# Inhibition ofPseudomonas aeruginosa Biofilm Formation by Traditional Chinese Medicinal HerbHerba patriniae

Authors: Bo Fu; Qiaolian Wu; Minyan Dang; Dangdang Bai; Qiao Guo; Lixin Shen; Kangmin Duan
Journal: BioMed Research International
 (2017)

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## Abstract

New antimicrobial agents are urgently needed to treat infections caused by drug-resistant pathogens and by pathogens capable of persisting in biofilms. The aim of this study was to identify traditional Chinese herbs that could inhibit biofilm formation ofPseudomonas aeruginosa, an important human pathogen that causes serious and difficult-to-treat infections in humans. AluxCDABE-based reporter system was constructed to monitor the expression of six key biofilm-associated genes inP. aeruginosa. The reporters were used to screen a library of 36 herb extracts for inhibitory properties against these genes. The results obtained indicated that the extract ofHerba patriniaedisplayed significant inhibitory effect on almost all of these biofilm-associated genes. Quantitative analysis showed thatH. patriniaeextract was able to significantly reduce the biofilm formation and dramatically altered the structure of the mature biofilms ofP. aeruginosa. Further studies showedH. patriniaeextract decreased exopolysaccharide production byP. aeruginosa and promoted its swarming motility, two features disparately associated with biofilm formation. These results provided a potential mechanism for the use ofH. patriniaeto treat bacterial infections by traditional Chinese medicines and revealed a promising candidate for exploration of new drugs againstP. aeruginosa biofilm-associated infections.


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## Body

## 1. Introduction

Pseudomonas aeruginosa is a remarkably adaptive bacterial pathogen which can cause persistent infections in burn patients, immune-compromised patients, and individuals with the genetic disease cystic fibrosis. It is one of the most prevalent nosocomial pathogens, and the infections caused byP. aeruginosa can be very serious and life-threatening [1].Biofilm formation is a major characteristic ofP. aeruginosachronic infections [2, 3].P. aeruginosacells in biofilms are surrounded by exopolysaccharides and form a structured aggregates, and these cells exhibit increased resistance to antibiotics and other adversary agents [3–6]. Infections caused by biofilm-formingP. aeruginosa,such as those in cystic fibrosis lung, are almost impossible to eradicate [7]. There is an urgent need to find novel antimicrobial agents to control such infections [8, 9].Traditional Chinese medicines (TCMs) have been widely used to treat infectious diseases for more than a thousand years in China. Many components constituents of TCMs have been found to be very effective in treating bacterial infections such as gastritis, stomatitis, dermatitis, and bacterial pneumonia [10]. However, the mechanisms of these herbs in treatment infectious diseases are mostly unknown.Herba patriniaeis a perennial herbal of TCM, which contains various beneficial ingredients such as amino acids, vitamins, minerals, alkaloids, tannins, and saponins. It has been reported to have functions of antioxidant, antibacterial, antiviral, blood-activating and stasis-eliminating, promoting regeneration of liver cells, and anxiety-alleviating. The boiling water extracts ofH. patriniaehad been identified having anticyanobacteria activity againstMicrocystis aeruginosa[11].In this study, 36 extracts of 18 Chinese herbs that are commonly used for treating infection-like symptoms were screened for inhibitory effect againstP. aeruginosa biofilms. We found that the extract of the Chinese herbH. patriniaesignificantly inhibited the expression of the genes associated with biofilm formation inP. aeruginosa PAO1. It reduced exopolysaccharide production and biofilm formation and then altered the structure of the mature biofilms.


## 2. Materials and Methods

### 2.1. Bacterial Strains and Culture Conditions

Bacterial strains used in this study are listed in Table1. All the strains were cultured in LB (Luria Bertani) broth at 37°C with orbital shaking at 200 rpm or on LB agar plates supplemented with antibiotic of kanamycin (Kan, 50 μg/mL) or trimethoprim (Tmp, 300 μg/mL) where appropriate.Table 1
Bacterial strains used in this study.
Strains/plasmids
Description
Source
E. coliDH10B
F-mcrA(mrr-hsdRMS-mcrBC)80dlacZΔM15ΔlacX74 deoRrecA1 endA1 araD139 Δ(ara leu)7697 galU galKλ-rpsl nupG
Invitrogen
P. aeruginosa PAO1
Wild type
This lab
pMS402
Expression reporter plasmid carrying the promoterlessluxCDABE; Kanr, Tmpr
This lab
pKD-pslM
pMS402 containingpslM promoter region; Kanr, Tmpr
This study
pKD-pelA
pMS402 containingpelA promoter region; Kanr, Tmpr
This study
pKD-algU
pMS402 containingalgU promoter region; Kanr, Tmpr
This study
pKD-ppyR
pMS402 containingppyR promoter region; Kanr, Tmpr
This study
pKD-algA
pMS402 containingalgA promoter region; Kanr, Tmpr
This study
pKD-bdlA
pMS402 containingbdlA promoter region; Kanr, Tmpr
This study


### 2.2. Traditional Chinese Medicine Extraction

Traditional Chinese medicinal herbs were selected according to their efficacy in treatment of infection-like symptoms in Chinese medicine. They were obtained from local pharmacy (Yikang Pharm chain store, China). The pulverized herb was firstly immerged in 75% ethanol or deionized water, respectively, for 2 h and then boiled for 2 h additionally (weight to solvent volume was 1 : 5). The extracts were filtered by filter paper and were evaporated under vacuum at 35°C using a rotary evaporator (Buchi, Switzerland). The concentrated extracts were then freeze-dried using a lyophilizer and stored at −80°C. The water extracts were redissolved in deionized water and ethanol extracts were redissolved in methanol and then sterilized immediately with 0.22μm Iwaki filter before use.


### 2.3. Gene Expression Assay

Key genesalgU[12],pslM[13, 14],pelA[15],algA[16],ppyR[17], andbdlA[18] that are known to be involved in biofilm formation inP. aeruginosa were selected to constructluxCDABE-based promoter-reporter fusions (Table 2). The reporters were constructed as previously described [19, 20] and were subsequently transformed into PAO1. UsingluxCDABE-based reporters, gene expression in liquid cultures was measured by light luminescence (in counts per second) in a Victor3 multilabel plate reader (Perkin-Elmer). Strains containing different reporters were cultivated overnight in LB broth supplemented with Tmp (300 μg/mL) and then diluted to an optical density at 600 nm of 0.2. The diluted cultures were used as inoculants. After additional 2 h incubation, 5 μL cultures were inoculated into parallel wells on a 96-well black plate containing 93 μL medium and 2 μL herbal extract in different concentrations. 50 μL mineral oil was added to the wells to prevent evaporation. Promoter activities were measured every 30 min for 24 h in a Victor multilabel plate reader and bacterial growth was monitored by measuring OD600 at the same time.Table 2
Reporter genes and primer sequences used.
PAnumber
Gene
Function
Primer
Sequence(5′→3′)
PA0762
algU
RNA polymerase sigma factor
pKD-algU-S
GCACTCGAGAGGATGCCTGAAGACCTC
pKD-algU-A
GTAGGATCCGATGGCGATCCGATACAG
PA2243
pslM
Succinate dehydrogenase;fumarate reductase flavoprotein
pKD-pslM-S
ATCCTCGAGCGGTGCGCAAGAAGACC
pKD-pslM-A
GTTGGATCCCGTAACGCTCGCCCAGTT
PA3064
pelA
Glycoside hydrolase;deacetylase
pKD-pelA-S
CGTCTCGAGCTTTCCACTTTGCCACAG
pKD-pelA-A
TACGGATCCTACCAGAACGCCACGCT
PA3551
algA
Phosphomannose isomerase; guanosine 5′-diphospho-D-mannose pyrophosphorylase
pKD-algA-S
TGACTCGAGTGAAGGCGGACTGAGAC
pKD-algA-A
TATGGATCCGCGACCAGTTTCATC
PA2663
ppyR
psl and pyoverdine operon regulator
pKD-ppyR-S
GCACTCGAGCACTTCTTCTGCTACAGC
pKD-ppyR-A
GTAGGATCCAGCACTTGCACAGCAGAC
PA1423
bdlA
Biofilm dispersion locus A
pKD-bdlA-S
GCACTCGAGCGTCATATTTCCGACGAA
pKD-bdlA-A
TCAGGATCCGTAGTCTTCCGATTGCG


### 2.4. Gene Expression Measurement by Real-Time Quantitative PCR (RT-qPCR)

Bacterial RNA was extracted using RNAprep Pure Cell/Bacteria Kit (TIANGEN). 1μg of RNA sample was reverse transcribed to cDNA using 1st Strand cDNA Synthesis Kit (Takara). Real-time qPCR was performed using SuperReal PerMix Plus (SYBR Green/probe) (TIANGEN) and primers specific foralgU (Forward: 5′-AACACCGCGAAGAACCACCT-3′, Reverse: 5′-ATCTCATCCCGCAACATCGCG-3′),algA(Forward: 5′-AGAACTGAAGAAGCACGACG-3′, Reverse: 5′-TTCTCCATCACCGCGTAGT-3′),pelA(Forward: 5′-ATGGCTGAAGGTATGGCTG-3′, Reverse: 5′-AGGTGCTGGAGGACTTCATC-3′), andpslM (Forward: 5′-CTATGACGCACGGCAACTGG-3′, reverse: 5′-CGCCATTGACCAGGTGCAT-3′). The values obtained were normalized to the housekeeping geneproC (Forward: 5′-CAGGCCGGGCAGTTGCTGTC-3′, reverse: 5′-GGTCAGGCGCGAGGCTGTCT -3′).


### 2.5. Quantification of the Biofilm Formation

Biofilm formation was measured in 96-well polystyrene microtiter plate as described previously with minor modifications [21]. 10 μL of overnight cultures of PAO1 was added to 90 μL fresh LB broth containing 160 μgH. patriniaewater extract. Same volume of deionized water was used as control. After being cultured with shaking at 200 rpm for 1 h the plates were kept at stationary state at 37°C for 3 days and 7 days with a medium replacement at every 24 h interval. Then wells were washed twice with deionized water gently to remove the planktonic cells. The sedentary cells were stained with 1% (v/v) crystal violet solution for 15 min. Unbound dye in the wells were washed off by deionized water before 200 μL of 95% ethanol was added to dissolve the crystal violet stains. The absorbance of the solutions was then measured at 570 nm [7, 22].


### 2.6. Biofilm Imaging by Silver Staining Method

To examine the biofilm structure, silver staining method was used as described previously with some modifications [23, 24]. Similar to the above described biofilm assay, biofilms were grown on cover slip (8 mm diameter) placed at bottom of the wells of 24-well plates. 200 μL of fresh bacterial culture was inoculated to 1.8 mL TSB supplemented with 3.2 mgH. patriniae. The plates were incubated at 37°C with TSB medium replaced every 24 h. After 3 days or 7 days of cultivation, the cover slip was taken out and washed three times with saline water to remove the planktonic cells. The cover slip was immersed in 2.5% glutaraldehyde for 1 h for the cells fixation and rinsed with distilled water for 1 min. Then the cover slip was immersed in saturated calcium chloride solution for 15 min and rinsed with distilled water for 5 min. The biofilms on the cover slip were then stained with 5% silver nitrate for 15 min, followed by 1% hydroquinone colour-rendering for 2 min and then rinsed with distilled water for 1 min. Fixation was treated with 5% sodium thiosulfate for 1 min, followed by a final water rinse. The cover slip was placed on an inverted optical microscope for biofilm structure observation.


### 2.7. Swarming Motility Assay

Swarming assay was carried out as previously reported [25]. The medium used for swarming motility assay consists of nutrient broth (0.8%), glucose (0.5%), and agar (0.5%). The plates were dried at room temperature overnight before being used. 2 μL ofP. aeruginosa PAO1 culture (OD600 = 0.5) mixed with 106.67 μg ofH. patriniae water extract was spotted onto the swarming plate and the one with deionized water was used as blank control. Plates were incubated at 37°C for 24 h before the swarming diameter was measured.


### 2.8. Measurement of Exopolysaccharide Production

Overnight culture of PAO1 (OD600 = 0.005) was spotted onto Congo red plates (1% Tryptone, 1% agar, 4% Congo red, and 2% Coomassie blue) with or withoutH. patriniae water extract. The amount ofH. patriniae water extract was 64 μg. The colony morphology and staining were observed after 3 days of incubation at 37°C [22].


## 2.1. Bacterial Strains and Culture Conditions

Bacterial strains used in this study are listed in Table1. All the strains were cultured in LB (Luria Bertani) broth at 37°C with orbital shaking at 200 rpm or on LB agar plates supplemented with antibiotic of kanamycin (Kan, 50 μg/mL) or trimethoprim (Tmp, 300 μg/mL) where appropriate.Table 1
Bacterial strains used in this study.
Strains/plasmids
Description
Source
E. coliDH10B
F-mcrA(mrr-hsdRMS-mcrBC)80dlacZΔM15ΔlacX74 deoRrecA1 endA1 araD139 Δ(ara leu)7697 galU galKλ-rpsl nupG
Invitrogen
P. aeruginosa PAO1
Wild type
This lab
pMS402
Expression reporter plasmid carrying the promoterlessluxCDABE; Kanr, Tmpr
This lab
pKD-pslM
pMS402 containingpslM promoter region; Kanr, Tmpr
This study
pKD-pelA
pMS402 containingpelA promoter region; Kanr, Tmpr
This study
pKD-algU
pMS402 containingalgU promoter region; Kanr, Tmpr
This study
pKD-ppyR
pMS402 containingppyR promoter region; Kanr, Tmpr
This study
pKD-algA
pMS402 containingalgA promoter region; Kanr, Tmpr
This study
pKD-bdlA
pMS402 containingbdlA promoter region; Kanr, Tmpr
This study


## 2.2. Traditional Chinese Medicine Extraction

Traditional Chinese medicinal herbs were selected according to their efficacy in treatment of infection-like symptoms in Chinese medicine. They were obtained from local pharmacy (Yikang Pharm chain store, China). The pulverized herb was firstly immerged in 75% ethanol or deionized water, respectively, for 2 h and then boiled for 2 h additionally (weight to solvent volume was 1 : 5). The extracts were filtered by filter paper and were evaporated under vacuum at 35°C using a rotary evaporator (Buchi, Switzerland). The concentrated extracts were then freeze-dried using a lyophilizer and stored at −80°C. The water extracts were redissolved in deionized water and ethanol extracts were redissolved in methanol and then sterilized immediately with 0.22μm Iwaki filter before use.


## 2.3. Gene Expression Assay

Key genesalgU[12],pslM[13, 14],pelA[15],algA[16],ppyR[17], andbdlA[18] that are known to be involved in biofilm formation inP. aeruginosa were selected to constructluxCDABE-based promoter-reporter fusions (Table 2). The reporters were constructed as previously described [19, 20] and were subsequently transformed into PAO1. UsingluxCDABE-based reporters, gene expression in liquid cultures was measured by light luminescence (in counts per second) in a Victor3 multilabel plate reader (Perkin-Elmer). Strains containing different reporters were cultivated overnight in LB broth supplemented with Tmp (300 μg/mL) and then diluted to an optical density at 600 nm of 0.2. The diluted cultures were used as inoculants. After additional 2 h incubation, 5 μL cultures were inoculated into parallel wells on a 96-well black plate containing 93 μL medium and 2 μL herbal extract in different concentrations. 50 μL mineral oil was added to the wells to prevent evaporation. Promoter activities were measured every 30 min for 24 h in a Victor multilabel plate reader and bacterial growth was monitored by measuring OD600 at the same time.Table 2
Reporter genes and primer sequences used.
PAnumber
Gene
Function
Primer
Sequence(5′→3′)
PA0762
algU
RNA polymerase sigma factor
pKD-algU-S
GCACTCGAGAGGATGCCTGAAGACCTC
pKD-algU-A
GTAGGATCCGATGGCGATCCGATACAG
PA2243
pslM
Succinate dehydrogenase;fumarate reductase flavoprotein
pKD-pslM-S
ATCCTCGAGCGGTGCGCAAGAAGACC
pKD-pslM-A
GTTGGATCCCGTAACGCTCGCCCAGTT
PA3064
pelA
Glycoside hydrolase;deacetylase
pKD-pelA-S
CGTCTCGAGCTTTCCACTTTGCCACAG
pKD-pelA-A
TACGGATCCTACCAGAACGCCACGCT
PA3551
algA
Phosphomannose isomerase; guanosine 5′-diphospho-D-mannose pyrophosphorylase
pKD-algA-S
TGACTCGAGTGAAGGCGGACTGAGAC
pKD-algA-A
TATGGATCCGCGACCAGTTTCATC
PA2663
ppyR
psl and pyoverdine operon regulator
pKD-ppyR-S
GCACTCGAGCACTTCTTCTGCTACAGC
pKD-ppyR-A
GTAGGATCCAGCACTTGCACAGCAGAC
PA1423
bdlA
Biofilm dispersion locus A
pKD-bdlA-S
GCACTCGAGCGTCATATTTCCGACGAA
pKD-bdlA-A
TCAGGATCCGTAGTCTTCCGATTGCG


## 2.4. Gene Expression Measurement by Real-Time Quantitative PCR (RT-qPCR)

Bacterial RNA was extracted using RNAprep Pure Cell/Bacteria Kit (TIANGEN). 1μg of RNA sample was reverse transcribed to cDNA using 1st Strand cDNA Synthesis Kit (Takara). Real-time qPCR was performed using SuperReal PerMix Plus (SYBR Green/probe) (TIANGEN) and primers specific foralgU (Forward: 5′-AACACCGCGAAGAACCACCT-3′, Reverse: 5′-ATCTCATCCCGCAACATCGCG-3′),algA(Forward: 5′-AGAACTGAAGAAGCACGACG-3′, Reverse: 5′-TTCTCCATCACCGCGTAGT-3′),pelA(Forward: 5′-ATGGCTGAAGGTATGGCTG-3′, Reverse: 5′-AGGTGCTGGAGGACTTCATC-3′), andpslM (Forward: 5′-CTATGACGCACGGCAACTGG-3′, reverse: 5′-CGCCATTGACCAGGTGCAT-3′). The values obtained were normalized to the housekeeping geneproC (Forward: 5′-CAGGCCGGGCAGTTGCTGTC-3′, reverse: 5′-GGTCAGGCGCGAGGCTGTCT -3′).


## 2.5. Quantification of the Biofilm Formation

Biofilm formation was measured in 96-well polystyrene microtiter plate as described previously with minor modifications [21]. 10 μL of overnight cultures of PAO1 was added to 90 μL fresh LB broth containing 160 μgH. patriniaewater extract. Same volume of deionized water was used as control. After being cultured with shaking at 200 rpm for 1 h the plates were kept at stationary state at 37°C for 3 days and 7 days with a medium replacement at every 24 h interval. Then wells were washed twice with deionized water gently to remove the planktonic cells. The sedentary cells were stained with 1% (v/v) crystal violet solution for 15 min. Unbound dye in the wells were washed off by deionized water before 200 μL of 95% ethanol was added to dissolve the crystal violet stains. The absorbance of the solutions was then measured at 570 nm [7, 22].


## 2.6. Biofilm Imaging by Silver Staining Method

To examine the biofilm structure, silver staining method was used as described previously with some modifications [23, 24]. Similar to the above described biofilm assay, biofilms were grown on cover slip (8 mm diameter) placed at bottom of the wells of 24-well plates. 200 μL of fresh bacterial culture was inoculated to 1.8 mL TSB supplemented with 3.2 mgH. patriniae. The plates were incubated at 37°C with TSB medium replaced every 24 h. After 3 days or 7 days of cultivation, the cover slip was taken out and washed three times with saline water to remove the planktonic cells. The cover slip was immersed in 2.5% glutaraldehyde for 1 h for the cells fixation and rinsed with distilled water for 1 min. Then the cover slip was immersed in saturated calcium chloride solution for 15 min and rinsed with distilled water for 5 min. The biofilms on the cover slip were then stained with 5% silver nitrate for 15 min, followed by 1% hydroquinone colour-rendering for 2 min and then rinsed with distilled water for 1 min. Fixation was treated with 5% sodium thiosulfate for 1 min, followed by a final water rinse. The cover slip was placed on an inverted optical microscope for biofilm structure observation.


## 2.7. Swarming Motility Assay

Swarming assay was carried out as previously reported [25]. The medium used for swarming motility assay consists of nutrient broth (0.8%), glucose (0.5%), and agar (0.5%). The plates were dried at room temperature overnight before being used. 2 μL ofP. aeruginosa PAO1 culture (OD600 = 0.5) mixed with 106.67 μg ofH. patriniae water extract was spotted onto the swarming plate and the one with deionized water was used as blank control. Plates were incubated at 37°C for 24 h before the swarming diameter was measured.


## 2.8. Measurement of Exopolysaccharide Production

Overnight culture of PAO1 (OD600 = 0.005) was spotted onto Congo red plates (1% Tryptone, 1% agar, 4% Congo red, and 2% Coomassie blue) with or withoutH. patriniae water extract. The amount ofH. patriniae water extract was 64 μg. The colony morphology and staining were observed after 3 days of incubation at 37°C [22].


## 3. Results and Discussion

### 3.1. Screening for Herbs with Inhibitory Effect onP. aeruginosa Biofilm-Associated Genes

Eighteen traditional Chinese medicinal herbs were selected because of their common usage for infection-like symptoms. Both boiling water extracts and ethanol extracts were obtained and used to screen for antibiofilm activities. SinceP. aeruginosa biofilm formation is directly associated with the activity of several known genes, we constructedluxCDABE-based reporters to examine the effect of herb extracts on these genes. The effects of the crude extracts on the expression of these biofilm-associated genes (algU,pslM,pelA,algA,ppyR, andbdlA) are presented in Table 3. The results indicate that different herbal extracts exhibited various degrees of inhibitory effects on these genes. The water extract ofH. patriniae showed the most significant effect on the expression ofalgU,algA,pslM, andbdlA. Examples of the gene expression profiles in the presence ofH. patriniae are shown in Figure 1.Table 3
Effect of water and ethanolic herb extracts on biofilm-associated genes expression.
The English name of herbs
Water extract
The English name of herbs
Ethanolic extract
algU
pslM
pelA
algA
ppyR
bdlA
algU
pslM
pelA
algA
ppyR
bdlA
Atractylodes lancea
+1.5
0
−2.5
0
0
0
Atractylodes lancea
0
0
0
0
0
0
Cortex fraxini
0
−3
−1.5
−1
0
0
Cortex fraxini
−1.8
−2.5
−0.5
−1.5
0
0
Cyrtomium fortunei
0
−1.5
−2
−1
0
0
Cyrtomium fortunei
0
0
0
0
0
0
Erodium stephanianumWilld.
a
a
a
a
a
a
Erodium stephanianum willd
a
a
a
a
a
a
Folium artemisiae Argyi
−2
0
−2
0
−1.5
0
Folium artemisiae argyi
0
0
−2
0
−1.5
0
Fructus quisqualis
a
a
a
a
a
a
Fructus quisqualis
a
a
a
a
a
a
Herba agrimoniae
a
a
a
a
a
a
Herba agrimoniae
a
a
a
a
a
a
Herba patriniae
−2
−2.5
−0.5
−2.5
0
−2
Herba patriniae
0
0
0
0
−1.5
−1
Herba scutellariae barbatae
0
0
0
−1
0
0
Herba scutellariae barbatae
0
0
0
0
0
−1.5
Pomegranate rind
−1.5
−1.2
0
−1
0
0
Pomegranate rind
−1.5
−1
−1
−0.5
0
0
Portulacae herba
+1
0
0
+1.5
0
0
Portulacae herba
0
0
0
0
0
0
Radix paeoniae
−0.5
−0.5
0
0
−1
0
Radix paeoniae
0
0
0
0
0
0
Radix sanguisorbae
−1.5
−1
−1.5
−1
−0.5
0
Radix sanguisorbae
−1.6
−1.5
0
0
−0.5
0
Radix lithospermi
0
−1.2
−2
0
−1
0
Radix lithospermi
0
0
0
0
0
0
Scrophulariae
−2
+1.5
0
0
0
0
Scrophulariae
−0.5
0
−1
0
0
0
Smoked plum
a
a
a
a
a
a
Smoked plum
a
a
a
a
a
a
Taraxacum mongolicum
0
0
0
0
0
0
Taraxacum mongolicum
0
+2
0
0
0
0
Tripterygium
−1.5
0
−2.5
0
0
0
Tripterygium
0
0
−2
0
−2.5
0
Note: herbal extracts were used at 1.6 mg/mL. The values shown represent maximal inhibition (fold changes between experiments with and without herb extract). “+” represents induction of gene expression by herb extracts; “−” represents inhibition of biofilm gene expression by herb extracts; “0” represents no effect of biofilm gene expression by herb extracts; “a” represents inhibition of the growth of bacteria. Numbers represent fold changes at the time points where maximal expression was reached in the absence of herbal extract.Figure 1
Expressions ofalgU,algA,pslM, andbdlA in medium with water extract ofHerba patriniae. Expressions ofalgU (a),algA (b),pslM(c), andbdlA (d) in medium (total volume of 100 μL) with 160 μgH. patriniaeextract and the controls were the ones without herbal extract.
(a)
(b)
(c)
(d)To confirm the results obtained from thelux-based reporter assay, real-time qPCR was carried out using bacterial RNA samples isolated in the presence and absence ofH. patriniae. The results are shown in Table 4. In agreement with the results from the reporter assay, the mRNA levels ofalgU,algA,pslM, andbdlA in PAO1 were all significantly decreased in the presence ofH. patriniae extract (at 1.6 mg/mL) compared with those in the absence ofH. patriniae extract. It is noted that the inhibition ofalgU andalgA was more pronounced in the qPCR assay.Table 4
The transcriptional levels of selected genes in PAO1 and PAO1 treated withH. patriniae.
Gene
Relative mRNA level determined by ΔCt calculation
WithoutH. patriniae
WithH. patriniae
P value
algU
1.00
0.192
0.014
algA
1.00
0.090
0.011
pslM
1.00
0.695
0.043
bdlA
1.00
0.412
0.036
Note: the mRNA level of each gene was normalized to that ofproC. The values shown represent the mean of three different tests.The results indicate that many of these herbs have an inhibitory effect on the genes associated with biofilm formation inP. aeruginosa. This is somewhat not surprising because they all have been used for treatment of chronic bacterial infections in traditional Chinese medicine.The effect from the water extract ofH. patriniaewas remarkable as it inhibited five genes tested. It has been reported that the herb had antibacterial and antiviral activity. However, it was noted that the water extract did not inhibit the growth ofP. aeruginosa (Figure 1). Even though conventional antibiotic compounds may exist inH. patriniaeagainst other bacteria, this result indicates no such component was present againstP. aeruginosa, at least not at the concentrations used in our experiments.Lacking of bacterial killing or growth inhibition activity, however, may not be a weakness of such herbs in treating infectious diseases. As discussed in previous reports [26–29], a promising new class of antipathogenic drugs that target virulence factors and/or biofilm formation instead of killing the pathogens has many advantages in clinical use. First, these antipathogenic drugs theoretically are less likely to render drug resistance in the pathogens because they do not assert a selective pressure on the pathogen’s survival. Second, such therapeutics would unlikely affect other nonpathogenic or beneficial bacteria, that is, the microbiome in the host.


### 3.2. Extract ofH. patriniae InhibitsP. aeruginosa Biofilm Formation

From the gene expression results, the extract ofH. patriniaepresumably would inhibit the biofilm formation ofP. aeruginosa. To verify such an effect,P. aeruginosa biofilm formation was compared in the presence and absence ofH. patriniaeextract. As shown in Figure 2(a), significantly less biofilm was formed in the presence of the herb extract than that without the extract at the irreversible attachment stage (1 d) and mature stage (3 d and 7 d) after inoculation. The result is in agreement with the gene expression data, suggesting theH. patriniaeextract could reduce biofilm formation through inhibiting the genes associated with biofilm formation.Figure 2
(a) Inhibition of biofilm production of PAO1 byH. patriniaeextract. The control did not contain any herb extract. “∗” indicates significant difference between the herb group and the control group (P<0.05). “∗∗” indicates very significant difference (P<0.01). (b) Micrographs of biofilms formed with and withoutH. patriniae. Top row, PAO1 silver stained biofilms in the presence of extract ofH. patriniaeafter 3 days and 7 days of incubation. Bottom row, PAO1 silver stained biofilms withoutH. patriniae.
(a)
(b)Importantly, the addition of theH. patriniaealso dramatically altered the structure of the biofilms (Figure 2(b)). It appears that the herb extract prevented the formation of mature biofilms, only allowingP. aeruginosato form smaller cell clusters. These results indicate that the water extract ofH. patriniaeindeed was able to inhibitP. aeruginosa biofilm formation.


### 3.3. Water Extract ofH. patriniae InhibitedP. aeruginosa Exopolysaccharide Production

The exopolysaccharide (EPS) matrix is an important component of biofilm structure [30]. We compared the exopolysaccharide production in the presence ofH. patriniaeextract to those withoutH. patriniaeextract. As shown in Figure 3,P. aeruginosa PAO1 cells produced more EPS shown in red by Congo red staining than the cells withH. patriniaeextract. This result indicates thatH. patriniaeinhibitedP. aeruginosa exopolysaccharide production and hence affected biofilm formation.Figure 3
Photograph of exopolysaccharide production on Congo red plates. (a) WithoutH. patriniae. (b) WithH. patriniae. The colony morphology and staining were observed after 3 days of incubation at 37°C.
(a)
(b)


### 3.4.H. patriniae Water Extract Promoted PAO1 Swarming Motility

Upon encountering a surface, the surface-associated behaviors ofP. aeruginosa, such as biofilm formation and swarming, are often coregulated [31]. InP. aeruginosa, swarming motility is reversely correlated with biofilm formation. Examination of the swarming motility of PAO1 in the presence and absence of the water extract ofH. patriniaeshowed thatH. patriniaepromotedP. aeruginosa swarming motility (Figure 4(a)). The diameter of PAO1 grown with 106.67 μgH. patriniaewas almost 5.60 cm, while the control was less than 2.0 cm (Figure 4(b)). Considering the reverse relationship between swarming motility and biofilm formation, the enhanced swarming motility byH. patriniae extract could have contributed the inhibition of biofilm formation.Figure 4
Swarming motility of PAO1. (a) PAO1 grown withH. patriniaewas at right; the one without herb was at left. (b) The swarming diameter of PAO1 withH. patriniae, the control was the one without herb. “∗∗” indicates significant difference (P<0.01).
(a)
(b)Taken together, the extract ofH. patriniaeclearly inhibited the biofilm formation ofP. aeruginosa. It inhibited several key genesalgU,pslM,pelA,algA, andbdlA that are involved in biofilm formation.H. patriniaereduced exopolysaccharide production and promoted swarming motility. Increased motility may reduce adhesion and enable bacteria to actively escape the biofilm matrix to become planktonic bacteria [13, 31, 32]. As depicted in Figure 5, multiple factors/pathways probably have contributed to the reduction of biofilm formation and the altered biofilm structure in the presence ofH. patriniae.Figure 5
P. aeruginosa biofilm formation and swarming motility in the influence ofH. patriniae.In a time of resistance to multiple antimicrobial agents in pathogenic bacteria being spread, there is an urgent need to develop new antibacterial agents [1, 32]. Drugs against infections that involve biofilms are particularly required. Pathogens in biofilm formation are more resistant to conventional antibiotics and other adversary conditions such as nutritional stress. Biofilms also protect bacterial cells from the activity of host immune response [33, 34].Traditional Chinese medicines are a valuable source for novel antibacterial agents [35–37]. The inhibitory effect of the water extract ofH. patriniaeagainstP. aeruginosa biofilms and biofilm-related phenotypes signifies thatH. patriniaeis a promising candidate for treatment of infections caused byP. aeruginosa biofilms. It could be used in the way of traditional Chinese medicine or it can be explored for active compounds.


## 3.1. Screening for Herbs with Inhibitory Effect onP. aeruginosa Biofilm-Associated Genes

Eighteen traditional Chinese medicinal herbs were selected because of their common usage for infection-like symptoms. Both boiling water extracts and ethanol extracts were obtained and used to screen for antibiofilm activities. SinceP. aeruginosa biofilm formation is directly associated with the activity of several known genes, we constructedluxCDABE-based reporters to examine the effect of herb extracts on these genes. The effects of the crude extracts on the expression of these biofilm-associated genes (algU,pslM,pelA,algA,ppyR, andbdlA) are presented in Table 3. The results indicate that different herbal extracts exhibited various degrees of inhibitory effects on these genes. The water extract ofH. patriniae showed the most significant effect on the expression ofalgU,algA,pslM, andbdlA. Examples of the gene expression profiles in the presence ofH. patriniae are shown in Figure 1.Table 3
Effect of water and ethanolic herb extracts on biofilm-associated genes expression.
The English name of herbs
Water extract
The English name of herbs
Ethanolic extract
algU
pslM
pelA
algA
ppyR
bdlA
algU
pslM
pelA
algA
ppyR
bdlA
Atractylodes lancea
+1.5
0
−2.5
0
0
0
Atractylodes lancea
0
0
0
0
0
0
Cortex fraxini
0
−3
−1.5
−1
0
0
Cortex fraxini
−1.8
−2.5
−0.5
−1.5
0
0
Cyrtomium fortunei
0
−1.5
−2
−1
0
0
Cyrtomium fortunei
0
0
0
0
0
0
Erodium stephanianumWilld.
a
a
a
a
a
a
Erodium stephanianum willd
a
a
a
a
a
a
Folium artemisiae Argyi
−2
0
−2
0
−1.5
0
Folium artemisiae argyi
0
0
−2
0
−1.5
0
Fructus quisqualis
a
a
a
a
a
a
Fructus quisqualis
a
a
a
a
a
a
Herba agrimoniae
a
a
a
a
a
a
Herba agrimoniae
a
a
a
a
a
a
Herba patriniae
−2
−2.5
−0.5
−2.5
0
−2
Herba patriniae
0
0
0
0
−1.5
−1
Herba scutellariae barbatae
0
0
0
−1
0
0
Herba scutellariae barbatae
0
0
0
0
0
−1.5
Pomegranate rind
−1.5
−1.2
0
−1
0
0
Pomegranate rind
−1.5
−1
−1
−0.5
0
0
Portulacae herba
+1
0
0
+1.5
0
0
Portulacae herba
0
0
0
0
0
0
Radix paeoniae
−0.5
−0.5
0
0
−1
0
Radix paeoniae
0
0
0
0
0
0
Radix sanguisorbae
−1.5
−1
−1.5
−1
−0.5
0
Radix sanguisorbae
−1.6
−1.5
0
0
−0.5
0
Radix lithospermi
0
−1.2
−2
0
−1
0
Radix lithospermi
0
0
0
0
0
0
Scrophulariae
−2
+1.5
0
0
0
0
Scrophulariae
−0.5
0
−1
0
0
0
Smoked plum
a
a
a
a
a
a
Smoked plum
a
a
a
a
a
a
Taraxacum mongolicum
0
0
0
0
0
0
Taraxacum mongolicum
0
+2
0
0
0
0
Tripterygium
−1.5
0
−2.5
0
0
0
Tripterygium
0
0
−2
0
−2.5
0
Note: herbal extracts were used at 1.6 mg/mL. The values shown represent maximal inhibition (fold changes between experiments with and without herb extract). “+” represents induction of gene expression by herb extracts; “−” represents inhibition of biofilm gene expression by herb extracts; “0” represents no effect of biofilm gene expression by herb extracts; “a” represents inhibition of the growth of bacteria. Numbers represent fold changes at the time points where maximal expression was reached in the absence of herbal extract.Figure 1
Expressions ofalgU,algA,pslM, andbdlA in medium with water extract ofHerba patriniae. Expressions ofalgU (a),algA (b),pslM(c), andbdlA (d) in medium (total volume of 100 μL) with 160 μgH. patriniaeextract and the controls were the ones without herbal extract.
(a)
(b)
(c)
(d)To confirm the results obtained from thelux-based reporter assay, real-time qPCR was carried out using bacterial RNA samples isolated in the presence and absence ofH. patriniae. The results are shown in Table 4. In agreement with the results from the reporter assay, the mRNA levels ofalgU,algA,pslM, andbdlA in PAO1 were all significantly decreased in the presence ofH. patriniae extract (at 1.6 mg/mL) compared with those in the absence ofH. patriniae extract. It is noted that the inhibition ofalgU andalgA was more pronounced in the qPCR assay.Table 4
The transcriptional levels of selected genes in PAO1 and PAO1 treated withH. patriniae.
Gene
Relative mRNA level determined by ΔCt calculation
WithoutH. patriniae
WithH. patriniae
P value
algU
1.00
0.192
0.014
algA
1.00
0.090
0.011
pslM
1.00
0.695
0.043
bdlA
1.00
0.412
0.036
Note: the mRNA level of each gene was normalized to that ofproC. The values shown represent the mean of three different tests.The results indicate that many of these herbs have an inhibitory effect on the genes associated with biofilm formation inP. aeruginosa. This is somewhat not surprising because they all have been used for treatment of chronic bacterial infections in traditional Chinese medicine.The effect from the water extract ofH. patriniaewas remarkable as it inhibited five genes tested. It has been reported that the herb had antibacterial and antiviral activity. However, it was noted that the water extract did not inhibit the growth ofP. aeruginosa (Figure 1). Even though conventional antibiotic compounds may exist inH. patriniaeagainst other bacteria, this result indicates no such component was present againstP. aeruginosa, at least not at the concentrations used in our experiments.Lacking of bacterial killing or growth inhibition activity, however, may not be a weakness of such herbs in treating infectious diseases. As discussed in previous reports [26–29], a promising new class of antipathogenic drugs that target virulence factors and/or biofilm formation instead of killing the pathogens has many advantages in clinical use. First, these antipathogenic drugs theoretically are less likely to render drug resistance in the pathogens because they do not assert a selective pressure on the pathogen’s survival. Second, such therapeutics would unlikely affect other nonpathogenic or beneficial bacteria, that is, the microbiome in the host.


## 3.2. Extract ofH. patriniae InhibitsP. aeruginosa Biofilm Formation

From the gene expression results, the extract ofH. patriniaepresumably would inhibit the biofilm formation ofP. aeruginosa. To verify such an effect,P. aeruginosa biofilm formation was compared in the presence and absence ofH. patriniaeextract. As shown in Figure 2(a), significantly less biofilm was formed in the presence of the herb extract than that without the extract at the irreversible attachment stage (1 d) and mature stage (3 d and 7 d) after inoculation. The result is in agreement with the gene expression data, suggesting theH. patriniaeextract could reduce biofilm formation through inhibiting the genes associated with biofilm formation.Figure 2
(a) Inhibition of biofilm production of PAO1 byH. patriniaeextract. The control did not contain any herb extract. “∗” indicates significant difference between the herb group and the control group (P<0.05). “∗∗” indicates very significant difference (P<0.01). (b) Micrographs of biofilms formed with and withoutH. patriniae. Top row, PAO1 silver stained biofilms in the presence of extract ofH. patriniaeafter 3 days and 7 days of incubation. Bottom row, PAO1 silver stained biofilms withoutH. patriniae.
(a)
(b)Importantly, the addition of theH. patriniaealso dramatically altered the structure of the biofilms (Figure 2(b)). It appears that the herb extract prevented the formation of mature biofilms, only allowingP. aeruginosato form smaller cell clusters. These results indicate that the water extract ofH. patriniaeindeed was able to inhibitP. aeruginosa biofilm formation.


## 3.3. Water Extract ofH. patriniae InhibitedP. aeruginosa Exopolysaccharide Production

The exopolysaccharide (EPS) matrix is an important component of biofilm structure [30]. We compared the exopolysaccharide production in the presence ofH. patriniaeextract to those withoutH. patriniaeextract. As shown in Figure 3,P. aeruginosa PAO1 cells produced more EPS shown in red by Congo red staining than the cells withH. patriniaeextract. This result indicates thatH. patriniaeinhibitedP. aeruginosa exopolysaccharide production and hence affected biofilm formation.Figure 3
Photograph of exopolysaccharide production on Congo red plates. (a) WithoutH. patriniae. (b) WithH. patriniae. The colony morphology and staining were observed after 3 days of incubation at 37°C.
(a)
(b)


## 3.4.H. patriniae Water Extract Promoted PAO1 Swarming Motility

Upon encountering a surface, the surface-associated behaviors ofP. aeruginosa, such as biofilm formation and swarming, are often coregulated [31]. InP. aeruginosa, swarming motility is reversely correlated with biofilm formation. Examination of the swarming motility of PAO1 in the presence and absence of the water extract ofH. patriniaeshowed thatH. patriniaepromotedP. aeruginosa swarming motility (Figure 4(a)). The diameter of PAO1 grown with 106.67 μgH. patriniaewas almost 5.60 cm, while the control was less than 2.0 cm (Figure 4(b)). Considering the reverse relationship between swarming motility and biofilm formation, the enhanced swarming motility byH. patriniae extract could have contributed the inhibition of biofilm formation.Figure 4
Swarming motility of PAO1. (a) PAO1 grown withH. patriniaewas at right; the one without herb was at left. (b) The swarming diameter of PAO1 withH. patriniae, the control was the one without herb. “∗∗” indicates significant difference (P<0.01).
(a)
(b)Taken together, the extract ofH. patriniaeclearly inhibited the biofilm formation ofP. aeruginosa. It inhibited several key genesalgU,pslM,pelA,algA, andbdlA that are involved in biofilm formation.H. patriniaereduced exopolysaccharide production and promoted swarming motility. Increased motility may reduce adhesion and enable bacteria to actively escape the biofilm matrix to become planktonic bacteria [13, 31, 32]. As depicted in Figure 5, multiple factors/pathways probably have contributed to the reduction of biofilm formation and the altered biofilm structure in the presence ofH. patriniae.Figure 5
P. aeruginosa biofilm formation and swarming motility in the influence ofH. patriniae.In a time of resistance to multiple antimicrobial agents in pathogenic bacteria being spread, there is an urgent need to develop new antibacterial agents [1, 32]. Drugs against infections that involve biofilms are particularly required. Pathogens in biofilm formation are more resistant to conventional antibiotics and other adversary conditions such as nutritional stress. Biofilms also protect bacterial cells from the activity of host immune response [33, 34].Traditional Chinese medicines are a valuable source for novel antibacterial agents [35–37]. The inhibitory effect of the water extract ofH. patriniaeagainstP. aeruginosa biofilms and biofilm-related phenotypes signifies thatH. patriniaeis a promising candidate for treatment of infections caused byP. aeruginosa biofilms. It could be used in the way of traditional Chinese medicine or it can be explored for active compounds.


## 4. Conclusions

Our results indicateH. patriniaeextract could significantly inhibit the expression ofP. aeruginosa genes associated with biofilm formation, alter the structure, and prevent the formation of mature biofilms. It also decreased exopolysaccharide production and promoted swarming motility. These results provided a potential underlying mechanism for the use ofH. patriniaeto treat bacterial infections in traditional Chinese medicine and revealed a promising candidate for exploration of new drugs againstP. aeruginosa biofilm-associated infections.



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*Source: 9584703-2017-03-09.xml*