Comparison Of The Fitness Cost Of Tet(X4) Positive Plasmids In Swine And Avian Derived Escherichia Coli

Tigecycline has been regarded as the last resort to control multi-drug resistant Gram-negative bacteria infections.However,with the emergence of the high-level tigecycline resistance gene tet(X4)in the livestock and poultry breeding industry,tigecycline resistance has attracted the attentions of researchers.Simultaneously,we found that the isolation rate of tet(X4)in Escherichia coli(E.coli)from swine was higher than that from avian.Therefore,we conducted related experiments to explore the reasons that account for the difference in the prevalence rate,which included fitness cost,plasmid stability and conjugation frequency of transformed strains in different temperatures.Firstly,we constructed the recombinant expression vector pUC19::tet(X4),subsequently transferred it into TOP 10,swine-derived E.coli SEC 10 and avian-derived E.coli E847.Growth curve assay,competition assay,virulence test,biofilm formation test and swimming assay were carried out to compare the fitness cost of tet(X4)gene posed to these bacteria.The study found that tet(X4)could imposed high fitness cost to TOP 10.In the contrary,tet(X4)did not cause fitness cost to avian-derived E.coli,whereas it improved the fitness of the strain,which was manifested in increasing growth ability,increasing relative fitness value and higher virulence.Meanwhile,tet(X4)posed slight fitness cost to swine-derived E.coli,although it did not affect growth rate and virulence,it decreased relative fitness value.Under the selection pressure of tigecycline,pUC19::tet(X4)can be maintained stably in the transformed strains.The expression of tet(X4)in E.coli of avian and swine is less burdensome and the tet(X4)-bearing plasmid could be stably maintained,indicating that this gene is capable of spreading in either avian or swine source strains,especially in E.coli from avian.In order to further study the reasons of the differences in isolation rate of tet(X4)in E.coli from swine and avian,we selected six tet(X4)plasmids(belong to 5 different replicon types)and transformed these plasmids into TOP10,swine-derived E.coli SEC 10,SEC78 and avian-derived E.coli E847,E901.Subsequently,we studied the fitness cost of these transformed strains.The results showed that there was no correlation between the size of the tet(X4)plasmids and the fitness cost.The tet(X4)positive plasmid posed a low fitness cost in avian-derived E.coli,which was manifested in increasing relative fitness value,virulence and motility.However,tet(X4)plasmids caused high fitness cost to swine-derived E.coli,with decreased relative fitness value and lower virulence.It is predicted that the isolation rate of tet(X4)in avian-derived E.coli will gradually increase due to the low fitness cost.At the same time,we detected the plasmid stability under the pressure of tigecycline,these tet(X4)plasmids can be maintained stably in transformed strains.Secondly,in order to explore whether the high prevalence of tet(X4)in the breeding industry is associated with the use of tetracycline antibiotics.The previously obtained transformed strains were subjected to serial passaging in antibiotic-free,oxytetracycline and tetracycline broth,respectively,and then to determine the plasmid stability.For tet(X4)positive plasmids which were unstable under antibiotic-free pressure,such as IncXl,IncFII,IncA/C2 and IncFIA(HI1),IncHI1A,IncHI1B(R27),IncX1,the stability of tet(X4)plasmids were improved to some extent,under the pressure of oxytetracycline and tetracycline.Because of the difference of body temperature between swine and avian,it was worth to explore whether the transmission of tet(X4)gene is related to temperature.The transformed strains containing IncFII plasmid and IncX1 plasmid were used as donor strains,and the EC600 strain was used as recipient strain.The conjugation assay was conducted at 37? and 42?,respectively.It was found that there was no difference in conjugation frequency between 37? and 42? for IncFII plasmid.However,IncX1 had a higher conjugation frequency at 42?.It means that this plasmid will spread fast in avian derived E.coli.In conclusion,this study found that the fitness cost of tet(X4)plasmids in avian-derived E.coli is lower than that in swine-derived E.coli.Hence,we predicted that the number of tet(X4)positive strains in avian environment will increase.IncFII had lower fitness cost after transferring to swine and avian derived strains and could persist in both antibiotic free and antibiotic containing broth.Moreover,it could exhibit high conjugation frequency at 37? and 42?,which indicated that tet(X4)-bearing IncFII type plasmid may evolve as the dominant epidemic plasmid in breeding industry.In addition,the abuse of tetracycline drugs in breeding industry can enrich and facilitate the dissemination of tet(X4)gene.