Study On The Genes Involved In Escherichia Coli Persistence To Tosufloxacin

Persister are a sub-population of dormant cells who can tolerant stresses(antibiotic,drug,heat,acidic p H and oxidative,etc.)and upon removing stresses persister can restore the ability of growth and reproduction.More and more evidence shows that persister are the root cause of treatment problems such as recurrence of chronic infection,bacterial drug resistance and biofilm formation.At present,there have been many reports on the genes and mechanisms related to the formation of persister.Due to its complexity,the mechanism of persister formation and survival are not entirely clear.Our previous study found that,compared with other quinolones,tosufloxacin(50μM)had high antibacterial activity against Escherichia coli(E.coli)and Staphylococcus aureus in vitro,but could not completely eliminate infected bacteria in vivo,suggesting that persister resistant to tosufloxacin still exist.However,the mechanism of bacteria forming persister under the tosufloxacin is not clear.By screening the KEIO mutant library of Escherichia coli,we found that the genes lpcA,surA,gltS,gltI and gltP may be related to forming persister of Escherichia coli under the action of tosufloxacin.Sur A encodes periplasmic chaperone,lpcA encodes heptose phosphate isomerase,gltS,gltI and gltP all encode glutamate transporters.Objective: study the importance and role of lpcA,surA,gltS,gltP and gltI genes in Escherichia coli persistence.Methods: Firstly,the single gene knockout mutants of E.coli BW25113 ΔlpcA,ΔgltS,ΔgltI and ΔgltP were constructed in E.coli BW25113 by homologous recombination ofλ-Red,and the persistence of E.coli BW25113 ΔlpcA,ΔgltS,ΔgltI and ΔgltP under antibiotic and stresses were evaluated in vitro.Secondly,the corresponding complementary strains ΔlpcA-p Trc99a-lpcA,ΔgltS-p Trc99a-gltS,ΔgltI-p Trc99a-gltI and ΔgltP-p Trc99a-gltP were constructed by plasmid p Trc99 a to evaluate whether the persistence of the complementary strain returned to the level of the wild strain under the same antibiotic and stresses.The lpcA,gltS,gltI and gltP single knockout mutants E.coli UTI89 ΔlpcA,ΔsurA,ΔgltS,ΔgltI and ΔgltP were constructed in uropathogenic E.coli UTI89 strains to evaluate persistent ability of E.coli UTI89ΔlpcA,ΔsurA,ΔgltS,ΔgltI and ΔgltP in BALB/c female mice,and their biofilm formation ability on 96-well plate.Finally,the adhesion and invasion ability of E.coli UTI89ΔlpcA,ΔsurA,ΔgltS,ΔgltP and ΔgltI to bladder epithelial cells(HTB-9)were detected.Results: the persistent capacity of E.coli BW25113ΔlpcA,ΔgltS,ΔgltI and ΔgltP in vitro was significantly lower than that of wild strain E.coli BW25113 and the corresponding complementary strains returned to the level of wild strains.Suggesting that lpcA,surA,gltS,gltI and gltP genes were related to Escherichia coli persistence in vitro.The persistent of pathogenicity of E.coli UTI89ΔlpcA,ΔsurA,ΔgltS,ΔgltI andΔgltP in mice were significantly lower than those of wild strain of E.coli UTI89.Suggesting that lpcA,surA,gltS,gltI and gltP genes are associated with the persistence of uropathogenic Escherichia coli.Compared with the wild strain E.coli UTI89,the biofilm forming ability of E.coli UTI89ΔlpcA,ΔsurA,ΔgltS,ΔgltI and ΔgltP decreased significantly,and the biofilm forming ability of the corresponding supplementary strains recovered to the level of the wild strain.Suggesting that lpcA,surA,gltS,gltI and gltP genes are related to the formation of bacterial biofilm.The adhesion and invasion ability of E.coli UTI89ΔlpcA,ΔsurA,ΔgltS,ΔgltP and ΔgltI to human bladder epithelial cell HTB-9 was significantly lower than the wild strain E.coli UTI89.Suggesting that lpcA,surA,gltS,gltI and gltP genes are related to the persistence and survival of uropathogenic Escherichia coli in cells.Conclusion: E.coli genes lpcA,surA,gltS,gltI and gltP are closely related to forming persister under stresses(antibiotic,heat,acidic p H and oxidative,etc.)in vitro,biofilm formation in vitro,pathogenicity and persistence in mice,adhesion and invasion to bladder epithelial cells.Suggesting that the periplasmic chaperones and glutamate transporters of Gram-negative bacteria may be another kind of important molecules related to the formation and survival of Escherichia coli persister.It has become a new target for the design of anti persister drugs.