Study The Cross-Resistance Mechanism Of Tylosin And Florfenicol Against Staphylococcus Xylosus

In dairy farms,mastitis is a serious disease with significant economic losses.The etiological agents of mastitis include Staphylococcus aureus,Streptococcus agalactiae,Escherichia coli and among others.In addition,the average proportion of mastitis due to coagulase-negative staphylococci(CNS)is on a constant increase anually,which makes them the most prevalent causal agents.Staphylococcus xylosus(S.xylosus)is a kind of CNS,and it is the main etiological agent of CNS mastitis.Mastitis caused by CNS seems to respond well to antibiotic treatment.When bacteria are under antibiotic stress for long time,they would evolve to resistance and even to cross-resistance,thereby hindering prescribed medication.Thus,it is necessary to demonstrate the mechanism of cross-resistance in S.xylosus in order to guide clinical medication.Tylosin has been widely used in veterinary clinics for a long time as a kind of macrolide in mitigating bacterial infections.It can reach into the PTC cavity and inhibit peptidyl transfer by competing with binding of the A-site substrates thereby helping to prevent the protein synthesis.Flornicol has also been widely used by vetenarians.It was a classic peptidyl transferase inhibitor,which binds at that A-site to block the binding of t RNA protein.Previous studies have shown that when bacteria are under one antibiotic,they would evolve into cross resistance for another antibiotic.However,the cross-resistance mechanism of tylosin and florfenicol against S.xylosus had not been reported.So,proteomics was applied to detect the proteins related to cross-resistance.Furthermore,the results of real time PCR demonstrated the effection of resistant proteins in evolution of cross resistance.In order to explore the molecular evolution mechanism,we used cloning,sequencing,homology modeling and molecular docking to detect mutations in the ribosomes.And the mutant strains were constructed to verify the cross-resistance mechanism of tylosin and florfenicol.The main results of this study are as follows:(1)The cross-resistance of S.xylosus to tylosin and florfenicol may relate to “stress-response proteins” and “translation related proteins”.We compared the differential expression of S.xylosus in response to tylosin stress by i TRAQ.We found a total of 155 differentially expressed proteins(59 up-regulated,96 down-regulated)with the fold-change of >1.2 or <0.8 change and p value ?0.05 between S.xylosus treated with 1/2 MIC(0.25 ?g/m L)tylosin and untreated.The chloramphenicol resistant protein was up-regulated,indicating that S.xylosus under tylosin selection would evolve into cross-resistance to florfenicol.Bioinformatics analysis showed that the changed proteins played important roles in stress-response(thioredoxin(trx A),Aldehyde dehydrogenase A(ald A-1)chaperonin(gros)and catalase(KAT))and translation(50S ribosomal protein L23(rpl W),translation initiation factor IF-2(inf B)).(2)The tylosin resistant S.xylosus induced in vitro showed cross-resistance to florfenicol.The strong selection was chosen to induce tylosin-resistant S.xylosus in vitro.Compared with S.xylosus,the MIC of tylosin-resistant S.xylosus to florfenicol from 0.5 ?g/m L to 8 ?g/m L.(3)The “stress-response related proteins”,“translation related preoteins” and chloramphenicol resistant protein played important role in cross-resistance of S.xylosus.The resistant proteins of tylosin-resistant S.xylosus(8 ?g/m L,32 ?g/m L and 128 ?g/m L)were determined at m RNA levels.The data showed that apart from ldh(2.5 times)showing a diminishing level,all other genes,inculding rpl W(2.8 times),ald A(2.4 times),trx A(4.2times),gros(5.93 times)and cl(1.5 times),gradually increased in the evolution of tylosin resistant S.xylosus.However,inf B and KAT did not significantly change.(4)The cross-resistance of S.xylosus against tylosin and florfenicol may relate to mutant ribosomes,including N135 G,A137G,S142 A and R162 K of ribosomal proteins L3,S158 N and A164 E of ribosomal proteins L4,97KRTSAIN98 insertion of ribosomal proteins L22,A2662 C,C1305A,T2560 C of 23 S rRNA.PCR amplification,sequencing and DNAMAN analysis were applied to determine the ribosomal mutations.Based on amino acid sequence of Staphylococcus aureus in PDB,the structures of ribosomal proteins were constructed and evaluated.Furthermore,molecular docking of ribosomal proteins with tylosin and florfenicol was used to infer the ribosomal mutations.The above results showed that the mutations included N135 G,A137G,S142 A and R162 K of ribosomal proteins L3,S158 N and A164 E of ribosomal proteins L4,97KRTSAIN98 insertion of ribosomal proteins L22,A2662 C,C1305A,T2560 C of 23 S rRNA.(5)The mutant strains were constructed to demonstrate that one of the cross-resistance mechanisms was due to ribosome mutations(97KRTSAIN98 insertion of ribosomal proteins L22 and A2662 C,C1305A,T2560 C of 23 S rRNA).The mutant strains were constructed by homologous recombination,and gfp gene was used as the maker to select the mutant strains by flow cytometry.Furthermore,the mutant fragments were sequenced to verify the mutant strains(L3(rpl C)mutant S.xylosus,L4(rpl D)mutant S.xylosus,L22(rpl V)mutant S.xylosus,23 S rRNA A2662 C mutant S.xylosus,23 S rRNA C1305 mutant S.xylosus and 23 S rRNA T2560 C mutant S.xylosus).In addition,the MIC of mutant strains revealed that compared with S.xylosus,the resistance of L22(rpl V)mutant S.xylosus,23 S rRNA A2662 C mutant S.xylosus,23 S rRNA C1305 mutant S.xylosus and 23 S rRNA T2560 C mutant S.xylosus to tylosin increased from 0.5 ?g/m L to 128 ?g/m L and the resistance of the four mutant strains mentioned above,to florfenicol increased from 0.5 ?g/m L to 2 ?g/m L.(6)Based on the rRNA of S.xylosus constructed by computer molecular simulation,the molecular docking of rRNA with tylosin and florfenicol was implemented.The result showed that 23 S rRNA A2275 was the cross-binding site.The spatial structure of cross-binding site(A2275)and mutant sites indicated that the above results were reliable.Namely,ribosome mutations(97KRTSAIN98 insertion of ribosomal proteins L22 and A2662 C,C1305A,T2560 C of 23 S rRNA)were related to cross-resistance.In view of 23 S rRNA sequence of Staphylococcus aureus,rRNA of S.xylosus was construted by DS 3.0 including ribosomal protein L3,L4,L22 and 23 S rRNA.The result of the molecular docking of rRNA with tylosin and florfenicol respectively showed that A2275,C2276,G2281,U2466,C2470 and A2275,G2095.The cross-binding site was A2275.(7)One of the cross-resistance mechanisms was the differential expression of resistant proteins which were regulated by 97KRTSAIN98 insertion of ribosomal proteins L22 and C1305 A,T2560C of 23 S rRNA.We explored the m RNA level of resistant proteins in mutant strains,and the results showed that resistant proteins changed in L22(rpl V)mutant S.xylosus,23 S rRNA C1305 mutant S.xylosus and 23 S rRNA T2560 C mutant S.xylosus,demonstrating that the above mutant strains led to crossresistance by adjusting the expression of resistant proteins.In conclusion,cross-resistance of S.xylosus against tylosin and florfenicol was regulated by multiple mechanisms,including ribosomal mutations(97KRTSAIN98 insertion of ribosomal proteins L22,A2662 C,C1305A,and T2560 C of 23 S rRNA)and the differential expression of “stress-response related proteins”,“translation related preoteins” and chloramphenicol resistant protein.