Effect of Coptis chinensis on Biofilm Formation and Antibiotic Susceptibility in Mycobacterium abscessus

Mycobacterium abscessus infections are notoriously difficult to be treated and newer treatment options are required. Coptis chinensis (C. chinensis) and its main compound berberine are frequently used to treat bacterial and viral infections. In this study, the susceptibility of M. abscessus to C. chinensis extract and berberine was assessed by minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) evaluation. The effects of C. chinensis and berberine on biofilm formation and antibiotic susceptibility in M. abscessus were observed. C. chinensis at concentrations of MIC (1.5 mg/mL) and 2 × MIC (3.0 mg/mL) and berberine at ½ × MIC (0.125 mg/mL) demonstrated a strong inhibition of biofilm formation. Concentration of C. chinensis at ½ × MIC resulted in a significant reduction in MICs of trimethoprim/sulfamethoxazole (TMP/SXT), clarithromycin (CLA), and linezolid (LZD). Similarly, ½ × MIC berberine had a significant effect on the MIC reductions of nine antibiotics including TMP/SXT, CLA, and LZD. Notably, the resistance level MIC of LZD against M. abscessus was reversed to a susceptible level by treatment with either C. chinensis or berberine. Therefore, C. chinensis and berberine have the potential to produce a synergistic antimycobacterial effect, reduce biofilm formation, and decrease antibacterial resistance to LZD in M. abscessus.

AIDS patients and transplant recipients, is growing worldwide [2,[9][10][11]. e number of NTM disease-related deaths is also rising, with higher mortality rates seen among adults over the age of 55 years and those with chronic obstructive pulmonary disease, bronchiectasis, HIV, interstitial lung diseases, and/or tobacco use [12].
While there is a wide range of treatment strategies available with multidrug antibiotic therapy being the current treatment of choice [13,14], the results of antibiotic treatment are often discouraging [14]. Patients require a prolonged antibiotic course, which often results in side effects, and/or acquired antibiotic resistance. Suggested treatment is antibiotic therapy lasting 9-18 months, or even up to 24 months [15]. Interestingly, M. abscessus infections are widely seen hard to be treated despite the use of antibiotics. In a study of antibiotic therapy for pulmonary M. abscessus infections over a median duration of 12 months (range 9-18 months) by Novosad et al., 62% of patients experienced side effects that resulted in the need to adjust the therapeutic regimen [14]. Adjuvant or alternative therapies may be required, and a response to antibiotic side effects is needed. Adding Chinese herbal medicine (CHM) to antibiotic treatments may provide an effective solution for treating antibiotic side effects.
CHM has been used to treat infectious diseases since ancient times in China. Over thousands of years, many mechanisms have been proposed and countless reports of CHM antimicrobial therapy have been published in scientific publications and textbooks, such as the classic Chinese medical monographs "Treatise on Febrile Diseases (Sh� ang Hán Lùn)" and "Systematic Differentiation of Warm Diseases (W� en Bìng Tiáo Biàn)." In recent decades, a large number of studies have reported on the anti-inflammatory and antimicrobial effects of heat-clearing Chinese herbs (HCCHs) [16][17][18]. Currently, HCCHs and CHM formulations are frequently used as monotherapy or combined therapy components to treat common infectious diseases such as pulmonary tuberculosis, methicillin-resistant Staphylococcus aureus (MRSA) infection, and sepsis [19][20][21]. CHM formulations with bacteriostatic activity may be useful in treating M. abscessus infections.
Coptis chinensis (C. chinensis; Huang Lian) is a traditional CHM frequently used to treat bacterial and viral infections. It has proven efficacy against many microorganisms including Staphylococcus spp. [18]. In 2006, Lin et al. found that Coptidis Rhizoma (the dried rhizome of C. chinensis Franch) provided 100% in vitro inhibitory effects on S. aureus and Escherichia coli [22]. Of note, C. chinensis was reported to have an effect comparable to that of the commercial antibiotic vancomycin against coagulase-negative Staphylococcus [23]. Recently, C. chinensis was found to produce significant antimicrobial activity against a variety of microorganisms including Gram-positive and Gram-negative bacteria [16]. Over 120 chemical components have been isolated from C. chinensis, with alkaloids as the main active ingredients and berberine as the most representative component [24]. Berberine's broadspectrum antibacterial effect is similar to that of C. chinensis, with effects manifesting proportional to concentrations. Higher concentrations of berberine are reported to kill bacteria more quickly [25]. In this era of increasing antibiotic resistance, the potential benefits of using C. chinensis and berberine in the treatment of M. abscessus infections warrant investigation. e aims of this study were to examine in vitro the antibacterial effect of C. chinensis and berberine (its major constituent) in treating M. abscessus infections. e researchers studied the potential of this herb to inhibit biofilm formation and amplify the activity of antibiotics in managing M. abscessus infections.

Materials and Methods
For this study, the M. abscessus strain, which was purchased from the College of American Pathologists for qualification of Medical Technologist, from the stock of Medical Laboratory of the Tamshui Branch of Mackay Memorial Hospital in Taiwan, was chosen to represent rapidly growing mycobacterium (RGM). An incubation broth (7H9) and commercially available antimycobacterial susceptibility test kit (Sensititre TM RAPMYCOI, Trek Sensititre, ermoFisher Scientific) were used to test the susceptibility of the RGM to a panel of 15 antibiotics which is recommended by the Clinical and Laboratory Standards Institute (CLSI) [26]. For the purpose of comparing the actual inhibitory activities of pretreatment (antibiotic alone) and combination treatment (antibiotic plus C. chinensis or berberine), the Sensititre TM RAPMYCOI was further used to test the synergistic antibacterial effects of C. chinensis and berberine. Based on our review of Chinese Medicine books, research reports, and discussions with traditional Chinese medicine physicians, we selected the CHM C. chinensis for this study, an herb containing 6.19% berberine. In order to overcome the possible issues, such as sample preparation or different batch of raw material, and to make the results more reliable, a pharmacy type of concentrated C. chinensis paste which was approved for clinical use by the Ministry of Health and Welfare, Taiwan (provided by Kaiser Pharmaceutical Co., Ltd; product: 8010) was used. e herbal materials provided by Kaiser Pharmaceutical conformed to the standards of Taiwan Herbal Pharmacopeia 2. e procedures complied with the drugs registration license approved by the Taiwan Food and Drug Administration, in which the extraction of the raw material is performed exclusively using water. Additionally, the concentrations of commercially available berberine and berberine-HCl obtained from the products of C. chinensis were tested by Kaiser Pharmaceutical by HPLC following the protocol listed in Taiwan Herbal Pharmacopeia 2. e antimicrobial activities of commercially available berberine and berberine-HCl were identical as examined with its antibacterial activities against M. abscessus. us, the erstwhile described berberine and berberine-HCl were purchased to enable a detailed analysis of its potential to enhance antibacterial effects (ISO 9001: 2015 certification; http://www.chemfaces.com).
2.1. M. abscessus Susceptibility to C. chinensis and Berberine. C. chinensis was dissolved in 7H9 broth and then serially diluted twofold from 12.0 mg/mL for testing concentration from 12.0 mg/mL to 0.375 mg/mL. e antibacterial effects of berberine against M. abscessus were determined in the same manner, from 2.0 mg/mL to 0.0625 mg/mL. e antibacterial activity of C. chinensis and berberine was evaluated by counting the colony forming unit (CFU) of M. abscessus found after their administration. As the protocol of TREK Diagnostic Systems, M. abscessus suspension (optical OD 600 ∼0.2) was then added to the indicated concentrations of C. chinensis and berberine-HCl to observe the bacterial growth at 30°C for 5 days [27]. Relative percentage of survival rate was expressed as CFU of viable bacteria present after treatment with the indicated concentrations of C. chinensis or berberine-HCl relative to the number of viable bacteria present at untreatment and then multiplied by 100.

Effects of C. chinensis and Berberine on the Biofilm Formation of M. abscessus.
To evaluate the prevention effect before biofilm development, serial indicated concentrations of C. chinensis and berberine-HCl were individually added to the glass test tubes in the same time of preparation of M. abscessus solution to calculate optical density differences. Optical densities before and after the addition of C. chinensis and berberine-HCl were measured and compared.
Biofilm formation was assessed by the ability of cells to adhere to the glass tubes, with some modification of the reported protocol [28]. For testing, the concentration of C. chinensis ranging between 3 and 0.375 mg/mL, and the concentration of berberine ranged between 0.5 and 0.0625 mg/mL. e glass test tube contained a 1 : 10 diluted aliquot of M. abscessus overnight culture, which was incubated in 7H9 broth with different concentrations of C. chinensis and berberine-HCl at 30°C for 14 days. After 14 days, nonadherent M. abscessus was triple washed with 3 mL PBS, and the adherent M. abscessus was stained with 3 mL of 0.1% safranin solution at room temperature for 30 min. e glass tube was rinsed twice with deionized water to remove excess stain. Finally, the safranin stained biofilm was solubilized in 1 mL of 33% acetic acid, and then 100 μL of the solution was removed to 96-well PVC plates (TPP 96 flat) for measuring the absorbance at λ � 492 nm.

Statistical Analysis.
e CFU numbers of M. abscessus were individually calculated after the indicated concentrations of C. chinensis and berberine-HCl were added. e results were expressed as relative percentages ± standard deviations (SD). e effects on biofilm formation after C. chinensis and berberine-HCl treatment were also analyzed. e results were expressed as mean scores ± SD. e Mann-Whitney Rank Sum Test was conducted to examine any differences between control and experimental groups with indicated concentrations. e level of statistical significance was set at p < 0.05.

Susceptibility of M. abscessus to C. chinensis and Berberine.
For evaluation of the effects of C. chinensis on the growth of M. abscessus, the number of M. abscessus CFU formed in 7H9 medium with different concentrations of C. chinensis was calculated and compared to the number of M. abscessus colonies formed in 7H9 medium alone (the control group). e relative survival percentages of M. abscessus were 59.95 ± 13.70%, 34.56 ± 6.58%, 15.20 ± 4.74%, and 2.55 ± 0.27% at concentrations of 0.375 mg/mL, 0.75 mg/mL, 1.5 mg/mL, and 3.0 mg/mL, respectively (Figure 1(a)). erefore, the MIC value of C. chinensis was around 1.5 mg/ mL for M. abscessus. Additionally, we observed that the growth of M. abscessus approached zero at C. chinensis concentration of 6.0 mg/mL (0.31 ± 0.34%). is implies that minimal bactericidal concentration (MBC) was 4 times higher than MIC of C. chinensis. e reductions in relative percentage of survival CFU of M. abscessus were statistically significant for 1/4 × MIC, 1/2 × MIC, MIC, 2 × MIC, and 4 × MIC following the addition of C. chinensis. Our findings revealed that C. chinensis has a conspicuous bacteriostatic effect on M. abscessus.
Berberine is the main active component of C. chinensis. To further observe the effects of berberine on M. abscessus growth, the CFU numbers of M. abscessus with different concentrations of berberine-HCl were calculated and compared to the numbers of M. abscessus grown in 7H9 medium alone. e relative survival percentages of M. abscessus were 69.45 ± 9.68%, 31.41 ± 2.50%, 16.27 ± 4.72%, and 0.57 ± 0.62% at berberine-HCl concentrations of 0.0625 mg/mL, 0.125 mg/mL, 0.25 mg/mL, and 0.5 mg/mL, respectively (Figure 1(b)).
e MIC value of berberine-HCl was around 0.25 mg/mL for M. abscessus.

Evidence-Based Complementary and Alternative Medicine
Likewise, nearly no M. abscessus growth was observed with berberine-HCl concentrations in 0.5 mg/mL and in excess of 1.0 mg/mL. e MBC of berberine-HCl was 2-4 times higher than MIC. As determined by the Mann-Whitney Rank Sum Test, the differences in relative percentage of survival CFU of M. abscessus between pretreatment and the addition of 1/4 × MIC, 1/2 × MIC, MIC, and 2 × MIC berberine were statistically significant. Our observations indicate that berberine-HCl also showed an obvious bacteriostatic effect against M. abscessus growth.

Effects of C. chinensis and Berberine on the Biofilm Formation of M. abscessus.
e biofilm formation of M. abscessus on the glass tubes was reviewed and compared. e biofilm of M. abscessus declined after incubation with C. chinensis and berberine-HCl for 14 days. e effects manifested were proportional to the added concentrations. Higher concentrations of C. chinensis and berberine-HCl exhibited poor abilities of adherence. To analyze whether C. chinensis and berberine-HCl affect biofilm formation of M. abscessus, the biofilm was determined at OD 492 by staining with safranin. As shown in Figure 2 ese findings reveal that biofilm formation of M. abscessus could be consistently inhibited using berberine-HCl concentrations in excess of ½ × MIC.

Effects of C. chinensis on the MICs of Antibiotics against M. abscessus.
According to the reference MICs of antibiotics for M. abscessus, this study strain of M. abscessus was primarily resistant to most studied antibiotics except clarithromycin, tigecycline, and amikacin [26,29]. To observe whether C. chinensis would affect the MICs of antibiotics against M. abscessus, combinations of antibiotics and C. chinensis were tested. e MICs of antibiotics alone and antibiotics combined with C. chinensis were then compared.
e addition of C. chinensis in concentrations of ¼ × MIC, ½ × MIC, MIC, and 2 × MIC were tested to measure the effects on MICs of antibiotics. As shown in Table 1, the addition of ¼ × MIC C. chinensis provided a reduction of only MIC in linezolid. C. chinensis concentrations of ½ × MIC and MIC resulted in significant synergistic effects measured for trimethoprim/sulfamethoxazole, clarithromycin, and linezolid. Importantly, the synergistic effect of C. chinensis on these three antibiotics is highly related to the concentration of C. chinensis. Furthermore, the concentration of C. chinensis at MIC could also reduce the MICs * * * * p<0.05 * * p<0.001 * * * * * *   (ii) The C. chinensis solution was removed.
(iii) The adherent M. abscessus was stained with 3mL of 0.1% safranin solution at room temperature for 30min.
(iv) The safranin stained biofilm was measured the differences in λ = 492 nm at plates. (ii) The berberine-HCL solution was removed.
(iii) The adherent M. abscessus was stained with 3 mL of 0.1% safranin solution at room temperature for 30 min.
(iv) The safranin stained biofilm was measured the differences in λ = 492nm at plates. Evidence-Based Complementary and Alternative Medicine 15-antibiotic well plates. As shown in Table 2, berberine-HCl concentration at ¼ × MIC only reduced the MIC of trimethoprim/sulfamethoxazole. Interestingly, we observed reductions of MICs in 9 of the 15 (60%) antibiotics that were tested (trimethoprim/sulfamethoxazole, clarithromycin, linezolid, amikacin, tigecycline, imipenem, cefepime, cefoxitin, and ciprofloxacin) when combined with berberine-HCl at a concentration of ½ × MIC. Similar to the effects of C. chinensis, the synergistic effects of berberine-HCl were more significant with trimethoprim/sulfamethoxazole, clarithromycin, and linezolid. ese observations indicate that berberine-HCl can enhance the antimycobacterial activity of most antibiotics against M. abscessus.

Discussion
e development of novel antimycobacterial drugs is ongoing. e antimicrobial, anti-inflammatory, and synergistic antibacterial effects of CHMs have been demonstrated in several studies [16,20,21,30,31]. Single extracts or formulations of CHM alone, or in combination with antibiotics, may be used to treat common bacterial infections such as MRSA, Ureaplasma urealyticum, and genital Chlamydia trachomatis infections [20,32]. In 2007, Liu et al. reported that 26 of 58 (44.8%) CHMs used in Taiwan exhibited antibacterial activity against antibiotic-resistant Pseudomonas aeruginosa, and one of the studied herbal medicines (Ramulus Cinnamomi) could yield a synergistic effect with antibiotics [30]. In the current study, we demonstrated that C. chinensis and its major constituent, berberine, can inhibit the growth of M. abscessus. e MICs of C. chinensis and berberine-HCl were 1.5 mg/mL and 0.25 mg/mL, respectively. e formation of biofilm is a key mechanism in the pathogenicity and resistance of human NTM infections. Bacteria within a biofilm exhibit higher virulence to human and resistance to antimicrobials and disinfectants. Complete differentiation, achievement of maximal replication, horizontal gene exchanges and mutations, and more adaption to environment were reported [2,33]. In addition, it influences the effectiveness of endoscope and invasive device disinfection by automated processors in hospitals [34,35]. Biofilm formation is usually an important factor in antimicrobial resistance and can lead to treatment failure. In 2011, Lin et al. reported that an active ingredient in commonly used Chinese herbal plants had inhibitory effects on S. aureus biofilm formation [36].
e capability of C. chinensis and berberine to reduce biofilm formation by M. abscessus was proven in this study. Biofilm is a complex multicellular structure that forms on bacterial surfaces. e complexity of mycobacterial biofilm, the synthesis of an extracellular matrix, usually involves various molecules including glycopeptidolipids, DNA such as groEL1 gene, and mycolic acids. e process of biofilm development is also influenced and regulated by nutrients, ions, and carbon sources [37]. Some proteins, such as adhesins, are believed to potentiate the aggregation of mycobacteria and their ability to bind to other cells [38]. Mycolic acids were reported to contribute to the resistance of mycobacteria to antibiotics and disinfectants [39]. e detailed mechanisms of reducing biofilm formation in M. abscessus for C. chinensis and berberine need further investigation. Antimicrobial therapies for M. abscessus infections have been investigated for decades. However, treatment regimens vary in duration, tolerability, and success. Clarithromycin, amikacin, and cefoxitin are the most commonly reported antimycobacterial choices for the treatment of M. abscessus infections [14]. Macrolide-based regimens in combination with intravenously administered antimicrobials are the currently recommended therapy [40]. However, poor responsiveness to antimicrobial chemotherapy, including macrolide-based regimens for M. abscessus infections, remains a challenging problem. Furthermore, side effects such as gastrointestinal upset, skin changes, and renal insufficiency are common and frequently require changing or discontinuing antibiotic therapy [14].
Previous literature has reported that combining antibiotics with CHM can produce better curative effects when treating pulmonary tuberculosis [19]. Treating intractable mycobacteriosis with antibiotics and CHMs that exhibit immunoadjunctive effects has been reported with favorable outcomes [41]. Likewise, the synergistic effects of the herbal plant extract ymbra spicata L. and antibiotics against common, multidrug-resistant S. aureus, and Klebsiella pneumoniae strains were reported in 2016 [31]. Similar antibacterial effects of antibiotics in combination with Chinese herbal remedies for M. abscessus infections can be expected, but currently there are no such reports in the literature.
C. chinensis and berberine-HCl were proven to inhibit M. abscessus growth in a dose-dependent manner. Additionally, both C. chinensis and berberine-HCl significantly strengthened the antimicrobial activities of trimethoprim/ sulfamethoxazole, clarithromycin, and linezolid against M. abscessus. According to measurements taken in this study, the MIC of linezolid was 32 μg/mL, defined as resistant to antimicrobial susceptibility for M. abscessus. e additions of both of these CHMs reduced its MIC to 4 μg/ mL, which is categorized as a susceptible level of linezolid against M. abscessus. Both parenteral and oral linezolid preparations are available for clinical use. e clinical implications of the combination of linezolid and C. chinensis or berberine may be prospective and therefore are recommended for long-term treatment of M. abscessus infections.
Although we found that berberine demonstrated an antimycobacterial effect on M. abscessus, the antibacterial mechanism of berberine on M. abscessus growth remains unknown, although previous studies have provided some useful information. Peng et al. reported that berberine could damage the cell membrane and walls of Streptococcus agalactiae, thereby increasing membrane permeability, and inhibit protein and DNA synthesis in cells [25]. Amin et al. also reported that berberine could inhibit RNA and protein synthesis in E. coli and exhibits antimicrobial effects [42]. e authors suggest that both Gram-positive and Gramnegative bacteria are susceptible to berberine. erefore, whether berberine could inhibit the growth of M. abscessus due to membrane integrity disruption or nucleotide acid or protein synthesis inhibition needs to be investigated further. e antimicrobial activity of trimethoprim/sulfamethoxazole is due to its interference with folate synthesis. e antimicrobial activity of clarithromycin and linezolid is due to their inhibition of protein synthesis by binding to 50S subunits of bacteria [43,44]. erefore, the synergistic effects of both C. chinensis and berberine-HCl in combination with these three antibiotics may be because of their interference with protein and RNA synthesis, as well as the cellular membrane. In addition, combining berberine-HCl with antibiotics could amplify the antimicrobial activities of amikacin and tigecycline through the mechanisms mentioned above. In contrast, the MIC reductions of amikacin, tigecycline, ciprofloxacin, and cefepime, measured when they were combined with 0.125 mg/mL (½ × MIC) berberine-HCl were not observed with C. chinensis. When compared with berberine, the attenuation of antimycobacterial activity in antibiotics is hypothesized to be antagonistic to other C. chinensis components. Moreover, we found that C. chinensis increases the MIC of tigecycline, which acts as a protein synthesis inhibitor by binding with 30S ribosomal subunits of bacteria [45].
e relationship between C. chinensis and the 30S ribosomal protein subunits of bacteria needs to be investigated further. Integrating the pleomorphic effects of C. chinensis and its active component berberine may contribute to our knowledge of pathways and enhance the susceptibility of M. abscessus to multiple antibiotics. erefore, this herb and its main component could be used and tested as an adjunct therapy for M. abscessus infections.

Conclusions
e results of this study demonstrate that combination treatment can enhance the effects of certain antibiotics against M. abscessus. Chinese and Western combination treatments deserve more attention because they can be convenient and safe. e combinations of C. chinensis or berberine with the antibiotics identified in this study may represent a novel strategy for the treatment of patients with M. abscessus infections.
Data Availability e data used and/or analyzed during the current study are available from the authors upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.