| Objective:To investigate the effect of Pseudomonas aeruginosa pvdQ gene on altering antibiotic susceptibility under swarming conditions and the mechanisms.Methods:The plasmid pME6032 with pvdQ gene was constructed and identified, then transformed into P. aeruginosa PAO1 wild type by using the electroporation, building pvdQ-overexpression strain, PAO1pMEpvdQ. The pME6032-PAO1 strain was constructed with the same method. The cloning plasmid pEX18Gm containing sacB was successfully used to construct unmarked deletion mutant of pvdQ gene, pvdQ-mutant strain, PAO1△pvdQ. Bacteria were inoculated in LB overnight, measuring the colony diameter of the swarming zone. E-test strips were used to test the PAO1 and PAO1pMEpvdQ MIC PAO1 and PAO1pMEpvdQ cultures were grown on BM2-swarming agar plates with the appropriate antibiotics for 24 h under swarming conditions. We used the TEM assay to compare the PAO1 wild type with PAO1pMEpvdQ swarmer cell morphology. Biofilm formation was quantified using crystal violet (CV) assay, biofilm development was monitored in different time intervals,24h,48h, and 72h. Using CLSM assay detect the biofilm formation at different time intervals 12h,24h, and 72h. PAOl and PAO1pMEpvdQ strains outer membrane barrier function was determined using the 1-N-phenyl naphthylamine (NPN) assay. Real-time PCR was performed to compare the expression of the multidrug efflux pumps MexAB-OprM oprM gene and MexGHI-OpmD mexl gene in swarming cells and planktonic bacteria cells between the PAO1 wild type and PAO1pMEpvdQ strains.Results:Strains of PAO1pMEpvdQ, pME6032PAO1, and PAO1△pvdQ were successfully constructed by real-time PCR. Comparison of four strains of the swarming motility of change in diameter:pME6032-PAO1 strain compared with the wild strain PAO1 wild type, diameter no significant change (P>0.05); PAO1△pvdQ strain compared with the wild strain PAO1, diameter decreased (P<0.05); PAO1pMEpvdQ strain compared with the wild strain PAO1, diameter increased (P<0.05).The PAO1 and PAO1pMEpvdQ strains were both susceptibility to the above mentioned antibiotics. Comparison of two strains of the swarming motility of change in diameter:The result showed that PAO1 wild type and PAO1pMEpvdQ strains can both improve the antibiotics resistance. The PAO1 wild type strain when the ceftazidime concentration over 8μg/ml, ciprofloxacin concentration over 0.4μg/ml, merpenem concentration over 4μg/ml, ploymyxinB concentration over 16μg/ml, gentamicin concentration over 8μg/ml, the swarming motility was inhibited. The PAO1pMEpvdQ strain when the ceftazidime concentration over 32μg/ml, ciprofloxacin concentration over 1.6μg/ml, merpenem concentration over 16μg/ml, ploymyxinB concentration over 64μg/ml, gentamicin concentration over 64μg/ml, the swarming motility was inhibited. This means that the swarmer cells of PAO1pMEpvdQ strain exhibited a 2-to 8-fold increase in antibiotic resistance toward ceftazidime, ciprofloxacin, meropenem, polymyxin B and gentamycin compared to PAO1 wild type on BM2-swarming agar plates. We analyzed the PAO1pMEpvdQ swarmer cell was long and multinucleate compared to PAO1 wild type. The PAO1pMEpvdQ strain was successfully constructed by PCR. After 24h,48h, and 72h hour incubation, the quantitative data showed that at 24h there were no significant difference in PAO1 wild type, PAO1pMEpvdQ strains, and PAO1△pvdQ (P> 0.05), but at 48h and 72h hour we found thicker biofilm was observed in PAOlApvdQ strains cpmpared to PAO1 wild type (P<0.05), where thinner biofilm formation was seen in PAO1pMEpvdQ strains cpmpared to PAO1 (P<0.05). The results of CLSM have shown that at 72h we found that thinner biofilm formation was seen in PAO1pMEpvdQ strains cpmpared to PAO1 wild type (P<0.05). Our data suggest that pvdQ also taken part in decreasing the P. aeruginosa outer membrane permeability. The mRNA expression of oprM gene and mexl gene in PAO1 wild type and PAOlpMEpvdQ swarming conditions respectively were higher than in the control strain PAO1 wild type planktonic bacteria conditions. But there was no significant difference between the PAO1 wild type and PAO1pMEpvdQ swarming conditions(P>0.05).Conclusion:The strain of PAO1pMEpvdQ can promote the swarming motility diameter, the PAO1△pvdQ strain inhibits the swarming motility diameter, this means pvdQ gene may involve in regulating the swarming motility of P. aeruginosa. The PAO1pMEpvdQ strain exhibited a 2-to 8-fold increase in antibiotic resistance towards ceftazidime, ciprofloxacin, merpenem, ploymyxinB, and gentamicin compared to what was observed for PAO1. We found that the antibiotic concentrations tolerated by the bacteria under swarming motility were significantly higher than the MIC, this means that pvdQ gene may do not participate in changing antibiotic resistance in the planktonic bacteria. PvdQ gene may through prarticipate in the swanner cell differentiation and take part in decreasing the P. aeruginosa outer membrane permeability, whether the multidrug efflux pumps MexAB-OprM and MexGHI-OpmD was involved in elevating antibiotics resistance...
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