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Combination of anti-tuberculosis drugs with vitamin C or NAC against different Staphylococcus aureus and Mycobacterium tuberculosis strains
Microbial Pathogenesis 93 (2016) 83e87
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Combination of anti-tuberculosis drugs with vitamin C or NAC against different Staphylococcus aureus and Mycobacterium tuberculosis strains Bahman Khameneh a, Bibi Sedigheh Fazly Bazzaz b, Alireza Amani c, Javad Rostami c, Nasser Vahdati-Mashhadian d, e, * a
Department of Pharmaceutical Control, Students Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Students Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran d Medical Toxicology Research Center, Mashhad University of Medical Sciences, Mashhad, IR Iran e Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 October 2015 Received in revised form 27 October 2015 Accepted 9 November 2015 Available online 19 November 2015
Backgrounds: Hepatotoxicity due to anti tuberculosis drugs, rifampin and isoniazid, is a major problem in tuberculosis patients. Vitamin C, an antioxidant, and N-acetyl cysteine (NAC), a scavenger of active metabolites, reduce the hepatotoxicity. The aim of present study was to investigate the effect of vitamin C and NAC individually on the antibacterial activity of anti tuberculosis drugs against Mycobacterium tuberculosis and Staphylococcus aureus strains. Methods: The MICs of each compound against all strains were determined in 96 wells plate. Rifampin was tested at serial two fold concentrations alone or in combination with NAC or vitamin C. Results: The MIC of rifampin against different strains of S. aureus was 0.008e0.032 mg/ml. The MIC of rifampin and isoniazid against M. tuberculosis strains were 40 and 0.2 mg/ml, respectively. Vitamin C and NAC had no antibacterial activity against all strains. MIC of rifampin was reduced two fold by combination with vitamin C for all S. aureus strains, while NAC did not affect the antibacterial activity of rifampin. Vitamin C and NAC had remarkable effects on the antibacterial activity of anti-tuberculosis drugs against M. tuberculosis. Conclusions: Synergistic effects were observed between rifampin or isoniazid and vitamin C against all tested strains. However, combination therapy of rifampin and isoniazid with NAC was not being effective. This study highlighted the advantages of combination of anti-tuberculosis drugs and vitamin C to eradicate the microbial infections. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Mycobacterium tuberculosis N-acetyl cysteine Rifampin Isoniazid Staphylococcus aureus Vitamin C
1. Introduction Staphylococcus aureus causes severe life-threatening infections and has become increasingly common, particularly methicillinresistant strains (MRSA) [1,2]. Rifampin is often used as adjunctive therapy to treat S. aureus infections. Staphylococcal infections are among the first non-mycobacterial infections treated with rifampin; however, it was soon discovered in order to prevent the emergence of rifampin-resistant isolates, at least another effective antimicrobial agent was required to be used in combination with
* Corresponding author. Department of Pharmacodynamics and Toxicology, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. E-mail addresses: [email protected], [email protected] (N. VahdatiMashhadian). http://dx.doi.org/10.1016/j.micpath.2015.11.006 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
rifampin [3,4]. Some human studies have addressed the role of adjunctive rifampin therapy. Adjunctive therapy in combination with fusidic acid seems most promising for the treatment of difficult to treat S. aureus infections in osteomyelitis and prosthetic joint infections, although studies were typically underpowered and benefits were not always seen [5]. From 1968 isoniazid and rifampin have been widely used for the treatment of tuberculosis; they kill more than 99% of tubercular bacilli within two months of start of therapy. Unfortunately, rifampin resistance started soon and the urgent need for multi therapy was recognized [6]. At that time and by development and introduction of combination therapy of these agents, drug resistant tuberculosis was overcome. This approach provided a good chance for the treatment as it killed rapidly both dividing and long-lived persistent cells of Mycobacterium tuberculosis. Despite the great
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efficacy of this combination therapy, serious reactions have been reported including hepatotoxicity and nephrotoxicity [7]. Other side effects such as unexplained fevers, cytopenias, and neurological disturbances have also been reported and lead to negatively affect treatment compliance and clinical outcome [8,9]. Even the use of rifampin for patients with underlying hepatitis infection may increase the risk of hepatotoxicity [10]. Towards these observations, combination therapy is a suitable approach to overcome bacterial resistance and these limitations [11,12]. Vitamin C is a potent and standard antioxidant with a satisfactory history of protective actions against oxidative stress by many agents in cell culture [13,14], animal [15,16] and human studies [17]. Evidence suggests that vitamin C may have promising effect as an adjuvant in the treatment of cancer [18,19]. It has shown that vitamin C alone or in combination with other antibacterial agents exhibited promising antibacterial activity against different pathogens such as Helicobacter pylori, S. aureus and also multidrug resistant Pseudomonas aeruginosa [20e22]. This can be explained by two mechanisms: On one hand, by transferring vitamin C into the bacterial cells, it is metabolized in the presence of oxygen and consequently bacteria are exposed to oxidative stress. On the other hand, acetic and lactate acids are generated from vitamin C [20]. N-acetyl cysteine (NAC) is also an antioxidant and free radical scavenger that is used as a standard and effective drug for the treatment of acetaminophen overdose [23]. Its protective effect against organ toxicity due to oxidative stress is also evident based on huge array of in vitro [14,24] and in vivo studies [25,26]. This compound is also considered as a non-antibiotic drug that has antibacterial properties. NAC can also impede the biofilm formation by Staphylococcus epidermidis [27]. The antibacterial activity of NAC is might be due to destruction of intermolecular or intramolecular disulfide bonds in bacterial proteins. Previously, the protective effects of vitamin C and NAC against the toxicity of rifampin on HepG2 and ICHN cells were demonstrated [14]. To answer the question of possible decrease in antibacterial activity of rifampin, along with hepatoprotection, the present study was designed. Consequently, in this study the antibacterial activity of vitamin C and NAC individually and in combination with rifampin and isoniazid were tested against different strains of S. aureus and M. tuberculosis. 2. Materials and methods 2.1. Bacterial strains Four clinically resistant strains of S. aureus and six isolated of M. tuberculosis; three susceptible to rifampin and isoniazid, and three resistance to these drugs, were received from the Clinical Microbiology Laboratory, University hospital of Imam Reza, in Mashhad, Iran, as multidrug resistant isolates. They were subjected again to disk diffusion method for confirmation of their resistance. The antibiotic disks used were methicillin (30 mg/ml), ciprofloxacin (5 mg/ml) and tetracycline (30 mg/ml) [Pad Tan Teb, Iran]. The standard strain of S. aureus ATCC25923 (American Type Culture Collection) which is MSSA (methicillin sensitive S. aureus) and ATCC43300 which is MRSA (methicillin resistance S. aureus) and standard strain of M. tuberculosis, H37Rv (control), were used as control strains. 2.2. Cultivation of microorganisms and bacterial preparation Prior to the test for S. aureus strains, the surface of caso agar medium plate (Merck, Germany) was inoculated from recently grown stock culture of each of the specified microorganisms already on caso agar and incubated at 35e37 C for 18e24 h. Four
colonies of the cells were inoculated into 5 ml Muller Hinton broth (MHB) (Merck, Germany), and incubated at 37 C for 3e4 h. When turbidity of about 0.5 McFarland was attained, the cells were diluted with cation-supplemented Mueller-Hinton broth (CSMHB) (BBL Microbiology Systems, Cockeysville, MD) to get a cell suspension of 3 105 cfu/ml [28]. For preparation of M. tuberculosis strains, all these steps were € wensteinrepeated except that the bacteria were cultured on Lo Jensen medium in the presence or absent of rifampin or isoniazid in McCartney bottles. 2.3. Susceptibility testing MICs of rifampin (Sandoz company, Switzerland), isoniazid (Hakim company, Iran) NAC (Exir company, Iran) and vitamin C (Osveh Company, Iran) against bacteria were determined according to the National Committee for Clinical Laboratory Standards (NCCLS) with some modifications [29]. All S. aureus strains, in two fold broth dilution method, with an inoculum size of approximately 106 CFU/ml (20 ml) were added to each wells of 96-well plate already contains 180 ml of serial two fold concentrations of rifampin (final concentration of 2e0.001 mg/ml), NAC (final concentration of 40.0e0.0.04 mg/ml) or vitamin C (final concentration of 40.0e0.04 mg/ml) dissolved in MHB. Negative control wells with no bacteria and positive control wells contain bacteria and no drug included. Plates were incubated at 37 C for 18 h. MIC was determined by adding TTC (2,3,5triphenyltetrazolium chloride, Sigma, St Louis, MO) to the wells at a concentration of 0.05% and incubating for 30 min at 37 C. MICs were defined as the lowest concentration of antimicrobial agent that the red formazan of TTC reduction was not observed. Each experiment has been performed in triplicate. In the case of M. tuberculosis strains, different concentrations of €wensteineJensen drugs were incubated with 3 105 bacteria in Lo media and incubated at 37 C for 28e42 days, after which the colonies were counted. The final concentration of rifampin, isoniazid, vitamin C and NAC were 10e80 mg/ml, 0.05e0.4 mg/ml, 0.025e0.2 mg/ml and 0.05e0.2 mg/ml, respectively. 2.4. Synergistic studies The antibacterial effects of a combination of rifampin with NAC or vitamin C against S. aureus were assessed by checkerboard micro-titer tests [1]. The antimicrobial combinations assayed included 100 ml of different serial two fold dilution of NAC (final concentration in a well 3.2e0.05 mg.ml1) or vitamin C (final concentration in a well 1.6e0.006 mg.ml1) plus 100 ml of serial two fold dilutions of rifampin (final concentration in a well 2e0.001 mg ml1) so that each row (and column) contained a fixed amount of one agent and increasing amounts of the second agent. Serial dilutions of antibiotics and NAC or vitamin C were mixed in Soybean Casein Digest Agar (SCDB) (HIMEDIA, India). Inocula were prepared from colonies grown on SCDA after overnight culture. 20 ml of 106 of each bacterial suspension was added to each well (final bacterial concentration after inoculation was 5 104 CFU/ml). Negative control wells had no bacteria and positive control wells contained bacteria and no drug included. After 18 h incubation at 37 C, MIC was determined by adding TTC to the wells at a concentration of 0.05% and incubating for 30 min at 37 C. Each experiment has been performed in triplicate. The FIC index was calculated according to the equation: FIC index ¼ FICA þ FICB ¼ (MIC of drug A in combination/MIC of drug A alone) þ (MIC of drug B in combination/MIC of drug B alone). The interaction was defined as synergistic if the FIC index was 0.5, additive if the FIC index was 0.5 and 1.0, indifferent if the FIC
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index was >1.0 and 2.0, and antagonistic if the FIC index was >2.0 [2]. To check the possible effect of vitamin C and NAC on the activity of anti-TB drugs against M. tuberculosis strains, IC50 values of rifampin and isoniazid were determined by clony forming unit (CFU) assay. Sample cultures of H37Rv were also tested with vitamin C or NAC alone (without antibacterial agents).
Table 2 Interaction of vitamin C on the inhibitory effects of rifampin against Staphylococcus aureus standard and hospital isolates. MIC Strains
Agent
Alone
Combination
FICI
Outcome
A
Rifampin Vit C Rifampin Vit C Rifampin Vit C Rifampin Vit C Rifampin Vit C Rifampin Vit C
0.016 40 0.016 40 0.008 40 0.032 40 0.008 40 0.032 40
0.008 0.025 0.008 0.025 0.004 0.025 0.016 0.025 0.004 0.025 0.016 0.025
0.5
Synergistic
0.5
Synergistic
0.5
Synergistic
0.5
Synergistic
0.5
Synergistic
0.5
Synergistic
B
2.5. Statistical analysis
C
Statistical significance of the observed differences was assessed with analysis of variance (one-way ANOVA).
D E
3. Results
F
3.1. Susceptibility testing From different S. aureus isolates tested, 4 isolates of S. aureus were resistant to ciprofloxacin, methicillin or tetracycline, which were considered for the continuation of the experiments. These are listed in Table 1 with the sample source and gender of the patients. All tested bacteria were resistant to NAC (up to 40 mg/ml) and vitamin C (up to 40 mg/ml). MICs of rifampin (mg/ml) against different isolates and standard strains of S. aureus were ranged from 0.008 to 0.032. MIC of rifampin and isoniazid against H37Rv strain of M. tuberculosis was found to be 40 and 0.2 mg/ml, respectively. Vitamin C and NAC alone did not affect the growth of M. tuberculosis. The experiments with sensitive and resistant strains were carried out with their MIC of the antibacterial agents only.
MIC: minimum inhibitory concentration. FICI: Fraction inhibitory concentration index. The experiments were repeated two times, each sample in triplicate, and no MIC value variation.
Table 3 Interaction of NAC on the inhibitory effects of rifampin against Staphylococcus aureus standard and hospital isolates. MIC Strains
Agent
Alone
Combination
FICI
Outcome
A
Rifampin NAC Rifampin NAC Rifampin NAC Rifampin NAC Rifampin NAC Rifampin NAC
0.016 40 0.016 40 0.008 40 0.032 40 0.008 40 0.032 40
0.016 0.05 0.016 0.05 0.008 0.05 0.032 0.05 0.008 0.05 0.032 0.05
1
Additive
1
Additive
1
Additive
1
Additive
1
Additive
1
Additive
B C D E
3.2. Synergistic studies F
In the case of S. aureus, NAC or vitamin C had no antimicrobial effect themselves. Checkerboard microdilution results indicated that vitamin C reduced two-times the MIC of rifampin against all isolates at concentrations of 1.6e0.025 mg/ml, while at concentrations below 0.025 mg/ml (0.012e0.006 mg/ml) did not have any effect on reduction of MIC of rifampin against any of the isolates. MIC of rifampin against A and B isolates changed from 0.016 to 0.008 and against C and E from 0.008 to 0.004 and against D and F from 0.32 to 0.016 mg/ml. NAC at concentrations of 3.2e0.05 mg/ml could not change the MIC of rifampin against any of the isolates. These data represented in Tables 2 and 3. In the case of M. tuberculosis, vitamin C and NAC in a dosedependent manner, reduced the number of colonies of H37Rv strain, grown in the culture medium containing rifampin in the concentrations less than MIC (Table 4). However, the effectiveness
Table 1 Demographic data of patients and sample source. Strain
A B C D E F
Sample source
Acne Eye Urine Acne ATCC43300a ATCC29737b
Gender
F M F M S S
85
MIC: minimum inhibitory concentration. FICI: Fraction inhibitory concentration index. The experiments were repeated two times, each sample in triplicate, and no MIC value variation.
of NAC was less than vitamin C. For resistant pathogens, NAC did not show remarkable effects, while, vitamin C showed similar effects on sensitive and resistant strains (Supplementary data). These observations were confirmed by statistical analysis. Vitamin C reduced the number of colonies of H37Rv strain of M. tuberculosis, grown in the culture medium containing isoniazid, in a dose-dependent manner (Table 5). Similar effects were also observed with sensitive and resistant strains. However, vitamin C showed a weaker effect against resistant strains of M. tuberculosis. NAC did not change the number of colonies of M. tuberculosis grown in the culture medium containing isoniazid in the concentrations less than MIC (Supplementary data). These results have been confirmed by statistical analysis.
Susceptibility C
M
T
þ þ þ þ þ þ
þ þ þ þ þ þ
þ þ þ þ þ þ
M: male, F: female, S: standard, C: ciprofloxacin, M: methicillin, T: tetracycline, þ: resistant, : sensitive. a MRSA: methicillin resistance Staphylococcus aureus. b MSSA: methicillin sensitive Staphylococcus aureus.
4. Discussion Today, in spite of remarkable advances in treatment of TB, hepatotoxicity caused by anti-tuberculosis drugs (Isoniazid, rifampin and pyrazinamide) remains a worldwide health concern. This side effect necessitates temporary stopping of antibiotic therapy or changing them to other medications with less confidential effects. Consequently, finding solution to reduce the hepatotoxicity of these drugs without changing their efficacy would be desirable. Previously, it was shown that combination of vitamin C and NAC
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Table 4 The effects of vitamin C or NAC on the percent of colony counts of Mycobacterium tuberculosis grown in the presence of rifampin. M.tuberculosis strains
H37Rv H37Rv Sensitive strain Resistant strain
Rifampin concentration (mg/ml)
10 20 40 40
Blank
100 100 100 100
0.025 mg/ml
0.05 mg/ml
Vitamin C
Vitamin C
NAC
0.1 mg/ml Vitamin C
NAC
0.2 mg/ml NAC
50 50 26 86.6
20 40 0 74.1
50 10 12.5 96.9
5 10 0 65.5
75 150 3 76.9
50 0 0 73.8
Table 5 The effects of vitamin C or NAC on the percent of colony counts of Mycobacterium tuberculosis grown in the presence of isoniazid. M. tuberculosis strains
H37Rv H37Rv Resistant strain
Isoniazid concentration (mg/ml)
0.05 0.1 0.2
Blank
100 100 100
0.025 mg/ml
0.05 mg/ml
Vitamin C
Vitamin C
NAC
Vitamin C
NAC
NAC
92.5 89.4 101.1
88.8 89.4 97.2
94.4 100 96.9
70.3 68.4 87.3
88.8 104 76.9
77.7 95 73.8
was able to reduce the hepatotoxicity of the anti-tuberculosis drugs [14]. It is important to note that this study was designed to see if vitamin C or NAC may decrease the antibacterial effects of rifampin and isoniazid, and their hepatoprotective effects might be ignored. In this study, vitamin C and NAC alone and in combination with rifampin and isoniazid were used to eradicate the S. aureus and M. tuberculosis. It was shown that deficiency of vitamin C severely depresses immune responses and results in decreased resistance to disease [30]. During infections and stress, concentrations of vitamin C in the plasma and leukocytes rapidly decline, and supplementation of it improve components of the human immune system such as antimicrobial and natural killer cell activities [31]. The other possible reason for enhancing the antibacterial activity of antibiotics in combination with vitamin C might be due to the effect of this substance on the ability of neutrophils in combating infections [32]. Results of the present study showed that vitamin C increased the antibacterial activity of rifampin against S. aureus at least twotimes (Table 2). Additionally, vitamin C increased the antibacterial effects of rifampin and isoniazid against M. tuberculosis as the same manner (Tables 4 and 5). The improved antibacterial activity of antibacterial agents in combination with vitamin C was previously described [33,34]. Vitamin C increased the effects of claritromycin against H. pylori in microbial culture. The mechanism of enhancing the antibacterial activity of antibiotics in combination of vitamin C is not fully understood, although, it was suggested that in pharmacologic doses, vitamin C can act as a prodrug for hydrogen peroxide (H2O2) formation, thereby helping the antibacterial agents to combat against bacteria. This effect is clearly independent of immune-enhancing action of vitamin C, as occurs even in vitro, and may contribute to its antibacterial and anticancer effects [35]. It was also suggested that some antibiotics like ciprofloxacin stimulate the induction of reactive oxygen species (ROS) in many bacterial strains and vitamin C because of the antioxidant activity affect antibiotic susceptibility, however, it cannot be observed for other antioxidants [36]. NAC showed similar results, but was less effective than vitamin C (Tables 3e5). NAC is a non-antibiotic compound which exerts antibacterial properties [37,38]. There are some studies, in agreement with our results, showing that high concentrations of NAC could inhibit biofilm formation due to the growth of some bacterial species, such as P. aeruginosa, S. epidermidis and other bacteria, in medical devices [39e41]. The antibacterial mechanism of NAC is not clear, but it was suggested that NAC is likely to be achieved by preventing amino acid (cysteine) utilization in bacteria or reaction
0.1 mg/ml
0.2 mg/ml
of sulfhydryl group of NAC with bacterial cell proteins [36]. 5. Conclusion Observations of this study imply that vitamin C is more potent than NAC in supporting rifampin against both strains, S. aureus and M. tuberculosis, and also isoniazid against M. tuberculosis. These results highlighted that these two antioxidants not only do not decrease the antibacterial activity of rifampin, but also increase their effects, to some extent and merit for further investigations. Conflict of interest All authors have declared that there is no conflict of interest in this study. Acknowledgment This work was supported financially by a research grant from the Vice Chancellor for Research of Mashhad University of Medical Sciences (910054), Mashhad, Iran. The results reported in this paper were part of a Pharm.D. thesis. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micpath.2015.11.006. References [1] B. Khameneh, M. Iranshahy, M. Ghandadi, D. Ghoochi Atashbeyk, B.S. Fazly Bazzaz, M. Iranshahi, Investigation of the antibacterial activity and efflux pump inhibitory effect of co-loaded piperine and gentamicin nanoliposomes in methicillin-resistant Staphylococcus aureus, Drug Dev. Ind. Pharm. 41 (6) (2015) 989e994. [2] D.G. Atashbeyk, B. Khameneh, M. Tafaghodi, B.S. Fazly Bazzaz, Eradication of methicillin-resistant Staphylococcus aureus infection by nanoliposomes loaded with gentamicin and oleic acid, Pharm. Biol. 52 (11) (2014) 1423e1428. [3] R.H. Eng, S.M. Smith, M. Tillem, C. Cherubin, Rifampin resistance. Development during the therapy of methicillin-resistant Staphylococcus aureus infection, Arch. Intern. Med. 145 (1) (1985) 146e148. [4] C.U. Tuazon, M.Y. Lin, J.N. Sheagren, In vitro activity of rifampin alone and in combination with nafcillin and Vancomycin against pathogenic strains of Staphylococcus aureus, Antimicrob. Agents Chemother. 13 (5) (1978) 759e761. [5] M. Drancourt, A. Stein, J.N. Argenson, R. Roiron, P. Groulier, D. Raoult, Oral treatment of Staphylococcus spp. infected orthopaedic implants with fusidic acid or ofloxacin in combination with rifampicin, J. Antimicrob. Chemother. 39 (2) (1997) 235e240.
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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Combination of anti-tuberculosis drugs with vitamin C or NAC against different Staphylococcus aureus and Mycobacterium tuberculosis strains Bahman Khameneh a, Bibi Sedigheh Fazly Bazzaz b, Alireza Amani c, Javad Rostami c, Nasser Vahdati-Mashhadian d, e, * a
Department of Pharmaceutical Control, Students Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran Students Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran d Medical Toxicology Research Center, Mashhad University of Medical Sciences, Mashhad, IR Iran e Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 October 2015 Received in revised form 27 October 2015 Accepted 9 November 2015 Available online 19 November 2015
Backgrounds: Hepatotoxicity due to anti tuberculosis drugs, rifampin and isoniazid, is a major problem in tuberculosis patients. Vitamin C, an antioxidant, and N-acetyl cysteine (NAC), a scavenger of active metabolites, reduce the hepatotoxicity. The aim of present study was to investigate the effect of vitamin C and NAC individually on the antibacterial activity of anti tuberculosis drugs against Mycobacterium tuberculosis and Staphylococcus aureus strains. Methods: The MICs of each compound against all strains were determined in 96 wells plate. Rifampin was tested at serial two fold concentrations alone or in combination with NAC or vitamin C. Results: The MIC of rifampin against different strains of S. aureus was 0.008e0.032 mg/ml. The MIC of rifampin and isoniazid against M. tuberculosis strains were 40 and 0.2 mg/ml, respectively. Vitamin C and NAC had no antibacterial activity against all strains. MIC of rifampin was reduced two fold by combination with vitamin C for all S. aureus strains, while NAC did not affect the antibacterial activity of rifampin. Vitamin C and NAC had remarkable effects on the antibacterial activity of anti-tuberculosis drugs against M. tuberculosis. Conclusions: Synergistic effects were observed between rifampin or isoniazid and vitamin C against all tested strains. However, combination therapy of rifampin and isoniazid with NAC was not being effective. This study highlighted the advantages of combination of anti-tuberculosis drugs and vitamin C to eradicate the microbial infections. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Mycobacterium tuberculosis N-acetyl cysteine Rifampin Isoniazid Staphylococcus aureus Vitamin C
1. Introduction Staphylococcus aureus causes severe life-threatening infections and has become increasingly common, particularly methicillinresistant strains (MRSA) [1,2]. Rifampin is often used as adjunctive therapy to treat S. aureus infections. Staphylococcal infections are among the first non-mycobacterial infections treated with rifampin; however, it was soon discovered in order to prevent the emergence of rifampin-resistant isolates, at least another effective antimicrobial agent was required to be used in combination with
* Corresponding author. Department of Pharmacodynamics and Toxicology, Faculty of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. E-mail addresses: [email protected], [email protected] (N. VahdatiMashhadian). http://dx.doi.org/10.1016/j.micpath.2015.11.006 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
rifampin [3,4]. Some human studies have addressed the role of adjunctive rifampin therapy. Adjunctive therapy in combination with fusidic acid seems most promising for the treatment of difficult to treat S. aureus infections in osteomyelitis and prosthetic joint infections, although studies were typically underpowered and benefits were not always seen [5]. From 1968 isoniazid and rifampin have been widely used for the treatment of tuberculosis; they kill more than 99% of tubercular bacilli within two months of start of therapy. Unfortunately, rifampin resistance started soon and the urgent need for multi therapy was recognized [6]. At that time and by development and introduction of combination therapy of these agents, drug resistant tuberculosis was overcome. This approach provided a good chance for the treatment as it killed rapidly both dividing and long-lived persistent cells of Mycobacterium tuberculosis. Despite the great
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efficacy of this combination therapy, serious reactions have been reported including hepatotoxicity and nephrotoxicity [7]. Other side effects such as unexplained fevers, cytopenias, and neurological disturbances have also been reported and lead to negatively affect treatment compliance and clinical outcome [8,9]. Even the use of rifampin for patients with underlying hepatitis infection may increase the risk of hepatotoxicity [10]. Towards these observations, combination therapy is a suitable approach to overcome bacterial resistance and these limitations [11,12]. Vitamin C is a potent and standard antioxidant with a satisfactory history of protective actions against oxidative stress by many agents in cell culture [13,14], animal [15,16] and human studies [17]. Evidence suggests that vitamin C may have promising effect as an adjuvant in the treatment of cancer [18,19]. It has shown that vitamin C alone or in combination with other antibacterial agents exhibited promising antibacterial activity against different pathogens such as Helicobacter pylori, S. aureus and also multidrug resistant Pseudomonas aeruginosa [20e22]. This can be explained by two mechanisms: On one hand, by transferring vitamin C into the bacterial cells, it is metabolized in the presence of oxygen and consequently bacteria are exposed to oxidative stress. On the other hand, acetic and lactate acids are generated from vitamin C [20]. N-acetyl cysteine (NAC) is also an antioxidant and free radical scavenger that is used as a standard and effective drug for the treatment of acetaminophen overdose [23]. Its protective effect against organ toxicity due to oxidative stress is also evident based on huge array of in vitro [14,24] and in vivo studies [25,26]. This compound is also considered as a non-antibiotic drug that has antibacterial properties. NAC can also impede the biofilm formation by Staphylococcus epidermidis [27]. The antibacterial activity of NAC is might be due to destruction of intermolecular or intramolecular disulfide bonds in bacterial proteins. Previously, the protective effects of vitamin C and NAC against the toxicity of rifampin on HepG2 and ICHN cells were demonstrated [14]. To answer the question of possible decrease in antibacterial activity of rifampin, along with hepatoprotection, the present study was designed. Consequently, in this study the antibacterial activity of vitamin C and NAC individually and in combination with rifampin and isoniazid were tested against different strains of S. aureus and M. tuberculosis. 2. Materials and methods 2.1. Bacterial strains Four clinically resistant strains of S. aureus and six isolated of M. tuberculosis; three susceptible to rifampin and isoniazid, and three resistance to these drugs, were received from the Clinical Microbiology Laboratory, University hospital of Imam Reza, in Mashhad, Iran, as multidrug resistant isolates. They were subjected again to disk diffusion method for confirmation of their resistance. The antibiotic disks used were methicillin (30 mg/ml), ciprofloxacin (5 mg/ml) and tetracycline (30 mg/ml) [Pad Tan Teb, Iran]. The standard strain of S. aureus ATCC25923 (American Type Culture Collection) which is MSSA (methicillin sensitive S. aureus) and ATCC43300 which is MRSA (methicillin resistance S. aureus) and standard strain of M. tuberculosis, H37Rv (control), were used as control strains. 2.2. Cultivation of microorganisms and bacterial preparation Prior to the test for S. aureus strains, the surface of caso agar medium plate (Merck, Germany) was inoculated from recently grown stock culture of each of the specified microorganisms already on caso agar and incubated at 35e37 C for 18e24 h. Four
colonies of the cells were inoculated into 5 ml Muller Hinton broth (MHB) (Merck, Germany), and incubated at 37 C for 3e4 h. When turbidity of about 0.5 McFarland was attained, the cells were diluted with cation-supplemented Mueller-Hinton broth (CSMHB) (BBL Microbiology Systems, Cockeysville, MD) to get a cell suspension of 3 105 cfu/ml [28]. For preparation of M. tuberculosis strains, all these steps were € wensteinrepeated except that the bacteria were cultured on Lo Jensen medium in the presence or absent of rifampin or isoniazid in McCartney bottles. 2.3. Susceptibility testing MICs of rifampin (Sandoz company, Switzerland), isoniazid (Hakim company, Iran) NAC (Exir company, Iran) and vitamin C (Osveh Company, Iran) against bacteria were determined according to the National Committee for Clinical Laboratory Standards (NCCLS) with some modifications [29]. All S. aureus strains, in two fold broth dilution method, with an inoculum size of approximately 106 CFU/ml (20 ml) were added to each wells of 96-well plate already contains 180 ml of serial two fold concentrations of rifampin (final concentration of 2e0.001 mg/ml), NAC (final concentration of 40.0e0.0.04 mg/ml) or vitamin C (final concentration of 40.0e0.04 mg/ml) dissolved in MHB. Negative control wells with no bacteria and positive control wells contain bacteria and no drug included. Plates were incubated at 37 C for 18 h. MIC was determined by adding TTC (2,3,5triphenyltetrazolium chloride, Sigma, St Louis, MO) to the wells at a concentration of 0.05% and incubating for 30 min at 37 C. MICs were defined as the lowest concentration of antimicrobial agent that the red formazan of TTC reduction was not observed. Each experiment has been performed in triplicate. In the case of M. tuberculosis strains, different concentrations of €wensteineJensen drugs were incubated with 3 105 bacteria in Lo media and incubated at 37 C for 28e42 days, after which the colonies were counted. The final concentration of rifampin, isoniazid, vitamin C and NAC were 10e80 mg/ml, 0.05e0.4 mg/ml, 0.025e0.2 mg/ml and 0.05e0.2 mg/ml, respectively. 2.4. Synergistic studies The antibacterial effects of a combination of rifampin with NAC or vitamin C against S. aureus were assessed by checkerboard micro-titer tests [1]. The antimicrobial combinations assayed included 100 ml of different serial two fold dilution of NAC (final concentration in a well 3.2e0.05 mg.ml1) or vitamin C (final concentration in a well 1.6e0.006 mg.ml1) plus 100 ml of serial two fold dilutions of rifampin (final concentration in a well 2e0.001 mg ml1) so that each row (and column) contained a fixed amount of one agent and increasing amounts of the second agent. Serial dilutions of antibiotics and NAC or vitamin C were mixed in Soybean Casein Digest Agar (SCDB) (HIMEDIA, India). Inocula were prepared from colonies grown on SCDA after overnight culture. 20 ml of 106 of each bacterial suspension was added to each well (final bacterial concentration after inoculation was 5 104 CFU/ml). Negative control wells had no bacteria and positive control wells contained bacteria and no drug included. After 18 h incubation at 37 C, MIC was determined by adding TTC to the wells at a concentration of 0.05% and incubating for 30 min at 37 C. Each experiment has been performed in triplicate. The FIC index was calculated according to the equation: FIC index ¼ FICA þ FICB ¼ (MIC of drug A in combination/MIC of drug A alone) þ (MIC of drug B in combination/MIC of drug B alone). The interaction was defined as synergistic if the FIC index was 0.5, additive if the FIC index was 0.5 and 1.0, indifferent if the FIC
B. Khameneh et al. / Microbial Pathogenesis 93 (2016) 83e87
index was >1.0 and 2.0, and antagonistic if the FIC index was >2.0 [2]. To check the possible effect of vitamin C and NAC on the activity of anti-TB drugs against M. tuberculosis strains, IC50 values of rifampin and isoniazid were determined by clony forming unit (CFU) assay. Sample cultures of H37Rv were also tested with vitamin C or NAC alone (without antibacterial agents).
Table 2 Interaction of vitamin C on the inhibitory effects of rifampin against Staphylococcus aureus standard and hospital isolates. MIC Strains
Agent
Alone
Combination
FICI
Outcome
A
Rifampin Vit C Rifampin Vit C Rifampin Vit C Rifampin Vit C Rifampin Vit C Rifampin Vit C
0.016 40 0.016 40 0.008 40 0.032 40 0.008 40 0.032 40
0.008 0.025 0.008 0.025 0.004 0.025 0.016 0.025 0.004 0.025 0.016 0.025
0.5
Synergistic
0.5
Synergistic
0.5
Synergistic
0.5
Synergistic
0.5
Synergistic
0.5
Synergistic
B
2.5. Statistical analysis
C
Statistical significance of the observed differences was assessed with analysis of variance (one-way ANOVA).
D E
3. Results
F
3.1. Susceptibility testing From different S. aureus isolates tested, 4 isolates of S. aureus were resistant to ciprofloxacin, methicillin or tetracycline, which were considered for the continuation of the experiments. These are listed in Table 1 with the sample source and gender of the patients. All tested bacteria were resistant to NAC (up to 40 mg/ml) and vitamin C (up to 40 mg/ml). MICs of rifampin (mg/ml) against different isolates and standard strains of S. aureus were ranged from 0.008 to 0.032. MIC of rifampin and isoniazid against H37Rv strain of M. tuberculosis was found to be 40 and 0.2 mg/ml, respectively. Vitamin C and NAC alone did not affect the growth of M. tuberculosis. The experiments with sensitive and resistant strains were carried out with their MIC of the antibacterial agents only.
MIC: minimum inhibitory concentration. FICI: Fraction inhibitory concentration index. The experiments were repeated two times, each sample in triplicate, and no MIC value variation.
Table 3 Interaction of NAC on the inhibitory effects of rifampin against Staphylococcus aureus standard and hospital isolates. MIC Strains
Agent
Alone
Combination
FICI
Outcome
A
Rifampin NAC Rifampin NAC Rifampin NAC Rifampin NAC Rifampin NAC Rifampin NAC
0.016 40 0.016 40 0.008 40 0.032 40 0.008 40 0.032 40
0.016 0.05 0.016 0.05 0.008 0.05 0.032 0.05 0.008 0.05 0.032 0.05
1
Additive
1
Additive
1
Additive
1
Additive
1
Additive
1
Additive
B C D E
3.2. Synergistic studies F
In the case of S. aureus, NAC or vitamin C had no antimicrobial effect themselves. Checkerboard microdilution results indicated that vitamin C reduced two-times the MIC of rifampin against all isolates at concentrations of 1.6e0.025 mg/ml, while at concentrations below 0.025 mg/ml (0.012e0.006 mg/ml) did not have any effect on reduction of MIC of rifampin against any of the isolates. MIC of rifampin against A and B isolates changed from 0.016 to 0.008 and against C and E from 0.008 to 0.004 and against D and F from 0.32 to 0.016 mg/ml. NAC at concentrations of 3.2e0.05 mg/ml could not change the MIC of rifampin against any of the isolates. These data represented in Tables 2 and 3. In the case of M. tuberculosis, vitamin C and NAC in a dosedependent manner, reduced the number of colonies of H37Rv strain, grown in the culture medium containing rifampin in the concentrations less than MIC (Table 4). However, the effectiveness
Table 1 Demographic data of patients and sample source. Strain
A B C D E F
Sample source
Acne Eye Urine Acne ATCC43300a ATCC29737b
Gender
F M F M S S
85
MIC: minimum inhibitory concentration. FICI: Fraction inhibitory concentration index. The experiments were repeated two times, each sample in triplicate, and no MIC value variation.
of NAC was less than vitamin C. For resistant pathogens, NAC did not show remarkable effects, while, vitamin C showed similar effects on sensitive and resistant strains (Supplementary data). These observations were confirmed by statistical analysis. Vitamin C reduced the number of colonies of H37Rv strain of M. tuberculosis, grown in the culture medium containing isoniazid, in a dose-dependent manner (Table 5). Similar effects were also observed with sensitive and resistant strains. However, vitamin C showed a weaker effect against resistant strains of M. tuberculosis. NAC did not change the number of colonies of M. tuberculosis grown in the culture medium containing isoniazid in the concentrations less than MIC (Supplementary data). These results have been confirmed by statistical analysis.
Susceptibility C
M
T
þ þ þ þ þ þ
þ þ þ þ þ þ
þ þ þ þ þ þ
M: male, F: female, S: standard, C: ciprofloxacin, M: methicillin, T: tetracycline, þ: resistant, : sensitive. a MRSA: methicillin resistance Staphylococcus aureus. b MSSA: methicillin sensitive Staphylococcus aureus.
4. Discussion Today, in spite of remarkable advances in treatment of TB, hepatotoxicity caused by anti-tuberculosis drugs (Isoniazid, rifampin and pyrazinamide) remains a worldwide health concern. This side effect necessitates temporary stopping of antibiotic therapy or changing them to other medications with less confidential effects. Consequently, finding solution to reduce the hepatotoxicity of these drugs without changing their efficacy would be desirable. Previously, it was shown that combination of vitamin C and NAC
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Table 4 The effects of vitamin C or NAC on the percent of colony counts of Mycobacterium tuberculosis grown in the presence of rifampin. M.tuberculosis strains
H37Rv H37Rv Sensitive strain Resistant strain
Rifampin concentration (mg/ml)
10 20 40 40
Blank
100 100 100 100
0.025 mg/ml
0.05 mg/ml
Vitamin C
Vitamin C
NAC
0.1 mg/ml Vitamin C
NAC
0.2 mg/ml NAC
50 50 26 86.6
20 40 0 74.1
50 10 12.5 96.9
5 10 0 65.5
75 150 3 76.9
50 0 0 73.8
Table 5 The effects of vitamin C or NAC on the percent of colony counts of Mycobacterium tuberculosis grown in the presence of isoniazid. M. tuberculosis strains
H37Rv H37Rv Resistant strain
Isoniazid concentration (mg/ml)
0.05 0.1 0.2
Blank
100 100 100
0.025 mg/ml
0.05 mg/ml
Vitamin C
Vitamin C
NAC
Vitamin C
NAC
NAC
92.5 89.4 101.1
88.8 89.4 97.2
94.4 100 96.9
70.3 68.4 87.3
88.8 104 76.9
77.7 95 73.8
was able to reduce the hepatotoxicity of the anti-tuberculosis drugs [14]. It is important to note that this study was designed to see if vitamin C or NAC may decrease the antibacterial effects of rifampin and isoniazid, and their hepatoprotective effects might be ignored. In this study, vitamin C and NAC alone and in combination with rifampin and isoniazid were used to eradicate the S. aureus and M. tuberculosis. It was shown that deficiency of vitamin C severely depresses immune responses and results in decreased resistance to disease [30]. During infections and stress, concentrations of vitamin C in the plasma and leukocytes rapidly decline, and supplementation of it improve components of the human immune system such as antimicrobial and natural killer cell activities [31]. The other possible reason for enhancing the antibacterial activity of antibiotics in combination with vitamin C might be due to the effect of this substance on the ability of neutrophils in combating infections [32]. Results of the present study showed that vitamin C increased the antibacterial activity of rifampin against S. aureus at least twotimes (Table 2). Additionally, vitamin C increased the antibacterial effects of rifampin and isoniazid against M. tuberculosis as the same manner (Tables 4 and 5). The improved antibacterial activity of antibacterial agents in combination with vitamin C was previously described [33,34]. Vitamin C increased the effects of claritromycin against H. pylori in microbial culture. The mechanism of enhancing the antibacterial activity of antibiotics in combination of vitamin C is not fully understood, although, it was suggested that in pharmacologic doses, vitamin C can act as a prodrug for hydrogen peroxide (H2O2) formation, thereby helping the antibacterial agents to combat against bacteria. This effect is clearly independent of immune-enhancing action of vitamin C, as occurs even in vitro, and may contribute to its antibacterial and anticancer effects [35]. It was also suggested that some antibiotics like ciprofloxacin stimulate the induction of reactive oxygen species (ROS) in many bacterial strains and vitamin C because of the antioxidant activity affect antibiotic susceptibility, however, it cannot be observed for other antioxidants [36]. NAC showed similar results, but was less effective than vitamin C (Tables 3e5). NAC is a non-antibiotic compound which exerts antibacterial properties [37,38]. There are some studies, in agreement with our results, showing that high concentrations of NAC could inhibit biofilm formation due to the growth of some bacterial species, such as P. aeruginosa, S. epidermidis and other bacteria, in medical devices [39e41]. The antibacterial mechanism of NAC is not clear, but it was suggested that NAC is likely to be achieved by preventing amino acid (cysteine) utilization in bacteria or reaction
0.1 mg/ml
0.2 mg/ml
of sulfhydryl group of NAC with bacterial cell proteins [36]. 5. Conclusion Observations of this study imply that vitamin C is more potent than NAC in supporting rifampin against both strains, S. aureus and M. tuberculosis, and also isoniazid against M. tuberculosis. These results highlighted that these two antioxidants not only do not decrease the antibacterial activity of rifampin, but also increase their effects, to some extent and merit for further investigations. Conflict of interest All authors have declared that there is no conflict of interest in this study. Acknowledgment This work was supported financially by a research grant from the Vice Chancellor for Research of Mashhad University of Medical Sciences (910054), Mashhad, Iran. The results reported in this paper were part of a Pharm.D. thesis. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.micpath.2015.11.006. References [1] B. Khameneh, M. Iranshahy, M. Ghandadi, D. Ghoochi Atashbeyk, B.S. Fazly Bazzaz, M. Iranshahi, Investigation of the antibacterial activity and efflux pump inhibitory effect of co-loaded piperine and gentamicin nanoliposomes in methicillin-resistant Staphylococcus aureus, Drug Dev. Ind. Pharm. 41 (6) (2015) 989e994. [2] D.G. Atashbeyk, B. Khameneh, M. Tafaghodi, B.S. Fazly Bazzaz, Eradication of methicillin-resistant Staphylococcus aureus infection by nanoliposomes loaded with gentamicin and oleic acid, Pharm. Biol. 52 (11) (2014) 1423e1428. [3] R.H. Eng, S.M. Smith, M. Tillem, C. Cherubin, Rifampin resistance. Development during the therapy of methicillin-resistant Staphylococcus aureus infection, Arch. Intern. Med. 145 (1) (1985) 146e148. [4] C.U. Tuazon, M.Y. Lin, J.N. Sheagren, In vitro activity of rifampin alone and in combination with nafcillin and Vancomycin against pathogenic strains of Staphylococcus aureus, Antimicrob. Agents Chemother. 13 (5) (1978) 759e761. [5] M. Drancourt, A. Stein, J.N. Argenson, R. Roiron, P. Groulier, D. Raoult, Oral treatment of Staphylococcus spp. infected orthopaedic implants with fusidic acid or ofloxacin in combination with rifampicin, J. Antimicrob. Chemother. 39 (2) (1997) 235e240.
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