conversion of the side-product tylosin C (macrocin) to the primary component tylosin A (C/A conversion). This conversion is the rate-limiting step in the biosynthesis of tylosin, and affects the quality of the end product. To find a high activity and environment-adapted TylF enzyme, a TylF variant pool has been constructed via protein evolution approach in our previous study (Fan et al., 2023 [41]). In this study, the TylF variants with higher C/A conversion rates were expressed in E. coli and purified. The variants TylFY139F, TylFQ138H, F232Y and TylFT36S, V54A were shown to have a higher C/A conversion rate at 30 °C than that of TylF at 38 °C. Moreover, they had a greater acid resistance and showed more adaptable to the pH change during fermentation. Further protein structural and substrate-binding affinity analyses revealed that the T36S, V54A, Q138H, Y139F, and F232Y mutations enlarged the volume of the substrate-binding pocket, thereby increasing the affinity of enzyme variants for their substrates of SAM and macrocin, and decreasing the inhibition of SAH. Three of the TylF variants were overexpressed in the industrial tylosin-producing S. fradiae strain, and the recombinant strains showed the highest C/A conversion at 30 °C without heating up to 38 °C during the last 24 h of fermentation. This is of great energy-saving significance for tylosin industrial production.

Oral Fecal Microbiota Transplantation in Dogs with Tylosin-Responsive Enteropathy-A Proof-of-Concept Study.

A clinical trial was conducted to evaluate the effect of fecal microbiota transplantation (FMT) on the canine chronic enteropathy clinical activity index (CCECAI), fecal consistency, and microbiome of dogs with tylosin-responsive enteropathy (TRE). The trial consisted of four phases: (1) screening with discontinuation of tylosin for 4 weeks, (2) inclusion with re-introduction of tylosin for 3-7 days, (3) treatment with FMT/placebo for 4 weeks, and (4) post-treatment with follow-up for 4 weeks after treatment cessation. The study found that the treatment efficacy of FMT (71.4%) was slightly higher than that of placebo (50%), but this difference was not statistically significant due to underpowering. The most abundant bacterial species detected in the fecal microbiomes of dogs with TRE before FMT or placebo treatment were Blautia hansenii, Ruminococcus gnavus, Escherichia coli, Clostridium dakarense, Clostridium perfringens, Bacteroides vulgatus, and Faecalimonas umbilicata. After FMT, the microbiomes exhibited increases in Clostridium dakarense, Clostridium paraputrificum, and Butyricicoccus pullicaecorum. The microbiome alpha diversity of TRE dogs was lower when on tylosin treatment compared to healthy dogs, but it increased after treatment in both the FMT and placebo groups. Comparisons with the stool donor showed that, on average, 30.4% of donor strains were engrafted in FMT recipients, with the most common strains being several Blautia sp., Ruminococcus gnavus, unclassified Lachnoclostridium, Collinsella intestinalis, and Fournierella massiliensis.

Gut microbiome perturbation and its correlation with tylosin pharmacokinetics in healthy and infected pigs.

Tylosin, an antibiotic with a long history in treating respiratory bacterial infections, has unknown effects on the gut microbiota of healthy and infected pigs. The study aimed to investigate the effect of a therapeutic dose of tylosin on swine gut microbiota and explored the relationship between this effect and tylosin pharmacokinetics (PK). We also assessed whether changes in gut microbiota after tylosin administration differ between healthy animals (n = 7) and animals intranasally co-infected (n = 7) with Actinobacillus pleuropneumoniae and Pasteurella multocida. Both groups were intramuscularly administered with tylosin (20 mg/kg). The 16S rRNA gene analyses revealed a significantly lower species richness and diversity, after tylosin treatment, in the infected than the healthy pigs, with infected pigs having lower levels of Bacteroidetes and Firmicutes and higher levels of Proteobacteria. Greater tylosin exposure (greater area under curve (AUC) and maximum plasma concentration (Cmax), and slower elimination (longer terminal half-life, T1/2) were observed in healthy than infected pigs. Relative abundance of Lactobacillus, Oscillibacter, Prevotella, and Sporobacter was positively and significantly correlated with AUC and Cmax, whereas the abundance of Acinetobacter, Alishewanella, and Pseudomonas was positively and significantly correlated with T1/2 and mean residence time (MRT) of tylosin. Our findings, for the first time, demonstrated significant changes in swine gut microbiota after a single therapeutic dose of tylosin was administered, whereas the effect of these changes on tylosin PK was not evident.

Photodegradation of tylosin tartrate by advanced oxidation processes.

Tylosin tartrate, a macrolide antibiotic, is one of a class of emerging contaminants that have been detected in natural bodies of water since they are not easily removed by conventional treatment processes. In this study, the direct and indirect photodegradation of tylosin tartrate was analyzed to understand the role of reactive oxygen species and organic matter that may be present in surface waters. While direct photolysis caused negligible degradation (k = (9.4 ± 1.8) × 10-5 s-1), the addition of 0.4 M hydrogen peroxide (k = (2.18 ± 0.01) × 10-4 s-1) or usage of the photo-Fenton process (k = (2.96 ± 0.02) × 10-4 s-1) resulted in greater degradation. The degradation was maximized by combining tylosin tartrate with an experimentally determined optimal concentration of humic acid (15 mg/L), which readily produced singlet oxygen and increased the overall degradation (k = 1.31 ± 0.05) × 10-3 s-1) by means of indirect photolysis. Absolute pseudo-first-order bimolecular reaction rate constants for tylosin tartrate were measured with singlet oxygen [(4.7936 ± 0.0001) × 105 M-1 s-1] and hydroxyl radical [(5.2693 ± 0.0002) × 109 M-1 s-1] using competition kinetics, and when combined with data on concentration of the reactive oxygen species, showed that the hydroxyl radical makes a contribution to the degradation that is approximately eleven orders of magnitude greater than singlet oxygen.

Enhanced adsorption of tylosin by ordered multistage porous carbon and efficient in-situ regeneration of saturated adsorbents by activated persulfate oxidation: Performance, mechanism and multiple cycles.

In this study, a novel ordered multistage porous carbon (OMPC) with a micro-mesoporous structure was prepared and used for the removal of tylosin (TYL). The porous material, carbonized at 900 °C (OMPC-900), exhibited micro-mesoporous structures with pore sizes of 0.71 nm and 3.63 nm, while had a specific surface area of 1300.02 m2 g-1. OMPC-900 demonstrated a maximum adsorption capacity of 341.28 mg g-1 for TYL in water by electrostatic attraction, hydrogen bonding, π-π interactions, and pore-filling mechanisms, which is 6.41 times higher than that of activated carbon. The TYL-saturated adsorbents could be efficiently regenerated by in-situ oxidation through the activation of persulfate (PDS), achieving a regeneration rate of 94.17%, significantly higher than that of activated carbon (55.22%). The excellent regeneration performance may be attributed to the presence of -C=O and graphitic carbon in the adsorbent, which promotes the production of free radicals (•OH, SO4•- and •O2-) and non-free radicals. Among these, the non-radical pathways (1O2 and electron transfer) played a key role in the degradation of TYL loaded on the adsorbent. OMPC-900 maintained stable regenerative adsorption performance of 80.85% after five in-situ regeneration, and the normalized adsorption capacity per unit surface area increased from 0.21 to 0.39 mg m-2, which may be due to that the increase in oxygen-carbon ratio and surface defects improved the adsorption sites activity of the regenerated adsorbent. In comparison to conventional pyrolysis and organic solvent elution, oxidative regeneration through the activation of PDS is a more efficient and sustainable method.

Structural and computational insights into the substrate specificity of acyltransferase domains from modular polyketide synthases.

Polyketides are natural products synthesized by polyketide synthases (PKSs), where acyltransferase (AT) domains play a crucial role in selection of extender units. Engineering of AT domains enables the site-specific incorporation of non-natural extender units, leading to generation of novel derivatives. Here, we determined the crystal structures of AT domains from the fifth module of tylosin PKS (TylAT5) derived from Streptomyces fradiae and the eighth module of spinosad PKS (SpnAT8) derived from Saccharopolyspora spinosa, and combined them with molecular dynamics simulations and enzyme kinetic studies to elucidate the molecular basis of substrate selection. The ethylmalonyl-CoA-specific conserved motif TAGH of TylAT5 and the MMCoA-specific conserved motif YASH of SpnAT8 were identified within the substrate-binding pocket, and several key residues close to the substrate acyl moiety were located. Through site-directed mutagenesis of four residues near the active site, we successfully reprogrammed the specificity of these two AT domains toward malonyl-CoA. Mutations in TylAT5 enhanced its catalytic activity 2.6-fold toward malonyl-CoA, and mutations in SpnAT8 eliminated the substrate promiscuity. These results extend our understanding of AT substrate specificity and would benefit the engineering of PKSs.

Utilizing two different sustainable and green spectrophotometric approaches using derivative ratio spectra for the determination of a ternary severely overlapped mixture: application to veterinary formulation.

Mathematical manipulation technique has proven to be a very powerful tool for efficient processing and handling of highly overlapped spectra. This work introduced two green and sustainable approaches for the successful recovery of the ternary mixture, Tylosin tartarate (TYL), Sulfadimdine (SLD), and Trimethoprim (TRI). The approaches were constructed using three different methods, derivative ratio spectrum zero-crossing method (DRSZ), double divisor ratio spectra derivative method (DDRD), and factorized derivative ratio method coupled with spectrum subtraction (FDRM-SS). The two approaches succeeded in recovering the three drugs (Linearity range achieved were 0.5-5 µg/mL for TYL, 0.3-1.3 µg/mL for SLD, and 0.3-5 µg/mL for TRI), giving convenient standard deviations and satisfactory recovery percentages. The recommended methods have been verified in accordance with (ICH) guidelines. When the results were statistically compared to the official methods, no significant difference was found. Both AGREE-Analytical GREEnness Metric Approach and Software, and White Analytical Chemistry (WAC) RGB model gave scores of 0.93 and 97.2%, respectively, which proved that the approaches were eco-friendly and abiding by the sustainability principles.

Effect of theophylline on serum and milk pharmacokinetics of tylosin following intramuscular administration in lactating goats.

AIM OF THE WORK: The study was conducted to evaluate the influence of theophylline pre-treatment on serum pharmacokinetics and milk elimination of tylosin following single intramuscular (IM) administrations in lactating goats. METHODS AND RESULTS: In a cross-over study, tylosin was injected via intramuscular (IM) at a single dose of 15 mg/kg b.wt. After a one-month washout period goats received theophylline at a daily IM dose of 2 mg/kg b.wt. for seven consecutive days then tylosin was injected IM dose of 15 mg/kg b.wt. two hours after the last theophylline dosing. Blood samples were collected before and at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12, and 24 h post-injection. Samples were left to clot and then centrifuged to yield serum. Milk samples were collected before and at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 72 h post-injection from each goat by hand milking. Tylosin serum concentrations were determined by high-performance liquid chromatography (HPLC). Tylosin concentrations versus time were analyzed by a noncompartmental method. Tylosin Cmax significantly declined from 1.73 ± 0.10 to 1.01 ± 0.11 µg/ml, and attained Tmax values of 2 and 1 h, respectively in theophylline-pretreated goats. Moreover, theophylline pretreatment significantly shortened the elimination half-life (t1/2el) from 6.94 to 1.98 h, t1/2ka from 0.62 to 0.36 h and the mean residence time (MRT) from 8.02 to 4.31 h, also Vz/F and AUCs decreased from 11.91 to 7.70 L/kg and from 12.64 to 4.57 µg*h/ml, respectively, consequently, theophylline enhanced the clearance (Cl/F) of tylosin from the body. Similarly, tylosin milk concentrations were significantly lower in theophylline-pretreated goats than in goats that received tylosin alone and were detected up to 24 and 72 h in both groups, respectively. Moreover, the t1/2el and AUCs were significantly decreased from 14.68 ± 1.97 to 4.72 ± 0.48 h, and from 181 to 67.20 µg*h/ml, respectively. CONCLUSIONS: The withdrawal period for tylosin in goat milk is at least 72 h. Theophylline pretreatment significantly decreases serum and milk tylosin concentrations to subtherapeutic levels, which could have serious clinical consequences such as failure of therapy. This means that after administering tylosin to goats, milk from these animals should not be consumed for at least 96 h to ensure that the milk is free from residues of the antibiotic.

Bioconversion of cow manure through vermicomposting: effects of tylosin concentration on the weight of worms and manure quality.

This study investigated batch-fed vermicomposting of cow manure, with a specific focus on assessing the effects of tylosin on the weight of earthworms and the overall quality of the resulting manure. Five reactors, including three concentrations of tylosin (50, 100, and 150 mg/kg) and two control reactors, were employed. Residual tylosin concentrations were measured using high-performance liquid chromatography (HPLC). Quality parameters such as pH, temperature, volatile solids (VS), organic carbon content (OCC), electrical conductivity (EC), ash content, C/N ratio, total Kjeldahl nitrogen (TKN), and microbial content were evaluated. The toxicity and maturity of vermicompost were assessed by determining the germination index (GI). The study also monitored variations in the earthworm's weight. The results demonstrated a decreasing trend in VS, OCC, C/N, and fecal coliforms, along with increased pH, EC, ash content, and TKN during the vermicomposting process. Furthermore, investigations revealed significant reductions in the reactors with tylosin concentrations of 50, 100, and 150 mg/kg, resulting in the removal of 98%, 90.48%, and 89.38% of the initial tylosin, respectively. This result confirms the faster removal of tylosin in reactors with lower concentrations. Degradation of tylosin also conforms to first-order kinetics. The findings showed a significant influence of tylosin on the weight of Eisenia fetida earthworms and the lowest antibiotic concentration led to the highest weight gain. Finally, the high percentage of germination index (90-100%) showed that the quality and maturity of vermicompost is by national and international standards.

Exploration of Streptomyces fradiae J1-021 as a Potential Host for the Heterologous Production of Spinosad.

Spinosad is a potent insecticide produced by Saccharopolyspora spinosa. However, it harbors certain limitations of a low growing rate and unfeasible genetic manipulation that can be overcome by adopting a superior platform, such as Streptomyces. Herein, we exploited the industrial tylosin-producing Streptomyces fradiae J1-021 for the heterologous production of spinosad. An engineered strain (HW01) with deletion of the tylosin biosynthetic gene cluster (BGC) was constructed and then transformed with the natural spinosad BGC. The distribution and expression levels of the tylosin BGC operons were assessed to construct a natural promoter library. The rate-limiting steps of spinosad biosynthesis were identified by analyzing the transcriptional expression of the spinosad biosynthetic genes. The stepwise engineering work involved the overexpression of the biosynthetic genes participating in rate-limiting pathways using strong promoters, affording an increase in spinosad production to 112.4 µg/L. These results demonstrate that strain HW01 has the potential to be used as a chassis for the heterologous production of polyketides.

Comparison of tet(X4)-containing contigs assembled from metagenomic sequencing data with plasmid sequences of isolates from a cohort of healthy subjects.

Recently discovered tet(X) gene variants have provided new insights into microbial antibiotic resistance mechanisms and their potential consequences for public health. This study focused on detection, analysis, and characterization of Tet(X4)-positive Enterobacterales from the gut microbiota of a healthy cohort of individuals in Singapore using cultivation-dependent and cultivation-independent approaches. Twelve Tet(X4)-positive Enterobacterales strains that were previously obtained from the cohort were fully genome-sequenced and comparatively analyzed. A metagenomic sequencing (MS) data set of the same samples was mined for contigs that harbored the tet(X4) resistance gene. The sequences of tet(X4)-containing contigs and plasmids sequences were compared. The presence of the resistance genes floR and estT (previously annotated as catD) was detected in the same cassette in 10 and 12 out of the 12 tet(X4)-carrying plasmids, respectively. MS detected tet(X4)-containing contigs in 2 out of the 109 subjects, while cultivation-dependent analysis previously reported a prevalence of 10.1%. The tet(X4)-containing sequences assembled from MS data are relatively short (~14 to 33 kb) but show high similarity to the respective plasmid sequences of the isolates. Our findings show that MS can complement efforts in the surveillance of antibiotic resistance genes for clinical samples, while it has a lower sensitivity than a cultivation-based method when the target organism has a low abundance. Further optimization is required if MS is to be utilized in antibiotic resistance surveillance.IMPORTANCEThe global rise in antibiotic resistance makes it necessary to develop and apply new approaches to detect and monitor the prevalence of antibiotic resistance genes in human populations. In this regard, of particular interest are resistances against last-resort antibiotics, such as tigecycline. In this study, we show that metagenomic sequencing can help to detect high abundance of the tigecycline resistance gene tet(X4) in fecal samples from a cohort of healthy human subjects. However, cultivation-based approaches currently remain the most reliable and cost-effective method for detection of antibiotic-resistant bacteria.

Simultaneous determination of sulfonamides, trimethoprim, amoxicillin and tylosin in medicated feed by high performance liquid chromatography with mass spectrometry.

Introduction: The article presents a rapid and simple analytical procedure for determination of four sulfonamides (sulfadiazine, sulfamerazine, sulfamethazine and sulfamethoxazole), trimethoprim, tylosin and amoxicillin in animal medicated feed. Material and Methods: Eighteen medicated feed samples were analysed for active substances. The analytical protocol used a mixture of acetonitrile and 0.05 M phosphoric buffer, pH 4.5 for the extraction of seven antibacterial substances. After extraction, the samples were diluted in Milli-Q water and analysed by liquid chromatography with mass spectrometry. The developed procedure was subjected to validation in terms of linearity, selectivity, limits of quantification and determination, repeatability, reproducibility and uncertainty. Results: The validation of the method was carried out in accordance with the criteria set out in Commission Implementing Regulation (EU) 2021/808 and ICH guidelines. This method provided average recoveries of 90.8 to 104.5% with coefficients of variation for repeatability and reproducibility in the ranges of 3.2-6.9% and 5.2-8.3%, respectively for all analysed antibacterial substances. The limit of detection and limit of quantification for all seven analytes ranged from 5.4 mg/kg to 48.3 mg/kg and from 10.4 mg/kg to 119.3 mg/kg, respectively. The uncertainty of the method depending on the compound varied from 14.0% to 24.0%. The validated method was successfully applied to the 18 medicated feeds. Conclusion: The developed method can be successfully used to routinely control the content and homogeneity of seven antibacterial substances in medicated feed.

Application of salting-out assisted liquid-liquid extraction for the determination of oxytetracycline, tetracycline, tilmicosin, and tylosin in cow milk by liquid chromatography with photodiode array detection.

This paper introduces a novel, sensitive, and rapid method for the quantification of oxytetracycline, tetracycline, tilmicosin, and tylosin residues in cow's milk. The method involves a two-step process of extraction and detection. The extraction process uses acetonitrile and salting-out assisted liquid-liquid extraction to isolate the antibiotics from the milk. The detection process employs Liquid Chromatography coupled with photo-diode array detector (PDA) to quantify the antibiotics. The method has been successfully applied to milk samples, demonstrating its effectiveness and potential for widespread use in residue analysis.•The calibration curves for the antibiotics were found to be linear within the range of 0.06-3.0 µg/mL to 0.1-3.0 µg/mL.•The limits of detection for oxytetracycline, tetracycline, tilmicosin, and tylosin were 0.03 µg/mL, 0.02 µg/mL, 0.04 µg/mL, and 0.02 µg/mL respectively.•The method demonstrated an average recovery rate of over 90% from milk samples with peak values reaching up to 0.100-0.200 µg/mL.

Transcriptomic analysis of the effects of tylosin on the protective immunity provided by the Mycoplasma gallisepticum vaccine Vaxsafe MG ts-304.

The antimicrobial tylosin is commonly used to control mycoplasma infections, sometimes in combination with vaccination. However, the efficacy of a live mycoplasma vaccine, when combined with subsequent antimicrobial treatment, against the effects of subsequent infection with a virulent strain is unknown. This study employed differential gene expression analysis to evaluate the effects of tylosin on the protection provided by the live attenuated Vaxsafe MG ts-304 vaccine, which has been shown to be safe and to provide long-term protective immunity against infection with Mycoplasma gallisepticum. The transcriptional profiles of the tracheal mucosa revealed significantly enhanced inflammation, immune cell proliferation and adaptive immune responses in unvaccinated, untreated birds and in unvaccinated birds treated with tylosin 2 weeks after infection with virulent M. gallisepticum. These responses, indicative of the typical immune dysregulation caused by infection with M. gallisepticum, were less severe in the unvaccinated, tylosin-treated birds than in the unvaccinated, untreated birds. This was attributable to the effect of residual levels of tylosin in the tracheal mucosa on replication of virulent M. gallisepticum. These responses were not detected in vaccinated, tylosin-treated birds or in vaccinated, untreated birds after infection. The tracheal mucosal transcriptional profiles of these birds resembled those of unvaccinated, untreated, uninfected birds, suggesting a rapid and protective secondary immune response and effective vaccination. Overall, these results show that, although tylosin treatment reduced the duration of immunity, the initial protective immunity induced by Vaxsafe MG ts-304 lasted for at least 22 weeks after vaccination, even after the administration of tylosin for 16 weeks following vaccination.

Anti-Toxoplasma gondii effect of tylosin in vitro and in vivo.

BACKGROUND: Toxoplasma gondii is an important protozoan pathogen with medical and veterinary importance worldwide. Drugs currently used for treatment of toxoplasmosis are less effective and sometimes cause serious side effects. There is an urgent need for the development of more effective drugs with relatively low toxicity. METHODS: The effect of tylosin on the viability of host cells was measured using CCK8 assays. To assess the inhibition of tylosin on T. gondii proliferation, a real-time PCR targeting the B1 gene was developed for T. gondii detection and quantification. Total RNA was extracted from parasites treated with tylosin and then subjected to transcriptome analysis by RNA sequencing (RNA-seq). Finally, murine infection models of toxoplasmosis were used to evaluate the protective efficacy of tylosin against T. gondii virulent RH strain or avirulent ME49 strain. RESULTS: We found that tylosin displayed low host toxicity, and its 50% inhibitory concentration was 175.3 µM. Tylsoin also inhibited intracellular T. gondii tachyzoite proliferation, with a 50% effective concentration of 9.759 µM. Transcriptome analysis showed that tylosin remarkably perturbed the gene expression of T. gondii, and genes involved in "ribosome biogenesis (GO:0042254)" and "ribosome (GO:0005840)" were significantly dys-regulated. In a murine model, tylosin treatment alone (100 mg/kg, i.p.) or in combination with sulfadiazine sodium (200 mg/kg, i.g.) significantly prolonged the survival time and raised the survival rate of animals infected with T. gondii virulent RH or avirulent ME49 strain. Meanwhile, treatment with tylosin significantly decreased the parasite burdens in multiple organs and decreased the spleen index of mice with acute toxoplasmosis. CONCLUSIONS: Our findings suggest that tylosin exhibited potency against T. gondii both in vitro and in vivo, which offers promise for treatment of human toxoplasmosis.

Re-evaluation of a microbiological acceptable daily intake for tylosin based on its impact on human intestinal microflora.

As veterinary drugs available for fish is very restricted, there is growing trials for repurposing livestock drugs as aquatic animal drugs. Tylosin is one of the most effective antibiotics to treat bacterial infections approved for livestock, and would be used in fish. Hence, we investigated the toxicological and microbiological aspects of tylosin to establish health-based guidance value (HBGV) and maximum residue limit (MRL) in fishes, and reevaluated the microbiological acceptable daily intake (mADI) based on updated relevant data and international guildeline. Lastly, exposure assessment was performed to confirm the appropriateness of MRL. By investigating available microbiologcial studies on tylosin, the microbiological point of departure was determined as 0.308 µg/mL, which was mean 50% minimum inhibitory concentration (MIC50), obtained from the Food Safety Committee of Japan (FSCJ) evaluation report. Furthermore, as a factor for the derivation of mADI, the volume of colon content was recently changed to 500 mL in compliance with the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) guidelines. This was previously defined as the mass of colon content (220 g). We applied correction factor 0.224 to the mean MIC50 for tylosin in the equation of mADI, since the drug is transformed to metabolites with reduced activity prior to entering the colon and bound to fecal materials within the colon of human. The mADI was evaluated as 0.01 mg/kg bw/day. Finally, the hazard index, calculated by dividing the estimated chronic dietary exposure by mADI, did not exceed 100%, suggesting that chronic dietary exposure to tylosin residues from veterinary use was unlikely to be a public health concern. Overall, this study contributes significantly in updating HBGV by application of the concept of mADI for the first time in Korea based on the revised microbiological risk assessment guidelines and in providing scientific rationale for the risk management of veterinary drug residues in food. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00179-z.

Production of monoclonal antibody against tylosin and tilmicosin with homogeneous cross-reactivity and its application in lateral flow immunoassay.

To avoid false negative results due to the low cross-reactivity rate (CR) in rapid immunoassay, a group-specific antibody with homogeneous CR toward target compounds is needed for accuracy. In this study, tylosin (TYL) and tilmicosin (TM) were selected as model molecules. Firstly, two-dimensional similarity, electrostatic potential energy, spatial conformation and charge distribution of the haptens TYL-CMO, TYL-6-ACA, TYL-4-APA, TYL-CHO and DES-CMO and target compounds of TYL and TM were obtained using Gaussian 09W and Discovery Studio. The optimal hapten was DES-CMO because it is the most similar to TYL and TM. Subsequently, the mAb 14D5 cell line was obtained with IC50 values of 1.59 and 1.72 ng/mL for TYL and TM, respectively, and a CR of 92.44%. Finally, amorphous carbon nanoparticles (ACNPs) were conjugated with mAb 14D5 to develop an accurate lateral flow immunoassay (LFA) for detection of TYL and TM by the reflectance value under natural light. The recoveries of TYL and TM ranged from 77.18 to 112.04% with coefficient of variation < 13.43%. The cut-off value in milk samples was 8 ng/mL, and the limits of detection were 11.44, 15.96, 22.29 and 25.53 µg/kg for chicken muscle, bovine muscle, porcine muscle and porcine liver samples, respectively, and the results being consistent with HPLC-UV. The results suggest that the developed LFA is accurate and potentially useful for on-site screening of TYL and TM in milk and animal tissue samples.

Simultaneous Screening of Six Families of Antibiotic Residues in Milk Samples by Biochip Multi-array Technology.

Background: Antimicrobial compounds are used in animal husbandry to prevent and treat bacterial diseases and as illegal growth-promoting agents. Due to the excessive and inappropriate use of antibiotics, the antibiotic residues in milk can cause allergic reactions and antibiotic resistance. A rapid biochip-based method for the multi-analyte screening of 6 families of antibiotic residues (quinolones, ceftiofur, florfenicol, streptomycin, tylosin, and tetracyclines) in milk was validated based on Commission Decision 2002/657 and the European guidance for screening methods for veterinary medicinal products. Methods: This methodology allows the 6 antibiotic families to be detected simultaneously, increasing the screening capacity and reducing costs in test settings. The method's applicability was shown by screening 38 UHT cow milk samples taken from Tehran province, IR Iran. Results: The results showed that the positive threshold T was above Fm, and the CCß was below the European Commission's Maximum Residue Limit (MRL) (100 ppb for ceftiofur and tetracycline and 50 ppb for tylosin in milk). Norfloxacin was detected in about 8% of the samples and tylosin in 2.63%. The total antibiotic concentration in UHT cow milk samples was lower than the European Commission's MRL. Conclusions: This study showed that the biochip technique is valid for screening tylosin, ceftiofur, streptomycin, tetracycline, norfloxacin, and florfenicol in milk. It was found that the method was easy, quick, and capable of detecting 6 families of antibiotic residues simultaneously from a single milk sample without sample preparation.

The photocatalytic degradation of tylosin by a trimetallic ZnCrNi/GO-layered double hydroxide in the conformation of a clustered crumb sheet.

Industrial wastewater from drug production is one of the contributors to water pollution. For drug wastewater treatment, photodegradation-based chemical technology has gained more attention because of the drug's microbicidal nature and stability. A zinc-chromium-nickel trimetallic-layered double hydroxide compounding with graphene oxide catalyst (ZnCrNi/GO) was synthesized and exhibited a clustered crumb sheet morphology. The prepared catalyst was characterized by a scanning electron microscope (SEM) equipped with an energy-dispersive X-ray spectrometer (EDS), Fourier transform infrared (FTIR) spectra, and X-ray photoelectron spectroscopy (XPS). The results of material analysis established the crystallographic structures of catalysts and evidenced the successful synthesis. The ZnCrNi/GO nanohybrid revealed a higher activity of approximately 90% degradation of tolysin under high-pressure mercury lamp irradiation. The optimized condition of the catalyst dosage of 500 mg/L and the natural pH of the solution at 7.0 under the tylosin concentration of 10 mg/L with high photocatalytic efficiency was explored. In addition, the main reactive species involved in this photocatalysis degradation were explored as the active cavity h+ and ·O2- to a certain extent by the radical trapping experiments. Reuse experiments have shown that as-prepared catalysts possessed the properties of high efficiency and long-lasting catalytic performance, which could meet pharmaceutical wastewater treatment. A three-metal-layered double hydroxide composed by the metal of Ni, Zn, and Cr was synthesized and attached onto graphene oxide. The catalytic materials obtained in this way have a significant catalysis efficiency to tylosin with the likely degradation mechanism of the active cavity h+ and the oxidative capacity of hydroxyl radials.

Structural basis of Cfr-mediated antimicrobial resistance and mechanisms for its evasion.

The ribosome is an essential drug target as many classes of clinically important antibiotics bind and inhibit its functional centers. The catalytic peptidyl transferase center (PTC) is targeted by the broadest array of inhibitors belonging to several chemical classes. One of the most abundant and clinically prevalent mechanisms of resistance to PTC-acting drugs is C8-methylation of the universally conserved adenine residue 2503 (A2503) of the 23S rRNA by the methyltransferase Cfr. Despite its clinical significance, a sufficient understanding of the molecular mechanisms underlying Cfr-mediated resistance is currently lacking. In this work, we developed a method to express a functionally-active Cfr-methyltransferase in the thermophilic bacterium Thermus thermophilus and report a set of high-resolution structures of the Cfr-modified 70S ribosome containing aminoacyl- and peptidyl-tRNAs. Our structures reveal that an allosteric rearrangement of nucleotide A2062 upon Cfr-methylation of A2503 is likely responsible for the inability of some PTC inhibitors to bind to the ribosome, providing additional insights into the Cfr resistance mechanism. Lastly, by determining the structures of the Cfr-methylated ribosome in complex with the antibiotics iboxamycin and tylosin, we provide the structural bases behind two distinct mechanisms of evading Cfr-mediated resistance.