头孢哌酮钠舒巴坦钠诱导对铜绿假单胞菌的耐药性影响

The effect of in vitro-selection by cefoperzone-sulbactam on antibiotics resistance of Pseudomonas aeruginosa

目的探讨体外高浓度头孢哌酮钠舒巴坦钠(SCF)诱导铜绿假单胞菌后耐药谱的变化。方法收集4株SCF敏感的临床铜绿假单胞菌以及1株实验室标准株,经过体外3.2倍最低抑菌浓度(MIC)的SCF诱导50代后,得到耐药铜绿假单胞菌。采用微量肉汤稀释法测定SCF、美罗培南等12种抗菌药物对诱导耐药株的MIC,根据2017年临床与实验室标准化所(CLSI)M100-S29的药物敏感性标准对结果进行判定。利用实时定量PCR(RT-qPCR)对铜绿假单胞菌的耐药相关基因的表达进行检测。结果经过3.2倍MIC的SCF诱导50代后,铜绿假单胞菌对抗假单胞菌的β-内酰胺类或β-内酰胺酶抑制剂抗菌药物的MIC平均增加14.0倍,对抗假单胞菌的头孢菌素类抗菌药物MIC平均增加9.0~16.0倍,对美罗培南的MIC至少增加4.0倍,但是对亚胺培南的MIC影响很小,对单环-β-内酰胺类抗菌药物MIC平均增加6.0倍,对喹诺酮类和氨基糖苷类抗菌药物的MIC大多保持不变。RT-qPCR结果显示,SCF诱导后外排泵基因mexD表达升高,孔道蛋白OprD表达降低(P均<0.05)。结论 SCF使用可引起敏感的铜绿假单胞菌耐药性改变,诱导成对多种β-内酰胺类抗菌药物耐药的铜绿假单胞菌,甚至耐碳青霉烯铜绿假单胞菌。外排泵mexD的升高和孔道蛋白OprD的降低是体外SCF诱导铜绿假单胞菌耐药的重要机制。临床使用SCF需要谨慎合理,以减少多重耐药铜绿假单胞菌的产生。

Objective To investigate the variations in the drug susceptibility of Pseudomonas aeruginosa(P.aeruginosa) isolates afier the in vitro-selection with high-dose cefoperazone-sulbactam(SCF).Methods Four clinical isolates of SCF-sensitive P.aeruginosa and 1 standard laboratory isolate were collected.The drug-resistant P.aeruginosa isolates were obtained after 50 generations of SCF induction at a concentration of 3.2×MIC.The antimicrobial susceptibility of 12 antibiotics,such as SCF and meropenem,was determined by disk diffusion method,and the results were judged according to the Clinical and Laboratory Standards Institute(CLSI) M100-S29 guideline in 2017.The expression of resistance-related genes in P.aeruginosa was detected by real-time quantitative PCR(RT-qPCR).Results After 50 generations of SCF induction at a concentration of 3.2×MIC,the MIC of P.aeruginosa against anti-pseudomonal β-lactam+β-lactamase inhibitors was increased by 14 times on average.In addition,the MIC to anti-pseudomonal cephalosporins was elevated by 9.0-16.0 times on average.Moreover,the resistance rate to meropenem was increased by at least 4.0 times.However,the MIC to imipenem was not significantly affected.For monobactams,the MIC was increased by 6.0 times on average.The susceptibility to anti-pseudomonal fluoroquinolones and aminoglycoside remained largely unchanged.RT-qPCR showed that the expression of the efflux pump gene mexD was significantly upregulated,whereas that of the outer membrane porin OprD was considerably down-regulated after SCF induction(both P<0.05).Conclusions The use of SCF can cause changes in the drug resistance of P.aeruginosa,induce multi-P-lactam antibiotics-resistant and even carbapenem-resistant P.aeruginosa.The increase of efflux pump MexCD-OprJ and the decrease of outer membrane porin OprD are the mechanisms underlying the role of SCF in the selection of P.aeruginosa.In clinical practice,physicians should be cautious about the use of SCF to reduce the occurrence of multidrug-resistant P.aeruginosa.