Introduction
Materials and methods
Plant materials
Preparation of extracts
UPLC–Q-TOF–MS conditions
Bacterial strain
Phylogenetic classification and virulence-associated genes carriage
Gene | Primer name | Sequence (5′–3′) | Amplicon size (bp) | Reference or gene bank accession no (genome region) |
---|---|---|---|---|
chuA
| ChuA.1 | GACGAACCAACGGTCAGGAT | 279 | [5] |
ChuA.2 | TGCCGCCAGTACCAAAGACA | |||
yjaA
| YjaA.1 | TGAAGTGTCAGGAGACGCTG | 211 | [5] |
YjaA.2 | ATGGAGAATGCGTTCCTCAAC | |||
TspE4.C2
| TspE4C2.1 | GAGTAATGTCGGGGCATTCA | 152 | [5] |
TspE4C2.2 | CGCGCCAACAAAGTATTACG | |||
papC
| pap1 | GACGGCTGTACTGCAGGGTGTGGCG | 328 | [6] |
pap2 | ATATCCTTTCTGCAGGGATGCAATA | 336 | ||
pap3 | GCAACAGCAACGCTGGTTGCATCAT | |||
pap4 | AGAGAGAGCCACTCTTATACGGACA | |||
sfa
| sfa1 | CTCCGGAGAACTGGGTGCATCTTAC | 410 | [6] |
sfa2 | CGGAGGAGTAATTACAAACCTGGCA | |||
afa
| afa1 | GCTGGGCAGCAAACTGATAACTCTC | 750 | [6] |
afa2 | CATCAAGCTGTTTGTTCGTCCGCCG | |||
csgA
| csgAF | GTAGCAGCAATTGCAGCAATCG | 383 | AE014075 |
csgAR | TTAGATGCAGTCTGGTCAACAG | (1247944..1248402) | ||
aer
| aer1 | TACCGGATTGTCATATGCAGACCG | 602 | [6] |
aer2 | AATATCTTCCTCCAGTCCGGAGAAG | |||
hlyA
| hlyA1.10f | GCTGCAAATAAATTGCACTCAG | 665 | [7] |
hlyA2.10r | CCCTGCACCGATATTATCAAG | |||
cnf1
| cnf1 | AAGATGGAGTTTCCTATGCAGGAG | 498 | [6] |
cnf2 | CATTCAGAGTCCTGCCCTCATTATT | |||
ant43
| ant43_F | TGGCACCATCAGCCTGCGTG | 127 | AE014075 |
ant43_R | CGTACCACTGTTGCCGGCGT | (1225454..1228729) | ||
luxS
| luxS_F | CGGCAGCCCATTGGCGAGAT | 178 | AE014075 |
luxS_R | TGAACACCCCGCATGGCGAC | (3096814..3097329) | ||
mcbA
| mcbA_F | CGCCTTGTTCGCGCGCTTTT | 138 | NC_000913 |
mcbA_R | TCACGGCTTATGCCGCGCAA | (841019..841279) | ||
mqsR
| mqsR_F | GCCTGTAACAAGCCTGGGTCTGT | 187 | U00096 |
mqsR_R | TGTCAATGCCGGGCAAGTTCGT | (3166270..3166566) | ||
sdiA
| sdiA_F | ATGGTACCGGGTGGCGGACA | 130 | AE014075 |
sdiA_R | TGGCGTCGCACGATGCTGTT | (2144786..2145520) | ||
16srRNA
| rRNA16SF | AGAGTTTGATCATGGCTCAG | 919 | [8] |
rRNA16SR | CCGTCAATTCATTTGAGTTT |
PCR products visualization and analysis
Antibacterial activity of plant extracts
Effect of plant extracts on hydrophobicity of bacterial cells
Effect of plant extracts on swimming motility
Effect of plant extracts on hemagglutination and expression of P fimbriae
Effect of plant extracts on curli expression
Biofilm formation assay and quantification
Effect of plant extracts on biofilm formation
Statistical analysis
Results
Qualitative and quantitative analysis of plant extracts
Parent ion [M−H]− (m/z) | Daughter ion MS/MS (m/z) | Compound |
B. pendula
|
E. arvense
|
H. glabra
|
G. odoratum
|
U. dioica
|
V. vitis-idaea
|
---|---|---|---|---|---|---|---|---|
Flavonols and derivatives | ||||||||
269.1342 | Apigenin derivative | x | ||||||
433.065 | 300.0171/271.0211/255.0303/179.0020 | Quercetin-3-arabinopyranoside | x | x | ||||
433.1033 | 300.049 | Quercetin-xyloside | x | x | ||||
447.0743 | 301.0319/300.0207 | Quercetino-3-rhamnoside | x | x | ||||
463.0887 | 30.0319/300.0313 | Quercetin-glucoside | x | x | ||||
463.0931 | 300.0242/301.0354 | Quercetin-galactoside | x | |||||
477.1022 | 301.0354 | Quercetin-glucuronide | x | |||||
477.1263 | 175.0372/301.0273/300.0348 | Quercetin derivative | x | |||||
591.1436 | 529.1354/489.1132/447.1003/301.0461/300.0242 | Quercetin-3-O-(4 bis-3-hydroxy-3-methylglutaryl)-α-rhamnoside | x | |||||
593.1606 | 285.0429 | Kaempherol ramnohexoside | x | |||||
609.1331 | 447.0916/285.0083 | Kaempherol diglycoside | x | |||||
609.1533 | 463.0931/301.0319 | Rutin | x | x | x | |||
623.1342 | 497.1168/315.0440/107.4882 | Isorhamnetin rhamnose-hexose | x | |||||
625.1591 | 463.0623/301.0176 | Quercetin dihexoside | x | |||||
737.1927 | 596.1407/284.0348 | Hexoside-rhamnoside kaempferol and hydroxyferulic acid derivative | x | |||||
755.3019 | 593.2354/447.0786/285.0498 | Kaempherol-di-rhamnosyl-hexoside | x | x | ||||
771.2246 | 609.1735/285.0049 | Kaempherol-trihexoside | x | |||||
771.3156 | 609.2391/463.1460/301.0603 | Quercetin-hexoside-rhamnoside-hexoside | x | |||||
Flavan-3-ols and procyanidins | ||||||||
289.0688 | 245.0659/165.0118/137.0206/125.0218 | Epicatechin | ||||||
289.0827 | 245.0787/203.0635/165.0065 | Catechin | ||||||
575.1207 | 539.0969/449.0889/407.0727/289.0688/285.0325 | Dimer procyanidin A | x | |||||
577.1201 | 407.0645/289.0758 | Dimer procyanidin B | x | |||||
577.1594 | 289.0584/245.0370/125.0241 | Dimer procyanidin B | x | |||||
577.1933 | 289.0897 | Dimer procyanidin B | x | |||||
863.163 | 711.1367/693.1317/573.0953/451.1297/411.0632/289.0723 | Trimer procyanidin A/B2 | x | |||||
Iridoids | ||||||||
389.0818 | 271.0616/124.9967/107.9938 | Iridoide | x | |||||
389.0858 | 217.0116/198.9950/155.0041 | Iridoide | x | |||||
389.0939 | 191.0100/147.0193 | Aucubioside | x | |||||
389.0939 | 209.0213/183.0417/165.0302 | Iridoide | x | |||||
499.1633 | 337.1049/235.0395 | Iridoide | x | |||||
535.1362 | 371.1109/329.1297/311.0642/191.0326/163.0406 | Coumaroyl iridoide | x | |||||
553.1731 | 389.11 | Iridoide | x | |||||
Phenolic acids | ||||||||
153.0027 | 109.0022/116.8969 | Protocatechuic acid | x | x | ||||
163.038 | 119.0533 |
p-Coumaric acid | x | x | ||||
173.0419 | 134.0375 | 3-FQA feruloylquinic acid | x | |||||
191.0072 | 167.9749/155.9433/110.9921 | Quinic acid derivative | x | |||||
191.0524 | 179.0513/113.0108/119.0220/105.0031 | Quinic acid | x | x | ||||
311.057 | 227.9305/179.0267/148.9988/135.0298 | Caffeoyl tartrate | x | |||||
315.0549 | 152.9976/109.0022 | Protocatechuic acid glucoside | x | |||||
325.0358 | 193.0236/135.0131 | Ferulic acid pentose derivative | x | |||||
325.1244 | 163.0275/119.0309 | Coumaroylglucose | x | |||||
337.0937 | 191.0468/163.0406/119.0533 | 3-p-Coumaroylquinic acid | x | x | ||||
337.1012 | 173.0419 | 5-p-Coumaroylquinic acid | x | |||||
337.1049 | 191.0496 |
p-Coumaric acid derivative | x | |||||
337.1162 | 191.0524/163.0197 | Coumaroylquinic acid | x | |||||
337.1162 | 173.0365/163.0249 | Coumaroylquinic acid | x | |||||
341.1113 | 195.0507/163.0249/119.0354 |
p-Coumaric acid derivative | x | |||||
345.1148 | 193.0521/146.9424 | Ferulic acid derivative | x | x | ||||
353.0648 | 191.0298/147.0367 | 4′-Caffeoylquinic acid | x | x | x | x | ||
353.0918 | 191.0666/179.0321/173.0419 | Caffeoylquinic acid | x | x | x | |||
353.1033 | 191.0439/179.0458 | 5′-Caffeoylquinic acid | x | x | x | x | x | |
367.0899 | 173.0419 | 4 FQA tri-feruloylquinic acid trans
| x | |||||
367.0923 | 193.0464/191.0666 | Feruloylquinic acid isomer | x | |||||
367.1002 | 173.0419 | 4 FQA tri-feruloylquinic acid cis
| x | |||||
367.108 | 191.0524 | 5 FQA tri-feruloylquinic acid | x | |||||
417.1147 | 307.0882/187.0564/163.0302/145.0259/119.0533 | Coumaroyl-hexose hydroxyphenol | x | |||||
417.1231 | 307.0739/163.0354/145.0284/119.0465 | Coumaroyl-hexose-hydroxyphenol | x | |||||
433.1075 | 323.0721/203.0314/179.0294/161.0241/135.0393 | Caffeoyl-hexose-hydroxyphenol | x | |||||
473.065 | 311.0389/179.0431/149.0138/135.0440 | Caftaric acid and hexose derivative | x | |||||
475.1194 | 179.0404/161.0293/135.0488 | Caffeic acid derivative | x | |||||
475.1328 | 301.0390/179.0349/161.0215 | Caffeic acid derivative | x | |||||
515.101 | 353.0802/191.0553/179.0404/173.0419 | Dicaffeoylquinic acid | x | |||||
515.1289 | 353.0802/191.0581/179.0349/173.0446 | Dicaffeoylquinic acid | x | |||||
515.2682 | 191.0637/179.0515 | Dicaffeoylquinic acid | x | |||||
591.1038 | 439.9709/295.0253/179.0075 | Caffeic acid derivative | x | |||||
623.078 | 311.0281/179.0075 | Dicaftaric acid | x | |||||
Propiophenone | ||||||||
327.1203 | 147.0367 | DHPPG (3,4′-dihydroxypropiophenone-3-β-d-glucoside) | x |
Flavonols (mg QG/g) | Phenolic acids (mg CQA/g) | DHPPG (mg CQA/g) | Iridoids (mg LA/g) | Flavanols/procyanidins (mg C/g) | Sum of main phenolics (mg/g) | |
---|---|---|---|---|---|---|
B. pendula
| 92.9 | 15.4 | 34.4 | Nd | Nd | 142.6 |
E. arvense
| 8.2 | 19.6 | Nd | 11.0 | Nd | 38.8 |
G. odoratum
| 69.7 | 53.2 | Nd | 58.1 | Nd | 181.0 |
H. glabra
| 6.9 | 10.2 | Nd | 11.8 | Nd | 28.8 |
U. dioica
| Nd | 2.1 | Nd | Nd | Nd | 2.1 |
V. vitis-idaea
| 134.6 | 10.7 | Nd | 12.6 | 41.1 | 199.1 |
Molecular characterization of bacterial strain
Antibacterial activity of plant extracts
Effect of plant extracts on hydrophobicity of bacterial cells
Plants | Extract concentrations (mg/mL) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Control | 0.125 | 0.25 | 0.5 | 1.0 | 5.0 | 10.0 | 15.0 | 20.0 | |
B. pendula
| Autoaggregative#
| Very strong hydrophobic (0.1)§
| Very strong hydrophobic (0.1) | Very strong hydrophobic (0.1) | Very strong hydrophobic (0.2) | Strong hydrophobic (0.4) | Strong hydrophobic (0.4) | Strong hydrophobic (0.4) | Strong hydrophobic (0.4) |
E. arvense
| Autoaggregative | Strong hydrophobic (0.8) | Strong hydrophobic (0.8) | Strong hydrophobic (0.8) | Strong hydrophobic (0.8) | Strong hydrophobic (0.8) | Strong hydrophobic (0.8) | Strong hydrophobic (0.8) | Strong hydrophobic (0.8) |
G. odoratum
| Autoaggregative | Strong hydrophobic (0.4) | Strong hydrophobic (0.4) | Strong hydrophobic (0.8) | Strong hydrophobic (0.8) | Strong hydrophobic (0.8) | Strong hydrophobic (0.8) | Hydrophilic (3.2) | Hydrophilic (3.2) |
H. glabra
| Autoaggregative | Strong hydrophobic (1.0) | Strong hydrophobic (1.0) | Strong hydrophobic (1.0) | Nt | Nt | Nt | Nt | Nt |
U. dioica
| Autoaggregative | Strong hydrophobic (0.4) | Strong hydrophobic (0.4) | Strong hydrophobic (0.4) | Strong hydrophobic (1.0) | Strong hydrophobic (1.0) | Strong hydrophobic (1.0) | Hydrophilic (3.2) | Hydrophilic (3.2) |
V. vitis-idaea
| Autoaggregative | Autoaggregative | Autoaggregative | Autoaggregative | Autoaggregative | Autoaggregative | Nt | Nt | Nt |
Effect of plant extracts on swimming motility
Plants | Extract concentrations (mg/mL) | ||||||||
---|---|---|---|---|---|---|---|---|---|
Control | 0.125 | 0.25 | 0.5 | 1.0 | 5.0 | 10.0 | 15.0 | 20.0 | |
B. pendula
| 16.2 (±2.0) | 18.7 (±3.1) | 18.3 (±1.5) | 19.3 (±4.2) | 18.7 (±5.5) | 15.0 (±1.7) | 12.7 (±2.1) | 11.7 (±3.1)* | 12.7 (±0.6)* |
E. arvense
| 16.2 (±2.0) | 18.7 (±2.1) | 20.3 (±0.6)* | 25.0 (±2.6)* | 34.3 (±7.5)* | 28.3 (±2.9)* | 36.7 (±7.4)* | 26.7 (±4.2)* | 23.0 (±1.7) |
G. odoratum
| 16.2 (±2.0) | 16.3 (±4.2) | 16.3 (±4.0) | 21.0 (±3.6)* | 17.0 (±3.5) | 23.3 (±1.5)* | 31.3 (±1.5)* | 32.7 (±5.5)* | 30.0 (±3.5)* |
H. glabra
| 16.2 (±2.0) | 15.7 (±4.0) | 15.7 (±3.2) | 16.0 (±2.6) | Nt | Nt | Nt | Nt | Nt |
U. dioica
| 16.2 (±2.0) | 21.3 (±3.1)* | 17.3 (±2.5) | 16.7 (±4.6) | 20.0 (±2.0)* | 12.7 (±4.2) | 15.3 (±2.1) | 14.0 (±2.0) | 10.7 (±1.5)* |
V. vitis-idaea
| 16.2 (±2.0) | 15.7 (±2.5) | 14.3 (±2.5) | 14.3 (±0.6) | 14.0 (±2.0) | 15.3 (±1.5) | Nt | Nt | Nt |
Effect of plant extracts on hemagglutination and expression of P fimbriae
Plants | Extract concentrations (mg/mL) | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Control | 0.125 | 0.25 | 0.5 | 1.0 | 5.0 | 10.0 | 15.0 | 20.0 | |||||||||
P | C | P | C | P | C | P | C | P | C | P | C | P | C | P | C | ||
B. pendula
| + | + | + | + | + | + | + | + | + | + | + | − | + | − | + | − | + |
E. arvense
| + | + | + | + | + | + | + | + | + | + | + | + | − | + | − | + | − |
G. odoratum
| + | + | + | + | + | + | + | + | + | − | + | − | + | − | + | − | + |
H. glabra
| + | + | + | + | + | + | + | Nt | Nt | Nt | Nt | Nt | Nt | Nt | Nt | Nt | Nt |
U. dioica
| + | + | + | + | + | + | + | + | + | + | + | − | + | − | + | − | + |
V. vitis-idaea
| + | + | − | + | − | + | − | − | − | − | − | Nt | Nt | Nt | Nt | Nt | Nt |
Effect of plant extracts on curli expression
Effect of plant extracts on biofilm formation
Time of incubation (days) | Biofilm formation | |||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
control |
B. pendula
|
E. arvense
|
G. odoratum
|
H. glabra
|
U. dioica
|
V. vitis-idaea
| ||||||||
OD (±SD) | (%) | OD (±SD) | (%) | OD (±SD) | (%) | OD (±SD) | (%) | OD (±SD) | (%) | OD (±SD) | (%) | OD (±SD) | (%) | |
1 | 0.004 (±0.001) | 100.0 | 0.002* (±0.002) | 50.0 | 0.0001* (±0.000) | 2.5 | 0.0001* (±0.000) | 2.5 | 0.001* (±0.0005) | 25.0 | 0.0001* (±0.000) | 2.5 | 0.0001* (±0.000) | 2.5 |
2 | 0.005 (±0.002) | 100.0 | 0.004 (±0.003) | 80.0 | 0.0001* (±0.000) | 2.0 | 0.004 (±0.002) | 80.0 | 0.002* (±0.001) | 40.0 | 0.002* (±0.001) | 40.0 | 0.003* (±0.002) | 60.0 |
3 | 0.01 (±0.002) | 100.0 | 0.01 (±0.003) | 100.0 | 0.002* (±0.0002) | 20.0 | 0.007 (±0.003) | 70.0 | 0.003* (±0.001) | 30.0 | 0.007 (±0.003) | 70.0 | 0.01* (±0.003) | 100.0 |
4 | 0.005 (±0.002) | 100.0 | 0.0001* (±0.000) | 2.0 | 0.0001* (±0.000) | 2.0 | 0.0001* (±0.000) | 2.0 | 0.0001* (±0.000) | 2.0 | 0.0001* (±0.000) | 2.0 | 0.0001* (±0.000) | 2.0 |
5 | 0.005 (±0.001) | 100.0 | 0.0001* (±0.000) | 2.0 | 0.0001* (±0.000) | 2.0 | 0.0001* (±0.000) | 2.0 | 0.0001* (±0.000) | 2.0 | 0.0001* (±0.000) | 2.0 | 0.0001* (±0.000) | 2.0 |
6 | 0.007 (±0.001) | 100.0 | 0.003* (±0.001) | 42.8 | 0.0001* (±0.000) | 1.4 | 0.0001* (±0.000) | 1.4 | 0.0001* (±0.000) | 1.4 | 0.0001* (±0.000) | 1.4 | 0.0001* (±0.000) | 1.4 |
7 | 0.01 (±0.002) | 100.0 | 0.006* (±0.002) | 60.0 | 0.002* (±0.001) | 20.0 | 0.005* (±0.002) | 50.0 | 0.003* (±0.001) | 30.0 | 0.004* (±0.001) | 40.0 | 0.0001* (±0.000) | 1.0 |
8 | 0.014 (±0.002) | 100.0 | 0.009* (±0.003) | 64.3 | 0.003* (±0.001) | 21.4 | 0.008* (±0.002) | 57.1 | 0.004* (±0.002) | 28.6 | 0.008* (±0.002) | 57.1 | 0.01 (±0.003) | 71.4 |
9 | 0.008 (±0.002) | 100.0 | 0.008 (±0.003) | 100.0 | 0.0001* (±0.000) | 1.3 | 0.003* (±0.001) | 37.5 | 0.0001* (±0.000) | 1.3 | 0.003* (±0.001) | 37.5 | 0.007 (±0.0025) | 87.5 |
10 | 0.005 (±0.001) | 100.0 | 0.005 (±0.003) | 100.0 | 0.0001* (±0.000) | 2.0 | 0.001* (±0.0004) | 20.0 | 0.0001* (±0.000) | 2.0 | 0.002* (±0.001) | 40.0 | 0.0001* (±0.000) | 2.0 |