Antimicrobial pattern analysis and mechanism of mode of action of chemically synthesized Aurone derivatives

Dr. Manikandan Madheslu

Sri Krishna Arts and Science College, Coimbatore-641008

Dr V. Kesavan

Department of Biotechnology, IIT Madras, Chennai-600036

Abstract

The present investigation was proposed with the main objective of identifying a potential biomolecule, which can eliminate the multi drug resistant bacterial pathogens. The microbial pathogens are fast learners, which can undergo evolution rapidly and getting resistance against antibiotics. Hence, the invention of new biomolecules against pathogens every time is not possible as most of the investigations resulting the existing molecules. The problem can overcome by engineering the pharmacophore of the antimicrobial molecules with the target of increasing the efficiency of bacterial killing. Considering the above issue, the two Aurone compounds were selected for this study and carried some morphological changes through structural engineering. The developed molecules named as SMR 18 & SMR 19 were subjected for 96 well plate assay to find the minimal inhibitory concentration against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus, Candida albicans and Candida parapsilopsis. The result suggested that, the compound has some broad spectrum activity against microbial strains with significant value of cell death percentage. Further exploration of molecule will be carried with techniques like, time kill assay, checker board assay and FACS.

Keywords: Aurone compounds, Pharmacophore, Structural engineering, Multidrug resistant bacteria, Minimal inhibitory concentration.

Abbreviations

INTRODUCTION (style it as Section Level 1)

Background/Rationale

The emergence of multidrug-resistant Gram-negative bacteria is a major concern in hospital settings in many parts of the world. Infections caused by these pathogens have become significantly challenging over the past two decades, particularly in the developing countries, and are associated with high morbidity and mortality rates as well as protracted hospital stay. Enterobacteriaceae including Klebsiella pneumoniae, Escherichia coli as well as Enterobacter spp. and non-lactose fermenting bacteria such as Pseudomonas aeruginosa and Acinetobacter spp. have been identified as major cause of multi-drug resistant bacterial infections . In many developing countries including Africa conducted studies, indicating high antibiotic resistance among bacteria i.e (Gram-negative) to commonly used antibiotics that leads to a loss of efficacy for treatment of common infections. These resistant bacterial pathogens are a major causative agent of both community and hospital-acquired infections. Most commonly reported infections attributable to these pathogens in many hospitals are Respiratory tract, urinary tract, bloodstream (septic), postsurgical (wound) infections and pneumonia. Although, the impact of antibiotic resistance caused by multidrug resistant Gram-negative bacteria has been recognized in hospitals in Ghana, measures such as surveillance studies that provide reliable data to mitigate the problem are not in place. Therefore studies are necessary in which it establishes to bridge the information gap and provide the basis to guide empiric therapy on the prevalence and extent of resistance are necessary. Infections were common among the elderly and infants, and predominantly caused by E. coli, K. pneumoniae and P. aeruginosa (Agyepong et al. 2018).

Pharmacophores is an intellectual description of the molecular features that are essential for the molecular description of a ligand by a biological macro molecule. IUPAC states it as “an ensemble of the electronic and steric features that are necessary to affirm the optical supramolecular communications with a specific biological target and to stimulates in triggering or to block its response biologically These Pharmacophores includes some typical features such as the hydrophobic centroids, aromatic rings, hydrogen bond acceptors or donors, cations and anions that may be located as the part on the ligand or in projected points of the receptor. These Pharmacophores plays a vital part in the drugs. As drug discovery projects have already reached great heights and advancements before the detailed data on the structure of the target has become available. The approaches made using these pharmacophores has proven to be successful which allows the perception and understanding of the key interactions between a target and a ligand; also the enrichment of hit rates obtained during the experimental screening of subsets obtained from in silico screening experiments (Wermuth et al. 1998).

In 1928, the microbial drug era in which Alexander Fleming discovered an antibiotic mold seeded with Staphylococcus aureus, a compound produced by a mold killed the bacteria in the Petri dish. The mold, identified as Penicillium notatum, produced an active agent named penicillin. Later, penicillin was isolated as a yellow powder and used as a potential antibacterial compound during World War II. By Fleming's method, other naturally occurring substances, such as chloramphenicol and streptomycin were also isolated.

Based on natural product structures, drugs of natural origin have been classified as (i) original natural products, (ii) products derived or chemically synthesized from natural products or (iii) synthetic products. for the treatment of human diseases is provided by the fact acts as an evident of the importance of natural products in the discovery of leads for the development of drugs that close to half of the bestselling pharmaceuticals in 1991 were either natural products or their derivatives (Cragg et al. 1997). In this regard, of the 25 top-selling drugs reported in 1997, 42% were natural products or their derivatives and of these, 67% were antibiotics. Today, the structures of around 140 000 secondary metabolites have been elucidated. It is important to understand that many chemically synthesized drugs owe their origin to natural sources. Applications of chemically synthesized natural metabolites include the use of derivatives from white willow, wintergreen and meadowsweet to relieve pain and suffering, a natural product derived from plant called salicyclic acid. In the year 500 BC, Concoctions of these plants were administered by Hippocrates whereas it was used for fever, pain and childbirth earlier in Egypt and Babylonia. In 1897, Arthur Eichengrun at Bayer discovered that an acetyl derivative (aspirin) which are Synthetic salicylates, reduced acidity, bad taste and stomach irritation. Thus plant-based systems continue to play an essential role in health care, and it has been estimated by the World Health Organization (WHO) in which approximately 80% of the world's inhabitants rely mainly on the traditional medicines are used for their primary health care (Farnsworth et al.1985). The active constituent of the ‘fever tree’ Cinchona succirubra, the alkaloid quinine has been known to control malaria for centuries by South American Indians. During the twentieth century, based on the quinine prototype, massive programs were made to synthesize quinoline derivatives were carried out. A large chemical synthesis program developed at the Sterling Winthrop Research Institute in which the first of the new quinolones to be used clinically as an antibacterial agent known as nalidixic acid emerged, the best compound found in the program that had a remarkable activity against Gram-negative bacteria and was shown to be an inhibitor of DNA gyrase. (Cushman and Ondetti 1999). Its discovery led to a whole series of synthetic quinolone and fluoroquinolone antibiotics (pefloxacin, norfloxacin, ciprofloxacin, levofloxacin, ofloxacin, lomefloxacin, sparfloxacin, etc.), which have been very successful in medicine and have achieved major commercial success. It is important to appreciate that all quinolones, though synthetic, are based on the structure of the natural plant product quinine.

A nonapeptide, designated teprotide, synthesized compounds originating from natural products isolated from the venom of the Brazilian pit viper Bothrops jararaca led to the design and synthesis of angiotensin-converting enzyme (ACE) inhibitors such as captopril, which was the first marketed, orally active ACE inhibitor. Another ACE inhibitor called Enalapril, used in the treatment of cardiovascular disease approved for marketing by the Food and Drug Administration (FDA) in 1985.

Bacteria have lived on the Earth for several billion years in which they are encountered in nature by a wide range of naturally occurring antibiotics. To survive, bacteria developed antibiotic resistance mechanisms. Therefore, it is not surprising that they have become resistant to most of the natural antimicrobial agents that have been developed over the past 50 years. New antibiotics that are active against resistant bacteria are required (Hancock 2007). This resistance increasingly limits the effectiveness of current antimicrobial drugs. The problem is not just antibiotic resistance but also multidrug resistance. More than 70% of pathogenic bacteria were estimated to be resistant to at least one of the currently available antibiotics in 2004. The so-called ‘superbugs’ (organisms that are resistant to most of the clinically used antibiotics) are emerging at a rapid rate (Katz et al. 2006). Methicillin resistant organism, S. aureus is responsible for many cases of infections each year. The incidence of multidrug-resistant pathogenic bacteria is increasing. The bacteria produce a biofilm to encase and protect them from the environment and they can grow on wounds, scar tissues and medical implants or devices, such as joint prostheses, spinal instrumentations, catheters, vascular prosthetic grafts and heart valves. The bacterial species producing such biofilms are more than 70% and are likely to be resistant to at least one of the drugs commonly used in anti-infectious therapy. The hospital-inhabiting microbes are called ‘nosocomial bacteria.’ Among them, Pseudomonas aeruginosa accounts for almost 80% of these opportunistic infections.

Aurones, (2-benzylidenebenzofuran-3(2H)-ones) are structurally isomeric of flavones, are obtained in vegetables, especially in flowers as they are responsible for the gold-yellow color. It was recently reported that, they also occur in marine organisms (Choudhary et al. 2007). They are also known as phytoalexins, in which they are used by plants as defense agents against various infections. The medicinal literature on aurones are in its infancy with trends and design compared to flavones and it’s just now emerging (Lawrence et al. 2003). Aurones are mostly found in a hydroxylated form like all subclasses of flavonoids and especially at C-4, C-6, and C-4′ (in flavones, these positions correspond to C-5, C-7, and C-4′).

A drug is a chemical substance used in the treatment, cure, prevention or diagnosis of disease or used to otherwise enhance physical or mental wellbeing. Rational drug designs are also known as drug designing. It is nothing but finding new medications based on the knowledge of a biological target it is an inventive process. Drug design defines the design of molecules that are complementary in shape and charge to the bimolecular target with which they interact and therefore will bind to it. The process of bringing a new pharmaceutical drug to the market is called drug development once the lead compound has been identified through the process of drug discovery they are brought it to the market. Drug designs are followed by some methods which are given below:

• Ligand based drug design

• Structure based drug design

• Pre- clinical phase

• Clinical phase

• Cost

• Valuation

• Novel initiatives

• Success rate

The process of drug development and drug discovery is very challenging, expensive and time consumable. It has been faster due to development of computational tools and methods. Very complex and includes an interdisciplinary effort in the process of drug discovery. In medical history in order to yield significant therapeutic response they invented this drug design. Drug is an organic molecule it can inhibit or activate when it is bind to the targeted site it activates the function of a biomolecule which results in a therapeutic benefit. There are three main periods in the drug discovery. 19th century is the first notable period where the basis of drug discovery relied on the serendipity of the medicinal chemists in early twentieth century the second period commenced and new drug structures were found which initiated a new era of antibiotics discovery based on this known structure and with the powerful new techniques such as combinatorial chemistry, molecular modeling and automated high throughput screening. During end of the century rapid advances occurred in drug discovery. Drug design is usually achieved through molecular modification of the lead compound. In the course of drug design the two major types of chemical modifications are achieved through the formation of analogues and prodrugs. The term prodrug is applied to either an appropriate derivative of a drug that undergoes in vivo hydrolysis of the parent drug. Analogue which is metabolically transformed to a biologically active drug.

Statement of the Problems

Due to prolonged exposure of microorganisms with the antimicrobials the microorganism undergoes changes and mutations against the antimicrobials that make them resistant towards it. Thus, it is found that the microorganisms are resistant towards almost all the traditional antimicrobial agents. Hence, it is necessary to find some novel drugs to combat these pathogens. Instead of finding novel molecules from the environment, modifying the existing molecules and increasing the efficiency will save the time and human resource.

Objectives of the Research

Aim and Objective

The current investigation was proposed with the following objectives:

To identify the minimal inhibitory concentration of SMR18 & SMR 19 against microbial strains using 96 well microtitre plate assay.

Scope

The improper usage of antibiotics has created a vulnerable situation in the medical history. It leads to the emergence of many antimicrobial resistance bacterial strains, which causes a major threat to human beings in the developing country. The problem of antimicrobial resistance is rapidly increasing, because of the lack of knowledge and research. With the seemingly exponential emergence of microorganisms resistant to the available antibiotics, the need for the discovery of novel drugs becomes real. The current investigation will give a chance to explore some novel molecules, which can be a key to solve the mystery of antimicrobial resistance. The further investigation on potential molecules can be taken to the next level and may result with some pharmaceutically significant molecules.

LITERATURE REVIEW

Antimicrobials

The discovery of antimicrobial drugs has proved remarkably effective for the control of bacterial infections. However, it was soon evident that bacterial pathogens were unlikely to surrender unconditionally, because some pathogens rapidly became resistant to many of the first effective drugs. Examples such as, the development of resistance to penicillin in Staphylococcus aureus by the production of a ß-lactamase has quickly decreased the efficient usage of penicillin for serious staphylococcal infections, especially among hospitalized patients, in whom resistant strains are frequently found where they spread it to the community. Bacterial resistance to antimicrobial drugs caused a problem in which it was solved by the discovery of new classes of drugs, such as the aminoglycosides, macrolides, and glycopeptides, as well as by the chemical modification of previously existing drugs. Unfortunately, there is no assurance that the development of new antimicrobial drugs can keep pace with the ability of bacterial pathogens to develop resistance. In note to the resistance to antimicrobial drugs, more mechanisms and epidemiology was found and it has become clear that bacteria have a remarkable array of tools at their disposal to overcome antibiotics. The pathogenicity or viability of a bacterial strain without getting altered can induce resistance by a single genetic mutation. For example, the development of resistance to antituberculous drugs such as streptomycin Theoretically, it should be possible to overcome mutational resistance by sufficient dosage of administering a combination of drugs long enough to eradicate the infection, thus preventing person-to-person dissemination of resistant bacteria.

Streptococcus pneumoniae were exquisitely susceptible to penicillin by 1940s. Not only inhibition in the growth of these organisms are found at concentrations of penicillin of less than 0.1 mg per milliliter but also killed them by rapid lysis. Strains of pneumococci with intermediate levels of penicillin resistance (minimal inhibitory concentrations of 0.1 to 0.6 mg per milliliter) began to appear after the 1960s recorded by reports (Appelbaum 1992). In the mid-1970s, more highly resistant pneumococci (requiring minimal inhibitory concentrations of penicillin of up to 4 to 8 mg per milliliter and often resistant to other antimicrobial drugs) were described in South Africa (Jacobs et al. 1978). Subsequently, penicillin-resistant S. pneumoniae have been found virtually worldwide. In a study where, S. pneumoniae collected from outpatients at medical centres in the United States between 1994 and 1995, in which only 6% of isolates from 1500 were not susceptible to penicillin with 1 percent intermediate resistance (minimal inhibitory concentration of penicillin, 0.1 to 1.0 mg per milliliter), and 9.5 percent highly resistant (minimal inhibitory concentration of penicillin, 2.0 mg per milliliter). The geographic variation in the rates of penicillin resistance among pneumococcal strains, ranging from 2.1 to 53 percent were marked. Unfortunately, it appears that the overall prevalence of resistant strains in the United States is rising steadily, with the most dramatic increases among highly resistant pneumococci. To complicate matters, many of these penicillin-resistant strains are resistant to other antimicrobial drugs, including erythromycin, tetracycline, chloramphenicol, and trimethoprim–sulfamethoxazole (Doern et al. 1996).

Vancomycin was virtually the only drug that could be consistently relied on for the treatment of infections caused by multidrug resistant enterococci. Without the emergence of marked resistance, Vancomycin had been in clinical use for more than 30 years. The other glycopeptide antibiotic in clinical use is Teicoplanin not available in the United States, but has been used in Europe due to their activity against methicillin-resistant staphylococci and other gram-positive bacteria. Drugs have been widely used for therapy and prophylaxis against infections due to these organisms. Also poorly absorbed oral vancomycin, has been used extensively for the treatment of Clostridium difficile enterocolitis. The use of glycopeptides is escalating and vancomycin use increased 20-fold from 1981 to 1991(Ena et al. 1993). It was against this backdrop that reports of acquired vancomycin resistance in enterococci began to appear in the mid-1980s (Uttley et al. 1988). Resistant organisms have become an important problem in an ever-growing number of centers in the United States, reported in Europe. Less than 0.5 percent in 1989 to more than 10 percent in 1995 vancomycin resistance had increased more than 20-fold among nosocomial isolates of enterococci by the data reported to the National Nosocomial Infection Survey of the Centers for Disease Control and Prevention. These results are alarming as the vancomycin resistant organisms, initially concentrated in intensive care units, have spread throughout hospitals. Due to the Vancomycin-resistant enterococci , patients gets affected with bacteremia, many patients have serious underlying disease and the mortality rate is attributed the bacteremia that may approach 50 percent (Shay et al. 1995). The length of hospital stay, underlying disease, intensity of antibiotic exposure, and exposure to particular antibiotics, including broad-spectrum drugs and oral vancomycin are associated with the acquisition of vancomycin-resistant enterococcae by the hospital patients (Handwerger et al. 1993). Outbreaks of vancomycin-resistant enterococci may be monoclonal or due to multiple strains. Isolates of vancomycin-resistant enterococci have been genetically diverse, but dissemination of single strains between hospitals has been reported in United States. These enterococcal bacteria have been isolated from farm animals, poultry for human consumption, and sewage but the original source of vancomycin-resistant enterococci remains unknown (Klare et al. 1995).

The treatment of multidrug-resistant Gram-negative bacteria (MDR-GNB) infections in critically ill patients presents many challenges. Since an effective treatment should be administered as soon as possible, resistance to many antimicrobial classes almost invariably reduces the probability of adequate empirical coverage, with possible unfavorable consequences. In this light, readily available patient's medical history and updated information about the local microbiological epidemiology remain critical for defining the baseline risk of MDR-GNB infections and firmly guiding empirical treatment choices, with the aim of avoiding both under treatment and overtreatment. Rapid diagnostics and efficient laboratory workflows are also of paramount importance both for anticipating diagnosis and for rapidly narrowing the antimicrobial spectrum, with de-escalation purposes and in line with antimicrobial stewardship principles. Carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii are being reported with increasing frequencies worldwide, although with important variability across regions. In the past few years, new treatment options, such as ceftazidime/avibactam, meropenem/vaborbactam, ceftolozane/tazobactam, plazomicin, and eravacycline have become available, and others will become soon, which have provided some much-awaited resources for effectively counteracting severe infections due to these organisms. However, their optimal use should be guaranteed in the long term, for delaying as much as possible the emergence and diffusion of resistance to novel agents. Despite important progresses, pharmacokinetic/pharmacodynamic optimization of dosages and treatment duration in critically ill patients has still some areas of uncertainty requiring further study, that should take into account also resistance selection as a major endpoint. Treatment of severe MDR-GNB infections in critically ill patients in the near future will require an expert and complex clinical reasoning, of course taking into account the peculiar characteristics of the target population, but also the need for adequate empirical coverage and the more and more specific enzyme-level activity of novel antimicrobials with respect to the different resistance mechanisms of MDR-GNB.

Polymyxins acts as detergents of the outer membrane of GNB, exerting bactericidal activity. Among those available for use in humans are colistin (polymyxin E) and polymyxin B. They were frequently used for the treatment of MDR-GNB infections in the past few years, when they often remained among the few (sometimes the only one) dependable options for CRE, CRPA, and CRAB . Nowadays, they are still among the first-line treatment options for CRAB infections (pending approval of more effective agents), whereas for CRE and CRPA already available novel agents should be preferred whenever possible, owing to the potential polymyxin-associated risks either of nephrotoxicity or of suboptimal concentrations (especially in the lung). Furthermore, worrisome trends of increasing resistance have been reported in some countries (e.g., Italy and Greece). Consequently, the use of polymyxins should be optimized as much as possible in terms of dosages and indications, in order both to maximize effectiveness and to curb the emergence of further polymyxin resistance. For this reason, an international consensus document has been developed very recently, for guiding the proper use of polymyxins on all those occasions (e.g., CRAB infections, CRE and CRPA resistant to novel BL-BLI) when they still remain essential.

Variable in vitro synergy has been reported for rifampin in combination with other agents for the treatment of MDR-GNB infections. Possible improvements in microbiological response by adding rifampin were observed in an RCT trial.

Omadacycline is an aminomethylcycline recently approved by the FDA for the treatment of community-acquired bacterial pneumonia and acute skin and skin structure infections. In vitro activity against some MDR-GNB has been reported, although clinical post-marketing experience is needed to clarify as to whether this drug will have a role in future treatment algorithms for MDR-GNB.

Aurones, a class of flavonoids have been investigated and is known for its biological activities in different areas. The structural similarity existing between aurones, chalcones and flavones has led to the investigation of aurones on the biological activities that have been reported for various flavonoids subclasses. As a well-known biologically pertinent molecule Aurones shows its potency as an anticancer, anti-anginal, anti-inflammatory, anti-obesity, anti-diabetic, anti-hepatitis, anti-thyroxinase, anti-fungal, anti-microbial activities. It also shows a good nutritional value and anti-hormonal activity and this article constitutes the complete information of all the above activities. Aurones acts as an anticancer agent by its properties such as CDK kinase inhibition, antioxidant property, its cytotoxicity towards cancer cells, inhibition of angiogenesis and anti-proliferative property towards cancer therapy. Aurones have also shown its significant use in cancer chemotherapy.

A series of synthesized 4, 6-dimethoxyaurones tested for their ability to modulate ABCG2 (breast cancer resistance protein)-mediated multidrug resistance in vitro. Several members (0.5µM) increased the accumulation of mitoxantrone (MX) in human breast cancer cells (MDA-MB-231) transfected with ABCG2 and re-sensitized these cells to the cytotoxic effects of MX. The re-sensitization assay showed that the aurones at 0.5µM reduced the resistance of the transfected cells to MX to double that of the parental cells, exceeding fumitremorgin C (FTC) tested at the same concentration. The aurones (10µM) also increased calcein-AM accumulation in MDCKII/MDR1 cells, comparable to verapamil tested at the same concentration. The substitution 3` position of ring B showed better inhibition of ABCB1. The data obtained indicated that aurones with good ABCG2 inhibitory activity were poor ABCB1 inhibitors and vice versa, further confirmation would be required. This proves that functionalized 4, 6-dimethoxyaurones are promising ABCG2 inhibitors. Aurones with disubstitution on ring B with methoxy or a combination of methoxy and hydroxy groups (4, 6, 3’, 4’- or 4, 6, 3’, 5’-tetramethoxylated Aurones) highlighted the versatility of the aurone template as a lead scaffold for the design of dual-targeting ABCG2 and ABCB1 modulators.

Summary

Considering all the above said facts, the existing biomolecules become sensitive to the dangerous microbial pathogens and finding the new molecules are the need of the hour. Hence, he two compounds named as SM18 & SM 19 derived from Aurone, was chemically engineered and prepared in Dimethyl sulfoxide. Further, the compounds were subjected for 96 well microtiter assay to find the MIC values against selected bacterial and yeast pathogens, such as, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus, Candida albicans and Candida parapsilopsis.

The intrinsic resistance to several commonly used antibiotics is one of the major reasons these organisms have thrived in the hospital environment perhaps more important, their ability to acquire resistance to all currently available antibiotics by mutation (Clewell et al. 1990). Most enterococci are tolerant to the bactericidal activity of ß-lactam and glycopeptide antibiotics, bactericidal synergy between one of these antibiotics and an aminoglycoside is needed to treat the most serious enterococcal infections, such as endocarditis and meningitis. This effect is lost if there is high-level resistance to either class of drug (Krogstad and Parquette 1980). Resistance to high concentrations of aminoglycoside antibiotics, usually due to aminoglycoside-modifying enzymes, is widespread among enterococci (more than 50 percent of isolates in some centers). Also, many isolates of Enterococcus faecium are highly resistant to penicillin by virtue of their low-affinity penicillin-binding proteins (Grayson et al. 1991).

METHODOLOGY

Concepts

The test bacterial strains were obtained from American Type Culture Collection and other local laboratories.

The compounds SMR18 & SMR 19 were taken from the previous investigation carried on Aurone compounds.

The Solubility of compounds checked with different solvents.

The compounds were subjected for antimicrobial activity.

The growth pattern of test organisms were studies by spectrophotometric method.

The MIC of compounds was identified using 96 well microtiter assay.

Methods

Strain cultivation

Bacterial culture

The bacterial cultures Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus was cultured on the Nutrient agar plates with quadrant streaking and also inoculated into the nutrient broth for carrying out the procedures. The plates were incubated in the incubator under 37⁰ C for 24 hrs. The growth was observed on plate, and a single colony was picked and streaked into the nutrient agar slant for storage purpose.

Fungal Culture

The fungal cultures like Candida albicans and Candida parapsilopsis were bought from MTCC (Microbial Type Culture Collection). The cultures were revived according to protocols suggested by MTCC. The Ampules containing cultures was opened in sterile condition, for fungal cultures, 0.4 ml of sterile water was added into lyophilized culture and left for 20 minutes before transferring into solid YEME media. After 20 minutes, the culture was quadrant streaked on YEME media and it was inoculated into liquid broth for further experiment. It is kept in incubator under 37⁰C for 72 hrs for the growth, and it was further stored in agar slant streaked with pure culture.

Growth Curve

The growth of the microbes was measured using spectrophotometer. The bacterial culture was inoculated with Muller Hinton broth while fungal strains were inoculated with YEME broth. The cultures were kept in shaker under optimal conditions. After every 4 hour the growth curve was measured to know the growth pattern of microbial strains.

Compound Stock Preparation

Vancomycin

The 256 mM of vancomycin stock solution was prepared by dissolving 37.102 g in 100 ml of water, it makes the vancomycin stock solution.

Colistin

The 512 mM of Colistin stock solution was prepared by dissolving 59.15 g in 100 ml of water. It is the stock solution for colistin.

Fluconazole

The 256 mM of Fluconazole stock solution was prepared by dissolving 7.84g in 0.1% of DMSO. That is the stock solution for Fluconazole.

Solubility Analysis of Compounds SMR18 & SMR19

The solubility of the compounds SMR 1-9, SMR 18 and SMR 19 were checked with water and various organic solvents such as acetone, ethanol, methanol, tetrahydrofuran (THF), Dimethylsulphoxide (DMSO). The solubility of the compounds was checked with different concentrations of DMSO such as 1-10%, 25%, 50% and 100%. The solvent in which the compound was completely soluble was chosen.

Preparation of compound SMR18 & SMR19

1

• 1

Fig 1 . Solubility analysis of test compounds using various solvents

The 1000 µM concentrations of compounds were prepared by calculating the molarity of compounds and dissolved in 1% DMSO. As per the need the working volume was diluted with diluted DMSO solution.

Microtiter Plate Assay

Inoculum preparation

The bacterial cultures inoculated into MHA broth and Fungi is inoculated in YEME media and kept in shaker. To standardize the microbial testing, the turbidity of microbial suspension was adjusted using McFarland Standards (i.e, Barium chloride and sulphuric acid together) as a reference. First McFarland Standard was measured at 600 nm wavelength, the OD value obtained as 0.5. Then the culture was measured for OD, if the bacterial turbidity was high it should be diluted with uninoculated sterile broth, if the turbidity was too low the culture was additionally inoculated, and the OD was initially now fixed as 0.5 at 600 nm. Similar it denotes that the concentration of the cell suspension is around 10⁸ CFU/ml. At that point of time the culture was taken for microtiter plates (Wiegand et al. 2008).

Solvents Activity

In 96 well microtiter plate 100 µl of culture was first added in all the wells. The first column of the plate was kept as growth control well, so the culture Staphylococcus aureus alone added. In the second column DMF was taken, in fourth ethanol and in sixth column DMSO were taken in the concentration from 2 µl to 10 µl, Assay was done in duplicates for each solvent. The plates were kept in 37^o C for 24 hrs and the OD value was measured using plate reader (Wiegand et al. 2008). According to the values obtained graph was plotted, it shows the activity of solvents against the Staphylococcus aureus.

Fig 2 96 well microtiter plate used for assays

Effect of DMSO alone against test strains

In 96 well microtiter plate 100 µl of culture was first added in all the wells. The first column of the plate was kept as growth control well, so the culture alone added. In the second and third column E. coli was added with DMSO, in the fourth and fifth column pseudomonas aeruginosa were added with DMSO, to check the antimicrobial activity of DMSO against the bacteria E. coli and Pseudomonas aeruginosa. The concentration of DMSO was taken from 50 to 0.3828. The plates were kept in 37^o C for 24 hrs and the OD value was measured using plate reader (Andrews 2001). According to the values obtained graph was plotted, it shows the activity of DMSO against E. coli and Pseudomonas aeruginosa.

Antimicrobial activity of compounds against Staphylococcus aureus

In 96 well microtiter plate 100 µl of Staphylococcus aureus was first added in all the wells. The first column of the plate was kept as growth control well, so the culture alone added. In the next column DMSO was added with culture, in the other Vancomycin was added with culture. The DMSO and Vancomycin act as positive control. SMR 18 and SMR 19 were added in up next column with triplets, from 128 µM to 1 µM Concentration. The antimicrobial compounds were added in decreasing concentration from the last well of the column to the first well by serial dilutions. The plates were kept in 37^o C for 24 hrs and the OD value was measured using plate reader. According to the readings graph was plotted, it shows the activity of compounds against of Staphylococcus aureus.

Antimicrobial activity of compounds against Candida parasilopsis

In 96 well microtiter plate 100 µl of Candida parasilopsis was first added in all the wells. The first column of the plate was kept as growth control well, so the culture alone added. In the next column DMSO and Fluconazole was added with culture, DMSO and Fluconazole act as a positive control. SMR 18 and SMR 19 were added in up next column with triplets, from 128 µM to 1 µM Concentration. The antimicrobial compounds were added in decreasing concentration from the last well of the column to the first well by serial dilution. The plates were kept in 37^o C for 36 hrs and the OD value was measured using plate reader (Wiegand et al. 2008). According to the readings graph was plotted, it shows the activity of compounds against Candida parasilopsis.

Compounds activity against Pseudomonas aeruginosa

The MIC was tested using 96 well microtiter plate 100 µl of Pseudomonas aeruginosa was first added in all the wells. The first column of the plate was kept as growth control well, so the culture was left alone. In the next column Colistin was added with culture, Colistin act as positive control for Pseudomonas aeruginosa. SMR 18 and SMR 19 were added in up next column with triplets, from 256 µM to 2 µM Concentration. compounds were added in decreasing concentration from the last well of the column to the first well by serial dilution. The plates were kept in 37^o C for 24 hrs and the OD value was measured using plate reader (Wiegand et al. 2008). According to the readings graph was plotted, it shows SMR 18 and SMR 19 activity against Pseudomonas aeruginosa with respect to Colistin.

Compounds activity against E. coli

The MIC was tested using 96 well microtiter plate 100 µl of E. coli was first added in all the wells. The first column of the plate was kept as growth control well, so the culture was left alone. In the next column Colistin was added with culture, Colistin act as positive control for E. coli. SMR 18 and SMR 19 were added in up next column with triplets, from 256 µM to 2 µM Concentration. compounds were added in decreasing concentration from the last well of the column to the first well by serial dilution. The plates were kept in 37^o C for 24 hrs and the OD value was measured using plate reader (Wiegand et al. 2008). According to the readings graph was plotted, it shows SMR 18 and SMR 19 activity against E. coli with respect to Colistin.

RESULTS AND DISCUSSION

Growth curve

The growth curve was plotted against the optical density in Y axis and time in X axis. It denotes that if the density is more growth has occurred. The growth curve of the bacteria & fungi was observed. the culture growth was optimal and both fungi and bacteria responded well in the media utilized for growth. the organisms were in lag phase for initial five hours, later the graph getting elevated indicates the growth leads towards exponential phase.

Fig 3 Growth curve analysis of E. coli

Fig 4 Growth curve analysis of Pseudomonas aeruginosa

Fig 5 Growth curve analysis of Staphylococcus aureus

Fig 6 Growth curve analysis of Candida albicans

Fig 7 Growth curve analysis of Candida parasilopsis

Solvent Assay

The Minimum inhibitory concentration of solvents DMSO, Ethanol and DMF was determined by reading the OD of the Microtiter plate that has been kept for incubation.

The MIC of the solvents against Staphylococcus aureus are found by plotting concentration against optical density in the graph and it was found that DMF and Ethanol has more or less same activity. DMSO has high antagonistic activity than DMF and Ethanol.

Fig 8 Effect of different solvents against Staphylococcus aureus

Effect of DMSO against pathogens

The Minimum inhibitory concentration of DMSO was determined against E. coli & Pseudomonas aeruginosa. By reading the OD of the Microtiter plate that has been kept for incubation. The values plotted in graph as concentration in X axis and Optical density in Y axis.

Fig 9 Effect of DMSO against E. coli & Pseudomonas aeruginosa

The results in graph confirm that at the concentration 1.531 microM the growth of Pseudomonas aeruginosa gets slow down. From 3.0625 microM concentration, the growth of E. coli gets inhibited and its falls down in the graph.

  Antimicrobial activity of compounds against Staphylococcus aureus

The antimicrobial activity of compounds SMR18 & SMR 19 was checked by microtiter plate method against Staphylococcus aureus with a positive control Vancomycin & solvent control DMSO. Graph was plotted with concentration in X axis and OD value in Y axis.

Fig 10 Effect of SMR 18 & SMR 19 against Staphylococcus aureus

From the graph interpretation that, the SMR 18 & SMR 19 has more or less same activity, higher than DMSO, but comparing with Vancomycin the compounds SMR 18 & SMR 19 was more or less equal.

Antimicrobial activity of compounds against Candida parasilopsis

The antimicrobial activity of compounds SMR18 & SMR 19 was checked by microtiter plate method against Candida parasilopsis. Graph was plotted with concentration in X axis and OD value in Y axis.

Fig 11 Effect of SMR 18 & SMR 19 against Candida parasilopsis

 The graph shows SMR 18 has more activity than SMR 19, at concentration 2 mM suddenly the growth of organism gets suppressed Besides, compared with positive control and DMSO, the SMR 18 has good antifungal activity. while comparing with Fluconazole, SMR 19 has very low activity against Candida parasilopsis.

Antimicrobial activity of compounds against Pseudomonas aeruginosa

The antimicrobial activity of compounds SMR18 & SMR 19 was checked by microtiter plate method against Pseudomonas aeruginosa. Graph was plotted with concentration in X axis and OD value in Y axis.

Fig 12 Effect of SMR 18 & SMR 19 against Pseudomonas aeruginosa

From the graph, it can be interpreted SMR 18 and SMR 19 has low antibacterial activity than positive control Colistin. At the concentration 16 microM SMR 19 shows good results, and control the growth of Pseudomonas aeruginosa.

Antimicrobial activity of compounds against E. coli

Fig 13 Effect of SMR 18 & SMR 19 against E. coli

The antimicrobial activity of compounds SMR18 & SMR 19 was checked by micro titer plate method against E. coli. Graph was plotted with concentration in X axis and OD value in Y axis. The results indicated that the Colistin has higher antmicrobial potential than SMR 18 & SMR 19. Still, both the compounds has the ability to control microbial growth at 32 microM concentration.

The results suggested that the compounds SMR18 & SMR 19 are having potential against multidrug resistance pathogenic micorbes and it could be a way to combat these organisms. These organisms are very severe pathogens to humans. In the United States, Enterococci are ascendant nosocomial pathogens (Grayson et al. 1991) has become the second most common organisms recovered from nosocomial urinary tract and wound infections and the third most common cause of nosocomial bacteremia (Schaberg et al. 1991). The aurone compounds are previuosly reported for various activities. Aurones, (2-benzylidenebenzofuran-3(2H)-ones) are structurally isomeric of flavones, are obtained in vegetables, especially in flowers as they are responsible for the gold-yellow color.It was recently reported that, they also occur in marine organisms (Choudhary et al. 2007). They are also known as phytoalexins, in which they are used by plants as defense agents against various infections. The medicinal literature on aurones are in its infancy with trends and design compared to flavones and it’s just now emerging (Lawrence et al. 2003). Aurones are mostly found in a hydroxylated formlike all subclasses of flavonoids and especially at C-4, C-6, and C-4′ (in flavones, these positions correspond to C-5, C-7, and C-4′).

CONCLUSION AND RECOMMENDATIONS

Conclusion

The compounds SMR 18 & SMR 19 has low antibacterial activity than positive control Colistin and it was observed SMR 19 shows high activity than SMR 18. The MIC of the compounds SMR 18 and 19 against the various microorganisms are found by plotting concentration vs optical density graph and was found that the MIC value of SMR 18 is 32µg/ml and that of SMR 19 is 16µg/ml. The future course of action is taking the molecules to other assay procedures like time kill assay, checker board assay and FACS.

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ACKNOWLEDGEMENTS

I express my sincere gratitude to my guide Prof. V. Kesavan, Department of Biotechnology, IITM, for giving me an opportunity to explore the various dimensions of the topic chosen. I thank scholar Mr. Ankur Sood, Mr. Lokesh, Mr . Rajni and summer fellow Ms. Subanidhi for helping me to learn the laboratory techniques. I thank the Indian Institute of Technology Madras for this valuable opportunity.