agr系统在单核增生细胞李斯特菌耐药及生物被膜形成中的作用

doi:10.13560/j.cnki.biotech.bull.1985.2022-0421

摘要/Abstract

摘要：

研究agr系统在单核增生细胞李斯特菌（Lm）对不同抗菌剂耐药及生物被膜形成中的作用。构建agr基因缺失突变株，测定突变株对抗菌药物的敏感性；微孔板定量法检测Lm在不同温度下生物被膜的形成量并利用倒置显微镜观察生物被膜结构；采用软平板法测定细菌的群集及泳动运动能力；通过实时荧光定量PCR检测运动相关基因的转录水平。缺失agr系统导致Lm对头孢噻肟、环丙沙星及苯扎氯铵的耐药性降低。与野生株EGD-e相比，突变株在37℃、20℃以及4℃条件下生物被膜形成量均明显降低；∆agrC的群集运动能力增强。RT-qPCR结果显示，缺失agr系统后flaA的转录水平下降。agr系统参与Lm耐药及生物被膜的形成。

关键词: 单核细胞增生李斯特菌, agr系统, 耐药, 生物被膜

Abstract:

This work aims to investigate the role of agr system in antimicrobial resistance and biofilm formation of Listeria monocytogenes（Lm）. Gene deletion mutants of agr system were constructed and antimicrobial susceptibility of the mutants was determined. The microplate method was used to measure the biofilm biomass at different temperature, and the inverted microscope was applied to observe the biofilm structure. Plate migration method was to investigate the swimming and swarming motilities of strains. RT-qPCR was adapted to detect the relative expressions of motility-associated genes. Reduced antimicrobial resistances to cefotaxime, ciprofloxacin and benzalkonium chloride were observed in the agr deletion mutants. Compared with the wild type EGD-e, the mutant strains showed decreasing biofilm biomass at 37℃, 20℃ and 4℃; and increased swarming motility was observed in ∆agrC. Results from RT-qPCR showed that the transcription level of flaA in the agr deletion mutants decreased. agr system is involved in antimicrobial resistance and biofilm formation of L. monocytogenes.

Key words: Listeria monocytogenes, agr system, antimicrobial resistance, biofilm

任思雨, 姜聪一, 于涛, 康瑞, 姜晓冰. agr系统在单核增生细胞李斯特菌耐药及生物被膜形成中的作用[J]. 生物技术通报, 2023, 39(2): 254-262.

REN Si-yu, JIANG Cong-yi, YU Tao, KANG Rui, JIANG Xiao-bing. Role of agr System in the Antimicrobial Resistance and Biofilm Formation of Listeria monocytogenes[J]. Biotechnology Bulletin, 2023, 39(2): 254-262.

图/表 15

图1 Lm中的agr系统

Fig. 1 agr system in L. monocytogenes

表1 PCR扩增引物序列

Table 1 Primer sequences for PCR amplification

引物名称 Primer name	序列 Sequence（5'-3'）	长度 Length/bp
agrA-P1	NNNNNNGGATCCGCTCTTACTGTCTTA- GCGTTCT	882
agrA-P2	CCTGTGGCACCGATAAAATTCGCTGC- ATTCTGTTATCTTC	882
agrA-P3	GAAGATAACAGAATGCAGCGAATTTT- ATCGGTGCCACAGG	568
agrA-P4	NNNNNNACGCGTAGTATTCCCATCGG- CATTT	568
agrB/D-P1	NNNNNNGGATCCGTCTACAAAGTTGA- TGGGATT	680
agrB/D-P2	ACTCATAGAAGAATCCGCAAATCATCT- TTCCAGCGGTC	680
agrB/D-P3	GACCGCTGGAAAGATGATTTGCGGATT- CTTCTATGAGT	608
agrB/D-P4	NNNNNNACGCGTGCTAAGACAGTAAG- AGCAACG	608

图2 agrA和agrB/D基因缺失株的构建 A：扩增上下游同源臂（M：DL2000 Maker；1：agrA上游同源臂；2：agrA下游同源臂；3：agrA融合片段；4：agrB/D上游同源臂；5：agrB/D下游同源臂；6：agrB/D融合片段）；B：双酶切验证（M：DL2000；1，2：pMAD-agrA双酶切验证；3，4：pMAD-agrB/D双酶切验证）；C：敲除验证（M：DL2000；1，2：ΔagrA；3：ΔagrB/D）

Fig. 2 Construction of ?agrA and ?agrB/D A: Amplification of upstream and downstream homology arms（M: DL2000 maker; 1: agrA upstream homology arm; 2: agrA downstream homology arm; 3: agrA fusion fragment; 4: agrB/D upstream homology arm; 5: agrB/D downstream homology arm; 6: agrB/D fusion fragment）. B: Double digestion verification（M: DL2000; 1, 2: pMAD-agrA double digestion verification; 3, 4: pMAD-agrB/D double digestion verification）. C: Knockout validation（M: DL2000; 1, 2: ΔagrA; 3: ΔagrB/D）

表2 抗菌物质对Lm菌株的MIC值

Table 2 MICs of antimicrobial agents against L. monocytogenes

抗菌剂Antibacterial agents		EGD-e	ΔagrA	ΔagrB/D	ΔagrC
抗生素 Antibiotic	氨苄西林 Ampicillin	1	1	1	1
	头孢噻肟 Cefotaxime	12	8	12	12
	头孢噻吩 Cephalothin	12	12	12	12
	红霉素 Erythromycin	0.125	0.125	0.125	0.125
	卡那霉素 Kanamycin	4	4	4	4
	四环素 Tetracycline	0.5	0.5	0.5	0.5
	氯霉素 Chloramphenicol	4	4	4	4
	环丙沙星 Ciprofloxacin	1	1	0.5	0.5
抗菌肽 Antimicrobial peptides	乳酸链球菌素 Nisin	10	10	10	10
消毒剂 Disinfectant	BC Benzalkonium chloride	6	6	6	6

图3 Lm的生长曲线

Fig. 3 Growth curves of L. monocytogenes

表3 生长曲线分析

Table 3 Growth curve analysis

菌株 Strain	迟缓期LPD Lag phase duration/h		平均最大生长速率MMGR Mean maximum growth rate/（Units·h^-1）		平均最大光密度值MMOD Mean maximum optical density/Units
菌株 Strain	BHI	BC/（1 μg·mL^-1）	BHI	BC/（1 μg·mL^-1）	BHI	BC/（1 μg·mL^-1）
EGDe	3.488±0.116	22.246±0.249	0.205±0.0115	0.0842±0.00641	0.7125±0.00200	0.615±0.045
ΔagrA	3.348±0.196	16.774±0.352***	0.185±0.0167	0.1300±0.01970*	0.7108±0.00062	0.566±0.005
ΔagrB/D	3.311±0.143	27.401±0.208**	0.199±0.0129	0.0484±0.00171**	0.7047±0.00960	0.577±0.063
ΔagrC	3.503±0.154	40.803±0.111***	0.196±0.0147	0.0646±0.00321*	0.7005±0.00710	0.211±0.155*

图4 Lm对BC的致死曲线

Fig. 4 Death curves of L. monocytogenes exposed to BC

图5 Lm菌株在37℃下形成的生物被膜 A：生物被膜形成量；B：倒置显微镜观察生物被膜（37℃培养2 d）

Fig. 5 Biofilm formation of L. monocytogenes strains at 37℃ A: The biofilm biomass. B: Biofilm assay by inverted microscope（incubated at 37℃ for 2 d）

表4 野生株和突变株在37℃下形成的生物被膜量

Table 4 Biofilm biomasses of wild type and mutant strains at 37℃

菌株 Strain	生物被膜量Biofilm mass
菌株 Strain	1 d	2 d	3 d
EGDe	0.219±0.044	1.408±0.114	0.611±0.086
?agrA	0.106±0.017	0.968±0.041	0.342±0.040
?agrB/D	0.103±0.025	1.043±0.070	0.330±0.057
?agrC	0.162±0.052	1.509±0.102	0.263±0.030

图6 Lm菌株在20℃下形成的生物被膜 A：生物被膜形成量；B：倒置显微镜观察生物被膜（20℃培养6 d）

Fig. 6 Biofilm formation of L. monocytogenes strains at 20℃ A: The biofilm biomass. B: Biofilm assay by inverted microscope（incubated at 20℃ for 6 d）

表5 野生株和突变株在20℃下形成的生物被膜量

Table 5 Biofilm biomasses of wild type and mutant strains at 20℃

菌株 Strain	生物被膜量Biofilm mass
菌株 Strain	3 d	6 d	9 d
EGDe	0.145±0.012	0.630±0.070	0.436±0.023
?agrA	0.102±0.004	0.612±0.033	0.425±0.019
?agrB/D	0.109±0.007	0.706±0.064	0.389±0.017
?agrC	0.099±0.003	0.621±0.009	0.474±0.013

图7 Lm菌株在4℃下形成的生物被膜 A：生物被膜形成量；B：倒置显微镜观察生物被膜（4℃培养20 d）

Fig. 7 Biofilm formations of L. monocytogenes strains at 4℃ 20 d A: The biofilm biomass. B: Biofilm assay by inverted microscope（incubated at 4℃ for 20 d）

表6 野生株和突变株在4℃下形成的生物被膜量

Table 6 Biofilm biomasses of wild type and mutant strains at 4℃

菌株 Strain	生物被膜量Biofilm mass
菌株 Strain	10 d	20 d	30 d
EGDe	0.568±0.017	0.794±0.043	0.229±0.012
?agrA	0.371±0.011	0.430±0.082	0.191±0.007
?agrB/D	0.336±0.022	0.540±0.073	0.204±0.008
?agrC	0.336±0.039	0.485±0.080	0.196±0.007

图8 野生株与agr基因缺失突变株的泳动和群集运动性 A：泳动运动性；B：群集运动性

Fig. 8 Swimming and swarming of the wild-type strain and agr gene deletion mutants A: Swimming motilities；B: Swarming motilities

图9 野生株与agr基因缺失突变株中运动相关基因的转录水平

Fig. 9 Transcription levels of motility-associated genes in the wild type and agr gene deletion mutants

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