The bacteriostatic effect and mechanism of berberine on Methicillin resistant Staphylococcus aureus in vitro

Background : To observe the bacteriostatic effect of berberine (BBR) and BBR combined with gentamicin (GEN), levofloxacin (LEV) and amikacin (AMI) on Methicillin resistant Staphylococcus aureus (MRSA), while also exploring the bacteriostatic mechanism of BBR on MRSA. Methods : The minimal inhibitory concentration (MIC) of BBR, GEN, LEV and AMI on 26 clinical MRSA strains was determined by broth microdilution, while the MICs of BBR combined with GEN, LEV and AMI against MRSA were determined using a microdilution checkerboard. Time-killing curves were used to determine the kinetics of BBR combined with antibiotics for MRSA. We used conductivity tests to assess the changes in membrane permeability in response to BBR on MRSA, while also investigating the changes in MRSA morphology by transmission electron microscopy. RNA-sequencing was used to analyze the expression of differentially expressed genes in reference strain USA300 after its treatment with BBR at different concentrations. Results : The MICs range of BBR on 26 strains of MRSA was 32-256 µg/mL. BBR combined with GEN, LEV and AMI had obvious bacteriostatic effect on MASA. After co-culturing MRSA with BBR at 512 ug/mL, 64 ug/mL and 8 ug/mL, respectively, the electrical conductivity increased, compared with the control group, by 8.14%, 13.08% and 12.01%, respectively. Using transmission electron microscopy, we found that low concentration of BBR (8 ug/mL) had no significant effect on MRSA structure (keeping intact), medium concentration of BBR (64 ug/mL) thinned the cell wall of MRSA, while high concentration of BBR (512 ug/mL) induced the destruction and dissolution of MRSA cell wall structure and the leakage of bacterial contents, leading to bacterial lysis. RNA-sequencing results showed that there were 754 differentially expressed genes in the high concentration group compared with the normal control group. Compared with the low concentration group, there were 590

3 differentially expressed genes in the high concentration group. Compared with the control group, only 19 genes were differentially expressed in the low concentration group. The upregulated genes are mainly related to the cell wall hydrolysis regulatory genes, while the down-regulated genes are mainly related to the serine protease family. pneumonia, pseudomembranous enteritis, pericarditis and sepsis [1]. In recent years, the emergence of Methicillin-resistant Staphylococcus aureus (MRSA), which is resistance to most beta-lactam, has severely limited treatment options [2][3]. Vancomycin is commonly used to treat MRSA infections [4]. However, Vancomycin-intermediate Staphylococcus aureus (VISA) appeared in the 1990s, and Vancomycin-resistant Staphylococcus aureus (VRSA) was first reported in the United States in 2002 [5]. The emergence of VISA and VRSA has raised the importance of discovering new treatments for MRSA infections.

Conclusions
Berberine (BBR) is the main component of the traditional Chinese medicine Coptis chinensis and Cortex Phellodendri, with an isoquinoline alkaloid structure. BBR is one of the commonly used drugs in the treatment of intestinal infections in China [6]. In addition to its broad spectrum bacteriostatic effects [7][8][9], BBR is reported to have anti-4 inflammatory, anti-oxidant, anti-tumor, hypoglycemic and anti-cardiac arrhythmia qualities [10][11][12][13][14]. Studies also reported that BBR had good bacteriostatic effect on E. coli and Bacillus subtilis [15]. Subsequent studies reported that the MIC of BBR against the MRSA reference strain ATCC33591 was 128 µg/mL. BBR can affect the aggregation of amyloid fibers in PSMs of MRSA biofilm, thus inhibiting the formation of MRSA biofilm and increasing the bactericidal activity of antibiotics [9]. Recent studies have reported that BBR had bacteriostatic effects on MRSA, yet no specific study on its bacteriostatic mechanism has been reported. In our hospital, aminoglycoside antibiotics (GEN, AMI) and quinolone antibiotics (LEV)  Co., LTD, batch number: 16080231) were all purchased from the pharmacy of Shanghai Eighth People's hospital. These antibiotics were freshly prepared with sterile water to a concentration of 1024 µg/mL.

Multilocus sequence typing (MLST)
Isolates were screened using a previously described method [16] [17],and the allelic number was determined for each sequence.

Conductivity test
MRSA02 strain was selected for the conductivity test. Measuring the conductivity of culture medium with a conductivity meter is to measure the ionic concentration of culture 8 medium. The higher the ion concentration is, the greater the conductivity. BBR solution at 16, 128, and 1024 µg/mL were added to the bacterial suspension culture at logarithmic phase. The final concentrations of BBR solution were 8, 64, and 512 µg/mL, respectively. 5 mL of suspension was taken and centrifuged at 2800×g for 10 min at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 h, respectively. Conductivity of the supernatant was measured by DDS-11A conductivity meter (Leici, Shanghai) after a 20-time dilution of the supernatant. Absolute ethanol was taken as the control group. The test was repeated 3 times, and the average value was obtained.

RNA isolation, mRNA enrichment and sequencing
After culturing S. aureus USA300 strain to logarithmic phase, USA300 was cultured with BBR of different concentration for 3 h, and total RNA was extracted. The samples were divided into 3 groups: normal control group (group A), high concentration group (group B, 1/2 MIC, 64 ug/mL), and low concentration group (group C, 1/8 MIC, 16 ug/mL). Each group 9 had 3 repetitive samples. Total RNA was extracted from bacterial cells using RNeasy Mini kit (Qiagen) [20]. Qubit 2.0 RNA detection kit was used to quantify total RNA accurately to determine the amount of total RNA added to the library. rRNA was removed by kit and fragmentation buffer was added to the obtained mRNA to make the fragments short. The fragmented RNA was used as template to synthesize the first strand of the DNA with random hexamers, and the second strand was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I. The product was purified by QiaQuick PCR kit and eluted by EB buffer. After terminal repair, base A and sequencing connector were added, the target fragments were collected by agarose gel electrophoresis, and amplified by PCR. The whole library was prepared and the library was sequenced by Illumina HiSeq2500 [21].

Antimicrobial susceptibility profiles
Reference strains USA300 and 26 clinical isolates of MRSA strains were identified by automatic identification and drug susceptibility system (VITEK 2 compact microbiology analysis system, BioMérieux). According to the 2018 version of the Clinical and Laboratory Standards Institute (CLSI) [18], all strains were identified as MRSA. Drug susceptibility test showed that 26 MRSAs were all resistant to oxacillin, 5 to gentamicin, 17 to levofloxacin, 6 to amikacin, 22 to clindamycin, 19 to ciprofloxacin, 21 to erythromycin, 18 to moxifloxacin, 1 to Trimethoprim-sulfamethoxazole(SMZ-TMP), and 17 to tetracycline. All 26 strains were sensitive to rifampicin, linezolid and vancomycin (Table 1). In addition, In addition, 26 clinical isolated strains were identified as MRSA by mecA gene using PCR.

Bacteriostatic effect of BBR, GEN, LEV and AMI on MRSA
The MIC of BBR on reference strain USA300 is 128 ug/mL, while the MIC of BBR on 26 clinical isolated MRSA strains is 32-256 ug/mL, indicating that BBR has strong bacteriostatic effect both on USA300 and clinical isolated strains, which is consistent with the previous research results of Chu, M. etc. [9], who reported that the MIC of BBR on reference strain ATCC33591 was 128 ug/mL ( Table 2).

BBR combined with antibiotics time-kill analysis
Of the 26 clinical MRSA strains, strain MRSA02, which was resistant to GEN, LEV, and AMI, was selected to assess the bacteriostatic effect of BBR combined with these 3 antibiotics.
With the concentration of BBR and antibiotics increasing, the amount of bacteria and the OD value decreased gradually. The the concentration of 50% reduction of OD value was taken as MIC. The MICs of BBR, GEN, LEV, and AMI for MRSA02 were 128 µg/mL, 64 µg/mL, 128 µg/mL, and 256 µg/mL, respectively ( Fig. 2A). In the time sterilization curve, compared with the control group, the OD value of BBR combined with GEN decreased by 75% (Fig. 2B); the OD value of BBR combined with LEV decreased by 98% (Fig. 2C); the OD value of BBR combined with AMI decreased most obviously, with a OD value almost to zero. Its bacteriostatic effect was better than that of GEN and LEV group, achieving bactericidal effect ( Figure 2D)

BBR increased membrane permeability of MRSA
In bacteria, cell membrane is a selective barrier. Bacterial cell membranes protect bacteria from harmful compounds such as drugs, toxins, detergents and degrading enzymes, and allow nutrients to penetrate to promote bacterial growth [22].The integrity of cell membrane will affect the life activity of bacteria. In this study, the results showed that the conductivity of bacterial solution increased rapidly after BBR treatment.
Therefore, BBR can increase the permeability of bacterial cell membrane and induce cytoplasmic leakage in a short time. MRSA02 strain, which was resistant to 3 antibiotics, was selected to observe the changes in conductivity of the culture medium after BBR treatment. The effect of BBR solution on the conductivity of MRSA02 medium is shown in  3A). When 512 µg/mL of BBR was applied to the MRSA02 for 0.5 h, the conductivity of the culture medium was almost unchanged (Fig. 3B). After 64 µg/mL of BBR was applied to the MRSA02, the conductivity increased significantly and increased with time within a 3.5 hours period (Fig. 3C). After 8 µg/mL of BBR was applied to the MRSA02, the conductivity also increased significantly and increased with time within a 3.5 hours period (Fig. 3D).

Effect of BBR on cell wall of MRSA
Among the 26 clinical MRSA strains, the MRSA02 strain, which was resistant to 3 antibiotics, was selected to observe cell wall damage after BBR treatment. The cell wall of the MRSA02 strain was damaged to varying degrees after being cultured in low (8 µg/mL; 1/8 MIC), medium (64 µg/mL; 1 MIC) and high (512 µg/mL; 8 MIC) BBR solutions (Fig. 4A-F). Low concentration (8 µg/mL) BBR did not result in any clear damage to the MRSA02 strain. The MRSA02 strain kept an intact structure, with BBR crystals around the cell wall ( Fig. 4A,4B). A medium concentration (64 µg/mL) of BBR thinned the cell wall of the MRSA02 strain, with BBR crystals adhering to the cell wall of the MRSA strain (Fig. 4C,4D).
A high concentration (512 µg/mL) of BBR destroyed the cell wall structure of MRSA02 strain, and the contents of the MRSA02 strain leaked out, leading to bacterial lysis and death (Fig. 4E,4F).

Analysis of RNA-seq results
RNA sequencing was performed after USA300 was exposed to BBR of high concentration (64 ug/mL) and low concentration (16 ug/mL) for 3 hours. Sequencing results showed that there were 754 differentially expressed genes in the high concentration group compared with the normal control group, of which 561 genes were up-regulated and 193 genes were down-regulated. Compared with the control group, only 19 genes were differentially expressed in the low concentration group, of which 11 genes were up-regulated and 8 genes were down-regulated. Compared with the low concentration group, there were 590 differentially expressed genes in the high concentration group, of which 402 genes were up-regulated and 188 genes were down-regulated (Fig. 5A,5B,6A-C). Among them, ssaA and lytM were significantly up-regulated. The down-regulated genes were mainly serine protease family genes ( Table 4). There are 14 overlaps in the differential regulation genes of USA300 at high and low concentrations, among which 5 are up-regulated overlaps, including speG, phage integrase family protein, scpB and some proteins with unknown functions, while 6 are down-regulated overlaps, including putative membrane protein, staphylococcal accessory regulator R, choline dehydrogenase and some proteins with unknown functions. How BBR regulates the function of these proteins will be further explored. The expression of differentially expressed genes in reference strain USA300 was significantly increased by high concentration BBR, while the expression of differentially expressed genes in low concentration BBR group was not significantly changed.  [23]. Related studies confirmed that BBR has obvious bacteriostatic effect on MRSA, with a MIC range of 32-128 µg/mL [1,24]. Similarly, our study found that BBR had an obvious bacteriostatic effect on 26 different MRSA strains in vitro, with a MIC range of 32-256 µg/mL. The concentration-killing curve ( Fig.2A) demonstrated that the bacteriostatic effect gradually enhanced as the concentration of BBR increased. Concentration of 128 µg/mL BBR inhibited 90% of MRSA growth.

Discussion
Currently, antibiotic resistance is becoming more and more serious. The treatment of infections with single antibiotics has faced many obstacles. Antibiotic combination therapy is an attractive option for preventing the emergence of resistance and overcoming resistance to single antibiotics [25]. In this study, the results of BBR combined with antibiotics showed that BBR combined with AMI had the best bacteriostatic effect on MRSA 14 and almost had a bactericidal effect. AMI is a kind of aminoglycoside antibiotics and is mainly used for Gram-negative bacilli infection treatment. The bacteriostatic mechanism is that the antibacterial drugs enter the bacteria through the cell wall of Gram-negative bacilli, irreversibly binding to the 30S ribosomal subunit. This results in inhibition of the synthesis of bacterial proteins thus achieving a bacteriostatic effect. However, MRSA are Gram-positive cocci with thicker cell walls. How does AMI enter MRSA through the cell wall? Why does BBR combined with AMI enhance the bacteriostatic effect of AMI on MRSA?
How does BBR play a role in this process? For these questions, we performed conductivity tests and TEM examination, to further investigate the possible bacteriostatic mechanism of BBR.
By conductivity tests and TEM examination, we found that low concentration BBR just changed the permeability of the cell wall of MRSA, resulting in increased release of small molecules from the bacteria while maintaining bacterial cell integrity, while high concentration (512ug/mL) BBR rapidly and directly destroyed the structure of cell wall, resulting in the dissolution of the cell wall and bacterial death. MRSA is a Gram-positive coccus, its cell wall is mainly composed of peptidoglycan and teichoic acid. Cell wall inhibitors such as β-lactam and vancomycin are beneficial to bacterial uptake of AMI [26,27]. Our study further confirmed that BBR can inhibit the synthesis of MRSA cell wall. High concentration BBR destroyed the structure of the MRSA cell wall, resulting in thinning or even lysis of the bacterial cell wall, enabling AMI to penetrate the cell wall more easily, with consequent action on the synthesis of DNA in bacteria. This resulted in further inhibition of synthesis of bacterial protein, leading to bacterial death.
In this study, we found that the expression of ssaA and lytM genes in the up-regulated genes of high concentration group was significantly different. According to the literature, these genes were all related to cell wall hydrolysis [28]. SsaA and lytM have potential WalKR binding sites. WalKR system directly regulates the hydrolysis of bacterial cell wall [29]. It is speculated that high concentration of BBR increases the binding sites of ssaA and lytM to WalKR, and enhances the ability of WalKR system to hydrolyze bacterial cell wall, thus causing bacterial cell wall lysis and bacterial death. In the high concentration group, the expression of splB~splF gene of serine protease family was significantly down-regulated. The serine protease family plays a hydrolytic role in protein metabolism, breaking the peptide bonds of macromolecules and forming small-molecule propeptides [30], which are important component of bacterial cell wall [31]. Lack of smallmolecule propeptides would affect the synthesis of bacterial cell wall, resulting in thinning and dissolution of bacterial cell wall. High concentration BBR increased the expression of ssaA and lytM genes in USA300 and down-regulated serine protease family genes, thus enhancing the damage of BBR to bacterial cell wall. However, the expression of ssaA, lytM and serine protease family genes in USA300 was not changed by low concentration of BBR.
These results further validated the results of TEM and conductivity: high concentration of BBR induced cell wall lysis and change of cell wall permeability. The regulation mechanism of BBR on ssaA, lytM and serine protease genes will be further explored in our future study.
This study found that BBR had an excellent bacteriostatic effect on MRSA. The combination of BBR and aminoglycoside antibiotics significantly reduced the resistance of aminoglycoside antibiotics. In addition, BBR enhanced antibiotic activity by inhibiting the synthesis of MRSA cell wall. We will study further on the bacteriostatic mechanism of BBR against bacteria.

Ethical approval and consent to participate
This study was performed in accordance with the ethical standards detailed in the Declaration of Helsinki. The authors' institutional ethics committee has approved this study and all patients have provided written informed consent.

Consent to publish
Publication has been approved by all authors and the responsible authorities at the institution where the work is carried out. The authors confirm that the work described has not been published before and it is not under consideration for publication elsewhere.