Investigation Of The Relationship Of Pilus And Carbenicillin Resistance In Pseudomonas Aeruginosa

In recent years,with the emergence of Multiple Drug-resistant Strains(MDR),more and more antibiotics are gradually losing their effectiveness;therefore human health faces a serious threat from infectious diseases once again.Pseudomonas aeruginosa(PA)is an opportunistic human pathogen.It can cause all kinds of serious infection and often pose a threat to the life of the patients.Infections caused by MDR PA in MDR are very difficult to treat in the clinic,which makes PA one of the dangerous drug resistant pathogens.It is clear that the study of the mechanism of PA drug resistance is not only important theoretically but also of urgent clinical significance.Our previous study showed that the the transposon mutant of pilY1,a Type IV Pili(T4P)gene,in PA was insusceptibility to carbenicillin(Car),with a MIC 128 times higher than that of the wild type.The Car MIC of another T4P gene pilE mutant also increased 16 times than the wild type.These observations suggest that PA T4P may be related to carbenicillin resistance.Therefore,in this study,the relationship between T4P and carbenicillin resistance was studied.First,the PAO1 mutant library of in our laboratory was screened,and we identified 5 knockout mutants and 7 transposon mutants with compromised twitching motility.Test of Car susceptibility with these mutants indicated that 5 of them had increased susceptibility and 7 had decreased susceptibility compared with the wild type,further indicating a potential connection between T4P and antibiotic resistance.Then,sixteen different antibiotics were used to assess the antibiotic resistance of the above 12 mutants,and the results suggested that only Car susceptibility was changed,which implied that T4P has unique association with Car.Then,we analyzed the relationship between antibiotic resistance and twitching motility in clinical isolates of PA.A total of 89 isolates of PA were collected from a hospital in Xi'an.Their twitching motility and carbenicillin antibiotic resistance were investigated.The results show that 70.78%of the clinical isolates had decreased twitching motility compared to PAO1,and 29.22%unchanged.Two isolates were more sensitivity to Car,accounting for 2.24%of the total isolates,and 3 strains remained unchanged,accounting for 3.37%while the remaining 84 strains were more resistant to Car,accounting for 93.38%.The MIC of some clinical isolates to Car reached 8 mg/ml.These results further suggest that there seems to be a potential association between T4P and carbenicillin resistance in PA.To reveal the mechanisms underlying the connection between motility and antibiotic resistance,we constructed the following knockout mutants:?pilZ,?pilB??pilJ,?pilS,?pilE,?fimX.?pilD and ApilYi1,which are involve in the synthesis of T4P.Although twitching motility of these mutants were severely disrupted,there were no significant changes in Car susceptibility.It is speculated that perhaps the Car resistance of the pilYi transposon mutant may depends on the specific site of transposon insertion.Simply knockout of the structure genes may not be able to cause Car resistance.Further investigation is needed to understand the T4P associated Car resistance.These results obtained provide a basis for further study on the potential mechanism of carbenicillin resistance in PA.