体外诱导多黏菌素耐药肺炎克雷伯菌的适合度代价相关研究

The fitness cost of clinical isolates of induced polymyxin-resistant Klebsiella pneumoniae in vitro

目的研究体外诱导多黏菌素耐药肺炎克雷伯菌的适合度代价,并分析耐药株的分子特性。方法采用体外诱导方法将临床分离的3株多黏菌素敏感肺炎克雷伯菌诱导成多黏菌素耐药株;微量肉汤稀释法检测体外诱导耐药前后菌株对临床常用抗菌药物的最低抑菌浓度(minimal inhibititory concentration,MIC);采用PCR方法检测多黏菌素耐药相关基因pmr A、pmr B、mgr B、pho P和pho Q,并对阳性片段进行测序比对分析;对诱导耐药菌株及其对应敏感菌株进行生长曲线分析、生物膜形成能力检测及体外竞争和抗血清试验,分析研究多黏菌素耐药肺炎克雷伯菌的适合度代价。结果 3株诱导耐药株中均检出pmr B(T157P)突变,在诱导株FK713R和FK729R携带的mgr B基因中分别检出ISkpn14和IS5like插入序列。诱导耐药株与敏感株相比,24h生长曲线未发现明显差异;生物膜形成能力检测结果发现诱导耐药株与敏感株生物膜形成能力无明显差异;3株菌获得多黏菌素耐药后均表现出一定程度的体外竞争缺陷,竞争指数(CI值)分别为0.01、0.54和5×10-4;3株诱导耐药菌中有2株(FK713R和FK729R)与其敏感菌相比出现抗血清作用增强现象。结论肺炎克雷伯菌获得多黏菌素耐药后会出现一定的适合度改变,不同的耐药机制可能会引起不同的适合度表现。

Objective To investigate the fitness cost ofpolymyxin-resistant Klebsiellapneumoniae which were acquired from polymyxin B susceptible strains through induction in vitro, and analyze the molecular characteristics of resistant strains. Methods Three polymyxin-resistant Klebsiella pneumoniae which were acquired from in vitro induction were collected. The six strains were tested the minimum inhibitory concentration （MIC） of clinical commonly used antimicrobial agents by the broth dilution method, and the polymyxin-resistant genes pmrA, pmrB, mgrB, phoP andphoQ were screened by polymerase chain reaction （PCR）. Growth curves, biofilm formation, serum resistance and competition assays in vitro were performed to detect the fitness costs of polymyxin-incduced resistant strains compared with their susceptible parental strains. Results For the in vitro selected mutants, all mutants revealed mutations in pmrB （T157P）. The ISkpnl4 and IS51ike insertions were detected in the mgrB gene in FK713R and FK729R, respectively. No differences were observed in growth curves and biofilm formation ability among three polymyxin B mutants and the corresponding susceptible parental strains. Three polymyxin B mutants exhibited significant fitness defects when co-cultured with the parental strains, competitive index （C! values） were 0.01, 0.54 and 5×10^-4, respectively. FK713R and FK729R exhibited more resistance to serum compared with their susceptible bacteria. Conclusion A certain degree of fitness cost would occur when Klebsiella pneumoniae acquired polymyxin B resistance, and different resistance mechanisms may cause different fitness costs.