哌拉西林他唑巴坦对多重耐药铜绿假单胞菌外排泵表达的影响

Effect of piperacillin/tazobactam on the expression of efflux pump of multi drug resistant Pseudomonas aeruginosa

目的研究哌拉西林他唑巴坦对多重耐药铜绿假单胞菌(MDRP)的MexAB-OprM、MexXY-OprM外排泵表达的影响。方法选择2013-2015年临床分离的28株MDRP,用泵抑制剂——碳酰氰基-对-氯苯腙(CCCP)100μmol·L^(-1)干预后,测定各种常见抗菌药物的最低抑菌浓度(MIC)值,筛选出主动外排表型阳性菌株;用实时定量RT-PCR法测定并筛选出的主动外排表型阳性菌株的mexA、mexX表达水平,用1μg·mL^(-1)哌拉西林他唑巴坦干扰12 h,再次测定mexA、mexX表达水平。结果用CCCP干预后,共有10株MDRP对美罗培南、头孢他啶、环丙沙星以及左氧氟沙星的敏感性有显著变化,MDRP05对美罗培南、头孢他啶、环丙沙星、左氧氟沙星的MIC值为16,256,32,64μg·m L-1,MDRP05对美罗培南+CCCP、头孢他啶+CCCP、环丙沙星+CCCP、左氧氟沙星+CCCP的MIC值降为4,64,2,4μg·mL^(-1)。在10株主动外排泵表型阳性菌中,有9株(除MDRP07外)MDRP表现为mexA高表达,其中MDRP08、MDRP12、MDRP18、MDRP20表现为mexX高表达。用哌拉西林他唑巴坦干预后,除MDRP07、MDRP24外,其余各菌株mexA相对表达量均较干预前显著降低(P<0.05)。干预前,PAO1、MDRP05、MDRP07、MDPR08、MDRP09、MDPR12、MDPR15、MDRP18、MDRP20、MDRP24、MDRP25菌株的mexA表达水平分别为9544±336,18473±821,6405±197,22395±796,15062±416,19582±356,11294±764,10291±876,34950±1186,9874±327,11215±1183;干预后,这11株MDRP的mexA表达水平分别为7856±216,14963±613,6205±108,17485±733,13052±215,15063±453,9320±521,8940±728,30049±638,8920±426,9201±730。用哌拉西林他唑巴坦干预后,MDRP08、MDPR12、MDRP18以及MDRP20的mexX相对表达量较干预前差异有统计学意义(P<0.05);而其余菌株干预前后mexX相对表达量变化差异无统计学意义(P>0.05)。干预前,这11株MDRP的mexX表达水平分别为73±6,35±3,27±4,186±21,17±3,387±37,65±12,734±70,395±24,25±5,36±6;干预后,这11株MDRP的mexX表达水平分别为70±10,37±2,28±5,148±15,17±5,312±46,60±11,658±30,332±41,28±8,28±8。结论哌拉西林他唑巴坦能够影响外排泵系统MexAB-OprM、MexXY-OprM,这可能是哌拉西林他唑巴坦对多重耐药铜绿假单胞菌的作用机制之一。

Objective To study the influence of piperacillin tazobactam in multi drug resistant Pseudomonas aeruginosa （MDRP） bacteria on the OprM, mexxy OprM efflux pump expression. Methods The clinical isolated 28 strains of MDRP bacteria were selected from 2013 to 2015. The value of the minimum inhibitory concentration （MIC） for a variety of common antibiotics was determined after intervention by 100 μg · mL-1 pump inhibitor carbonylcyanide, p -chlorophenylhydrazone （CCCP） and screening of active efflux phenotype positive strains. The active row positive phenotype strains the mexA, mexX expression level was screen out using real - time quantitative RT - PCR assay. The mexA, mexX expression levels was again determined using 1μg · mL-1 piperacillin tazobactam interference 12 h. Results After treatment of CCCP, among which there were 10 strains of MDRP had significant changes in sensitivity of meropenem, ceftazidime, ciprofloxacin and levofloxa- cin,such as the MIC of MDRP05 to meropenem, ceftazidime, ciprofloxacin and levofloxacin are 16,256,32,64 μg · mL-1, but the MIC of MDRP05 to meropenem ＋CCCPceftazidime ＋CCCPciprofloxacin ＋CCCPand levofloxacin ＋CCCPare decreased with 4,64,2,4μg · mL-1. Ten strains of active efflux phenotype positive strains, including 9 strains （ except MDRP07 ） MDRP showed high expression of mexA, of which 4 strains （ MDRP08, MDRP, MDRP18, MDRP12 MDRP20 ） showed high expression of mexX. After using piperacillin tazobactam intervention, except MDRP07 and MDRP24, the relative expression of strain mexA were significantly lower than those before inter- vention（P 〈 0. 05）. The expression levels of PAO1, MDRP05, MDRP07, MDPR08, MDRP09, MDPR12, MDPR15, MDRP18, MDRP20, MDRP24 and MDRP25 strains mexA before intervention were 9544 ± 336, 18473 ± 821, 6405 ± 197,22395 ± 796, 15062 ± 416, 19582 ± 356, 11294 ± 764, 10291 ± 876, 34950 ± 1186, 9874 ± 327, 11215 ± 1183, respeetively; and after intervention that were 7856 ± 216, 14963 ± 613, 6205 ± 108, 17485 ± 733, 13052 ±215,15063 ±453, 9320 ±521, 8940 ±728, 30049 ±638, 8920 ±426, 9201 ±730, respectively. After using piperaeillin tazobaetam intervention, MDRP08, MDPR12, MDRP18 and MDRP20 mexX relative expression was signifi- cantly lower than before intervention （ P 〈 0.05 ）. And the rest of strains after the intervention of mexX mRNA expre- ssion was no difference than before intervention （ P 〉 0.05 ）. The expression levels of PAO1, MDRP05, MDRP07, MDPR08, MDRP09, MDPR12, MDPR15, MDRP18, MDRP20, MDRP24 and MDRP25 strains mexX before interven- tion were 73 ±6,35 ±3,27 ±4,186 ±21,17 ±3,387 ±37,65 ± 12,734 ±70,395 ±24,25 ±5,36 ±6, and after inter- vention were 70 ± 10,37 ±2,28 ±5,148 ± 15,17 ±5,312 ±46,60 ± 11,658 ±30,332 ±41,28 ±8,28 ±8. Coneluslon The piperaeillin tazobactam can affect pump system MexAB - OprM, MexXY - OprM efflux, it may is one of mechanism on piperacillin tazobactam against multi drug resistant Pseudomonas aeruginosa.