Evaluating the Potentiating Effect of Galbanic acid from Ferula szowitsiana on three common antibiotics against resistant hospital isolates of Staphylococcus aureus

Authors

Abstract

The plant kingdom constitutes a source of new chemical compounds, which may be important due to their potential uses in medicine, or their other biological properties. In this study, the effects of Galbanic acid (GA), a sesquiterpene coumarin from roots of Ferula szowitsiana, were investigated as a modulator of antibiotic resistance in clinical isolates of Staphylococcus aureus. Isolates of S. aureus clinically resistant to methicillin, tetracycline and ciprofloxacin were isolated using disk diffusion method. The MICs of antibiotics were determined using broth macrotiter plate method against isolated bacteria. The inhibitory effects of GA alone and in combination with each antibiotic were investigated by macrotiter plate. None of the tested bacteria were affected by GA (up to 800 µg/mL). MICs of antibiotics against S. aureus (in µg/mL) were 10-80 for methicillin, 40->80 for tetracycline, and10-20 for ciprofloxacin. The MICs of antibiotics in presence of 400 µg/mL GA were less than 1.25 µg/mL for S. aureus, which is usually much less than the MICs against sensitive isolates. This study provides interesting results suggesting a potentiating activity of GA on antibiotics against the resistant strains of S. aureus.

Keywords


Acute and Subchronic Toxicity?of Teucrium polium Total Extract in Rats
Iranian Journal of Pharmaceutical Research  (2009), 8 (3): 217-221
Received: June 2008
Accepted: October 2008

Copyright ? 2009 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services

Original Article

Evaluating the Potentiating Effect of Galbanic acid from
Ferula szowitsiana on three common antibiotics against
resistant hospital isolates of Staphylococcus aureus

 

Bibi Sedigheh Fazly Bazzaza,b*, Ali Rezaei Dua, Mehrdad Iranshahia,b,
Mahboobeh Naderinasabc and Mehrangiz Khajeh Karamodind

aSchool of Pharmacy, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran. bBiotechnology Research Center, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran. cSchool of Medicine, Imam Reza Hospital, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran. dSchool of Medicine, Ghaem Hospital, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran.

 

Abstract

The plant kingdom constitutes a source of new chemical compounds, which may be important due to their potential uses in medicine, or their other biological properties. In this study, the effects of Galbanic acid (GA), a sesquiterpene coumarin from roots of Ferula szowitsiana, were investigated as a modulator of antibiotic resistance in clinical isolates of Staphylococcus aureus. Isolates of S. aureus clinically resistant to methicillin, tetracycline and ciprofloxacin were isolated using disk diffusion method. The MICs of antibiotics were determined using broth macrotiter plate method against isolated bacteria. The inhibitory effects of GA alone and in combination with each antibiotic were investigated by macrotiter plate. None of the tested bacteria were affected by GA (up to 800 ?g/mL). MICs of antibiotics against S. aureus (in ?g/mL) were 10-80 for methicillin, 40->80 for tetracycline, and10-20 for ciprofloxacin. The MICs of antibiotics in presence of 400 ?g/mL GA were less than 1.25 ?g/mL for S. aureus, which is usually much less than the MICs against sensitive isolates. This study provides interesting results suggesting a potentiating activity of GA on antibiotics against the resistant strains of S. aureus.

 

Keywords: Antibiotics; Resistant bacterial isolates; Galbanic acid; Potentiation; Staphylococcus aureus.

Introduction

Staphylococcus aureus is an important community and major hospital-acquired pathogen (1, 2). This organism is of considerable concern due to its ability to acquire resistance to antibiotics. The problem of resistant Gram-positive bacteria highlights the urgent need for new drugs or combination therapies to treat the infections caused by resistant pathogens.
The plant kingdom is a rapidly recognized source of new chemical compounds, which may be important due to their potential uses in medicine, or their other biological properties. Galbanic acid (Figure 1), a major plant sesquiterpene coumarin presents in Ferula szowitsiana, has been reported to have antibacterial enhancing effect on penicillin G and cephalexin against S. aureus (3). In addition, galbanic acid and its derivatives have been reported to possess anticoagulant and hepatoprotective activities (4).

 

 

In this paper, the effects of galbanic acid as a modulator of antibiotic resistance among clinical isolates of S. aureus are reported.

Experimental

Galbanic acid (GA)

This compound has been previously isolated from F. szowitsiana DC., a plant from Apiaceae family, and characterized (5). The plant was identified by Dr Akhani, Faculty of Sciences, University of Tehran, Tehran, Iran.

 

Confirmation of resistant isolates of S. aureus

Isolated samples of S. aureus (9 isolates) were obtained from the University Hospitals Imam Reza and Ghaem, Mashhad, Iran as resistant isolates. They were already subjected to disk diffusion method according to NCCLS (6) in the hospitals in order to obtain resistant strains. The antibiotics used for S. aureus susceptibility test were methicillin (30 ?g), tetracycline (30 ?g) and ciprofloxacin (5 ?g). Antibiotic disks were purchased from Padtan Teb, Iran.

 

Determination of the minimum inhibitory concentration of antibiotics or GA

Broth macrodilution method was carried out on 24 well tissue culture plates (Orange Scientific, E. U.). In two fold broth dilution method, each 0.1 mL of each bacterial cell suspension (107 cfu/mL) was added to each well already containing 1 ml of different dilutions of either antibiotics or galbanic acid (GA) in Mueller-Hinton Broth (MHB) (Himedia, India), plus 1 mL of MHB and then was mixed. Antibiotics were tetracycline, ciprofloxacin and methicillin (Sigma, Poole, UK).

In each plate, wells of negative control (2 mL media) for sterility and positive control (2 mL media +0.1 mL resistant/standard bacteria) were included. The Plates were incubated at 37?C for 18 h.

The presence or absence of growth of the microorganisms was assessed by MTT (2, 3, 5-tripheny tetrazolium chloride) assay (7). Briefly, 0.5 mL MTT (5 mg/mL) (Merck) was added to each well and the plates were incubated for 3 h at 37?C. The minimum inhibitory concentration (MIC) was defined as the lowest concentration able to inhibit any red dye production. The experiments were repeated two times, each sample in triplicate. According to NCCLS (8) for ciprofloxacin, if the MIC is more than or equal to 4 ?g/mL, the bacteria are considered resistant. This was 16 ?g/mL for tetracycline and methicillin against S. aureus.
 

Interaction of antibiotics and GA

Interaction studies were performed by a broth checkerboard method (9). The concentrations ranged from 1.25 ?g/mL to 80 ?g/mL for all antibiotics and from 100 ?g/mL to 400 ?g/mL for GA. The final bacterial inoculums in each well was 5?104 cfu/mL. The plates were incubated at 37?C for 18 h. The growth of microorganisms was assessed by MTT assay as described above. The experiments were repeated two times, each sample in triplicate.

Results

From different S. aureus isolates tested, 9 isolates were resistant to at least one of the antibiotics (ciprofloxacin, methicillin or tetracycline), and they were considered for the continuation of the experiments. These are listed in Table 1 with their source sample and gender of the patients.

 

 

All tested bacteria were resistant to GA (up to 800 ?g/mL). MICs of antibiotics (?g/mL) against S. aureus were as follows: 10-80 for methicillin, 40->80 for tetracycline, and10-20 for ciprofloxacin (Table 2). The effective concentrations of ciprofloxacin in combination with 100, 200 and 400 ?g/mL GA against S. aureus were 10-<1.25, 10-<1.25 and <1.25 ?g/mL, respectively (Table 3). The effective concentrations of methicillin in combination with 100, 200 and 400 ?g/mL GA against S. aureus, were 10, 5-<1.25 and <1.25 ?g/mL, respectively (Table 4). The effective concentrations of tetracycline in combination with 100, 200 or 400 ?g/mL GA against S. aureus were 80, <1.25 and <1.25 ?g/mL, respectively (Table 5).

 

 

 

 

Discussion

To overcome the emerging resistance problem, studies on a combination of plant extracts and antibiotics against clinical isolates have been reported (10-12). The present study shows that GA, a new sesquiterpene coumarin isolated from the acetone extract of F. szowitsiana roots, reduces the MIC of the antibiotics ciprofloxacin, methicillin and tetracycline against resistant isolates of S. aureus. GA alone had no inhibitory effect on tested isolates in concenctrations up to 800 ?g/mL (Table 2). Therefore, the observed effect in this case could be due to the antibiotic ? GA combination. While GA at 100 ?g/mL usually had no effect on MIC of antibiotics, increasing its concentration caused a decrease in MIC. This effect was concentration dependent.

As a group, coumarins have been found to stimulate macrophages, which could have an indirect effect on infections. Hydroxycinnamic acids, related to coumarins, seem to be inhibitory to Gram ? positive bacteria. However, data about specific antibiotic properties of coumarins are scarce, although many reports give reason to believe that some utilities may reside in these phytochemicals (13).

Studies on antibiotic activity modulation effects of an extract or a crude fraction of a plant are usually performed using sub-inhibitory concentrations of the materials. The maximum concentration of GA used in this report for its potentiating effect was 400 ?g/mL which is at least 3-fold lower than its MIC; as GA alone, even at 800 ?g/mL, had no effect on these isolates (Table 2). However, it is believed that a concentration of 4-fold lower than the MIC is suitable when performing a potentiating assay (14).

GA having no inhibitory action on S. aureus at 400 ?g/mL reduced the MIC of ciprofloxacin from 10-20 ?g/mL to less than 1.25 ?g/mL (8- to 16-fold reduction) which is close to the MIC against sensitive isolates (8). At 200 ?g/mL of GA, 50% of isolates became sensitive to the action of ciprofloxacin. This effect was not considerable at 100 ?g/mL of GA.

GA at 400 ?g/mL reduced the MIC of methicillin from 10-80 ?g/mL to less than 1.25 ?g/mL which is close to the MIC against sensitive strains. At 200 ?g/mL of GA, 50% of isolates became sensitive to the action of methicillin. This effect was not considerable at a concentration of 100 ?g/mL.

At 200 ?g/mL GA, the MIC of tetracycline against S. aureus was reduced from 40- 80 ?g/mL to less than 1.25 ?g/mL, i.e. 32- to 64-fold reduction in MIC.

This study shows that GA could act as a plant derived modulator of antibiotic activity on resistant isolates of S. aureus. The mechanism of this sensitization effect is not clear and merits investigation. A general role for sesquiterpenoid compounds as enhancers of nonspecific bacterial permeability to antibiotics and antimicrobials have been suggested (15). There is evidence on inhibition of efflux pump proteins by sesquiterpene coumarins (16). A coumarin derivative has been characterization after fractionation of grapefruit oil, which has been shown to enhance the activity of ethidium bromide and norfloxacin (17). This study provides interesting information on potentiating effect of GA on antibiotics against the resistant strains of S. aureus. Since all these three antibiotics can act as substrates for some MDR pumps of the pathogens (18), and GA as a sesquiterpene coumarin, reduced the MICs of these antibiotics against S. aureus isolates, this effect may be due to the inhibition of efflux pump proteins thus preventing extrusion of antibiotics. However, this hypothesis needs more investigation.

Acknowledgement

This study was supported by a grant from Deputy of Research, Mashhad University of Medical Sciences, Mashhad, I R Iran (Project no. 84175).

References

        (1) Levy SB. Active efflux mechanisms for antimicrobial resistance. Antimicrob. Agents Chemother. (1992) 36: 695-703.
(2) Nikaido H. The role of outer membrane and efflux pumps in the resistance of Gram-negative bacteria. Can we improve drug access? Drug Research Updates (1998) 1: 93-98.
(3) Shahverdi AR, Fakhimi A, Zarrini Gh, Dehghan Gh and Iranshahi M. Galbanic acid from Ferula szowitsiana enhanced the antibacterial activity of penicillin G and cephalexin against Staphylococcus aureus. Biol. Pharm. Bull. (2007) 30: 1805-1807.
(4) Grably S and Thiericke R. Drug Discovery from Nature. Springler-verlag, Berlin (1999) 99.
(5) Iranshahi M, Arfa P, Ramezani M, Jaafari MR, Sadeghian H, Bassarello C, Piacente S and Pizza C. Sesquiterpene coumarins from Ferula szowitsiana and in vitro antileishmanial activity of 7-prenyloxycoumarins against promastigotes. Phytochem. (2007) 68: 554-561.
(6) National Committee for Clinical Laboratory Standards, Performance standards for antimicrobial susceptibility testing. Tenth informational supplement (Disk Diffusion). (NCCLS), M100-S10 (M): (January 2000): 14-21.
(7) Eloff JN. A sensitive and quick method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med. (1998) 64: 711-713.
(8) National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing. 14th informational supplement. Document M100-S14. NCCLS Publisher, Wayne (2004).
(9) Eliopoulus GM and Moellering RCJ. Antimicrobial combinations. In: Lorian V. (ed.) Antibiotics in Laboratory Medicine, 4th Ed. The Williams & Wilkins Co. Baltimore (2007) 330-396.
(10) Gibbons S. Plants as a source of bacterial resistance modulators and anti-infective agents. Phytochem. Rev. (2005) 4: 63-78.
(11) Aqil F, Khan MS, Owais M and Ahmad I. Effect of certain bioactive plant extracts on clinical isolates of beta-lactamase producing methicillin resistant Staphylococcus aureus. J. Basic Microbiol. (2005) 45: 106-114.
(12) Ahmad I and Aqil F. In vitro efficacy of bioactive extracts of 15 medicinal plants against ESβL-producing multidrug-resistant enteric bacteria. Microbiol. Res. (2007) 162: 264-75.
(13) Cowan MM. Plant products as antimicrobial agents. Clin. Microbiol. Rev. (1999) 12: 564-582.
(14) Stavri M, Piddock LJV and Gibbons S. Bacterial efflux pump inhibitors from natural sources. J. Antimicrob. Chemother. (2007) 59: 1247-1260.
(15) Brehm-Stecher BF and Johnson EA. Sensitization of Staphylococcus aureus and Escherichia coli to antibiotics by sesquiterpenoids neroliodol, farnesol, bisabolol and apritone. Antimicrob. Agents Chemother. (2003) 47: 3357-3360.
(16) Madureira AM, Molnar A, Abreu PM, Molnar J and Ferreira MU. A new sesquiterpene-coumarin ether and a new abietane diterpene and their effect as inhibitors of P-glycoprotein. Planta Med. (2004) 70: 828-833.
(17) Abulrob AN, Suller MTE, Gumbleton M, Simons S and Russell AD. Identification and biological evaluation of grapefruit oil components as potential novel efflux pump modulators in methicillin-resistant Staphylococcus aureus bacterial strains. Phytochem. (2004) 65: 3021-3027.
(18) Lomovskaya O and Watkins WJ. Efflux pumps: their role in antibacterial drug discovery. Cur. Med. Chem. (2001) 8: 1699-1711.