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.

Insights into the effects of pulsed antimicrobials on the chicken resistome and microbiota from fecal metagenomes

Antimicrobial resistance has become a global problem that poses great threats to human health. Antimicrobials are widely used in broiler chicken production and consequently affect their gut microbiota and resistome. To better understand how continuous antimicrobial use in farm animals alters their microbial ecology, we used a metagenomic approach to investigate the effects of pulsed antimicrobial administration on the bacterial community, antibiotic resistance genes(ARGs) and ARG bacterial hosts in the feces of broiler chickens. Chickens received three 5-day courses of individual or combined antimicrobials, including amoxicillin, chlortetracycline and florfenicol. The florfenicol administration significantly increased the abundance of mcr-1 gene accompanied by floR gene, while amoxicillin significantly increased the abundance of genes encoding the AcrAB-tolC multidrug efflux pump(marA, soxS, sdiA, rob, evgS and phoP).These three antimicrobials all led to an increase in Proteobacteria. The increase in ARG host, Escherichia, was mainly attributed to the β-lactam, chloramphenicol and tetracycline resistance genes harbored by Escherichia under the pulsed antimicrobial treatments. These results indicated that pulsed antimicrobial administration with amoxicillin,chlortetracycline, florfenicol or their combinations significantly increased the abundance of Proteobacteria and enhanced the abundance of particular ARGs. The ARG types were occupied by the multidrug resistance genes and had significant correlations with the total ARGs in the antimicrobial-treated groups. The results of this study provide comprehensive insight into pulsed antimicrobial-mediated alteration of chicken fecal microbiota and resistome.

Identification of blaIMI-mediated carbapenem-resistant Enterobacter from a duck farm in China

Carbapenem-and colistin-resistant Enterobacter has been a clinical and therapy problem in recent years.Here,we report the carbapenem-and colistin-resistant Enterobacter harboring bla_(IMI) isolated from intestinal samples and the environment of a duck farm in China.Four bla_(IMI)-positive Enterobacter isolates were resistant to carbapenem and colistin.Three bla_(IMI) subtypes were detected in different molecular categories of Enterobacter.The detection of the various IMI producers highlights the diversity of carbapenemases in a duck farm.Whole-genome sequencing demonstrated the bla_(IMI) genes were present in chromosomes or plasmids in these strains.The conjugation experiment demonstrated the ability of bla_(IMI)-carrying plasmid to transmit horizontally.The molecular evolution characteristics were examined through comparative genetic analysis.The study demonstrated the presence of chromosomal and plasmid bla_(IMI) and the bla_(IMI)-carrying plasmid exhibits a horizontal transmission between Enterobacter and Escherichia coli C600.The similar genetic content was discovered between two bla_(IMI-16)-positive Enterobacter asburiae.In addition,a bla_(IMI-16)-carrying plasmid is an IncFII(Yp)plasmid,and a substantial amount of mobile genetic elements were identified around bla_(IMI-16).The IS-like elements and IncFII(Yp)plasmid are significant in the propagation of bla_(IMI).Our study provides evidence for the transmission of diverse bla_(IMI) genes in China and supplies additional reference data for bla_(IMI)-positive antimicrobialresistant Enterobacter.Routine surveys of bla_(IMI)-positive Enterobacter from animal-raising environments must be given more focus.

The Estimation of Soil Trace Elements Distribution and Soil-Plant-Animal Continuum in Relation to Trace Elements Status of Sheep in Huangcheng Area of Qilian Mountain Grassland,China

The purpose of the present study was to survey contents of trace elements of Cu, Mn, Fe, and Zn in the surface layer （0-20 cm） in the soil, pasture and serum of sheep in Huangcheng area of Qilian mountain grassland, China. Also the soil-plantanimal continuum was analyzed. Soil （n=300）, pasture （n=60）, and blood serum samples from sheep （n=480） were collected from Huangcheng area of Qilian mountain grassland, China. The contents of trace element in the samples were analyzed by atomic absorption spectrophotometer after digestion. The soil trace elements density distribution shows a ladder-like pattern distribution. Equations developed in the present study for prediction of Fe （R2=0.943） and Zn （R2=0.882） had significant R2 values.

Genetic characterization of antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis cases in Northwest China

Staphylococcus aureus is the most common etiological pathogen of bovine mastitis. The resistant strains make the disease difficult to cure. The aim of this study was to characterize the genetic nature of the antimicrobial resistance in S. aureus cultured from bovine mastitis in Northwest China in 2014. A total of 44 S. aureus were isolated for antimicrobial resistance and resistance-related genes. Antimicrobial resistance was determined by disc diffusion and the corresponding resistance genes were detected by PCR. Phenotype indicated that S. aureus isolates were resistant to penicillin （84.09%）, erythromycin （20.45%）, tetracycline （15.91%）, gentamicin （9.09%）, tobramycin （6.82%）, kanamycin （6.82%） and methicillin （2.27%）. 9.09% of the S. aureus isolates were classified as multidrug resistant. In addition, genotypes showed that the isolates were resistant to rifampicin （100%, rpoB）, penicillin （95.45%, blaZ）, tetracycline （22.73%, tetK, tetM, alone or in combination）, erythromycin （22.73%, ermB or ermC）, gentamicin/tobramycin/kanamycin （2.27%, aacA-aphD）, methicillin （2.27%, mecA） and vancomycin （2.27%, vanA）. Resistance to tetracycline was attributed to the genes tetK and tetM （r=0.558, P〈0.001）. This study noted high-level geno- and phenotypic antimicrobial resistance in S. aureus isolates from bovine mastitis cases in Northwest China.

Effects of 105 traditional Chinese medicines on the detection ofβ-agonists in medicine extracts and swine urine based on colloidal gold immunochromatographic assay

Colloidal gold immunochromatographic assay(CGIA)is commonly used for the on-site detection ofβ-agonists that are sometimes used illegally as feed additives in swine diets.However,few studies have evaluated the causes of false-positive results that sometimes occur when applying CGIA in agricultural settings.In this study,we investigated if this false-positive phenomenon is related to the addition of certain traditional Chinese medicines(TCMs)to swine feed.We established and verified an extraction method for TCMs,and then applied CGIA to detectβ-agonists in the extracts of 105 TCMs and in the urine of swine dosed with TCMs,respectively.Liquid chromatography-tandem mass spectrometry was used to validate the results of the urine samples tested positive forβ-agonists using CGIA.The results were also verified using TCMs and colloidal gold test strips produced by different manufacturers.The extracts of Citri Reticulatae Pericarpium Viride,Citri Reticulatae Pericarpium,Magnoliae Officinalis Cortex,Chaenomelis Fructus,and Rhodiolae Crenulatae Radix Et Rhizoma were tested positive forβ-agonists.Meanwhile,the addition of Citri Reticulatae Pericarpium Viride and Citri Reticulatae Pericarpium to swine feed resulted in false-positive results forβ-agonists in swine urine.The results provide a new way to explain false-positive CGIA results and provide valuable information for livestock feeding programs.

Characterization of NDM-5-producing Enterobacteriaceae isolates from retail grass carp(Ctenopharyngodon idella) and evidence of blaNDM-5-bearing IncHI2 plasmid transfer between ducks and fish

We aimed to characterize NDM-5-producing Enterobacteriaceae from aquatic products in Guangzhou,China.A total of 196 intestinal samples of grass carp collected in 2019 were screened for carbapenemase genes.Characterization of bla_(NDM-5) positive isolates and plasmids was determined by antimicrobial susceptibility testing,conjugation experiments,Illumina HiSeq,and Nanopore sequencing.One Citrobacter freundii and six Escherichia coli strains recovered from seven intestinal samples were verified as bla_(NDM-5) carriers(3.57%,7/196).The bla_(NDM-5) genes were located on the lncX3(n=5),lncHI2(n=1),or lncHI2-lncF(n=1)plasmids.All bla_(NDM-5)-bearing plasmids were transferred by conjugation at frequencies of~10^(-4)-10^(-6).Based on sequence analysis,the lncHI2 plasmid pHNBYF33-1 was similar to other bla_(NDM-5)-carrying lncHI2 plasmids deposited in GenBank from Guangdong ducks.In all lncHI2 plasmids,bla_(NDM-5)was embedded in a novel transposon,Tn7057(IS3000-△ISAba125-IS5-△ISAba125-bla_(NDM-5)-bleMBL-trpF-tat-△dct-IS26-△umuD-△ISKox3-IS3000),which was identical to the genetic structure surrounding bla_(NDM-5)found in some IncX3 plasmids.The lncHI2-lncF hybrid plasmid pHNTH9F11-1 was formed by homologous recombination of the bla_(NDM-5)-carrying lncHI2 plasmid and a heavy-metal-resistant IncF plasmid through△Tn1721 To the best of our knowledge,this is the first report on the characterization of bla_(NDM-5)-bearing plasmids in fish in China.The lncHI2 plasmid pHNBYF33-1 may be transmitted from ducks,considering the common duck-fish freshwater aquaculture system in Guangdong.Tn7051 is likely responsible for the transfer of bla_(NDM-5) from lncX3 to lncHI2 plasmids in Enterobacteriaceae,resulting in the expansion of transmission vectors of bla_(NDM-5).

Effect of Fuzheng Jiedu granule on immunological function and level of immune-related cytokines in immune-suppressed mice

Fuzheng Jiedu granule exhibits a number of health benefits and it is thought that the mechanisms involved in these effects are due to the modulation of immunity. In this article, we studied the effect of Fuzheng Jiedu granule on immunological function and the expression of immune-related cytokines in immune-suppressed mice. 72 mice were randomly divided into six groups, with 12 in each group. The control groups included an untreated group, a negative control group（Cyclophosphamide） and a positive control group（Astragalus polysaccharide）. There were three treated groups, which were given different doses of Fuzheng Jiedu granule： a low dose（100 mg kg^（–1））, a medium dose（400 mg kg^（–1）） and a high dose（600 mg kg^（–1））. With the exception of the untreated control animals, each group received an intraperitoneal injection of Cyclophosphamide（100 mg kg^（–1）） for 3 days to establish the immune-suppressed model. Mice were then treated for 19 consecutive days and, 24 h after the last treatment, blood was taken for the eyeballs and serum separation was performed. Analysis was made of the levels of related cytokines（IgA, IgG, IgM, IL-6, IFN-γ, C3, C4 and TNF-α）, the transformation of lymphocytes and the immune organ indexes. The results showed that Fuzheng Jiedu granule can improve the levels of cytokines, the rate of proliferation of lymphocytes and the immune organ indexes of immune-suppressed mice.

Comparative Proteomic Analysis Shows an Elevation of Mdh1 Associated with Hepatotoxicity Induced by Copper Nanoparticle in Rats

Copper nanoparticle is a new material widely used in biological medicine, animal husbandry and industrial areas, but its potential toxicity to human health and environment remains unclear. In order to study the hepatotoxic mechanisms of nanoparticles copper, two-dimensional gel electrophoresis （2-DE） and matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI/TOF MS） of proteomics technology were used to isolate and identify the differentially expressed proteins from liver, which associated with hepatotoxicity induced by copper nanoparticle in rats. In this study, we have screened 15 kinds of proteins related with hepatotoxicity, of which spot8212 was identified as Malate dehydrogenase （Mdhl）. The mRNA expression trend of Mdhl was consistent with the result of 2-DE by RT-PCR validation. Bioinformatics analysis showed that Mdhl was stable and no signal peptides, subcellular location was in endoplasmic reticulum; it contained many functional sites such as malate dehydrogenase activity signal sites 155LTRLDHNRAKSQI167; a helixes and random coils were the two main elements. Homologous analysis demonstrated high homologous of Mdhl in rats with mouse and human, and the phylogenetic tree of Mdhl was constructed. The result indicated that copper nanoparticle could regulate up the Mdhl protein expression so as to compensate the energy deficit. Energy metabolic disturbance may be a pathway for copper nanoparticle particles to exert the hepatotoxic effects in rats.

Syntheses,Crystal Structures and Antibacterial Evaluation of Two New Pleuromutilin Derivatives

Two new pleuromutilin derivatives, 14-O-[（4-amino-6-methoxyl-pyrimidine-2-yl）-thioacetyl] mutilin（4） and 14-O-[4-amino-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl）thioacetyl] mutilin（5）, were synthesized and structurally characterized by IR, NMR spectra, HRMS and single-crystal X-ray diffraction. These compounds contain a 5-6-8 tricyclic carbon skeleton and a pyrimidine ring. Compound 4 is in the monoclinic system, space group P1211 with a = 10.2517（4）, b = 12.5655（4）, c = 10.3435（4）A, V = 1315.69（8） A^3, Z = 2, Dc = 1.309 g/cm3, F（000） = 558, μ = 0.166 mm^-1, S = 1.047, R = 0.0457 and w R = 0.0934 for 4721 unique reflections（R（int） = 0.0322） with I 〉 2σ（I）. Compound 5 belongs to the orthorhombic system, space group P212121 with a = 7.3667（4）, b = 13.9990（7）, c = 29.0434（13） A, V = 2995.1（2） A^3, Z = 4, Dc = 1.250 g/cm^3, F（000） = 1216, μ = 0.153 mm^-1, S = 1.031, R = 0.0545 and wR = 0.0982 for 5242 unique reflections（R（int） = 0.0476） with I 〉 2σ（I））. The in vitro antibacterial activity study showed the title compounds 4 and 5 displayed slightly less activity against methicillin-resistant Staphylococcus aureus（MRSA） and methicillin-resistant Staphylococcus epidermidis（MRSE）, and lower potent against Escherichia coli（E.coli） and Bacillus subtilis（B.subtilis） when compared to those of tiamulin fumarate.

Toxicological effects of nanoselenium in animals

The productivity and sustainability of livestock production systems are heavily influenced by animal nutrition.To maintain homeostatic balance in the body of the animal at different phases of life,the percentage of organically active minerals in livestock feed must be optimized.Selenium(Se)is a crucial trace mineral that is required for the maintenance of many functions of the body.Se nanoparticles(SeNPs)attracted considerable interest from researchers for a variety of applications a decade ago,owing to their extraordinary properties.SeNPs offer significant advantages over larger-sized materials,by having a comparatively wider surface area,increased surface energy,and high volume.Despite its benefits,SeNP also has toxic effects,therefore safety concerns must be taken for a successful application.The toxicological effects of SeNPs in animals are characterized by weight loss,and increased mortality rate.A safe-by-strategy to certify animal,human and environmental safety will contribute to an early diagnosis of all risks associated with SeNPs.This review is aimed at describing the beneficial uses and potential toxicity of SeNPs in various animals.It will also serve as a summary of different levels of SeNPs which should be added in the feed of animals for better performance.

Preparation of Compound Ban Huang Oral Liquid

[Objectives] To determine the optimal preparation process of compound Ban Huang oral liquid. [Methods] Orthogonal test and other methods were used to optimize the preparation conditions of compound Ban Huang oral liquid. [Results] The optimal conditions determined were as follows: soaking for 12 h,decocting twice with 2 h per time,concentrating filtrate to 1. 18-1. 25 g/m L at 80 ℃,ethanol precipitating twice with ethanol contents of 600 and 750 ml/L,standing for 24 h. [Conclusions] The optimized preparation process is simple,feasible and stable,and can be used for preparing compound Ban Huang oral liquid.

Synthesis, Crystal Structure and Biological Evaluation of 6'-Chloro-4'-methylene-3H-spiro[isobenzofuran-1,3'-isochroman]

The title compound 6?-chloro-4?-methylene-3 H-spiro [isoben zofura-1,3?-isochroman], C_(17)H_(13)ClO_2, was prepared and characterized by IR, NMR, HRMS and single-crystal X-ray diffraction. This compound has a bisbenzannulated [5,5]-spiroketals skeleton. It crystallizes in orthorhombic, space group P212121 with a = 13.383(3), b = 7.6120(17), c = 13.793(3) ?, Z = 4, Dc= 1.275 g/cm^3, μ = 0.28 mm^(–1), F(000) = 592, wR = 0.1443 and R = 0.0557. The preliminary biological test showed that the title compound has good anti-bacterial activities in vitro.

An Efficient Novel Synthesis of 14-O-[(4-Amino-6-hydroxy-pyrimidine-2-yl)thioacetyl] Mutilin and the Antibacterial Evaluation

The title structure of 14-O-[（4-amino-6-hydroxy-pyrimidine-2-yl）thioacetyl] mutilin, C26H47N3O5S, has been synthesized using 22-O-tosyl pleuromutilin and 4-amine-6-hydroxy-2-mercatopyrimidine monohydrate, and its structure was characterized by IR, NMR, H RMS and single-crystal X-ray diffraction. This compound has a 5-6-8 tricyclic carbon skeleton and a pyrimidine ring. It crystallizes in orthorhombic, space group P212121 with a = 10.494（3）, b = 16.997（5）, c = 16.997 A, Z = 4, Dc = 1.275 Mg×m^–3, μ = 0.220 mm^–1, F（000） = 1248, wR（F^2） = 0.1159 and R = 0.0381. The preliminary biological test showed that the title compound has more potent inhibitions to Staphylococcus aureus, MRSA and MRSE than that of tiamulin fumarate in vitro.

Acute Toxicity of Oxyclozanide Suspension in Mice by Oral Administration

The safety of oxyclozanidc suspension was preliminarily evaluated through acute toxicity test in mice. Administration dose, formal trial grouping and group interval were determined in pre-trial using incremental method. Formal test was performed using simplified karber＇s method. Changes in sign of mice after ad- ministration were observed; the mortality rate was statistically calculated, and the time of death was recorded; the median lethal dose （LD50） and 95% confidence limit of oxyclozanide suspension were calculated. The results showed the LD50 of oxyclozanide suspension in mice by oral administration was 1. 679 g/kg, and the 95% confidence interval was 1. 439 - 1. 947 g/kg. According to toxicity grading of chemicals, oxyclozanide suspension was low toxic substance.

A New Pleuromutilin Derivative: Synthesis,Crystal Structure and Antibacterial Evaluation

A new pleuromutilin derivative, 14-O-[（4,6-diaminopyrimidine-2-yl） thioacetate] mutilin, was synthesized and structurally characterized by IR, NMR spectra and single-crystal X-ray diffraction. This compound contains a 5-6-8 tricyclic carbon skeleton and a pyrimidine ring. Its crystal is of orthorhombic system, space group P21 with a = 10.0237（6）, b = 12.6087（7）, c = 10.3749（8） A, fl = 101.48（1）°, V = 1284.99（14） A3, Z = 2, F（000） = 540, Oc（Mg/m3） = 1.299, # = 0.165 mm1, R = 0.0649 and wR = 0.0797. The in vitro antibacterial activity study using Oxford cup assay showed this compound displayed more potent activity than pleuromutilin and similar antibacterial activity to that oftiamulin.

Dynamic RNA profiles in the small intestinal epithelia of cats after Toxoplasma gondii infection

Background Felids are the only definitive hosts ofToxoplasma gondii. However, the biological features of the feline small intestine followingT. gondii infection are poorly understood. We investigated the changes in the expression of RNAs (including mRNAs, long non-coding RNAs and circular RNAs) in the small intestinal epithelia of cats followingT. gondii infection to improve our understanding of the life cycle ofT. gondii and cat responses toT. gondii infection.Methods Fifteen cats were randomly assigned to five groups, and the infection groups were inoculated with 600 tissue cysts of theT. gondii Pru strain by gavage. The small intestinal epithelia of cats were collected at 6, 10, 14, and 30 days post infection (DPI). Using high-throughput RNA sequencing (RNA-seq), we investigated the changes in RNA expression. The expression levels of differentially expressed (DE) genes and non-coding RNAs (ncRNAs) identified by RNA-seq were validated by quantitative reverse transcription PCR (qRT-PCR). Differential expression was determined using the DESeq R package.Results In total, 207 annotated lncRNAs, 20,552 novel lncRNAs, 3342 novel circRNAs and 19,409 mRNAs were identified. Among these, 70 to 344 DE mRNAs, lncRNAs and circRNAs were detected, and the post-cleavage binding sites between 725 ncRNAs and 2082 miRNAs were predicted. Using the co-location method, we predicted that a total of 235 lncRNAs target 1044 protein-coding genes, while the results of co-expression analysis revealed that 174 lncRNAs target 2097 mRNAs. Pathway enrichment analyses of the genes targeted by ncRNAs suggested that most ncRNAs were significantly enriched in immune or diseases-related pathways. NcRNA regulatory networks revealed that a single ncRNA could be directly or indirectly regulated by multiple genes or ncRNAs that could influence the immune response of cats. Co-expression analysis showed that 242 circRNAs, mainly involved in immune responses, were significantly associated withT. gondii infection. In contrast, 1352 protein coding RNAs, mainly involved in nucleic acid process/repair pathways or oocyte development pathways, were negatively associated withT. gondii infection.Conclusions This study is the first to reveal the expression profiles of circRNAs, lncRNAs and mRNAs in the cat small intestine followingT. gondii infection and will facilitate the elucidation of the role of ncRNAs in the pathogenesis ofT. gondii infection in its definitive host, thereby facilitating the development of novel intervention strategies againstT. gondii infection in humans and animals.

Acquisition of a novel conjugative multidrug-resistant hypervirulent plasmid leads to hypervirulence in clinical carbapenem-resistant Klebsiella pneumoniae strains

The co-occurrence of plasmid-mediated multidrug resistance and hypervirulence in epidemic carbapenem-resistant Klebsiella pneumoniae has emerged as a global public health issue.In this study,an ST23 carbapenem-resistant hyper-virulent K.pneumoniae(CR-HvKP)strain VH1-2 was identified from cucumber in China and harbored a novel hybrid plasmid pVH1-2-VIR.The plasmid pVH1-2-VIR carrying both virulence and multidrug-resistance(MDR)genes was likely generated through the recombination of a virulence plasmid and an IncFIIK conjugative MDR plasmid in clinical ST2318622 isolated from a sputum sample.The plasmid pVH1-2-VIR exhibited the capacity for transfer to the clinical ST11 carbapenem-resistant K.pneumoniae(CRKP)strain via conjugation assay.Acquisition of pVH1-2-VIR plasmid directly converted a CRKP into CR-HvKP strain characterized by hypermucoviscosity,heightened virulence for Galleria mellonella larvae,and increased colonization ability in the mouse intestine.The emergence of such a hybrid plasmid may expedite the spread of CR-HvKP strains,posing a significant risk to human health.

Cinnamaldehyde targets SarA to enhance β-lactam antibiotic activity against methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus(MRSA)is a current global public health problem due to its increasing resistance to the most recent antibiotic therapies.One critical approach is to develop ways to revitalize existing antibiotics.Here,we show that the phytogenic compound cinnamaldehyde(CIN)and β-lactam antibiotic combinations can functionally synergize and resensitize clinical MRSA isolates to β-lactam therapy and inhibit MRSA biofilm formation.Mechanistic studies indicated that the CIN potentiation effect on β-lactams was primarily the result of inhibition of the mecA expression by targeting the staphylococcal accessory regulator sarA.CIN alone or in combination with β-lactams decreased sarA gene expression and increased SarA protein phosphorylation that impaired SarA binding to the mecA promoter element and downregulated virulence genes such as those encoding biofilm,α-hemolysin,and adhesin.Perturbation of SarA-mecA binding thus interfered with PBP2a biosynthesis and this decreased MRSA resistance to β-lactams.Furthermore,CIN fully restored the anti-MRSA activities of β-lactam antibiotics in vivo in murine models of bacteremia and biofilm infections.Together,our results indicated that CIN acts as a β-lactam adjuvant and can be applied as an alternative therapy to combat multidrug-resistant MRSA infections.

Andrographolide and Its Derivative Potassium Dehydrographolide Succinate Suppress PRRSV Replication in Primary and Established Cells via Differential Mechanisms of Action

Porcine reproductive and respiratory syndrome virus(PRRSV) continues to cause significant economic loss worldwide and remains a serious threat to the pork industry. Currently, vaccination strategies provide limited protection against PRRSV infection, and consequently, new antiviral strategies are urgently required. Andrographolide(Andro) and its derivative potassium dehydrographolide succinate(PDS) have been used clinically in China and other Asian countries as therapies for inflammation-related diseases, including bacterial and viral infections, for decades. Here, we demonstrate that Andro and PDS exhibit robust activity against PRRSV replication in Marc-145 cells and primary porcine alveolar macrophages(PAMs). The two compounds exhibited broad-spectrum inhibitory activities in vitro against clinically circulating type 2 PRRSV GD-HD, XH-GD, and NADC30-like HNhx strains in China. The EC_(50)values of Andro against three tested PRRSV strain infections in Marc-145 cells ranged from 11.7 to 15.3 lmol/L, with selectivity indexes ranging from 8.3 to10.8, while the EC_(50)values of PDS ranged from 57.1 to 85.4 lmol/L, with selectivity indexes ranging from 344 to 515.Mechanistically, the anti-PRRSV activity of the two compounds is closely associated with their potent suppression on NFj B activation and enhanced oxidative stress induced by PRRSV infection. Further mechanistic investigations revealed that PDS, but not Andro, is able to directly interact with PRRSV particles. Taken together, our findings suggest that Andro and PDS are promising PRRSV inhibitors in vitro and deserves further in vivo studies in swine.