铜绿假单胞菌对环丙沙星异质性耐药的研究

doi:10.3969/j.issn.1674-8115.2022.07.001

摘要/Abstract

摘要：

目的·探究铜绿假单胞菌对环丙沙星的异质性耐药机制。方法·通过圆盘扩散法对临床分离鉴定的铜绿假单胞菌（n=227）的环丙沙星异质性耐药进行初筛，同时根据抑菌圈直径对这些菌株的耐药性进行划分。通过无抗生素条件下连续传代后测定敏感性来判断异质性耐药菌株的稳定性。利用群体谱型分析法（population analysis profiling，PAP）对初筛结果进行验证。通过全基因测序分析异质性耐药的产生机制，最后测定比较不同突变类型的耐药亚群对不同抗生素敏感性的影响。结果·在圆盘扩散法中，227株临床菌株中有142株（62.6%）对环丙沙星表现为敏感，26株（11.5%）表现为中介，59株（26.0%）表现为耐药。该方法筛选得到18株潜在异质性耐药菌株，其中11株经PAP及稳定性检测后确定为稳定异质性耐药菌。随机挑选的18株初筛鉴定为非异质性耐药的菌株中，有3株经PAP鉴定为异质性耐药菌株。通过全基因组测序发现11株耐药亚群中，有8株发生mexS突变，2株发生gyrA突变；除此之外，基因fleQ、PA2632和PAKAF_02255均分别在1株菌株中发生突变。mexS与gyrA这2类突变型均导致莫西沙星耐药，部分mexS突变株同时对氯霉素与亚胺培南耐药。结论·铜绿假单胞菌对环丙沙星的异质性耐药率为4.8%（11/227）。圆盘扩散法作为铜绿假单胞菌异质性耐药筛选方法灵敏度较低，存在一定假阴性率。mexS突变是耐药亚群中最常见的突变类型，该突变可能通过使外排泵MexEF-OprN过表达来介导对环丙沙星在内的多种抗生素耐药。

关键词: 铜绿假单胞菌, 环丙沙星, 异质性耐药, 圆盘扩散法, 群体谱型分析

Abstract:

Objective·To investigate ciprofloxacin heteroresistance in Pseudomonas aeruginosa (PA).

Methods·Ciprofloxacin heteroresistance in 227 clinical PA strains was initially identified by disk diffusion. Meanwhile, susceptibilities of these strains was classified according to the diameter of the inhibition zone. The stability of the identified heteroresistant strains was tested by measuring the sensitivity after serial passage under antibiotic-free conditions. Mechanisms mediating heteroresistance were analyzed by whole-genome sequencing. Susceptibilities of resistant subpopulations to different antibiotics were also detected.

Results·Based on disk diffusion, 142 (62.6%), 26 (11.5%), and 59 (26.0%) of 227 isolates were classified as susceptible, intermediate and resistant to ciprofloxacin respectively. Eighteen putative heteroresistant strains were detected through primary disk diffusion selection, and 11 among them were further verified as stable heteroresistant strains by population analysis profiling (PAP). By whole-genome sequencing, 8 and 2 among 11 resistant subpopulations carried mutations in mexS and mutations in gyrA individually. In addition, mutations in fleQ, PA2632 or PAKAF_02255 were detected respectively. Both mexS and gyrA mutants led to moxifloxacin resistance, and some mexS mutants also showed resistance to chloramphenicol and imipenem.

Conclusion·Ciprofloxacin heteroresistance rate inPA is 4.8% (11/227). As a screening method for heteroresistance of PA, the disk diffusion has low sensitivity and has a certain false negative rate. The mutation mexS is the predominant mutation type in resistant subpopulations and it might enhance the overexpression of efflux pump MexEF-OprN, thus mediating resistance to different antibiotics including ciprofloxacin.

Key words: Pseudomonas aeruginosa, ciprofloxacin, heteroresistance, disk diffusion, population analysis profiling (PAP)

中图分类号:

R378

李聪聪, 姚玉峰, 张传珍. 铜绿假单胞菌对环丙沙星异质性耐药的研究[J]. 上海交通大学学报（医学版）, 2022, 42(7): 839-845.

LI Congcong, YAO Yufeng, ZHANG Chuanzhen. Emergence of ciprofloxacin heteroresistance in clinical Pseudomonas aeruginosa[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(7): 839-845.

图/表 5

图1 圆盘扩散法筛选平板Note： A. P. aeruginosa with heteroresistance in disk diffusion had a large number of clones in its inhibition zone. B. P. aeruginosa showed non-heteroresistance in the disk diffusion, with obvious boundaries of the inhibition zone and no obvious clones inside.

Fig 1 Plates of disk diffusion

图2 检测流程与主要结果Note： A. The collected 227 strains of P. aeruginosa were screened by disk diffusion, of which 18 strains showed potential heteroresistance. B. The stability of resistant subpopulations of the 18 potential heteroresistant strains was tested, and 11 of them showed stable heteroresistance. C. Eleven potential stable heteroresistant strains were re-tested with PAP, and no false positives appeared. D. Randomly selected 18 strains identified as non-heteroresistance by disk diffusion were re-tested by PAP, among which 3 showed heteroresistance.

Fig 2 Summary of tests performed and main results

图3 PAP实验结果Note： A. The 11 potentially stable heteroresistant strains all met the definition of heteroresistance in PAP (orange line). The black line represents the control strain PAO1. B. Of the 18 strains identified as non-heteroresistant by disk diffusion, 3 met the definition of heteroresistance in PAP (orange lines), and the rest did not (blue lines). The black line represents the control strain PAO1.

Fig 3 Results of PAP

表1 异质性耐药菌株与其耐药亚群MIC （μg·mL-1）

Tab 1 Susceptibilities of heteroresistant strain and its subpopulation （μg·mL-1）

Strain	MIC
Strain	CIP	MXF	IPM	MEM	CHL
30	0.5	8	0.5	0.25	16
30R	4	64	0.5	0.25	>128
1595	8	64	>1 024	>1 024	>128
1595R	64	64	>1 024	>1 024	>128
6655	0.125	1	0.5	0.25	32
6655R	4	64	4	0.5	>128
7314	0.06	0.5	1	4	16
7314R	1	2	1	4	16
7318	0.125	0.5	1	0.125	8
7318R	2	64	4	0.125	>128
7500	0.125	1	0.5	0.125	16
7500R	2	16	4	0.5	>128
7637	0.06	0.5	0.5	0.25	16
7637R	1	8	2	0.5	>128
7755	0.06	0.06	2	0.06	8
7755R	1	0.5	2	0.06	>128
7866	0.5	2	8	8	32
7866R	4	64	8	4	>128
7895	0.06	0.5	0.5	<0.06	4
7895R	2	16	0.5	<0.06	>128
A601	0.125	1	1	1	32
A601R	1	4	1	1	32

表2 耐药亚群突变基因与突变类型

Tab 2 Mutant gene and mutant type of resistant subpopulation

Strain	ST	Mutation （based on genome and Sanger sequencing）
30	508
30R	508	988‒998 bp deletion （mexS， frameshift）， rg131His （PA2632）
6655	508
6655R	508	354 insert T （mexS， frameshift）， Glu18Lys （hypothetical protein）
7314	204
7314R	204	Thr83Ile （gyrA）
7318	498
7318R	498	Phe259Ser （mexS）
7500	3 610
7500R	3 610	740‒754 bp deletion （mexS， frameshift）， 554 bp deletion （fleQ）
7637	532
7637R	532	423 bp deletion （mexS， frameshift）
7755	‒
7755R	‒	Trp42Leu （mexS）
7866	498
7866R	498	Gln147Lys （mexS）
7895	1 238
7895R	1 238	Pro46Leu （mexS）
A601	1 212
A601R	1 212	Thr83Ile （gyrA）

参考文献 21

1	GAYNES R, EDWARDS J R, SYSTEM N N I S. Overview of nosocomial infections caused by gram-negative bacilli[J]. Clin Infect Dis, 2005, 41(6): 848-854.
2	KALIL A C, METERSKY M L, KLOMPAS M, et al. Executive summary: management of adults with hospital-acquired and ventilator-associated pneumonia: 2016 clinical practice guidelines by the infectious diseases society of America and the American thoracic society[J]. Clin Infect Dis, 2016, 63(5): 575-582.
3	LYCZAK J B, CANNON C L, PIER G B. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist[J]. Microbes Infect, 2000, 2(9): 1051-1060.
4	HOOPER D C. New uses for new and old quinolones and the challenge of resistance[J]. Clin Infect Dis, 2000, 30(2): 243-254.
5	ANDRIOLE V T. The quinolones: past, present, and future[J]. Clin Infect Dis, 2005, 41(Suppl 2): S113-S119.
6	LEE J K, LEE Y S, PARK Y K, et al. Alterations in the GyrA and GyrB subunits of topoisomerase II and the ParC and ParE subunits of topoisomerase Ⅳ in ciprofloxacin-resistant clinical isolates of Pseudomonas aeruginosa[J]. Int J Antimicrob Agents, 2005, 25(4): 290-295.
7	LLANES C, KÖHLER T, PATRY I, et al. Role of the MexEF-OprN efflux system in low-level resistance of Pseudomonas aeruginosa to ciprofloxacin[J]. Antimicrob Agents Chemother, 2011, 55(12): 5676-5684.
8	EL-HALFAWY O M, VALVANO M A. Antimicrobial heteroresistance: an emerging field in need of clarity[J]. Clin Microbiol Rev, 2015, 28(1): 191-207.
9	MORAND B, MÜHLEMANN K. Heteroresistance to penicillin in Streptococcus pneumoniae[J]. Proc Natl Acad Sci USA, 2007, 104(35): 14098-14103.
10	OKADO J B, AVACA-CRUSCA J S, OLIVEIRA A L, et al. Daptomycin and vancomycin heteroresistance revealed among CC₅-SCCmecII MRSA clone and in vitro evaluation of treatment alternatives[J]. J Glob Antimicrob Resist, 2018, 14: 209-216.
11	ANDERSSON D I, NICOLOFF H, HJORT K. Mechanisms and clinical relevance of bacterial heteroresistance[J]. Nat Rev Microbiol, 2019, 17(8): 479-496.
12	JIA X J, MA W J, HE J C, et al. Heteroresistance to cefepime in Pseudomonas aeruginosa bacteraemia[J]. Int J Antimicrob Agents, 2020, 55(3): 105832.
13	HE J C, JIA X J, YANG S S, et al. Heteroresistance to carbapenems in invasive Pseudomonas aeruginosa infections[J]. Int J Antimicrob Agents, 2018, 51(3): 413-421.
14	POURNARAS S, IKONOMIDIS A, MARKOGIANNAKIS A, et al. Characterization of clinical isolates of Pseudomonas aeruginosa heterogeneously resistant to carbapenems[J]. J Med Microbiol, 2007, 56(Pt 1): 66-70.
15	HERMES D M, PORMANN PITT C, LUTZ L, et al. Evaluation of heteroresistance to polymyxin B among carbapenem-susceptible and-resistant Pseudomonas aeruginosa[J]. J Med Microbiol, 2013, 62(8): 1184-1189.
16	MEI S C, GAO Y L, ZHU C T, et al. Research of the heteroresistance of Pseudomonas aeruginosa to imipenem[J]. Int J Clin Exp Med, 2015, 8(4): 6129-6132.
17	NICOLOFF H, HJORT K, LEVIN B R, et al. The high prevalence of antibiotic heteroresistance in pathogenic bacteria is mainly caused by gene amplification[J]. Nat Microbiol, 2019, 4(3): 504-514.
18	XU Y, ZHENG X K, ZENG W L, et al. Mechanisms of heteroresistance and resistance to imipenem in Pseudomonas aeruginosa[J]. Infect Drug Resist, 2020, 13: 1419-1428.
19	OH H, STENHOFF J, JALAL S, et al. Role of efflux pumps and mutations in genes for topoisomerases II and IV in fluoroquinolone-resistant Pseudomonas aeruginosa strains[J]. Microb Drug Resist, 2003, 9(4): 323-328.
20	GOLI H R, NAHAEI M R, REZAEE M A, et al. Contribution of mexAB-oprM and mexXY (-oprA) efflux operons in antibiotic resistance of clinical Pseudomonas aeruginosa isolates in Tabriz, Iran[J]. Infect Genet Evol, 2016, 45: 75-82.
21	JØRGENSEN K M, WASSERMANN T, JENSEN P Ø, et al. Sublethal ciprofloxacin treatment leads to rapid development of high-level ciprofloxacin resistance during long-term experimental evolution of Pseudomonas aeruginosa[J]. Antimicrob Agents Chemother, 2013, 57(9): 4215-4221.

[1]	余春波 1，卢明 2，邵雷 3，蒲甜 4，陈代杰 4，周薇 1, 5. relA基因敲除对多黏菌素抗鲍曼不动杆菌异质性耐药的影响[J]. 上海交通大学学报（医学版）, 2019, 39(9): 1004-.
[2]	徐彬，高晶，郭晓奎，等. 铜绿假单胞菌噬菌体D204的生物学特性和基因组学研究[J]. 上海交通大学学报（医学版）, 2016, 36(1): 8-.
[3]	张佳星，沈犁，华子瑜，等. 两种提取铜绿假单胞菌外膜囊泡的方法比较[J]. 上海交通大学学报（医学版）, 2014, 34(3): 279-.