WO2007088408A1

WO2007088408A1 - A method of curing antibiotic resistant plasmids

Info

Publication number: WO2007088408A1
Application number: PCT/IB2006/000628
Authority: WO
Inventors: Latha Chandramathi; Varsha Dubayya Shriram; Supada Rambhau Rojatkar; Sheetal Shripad Jahagirdar; Prashant Kamalakar Dhakephalkar
Original assignee: Council Of Scientific And Industrial Research; Agharkar Research Institute
Priority date: 2006-02-03
Filing date: 2006-03-22
Publication date: 2007-08-09

Abstract

The subject of our invention is to identify a novel plasmid curing/antiplasmid agent from the rhizomes of Alpinia galanga (L.) Swartz. The compound used in the present invention I 'acetoxychavicol acetate (ACA) with a formula C13H14O4 is not known before as an antiplasmid agent. ACA is capable of reducing the minimal inhibitory concentration (MIC) of antibiotic required to inhibit growth of bacteria thus making the antibiotic treatment more effective in vivo and in vitro. It is capable of curing plasmids from bacterial host by reducing the copy number of plasmids in the daughter cells or by completely eliminating the plasmids in the daughter cells. Thus, ACA has a potential of eliminating or curing antibiotic resistance in bacterial cells making the bacterial population sensitive to antibiotic.

Description

A METHOD OF CURING ANTIBIOTIC RESISTANT PLASMIDS

FIELD OF THE INVENTION

This invention relates to a method of curing antibiotic resistant plasmids. More particularly, the invention relates to a method of curing antibiotic resistance in bacteria and a composition therefor. It further relates to said composition comprising l '-acetoxychavicol acetate [ACA] optionally along with other bioactive agents such as antibiotics. The ACA in this composition can be derived from the rhizomes of Alpinia galanga (L.) Swartz used against different strains of commercially available bacteria. The method relates to administration of the antiplasmid agent ACA which has the ability to reduce number of plasmids from the bacterial cell thereby leading to the loss of plasmids from bacterial cells and sensitization of the bacteria to the drugs to which they were previously resistant.

BACKGROUND OF THE INVENTION

The euphoria produced by the discovery of penicillin during the Second World War led to the discovery of many antibiotics. Since then antibiotics have been the mainstay of treatment of infections caused by bacteria. However, despite the diverse type of antibiotics and the varied modes of action, many bacteria have developed means of resistance to the antibiotics. Multi drug resistant strains of bacteria such as Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Enterococcus (VRE) were first discovered in hospital settings, but many of them are to be identified infecting healthy individuals outside hospital atmosphere. The spread of VRE is particularly concerning when it is taken in to account that vancomycin is generally referred as the last line of defense against bacteria.

Most of the genetic determinants that confer resistance to antibiotics are located on plasmids or extra-chromosomal elements (Bloomfield, S.F., 2002, J. Applied Microbiol., 92, 144-157). Bacterial plasmids not only confer resistance to antibiotics but also pathogenicity to bacterial cells, which is particularly true for enteropathogens (Saunders, J.R., 1981, Nature, 290, 674-675). The antibiotic resistance plasmids allow the multiplication of bacteria in presence of antibiotics. The daughter cell will inherit this resistance, and this self transmissible property can be transferred to other kind of bacteria, which live in environment by so called horizontal gene transfer (Falknow, S., 1975, Infectious Multiple Drug Resistance). Such epidemic spread of antibiotic resistance in human flora has been reported in several cases (Molnar, J., 1988, Meth. and Find. Exptl. Clin. Pharmacol., 10, 467-474). Such antibiotic resistant bacteria can be transferred from animals to animals and from animals to people. In addition, transmissible antibiotic resistance also spreads in soil and water (Trevors, J.T. and Oddie, K.M., 1988, Can. J. Microbiol., 32, 610-614).

The impact of bacterial resistance to antibiotics on therapy is very serious since in vivo transfer can occur and this makes antibiotic treatment insufficient or inadequate. Theoretically in such cases new antibiotic can be effective. Alternatively, an already ineffective antibiotic will become effective if R- plasmid encoding antibiotic resistance is removed from the bacterial population. There exist compounds known as antiplasmid drugs or plasmid curing agents, which inhibit plasmid replication. Thus after multiplication of bacteria, the new bacterial cells will be plasmid free. Curing agents can be applied at concentrations that do not kill bacteria but destabilizes plasmids. Various physical and chemical agents called as curing agents have been used to increase the elimination rate of R-plasmids (Trevors, J. T., 1986, FEMS Microbial Rev., 32, 149-157). The currently available curing agents are toxic to both human beings and bacteria. It is known that acridine orange, ethidium bromide and sodium dodecyl sulphate (SDS) affect plasmid replication. However, acridine dyes and ethidium bromide cannot be used for clinical purpose because of their mutagenicity and toxicity (Amabile-Cuevas, CF. , and Heinemann, J.A., 2004, Drugs Discovery Today, 9 (11), 465-467).

There was a turning point in plasmid elimination experiments when it was observed that two well-known medicines applied in everyday medical practice, chlorpromazine and promethazine eliminated the tetracycline and sulphonamide resistance of an E. coli with a frequency of 30% (Molnar, J., Kirlay, J., and Mandi, Y., 1975, Experentia, 31, 444-447). The observation was theoretically important since it gave rise to the possibility of a basically new type of combination between antibiotics and potential drugs effective against plasmids. Thus the science and circumstances have changed dramatically for the identification of new era of plasmid curing agents with less toxicity and side effects. Higher plants represent a tremendous source of bioactive molecules with variety of applications. Mankind has recognized plants as renewable source of chemicals even before the dawn of the civilization. Most of the earlier natural product research was directed towards medicinally important plants. The ethnobotanical pharmacological knowledge accumulated over the generations has been serving as a base of development of pharmaceuticals of great importance (Hostettmann, K., Potterart, O. and Wolfender, J.L., 1997, Medicinal Chemistry: Today and Tomorrow, Proceedings of the International Medicinal Chemistry Symposium, 3-8, Sept., Tokyo, Japan).

The contribution of traditional medicine to human health in the 21^st century is of immense importance. The importance of medicinal plants and the constant need for phytochemical remedies have been discussed often in many international forums

(WHA, 1991 WHO Policy System Eighty-Seventh Session EB 87.R24, Traditional

Medicine and Modern Health Care, Geneva, January 14-25^th). Many countries of the world have a well-established system of traditional medicine. More than 80% of world population use plants as their primary source of medical care (WHO, 1995, Traditional

Practioners as Primary Health Care Workers, WHO/SHS/DHS/TRM/95.6, Geneva).

Traditional remedies have always been a source of important antibiotics and continue to provide novel effective treatments. The immense potential of natural products due to their unlimited bio- and chemo diversity is being increasingly realized by the chemists, biotechnologists and biologist and has created a new awareness in the field of drug discovery.

Researchers have carried out extensive chemical investigations on A. galanga. The important class of chemicals in A. galanga is aromatic compounds. The quantitatively dominating compound of this class is l'-acetoxychavicol acetate (Mitsui, Kobayashi, S., and Nagahora, H., 1976, Chem. Pharm. Bull., 24, 2377-2382; De Pooter, H.L., Omar, M.N., Coolsart, B. A., and Schamp, N.M., 1985, Phytochemistry, 24, 93-96; Bank, B.R., Kundu, A.B., and Dey, A.K., 1987, Phytochemistry, 26, 2126-2127).

It was widely accepted that l'-acetoxychavicol acetate possesses anti ulcer (Mitsui,

Kobayashi, S., and Nagahora, H., 1976, Chem. Pharm. Bull., 24, 2377-2382), anti tuberculosis (Palittapongarnpim, P., Kirdmanee, C, Kittakoop, P., Prathumthani,

Rukseree, K., US Patent No. US 2002192262), anti HIV (YE, Y., Patent CN 200300130876), anti oxidative (Kubota, K., Ueda, Y., Yasuda, M., and Masuda, A., 2001, Food Flavors and Chemistry, Spec. Publ.-R. Soc.Chem., 274, 601-607) and insecticidal (Lee, S., and Ando, T., 2001, J. Pestc. ScL, 26, 76-81). Pharmacological properties of l'-acetoxychavicol acetate have been thoroughly studied by many scientists all over the world. It is reported to inhibit various fungi (Janssen, A.M., and Scheffer, JJ., 1985, Planta Med., 16, 507-511). 1' -acetoxychavicol acetate can inhibit the function of the enzymes, xanthine oxidase and NADPH oxidase, involve superoxide anion production which is one of the spontaneously occurring toxic substances in the body (Noro, T., Sekiya, T., Katoh, M., Oda, Y., Miyase, T., Kuroyanagi, M., Ueno, A., and Fukushima, S., 1988, Chem. Pharm. Bull, 36, 244-248; Nakamura Y.,Marukami, A., Ohto, Y., Torikai, K., Tanaka, T., and Ohigashi, H., 1998, Cancer Res., 58, 4832- 4839). This compound can inhibit the activation of tumor virus such as Ebstein-Barr virus (Marukami A., Toyota, K., Ohura, S., Koshimizu, K., and Ohigashi, H., 2000, J. Agric.Food Chem., 48, 1518-1523 ). It was observed that l'-acetoxychavicol acetate can inhibit formation of many tumor and cancer (Murakami, A., Ohura, S., Nakamura, Y., Koshimizu, K., and Ohigashi, H., 1996, Oncology, 53, 386-391; Ohnishi, M., Tanaka, T., Makita, H., Kawamori, T. Mori, H., Satoh, K., Hara, A., Murakami, A., Ohigashi, H., and Koshimizu, K., 1996, Jpn. J. Cancer Research, 87, 349-356; Tanaka, T., Makita, H., Kawamori, T., Kawabata, K., Mori, H., Murakami, A., Satoh, K., Hara, A., Ohigashi, H., and Koshimizu, K., 1997, Carcinogenesis, 18, 1113-1118; Kobayashi, Y., Nakae, D., Akal, H., Kishida, H., Okajima, E., Kitayama, W., Denda, A., Tsujiuchi, T., Murakami, A., Koshimizu, K., Ohigashi, H., and Konishi, Y., 1998, Carcinogenesis, 19, 1809-1814). Thus, it has been well established that l'-acetoxychavicol acetate exhibits a wide spectrum of bioactivities.

The fact that A. galanga is abundant in India and contains significantly high quantity of

1 '-acetoxychavicol acetate as one of the ingredients, prompted the inventors to select this molecule as an ideal bioactive compound to study for its plasmid curing efficiency. Furthermore the toxicological studies conducted by the crude extract of the rhizomes of A. galanga did not induce any acute toxic effects in mice even at the dose as high as 3g/kg body weight and did not show any chronic toxicity when given to mice at the dose of 100 mg/kg body weight for 90 days (Qureshi, S., Shah, A.H., and Ageel, A.M., 1992, Planta Med., 58 (2), 124-127). In addition, studies carried out by Ohnishi, M., Tanaka, T., Makita, H., Kawamori, T., Mori, H., Satoh, K., Hara, A., Murakami, A., Ohigashi, H., and Koshimizu, K., 1996, Jpn. J. Cancer Research, 87, 349-356; Tanaka, T., Makita, H., Kawamori, T., Kawabata, K., Mori, H., Murakami, A., Satoh, K., Hara, A., Ohigashi, H., and Koshimizu, K., 1997, Carcinogenesis, 18, 1113-1118, and Tanaka, T., Kawabata, K., Kakumoto, M., Makita, H., Matsunaga, K., Mori, H., Satoh, K., Hara, A., Murakami, A., Koshimizu, K., and Ohigashi, H., 1997, Jap. J. Cancer Res., 88, 821-830, showed that ACA exhibited no detectable toxicity or marked body weight retardation in rodents by oral feeding. Such activity and toxicity profiles suggest that the mode of action of ACA is rather specific to biological systems rather than simple and non specific interaction with any nucleophilic groups of cellular components.

OBJECTS OF THE INVENTION

The main object of the present invention is to provide a method of curing antibiotic resistant plasmids in commercially available antibiotic resistant bacterial strains.

Another object of the present invention is to provide and a composition for curing the said antibiotic resistant plasmids in bacterial strains.

Still another object of the present invention is to provide a method that can cure antibiotic resistance in the pathogenic bacteria with curing efficiency ranging up to 92%.

A further object of the present invention is to provide l'-acetoxychavicol acetate, major component in A. galanga (L.) Swartz., as an antiplasmid drug or curing agent capable of eliminating or curing antibiotic resistance and plasmids from the bacterial strains tested.

Accordingly, the present invention provides a method of curing plasmids in commercially available antibiotic resistant bacterial strains characterized in that the compound l'-acetoxychavicol acetate is utilized for curing the plasmids, the said method comprising, growing an antibiotic resistant bacterial strain in the presence of 25 to 800 μg/ml of 1 '-acetoxychavicol acetate dissolved in an organic solvent optionally along with 4 to 800 μg/ml of an antibiotic to which the bacterial strain is resistant for a period of 24 to 72 hours at a temperature ranging from 30 to 37 degree C. In an embodiment the present invention provides a method of curing antibiotic resistant plasmids in commercially available antibiotic resistant bacterial strains by administering in the conventional forms the composition as claimed herein to the patients suffering from single/multiple drug resistance.

In a further embodiment of the present invention any antibiotic resistant plasmid can be cured by the process of the present invention.

In still another embodiment of the present invention the bacterial strains are grown in the presence of 25 to 800 μg/ml of l '-acetoxychavicol acetate dissolved in an organic solvent optionally along with 4 to 800 μg/ml of an antibiotic to which the bacterial strain is resistant.

In yet another embodiment of the present invention the antibiotic resistant bacterial strain is any commercially available antibiotic resistant bacterial strain.

In another embodiment of the present invention the antibiotic resistant bacterial strain is grown at a temperature preferably 37 degree C for a period of 24hours. In still another embodiment of the present invention l'-acetoxychavicol acetate may be obtained either from synthetic or natural sources.

In yet another embodiment of the present invention the curing efficacy of l'- acetoxychavicol acetate against the antibiotic resistant bacterial strains ranges up to 92%. In a further embodiment of the present invention the composition may be in the form of means orally administrable, injectable, or in a suspension form in pharmaceutically acceptable methods.

DETAILED DESCRIPTION OF THE INVENTION l'-acetoxychavicol acetate is capable of curing plasmid from bacterial host by reducing the copy number of plasmid in the daughter cells or by completely eliminating the plasmid from the daughter cells. This compound is even capable of eliminating or curing antibiotic resistance in bacterial cells making the bacterial population sensitive to antibiotic. ACA can reduce the MIC of antibiotic required to inhibit growth of bacteria thus making the antibiotic treatment more effective in vivo and in vitro. For this purpose the crude acetone extract derived from the rhizomes of A. galnaga (L.) Swartz, was screened on different bacterial strains to see the antibacterial and antiplasmid effect.

Further, the above mentioned composition of this compound can be employed for commercial production of new class of antibiotics and/or plasmid curing agents. Simple extraction and chromatography can be employed for its commercial production. In relation to the present invention, the term l'-acetoxychavicol acetate refers the compound obtained from any variety of Alpinia galanga or Languas galanga found any where in the world. The rhizomes of A. galanga were chopped and dried. Raw material for the crude extract was prepared from the dried rhizomes. This was dried in shade or under controlled temperature at 40° C. The dried rhizomes were crushed or pulverized or powdered. The above material was used immediately or stored at room temperature for 2 to 5 months before extraction.

Preparation of Crude Acetone Extract

The crude extract was prepared from 1.5 kg of above mentioned material in 2.5 L of acetone by cold percolation for 12 hr in a 5 L flat bottom flask at room temperature. The process of extraction was repeated five times with acetone. Each time the filtrate was concentrated in vacuo at 40⁰C and pooled together to obtain 56.25 gm of reddish syrup.

Bacterial strains and plasmids

Clinical isolates used in this invention were Salmonella typhi, Shigella flexnerii, Escherichia coli, Pseudomonas sp., Enterococciis faecalis and Staphylococcus aureus.

Also Bacillus cereus was used for this study. In addition to these strains a few reference plasmids were also used to test the putative curing agents. These bacterial hosts harboring plasmids were Bacillus subtilis (pUBHO), E. coli (RP4), E. coli (pKT230),

E. coli (pCHlOO), and E. coli (pUC18). The cultures were identified on the basis of morphological, cultural and biochemical characters according to the Bergey's Manual of Systematic bacteriology. The identity of cultures was further confirmed on the basis of the 16S rRNA gene sequencing. Antiplasmid Testing of the Crude Extract

The plasmid curing was performed as elucidated as follows. The cultured bacterial cells were grown in nutrient medium (Luria broth) in the presence of crude acetone extract at the specified concentration (25 - 800 μg/ml) for 24 h at 37 ⁰C and then plated on Luria agar plates to obtain isolated colonies. The isolated colonies were then replica plated simultaneously on to Luria agar and Luria agar containing antibiotics. The colonies that failed to grow in presence of antibiotics were considered as putative cured derivatives. The physical loss of plasmid in the cured derivative was confirmed by agarose gel electrophoresis of the plasmid DNA preparation of respective cultures. The percentage curing efficiency was expressed as number of colonies with cured phenotype per 100 colonies tested. Vancomycin sensitive cured colonies were also tested for loss of resistance to other antibiotics by disc diffusion assay.

Determination of Resistance to Antibiotics Antibiotic resistance profile was determined by the disc diffusion method as follows. About 10⁴ cells from overnight grown culture were spread on Luria agar plates. Multidiscs containing antibiotics (Don Whitley Scientific Equipments, Mumbai, India) were placed on the plates. The zones of inhibition around the antibiotic discs were measured after incubation at 37°C for 24 h. The cultures were then assigned as resistant or sensitive by referring to the manufacturer's interpretation table.

Plasmid Isolation

The cells used for plasmid isolation were first washed with 50 mM Tris hydrochloride buffer (pH 8.0). The cells were sedimented again, suspended in ice-cold acetone and kept on ice for 5 minutes. The cells were then resedimented, acetone was decanted, and residual acetone was removed with gentle stream of air. Plasmid DNA isolation was carried out by alkali lysis method (Sambrook, J., Fritsch, T., Maniatis, T., 1989, Molecular Cloning: A Laboratory Mannual, Second Ed. Cold Spring Harbour Laboratory Press). DNA was detected by horizontal agarose gel (0.7%) electrophoresis using Tris-boric acid-EDTA buffer (pH 8.0). As shown in Table 1, it was observed that the crude acetone extract was able to cure the plasmids of vancomycin resistant E. faecalis with an efficiency of 8%, gentamycin resistant S. typhi with an efficiency of 92% and E. coli with 82%. Even though, the curing efficiencies were low, this was very interesting observation.

Purification of l'-Acetoxychavicol Acetate

It was observed that the acetone extract of the rhizome of A. galanga exhibited antiplasmid activity. The inventors further analyzed the extract by GC with FID detector in order to confirm the purity. It was noted that a compound with the retention time of 10.54 minute comprised 87% of the crude. The retention time was matched with the reported retention time of l'-acetoxychavicol acetate.

First Chromatography

The crude acetone extract of 20 gm was chromatographed on a silica gel column. This was eluted with a stepwise gradient of hexane-acetone. The fractions were collected separately and concentrated in vacuo at 40⁰C. Similar fractions in the TLC were pooled together to obtain 5 fractions: LA (7.18 gm), 2.A (6.11 gm), 3.A (4.4 gm), 4.A (1.2 gm) and 5.A (1.09 gm). The high yield fractions, LA and 2.A, were further analyzed by

GC with FID detector. Inventors observed that these fractions were showing substantial quantity of 1 '-acetoxychavicol acetate with a retention time of 10.54 minute.

Second Chromatography

The LA of 7 gm was chromatographed on a silica gel column with hexane and acetone solvent systems. The fractions obtained were concentrated in vacuo at 40⁰C. According to the TLC pattern similar fractions were pooled together to obtain LlA (0.37 gm), 1.2A (4.87 gm), 1.3A (0.52 gm), 1.4A (0.32 gm), 1.5A (0.30 gm), 1.6A (0.21 gm) and 1.7 A (0.32 gm). It was found that the fraction 1.2A with the yield of 4.87 gm was pure with Rf value of 0.61 in a solvent system hexane: acetone (80:20).

The fraction 2A of 6 gm was rechromatographed on a silica gel column. The column was eluted with gradient of hexane and acetone. The fractions obtained were concentrated in vacuo at 40⁰C. Each fractions were checked by TLC and similar fractions were pooled together to obtain 2.1A (0.47 gm), 2.2A (3.83 gm), 2.3A (0.71 gm), 2.4A (0.21 gm), 2.5A (0.4 gm), 2.6A (0.28 gm). The fraction 2.2A with the yield of 3.83 gm was highly pure with Rf value of 0.61 similar to 1.2A isolated in the present invention.

It was found that according to TLC, ¹H NMR and ¹³C recorded on DRX-500 (Broker) MHz₁ NMR spectrometer in CDCl₃ solution with TMS as an interval standard, the fractions 1.2A and 2.2A were similar and the structure is elucidated as 1'- acetoxychavicol acetate. The Rf value of 0.61 was recorded by Camag Linomat IV HPTLC with Camag TLC Scanner -3, in hexane and acetone (80:20). Chemical shifts recorded were relative to residual CDCI₃ at 77.0 ppm for ¹³C and TMS at 0.00 ppm for ¹H. The IR spectrum was recorded on Perkin Elmer FTIR spectrometer. The specific rotation recorded on JASCO P 1020 in chloroform was [oto] -55.85. The purity of the compound was 99% checked by GC with FID detector.

The ¹H NMR yielded the following chemical shifts (δ in ppm): 2.01s and 2.30s (2CH₃), 5.17dd (J=17.0, 10.0 Hz, H-3'), 5.89ddd (J=I 7, 10, 6 Hz, (U-T), 6.18d (J= 10.0 Hz, H- 1'), 7.0Od (J= 8.5 Hz, H-3, H-5) and 7.25d (J= 8.5 Hz, H-2, H-6). The ¹³C NMR of the compound gave 13 carbon signals or chemical shifts (δ) as follows in (ppm): 135.8s (C- 1), 120.83d (C-2), 127.45d (C-3), 149.76s (C-4), 120.83d (C-5), 127.45d (C-6), 74.56d (C-I '), 135.84d (C-2'), 116.45t (C-3'), OCOCH₃: 20.42q, 20.51q and OCOCHs: 168.10s and 168.68s. The structure was further confirmed by COSY, NOESY and HETCOR. The presence of ester carbonyl groups was confirmed by IR spectrum at 1741 and 1750 cm^"1. UV absorption maxima in chloroform were 246 nm and 262 nm.

Gas chromatographic mass spectrum data showed that the compound gave molecular iron (M⁺) at m/z 234 with base peak at m/z 132. The fragments at m/z 192 (M⁺-42) and m/z 174 (M⁺ -60) were due to loss Of CH₂=C=O and acetic acid, respectively. The loss of [M⁺-(OCO(CH₃)+^]^"1" is shown at m/z 174. The base peak at m/z 132 is from the loss of (M⁺-(OCOCH₃ )+(O=C=CH₂)+. From these spectral data, the active compound isolated from A. galanga was identified as l'-Acetoxychavicol acetate (ACA) with molecular formula Of Ci₃HnO₄.

The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention. Example 1

Antiplasmid Activity of the Composition

The composition of ACA and antibiotics were dissolved in DMSO and tested for antibacterial and plasmid curing activities in commercially available bacterial starins of S. aureus # 94, E. faecalis, B.cereus, S. typhi, P. aeruginosa, S. soneii, E. coli, and B. subtilis (pUBHO), and E. coli (RP4) on Luria agar plates with concentrations ranging from 25μg/ml and 800 μg/ml. Antibiotic resistance profile was determined by disc diffusion method. The colonies that failed to grow in presence of antibiotics were considered as putative cured derivatives. The physical loss of plasmid in the cured derivative was confirmed by agarose gel electrophoresis of the plasmid DNA preparation of respective cultures. The percentage of curing efficiency was expressed as number of colonies with cured phenotype per 100 colonies tested. Table 3 describes the Minimal Inhibitory Concentration (MIC) and Sub Inhibitory Concentration (SIC) of this compound against several clinical isolates. Table 4 depicts the MIC and SIC of the antibiotics against the used bacterial strains. However, the SIC of the antibiotics was used for the purposes of the plasmid curing experiments.

Plasmid isolation

Cells for Plasmid isolation were first washed with 5OmM Tris hydrochloride buffer (pH 8.0). The sedimented cells were again suspended in ice cold acetone and kept on ice for 5 minutes. The cells were then resedimented, acetone was decanted, and residual acetone was removed with gentle stream of air. Plasmid DNA isolation was carried out by alkali lysis method. DNA was detected by horizontal agarose gel (0.7%) electrophoresis using Tris-boric acid EDTA buffer (pH 8.0)

Table 1: Bacterial strains and plasmids used

a- MACS Collection of Microorganisms, Agarkar Research Institute, G.G Agarkar Road, Pune-11 004. India.

b- Microbial Type Culture Collection, Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036. India.

Table 2: Curing of Antibiotic Resistance by Crude Acetone Extract of A. galanga

MIC SIC Curing Resistance cured

Strain

(μg.mr¹) (μg.ml efficiency *

S. aureus #94 800 400 0% —

E. faecalis 800 400 8% Vancomycin

B. cereus 800 400 0% —

S. typhi 800 400 92% Gentamicin

Ps. aeruginosa 800 400 0% —

Sh. soneii 800 400 0% --

E. coli 800 400 82% Gentamicin

PUBIlO >800 800 0% —

RP4 800 400 0% ~

@: Only the resistance phenotypes that were encoded by plasmid borne gene(s) in specific strains were tested.

Table 3: Curing of Antibiotic Resistance by ACA

*. :- Average of two independent experiments

The results were tabulated as shown in the Table 3. It was observed that the composition had an ability to eliminate antibiotic resistance in pathogenic bacteria with curing efficiency ranging between 6% and 75% when ACA is used in a pure form. However, the curing efficiency is up to 92% when ACA is used as ac crude extract [Table 2]. The concentrations of the composition used for plasmid curing in this invention were sub inhibitory concentrations. Thus, the bacteria were resistant to composition and therefore the question of bacteria developing resistance against them does not arise. It may be noted here that the efficiency of curing of antibiotic resistance by crude extracts of A. galanga, observed in S. typhi and E. coli were higher as compared to those observed with pure compound (Tables 1, 2). This observation may be due to the presence of additional compound(s) in crude extract, which are involved in curing of antibiotic resistance.

Table 4: MIC and SIC of antibiotics against bacterial strains.

* SIC concentration of antibiotic were taken for curing experiment.

ADVANTAGES OF THE INVENTION

The triumph of antibiotics over disease causing bacteria is one of the greatest success stories of modern medicine. After more than 50 years of wide spread of antibiotics use most of the bacteria have developed Multi Drug Resistance (MDR). Development of resistance in bacteria to multiple antibiotics leaves medical community with few therapeutic options. Today virtually all important bacterial infections throughout the world are becoming resistant to even strongest antibiotics. Now antibiotic resistance has been called one of world's most pressing public health problems. At the center of current concern is the antibiotic vancomycin, which is literally the last resort drug for many infections. The spread of VRE is particularly concerning when it is taken to account that vancomycin is generally referred as the last line of defense against bacteria. Thus, inexorable rise in antibiotic resistant bacteria and comparative dearth of new therapeutic agents needs to be taken seriously. One of the ways to overcome this problem is to eliminate genes encoding resistance in bacteria. This is the context of the present invention directed towards the identification of novel therapeutic plasmid curing agent. The antibiotic resistance in the pathogenic bacteria could be cured by composition of ACA with curing efficiency ranging between 6% and 75%. The frequency of spontaneous mutation was found to be less than one in 10⁸ cells. In comparison plasmid curing efficiencies obtained in the current invention were extremely high. Since the concentration applied in the present study is sub inhibitory concentrations there will be no possibility of bacteria developing resistant against the composition of ACA. Subsequently it is proposed that bacteria will not develop any mechanism to counter the plasmid curing property of ACA and its composition.

Most of the currently available plasmid curing agents are toxic to bacteria as well as human beings. The compound of the present invention l'-acetoxychavicol acetate exhibits a broad array of bioactivities in vivo and in vitro. Based on some of the previous studies of the toxicological nature of this compound it can be assumed that

ACA exhibits no detectable toxicity or marketed body weight retardation in rodents by oral feeding. Such activity and toxicity profiles suggest that the mode of ACA is rather specific to biological systems.

Thus, in conclusion, we may say that this investigation has provided a novel curing agent that has the ability to cure plasmid mediated resistance to multiple antibiotics. Hence it will have a tremendous potential in containing spread of antibiotic resistance especially in nosocomial environment.

Finally in the commercial and economical point of view the plasmid curing molecule of the present invention provides a tremendous potential to scale up while considering the abundance of A. galanga as a raw material and the presence of high percentage of l'- acetoxychavicol acetate in the rhizomes.

Claims

We claim:

1. A method of curing antibiotic resistant plasmids in commercially available antibiotic resistant bacterial strains characterized in that the compound T- acetoxychavicol acetate is utilized for curing the plasmids, the said method comprising, growing an antibiotic resistant bacterial strain in the presence of 25 to 800 μg/ml of 1' -acetoxychavicol acetate dissolved in an organic solvent optionally along with 4 to 800 μg/ml of an antibiotic to which the bacterial strain is resistant for a period of 24 to 72 hours at a temperature ranging from 30 to 37 degree C.

2. A method as claimed in claim 1, wherein the antibiotic resistant bacterial strain is any commercially available antibiotic resistant bacterial strain.

3. A method as claimed in claim 1, wherein 1 '-acetoxychavicol acetate may be obtained either from synthetic or natural sources.

4. A method as claimed in claim 1, wherein l'-acetoxychavicol acetate is preferably obtained from the rhizomes of Alpinia galanga.

5. A method as claimed in claim 1, wherein l'-acetoxychavicol acetate is used either in the pure form or as a crude extract.

6. A method as claimed in claim 1, wherein the organic solvent is selected from acetone or chloroform.

7. A method as claimed in claim 1, wherein the antibiotic resistant bacterial strain is grown at a temperature preferably 37 degree C.

8. A method as claimed in claim 1, wherein the antibiotic resistant bacterial strain is grown for a period preferably 24 hours.

9. A method as claimed in claim 1, wherein the curing efficacy of l'- acetoxychavicol acetate against the antibiotic resistant bacterial strains is up to 92%.

10. A method as claimed in claim 1, wherein the plasmids are selected from the group comprising of pUC 18 from E.coli, RP4 from E.coli, R751 from E.coli, pUBHO from Bacillus subtilis, pAR1811 from Staphylococcus aureus, pAR1812 from Enter -ococcus faecalis, pAR1813 from E.coli, pAR1814 from Salmonella typhi, pAR1815 from Shigella sonei, pAR1816 from Pseudomonas aeruginosa, pAR1817 from Bacillus cereus, pAR1818 from Staphylococcus aureus.

11. A pharmaceutical composition for curing antibiotic resistance in commercially available antibiotic resistant bacterial strains comprising of 25 to 800 μg/ml of l'-acetoxychavicol acetate dissolved in an organic solvent along with pharmaceutically acceptable carriers, diluents or additives, optionally along with 4 to 800 μg/ml of an antibiotic to which the bacterial strain is resistant.

12. A method of curing antibiotic resistance in antibiotic resistant bacterial strains substantially as herein described with reference to the foregoing examples.