Core and variable antimicrobial resistance genes in the gut microbiomes of Chinese and European pigs

Monitoring the prevalence of antimicrobial resistance genes(ARGs)is vital for addressing the global crisis of antibiotic-resistant bacterial infections.Despite its importance,the characterization of ARGs and microbiome structures,as well as the identification of indicators for routine ARG monitoring in pig farms,are still lacking,particularly concerning variations in antimicrobial exposure in different countries or regions.Here,metagenomics and random forest machine learning were used to elucidate the ARG profiles,microbiome structures,and ARG contamination indicators in pig manure under different antimicrobial pressures between China and Europe.Results showed that Chinese pigs exposed to high-level antimicrobials exhibited higher total and plasmid-mediated ARG abundances compared to those in European pigs(P<0.05).ANT(6)-Ib,APH(3')-IIIa,and tet(40)were identified as shared core ARGs between the two pig populations.Furthermore,the core ARGs identified in pig populations were correlated with those found in human populations within the same geographical regions.Lactobacillus and Prevotella were identified as the dominant genera in the core microbiomes of Chinese and European pigs,respectively.Forty ARG markers and 43 biomarkers were able to differentiate between the Chinese and European pig manure samples with accuracies of 100%and 98.7%,respectively.Indicators for assessing ARG contamination in Chinese and European pigs also achieved high accuracy(r=0.72-0.88).Escherichia flexneri in both Chinese and European pig populations carried between 21 and 37 ARGs.The results of this study emphasize the importance of global collaboration in reducing antimicrobial resistance risk and provide validated indicators for evaluating the risk of ARG contamination in pig farms.

Preparation and interaction mechanism analysis of single‑chain fragment variables against phenylethanolamine A

This is the first report on the screening,expression,and recognition mechanism analysis of single-chain fragment variable(scFv)against phenylethanolamine A(PEAA),a newly emergedβ-adrenergic agonist illegally used as a feed additive for growth promotion.The PEAA-specific scFv scFv,called scFv-32,was screened from hybridoma cell lines by phage display and was found to be optimally expressed in the E.coli system.The ic-ELISA results revealed an IC_(50)value of 10.34μg/L for scFv-32 and no cross-reactivity with otherβ-adrenergic agonists.Homology modeling and molecular docking revealed the key binding sites VAL178,TYP228,and ASP229.One hydrogen bond,two pisigma bonds,and one pi-pi bond maintain the formation of the antibody‒drug complex.Alanine scanning mutagenesis of the three predicted key binding sites showed that the mutants completely lost their recognition activity,which confirmed the accuracy of the theoretical analysis.These results are valuable for the preparation of scFvs and the analysis of the molecular recognition mechanism of antigen-antibodies.

Antimicrobial resistance and molecular typing of Salmonella in the chicken production chain in Hubei Province, China

Salmonella is a significant foodborne zoonotic pathogen that endangers both human and animal health.The goal of this research is to gain a preliminary understanding of Salmonella contamination and antimicrobial resistance in the chicken production chain in Hubei Province,China.1149 animal and environmental samples were collected from chicken farms,slaughterhouses,and retail markets in six cities across Hubei Province in China from 2019 to 2020,yielding Salmonella isolation rates of 4.68%(28/598),12.21%(47/385),and 9.64%(16/166),respectively.Seventeen distinct serotypes were detected among 53 non-clonal Salmonella strains,of which Meleagridis(26.42%,14/53)was the dominant serotype.Almost half of the strains(49.06%,26/53)were multi-drug resistant(MDR).Whole-genome sequencing(WGS)showed that 10 resistance genes(tetA,bla_(TEM),parC,qnrs1,floR,aac(6'-ly,aph(6)-ld,aph(3")-b,aac(6')-laa and sul2)and 7 categories of virulence genes were present in all three links in 22 non-clonal dominant serotype strains.It was shown that Salmonella in the chicken production chain in Hubei Province had a high resist-ance rate to Tetracycline(TET,73.58%),Ofloxacin(OFL,69.81%),Florfenicol(FFC,60.38%)and Ampicillin(AMP,39.62%)which was consistent with the widespread use of these drugs in the husbandry industry in China.Salmonella ST types determined by MLST and serotypes determined by WGS had a one-to-one correlation.Minimum spanning tree analysis revealed that there was cross contamination of Salmonella in farms and slaughterhouses,slaughterhouses and markets,animal samples and environmental samples.This work provides useful information for the prevention and control of contamination and antimicrobial resistance of Salmonella in the chicken production chain,as well as demonstrating the dependable role of WGS in Salmonella molecular typing.

The dose regimen formulation of doxycycline hydrochloride and florfenicol injection based on ex vivo pharmacokinetic-pharmacodynamic modeling against the Actinobacillus pleuropneumoniae in pigs

Doxycycline hydrochloride and florfenicol combination(DoxHcl&FF)is an effective treatment for respiratory diseases.In the study,our objective Was to evaluate the activity of DoxHcl&FF against Actinobacillus pleuropneumoniae(APP)in porcine pulmonary epithelial lining fluid(PELF)and the optimal dosage scheme to avoid the development of resistance.The DoxHcl&FF Was administered intramuscularly(IM)at 20mg/kg,and the PELF was collected at differ-ent time points.The minimum inhibitory concentration(MIC)and time-mortality curves were also included in the study.Based on the sigmoid Emax equation and dose equations,the study integrated the in vivo pharmacokinetic data of infected pigs and ex vivo pharmacodynamic data to obtain the area under concentration time curve(AUCo-24h)MIC values in PELF and achieve bacteriostatic activity,bactericidal activity and the virtual eradication of bacteria.The study showed that the combination of DoxHcl and FF caused no significant changes in PK parameters.The peak concentration(Cmax)of FF in healthy and diseased pigs was 8.87±0.08 and 8.67±0.07μg/mL,the_AUCo-24h were.172.75±2.52 and 18022±3.13 h-μg/mL,the Cmax of DoxHcl was 7.91±0.09 and 7.99±0.05μg/mL,and the AUCo-24h was 129.96±3.70 h-μg/mL and 169.82±4.38 h-μg/mL.DoxHcl&FF showed strong concentra-tion-dependent tendencies.The bacteriostatic,bactericidal,and elimination activity were calculated as 5.61,18.83 and 32.68 h,and the doses were 1.37(bacteriostatic),4.59(bactericidal)and 7.99(elimination)mg/kg.These findings indicated that the calculated recommended dose could assist in achieving more precise administration,increasing the effectiveness of DoxHcl&FF treatment for APP infections.

Development of High Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Detection of Tulathromycin in Swine Plasma

An accurate and precise method for the determination of tulathromycin in swine plasma was developed and validated.Plasma samples were analyzed by high-performance liquid chromatography with tandem mass spectrometry detection （HPLC-MS/MS） using electrospray ionization （ESI）.Tulathromycin was extracted from plasma by precipitation with acetonitrile and separated using a Phenomenex Luna 5 μm C18 column （150 mm×2.0 mm） at a flow rate of 0.25 mL min^-1.Solvent A consisted of 0.002 mol L^-1 ammonium acetate and formic acid （999：1,v/v）,and solvent B was acetonitrile.The mass spectrometer was operated in the selected-ion mode with atmospheric pressure chemical ionization to monitor the respective MH＋ ions,namely,m/z 577.3 for tulathromycin and m/z 679.3 for the internal standard roxithromycin.The calibration curves were linear in a dynamic range of 2.0-500 ng mL^-1 on the column.The accuracy was ranged from 95.25 to 109.75%,and the precision was ranged from 2.81 to 7.72%.The recoveries measured at 3 concentration levels （20,250,and 500 ng mL^-1） were higher than 98%.The method described above is efficient,and has the required accuracy and precision for rapid determination of tulathromycin in plasma.The method was applied to study the pharmacokinetics of tulathromycin in swine,and tulathromycin demonstrated a rapid absorption,wide distribution,and slow elimination after intramuscular administration.

Metabolism of Mequindox in Isolated Rat Liver Cells

Mequindox （MEQ）, 3-methyl-2-quinoxalinacetyl-l,4-dioxide, is widely used in Chinese veterinary medicine as an antimicrobial agent and feed additive. Its toxicity has been reported to be closely related to its metabolism. To understand the pathways underlying MEQ＇s metabolism more clearly, we studied its metabolism in isolated rat liver cells by using liquid chromatography coupled with electrospray ionization hybrid linear trap quadrupole orbitrap （LC-LTQ-Orbitrap） mass spectrometry. The structures of MEQ metabolites and their product ions were readily and reliably characterized on the basis of accurate MS2 spectra and known structure of MEQ. Eleven metabolites were detected in isolated rat liver cells, two of which were detected for the first time in vitro. The major metabolic pathways reported previously for in vitro metabolism of MEQ in rat microsomes were confirmed in this study, including N O group reduction, carbonyl reduction, and methyl monohydroxylation. In addition, we fotmd that acetyl hydroxylation was an important pathway of MEQ metabolism. The results also demonstrate that cellular systems more closely simulate in vivo conditions than do other in vitro systems such as microsomes. Taken together, these data contribute to our understanding of the in vivo metabolism of MEQ.

Effective protective agents against the organ toxicity of T-2 toxin and corresponding detoxification mechanisms:A narrative review

T-2 toxin is one of the most widespread and toxic fungal toxins in food and feed.It can cause gastrointestinal toxicity,hepatotoxicity,immunotoxicity,reproductive toxicity,neurotoxicity,and nephrotoxicity in humans and animals.T-2 toxin is physicochemically stable and does not readily degrade during food and feed processing.Therefore,suppressing T-2 toxin-induced organ toxicity through antidotes is an urgent issue.Protective agents against the organ toxicity of T-2 toxin have been recorded widely in the literature,but these protective agents and their molecular mechanisms of detoxification have not been comprehensively summarized.In this review,we provide an overview of the various protective agents to T-2 toxin and the molecular mechanisms underlying the detoxification effects.Targeting appropriate targets to antagonize T-2 toxin toxicity is also an important option.This review will provide essential guidance and strategies for the better application and development of T-2 toxin antidotes specific for organ toxicity in the future.

Linear Thanatin Is an Effective Antimicrobial Peptide against Colistin-Resistant <i>Escherichia coli in Vitro</i>

Colistin has been regarded as the last line antibiotic for treatment of infections caused by multidrug resistant gram-negative bacteria. Therefore, the increasing emergence of colistin resistance among gram-negative bacteria represents a serious problem. The objective of this study was to characterize the effectiveness of the chemically synthesized thanatin in linear form against colistin-resistant E. coli isolated from a pig farm in China. Agar diffusion assay and broth microdilution test were employed to analyze the susceptibility of colistin-sensitive E. coli (ATCC25922) and colistin-resistant E. coli (SHP45) to linear thanatin (L-thanatin). Combinatory effect of linear thanatin and colistin against E. coli was also determined by fractional inhibition concentration index (FICI) analysis. The results showed that L-thanatin at a concentration of 1 mg/ml produced larger inhibition zone on agar against ATCC25922 than SHP45. In the quantitative microdilution test, L-thanatin had the same MIC of 3.2 μg/ml for ATCC25922 and SHP45. Based on the FICI analysis, additive effect was obtained with 1.56 μg/ml of L-thanatin and 0.125 μg/ml of colistin for ATCC25922;but with 1.56 μg/ml of L-thanatin and 0.25 μg/ml of colistin or with 2 μg/ml of colistin and 0.39 μg/ml of L-thanatin for SHP45. These data proved that L-thanatin is an effective antimicrobial peptide against colistin-resistant E. coli.

Oxidative stress,the blood-brain barrier and neurodegenerative diseases:The critical beneficial role of dietary antioxidants

In recent years,growing awareness of the role of oxidative stress in brain health has prompted antioxidants,especially dietary antioxidants,to receive growing attention as possible treatments strategies for patients with neurodegenerative diseases(NDs).The most widely studied dietary antioxidants include active substances such as vitamins,carotenoids,flavonoids and polyphenols.Dietary antioxidants are found in usually consumed foods such as fresh fruits,vegetables,nuts and oils and are gaining popularity due to recently growing awareness of their potential for preventive and protective agents against NDs,as well as their abundant natural sources,generally non-toxic nature,and ease of long-term consumption.This review article examines the role of oxidative stress in the development of NDs,explores the‘two-sidedness’of the blood-brain barrier(BBB)as a protective barrier to the nervous system and an impeding barrier to the use of antioxidants as drug medicinal products and/or dietary antioxidants supplements for prevention and therapy and reviews the BBB permeability of common dietary antioxidant suplements and their potential efficacy in the prevention and treatment of NDs.Finally,current challenges and future directions for the prevention and treatment of NDs using dietary antioxidants are discussed,and useful information on the prevention and treatment of NDs is provided.

Functional characterization of novel NPRL3 mutations identified in three families with focal epilepsy

Focal epilepsy accounts for 60% of all forms of epilepsy, but the pathogenic mechanism is not well understood. In this study,three novel mutations in NPRL3(nitrogen permease regulator-like 3), c.937_945del, c.1514dup C and 6,706-bp genomic DNA(g DNA) deletion, were identified in three families with focal epilepsy by linkage analysis, whole exome sequencing(WES) and Sanger sequencing. NPRL3 protein is a component of the GATOR1 complex, a major inhibitor of m TOR signaling. These mutations led to truncation of the NPRL3 protein and hampered the binding between NPRL3 and DEPDC5, which is another component of the GATOR1 complex. Consequently, the mutant proteins enhanced m TOR signaling in cultured cells, possibly due to impaired inhibition of m TORC1 by GATOR1. Knockdown of nprl3 in Drosophila resulted in epilepsy-like behavior and abnormal synaptic development. Taken together, these findings expand the genotypic spectrum of NPRL3-associated focal epilepsy and provide further insight into how NPRL3 mutations lead to epilepsy.

Rapid detection of fluoroquinolone residues in aquatic products based on a gold-labeled microwell immunochromatographic assay

Objectives:Fluoroquinolones(FQs)are widely used in aquaculture,and their residues have caused many problems threatening human health.Here,this study aims to develop a colloidal gold immunochromatographic strip based on gold-labeled microwells to screen the residues of FQs on site.Materials and methods:The Protein A Magarose Beads affinity chromatography method was adopted to purify the ascites against FQs.By using a strategy of heterologous coating antigen,different coating antigens are applied to detect FQs.The gold-labeled microwell immunochromatographic assay was used to improve the sensitivity of the test strip by the advanced reaction of antigen and antibody.Results:The antibodies were verified to be of high purity up to 99%,and the titer reached 1:1024000.The combination(enoxacin-OVA and the antibody)detected the 4 banned FQs(pefloxacin,PEF;norfloxacin,NOR;lomefloxacin,LOM;ofloxacin,OFL)with the 50%inhibiting concentration(IC50)values ranging from 1.3 to 2.1 ng/mL and cross-reactions ranging from 67.3%to 106.1%.The analysis of spiked crucian carp,silver carp,grass carp,and shrimp samples showed that the limit of detection for PEF,NOR,LOM,and OFL was 4μg/kg.A comparative study with liquid chromatography tandem mass spectrometry(LC-MS/MS)demonstrated that the assay provided an effective screening tool for the rapid detection of FQs residues.Conclusions:The results indicated that the test strip can realize full coverage recognition of the 4 banned FQs and has good accuracy,specifi-city,reproducibility,and stability;therefore,they are more suitable for rapid detection of FQs in aquatic products.

G-quadruplexes in genomes of viruses infecting eukaryotes or prokaryotes are under different selection pressures from hosts

G-quadruplexes in viral genomes can be applied as the targets of antiviral therapies, which has attracted wide interest. However, it is still not clear whether the pervasive number of such elements in the viral world is the result of natural selection for functionality. In this study, we identified putative quadruplex-forming sequences(PQSs) across the known viral genomes and analyzed the abundance, structural stability, and conservation of viral PQSs. A Viral Putative G-quadruplex Database(http://jsjds.hzau.edu.cn/MBPC/Vi PGD/index.php/home/index) was constructed to collect the details of each viral PQS, which provides guidance for selecting the desirable PQS. The PQS with two putative G-tetrads(G2-PQS) was significantly enriched in both eukaryotic viruses and prokaryotic viruses, whereas the PQSs with three putative G-tetrads(G3-PQS) were only enriched in eukaryotic viruses and depleted in prokaryotic viruses. The structural stability of PQSs in prokaryotic viruses was significantly lower than that in eukaryotic viruses. Conservation analysis showed that the G2-PQS, instead of G3-PQS, was highly conserved within the genus. This suggested that the G2-quadruplex might play an important role in viral biology, and the difference in the occurrence of G-quadruplex between eukaryotic viruses and prokaryotic viruses may result from the different selection pressures from hosts.

Cytochrome P450 enzymes mediated by DNA methylation is involved in deoxynivalenol-induced hepatoxicity in piglets

Deoxynivalenol(DON)is an inevitable contaminant in animal feed and can lead to liver damage,then decreasing appetite and causing growth retardation in piglets.Although many molecular mechanisms are related to hepatoxicity caused by DON,few studies have been done on cytochrome P450(CYP450)enzymes and DNA methylation.To explore the role of CYP450 enzymes and DNA methylation in DONinduced liver injury,male piglets were fed a control diet,or diet containing 1.0 or 3.0 mg/kg DON for 4 weeks.DON significantly raised the activity of aspartate aminotransferase(AST),alanine aminotransferase(ALT),and glutamyl transpeptidase(GGT)(P<0.01),leading to liver injury.In vivo study found that DON exposure increased the expression of CYP450 enzymes(such as CYP1A1,CYP2E1,CYP3A29)(P<0.05),and disturbed the expression of nicotinamide N-methyltransferase(NNMT),galanin-like peptide(GALP)and insulin-like growth factor 1(IGF-1)(P<0.05),in which DNA methylation affected the expression of these genes.In vitro study(human normal hepatocytes L02)further proved that DON elevated the expression of CYP1A1,CYP2E1 and CYP3A4(P<0.05),and inhibited cell growth in a dose-dependent manner,resulting in cell necrosis.More importantly,knockdown of CYP1A1 or CYP2E1 could alleviate DON-induced growth inhibition by promoting IGF-1 expression.Taken together,increased CYP450 enzymes expression was one of the mechanisms of hepatoxicity and growth inhibition induced by DON,suggesting that the decrease of CYP450 enzymes can antagonize the hepatoxicity in animals,which provides some value for animal feed safety.