Role of agr System in the Antimicrobial Resistance and Biofilm Formation of Listeria monocytogenes

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

Abstract

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

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.

Figures/Tables 15

References 21

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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

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

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

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

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*