Investigation Of The Fitness Cost Of Tet(X4)-mediated High-Level Tigecycline Resistance

Tigecycline is the first anti-infective drug in the clinic among the glycyltetracycline antibiotics,for which no cross-resistance exists with traditional tetracyclines.Tigecycline has a wide antimicrobial spectrum and also has better antibacterial activity in vitro and in vivo for multidrug-resistant(MDR)bacteria,which is considered as the last line of defense in the treatment of drug-resistant bacterial infections.In recent years,with the massive use of various antibiotics,drug-resistant bacteria have increased dramatically that seriously threatens public health.The tet(X)gene,the first discovered in the 1980s on the R plasmid,which is directly inactivating tetracyclines novel resistance gene.The tet(X4)gene,as the tet(X)variant gene,was firstly discovered and identified in 2019,which is located on the plasmid and could mediate high-level resistance to tigecycline.It has been shown that the acquirement of drug resistance in bacteria would impose corresponding fitness costs.This is a key factor in the evolution of the epidemic of drug-resistant bacteria.However,it is still unclear whether the expression of tet(X4)causes fitness costs to the host bacteria,and whether there is a combination of low fitness costs,and the underlying molecular mechanism.Therefore,in this study,we will systematically explore the fitness cost of tet(X4)-mediated high-level tigecycline resistance and decipher the fitness cost regulation mechanisms.This work would provide scientific evidence for the prevention and control of tigecycline-resistant bacteria.First,the fitness cost of tet(X4)on different host bacteria was determined.The constructed plasmid pBAD-HisA-tet(X4)was introduced into Escherichia coli TOP 10,Salmonella Enteritidis ATCC13076 and Klebsiella pneumonia ATCC700603 by electric transformation method,respectively.And then,the fitness cost of tet(X4)on the host was determined including the growth curve and competition test in vitro.The result showed that the fitness cost of tet(X4)expression on TOP 10 was minimal than other hosts.Thus,the TOP 10 was selected for further research.Then,five different plasmids with different types were introduced into TOP 10 to screen the plasmid with the lowest fitness cost on the host.After plasmid transformation,the results showed that IncFⅡ type plasmid pC41 revealed minimal fitness cost.In vivo infection model of G.mellonella displayed comparable pathogenicity of TOP10/pC41 and TOP 10.The above results indicated that the combination of TOP 10 and pC41 exhibited minimal fitness cost.To evaluate the stability and potential hazards of this combination,the evolution of TOP10/pC41 was first evaluated by subculture with and without the antibiotic-containing medium.Then,the changes in drug resistance,conjugative transfer ability,biofilm formation,and virulence of the bacteria were evaluated after passages.The results showed that pC41 was stable in the TOP10 after evolution in both with and without antibiotic passages.The level of fitness was slightly increased after serial passaged in the antibiotic-containing medium.MIC values of TOP10/pC41 increased after passaging,the frequency of conjugative transfer and the bacterial biofilm-forming ability was increased,as well as bacterial virulence.No nonsynonymous mutation sites were identified by genomic sequencing of pC41 plasmids before and after passage,implying that changes in fitness-related phenotypes may be attributed to the changes in the expression of related genes.The above result of in vitro experiments confirmed that TOP10/pC41 had a highly stable relative fitness,but the in vivo fitness cost needs further investigation.To verify whether TOP10/pC41 has excellent fitness in vivo,we established a mouse gut flora clearance model that could exclude the interference of gut flora to determine the in vivo conjugation ability and competitive fitness of TOP10/pC41.The results demonstrated that the conjugative transfer ability of TOP10/pC41 in the mouse gut is consistent with the in vitro test,and the same trend happened in the fitness evaluation in vivo.In vivo competition assays demonstrated that TOP10/pC41 was highly pathogenic and colonizing,and it still retains a high conjugation frequency and fitness under a complex environment in vivo.In addition,we showed that the anti-HIV drug azidothymidine significantly reduced the conjugative transfer and competitive ability of TOP10/pC41 both in in vitro and in vivo assays,providing new insights into blocking the transmission of tet(X4)-positive plasmids.The above results indicated that E.coli and the IncFII type plasmid pC41 had the potential to become an epidemic strain,however,the molecular mechanism needs further investigation.To find which genes are involved in the regulation of fitness cost in pC41,we screened the IncFII type plasmid pC59 and assayed its relative fitness,biofilm-forming ability,and pathogenicity.It was found that the pC59 plasmid exhibited a diametrically opposite trend of fitness cost to pC41.Specifically,pC59 imposed a heavy fitness cost to host TOP10,at the same time,both biofilm formation ability and virulence were weaker than pC41.Two plasmids were aligned by Nanopore sequencing,there was about 3.5 kb DNA base in pC41,named 3K.In addition,a putative protein sequence was included in the 3K sequence(atlR).3K and atlR knocked out experiments were performed respectively using CRISPR-Cas9,and the complementation of 3K or atlR by vector.Knockdown of either 3K or atlR resulted in a significantly higher fitness cost in pC41,essentially equivalent to that of pC59.However,a significant decrease in fitness cost was observed for pC41 after plasmid complementation.These results indicated that the 3K and atlR genes in the IncFII type plasmid pC41 were directly involved in the regulation of the fitness cost.The changes in the expression levels of drug resistance genes were directly related to the fitness cost of the host bacteria,so we speculated whether the 3K and atlR genes regulated the expression level of tet(X4)gene.To test this hypothesis,we determined tet(X4)expression level in TOP10/pC41,TOP10/pC41 Δ3K,and TOP10/pC41ΔatlR.It was found that tet(X4)expression was 4-fold higher in TOP10/pC41Δ3K and TOP10/pC41ΔatlR than that in TOP10/pC41,indicating that the high fitness cost of TOP10/pC41Δ3K and TOP10/pC41ΔatlR was attributed to high tet(X4)expression level.Considering that tet(X4)gene expression is strongly correlated with plasmid copy number,we assayed the plasmid copy number changes in TOP10/pC41,TOP10/pC41 Δ3K and TOP10/pC41ΔatlR.The results showed that the plasmid copy number was significantly increased after 3K and atlR gene knockout,but was remarkably decreased after complementation,suggesting that the increase of tet(X4)gene expression may be due to the increase in plasmid copy number.Next,we measured the expression changes of repA,a replication gene in the IncFII plasmid,using lacZ as a reporter,and found that the expression of repA after the complementation of 3K and atlR genes appeared to decrease.In addition,mutation of four sites,+260,+261,+262,+263 in repA,which would disrupt the bind site of DNA,almost abolished the inhibitory effect of 3K and atlR on repA expression,indicating that these four sites might be the binding sites of regulator AtlR to the DNA of repA.These results indicated that the 3K and atlR genes reduced the expression of the drug resistance gene tet(X4)by inhibiting the replication of an IncFⅡplasmid,which ultimately regulated the fitness cost to the host.In summary,this study systematically explored the fitness cost of high-level tigecycline resistance mediated by tet(X4)gene on host bacteria and its regulatory mechanisms.The TOP10/pC41 displayed the lowest fitness cost in vitro and in vivo among others.It was a stable combination that after passaged the level of resistance and virulence could be further enhanced.Mechanistic studies revealed that an entirely new regulator AtlR on the plasmid,it could reduce the expression of drug resistance genes by inhibiting the plasmid copy number,thereby improving the fitness of the host.Our findings lay a scientific foundation for the subsequent prevention and control of the epidemic spread of the tet(X4)gene and the development of new bacterial resistance prevention and control strategies.