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BERBERINE EFFECTS BIOFILM FORMATION AND EXPRESSION OF LuxS AND VIRULENCE FACTORS IN Streptococcus suis

Chang Wang, Shuai Wang, Yanbei Yang, Yonghui Zhou, Jianqing Chen, Yanhua Li*
 College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030, PR China
Corresponding author: Yanhua Li, College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Xiangfang, Harbin, Heilongjiang 150030, P.R. China, Tel:+86045155191881 E-mail: liyanhua1970a@163.com
Received date: 08/21/2015 Accepted date: 12/10/2015 Published date: 12/17/2015
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Abstract

Streptococcus suis (S. suis) is an important pathogen of pigs, responsible for diverse diseases in swine and human. It is found to form biofilm in virtro and in vivo. luxS/AI-2 not only influences the formation of biofilm, but also bacterial virulence factors. Berberine is an isoquinoline-type alkaloid isolated from Copyidis rhizome and other herbs against bacteria. In this study, we observed that sub-minimal inhibitory concentrations (sub-MIC) of berberine (62.5μg•mL-1) were sufficient to exhibit an antibacterial effect and to inhibit biofilm formation significantly, as shown by the scanning electron microscopy. Real-time PCR showed that berberine decreased the amount of luxS-mRNA lower than that of negative control. Quantification of expression levels of known virulence genes by real-time PCR revealed that berberine on the transcription levels of the ef, sly and gapdh genes of biofilm formation were downregulated, while the gdh, cps and mrp genes were upregulated. To summarize the collective data demonstrated that berberine may regulate transcription levels of luxS/AI-2 and many virulence genes, and inhibit S. suis biofilm formation.

Keywords
Streptococcus suis, Biofilm, Berberine, AI-2, luxS, Virulence gene

INTRODUCTION
S. suis is an important pathogen responsible for important economic losses to the porcine industry worldwide. It causes a variety of life-threatening infections, causing meningitis, septicemia, endocarditis, arthritis and pneumonia in pigs. S. suis infections have been reported in over 30 countries, and 35 serotypes (1-34 and 1/2) have been identified on the basis of capsular antigerns [1]. S. suis has ability to form biofilms, which plays a key role in causing persistent infections [2].
Quorum sensing system plays an important role in the biofilm formation. Quorum sensing is an intercellular communication system by which bacterial cells are capable of indirectly monitoring their own population density through production and exchange of diffusible signal molecules [3]. This regulatory system involves the production of cell signaling molecules via the luxS-based autoinducer-2 (AI-2), the extracellular concentration of which increases as the cell density increases [4]. As the extracellular concentration of signaling molecules reaches a threshold, the bacteria can alter their gene expression in unison [5]. TheluxSgene plays a vital role in biofilm formation, gene expression, motility and drug resistance [6]. In previous study, higher concentrations of AI-2 inhibited S. suis biofilm formation and host-cell adherence [7]. The virulence factors such as cps, mrp, gapdh, sly, fbps, gdh and ef in the mutant strains, which media the adhesion, invasion, colonization and proliferation capacity of S. suis to host cells. Thus, understanding the association between biofilm, virulence and quorum-sensing may lead to the development of novel strategies to fight infections.
There is an urgent need to develop alternative treatments for S. suis biofilm infections that are safe, effective and inexpensive. Herbal medicines have been used for centuries to treat various diseases. They also exhibit antibacterial, antivirus [8], anti-inflammatory and anticancer activities [9]. Among all of the strategies that have been exploited to overcome drug resistance, the use of natural substances has shown particular promise, and many natural substances also have antibiofilm properties [10-12]. Previous reports showed that the extracts of Coptis chinensis Franch and Phellodendron amurense Ruprecht have antimicrobial effect [13]. One of the major compounds of C. chinensis and P. amurense is berberine. Berberine, one of the most important members of the protoberberine group of alkaloid, exhibits antimalarial, antisecretory, and anti-inflammatory as well as anticancer activities with relatively low cytotoxicity [14]. It has also been reported that berberine is useful in the treatment of gastroenteritis, diarrhea, and cholera diseases [15]. Previous studies have shown that berberine has antimicrobial activity against several bacterial species, and interferes with the adherence of Streptococcus pyogenes to host cells, either by preventing the complexing of lipoteichoic acid with fibronectin or by dissolution of such complexes once they are formed [16].
In most studies of the anti-infective activities of berberine, the focus has been on its antimicrobial effects, and little has been paid to the effects of berberine on the biofilm formation of S. suis. Here we focused on investigating the effect of berberine on the luxS/AI-2 QS system and virulence factors of biofilm formation of S. suis.

MATERIAL AND METHODS

Bacterial Strains and Culture Conditions
S. suis strain ATCC700794 was used in this study. It was grown in Todd-Hewitt broth (THB) containing 5% fetal bovine serum at 37°C with shaking at 150 rmp to obtain a planktonic culture in mid-exponential growth phase.

Minimal Inhibitory Concentration (MIC)
MIC was the lowest concentration that inhibited the visible growth. MIC was determined for each compound by using a microdilution assay, as previously described [17]. Briefly, 100 μl 1.0 × 106 cfu/ml S. suis (based upon McFarlane standards) was added to each well, microtiter plate was incubated for 24 h at 37°C.

Quantification of Biofilm Formation
Biofilm formation was performed on the Calgary biofilm device (CBD) according to previously described methods with modifications [18]. Briefly, 200 μl 1.0 × 106 cfu/ml S. suis (based upon McFarlane standards) was added to CBD. Incubated at 37°C for 3 days without shaking.

Effect of Berberine on Biofilm Formation
100 μl THB broth containing berberine were added to the wells of a CBD plate. An overnight culture of S. suis was diluted in fresh culture broth to 1.0 × 106 cfu/ml and 100 μl was inoculated into each well. All assays were performed in quintic. After incubation for 3 days at 37°C, the content of the wells were then discarded and the pegs were washed three times using 200 μl sterile PBS to remove loosely adherent cells. The plate was stained with crystal violet. After fixing of methanol and then staining was measured at 595 nm.

Determination of Colony Forming Units (CFU)
To test the bactericidal activity of berberine against the bacteria in biofilm, 100 μl S. suis (1.0 × 106 cells/ml) was added to the CBD and the method as described above. Then the microplate was sealed and sonicated for 10 min. The cells were plated on Petri dishes containing a solid medium in triplicate, and incubated at 37°C for 2 days before they were counted. The means ± standard deviations were calculated.

Observation on Biofilm Formation by Scanning Electron Microscopy
In addition, Scanning electron microscopy (SEM) images were taken to confirm the prevention of biofilm formation. Biofilm formation on CBD was undertaken as above. Briefly, the Pegs were washed twice with PBS and then initially fixed by 2.5% glutaraldehyde for 20 h at 4°C. The surfaces were washed twice with PBS. The bacteria were then dehydrated by replacing the buffer with increasing concentrations of ethanol (50%, 70%, 90%, 100%) for 10 min each. After critical point drying and coating by gold sputter, samples were examined with a scanning electron microscope.

Real-Time PCR Analysis
Total RNA was isolated from S. suis grown as biofilms cells for 3 days. cDNAs were generated by reverse transcription. 16sRNA was used as internal control in real-time PCR (RT-PCR) analysis. RT-PCR was carried out according to the method previously described [19]. RT-PCR was performed in a total volume of 20 μl containing 10 μl of FastStart DNA Master SYBR GreenI, 1.2 μl of cDNA, 7.6 μl of ddH2O, 0.6 μl of forward primer, and 0.6 μl of reverse primer. The related genes of primers used for the various RT-PCR assays are listed in Table 1.

Statistical Analysis
Data were expressed as the mean ± SD. AP value of <0.05 was considered significant. All statistical analyses were performed using a SPSS 20.0 Statistical Software. Data analysis was carried out using Prism Software.

RESULTS AND DISCUSSION

Effect of Berberine on Biofilm Formation
As biofilm formation is thought to be the leading to cause of persistent infection caused by S. suis. Therefore, we studied the influence of berberine on S. suis biofilm formation and biofilm CFU counts. Various concentrations of berberine were added to the biofilm plate to test whether berberine regulates S. suis biofilm formation. Wang et al. reported that berberine is active in vitro against S. epidermidis and that sub-MICs of berberine may play a role in the prevention of biofilm formation of S. epidermidis [20]. As well, we found the MIC of berberine against S. suis was determined as 62.5 μg/ml. After 3 days of exposure, we observed a decreased in the biofilm CFU counts. The biofilm was markly decreased by incubation with 31.25 μg/ml berberine for 72 h (P<0.01) (Figure 1). Decreased biomass was observed for berberine-treated bacteria compared to un-treated control in Figure 2. The S. suis cfu/ml decreased from 9.16 ± 0.90 cfu/ml in controls to 6.71 ± 0.11 cfu/ml in berberine-exposed isolates (P<0.05). The biofilm CFU counts significantly decreased when the cells were incubated with berberine.
The S. suis observed by SEM are shown in Figure 3. The strain formed a thick, heterogeneous layer with columnar clumps on the CBD when incubated for 72 h without berberine. When strain is treated with berberine at concentration of 31.25 μg/ml, no biofilm was obviously formed and only a few bacterial microcolonies could be seen. The results showed that berberine could inhibit biofilm formation in a dose-dependent manner.

Effect of Berberine on the luxS/AI-2 of S. suis Biofilm
Previous studies have found that the luxS/AI-2 QS system and virulence factors affects biofilm development in S. suis, and the results of these studies revealed that AI-2 and virulence factors limits biofilm formation [21]. It has been reported that berberine may play a role in the prevention of biofilm formation of S. epidermidis [20], but its effect on S. suis is not clear. In this study, we focused on investigating the effect of berberine on the luxS/AI-2 QS system and virulence factors of biofilm formation of S. suis.
Biofilm development and quorum sensing are closely interconnected processes. Quorum sensing is the cell population density-dependent regulation of gene expression by small signaling molecules, called AI [22]. When the AIs accumulate to a threshold concentration, the system is activated and directly or indirectly controls the transcription of target genes. A gene called luxS is required for the synthesis of AI-2 [23]. The result of real-time PCR showed that berberine decreased the amount of luxSmRNA to 4.35-fold lower than that of S. suis cells grown in THB (P<0.05) (Figure 4). In conclusion, berberine may regulate transcription levels of luxS/AI-2 and inhibit S. suis biofilm formation.

Effects of Berberine on Virulence gene Expression of S. suis Biofilm
Previous research on the virulence-associated factors of S. suis has focused mainly on the capsular polysaccharide (cps), muramidase-released protein (mrp) (mrp gene), extracellular protein factor (ef) and suilysin (sly) [24]. These molecules have been suggested to be virulence-related factors. Recently, a large number of putative virulence factors associated with S. suis 2 have been described [25-27]. These include fibronectin, fibrinogen-binding protein (fbps) [28], glyceraldehyde-3-phosphate dehydrogenase (gadph) [29], and glutamate dehydrogenas (gdh) [30]. Extensive research during the last two decades has identified many factors involved in the virulence of this pathogen including notably the cps, which plays a major protective role against host immunity [31]. So far, different genes have been identified as critical for S. suis virulence [32]. Hammerschmidt et al., [33] found invasive pneumococci exhibited an enhanced capacity to adhere and invade epithelial cells, appearing to cause a reduction in capsular material, which potentially increases biofilm formation. In this study, berberine increased the cps to 1.54-fold than negative control (Figure 5). The result showed that cps as a surface structure seems to be essential for regulation of biofilm development and virulence. mrp, originally associated with virulent strains, has been reported in serotype 2 strains [34].
The N-terminal region of BaoA1 is homologous to the mrp from S. suis. Previous study showed that the BaoA1 deficiency inhibited bacterial biofilm formation [35]. Yang et al. reported the expression level of the mrp gene was downregulated between biofilms and planktonic cells [36]. In this study the expression data demonstrated that berberine increased the mrp to 1.71-fold than negative control. Therefore, berberine could regulate the expression of mrp and inhibit S. suis biofilm formation. gapdh possesses a binding activity for albumin and the presence of albumin at the bacterial surface increases the virulence of S. suis [37]. S. suis gapdh also plays a role in the bacterial adhesion [29]. The expression data demonstrated that berberine decreased the gapdh to 3.85 fold. It stands to reason that berberine deal with gapdh reduced the bacterial adherence and therefore weakened the virulence of S. suis. sly belongs to the family of toxins known as thiolactivated toxins or related cholesterol-binding cytolytic toxins that can regulate hemolysin [4]. The expression data demonstrated that berberine decreased the sly to 3.13 fold. In this study, berberine may regulate the expression level of the sly, which was effect on the S. suis. Yang et al. reported three virulence genes were downregulated in the expression level of the gdh, cps2 and mrp genes between biofilms and planktonic cells, while gapdh and sly were upregulated in biofilms [36]. And in this study, three virulence genes (ef, sly, and gapdh) were downregulated and three virulence genes (gdh, cps, mrp) were upregualted addition of 31.25 μg/ml berberine. However, berberine did not dramatically increase the amount of fbps-mRNA compared to negative control. A similar result has been found by Yang et al. The results showed that berberine could regulate the expression of virulence genes of S. suis.

ACKNOWLEDGEMENT
This work was supported by the National Natural Science Foundation of China (No. 820055) and the Projects of Application Technology Research and Development in Heilongjiang (No. 2014G0173).

Tables at a glance
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Table 1

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References