Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (2): 254-262.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0421
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REN Si-yu1(), JIANG Cong-yi1, YU Tao2, KANG Rui1, JIANG Xiao-bing1()
Received:
2022-04-07
Online:
2023-02-26
Published:
2023-03-07
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.
引物名称 Primer name | 序列 Sequence(5'-3') | 长度 Length/bp |
---|---|---|
agrA-P1 | NNNNNNGGATCCGCTCTTACTGTCTTA- GCGTTCT | 882 |
agrA-P2 | CCTGTGGCACCGATAAAATTCGCTGC- ATTCTGTTATCTTC | |
agrA-P3 | GAAGATAACAGAATGCAGCGAATTTT- ATCGGTGCCACAGG | 568 |
agrA-P4 | NNNNNNACGCGTAGTATTCCCATCGG- CATTT | |
agrB/D-P1 | NNNNNNGGATCCGTCTACAAAGTTGA- TGGGATT | 680 |
agrB/D-P2 | ACTCATAGAAGAATCCGCAAATCATCT- TTCCAGCGGTC | |
agrB/D-P3 | GACCGCTGGAAAGATGATTTGCGGATT- CTTCTATGAGT | 608 |
agrB/D-P4 | NNNNNNACGCGTGCTAAGACAGTAAG- AGCAACG |
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 | |
agrA-P3 | GAAGATAACAGAATGCAGCGAATTTT- ATCGGTGCCACAGG | 568 |
agrA-P4 | NNNNNNACGCGTAGTATTCCCATCGG- CATTT | |
agrB/D-P1 | NNNNNNGGATCCGTCTACAAAGTTGA- TGGGATT | 680 |
agrB/D-P2 | ACTCATAGAAGAATCCGCAAATCATCT- TTCCAGCGGTC | |
agrB/D-P3 | GACCGCTGGAAAGATGATTTGCGGATT- CTTCTATGAGT | 608 |
agrB/D-P4 | NNNNNNACGCGTGCTAAGACAGTAAG- AGCAACG |
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)
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 |
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 |
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 | ||||||
---|---|---|---|---|---|---|---|---|---|
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* |
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 | ||||||
---|---|---|---|---|---|---|---|---|---|
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* |
菌株 Strain | 生物被膜量Biofilm mass | ||
---|---|---|---|
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 |
Table 4 Biofilm biomasses of wild type and mutant strains at 37℃
菌株 Strain | 生物被膜量Biofilm mass | ||
---|---|---|---|
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 |
菌株 Strain | 生物被膜量Biofilm mass | ||
---|---|---|---|
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 |
Table 5 Biofilm biomasses of wild type and mutant strains at 20℃
菌株 Strain | 生物被膜量Biofilm mass | ||
---|---|---|---|
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 |
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)
菌株 Strain | 生物被膜量Biofilm mass | ||
---|---|---|---|
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 |
Table 6 Biofilm biomasses of wild type and mutant strains at 4℃
菌株 Strain | 生物被膜量Biofilm mass | ||
---|---|---|---|
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 |
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