- Email: [email protected]
Stigmasterol: An adjuvant for beta lactam antibiotics against beta-lactamase positive clinical isolates
Steroids 128 (2017) 68–71
Contents lists available at ScienceDirect
Steroids journal homepage: www.elsevier.com/locate/steroids
Stigmasterol: An adjuvant for beta lactam antibiotics against beta-lactamase positive clinical isolates
MARK
⁎
Tong Woei Yenna, , Muhammad Arslan Khanb, Nur Amiera Syuhadaa, Leong Chean Ringa, Darah Ibrahimc, Wen-Nee Tand a Universiti Kuala Lumpur, Malaysian Institute of Chemical and Bioengineering Technology, Lot 1988 Kawasan Perindustrian Bandar Vendor, Taboh Naning, 78000 Alor Gajah, Melaka, Malaysia b The University of Lahore, 1-KM Defence Road, Lahore, Pakistan c Industrial Biotechnology Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia d School of Distance Education, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
A R T I C L E I N F O
A B S T R A C T
Keywords: Adjuvant Antibiotic Ampicillin Stigmasterol
The emergence of beta lactamase producing bacterial strains eliminated the use of beta lactam antibiotics as chemotherapeutic alternative. Beta lactam antibiotics can be coupled with non-antibiotic adjuvants to combat these multidrug resistant strains. We study the synergistic antibiotic effect of stigmasterol as adjuvant of ampicillin against clinical isolates. Ampicillin was used in this study as a beta lactam antibiotic model. All test bacteria were beta lactamase producing clinical isolates. The combination showed significantly better antibiotic activity on all bacteria tested. The two test substances have synergistic antibiotic activity, and the effect was observed in both Gram positive and Gram negative bacteria. The synergistic antibiotic effect of stigmasterol and ampicillin was evident by the low fractional inhibitory concentration (FIC) index on Checkerboard Assay. The results suggest that the combination of ampicillin and stigmasterol acts additively in the treatment of infections caused by beta-lactamase producing pathogens. In bacterial growth reduction assay, ampicillin and stigmasterol alone exhibited very weak inhibitory effect on the bacterial growth, relative to ethanol control. Comparatively, combination of stigmasterol-ampicillin greatly reduced the colony counts at least by 98.7%. In conclusion, we found synergistic effects of stigmasterol and ampicillin against beta lactamase producing clinical isolates. This finding is important as it shows potential application of stigmasterol as an antibiotic adjuvant.
1. Introduction Antibiotic resistance can be literally defined as the acquired ability of a microorganism to resist the effect of chemotherapeutic agent to which it is normally susceptible to ensure a successful life history [1]. This phenomenon is frequently affecting critically-illed or immunecompromised patients which usually associate with hospital-acquired pathogens in intensive care unit specifically [2]. However, the incidence of drug-resistant pathogens in community-acquired infection has been rising in recent years and this led to increase in morbidity, mortality rate and also health care spending [3]. Within the past half century, the pharmaceutical industry, academic institutions and the government are investing necessary resources to develop new antibiotics. However, the vast investments did not give a fruitful outcome where the numbers of approved antibiotics are declining [4]. Beta lactam antibiotics are the most frequently prescribed class of drugs worldwide. The antibiotic inhibits bacterial cell wall synthesis ⁎
and thus resulting in the lysis and deformation of the bacterium [5]. It is effective on both Gram positive and negative bacteria. The antibiotic binds to specific penicillin-binding proteins in the bacterial cell wall, and further inhibits the activity of trans-peptidase, an important enzyme in peptidoglycan cell wall synthesis [6]. The antibiotic is widely prescribed to treat skin and bladder infections, pneumonia, gonorrhoea, meningitis, and intestinal infections [7]. Beta lactam antibiotics are effective alternative to chloramphenicol, furthermore they are considered safe for neonates and children [5,6]. The efficacy of beta lactam antibiotics is reduced due to the beta lactamase enzyme produced by bacteria that hydrolyses the beta lactam ring of the antibiotics. It causes many failures of antimicrobial chemotherapy because it converts beta lactam to inert and ineffective structure [8]. Emergence of beta lactamase producing strains cause development of beta lactam resistant pathogens, which eliminated the use of beta lactam antibiotics as chemotherapeutic alternative. Many attempts have been made to combat these pathogens. The concept of
Corresponding author. E-mail address: [email protected] (T.W. Yenn).
http://dx.doi.org/10.1016/j.steroids.2017.10.016 Received 26 July 2017; Received in revised form 26 October 2017; Accepted 30 October 2017 0039-128X/ © 2017 Elsevier Inc. All rights reserved.
Steroids 128 (2017) 68–71
T.W. Yenn et al.
2.5. Checkboard assay
synergism can be utilized to combat infections caused by complex multidrug resistant pathogens [9]. Combinational therapy of several clinical approved antibiotics from different subclasses has been successful in treating multidrug-resistant pathogens. Ampicillin in combination with ceftaroline, cefepime and ceftriaxone has become a prime chemotherapeutic choice for Entericoccus faecalis infection [10]. To further enhance the biocidal property while minimizing necessary antibiotic concentrations, the antibiotic can be coupled with non-antibiotic adjuvants to combat multidrug resistant strains [9]. In this study, we study the synergistic antibiotic effect of stigmasterol with ampicillin against clinical isolates. Ampicillin was used in this study as a beta lactam antibiotic model to study the drug synergism effect.
This assay was performed to study the synergistic antibiotic effect of stigmasterol and ampicillin. Stigmasterol was serially diluted to final concentrations ranged from 3.1 to 200 µg/ml and mixed with fixed amount of ampicillin (20 µg/ml). The MIC and MBC of the drug combinations were determined according to the protocol described in Section 2.4. The fractional inhibitory concentration (FIC) index was calculated based on the following equation: MIC of stigmasterol/ MIC of stigmasterol-ampicillin combination. Synergism is defined if FIC index ≤0.5; while antagonism is defined if FIC index > 0.5. 2.6. Bacterial growth reduction assay
2. Methodology
Firstly, 0.1 ml of bacterial inoculum was added into 9.9 ml of Mueller Hinton broth. The test groups include: Stigmasterol (20 µg/ml), ampicillin at concentration of MIC, and stigmasterol-ampicillin combination at concentration of MIC. A negative control was included with 0.1 ml ethanol. The test substance was added into the flasks. After incubation with agitation speed 120 rpm at 37 °C for 24 h, aliquot was removed from each flask and serially diluted 10-fold with sterile saline. Then, 0.1 ml of the diluent was spread on Mueller Hinton agar plate. Colony counts were determined after incubation at 37 °C for 24 h. The percentage of growth reduction was calculated relative to ethanol control.
2.1. Chemicals and drugs Stigmasterol (95%) used in this study was purchased from SigmaAldrich. Ampicillin was purchased from Merck. The compound was dissolved in ethanol and filtered with membrane filter (PTFE, 0.2 µm) prior to use. 2.2. Test bacteria The test bacteria used in this study were previously isolated from clinical samples in Hospital Seberang Jaya, Penang, Malaysia. The test bacteria include 2 Gram positive bacteria (Staphylococcus aureus, Streptococcus pyogenes) and 2 Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa). All test bacteria were identified as beta-lactamase producers. The microbial inoculums were prepared as per protocols described by Yenn et al. [11].
3. Results and discussion Stigmasterol is a steroid derivative commonly found in plants with a molecular weight of 412.7 g/mol. The compound is commonly used as food additives due to its cholesterol-lowering property [14]. Stigmasterol also showed significant anticancer activity on several cell lines, including DMBA-induced skin carcinoma [15]. However, the potential of stigmasterol as antibiotic adjuvant is still unknown. Antibiotic is usually paired with non-antibiotic adjuvants to combat multidrug resistant strains [9]. Adjuvant is known as a substance that is not exhibiting antibiotic activity when administered alone, but increases the microbicidal effect when combines with antibiotic [16]. Beta lactam antibiotics are usually used with beta lactamase inhibitor, such as clavulanic acid. Clavulanic acid functions by inhibiting the beta lactamase enzyme produced by the bacteria, hence allows the antibiotic to inhibit bacterial cell wall synthesis [9,16]. This combination allows the continued used of beta lactam antibiotics in inhibiting the growth of beta lactamase producing pathogens. The test bacteria used in this study were clinical isolates with extended spectrum beta lactamases. The bacterial strains were resistant to beta lactam antibiotics, including penicillin and ampicillin. The resistance is due to the ability of the enzyme to digest the beta lactam ring in the antibiotic [8]. Of all test bacteria, chloramphenicol inhibits only S. pyogenes. The antibiotic spectrum of all test bacteria was determined to compare the antibiotic efficacy of the test substance. The antibiotic efficacy of stigmasterol was tested in the same assay. Only E. coli is susceptible to stigmasterol. The antibiotic activity of stigmasterol is previously reported by Sharma [17]. However, the antibiotic activity reported here is weak, judging on the size of inhibition zone (≤9 mm). The stigmasterol-ampicillin combination inhibits all test bacteria (Table 1). The combination showed significantly better (p ≤ .05) antibiotic activity on all bacteria tested. The two test substances have synergistic antibiotic activity. Zygmunt and Tavormina [18] first reported the interference of stigmasterol with polyene antibiotics. They noticed a mild antagonistic anti-Candida effect after addition of stigmasterol. Our results do not match their study as we noticed a significant synergistic antibiotic effect when stigmasterol was used with beta lactam antibiotics. The synergistic effect was observed in both Gram positive and Gram negative bacteria. Beta lactamase produced by the bacteria can be deactivated by adenylation, phosphorylation or acetylation of
2.3. Disc diffusion assay The assay was performed on Mueller Hinton agar (Merck) plates according to Panacek et al. [12]. Firstly, 100 µl of 106 cells/ml suspension was spread uniformly onto the agar plates. Then, 6 mm paper discs impregnated with ampicillin (Fisher) and stigmasterol alone at concentration 1 µg/disc, or in combination (1 µg/disc ampicillin + 1 µg/disc stigmasterol), were placed onto the inoculated medium. The plates were incubated at 37 °C for 24 h. The diameter of clear zone surrounding the paper disc were measured and recorded. 2.4. Antibiotic susceptibility test The minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of the test substances against all test bacteria were determined according to CLSI-recommended procedure described by Hamoud et al. [13]. The bacterial inoculum was diluted to 5 × 105 cells/ml with double strength Mueller Hinton broth (Merck). Ampicillin was serially diluted with ethanol and mixed with the bacterial inoculum in a sterile 96-well plate (Fisher). The tested concentrations of ampicillin were ranged from 3.1 to 200 µg/ml. A negative control was included by replacing ampicillin with ethanol for all the test bacteria. The plate was incubated at 37 °C for 24 h. After that, 40 µl of 0.2 mg/ml Iodonitrotetrazolium Violet (Sigma) was added into each well and incubated for 30 min as a growth indicator. The color change of the broth from yellow to pink indicates bacterial growth. The MIC is defined as the lowest concentration of the test compound to prevent bacterial growth (no color change). To judge the viability of bacterial cells, one loopful of the sample in each well was streaked on Muller Hinton agar plates. The plates were then incubated at 37 °C for 24 h. MBC is defined as the lowest concentration of the test compound to kill the bacterial cells. 69
Steroids 128 (2017) 68–71
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Table 1 Antibiotic efficacy of ampicillin and stigmasterol alone at concentration 1 µg/disc, or in combination (1 μg/disc ampicillin + 1 μg/disc stigmasterol) on disc diffusion assay. Test bacteria
Ampicilin
Stigmasterol
Combination
Chloramphenicol
Penicillin G
Negative control
S. aureus S. pyogenes E. coli P. aeruginosa
− − − −
− − + −
++ +++ ++ +
− + − −
− − − −
− − − −
The antimicrobial activity was determined based on the diameter of inhibition zone measured in mm. The result of screening test was recorded according to scale: +++ ≥18 mm, + + = 10–17 mm, + ≤9 mm, − = no inhibition zone observed. Table 2 The MIC, MBC and FIC index recorded for ampicillin alone, or in combination on Checkerboard Assay. Test bacteria
Ampicillin
Combination
MIC (µg/ ml)
MBC (µg/ ml)
MIC (µg/ ml)
MBC (µg/ ml)
S. aureus
> 200
> 200
3.13
6.25
S. pyogenes
> 200
> 200
1.56
3.13
E. coli
100
> 200
6.25
12.50
P. aeruginosa
> 200
> 200
6.25
25
Table 3 All test bacteria showed significant growth reduction with the combination therapy of stigmasterol and ampicillin.
FIC index
Test bacteria
S. aureus S. pyogenes E. coli P. aeruginosa
< 0.5, Synergism < 0.5, Synergism 0.125, Synergism < 0.5, Synergism
% of growth reduction (relative to ethanol control) Ampicillin
Stigmasterol
Combination
11.2 9.7 6.3 10.2
18.7 24.3 45.9 22.4
99.9 98.7 100 100
to ethanol control (Table 3). Mirroring the results of disc diffusion assay of stigmasterol, E. coli showed greatest percentage of growth reduction (45.9%) compared to other test bacteria, which indicates the high susceptibility of the bacterium to stigmasterol. We also noticed for stigmasterol and ampicillin alone, none of the bacterium showed greater than 3-log10 decrease in bacterial growth (bactericidal target). Comparatively, combination of stigmasterol-ampicillin greatly reduced the colony counts by 98.7–100%. The assay confirms that combining ampicillin with stigmasterol results in greater bacterial effect on all test bacteria. The results provide solid evidence of the synergistic antibiotic effect of stigmasterol and ampicillin. The combination shows bactericidal effect in this assay [22]. In conclusion, we found synergistic effects of stigmasterol and ampicillin in against beta lactamase producing clinical isolates. This finding is important as it shows potential application of stigmasterol as an antibiotic adjuvant. However, we do not have an explanation on the synergistic mechanism. To ascertain the mechanisms of drug synergy between stigmasterol and ampicillin, further investigations must be conducted to profile the changes in cellular environment after drug administration.
aminoglycosides in the beta lactam ring [9]. Stigmasterol may also acts as beta lactamase inhibitor, which restore their susceptibility to ampicillin. The antibiotic resistance can be caused by removal of antibiotics from the bacterial cells through membrane bound efflux pump protein [19]. Thus, we hypothesize that stigmasterol interference in the antibiotic activity can be essentially on a physiochemical basis, which involve the interference with protein pump. The susceptibility to antibiotic can be improved by blocking the mechanism of resistance. Elkins and Mullis [20] report that some steroids, including stigmasterol, affects the multiple drug efflux pump proteins in multidrug resistant E. coli, thus suppressing the emergence of resistance to first line antibiotics. The synergistic antibiotic effect of stigmasterol and ampicillin was evident by the FIC index (Table 2). In checkerboard assay, a low FIC index (< 0.5) indicates the synergism effect between two test substances [21]. Low FIC index was observed for all test bacteria. The results suggest that the combination of ampicillin and stigmasterol can be expected to act additively in the treatment of infections caused by betalactamase producing pathogens. In this assay, the concentration of stigmasterol was fixed as it acts as an antibiotic adjuvant to strengthen the efficacy of ampicillin. We observed differences between ampicillin and stigmasterol-ampicillin combination therapy. The MICs recorded for ampicillin alone was 100 µg/ml for E. coli, and > 200 µg/ml for others. The high MICs indicate high resistance to ampicillin. However, the MIC range for combination therapy was relatively wide, with a range of 3.13–6.25 µg/ml. The MIC and MBC of stigmasterol were > 200 µg/ml for all the test bacteria. We also noticed a significant improvement in ampicillin susceptibility when the stigmasterol was added as adjuvant, for all test bacteria. The MBC indicates the bacterial killing capability of the test substance. The stigmasterol-ampicillin combination showed significantly lower MBCs compared to ampicillin alone. We noticed that the combination showed bactericidal activity, based on the low MBC/MIC ratio. To further validate our results, a bacterial growth reduction assay was conducted. The bacterial growth was monitored in liquid medium supplemented with stigmasterol and ampicillin, either separately and jointly. The checkerboard and bacterial growth reduction assay results agreed for all test bacteria. Generally, ampicillin and stigmasterol alone exhibited very weak inhibitory effect on the bacterial growth, relative
Acknowledgements The authors are thankful to Universiti Kuala Lumpur and Fundamental Research Grant Scheme (FRGS/1/2017/STG05/UNIKL/ 02). References [1] H. Goossens, M. Ferech, R.V. Stichele, M. Elseviers, Outpatient antibiotic use in Europe and association with resistance: a cross-national database study, Lancet 365 (2005) 579–587. [2] S. Dzidic, J. Suskovic, V. Bedekovic, Horizontal gene transfer-emerging multidrug resistance in hospital bacteria, Acta Pharmacol. Sin. 24 (2003) 519–526. [3] I.S. Hooton, S.B. Levy, Antimicrobial resistance: a plan for action for community practice, Am. Fam. Physician 63 (2001) 1034–1036. [4] L.J. Piddock, The crisis of no new antibiotics—what is the way forward? Lancet Infect. Dis. 12 (2012) 249–253. [5] A. Kantele, T. Lääveri, S. Mero, K. Vilkman, S.H. Pakkanen, J. Ollgren, J. Kirveskari, Antimicrobials increase travelers' risk of colonization by extended-spectrum beta lactamase-producing Enterobacteriaceae, Clin. Infect. Dis. 60 (2015) 837–846. [6] H. Cho, T. Uehara, T.G. Bernhardt, Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery, Cell 159 (2014) 1300–1311. [7] N. Lakshmidevi, K.Y.A. Al-hetar, G.B. Kavishankar, Extended spectrum beta lactamase producing extra-intestinal pathogenic Escherichia coli (ESBL-ExPEC), J. Pharm. Biomed. Sci. 7 (2014) 343–355.
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[15] H. Ali, S. Dixit, D. Ali, S.M. Alqahtani, S. Alkahtani, S. Alarifi, Isolation and evaluation of anticancer efficacy of stigmasterol in a mouse model of DMBA-induced skin carcinoma, Drug Des. Devel. Ther. 9 (2015) 2793. [16] G.D. Wright, Antibiotic adjuvants: rescuing antibiotics from resistance, Trends Microbiol. 24 (2016) 862–871. [17] R.K. Sharma, Phytosterols: wide-spectrum antibacterial agents, Bioorg. Chem. 21 (1993) 49–60. [18] W.A. Zygmunt, P.A. Tavormina, Steroid interference with antifungal activity of polyene antibiotics, Appl. Microbiol. 14 (1966) 865–869. [19] H. Kawabata, Y. Habu, H. Tomioka, H. Kutsumi, M. Kobayashi, K. Oyasu, K. Kimoto, Effect of different proton pump inhibitors, differences in CYP2C19 genotype and antibiotic resistance on the eradication rate of Helicobacter pylori infection by a 1week regimen of proton pump inhibitor, amoxicillin and clarithromycin, Aliment. Pharmacol. Ther. 17 (2003) 259–264. [20] C.A. Elkins, L.B. Mullis, Mammalian steroid hormones are substrates for the major RND-and MFS-type tripartite multidrug efflux pumps of Escherichia coli, J. Bacteriol. 188 (2006) 1191–1195. [21] G. Orhan, A. Bayram, Y. Zer, I. Balci, Synergy tests by E test and checkerboard methods of antimicrobial combinations against Brucella melitensis, J. Clin. Microbiol. 43 (2005) 140–143. [22] P. Kaur, S. Agarwal, S. Datta, S. Delineating bacteriostatic and bactericidal targets in mycobacteria using IPTG inducible antisense expression, PLoS One 4 (2009) 5923–5927.
[8] M.A. Pence, N.M. Haste, H.S. Meharena, J. Olson, R.L. Gallo, V. Nizet, S.A. Kristian, Beta-lactamase repressor BlaI modulates Staphylococcus aureus cathelicidin antimicrobial peptide resistance and virulence, PLoS One 10 (2015) 136–140. [9] R.J. Worthington, C. Melander, Combination approaches to combat multidrug-resistant bacteria, Trends Biotechnol. 31 (2013) 177–184. [10] M.K. Luther, L.B. Rice, K.L. LaPlante, Ampicillin in combination with ceftaroline, cefepime, or ceftriaxone demonstrates equivalent activities in a high-inoculum Enterococcus faecalis infection model, Antimicrob. Agents Chemother. 60 (2016) 3178–3182. [11] T.W. Yenn, A.S. Ngim, D. Ibrahim, L. Zakaria, Antimicrobial activity of Penicillium minioluteum ED24, an endophytic fungus residing in Orthosiphon Stamineus benth, World J. Pharm. Pharm. Sci. 3 (2014) 121–132. [12] A. Panacek, M. Smekalova, M. Kilianova, R. Prucek, K. Bogdanova, R. Vecerova, R. Zboril, Strong and nonspecific synergistic antibacterial efficiency of antibiotics combined with silver nanoparticles at very low concentrations showing no cytotoxic effect, Molecules 21 (2015) 26–30. [13] R. Hamoud, S. Zimmermann, J. Reichling, M. Wink, M. Synergistic interactions in two-drug and three-drug combinations (thymol, EDTA and vancomycin) against multi drug resistant bacteria including E. coli, Phytomedicine 21 (2014) 443–447. [14] R.T. Ras, W.P. Koppenol, U. Garczarek, A. Otten-Hofman, D. Fuchs, F. Wagner, E.A. Trautwein, Increases in plasma plant sterols stabilize within four weeks of plant sterol intake and are independent of cholesterol metabolism, Nutr. Metab. Cardiovasc. Dis. 26 (2016) 302–309.
71
Contents lists available at ScienceDirect
Steroids journal homepage: www.elsevier.com/locate/steroids
Stigmasterol: An adjuvant for beta lactam antibiotics against beta-lactamase positive clinical isolates
MARK
⁎
Tong Woei Yenna, , Muhammad Arslan Khanb, Nur Amiera Syuhadaa, Leong Chean Ringa, Darah Ibrahimc, Wen-Nee Tand a Universiti Kuala Lumpur, Malaysian Institute of Chemical and Bioengineering Technology, Lot 1988 Kawasan Perindustrian Bandar Vendor, Taboh Naning, 78000 Alor Gajah, Melaka, Malaysia b The University of Lahore, 1-KM Defence Road, Lahore, Pakistan c Industrial Biotechnology Research Laboratory, School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia d School of Distance Education, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia
A R T I C L E I N F O
A B S T R A C T
Keywords: Adjuvant Antibiotic Ampicillin Stigmasterol
The emergence of beta lactamase producing bacterial strains eliminated the use of beta lactam antibiotics as chemotherapeutic alternative. Beta lactam antibiotics can be coupled with non-antibiotic adjuvants to combat these multidrug resistant strains. We study the synergistic antibiotic effect of stigmasterol as adjuvant of ampicillin against clinical isolates. Ampicillin was used in this study as a beta lactam antibiotic model. All test bacteria were beta lactamase producing clinical isolates. The combination showed significantly better antibiotic activity on all bacteria tested. The two test substances have synergistic antibiotic activity, and the effect was observed in both Gram positive and Gram negative bacteria. The synergistic antibiotic effect of stigmasterol and ampicillin was evident by the low fractional inhibitory concentration (FIC) index on Checkerboard Assay. The results suggest that the combination of ampicillin and stigmasterol acts additively in the treatment of infections caused by beta-lactamase producing pathogens. In bacterial growth reduction assay, ampicillin and stigmasterol alone exhibited very weak inhibitory effect on the bacterial growth, relative to ethanol control. Comparatively, combination of stigmasterol-ampicillin greatly reduced the colony counts at least by 98.7%. In conclusion, we found synergistic effects of stigmasterol and ampicillin against beta lactamase producing clinical isolates. This finding is important as it shows potential application of stigmasterol as an antibiotic adjuvant.
1. Introduction Antibiotic resistance can be literally defined as the acquired ability of a microorganism to resist the effect of chemotherapeutic agent to which it is normally susceptible to ensure a successful life history [1]. This phenomenon is frequently affecting critically-illed or immunecompromised patients which usually associate with hospital-acquired pathogens in intensive care unit specifically [2]. However, the incidence of drug-resistant pathogens in community-acquired infection has been rising in recent years and this led to increase in morbidity, mortality rate and also health care spending [3]. Within the past half century, the pharmaceutical industry, academic institutions and the government are investing necessary resources to develop new antibiotics. However, the vast investments did not give a fruitful outcome where the numbers of approved antibiotics are declining [4]. Beta lactam antibiotics are the most frequently prescribed class of drugs worldwide. The antibiotic inhibits bacterial cell wall synthesis ⁎
and thus resulting in the lysis and deformation of the bacterium [5]. It is effective on both Gram positive and negative bacteria. The antibiotic binds to specific penicillin-binding proteins in the bacterial cell wall, and further inhibits the activity of trans-peptidase, an important enzyme in peptidoglycan cell wall synthesis [6]. The antibiotic is widely prescribed to treat skin and bladder infections, pneumonia, gonorrhoea, meningitis, and intestinal infections [7]. Beta lactam antibiotics are effective alternative to chloramphenicol, furthermore they are considered safe for neonates and children [5,6]. The efficacy of beta lactam antibiotics is reduced due to the beta lactamase enzyme produced by bacteria that hydrolyses the beta lactam ring of the antibiotics. It causes many failures of antimicrobial chemotherapy because it converts beta lactam to inert and ineffective structure [8]. Emergence of beta lactamase producing strains cause development of beta lactam resistant pathogens, which eliminated the use of beta lactam antibiotics as chemotherapeutic alternative. Many attempts have been made to combat these pathogens. The concept of
Corresponding author. E-mail address: [email protected] (T.W. Yenn).
http://dx.doi.org/10.1016/j.steroids.2017.10.016 Received 26 July 2017; Received in revised form 26 October 2017; Accepted 30 October 2017 0039-128X/ © 2017 Elsevier Inc. All rights reserved.
Steroids 128 (2017) 68–71
T.W. Yenn et al.
2.5. Checkboard assay
synergism can be utilized to combat infections caused by complex multidrug resistant pathogens [9]. Combinational therapy of several clinical approved antibiotics from different subclasses has been successful in treating multidrug-resistant pathogens. Ampicillin in combination with ceftaroline, cefepime and ceftriaxone has become a prime chemotherapeutic choice for Entericoccus faecalis infection [10]. To further enhance the biocidal property while minimizing necessary antibiotic concentrations, the antibiotic can be coupled with non-antibiotic adjuvants to combat multidrug resistant strains [9]. In this study, we study the synergistic antibiotic effect of stigmasterol with ampicillin against clinical isolates. Ampicillin was used in this study as a beta lactam antibiotic model to study the drug synergism effect.
This assay was performed to study the synergistic antibiotic effect of stigmasterol and ampicillin. Stigmasterol was serially diluted to final concentrations ranged from 3.1 to 200 µg/ml and mixed with fixed amount of ampicillin (20 µg/ml). The MIC and MBC of the drug combinations were determined according to the protocol described in Section 2.4. The fractional inhibitory concentration (FIC) index was calculated based on the following equation: MIC of stigmasterol/ MIC of stigmasterol-ampicillin combination. Synergism is defined if FIC index ≤0.5; while antagonism is defined if FIC index > 0.5. 2.6. Bacterial growth reduction assay
2. Methodology
Firstly, 0.1 ml of bacterial inoculum was added into 9.9 ml of Mueller Hinton broth. The test groups include: Stigmasterol (20 µg/ml), ampicillin at concentration of MIC, and stigmasterol-ampicillin combination at concentration of MIC. A negative control was included with 0.1 ml ethanol. The test substance was added into the flasks. After incubation with agitation speed 120 rpm at 37 °C for 24 h, aliquot was removed from each flask and serially diluted 10-fold with sterile saline. Then, 0.1 ml of the diluent was spread on Mueller Hinton agar plate. Colony counts were determined after incubation at 37 °C for 24 h. The percentage of growth reduction was calculated relative to ethanol control.
2.1. Chemicals and drugs Stigmasterol (95%) used in this study was purchased from SigmaAldrich. Ampicillin was purchased from Merck. The compound was dissolved in ethanol and filtered with membrane filter (PTFE, 0.2 µm) prior to use. 2.2. Test bacteria The test bacteria used in this study were previously isolated from clinical samples in Hospital Seberang Jaya, Penang, Malaysia. The test bacteria include 2 Gram positive bacteria (Staphylococcus aureus, Streptococcus pyogenes) and 2 Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa). All test bacteria were identified as beta-lactamase producers. The microbial inoculums were prepared as per protocols described by Yenn et al. [11].
3. Results and discussion Stigmasterol is a steroid derivative commonly found in plants with a molecular weight of 412.7 g/mol. The compound is commonly used as food additives due to its cholesterol-lowering property [14]. Stigmasterol also showed significant anticancer activity on several cell lines, including DMBA-induced skin carcinoma [15]. However, the potential of stigmasterol as antibiotic adjuvant is still unknown. Antibiotic is usually paired with non-antibiotic adjuvants to combat multidrug resistant strains [9]. Adjuvant is known as a substance that is not exhibiting antibiotic activity when administered alone, but increases the microbicidal effect when combines with antibiotic [16]. Beta lactam antibiotics are usually used with beta lactamase inhibitor, such as clavulanic acid. Clavulanic acid functions by inhibiting the beta lactamase enzyme produced by the bacteria, hence allows the antibiotic to inhibit bacterial cell wall synthesis [9,16]. This combination allows the continued used of beta lactam antibiotics in inhibiting the growth of beta lactamase producing pathogens. The test bacteria used in this study were clinical isolates with extended spectrum beta lactamases. The bacterial strains were resistant to beta lactam antibiotics, including penicillin and ampicillin. The resistance is due to the ability of the enzyme to digest the beta lactam ring in the antibiotic [8]. Of all test bacteria, chloramphenicol inhibits only S. pyogenes. The antibiotic spectrum of all test bacteria was determined to compare the antibiotic efficacy of the test substance. The antibiotic efficacy of stigmasterol was tested in the same assay. Only E. coli is susceptible to stigmasterol. The antibiotic activity of stigmasterol is previously reported by Sharma [17]. However, the antibiotic activity reported here is weak, judging on the size of inhibition zone (≤9 mm). The stigmasterol-ampicillin combination inhibits all test bacteria (Table 1). The combination showed significantly better (p ≤ .05) antibiotic activity on all bacteria tested. The two test substances have synergistic antibiotic activity. Zygmunt and Tavormina [18] first reported the interference of stigmasterol with polyene antibiotics. They noticed a mild antagonistic anti-Candida effect after addition of stigmasterol. Our results do not match their study as we noticed a significant synergistic antibiotic effect when stigmasterol was used with beta lactam antibiotics. The synergistic effect was observed in both Gram positive and Gram negative bacteria. Beta lactamase produced by the bacteria can be deactivated by adenylation, phosphorylation or acetylation of
2.3. Disc diffusion assay The assay was performed on Mueller Hinton agar (Merck) plates according to Panacek et al. [12]. Firstly, 100 µl of 106 cells/ml suspension was spread uniformly onto the agar plates. Then, 6 mm paper discs impregnated with ampicillin (Fisher) and stigmasterol alone at concentration 1 µg/disc, or in combination (1 µg/disc ampicillin + 1 µg/disc stigmasterol), were placed onto the inoculated medium. The plates were incubated at 37 °C for 24 h. The diameter of clear zone surrounding the paper disc were measured and recorded. 2.4. Antibiotic susceptibility test The minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of the test substances against all test bacteria were determined according to CLSI-recommended procedure described by Hamoud et al. [13]. The bacterial inoculum was diluted to 5 × 105 cells/ml with double strength Mueller Hinton broth (Merck). Ampicillin was serially diluted with ethanol and mixed with the bacterial inoculum in a sterile 96-well plate (Fisher). The tested concentrations of ampicillin were ranged from 3.1 to 200 µg/ml. A negative control was included by replacing ampicillin with ethanol for all the test bacteria. The plate was incubated at 37 °C for 24 h. After that, 40 µl of 0.2 mg/ml Iodonitrotetrazolium Violet (Sigma) was added into each well and incubated for 30 min as a growth indicator. The color change of the broth from yellow to pink indicates bacterial growth. The MIC is defined as the lowest concentration of the test compound to prevent bacterial growth (no color change). To judge the viability of bacterial cells, one loopful of the sample in each well was streaked on Muller Hinton agar plates. The plates were then incubated at 37 °C for 24 h. MBC is defined as the lowest concentration of the test compound to kill the bacterial cells. 69
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Table 1 Antibiotic efficacy of ampicillin and stigmasterol alone at concentration 1 µg/disc, or in combination (1 μg/disc ampicillin + 1 μg/disc stigmasterol) on disc diffusion assay. Test bacteria
Ampicilin
Stigmasterol
Combination
Chloramphenicol
Penicillin G
Negative control
S. aureus S. pyogenes E. coli P. aeruginosa
− − − −
− − + −
++ +++ ++ +
− + − −
− − − −
− − − −
The antimicrobial activity was determined based on the diameter of inhibition zone measured in mm. The result of screening test was recorded according to scale: +++ ≥18 mm, + + = 10–17 mm, + ≤9 mm, − = no inhibition zone observed. Table 2 The MIC, MBC and FIC index recorded for ampicillin alone, or in combination on Checkerboard Assay. Test bacteria
Ampicillin
Combination
MIC (µg/ ml)
MBC (µg/ ml)
MIC (µg/ ml)
MBC (µg/ ml)
S. aureus
> 200
> 200
3.13
6.25
S. pyogenes
> 200
> 200
1.56
3.13
E. coli
100
> 200
6.25
12.50
P. aeruginosa
> 200
> 200
6.25
25
Table 3 All test bacteria showed significant growth reduction with the combination therapy of stigmasterol and ampicillin.
FIC index
Test bacteria
S. aureus S. pyogenes E. coli P. aeruginosa
< 0.5, Synergism < 0.5, Synergism 0.125, Synergism < 0.5, Synergism
% of growth reduction (relative to ethanol control) Ampicillin
Stigmasterol
Combination
11.2 9.7 6.3 10.2
18.7 24.3 45.9 22.4
99.9 98.7 100 100
to ethanol control (Table 3). Mirroring the results of disc diffusion assay of stigmasterol, E. coli showed greatest percentage of growth reduction (45.9%) compared to other test bacteria, which indicates the high susceptibility of the bacterium to stigmasterol. We also noticed for stigmasterol and ampicillin alone, none of the bacterium showed greater than 3-log10 decrease in bacterial growth (bactericidal target). Comparatively, combination of stigmasterol-ampicillin greatly reduced the colony counts by 98.7–100%. The assay confirms that combining ampicillin with stigmasterol results in greater bacterial effect on all test bacteria. The results provide solid evidence of the synergistic antibiotic effect of stigmasterol and ampicillin. The combination shows bactericidal effect in this assay [22]. In conclusion, we found synergistic effects of stigmasterol and ampicillin in against beta lactamase producing clinical isolates. This finding is important as it shows potential application of stigmasterol as an antibiotic adjuvant. However, we do not have an explanation on the synergistic mechanism. To ascertain the mechanisms of drug synergy between stigmasterol and ampicillin, further investigations must be conducted to profile the changes in cellular environment after drug administration.
aminoglycosides in the beta lactam ring [9]. Stigmasterol may also acts as beta lactamase inhibitor, which restore their susceptibility to ampicillin. The antibiotic resistance can be caused by removal of antibiotics from the bacterial cells through membrane bound efflux pump protein [19]. Thus, we hypothesize that stigmasterol interference in the antibiotic activity can be essentially on a physiochemical basis, which involve the interference with protein pump. The susceptibility to antibiotic can be improved by blocking the mechanism of resistance. Elkins and Mullis [20] report that some steroids, including stigmasterol, affects the multiple drug efflux pump proteins in multidrug resistant E. coli, thus suppressing the emergence of resistance to first line antibiotics. The synergistic antibiotic effect of stigmasterol and ampicillin was evident by the FIC index (Table 2). In checkerboard assay, a low FIC index (< 0.5) indicates the synergism effect between two test substances [21]. Low FIC index was observed for all test bacteria. The results suggest that the combination of ampicillin and stigmasterol can be expected to act additively in the treatment of infections caused by betalactamase producing pathogens. In this assay, the concentration of stigmasterol was fixed as it acts as an antibiotic adjuvant to strengthen the efficacy of ampicillin. We observed differences between ampicillin and stigmasterol-ampicillin combination therapy. The MICs recorded for ampicillin alone was 100 µg/ml for E. coli, and > 200 µg/ml for others. The high MICs indicate high resistance to ampicillin. However, the MIC range for combination therapy was relatively wide, with a range of 3.13–6.25 µg/ml. The MIC and MBC of stigmasterol were > 200 µg/ml for all the test bacteria. We also noticed a significant improvement in ampicillin susceptibility when the stigmasterol was added as adjuvant, for all test bacteria. The MBC indicates the bacterial killing capability of the test substance. The stigmasterol-ampicillin combination showed significantly lower MBCs compared to ampicillin alone. We noticed that the combination showed bactericidal activity, based on the low MBC/MIC ratio. To further validate our results, a bacterial growth reduction assay was conducted. The bacterial growth was monitored in liquid medium supplemented with stigmasterol and ampicillin, either separately and jointly. The checkerboard and bacterial growth reduction assay results agreed for all test bacteria. Generally, ampicillin and stigmasterol alone exhibited very weak inhibitory effect on the bacterial growth, relative
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