铜绿假单胞菌对青霉素类抗生素异质性耐药研究

Heteroresistance of Pseudomonas aeruginosa to penicillins

摘要:

【背景】铜绿假单胞菌是临床上常见的条件致病菌，其异质性耐药的发生常导致临床治疗失败。【目的】研究铜绿假单胞菌对青霉素类抗生素的异质性耐药情况，为相关临床感染治疗提供一定的依据。【方法】收集临床分离的50株铜绿假单胞菌，采用纸片扩散法(disk diffusion method)即Kirby-Bauer (K-B)法、菌落谱型分析(population analysis profile，PAP)法、生长实验以及传代稳定性实验探究铜绿假单胞菌的异质性耐药特征。【结果】K-B法初筛得到铜绿假单胞菌对哌拉西林(piperacillin，PIP)、哌拉西林/他唑巴坦(piperacillin/tazobactam，TZP)和替卡西林/克拉维酸(ticarcillin/clavulanic acid，TIM)的异质性耐药率分别为52%、52%和54%。PAP实验确认后有13株异质性耐药菌，其检出率占总实验菌株的26%。随机选取8株异质性耐药菌株，其耐药亚群的发生频率为7.3×10⁻⁷−1.2×10⁻⁵。通过无抗生素压力的生长实验发现，异质性耐药菌株PAS92、PAS57与其各自的3株最高PIP浓度平板上的耐药亚群的生长速度均差异无统计学意义(P>0.05)，但PAS92抑菌圈内的耐药亚群比PAS92的生长速度快(8−12 h)，有极显著差异(P=0.002 2<0.01)。抑菌圈内不同耐药水平的耐药亚群及从PAP实验中最高PIP浓度平板上挑取的高水平耐药亚群在无抗生素压力下传代培养，发现仅有一株耐药亚群不稳定且恢复到原始菌株的药物敏感水平，其他菌株耐药稳定性良好。【结论】铜绿假单胞菌对青霉素类抗生素的异质性耐药率相对偏低，但异质性耐药亚群的发生频率较高，而且对哌拉西林的耐药水平较高，大多耐药亚群传代稳定性良好。因此在使用该类抗生素治疗时应更加注意异质性耐药现象的发生，防止更高水平耐药菌的出现而导致治疗失败。

Abstract:

[Background] Pseudomonas aeruginosa (PA) is a common opportunistic pathogen in clinical practice, and its heteroresistance often leads to failure of clinical treatment. [Objective] To study the heteroresistance of PA to penicillins, and to provide a basis for the treatment of related clinical infections. [Methods] We collected 50 clinical isolates of PA and investigated their heteroresistance characteristics by using disk diffusion method (Kirby-Bauer (K-B) test), population analysis profile (PAP), growth assay and passage stability test. [Results] According to the preliminary screening results obtained with K-B test, 52%, 52%, and 54% of the PA isolates had heteroresistance to piperacillin (PIP), piperacillin/tazobactam (TZP) and ticarcillin/clavulanic acid (TIM), respectively. Thirteen (26%) strains were confirmed to have heteroresistance by PAP. Eight heteroresistant strains were randomly selected, and the frequency of resistant subpopulations ranged from 7.3×10⁻⁷ to 1.2×10⁻⁵. Without antibiotic pressure, the heteroresistant strains PAS92 and PAS57 and their three subpopulations on the plates with the highest PIP concentration showed no statistical difference in growth rate (P>0.05), while the growth of the subpopulations in the inhibition zone of PAS92 was faster than that of PAS92 within 8–12 h (P=0.002 2<0.01). The subpopulations with different levels of resistance in the inhibition zone and those with high levels of resistance selected from the plates with the highest PIP concentration in PAP were subcultured without antibiotic pressure. Only one of the subpopulations was found to be unstable and returned to the sensitivity level of the original strain, while the others had good stability of resistance. [Conclusion] In this study, PA isolates show low heteroresistance ratio while high frequency of resistant subpopulations to penicillins. Besides, they have high heteroresistance to PIP, and most of the resistant subpopulations had good passage stability. Therefore, when using this type of antibiotics, attention should be paid to the occurrence of heteroresistance to prevent the emergence of strains with higher drug-resistance from causing treatment failure.