- Email: [email protected]
Anti-biofilm activity of TanReQing, a Traditional Chinese Medicine used for the treatment of acute pneumonia
Journal of Ethnopharmacology 134 (2011) 165–170
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Anti-biofilm activity of TanReQing, a Traditional Chinese Medicine used for the treatment of acute pneumonia Yi Wang a , Tao Wang b , Jianjiang Hu a , ChuanYun Ren c , Hongtao Lei a , Yanming Hou a , Adelheid H. Brantner d,∗ a
Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing, China Academy of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine,Tianjin, China Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing, China d Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Graz, Austria b c
a r t i c l e
i n f o
Article history: Received 22 June 2010 Received in revised form 23 September 2010 Accepted 28 November 2010 Available online 21 December 2010 Keywords: Traditional Chinese Medicine TanReQing injection Penicillin Anti-biofilm activity
a b s t r a c t Aim of the study: TanReQing (TRQ) is a Traditional Chinese Medicine used to treat biofilm related upper respiratory infections. However, its anti-biofilm mechanism remains unknown. The aim of this study is to investigate the anti-biofilm activity of TRQ and to compare it with penicillin in vitro. Materials and methods: The effect of TRQ and penicillin on free state, biofilm formation and mature biofilms of Staphylococcus aureus was studied using the crystal violet and the XTT reduction assays. Confocal Laser Scanning Microscopy was used to generate the 3D-transmission-fluorescence images of drug treated Staphylococcus aureus biofilms. Results: The in vitro data showed that TRQ is less effective than penicillin in eradicating the planktonic bacteria. However, the anti-biofilm activity of TRQ is different from that of penicillin. TRQ not only does inhibit the formation of the Staphylococcus aureus biofilm, but also kills the viable cells embed in Staphylococcus aureus biofilm matrix. Conclusions: This study shows for the first time that TRQ possesses an antibiotic activity against biofilm bacteria. This activity is different from that of penicillin. The evaluation system applied in this study can be utilized for identifying new anti-biofilm products from Traditional Chinese Medicine. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Bacterial biofilms are responsible for several heavy infectious diseases that are difficult to treat (Scudiero et al., 1988; Lim et al., 2004). The well-known reason for this phenomenon is that biofilm forming bacteria show much greater resistance to antibiotics than their free-living counterparts. The reason therefore is the antibiotic penetration barrier which consists of a special polysaccharide complex surrounding the biofilm bacteria (Jackson et al., 2002). In previous papers the authors suggest different mechanisms for biofilm control such as enzymatic dispersion (Johansen et al., 1997), chelation between biofilm cells (Stoodley et al., 2001) or molecular regulation to genes responsible for biofilm formation (Conrady et al., 2008). However, the identification to anti-biofilm compounds with clinical efficacy and safety is still a challenge. TanReQing (TRQ) injection is the combination of water soluble natural products from five Traditional Chinese Medicines. It has been used as clinical reagent in China for several years. The
∗ Corresponding author. E-mail address: [email protected] (A.H. Brantner). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.11.066
main treatment of this reagent is for acute upper respiratory infections and early stage of pneumonia (Li et al., 2008; Jiang et al., 2009). It is well known that pneumonia and respiratory infections are closely related with the formation of biofilms on the surface of the respiratory tract (Martinez-Solano et al., 2008). In a previous study the anti-microbial activity of TRQ had been investigated against planktonic cells (Zhang et al., 2004) according to the method described in the National Committee for Clinical Laboratory Standards (NCCLS). TRQ shows a limited anti-microbial activity in its plasma concentration similar to most of the Traditional Chinese Medicines. However, the clinicians especially those from Traditional Chinese Medical hospitals argue that TRQ has a better activity than common antibiotics against biofilm related respiratory infections. TRQ can also improve the efficacy of common antibiotics when used together for the treatment (Wu et al., 2006). It can be supposed that the anti-microbial mechanism of TRQ is more complicated than it has been demonstrated by previous in vitro experiments. The antimicrobial mechanism of TRQ against Staphylococcus aureus biofilm in vitro was investigated. This study can be considered as basis for identifying further anti-biofilm products from Traditional Chinese Medicine.
166
Y. Wang et al. / Journal of Ethnopharmacology 134 (2011) 165–170
2. Materials and methods 2.1. Materials TanReQing (TRQ) injection, a second-class of new Traditional Chinese Medicine (Approval No. Z20030054), is a product of Shanghai Kaibao Pharmaceutical Company, China, Lot. No. 100406. TRQ is the aqueous extract of a formulation from Traditional Chinese Medicine containing five drugs (Scutellaria baicalensis Georgi (radix), Bear Gall powder, Goral horn, Lonicera japonica Thunb (flos) and Forsythia suspensa (Thunb.) Vahl. (fructus)). Baicalein, ursodeoxycholic acid, chenodeoxycholic acid, forsythin and chlorogenic acid are the main active compounds in the formulation. The content of these active compounds is also the standard of quality control. It has been used as clinical drug in China for several years. According to the Chinese Pharmacopoeia, the process of TRQ production and quality control strictly meets the GMP requirements. The dry residue of this injection was 33 mg/ml. The plasma concentration of the injection has to be 330–165 g/ml for an effective clinical treatment. Penicillin G-K-salt (Feinbiochemica GmbH & Co.) was dissolved in distilled water to get a stock solution of 20 g/ml. XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carbox anilide, Sigma) was dissolved in LB culture medium (60 ◦ C) to get a concentration of 1 g/l. It had to be prepared freshly before use. PMS (phenazine methyl sulfate, Sigma), an electron-coupling agent necessary for the reduction of XTT, was prepared as a stock solution in a concentration of 10 mM and sterile filtered. It had to be kept at 4 ◦ C before use. Crystal violet (Sigma, USA) was used at a concentration of 0.4%. LB medium (pH 7.3–7.4) contained 10 g/l tryptone (Sigma, USA), yeast extract 5 g/l (Merck, Germany) and NaCl 5 g/l (Merck, Germany). G-LB containing 0.25% d-glucose was used for a better growth of Staphylococcus aureus biofilm. Staphylococcus aureus ATCC 25923 was applied as bacterial strain. WST-8 kit (Dojindo, Japan) was used to load the live cells inside the biofilm. 35 mm glass bottom Petri dishes (Shengyou Biotechnology, China) were used for culturing the bacteria which had to be used for imaging by Confocal Laser Scanning Microscopy (Olympus FV1000, Japan). 2.2. Chemical staining and statistical analysis The quantity of whole biomass and living bacteria in the biofilm was monitored by the crystal violet (CV) and the XTT reduction assay. CV staining assay: the micro-titer plate wells were stained with 200 l of 0.4% CV for 10 min at room temperature. The un-bound CV was removed and washed twice with a micro-plate washer. The CV in each well was dissolved by adding 200 l of 99% ethanol. Finally the plate was read at 590 nm using a micro-titer plate reader (Bio-Red 530, USA). XTT assay: 200 l of culture medium was transferred into each well, then 50 l freshly prepared XTT-PMS solution was added (final concentrations: XTT 200 mg/ml; PMS 2 M). Then the plates were incubated at 37 ◦ C for 3 h. Finally the plates were read at a wavelength of 490 nm and 630 nm, respectively. All experiments were performed three times. All data are expressed as mean ± SEM. The significance of the difference between samples and control groups was analyzed by the t-test. 2.3. MICs of TRQ and penicillin against Staphylococcus aureus The MIC (minimum inhibitory concentration) of TRQ and penicillin against free Staphylococcus aureus was determined according to Giacometti (Li et al., 2003). Concentration series of TRQ (16,500–9 g/ml) and penicillin (2–0.001 g/ml) were mixed with the bacterial suspension in each well of the 96-well plates. Then the
plates were incubated 18 h at 37 ◦ C. Finally the number of bacteria in each well was determined by calculating the colony number of overnight cultured plates with 10 l × 10−6 dilution on the agar. MIC was the lowest drug concentration showing 50% inhibition to bacteria prolife compared to the control. 2.4. Biomass determination of forming Staphylococcus aureus biofilm In order to investigate the effect of TRQ on the formation of Staphylococcus aureus biofilm, the mixture of drug and bacterial suspension, TRQ (serial concentrations from 16,500 to 9 g/ml) and penicillin (serial concentrations from 2 to 0.001 g/ml) were added into a 96-well round bottom microplate and incubated overnight at 37 ◦ C. The same volume of culture medium was used as negative control. After incubation each plate was washed twice with 0.9% NaCl to remove planktonic cells. Finally the living cells and the total biomass of the attached cells were determined by the CV and the XTT method. 2.5. Biomass determination of mature Staphylococcus aureus biofilm The first step was the preparation of a mature biofilm. Staphylococcus aureus was cultivated with G-LB at 37 ◦ C for 24 h. After that, the culture was diluted to OD 0.05 with G-LB medium at 600 nm. 200 l of the dilution was added into each well of the 96-well micro-plate and incubated for 24 h at 37 ◦ C. Finally the planktonic cells containing supernatant were removed and each well was rinsed with 200 l 0.9% NaCl. After preparation of mature Staphylococcus aureus biofilm 200 l TRQ in a serial concentration from 9 to 16,500 g/ml diluted with G-LB was added into each well. The plates were incubated for 12 h (37 ◦ C). Penicillin was used as positive control in a serial concentration of 2–0.001 g/ml; G-LB was used as negative control. After 24 h of incubation the planktonic cells were removed from each well with 0.9% NaCl. The living and the total biomass in each well were determined by the CV and the XTT method. 2.6. Imaging observation of biofilm Staphylococcus aureus with CLSM In order to confirm the effect of TRQ against mature Staphylococcus aureus biofilm, the Confocal Laser Scanning Microscope was employed to generate 3D-transmission-fluorescence imaging of Staphylococcus aureus biofilms treated with drugs. The parameters for the optical sections were the following: 5 m steps in the Z-axial direction, 512 × 512 pixels and 12-bit intensity resolution. The objective was an UPLANSAPO 10×/0.4 NA with 270 m pinhole. All data were processed with the software Auto Quant (version X2.1.3). 2 ml Staphylococcus aureus dilution (OD 0.05) was cultured 24 h in a 35 mm glass bottom Petri dish for the experiment. Then TRQ and penicillin were added into parallel dishes. The final drug concentration was 6500 g/ml and 20 g/ml, respectively. G-LB was used as control. 24 h later, imaging data of the total biomass were collected and evaluated directly with CLSM. Living cells had to be stained with a WST-8 kit first before data with CLSM at a wavelength of 448 nm could be collected. 3. Results 3.1. Effect of TRQ and penicillin on free Staphylococcus aureus bacteria The MIC values of TRQ and penicillin against free Staphylococcus aureus were 16,500 g/ml and 2 g/ml. The antibiotic penicillin
Y. Wang et al. / Journal of Ethnopharmacology 134 (2011) 165–170 Table 1 Synergistic effect of TRQ and penicillin on free Staphylococcus aureus bacteria. TRQ (g/ml)
Penicillin MIC (g/ml)
2063 1032 516 258 129 65 33 17 9
0.001 0.063 2 2 2 2 2 2 2
showed a much stronger activity against Staphylococcus aureus in comparison to TRQ which showed a weaker effect similar to most of the TCM drugs with antibiotic activity. In addition, this MIC value of TRQ is much higher than the effective plasma concentration (330–165 g/ml) suggested by the manufacturer. The evaluation of the results showed that a certain concentration of TRQ (9–516 g/ml) could not decrease the MIC of penicillin significantly (Table 1). After increasing the TRQ concentration to 1032 g/ml synergistic phenomena between TRQ and penicillin could be observed. 3.2. Effect of TRQ and penicillin on the formation of Staphylococcus aureus biofilm In order to investigate the effect of TRQ and penicillin on the formation of Staphylococcus aureus biofilm, the chemical staining methods were employed for monitoring the relationship between drug doses and biomass of Staphylococcus aureus biofilm. The CV staining results showed that penicillin and TRQ could inhibit the
167
Table 2 Synergistic effect of TRQ and penicillin in the formation of Staphylococcus aureus biofilm. TRQ (g/ml) 65 33 17 9
Penicillin IC50 ; CV assay
Penicillin IC50 ; XTT assay
0.001 0.044 0.043 0.05
0.002 0.019 0.006 0.008
formation of Staphylococcus aureus biofilm (Fig. 1). The IC50 of penicillin and TRQ was 0.067 g/ml and 49 g/ml, respectively. XTT detection showed similar results (Fig. 2). Certain concentrations of TRQ and penicillin could reduce the amount of living bacterial cells in forming Staphylococcus aureus biofilm. The IC50 of penicillin and TRQ was 0.047 g/ml and 21.2 g/ml, respectively. It has to be mentioned that effective concentrations of TRQ in these tests are much lower than the clinical plasma concentrations (330–165 g/ml). Table 2 shows the synergistic effect of TRQ and penicillin in the formation of Staphylococcus aureus biofilm. TRQ can support the antibiotic penicillin to interfere the formation of Staphylococcus aureus biofilm. This effect could be proved by the CV as well as the XTT assay. 3.3. Effect of TRQ and penicillin on mature Staphylococcus aureus biofilm CV and XTT assays were employed to monitor the effect of TRQ and penicillin on mature Staphylococcus aureus biofilm. In the CV assay the results showed that penicillin and TRQ had no significant
Fig. 1. Effect of penicillin and TRQ on the formation of Staphylococcus aureus biofilms (CV assay) vs. control **P < 0.01, ***P < 0.001.
Fig. 2. Effect of penicillin and TRQ on the formation of Staphylococcus aureus biofilms (XTT assay) vs. control ***P < 0.001.
168
Y. Wang et al. / Journal of Ethnopharmacology 134 (2011) 165–170
Fig. 3. Effect of penicillin and TRQ on mature Staphylococcus aureus biofilms (CV assay).
Fig. 4. Effect of penicillin and TRQ on mature Staphylococcus aureus biofilms (XTT assay) vs. control ***P < 0.001.
effect on the total biomass in mature biofilms (Fig. 3). Penicillin showed similar results in both staining tests. This phenomenon is following literature data reporting that penicillin cannot penetrate the polysaccharide barriers of mature biofilm (Kim and Jones, 2004). However, the XTT assay showed that TRQ could decrease the number of living cells embed in the biofilm matrix (Fig. 4). The IC50 of TRQ for this activity was 58 g/ml, which was much higher than that in the biofilm forming test (21.2 g/ml). But it is still lower than its clinical plasma concentration. In order to investigate if TRQ could assist penicillin to penetrate the barrier of the biofilm matrix, certain concentrations of TRQ (9–65 g/ml) were mixed with different concentrations of penicillin. These mixtures were applied on mature Staphylococcus
aureus biofilm. Fig. 5 shows that no synergistic effect could be verified. Fig. 6 illustrates the transmission and 3D reconstruction imaging of mature Staphylococcus aureus biofilms generated by CLSM. The transmission imaging results proved that TRQ could reduce the biofilm thickness significantly but it could not disrupt the integrity of biofilm matrix. Moreover, the TRQ treated biofilms exhibited a smoother surface than that of penicillin or the control group. These results demonstrate the interaction between the biofilm and TRQ. In 3D reconstruction imaging the penicillin treated biofilm exhibited a status like that of the control group. However, TRQ treated biofilms had lost many living cells on its surface. Consequently, the TRQ treated biofilms exhibited a rough
Fig. 5. Synergistic effect of TRQ and penicillin on mature Staphylococcus aureus biofilms (XTT assay).
Y. Wang et al. / Journal of Ethnopharmacology 134 (2011) 165–170
169
Fig. 6. Transmission and 3D restruction imaging of Staphylococcus aureus biofilms generated by CLSM. (A) Transmission imaging for the total biomass. (B) 3D reconstruction of the surface of living cells. The thickness of the total biomass is defined from the bottom line (green line) to the high reflection line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
surface in the 3D reconstruction imaging. This is highly consistent with the results obtained by the XTT test.
4. Discussion The difficulty in treating biofilm related infectious diseases is that most of the commercial antibiotics are not able to penetrate the biofilm barrier to kill the living cells. The significance of reducing the size of biofilms is that the active components of TRQ could penetrate the biofilm matrix before interacting with living cells in the biofilm while penicillin could not. The MIC value (National Committee for Clinical Laboratory Standards, M7-A5, 2000; European Pharmacopoeia) has to be employed to quantify the activity of anti-infectious agents in clinical laboratory. The MIC values of drugs from Traditional Chinese Medicine (TCM) are usually much higher than those of common antibiotics (Nishibe et al., 1982). In order to explain this phenomenon some scientists argue that the concentrations of active compounds in Traditional Chinese Medicines are very low because of their simple manufacturing technology. The MIC value of active components would be competitive once isolated and purified from TCM mixtures. However, up to now, this hypothesis cannot be supported, even though lots of purification work had been done every year. In this experiment, the MIC value of TRQ is about 50–100 times higher than its clinical plasma concentration. So it is impossible to explain the competitive clinical performance of TRQ according to this evaluation method. However, the activity of TRQ is very competitive when Staphylococcus aureus was changed from planktonic status to biofilm and evaluated. The results in the experiment showed that TRQ not only did inhibit the formation of Staphylococcus aureus biofilm but also killed the living cells embed in Staphylococcus aureus biofilm matrix. Moreover, the effective concentrations for these activities were much lower than its clinical plasma concentration.
In clinics TRQ is mainly applied against acute upper respiratory infections and in the early stage of pneumonia. They are all typical biofilm related diseases (Gong and Li, 2009). The difficulty in treating these infectious diseases is that most of the commercial antibiotics are not able to penetrate the biofilm barrier to kill the living cells (Hadaway, 2005). In this study TRQ demonstrated the synergistic activity against forming and mature Staphylococcus aureus biofilm. This may be the reason why TRQ showed the competitive clinical performance against biofilm related diseases. In order to confirm and elucidate the anti-biofilm activity of TRQ, CLSM was used to investigate the effect of TRQ on the morphology of mature Staphylococcus aureus biofilms. The results supported the anti-biofilm activity of TRQ after applying the XTT staining test. The morphological data also indicated that TRQ could not destroy or disrupt the mature biofilm. This means that the active components of TRQ had to penetrate the biofilm matrix before interacting with living cells in the biofilm. In addition it has to be mentioned that the concentration of TRQ in sterilizing biofilm bacteria is much lower than that in killing planktonic cells (58 g/ml vs. 1650 g/ml). This suggests that the living cells in the biofilm should have different characters in comparison to those in the planktonic status. The living cells in the biofilm are much easier to be sterilized once the active components could penetrate through the biofilm matrix. This study demonstrated the anti-biofilm activity of TRQ, a clinical Chinese medicine. The research on the active compounds and their mechanism of penetration through the biofilm matrix is still ongoing. But on the basis of this study further anti-biofilm products from Traditional Chinese Medicine should be identified.
Acknowledgments Y.W. thanks the EURASIA Pacific Uninet for a postdoc scholarship. This work was supported by grants from the National Natural Science Foundation of China (30600842) and Foundation from Ministry of Finance People’s Republic of China (ZX-(ZZXT)-2006-005).
170
Y. Wang et al. / Journal of Ethnopharmacology 134 (2011) 165–170
References Conrady, D.G., Brescia, C.C., Horii, K., Weiss, A.A., Hassett, D.J., Herr, A.B., 2008. A zinc-dependent adhesion module is responsible for intercellular adhesion in staphylococcal biofilms. Proceedings of the National Academy of Sciences of the United States of America 105, 19456–19461. Gong, G., Li, X., 2009. Study of clinical effect on treatment of chronic obstructive pulmonary disease in acute aggravated stage with Tanreqing injection and cell factor level. Zhongguo Zhong Yao Za Zhi 34, 104–106. Hadaway, L.C., 2005. Skin flora: unwanted dead or alive. Nursing 35, 20. Jackson, D.W., Suzuki, K., Oakford, L., Simecka, J.W., Hart, M.E., Romeo, T., 2002. Biofilm formation and dispersal under the influence of the global regulator CsrA of Escherichia coli. Journal of Bacteriology 184, 290–301. Jiang, H.L., Mao, B., Zhong, Y.Q., Yang, H.M., Fu, J.J., 2009. Tanreqing Injection for community-acquired pneumonia: a systematic review of randomized evidence. Zhong Xi Yi Jie He Xue Bao 7, 9–19. Johansen, C., Falholt, P., Gram, L., 1997. Enzymatic removal and disinfection of bacterial biofilms. Applied and Environmental Microbiology 63, 3724–3728. Kim, H.J., Jones, M.N., 2004. The delivery of benzyl penicillin to Staphylococcus aureus biofilms by use of liposomes. Journal of Liposome Research 14, 123–139. Li, W., Mao, B., Wang, G., Wang, L., Chang, J., Zhang, Y., Wan, M.H., Guo, J., 2008. A study of the mechanism of Qingre Huatan therapy in treatment of acute exacerbation of chronic obstructive pulmonary disease by improving airway inflammation and mucus hypersecretion. Zhong Xi Yi Jie He Xue Bao 6, 799–805.
Li, X., Yan, Z., Xu, J., 2003. Quantitative variation of biofilm among strains in natural populations of Candida albicans. Microbiology 149, 353–362. Lim, Y., Jana, M., Luong, T.T., Lee, C.Y., 2004. Control of glucose- and NaCl-induced biofilm formation by rbf in Staphylococcus aureus. Journal of Bacteriology 186, 722–729. Martinez-Solano, L., Macia, M.D., Fajardo, A., Oliver, A., Martinez, J.L., 2008. Chronic Pseudomonas aeruginosa infection in chronic obstructive pulmonary disease. Clinical Infectious Diseases 47, 1526–1533. Nishibe, S., Okabe, K., Tsukamoto, H., Sakushima, A., Hisada, S., Baba, H., Akisada, T., 1982. Studies on the Chinese crude drug “Forsythiae Fructus”. VI. The structure and antibacterial activity of suspensaside isolated from Forsythia suspensa. Chemical & Pharmaceutical Bulletin (Tokyo) 30, 4548–4553. Scudiero, D.A., Shoemaker, R.H., Paull, K.D., Monks, A., Tierney, S., Nofziger, T.H., Currens, M.J., Seniff, D., Boyd, M.R., 1988. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Research 48, 4827–4833. Stoodley, P., Wilson, S., Hall-Stoodley, L., Boyle, J.D., Lappin-Scott, H.M., Costerton, J.W., 2001. Growth and detachment of cell clusters from mature mixed-species biofilms. Applied and Environmental Microbiology 67, 5608–5613. Wu, W.-m., Chen, L.-j., Chen, J., 2006. Clinical observation of intravenous Tanreqing and azithromycin in treating pneumonia. Journal of Pediatric Pharmacy 12, 37–39. Zhang, L.-L., Zhou, L., Xu, X., Li, Y., Zhou, J., 2004. Pharmacodynamic research of Tanreqing Capiscule. Chinese Journal of Experimental Traditional Medical Formulae 10, 37–40.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Anti-biofilm activity of TanReQing, a Traditional Chinese Medicine used for the treatment of acute pneumonia Yi Wang a , Tao Wang b , Jianjiang Hu a , ChuanYun Ren c , Hongtao Lei a , Yanming Hou a , Adelheid H. Brantner d,∗ a
Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing, China Academy of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine,Tianjin, China Dongzhimen Hospital Affiliated to Beijing University of Chinese Medicine, Beijing, China d Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz, Graz, Austria b c
a r t i c l e
i n f o
Article history: Received 22 June 2010 Received in revised form 23 September 2010 Accepted 28 November 2010 Available online 21 December 2010 Keywords: Traditional Chinese Medicine TanReQing injection Penicillin Anti-biofilm activity
a b s t r a c t Aim of the study: TanReQing (TRQ) is a Traditional Chinese Medicine used to treat biofilm related upper respiratory infections. However, its anti-biofilm mechanism remains unknown. The aim of this study is to investigate the anti-biofilm activity of TRQ and to compare it with penicillin in vitro. Materials and methods: The effect of TRQ and penicillin on free state, biofilm formation and mature biofilms of Staphylococcus aureus was studied using the crystal violet and the XTT reduction assays. Confocal Laser Scanning Microscopy was used to generate the 3D-transmission-fluorescence images of drug treated Staphylococcus aureus biofilms. Results: The in vitro data showed that TRQ is less effective than penicillin in eradicating the planktonic bacteria. However, the anti-biofilm activity of TRQ is different from that of penicillin. TRQ not only does inhibit the formation of the Staphylococcus aureus biofilm, but also kills the viable cells embed in Staphylococcus aureus biofilm matrix. Conclusions: This study shows for the first time that TRQ possesses an antibiotic activity against biofilm bacteria. This activity is different from that of penicillin. The evaluation system applied in this study can be utilized for identifying new anti-biofilm products from Traditional Chinese Medicine. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Bacterial biofilms are responsible for several heavy infectious diseases that are difficult to treat (Scudiero et al., 1988; Lim et al., 2004). The well-known reason for this phenomenon is that biofilm forming bacteria show much greater resistance to antibiotics than their free-living counterparts. The reason therefore is the antibiotic penetration barrier which consists of a special polysaccharide complex surrounding the biofilm bacteria (Jackson et al., 2002). In previous papers the authors suggest different mechanisms for biofilm control such as enzymatic dispersion (Johansen et al., 1997), chelation between biofilm cells (Stoodley et al., 2001) or molecular regulation to genes responsible for biofilm formation (Conrady et al., 2008). However, the identification to anti-biofilm compounds with clinical efficacy and safety is still a challenge. TanReQing (TRQ) injection is the combination of water soluble natural products from five Traditional Chinese Medicines. It has been used as clinical reagent in China for several years. The
∗ Corresponding author. E-mail address: [email protected] (A.H. Brantner). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.11.066
main treatment of this reagent is for acute upper respiratory infections and early stage of pneumonia (Li et al., 2008; Jiang et al., 2009). It is well known that pneumonia and respiratory infections are closely related with the formation of biofilms on the surface of the respiratory tract (Martinez-Solano et al., 2008). In a previous study the anti-microbial activity of TRQ had been investigated against planktonic cells (Zhang et al., 2004) according to the method described in the National Committee for Clinical Laboratory Standards (NCCLS). TRQ shows a limited anti-microbial activity in its plasma concentration similar to most of the Traditional Chinese Medicines. However, the clinicians especially those from Traditional Chinese Medical hospitals argue that TRQ has a better activity than common antibiotics against biofilm related respiratory infections. TRQ can also improve the efficacy of common antibiotics when used together for the treatment (Wu et al., 2006). It can be supposed that the anti-microbial mechanism of TRQ is more complicated than it has been demonstrated by previous in vitro experiments. The antimicrobial mechanism of TRQ against Staphylococcus aureus biofilm in vitro was investigated. This study can be considered as basis for identifying further anti-biofilm products from Traditional Chinese Medicine.
166
Y. Wang et al. / Journal of Ethnopharmacology 134 (2011) 165–170
2. Materials and methods 2.1. Materials TanReQing (TRQ) injection, a second-class of new Traditional Chinese Medicine (Approval No. Z20030054), is a product of Shanghai Kaibao Pharmaceutical Company, China, Lot. No. 100406. TRQ is the aqueous extract of a formulation from Traditional Chinese Medicine containing five drugs (Scutellaria baicalensis Georgi (radix), Bear Gall powder, Goral horn, Lonicera japonica Thunb (flos) and Forsythia suspensa (Thunb.) Vahl. (fructus)). Baicalein, ursodeoxycholic acid, chenodeoxycholic acid, forsythin and chlorogenic acid are the main active compounds in the formulation. The content of these active compounds is also the standard of quality control. It has been used as clinical drug in China for several years. According to the Chinese Pharmacopoeia, the process of TRQ production and quality control strictly meets the GMP requirements. The dry residue of this injection was 33 mg/ml. The plasma concentration of the injection has to be 330–165 g/ml for an effective clinical treatment. Penicillin G-K-salt (Feinbiochemica GmbH & Co.) was dissolved in distilled water to get a stock solution of 20 g/ml. XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carbox anilide, Sigma) was dissolved in LB culture medium (60 ◦ C) to get a concentration of 1 g/l. It had to be prepared freshly before use. PMS (phenazine methyl sulfate, Sigma), an electron-coupling agent necessary for the reduction of XTT, was prepared as a stock solution in a concentration of 10 mM and sterile filtered. It had to be kept at 4 ◦ C before use. Crystal violet (Sigma, USA) was used at a concentration of 0.4%. LB medium (pH 7.3–7.4) contained 10 g/l tryptone (Sigma, USA), yeast extract 5 g/l (Merck, Germany) and NaCl 5 g/l (Merck, Germany). G-LB containing 0.25% d-glucose was used for a better growth of Staphylococcus aureus biofilm. Staphylococcus aureus ATCC 25923 was applied as bacterial strain. WST-8 kit (Dojindo, Japan) was used to load the live cells inside the biofilm. 35 mm glass bottom Petri dishes (Shengyou Biotechnology, China) were used for culturing the bacteria which had to be used for imaging by Confocal Laser Scanning Microscopy (Olympus FV1000, Japan). 2.2. Chemical staining and statistical analysis The quantity of whole biomass and living bacteria in the biofilm was monitored by the crystal violet (CV) and the XTT reduction assay. CV staining assay: the micro-titer plate wells were stained with 200 l of 0.4% CV for 10 min at room temperature. The un-bound CV was removed and washed twice with a micro-plate washer. The CV in each well was dissolved by adding 200 l of 99% ethanol. Finally the plate was read at 590 nm using a micro-titer plate reader (Bio-Red 530, USA). XTT assay: 200 l of culture medium was transferred into each well, then 50 l freshly prepared XTT-PMS solution was added (final concentrations: XTT 200 mg/ml; PMS 2 M). Then the plates were incubated at 37 ◦ C for 3 h. Finally the plates were read at a wavelength of 490 nm and 630 nm, respectively. All experiments were performed three times. All data are expressed as mean ± SEM. The significance of the difference between samples and control groups was analyzed by the t-test. 2.3. MICs of TRQ and penicillin against Staphylococcus aureus The MIC (minimum inhibitory concentration) of TRQ and penicillin against free Staphylococcus aureus was determined according to Giacometti (Li et al., 2003). Concentration series of TRQ (16,500–9 g/ml) and penicillin (2–0.001 g/ml) were mixed with the bacterial suspension in each well of the 96-well plates. Then the
plates were incubated 18 h at 37 ◦ C. Finally the number of bacteria in each well was determined by calculating the colony number of overnight cultured plates with 10 l × 10−6 dilution on the agar. MIC was the lowest drug concentration showing 50% inhibition to bacteria prolife compared to the control. 2.4. Biomass determination of forming Staphylococcus aureus biofilm In order to investigate the effect of TRQ on the formation of Staphylococcus aureus biofilm, the mixture of drug and bacterial suspension, TRQ (serial concentrations from 16,500 to 9 g/ml) and penicillin (serial concentrations from 2 to 0.001 g/ml) were added into a 96-well round bottom microplate and incubated overnight at 37 ◦ C. The same volume of culture medium was used as negative control. After incubation each plate was washed twice with 0.9% NaCl to remove planktonic cells. Finally the living cells and the total biomass of the attached cells were determined by the CV and the XTT method. 2.5. Biomass determination of mature Staphylococcus aureus biofilm The first step was the preparation of a mature biofilm. Staphylococcus aureus was cultivated with G-LB at 37 ◦ C for 24 h. After that, the culture was diluted to OD 0.05 with G-LB medium at 600 nm. 200 l of the dilution was added into each well of the 96-well micro-plate and incubated for 24 h at 37 ◦ C. Finally the planktonic cells containing supernatant were removed and each well was rinsed with 200 l 0.9% NaCl. After preparation of mature Staphylococcus aureus biofilm 200 l TRQ in a serial concentration from 9 to 16,500 g/ml diluted with G-LB was added into each well. The plates were incubated for 12 h (37 ◦ C). Penicillin was used as positive control in a serial concentration of 2–0.001 g/ml; G-LB was used as negative control. After 24 h of incubation the planktonic cells were removed from each well with 0.9% NaCl. The living and the total biomass in each well were determined by the CV and the XTT method. 2.6. Imaging observation of biofilm Staphylococcus aureus with CLSM In order to confirm the effect of TRQ against mature Staphylococcus aureus biofilm, the Confocal Laser Scanning Microscope was employed to generate 3D-transmission-fluorescence imaging of Staphylococcus aureus biofilms treated with drugs. The parameters for the optical sections were the following: 5 m steps in the Z-axial direction, 512 × 512 pixels and 12-bit intensity resolution. The objective was an UPLANSAPO 10×/0.4 NA with 270 m pinhole. All data were processed with the software Auto Quant (version X2.1.3). 2 ml Staphylococcus aureus dilution (OD 0.05) was cultured 24 h in a 35 mm glass bottom Petri dish for the experiment. Then TRQ and penicillin were added into parallel dishes. The final drug concentration was 6500 g/ml and 20 g/ml, respectively. G-LB was used as control. 24 h later, imaging data of the total biomass were collected and evaluated directly with CLSM. Living cells had to be stained with a WST-8 kit first before data with CLSM at a wavelength of 448 nm could be collected. 3. Results 3.1. Effect of TRQ and penicillin on free Staphylococcus aureus bacteria The MIC values of TRQ and penicillin against free Staphylococcus aureus were 16,500 g/ml and 2 g/ml. The antibiotic penicillin
Y. Wang et al. / Journal of Ethnopharmacology 134 (2011) 165–170 Table 1 Synergistic effect of TRQ and penicillin on free Staphylococcus aureus bacteria. TRQ (g/ml)
Penicillin MIC (g/ml)
2063 1032 516 258 129 65 33 17 9
0.001 0.063 2 2 2 2 2 2 2
showed a much stronger activity against Staphylococcus aureus in comparison to TRQ which showed a weaker effect similar to most of the TCM drugs with antibiotic activity. In addition, this MIC value of TRQ is much higher than the effective plasma concentration (330–165 g/ml) suggested by the manufacturer. The evaluation of the results showed that a certain concentration of TRQ (9–516 g/ml) could not decrease the MIC of penicillin significantly (Table 1). After increasing the TRQ concentration to 1032 g/ml synergistic phenomena between TRQ and penicillin could be observed. 3.2. Effect of TRQ and penicillin on the formation of Staphylococcus aureus biofilm In order to investigate the effect of TRQ and penicillin on the formation of Staphylococcus aureus biofilm, the chemical staining methods were employed for monitoring the relationship between drug doses and biomass of Staphylococcus aureus biofilm. The CV staining results showed that penicillin and TRQ could inhibit the
167
Table 2 Synergistic effect of TRQ and penicillin in the formation of Staphylococcus aureus biofilm. TRQ (g/ml) 65 33 17 9
Penicillin IC50 ; CV assay
Penicillin IC50 ; XTT assay
0.001 0.044 0.043 0.05
0.002 0.019 0.006 0.008
formation of Staphylococcus aureus biofilm (Fig. 1). The IC50 of penicillin and TRQ was 0.067 g/ml and 49 g/ml, respectively. XTT detection showed similar results (Fig. 2). Certain concentrations of TRQ and penicillin could reduce the amount of living bacterial cells in forming Staphylococcus aureus biofilm. The IC50 of penicillin and TRQ was 0.047 g/ml and 21.2 g/ml, respectively. It has to be mentioned that effective concentrations of TRQ in these tests are much lower than the clinical plasma concentrations (330–165 g/ml). Table 2 shows the synergistic effect of TRQ and penicillin in the formation of Staphylococcus aureus biofilm. TRQ can support the antibiotic penicillin to interfere the formation of Staphylococcus aureus biofilm. This effect could be proved by the CV as well as the XTT assay. 3.3. Effect of TRQ and penicillin on mature Staphylococcus aureus biofilm CV and XTT assays were employed to monitor the effect of TRQ and penicillin on mature Staphylococcus aureus biofilm. In the CV assay the results showed that penicillin and TRQ had no significant
Fig. 1. Effect of penicillin and TRQ on the formation of Staphylococcus aureus biofilms (CV assay) vs. control **P < 0.01, ***P < 0.001.
Fig. 2. Effect of penicillin and TRQ on the formation of Staphylococcus aureus biofilms (XTT assay) vs. control ***P < 0.001.
168
Y. Wang et al. / Journal of Ethnopharmacology 134 (2011) 165–170
Fig. 3. Effect of penicillin and TRQ on mature Staphylococcus aureus biofilms (CV assay).
Fig. 4. Effect of penicillin and TRQ on mature Staphylococcus aureus biofilms (XTT assay) vs. control ***P < 0.001.
effect on the total biomass in mature biofilms (Fig. 3). Penicillin showed similar results in both staining tests. This phenomenon is following literature data reporting that penicillin cannot penetrate the polysaccharide barriers of mature biofilm (Kim and Jones, 2004). However, the XTT assay showed that TRQ could decrease the number of living cells embed in the biofilm matrix (Fig. 4). The IC50 of TRQ for this activity was 58 g/ml, which was much higher than that in the biofilm forming test (21.2 g/ml). But it is still lower than its clinical plasma concentration. In order to investigate if TRQ could assist penicillin to penetrate the barrier of the biofilm matrix, certain concentrations of TRQ (9–65 g/ml) were mixed with different concentrations of penicillin. These mixtures were applied on mature Staphylococcus
aureus biofilm. Fig. 5 shows that no synergistic effect could be verified. Fig. 6 illustrates the transmission and 3D reconstruction imaging of mature Staphylococcus aureus biofilms generated by CLSM. The transmission imaging results proved that TRQ could reduce the biofilm thickness significantly but it could not disrupt the integrity of biofilm matrix. Moreover, the TRQ treated biofilms exhibited a smoother surface than that of penicillin or the control group. These results demonstrate the interaction between the biofilm and TRQ. In 3D reconstruction imaging the penicillin treated biofilm exhibited a status like that of the control group. However, TRQ treated biofilms had lost many living cells on its surface. Consequently, the TRQ treated biofilms exhibited a rough
Fig. 5. Synergistic effect of TRQ and penicillin on mature Staphylococcus aureus biofilms (XTT assay).
Y. Wang et al. / Journal of Ethnopharmacology 134 (2011) 165–170
169
Fig. 6. Transmission and 3D restruction imaging of Staphylococcus aureus biofilms generated by CLSM. (A) Transmission imaging for the total biomass. (B) 3D reconstruction of the surface of living cells. The thickness of the total biomass is defined from the bottom line (green line) to the high reflection line. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
surface in the 3D reconstruction imaging. This is highly consistent with the results obtained by the XTT test.
4. Discussion The difficulty in treating biofilm related infectious diseases is that most of the commercial antibiotics are not able to penetrate the biofilm barrier to kill the living cells. The significance of reducing the size of biofilms is that the active components of TRQ could penetrate the biofilm matrix before interacting with living cells in the biofilm while penicillin could not. The MIC value (National Committee for Clinical Laboratory Standards, M7-A5, 2000; European Pharmacopoeia) has to be employed to quantify the activity of anti-infectious agents in clinical laboratory. The MIC values of drugs from Traditional Chinese Medicine (TCM) are usually much higher than those of common antibiotics (Nishibe et al., 1982). In order to explain this phenomenon some scientists argue that the concentrations of active compounds in Traditional Chinese Medicines are very low because of their simple manufacturing technology. The MIC value of active components would be competitive once isolated and purified from TCM mixtures. However, up to now, this hypothesis cannot be supported, even though lots of purification work had been done every year. In this experiment, the MIC value of TRQ is about 50–100 times higher than its clinical plasma concentration. So it is impossible to explain the competitive clinical performance of TRQ according to this evaluation method. However, the activity of TRQ is very competitive when Staphylococcus aureus was changed from planktonic status to biofilm and evaluated. The results in the experiment showed that TRQ not only did inhibit the formation of Staphylococcus aureus biofilm but also killed the living cells embed in Staphylococcus aureus biofilm matrix. Moreover, the effective concentrations for these activities were much lower than its clinical plasma concentration.
In clinics TRQ is mainly applied against acute upper respiratory infections and in the early stage of pneumonia. They are all typical biofilm related diseases (Gong and Li, 2009). The difficulty in treating these infectious diseases is that most of the commercial antibiotics are not able to penetrate the biofilm barrier to kill the living cells (Hadaway, 2005). In this study TRQ demonstrated the synergistic activity against forming and mature Staphylococcus aureus biofilm. This may be the reason why TRQ showed the competitive clinical performance against biofilm related diseases. In order to confirm and elucidate the anti-biofilm activity of TRQ, CLSM was used to investigate the effect of TRQ on the morphology of mature Staphylococcus aureus biofilms. The results supported the anti-biofilm activity of TRQ after applying the XTT staining test. The morphological data also indicated that TRQ could not destroy or disrupt the mature biofilm. This means that the active components of TRQ had to penetrate the biofilm matrix before interacting with living cells in the biofilm. In addition it has to be mentioned that the concentration of TRQ in sterilizing biofilm bacteria is much lower than that in killing planktonic cells (58 g/ml vs. 1650 g/ml). This suggests that the living cells in the biofilm should have different characters in comparison to those in the planktonic status. The living cells in the biofilm are much easier to be sterilized once the active components could penetrate through the biofilm matrix. This study demonstrated the anti-biofilm activity of TRQ, a clinical Chinese medicine. The research on the active compounds and their mechanism of penetration through the biofilm matrix is still ongoing. But on the basis of this study further anti-biofilm products from Traditional Chinese Medicine should be identified.
Acknowledgments Y.W. thanks the EURASIA Pacific Uninet for a postdoc scholarship. This work was supported by grants from the National Natural Science Foundation of China (30600842) and Foundation from Ministry of Finance People’s Republic of China (ZX-(ZZXT)-2006-005).
170
Y. Wang et al. / Journal of Ethnopharmacology 134 (2011) 165–170
References Conrady, D.G., Brescia, C.C., Horii, K., Weiss, A.A., Hassett, D.J., Herr, A.B., 2008. A zinc-dependent adhesion module is responsible for intercellular adhesion in staphylococcal biofilms. Proceedings of the National Academy of Sciences of the United States of America 105, 19456–19461. Gong, G., Li, X., 2009. Study of clinical effect on treatment of chronic obstructive pulmonary disease in acute aggravated stage with Tanreqing injection and cell factor level. Zhongguo Zhong Yao Za Zhi 34, 104–106. Hadaway, L.C., 2005. Skin flora: unwanted dead or alive. Nursing 35, 20. Jackson, D.W., Suzuki, K., Oakford, L., Simecka, J.W., Hart, M.E., Romeo, T., 2002. Biofilm formation and dispersal under the influence of the global regulator CsrA of Escherichia coli. Journal of Bacteriology 184, 290–301. Jiang, H.L., Mao, B., Zhong, Y.Q., Yang, H.M., Fu, J.J., 2009. Tanreqing Injection for community-acquired pneumonia: a systematic review of randomized evidence. Zhong Xi Yi Jie He Xue Bao 7, 9–19. Johansen, C., Falholt, P., Gram, L., 1997. Enzymatic removal and disinfection of bacterial biofilms. Applied and Environmental Microbiology 63, 3724–3728. Kim, H.J., Jones, M.N., 2004. The delivery of benzyl penicillin to Staphylococcus aureus biofilms by use of liposomes. Journal of Liposome Research 14, 123–139. Li, W., Mao, B., Wang, G., Wang, L., Chang, J., Zhang, Y., Wan, M.H., Guo, J., 2008. A study of the mechanism of Qingre Huatan therapy in treatment of acute exacerbation of chronic obstructive pulmonary disease by improving airway inflammation and mucus hypersecretion. Zhong Xi Yi Jie He Xue Bao 6, 799–805.
Li, X., Yan, Z., Xu, J., 2003. Quantitative variation of biofilm among strains in natural populations of Candida albicans. Microbiology 149, 353–362. Lim, Y., Jana, M., Luong, T.T., Lee, C.Y., 2004. Control of glucose- and NaCl-induced biofilm formation by rbf in Staphylococcus aureus. Journal of Bacteriology 186, 722–729. Martinez-Solano, L., Macia, M.D., Fajardo, A., Oliver, A., Martinez, J.L., 2008. Chronic Pseudomonas aeruginosa infection in chronic obstructive pulmonary disease. Clinical Infectious Diseases 47, 1526–1533. Nishibe, S., Okabe, K., Tsukamoto, H., Sakushima, A., Hisada, S., Baba, H., Akisada, T., 1982. Studies on the Chinese crude drug “Forsythiae Fructus”. VI. The structure and antibacterial activity of suspensaside isolated from Forsythia suspensa. Chemical & Pharmaceutical Bulletin (Tokyo) 30, 4548–4553. Scudiero, D.A., Shoemaker, R.H., Paull, K.D., Monks, A., Tierney, S., Nofziger, T.H., Currens, M.J., Seniff, D., Boyd, M.R., 1988. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Research 48, 4827–4833. Stoodley, P., Wilson, S., Hall-Stoodley, L., Boyle, J.D., Lappin-Scott, H.M., Costerton, J.W., 2001. Growth and detachment of cell clusters from mature mixed-species biofilms. Applied and Environmental Microbiology 67, 5608–5613. Wu, W.-m., Chen, L.-j., Chen, J., 2006. Clinical observation of intravenous Tanreqing and azithromycin in treating pneumonia. Journal of Pediatric Pharmacy 12, 37–39. Zhang, L.-L., Zhou, L., Xu, X., Li, Y., Zhou, J., 2004. Pharmacodynamic research of Tanreqing Capiscule. Chinese Journal of Experimental Traditional Medical Formulae 10, 37–40.