Molecules 2014, 19, 6583-6596; doi:10.3390/molecules19056583 OPEN ACCESS
molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article
Berberine Enhances the Antibacterial Activity of Selected Antibiotics against Coagulase-Negative Staphylococcus Strains in Vitro Robert D. Wojtyczka 1,*, Arkadiusz Dziedzic 2, Małgorzata Kępa 1, Robert Kubina 3, Agata Kabała-Dzik 3, Tomasz Mularz 1 and Danuta Idzik 1 1
2
3
Department and Institute of Microbiology and Virology, School of Pharmacy and Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Katowice, ul. Jagiellońska 4, 41-200 Sosnowiec, Poland; E-Mails: [email protected] (M.K.); [email protected] (T.M.); [email protected] (D.I.) Department of Conservative Dentistry with Endodontics, Medical University of Silesia, Katowice, Pl. Akademicki 17, 41-902 Bytom, Poland; E-Mail: [email protected] Department and Institute of Pathology, School of Pharmacy and Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Katowice, ul. Ostrogórska 30, 41-200 Sosnowiec, Poland; E-Mails: [email protected] (R.K.); [email protected] (A.K.-D.)
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +48-32-364-16-21. Received: 31 March 2014; in revised form: 18 May 2014 / Accepted: 19 May 2014 / Published: 22 May 2014
Abstract: Synergistic interactions between commonly used antibiotics and natural bioactive compounds may exhibit therapeutic benefits in a clinical setting. Berberine, an isoquinoline-type alkaloid isolated from many kinds of medicinal plants, has proven efficacy against a broad spectrum of microorganisms. The aim of the presented work was to assess the antibacterial activity of berberine chloride in light of the effect exerted by common antibiotics on fourteen reference strains of Staphylococccus spp., and to evaluate the magnitude of interactions of berberine with these antistaphylococcal antibiotics. In our study minimum inhibitory concentrations (MIC) of berberine chloride against CoNS ranged from 16 to 512 µg/mL. The most noticeable effects were observed for S. haemolyticus ATCC 29970, S. epidermidis ATCC 12228, S. capitis subsp. capitis ATCC 35661, S. galinarium ATCC 700401, S. hominis subsp. hominis ATCC 27844, S. intermedius ATCC 29663 and S. lugdunensis ATCC 49576. The most significant synergistic effect was noticed for berberine in combination with linezolid, cefoxitin and erythromycin. The
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synergy between berberine and antibiotics demonstrates the potential application of compound combinations as an efficient, novel therapeutic tool for antibiotic-resistant bacterial infections. Keywords: antimicrobial activity; coagulase-negative staphylococci (CoNS); berberine
1. Introduction The widespread abuse of antibiotics for the treatment of bacterial infections has led to the emergence and spread of drug resistant strains. The rise in the rate of infections from multi-drug resistant (MDR) bacteria is recognized worldwide as a major health crisis. A recent joint technical report of the European Centre for Disease Prevention and Control (ECDC) and the European Medicines Agency (EMA) in collaboration with Action on Antibiotic Resistance (ReAct) estimated that at least 25,000 patients die each year in the EU from infections due to multidrug resistant bacteria [1]. Antibiotic resistant staphylococci strains are the major public health concern since the bacteria can circulate in the environment without difficulty. Coagulase-negative staphylococci (CoNS) colonize different parts of the human skin and mucous membranes. Notably, every species of CoNS that has been characterized as a resident in humans (S. epidermidis, S. capitis, S. cohnii, S. haemolyticus, S. hominis, S. lugdunensis, S. saccharolyticus, S. saprophyticus, S.warneri), may also be responsible for nosocomial infections, particularly in immunocompromised patients. What is more, CoNS have become the leading cause of infections related to medical devices such as vascular catheters, prosthetic joints and artificial heart valves [2,3]. Plants are known to produce a variety of compounds as defenses against a wide range of microorganisms. Berberine is an isoquinoline-type alkaloid isolated from many kinds of medicinal plants such as Berberis aristata, Berberis aquifolium, Berberis vulgaris, Coptis chinensis, Coptis japonica, Hydrastis canadensis, Phellodendron amurense, Phellodendron chinense schneid and other species [4–6]. Berberine has antidiabetic, antidiarrhoeal, antimicrobial, immuno-stimulating, hypotensive and anti-inflammatory properties [4–8]. To evaluate its antibacterial activity, most studies have focused on the bacteriostatic and/or bactericidal activities of berberine toward different bacterial species [9,10]. It has been reported that berberine has weak activity against Gram-negative bacteria and is more active against Gram-positive bacteria including S. aureus and S. epidermidis [7]. Moreover, the toxicity and mutagenicity of berberine to human cells were relatively low in both in vitro and in vivo experiments. [8,11,12]. To our knowledge, isoquinoline-type alkaloids have not yet been investigated in the context of their possible synergistic effects with commonly used antibiotics against CoNS strains. It has been shown that pharmacological treatment by some phytochemicals is an inexpensive, readily applicable approach in the chemotherapy and management of various infections [5,6,8]. Due to the multi-drug resistance problem the use of combinations of antibiotics with the different mechanisms of action is often necessary for the treatment of severe staphylococcal infections. The augmented action of antibiotics along with natural substances may have positive synergistic effects toward specific, drug resistant microorganisms which are difficult to eradicate, particularly in hospital settings.
Molecules 2014, 19
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In this paper, we explored in vitro antimicrobial activity of berberine in combination with 10 different antibiotics (penicillin—P, erythromycin—E, clindamycin—DA, cefoxitin—FOX, ciprofloxacin—CIP, tobramycin—TOB, chloramphenicol—C, linezolid—LIN, tetracycline—TE, trimethoprim with sulfamethoxazole—SXT) against 14 reference strains of Staphylococccus spp. using minimum inhibitory concentration (MIC) and time-kill assays. All these antibiotics have been reported as antistaphylococcal drugs with different target points. 2. Results and Discussion 2.1. Activity of the Berberine The antibacterial activity of berberine against the tested CoNS strains varied, with MIC values that ranged from 16 to 512 µg/mL (median 126 µg/mL, Table 1). The highest MIC values were 16 µg/mL for S. capitis subsp. capitis and 32 µg/ mL for S. epidermidis ATCC 12228. In case of 9 examined strains MIC values were within the range from 64 µg/mL to 128 µg/mL. The lowest MIC values were observed for S. warneri ATCC 49454 strain (512 µg/mL), S. saprophyticus ATCC 15303 (512 µg/mL) and S. haemolyticus ATCC 29970 strain (256 µg/mL). Comparison of non-biofilm forming strain of S. epidermidis (ATCC 12228) and biofilm-forming strain (ATCC 35983), showed that the first one mentioned strain was four times less susceptible to berberine, with MIC values obtained as 32 µg/mL and 128 µg/mL respectively. The berberine used in this study significantly inhibited the growth of all examined bacterial strains. Table 1. MIC (expressed in µg/mL) of berberine against fourteen CoNS strains. Bacterial strain S. epidermidis ATCC 12228 S. epidermidis ATCC 35983 S. haemolyticus ATCC 29970 S. hominis subsp. hominis ATCC 27844 S. warneri ATCC 49454 S. saprophyticus ATTC 15303 S. capitis subsp. capitis ATCC 35661 S. intermedius ATCC29663 S. lentus ATCC 700403 S. lugdunensis ATCC 49576 S. simulans ATCC 27851 S. galinarium ATCC 700401 S. sciuri ATCC 29060 S. xylosus ATCC 700404
MIC (µg/mL) 32 128 256 64 512 512 16 64 64 64 128 128 128 128
2.2. Time-Kill Assay After 2 h of incubation no growth of the tested strains was detected, both with and without berberine addition to the medium (Figure 1B). There were no differences between culture growth when comparing time-kill curves for different experiment starting points (Figure 1A). After 6 h of incubation we noted for all CoNS strains a substantial decrease of the number of microorganisms (evidenced by OD value changes) when compared to growth control (GC, Figure 1C). In the 12th h of the study
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(Figure 1D) we observed an essential decrease of the number of microorganisms related to the berberine concentration within the range from 8 µg/mL to 512 µg/m. After 24 h of experiment (Figure 1E) within the range of berberine concentrations from 32 µg/mL to 512 µg/mL, a total growth inhibition was recorded for some strains, and no change of OD values was observed. The data obtained for S. warneri ATCC49454 strain showed that for all tested berberine concentrations, only small reduction of bacteria growth was observed, which may indicate an obvious strain resistance to berberine. In the case of the biofilm-forming strain S. epidermidis ATCC35983, a significant reduction of microorganism growth was observed for the first 12 h, but after 24 h of incubation we detected a subsequent increase of the OD value apparently related to the biofilm formation phenomenon. However, even for this non-biofilm forming strain a reduction of the number of microorganisms, expressed as an OD change, was noticeable. Figure 1. Growth kinetics of Staphylococcus strains in the presence of different berberine concentrations after 0 h of incubation (A); after 2 h of incubation (B); after 6 h of incubation (C); after 12 h of incubation (D) and after 24 h of incubation (E).
Molecules 2014, 19
6587 Figure 1. Cont.
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The ANOVA indicated that the growth kinetics of all staphylococcal strains was significantly affected by berberine incubation time (p < 0.001), type of strains (p < 0.001), interaction between strain and incubation time and concentration. The interaction between these factors was also significant (p < 0.001). The incubation time effect (68.35%), type of strain (7.43%), interaction between type of strain and incubation time (6.93%) and concentration (6.03%) explained most of variance (Table 2). Table 2. Multivariate analysis of variance by three-way ANOVA of CoNS susceptibility to berberine. Factors
df
strain (S) time (T) concentration (C) SxT SxC TxC SxTxC
13 4 11 52 143 44 572
Sum of Squares 26.8035 246.5799 21.7349 24.9890 8.8737 17.4305 13.4642
Mean Squares 2.0618 61.6450 1.9759 0.4806 0.0621 0.3961 0.0235
F 6016 179878 5766 1402 181 1156 69
% of Variance 7.43 68.35 6.03 6.93 2.46 4.83 3.73
p
molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article
Berberine Enhances the Antibacterial Activity of Selected Antibiotics against Coagulase-Negative Staphylococcus Strains in Vitro Robert D. Wojtyczka 1,*, Arkadiusz Dziedzic 2, Małgorzata Kępa 1, Robert Kubina 3, Agata Kabała-Dzik 3, Tomasz Mularz 1 and Danuta Idzik 1 1
2
3
Department and Institute of Microbiology and Virology, School of Pharmacy and Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Katowice, ul. Jagiellońska 4, 41-200 Sosnowiec, Poland; E-Mails: [email protected] (M.K.); [email protected] (T.M.); [email protected] (D.I.) Department of Conservative Dentistry with Endodontics, Medical University of Silesia, Katowice, Pl. Akademicki 17, 41-902 Bytom, Poland; E-Mail: [email protected] Department and Institute of Pathology, School of Pharmacy and Division of Laboratory Medicine in Sosnowiec, Medical University of Silesia, Katowice, ul. Ostrogórska 30, 41-200 Sosnowiec, Poland; E-Mails: [email protected] (R.K.); [email protected] (A.K.-D.)
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +48-32-364-16-21. Received: 31 March 2014; in revised form: 18 May 2014 / Accepted: 19 May 2014 / Published: 22 May 2014
Abstract: Synergistic interactions between commonly used antibiotics and natural bioactive compounds may exhibit therapeutic benefits in a clinical setting. Berberine, an isoquinoline-type alkaloid isolated from many kinds of medicinal plants, has proven efficacy against a broad spectrum of microorganisms. The aim of the presented work was to assess the antibacterial activity of berberine chloride in light of the effect exerted by common antibiotics on fourteen reference strains of Staphylococccus spp., and to evaluate the magnitude of interactions of berberine with these antistaphylococcal antibiotics. In our study minimum inhibitory concentrations (MIC) of berberine chloride against CoNS ranged from 16 to 512 µg/mL. The most noticeable effects were observed for S. haemolyticus ATCC 29970, S. epidermidis ATCC 12228, S. capitis subsp. capitis ATCC 35661, S. galinarium ATCC 700401, S. hominis subsp. hominis ATCC 27844, S. intermedius ATCC 29663 and S. lugdunensis ATCC 49576. The most significant synergistic effect was noticed for berberine in combination with linezolid, cefoxitin and erythromycin. The
Molecules 2014, 19
6584
synergy between berberine and antibiotics demonstrates the potential application of compound combinations as an efficient, novel therapeutic tool for antibiotic-resistant bacterial infections. Keywords: antimicrobial activity; coagulase-negative staphylococci (CoNS); berberine
1. Introduction The widespread abuse of antibiotics for the treatment of bacterial infections has led to the emergence and spread of drug resistant strains. The rise in the rate of infections from multi-drug resistant (MDR) bacteria is recognized worldwide as a major health crisis. A recent joint technical report of the European Centre for Disease Prevention and Control (ECDC) and the European Medicines Agency (EMA) in collaboration with Action on Antibiotic Resistance (ReAct) estimated that at least 25,000 patients die each year in the EU from infections due to multidrug resistant bacteria [1]. Antibiotic resistant staphylococci strains are the major public health concern since the bacteria can circulate in the environment without difficulty. Coagulase-negative staphylococci (CoNS) colonize different parts of the human skin and mucous membranes. Notably, every species of CoNS that has been characterized as a resident in humans (S. epidermidis, S. capitis, S. cohnii, S. haemolyticus, S. hominis, S. lugdunensis, S. saccharolyticus, S. saprophyticus, S.warneri), may also be responsible for nosocomial infections, particularly in immunocompromised patients. What is more, CoNS have become the leading cause of infections related to medical devices such as vascular catheters, prosthetic joints and artificial heart valves [2,3]. Plants are known to produce a variety of compounds as defenses against a wide range of microorganisms. Berberine is an isoquinoline-type alkaloid isolated from many kinds of medicinal plants such as Berberis aristata, Berberis aquifolium, Berberis vulgaris, Coptis chinensis, Coptis japonica, Hydrastis canadensis, Phellodendron amurense, Phellodendron chinense schneid and other species [4–6]. Berberine has antidiabetic, antidiarrhoeal, antimicrobial, immuno-stimulating, hypotensive and anti-inflammatory properties [4–8]. To evaluate its antibacterial activity, most studies have focused on the bacteriostatic and/or bactericidal activities of berberine toward different bacterial species [9,10]. It has been reported that berberine has weak activity against Gram-negative bacteria and is more active against Gram-positive bacteria including S. aureus and S. epidermidis [7]. Moreover, the toxicity and mutagenicity of berberine to human cells were relatively low in both in vitro and in vivo experiments. [8,11,12]. To our knowledge, isoquinoline-type alkaloids have not yet been investigated in the context of their possible synergistic effects with commonly used antibiotics against CoNS strains. It has been shown that pharmacological treatment by some phytochemicals is an inexpensive, readily applicable approach in the chemotherapy and management of various infections [5,6,8]. Due to the multi-drug resistance problem the use of combinations of antibiotics with the different mechanisms of action is often necessary for the treatment of severe staphylococcal infections. The augmented action of antibiotics along with natural substances may have positive synergistic effects toward specific, drug resistant microorganisms which are difficult to eradicate, particularly in hospital settings.
Molecules 2014, 19
6585
In this paper, we explored in vitro antimicrobial activity of berberine in combination with 10 different antibiotics (penicillin—P, erythromycin—E, clindamycin—DA, cefoxitin—FOX, ciprofloxacin—CIP, tobramycin—TOB, chloramphenicol—C, linezolid—LIN, tetracycline—TE, trimethoprim with sulfamethoxazole—SXT) against 14 reference strains of Staphylococccus spp. using minimum inhibitory concentration (MIC) and time-kill assays. All these antibiotics have been reported as antistaphylococcal drugs with different target points. 2. Results and Discussion 2.1. Activity of the Berberine The antibacterial activity of berberine against the tested CoNS strains varied, with MIC values that ranged from 16 to 512 µg/mL (median 126 µg/mL, Table 1). The highest MIC values were 16 µg/mL for S. capitis subsp. capitis and 32 µg/ mL for S. epidermidis ATCC 12228. In case of 9 examined strains MIC values were within the range from 64 µg/mL to 128 µg/mL. The lowest MIC values were observed for S. warneri ATCC 49454 strain (512 µg/mL), S. saprophyticus ATCC 15303 (512 µg/mL) and S. haemolyticus ATCC 29970 strain (256 µg/mL). Comparison of non-biofilm forming strain of S. epidermidis (ATCC 12228) and biofilm-forming strain (ATCC 35983), showed that the first one mentioned strain was four times less susceptible to berberine, with MIC values obtained as 32 µg/mL and 128 µg/mL respectively. The berberine used in this study significantly inhibited the growth of all examined bacterial strains. Table 1. MIC (expressed in µg/mL) of berberine against fourteen CoNS strains. Bacterial strain S. epidermidis ATCC 12228 S. epidermidis ATCC 35983 S. haemolyticus ATCC 29970 S. hominis subsp. hominis ATCC 27844 S. warneri ATCC 49454 S. saprophyticus ATTC 15303 S. capitis subsp. capitis ATCC 35661 S. intermedius ATCC29663 S. lentus ATCC 700403 S. lugdunensis ATCC 49576 S. simulans ATCC 27851 S. galinarium ATCC 700401 S. sciuri ATCC 29060 S. xylosus ATCC 700404
MIC (µg/mL) 32 128 256 64 512 512 16 64 64 64 128 128 128 128
2.2. Time-Kill Assay After 2 h of incubation no growth of the tested strains was detected, both with and without berberine addition to the medium (Figure 1B). There were no differences between culture growth when comparing time-kill curves for different experiment starting points (Figure 1A). After 6 h of incubation we noted for all CoNS strains a substantial decrease of the number of microorganisms (evidenced by OD value changes) when compared to growth control (GC, Figure 1C). In the 12th h of the study
Molecules 2014, 19
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(Figure 1D) we observed an essential decrease of the number of microorganisms related to the berberine concentration within the range from 8 µg/mL to 512 µg/m. After 24 h of experiment (Figure 1E) within the range of berberine concentrations from 32 µg/mL to 512 µg/mL, a total growth inhibition was recorded for some strains, and no change of OD values was observed. The data obtained for S. warneri ATCC49454 strain showed that for all tested berberine concentrations, only small reduction of bacteria growth was observed, which may indicate an obvious strain resistance to berberine. In the case of the biofilm-forming strain S. epidermidis ATCC35983, a significant reduction of microorganism growth was observed for the first 12 h, but after 24 h of incubation we detected a subsequent increase of the OD value apparently related to the biofilm formation phenomenon. However, even for this non-biofilm forming strain a reduction of the number of microorganisms, expressed as an OD change, was noticeable. Figure 1. Growth kinetics of Staphylococcus strains in the presence of different berberine concentrations after 0 h of incubation (A); after 2 h of incubation (B); after 6 h of incubation (C); after 12 h of incubation (D) and after 24 h of incubation (E).
Molecules 2014, 19
6587 Figure 1. Cont.
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The ANOVA indicated that the growth kinetics of all staphylococcal strains was significantly affected by berberine incubation time (p < 0.001), type of strains (p < 0.001), interaction between strain and incubation time and concentration. The interaction between these factors was also significant (p < 0.001). The incubation time effect (68.35%), type of strain (7.43%), interaction between type of strain and incubation time (6.93%) and concentration (6.03%) explained most of variance (Table 2). Table 2. Multivariate analysis of variance by three-way ANOVA of CoNS susceptibility to berberine. Factors
df
strain (S) time (T) concentration (C) SxT SxC TxC SxTxC
13 4 11 52 143 44 572
Sum of Squares 26.8035 246.5799 21.7349 24.9890 8.8737 17.4305 13.4642
Mean Squares 2.0618 61.6450 1.9759 0.4806 0.0621 0.3961 0.0235
F 6016 179878 5766 1402 181 1156 69
% of Variance 7.43 68.35 6.03 6.93 2.46 4.83 3.73
p
