Evaluation Of Collateral Susceptibility And Cross-resistance Of Berberine And Licochalcone A Against Clostridium Perfringens Type A

Clostridium perfringens is a widespread pathogen in livestock and poultry that causes gastrointestinal illnesses,with type A being the most frequent and dangerous.They can induce infections in the manufacture and processing of animal products,resulting in human gas gangrene and food poisoning.Antibiotic overuse has resulted in a serious problem of drug resistance,whereas traditional Chinese medicine（TCM）,as a good alternative to antibiotics,not only effectively limits the growth of bacterial infections but is also economical and environmentally beneficial.However,it is yet unclear if the risk of resistance and co-resistance will occur as a result of excessive TCM use.Firstly,we examined the antibacterial effects of two TCM monomers berberine（BBR）and licochalcone A（LCA）on Clostridium perfringens type A and the synergistic inhibition with common antibiotics in vitro.Then compared the strains before and after the induction of Clostridium perfringens type A with TCM monomers to explore possible resistance phenotypes through changes in bacterial morphology,growth capacity,biofilm formation ability,and co-resistance,collateral susceptibility and stability to other antibiotics,and combine genomic,transcriptomic and metabolomic analyses with the aim of investigating the mechanisms of resistance,co-resistance and collateral susceptibility of Clostridium perfringens type A under unreasonable application conditions of TCM.1.Antibacterial effect of TCM monomers and antibiotics on clinical Clostridium perfringens type A and combined in vitro inhibitionIn this study,the susceptibility of 102 clinical strains of Clostridium perfringens type A was tested by micro-broth method against 27 antibiotics and two TCM monomers.The MIC₉₀of the two TCM medicines against the clinical strains was used as the resistance breakpoints（32μg/m L（BBR）,8μg/m L（LCA））.Using the checkerboard method,BBR and LCA were tested in vitro in combination with common antibiotics for bacterial inhibition.LCA was analyzed to has synergistic inhibition with engramycin（FICI=0.375）,penicillin（FICI=0.375）,linezolid（FICI=0.25）,cefoxitin（FICI=0.5）,doxycycline（FICI=0.5）,LCA with tiamulin,gentamicin,clindamycin,kitasamycin,bacitracin,azithromycin flufenamicol and tilmicosin（FICI=0.75）have additive antibacterial effect;BBR with kitasamycin,bacitracin,cefoxitin and quinolone have additive antibacterial effect（FICI=0.75）.2.Phenotypes of cross-resistance and collateral susceptibility of Clostridium per fringens type A induced by TCM monomersIn this study,strains with different levels of adaptation/resistance were obtained by gradually increasing concentrations of BBR and LCA in vitro induction tests,in which the BBR-induced strains showed resistance to BBR,while the low-fold induced strains showed cross-resistance to ceftiofur,tilmicosin,engramycin,and quinolone,along with increased sensitivity to gentamicin.High-fold induced strains were highly cross-resistant to clindamycin,erythromycin,and guillamycin.Increased susceptibility to enramycin,doxycycline,and quinolone.The bacterial morphology of the induced strains showed elongated,irregularly shaped,and loose extracellular plasma membrane.The LCA-induced strains showed adaptive resistance to LCA and recovered easily,while the low-fold induced strains showed adaptive resistance to ceftiofur,quinolenone and nexium and recovered immediately,with increased sensitivity to doxycycline,tetracycline and engramycin,and the high-fold induced strains showed increased sensitivity to erythromycin,tiamulin,tetramycin and clindamycin.kitasamycin,clindamycin with low-fold cross-resistance,and adaptive resistance to quinolone,tetracycline,and natriuretic peptide with subsequent recovery.The morphology of the induced strains showed protruding outer membrane or blistering cell wall or cell membrane rupture with irregular shape and adherence of the bacterium.There were no significant changes in their growth ability and biofilm formation ability.3.Genomic sequencing analysis of adaptation/resistance Clostridium perfringens type A induced by TCM monomerIn this study,the genomic data of CVCC2030 parental strain（group A）vs CVCC2030-BBR-3MIC induced strain（group B）,YS5 parental strain（group D）vs YS5-BBR-8MIC induced strain（group E）,CVCC2030 parental strain（group A）vs CVCC2030-LCA-MIC induced strain（group C）,YS1 parental The genomic data of CVCC2030 parental strain（group F）vs YS1-LCA-7MIC induced strain（group G）were analyzed for comparison.Mainly analyzed the changes of resistance genes and virulence genes after induction.Regarding resistance genes,the tetracycline resistance genes tet A（P）and tet B（P）were lost on the plasmid of strain CVCC-LCA-MIC.The lincosamine resistance gene lnu D and the tetracycline resistance genes tet A（P）and tet B（P）were lost on the chromosome of strain YS5-BBR-8MIC,but the exocytosis pump gene arl R was present on the chromosome.The tetracycline resistance genes tet A（P）,tet B（P）and macrolide resistance gene erm Q were present on the plasmid of strain YS5-BBR-8MIC.Regarding virulence genes,bsh virulence gene was present on the plasmid of strain CVCC-LCA-MIC.The number of strain YS5-BBR-8MIC virulence genes increased with the presence of virulence genes pfo A,BAS3109,and slo on the chromosome;bsh virulence genes were present on the plasmid.4.Transcriptome sequencing analysis of adaptation/resistance Clostridium perfringens type A induced by TCM monomerThe transcriptome data of the four groups of strains were compared and analyzed.The results showed that in group A vs B there were 424 gene expression changes,of which 273 increased expression and 151 decreased expression;in group D vs E there were 1121 gene expression changes,479 increased expression and 642 decreased expression;in group A vs C there were 282 gene expression changes,of which 165increased expression and 117 decreased expression;in group F vs G there were 279 gene expression changes,169 were increased expression and 110 decreased expression.The expression of 279 genes was changed in the F vs G group,with 169 genes increased expression and 110 decreased expression.Expression of genes associated with drug efflux and membrane transport（mep A,opu CC,TP＿0036,yvg M,ykn V,yvg L）was upregulated;membrane proteins（Mpr F,cls2）,biofilms（lux S,sip W,gene538,gene536）,tetracycline efflux gene yxa M expression was downregulated in BBR-induced strains（A vs B and D vs E）.Expression of genes associated with drug efflux（mep A,nor M,yis Q,yxa M）,biofilm（abr B,arg G,rib D,rib E,lex A）,and tetracycline resistance gene tet P was downregulated;Expression of genes associated with regulation of 23S RNA methylation（rlm N,Rlm B,Rlm F,Rlm H,Rlm D）genes expression was upregulated in LCA-induced strains.These differential expression of genes may be associated with cross-resistance and collateral sensitivity.We also found increased expression of sugar-related transporter genes（aga C,M6＿Spy0801,man Z,ula C,gene1653,mdx G,yes O）in BBR-induced strains regarding the bacterial phosphotransferase system（PTS）.The increased expression of thi S、thi E、thi G、thi C、thi D、and thi M in BBR-induced strains which related to thiamin metabolism5.Metabolome sequencing analysis of adaptation/resistance Clostridium perfringens type A induced by TCM monomerThe metabolomic data of the four groups of strains were analyzed for comparison.The results showed that the number of significant metabolic pathways enriched in the two groups A vs B and D vs E was 21 and 44,respectively.14 metabolic pathways were enriched in both groups,mainly about protein digestion and absorption,the biosynthesis of aminoacyl-t RNA and amino acid（Leucine,isoleucine and valine）,mineral absorption,the metabolism of amino acids（Glycine,cysteine,beta-alanine and methionine,etc.）and m TOR signaling pathway,etc.Among them,metabolic pathways regarding ABC transport,galactose metabolism and carbohydrate digestion and uptake in low-fold resistant strains and metabolic pathways regarding TCA cycle,phosphotransferase system（PTS）,glycolysis/gluconeogenesis in high-fold resistant strains may be associated with cross-resistance,collateral susceptibility and compensatory metabolism.The number of significant metabolic pathways enriched in the two groups A vs C and F vs G treated by LCA was 28 and 48,respectively.22 metabolic pathways were enriched in both groups,mainly about protein digestion and absorption,aminoacyl-t RNA and amino acid（Leucine,phenylalanine,tryptophan,etc.）biosynthesis,mineral absorption,ABC outer membrane protein transport,amino acid（leucine,isoleucine and valine）degradation,amino acid（β-alanine,glycine,histidine,threonine,alanine,aspartic acid,serine,glutamic acid,tryptophan,phenylalanine）metabolism,glutathione metabolism,oxidative phosphorylation,carbon fixation,etc.Among them,metabolic pathways regarding ABC outer membrane protein transport,oxidative phosphorylation,and galactose metabolism and thiamine metabolism in high-fold adapted strains may be associated with cross-resistance,collateral susceptibility and compensatory metabolism.