Analyzing the synergistic effects of plant extracts with bacteriophages against Pseudomonas aeruginosa

TABLE OF CONTENTS

CHAPTER NO CONTENTS
Dedication
Table of contents
List of tables
List of figures
ABSTRACT

CHAPTER 1 INTRODUCTION
Antibiotic Resistance
Pseudomonas aeruginosa
Antibiotic resistance in Pseudomonas aeruginosa
Sources alternative to antibiotics
Phage therapy
Quorum sensing
Quorum sensing inhibitors
Secondary metabolites in plants and their functions
Mode of action of secondary metabolites
Covalent modification of proteins and DNA base
Non covalent modification of proteins
Interaction of secondary metabolites with cell membrane
Secondary metabolites as antioxidants
Aims and objectives

CHAPTER 2 MATERIAL AND METHODS
Bacterial strain
Bacteriophage propagation
Preparation of clove extract
Preparation of methanol extract of clove
Preparation of chloroform extract of clove
Antibacterial activity of plant extracts
Evaluation of synergistic antibacterial effect plant extracts and bacteriophages

CHAPTER 3 RESULTS
Antibacterial activity of plant extracts
Negative control
Synergism between plant extract and bacteriophages
Bacterial growth reduction assay using RL alone and in combination with plant extracts
Bacterial growth reduction assay using RS alone and in combination with plant extracts
Bacterial growth reduction assay using GPP alone and in combination with plant extracts
Bacterial growth reduction assay using JPA alone and in combination with plant extracts
Bacterial growth reduction assay using Iqpl alone and in combination with plant extracts
Bacterial growth reduction assay using Ittp alone and in combination with plant extracts
Bacterial growth reduction assay using Sal alone and in combination with plant extracts

CHAPTER 4 DISCUSSION
CONCLUSION

CHAPTER 5 REFRENCES

LIST OF TABLES

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LIST OF FIGURES

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ABSTRACT

By the passage of time the microorganisms that cause diseases become more resistant to antibiotics that are commonly used for treatment of diseases. The Pseudomonas aeruginosa causes nosocomial infections and is resistant to antibiotics due to presence of antibiotic resistance genes in chromosome. For the treatment of diseases caused by Pseudomonas aeruginosa, there is dire need of different alternative therapies to overcome the problem of antibiotic resistance and bacteriophage therapy is one among them. There are certain hurdles the bacteriophage therapy is facing for its acceptance as routine therapy which includes, narrow host range and emergence of phage resistance in bacteria. There are assumptions that bacteriophage therapy might work more effectively by using phages in combination with antibiotics and plant extracts. Pseudomonas aeruginosa uses quorum sensing (management of genetic expression) in reaction to changes in cell concentration. The quorum sensing inhibitors should have the characteristics like stability, nontoxicity, present in small amount and non-degradable. The plant extract that we used was in the crude form and can be used for prevention of bacterial infection. In combination with bacteriophages, plant extract can alter structure of bacterial enzymes and proteins, can increase the flexibility of the membrane and, can also degrade it. Some plant extracts can produce reactive oxygen species (ROS) which leads to death of bacteria. In this study, synergistic effect of both plant extract and bacteriophages was studied to find out how both plant extract and phages reduce the growth of bacteria. Bacterial inhibition was checked in liquid medium as well as on agar plates. Inhibitory effect was shown by all phages, and plant extracts at different levels with mixture of phage RL and RS. With methanol extract of clove indicated higher inhibitory effects against Pseudomonas aeruginosa as compared to phages and extracts alone. However, the results presented here indicates increased bacterial inhibition by combination of bacteriophages with plant extracts, which should be further explored for finding suitable combinations and can be used in future studies.

CHAPTER 1 INTRODUCTION

Antibiotic Resistance

The amount of people in this world is enhancing day by day. As the population is increased, the rate of disease establishment is also increased. The main agents for disease progression are “Microorganisms”. Primarily, usage of antibiotics was done to cure afflictions. Since all living organisms are accustomed to change, the microorganisms have adjusted themselves to this situation (Lindahl and Grace, 2015).

Antibiotic resistance is attained due to incredible genetic content of microorganisms. Plasmids are liable for this character (Davies and Davies, 2010). Roughly, all microbes have plasmids so they can adapt to changes. Some bacteria have genes for resistance on their chromosomal regions. Pseudomonas aeruginosa is one among them. Microorganisms are well known for antibiotic resistance. Resistance is also developed by using large amount of antibiotics. Hindrance against antibiotics is developed due to the following aspects. Microbes have innate hindrance against microbial killing factors because the agents have low accessibility towards cell wall. Many genetic resistant systems are present in bacteria. Plasmids are the main agents for hindrance. Due to enormous genes, Pseudomonas has flexible nature (Lambert, 2002).

There are some disease causing organisms that do not pass on diseases to hale and hearty people rather they infect those people who have weak immune system. These organisms are called opportunistic pathogens. One of them is Pseudomonas aeruginosa. This multi-drug resistant organism has put lives of all people in danger (Shaban et al., 2013). Nosocomial infections are caused by Pseudomonas aeruginosa and they are problematic to cure because of acquired resistance against antibiotics (Carmeli et al., 1999). Pseudomonas aeruginosa causes blood stream infections, cystic fibrosis, urinary tract infection and pneumonia. Catheters, respiratory equipment, hospital floor, fruits, flowers are contaminated with Pseudomonas, causing infections in persons with weakened immune system. When ailment passes through seriously, it can also lead to death (Kang et al., 2003).

Pseudomonas aeruginosa

Gram negative Pseudomonas aeruginosa is present everywhere in the environment (Soil, water, fruits, flowers etc.). It has the ability to grow and adapt itself to discrete range of environmental conditions such as temperature, pH, and osmotic conditions. It has a specific grape like odor. Pseudomonas aeruginosa is distinguished from other microbes by production of pigments such as pyocyanin, pyorubin and pyoverdine. Pyocyanin is responsible for cystic fibrosis. However, its mechanism is unknown. Pseudomonas aeruginosa is classified by IATS (International antigenic typing scheme) into twenty serotypes.

Genome size of Pseudomonas aeruginosa is large almost 5.5-7 Mb. Genome is encoded by a variety of enzymes and regulatory genes. Due to number of enzymes, it has many metabolic pathways conferring Pseudomonas nutritional diversity. Due to presence of regulatory genes Pseudomonas has flexible nature and has ability to adapt itself to changing environmental conditions. Being gram negative Pseudomonas has ability to release exotoxins. Its exotoxin A is used as virulence factor. However, oxidative stress also contributes to its virulence (Wu et al., 2015).

Pseudomonas, a multi-drug resistant bacteria, is found enormously in Pakistan especially in hospitals. Patients who are admitted in hospitals ICU are at major risk of developing infections. Following is data collected from Karachi by Zaheer Ali and his colleagues in 2012. Survey of different hospitals was done and Pseudomonas aeruginosa prevalence in different wards of hospitals was noted.

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Figure 1.1 Prevalence of Pseudomonas aeruginosa in different hospital wards in Karachi (Ali et al., 2015).

Antibiotic resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa has attained resistance because it has exclusive variations in chromosomal genes. The main system is variations in peptidoglycan- reclaiming of genes. There is remark increase in AmpC (Cabot et al., 2011). Any alteration in it can lead to increase in resistance (Berrazeg et al., 2015). However, due to cessation against porin OprD receptiveness against meropenem has decreased (Cabot et al., 2011). Variations in type IV topoisomerases and DNA gyrase are also responsible for resistance against microbial killing agents (Bruchmann et al., 2013). Alterations in Lipid A structure by changing LPS leads to resistance against polymyxin (Olaitan et al., 2014). However, expression of arnBCADTEF operon is main cause of MDR character. It leads to activation of MexXY and inhibition of OprD (Muller et al., 2011).

Pseudomonas aeruginosa is highly variable strain and due to its adaptive character, it is difficult to treat diseases. So many therapies are being used. One of them is phage therapy.

Sources alternative to antibiotics

Antibiotic resistance is increasing day by day and it has become a challenge for the scientific community. Increasing trend of antibiotic resistance has made scientists to think about alternative sources that can be used to treat infections. Among them plant extracts, bacteriophages and their products, monoclonal antibodies, probiotics, immune stimulation and antibacterial peptides are noticeable (François et al., 2016). Monoclonal antibodies have ability to bind to virulence factors of pathogens and neutralize them. This is also a way to treat infections but it is too expensive (Wang et al., 2008). Probiotics are healthy bacteria that have healthy effect on the health of people. They compete for pathogens and acquire nutrition and colonization space. However, the effectiveness of antibiotic therapy depends only on immune response. Bacteriophages and their products kill bacteria and can also be used as an alternative source to antibiotics.

Phage therapy

Bacteriophage therapy is useful against Pseudomonas aeruginosa. Bacteriophages are almost present everywhere in the environment. They require a living host for their replication (Holmfeldt et al., 2013). Lytic phages are used for curative purpose. Use of bacteriophages as microbial killing entities was done in 1917 (Dublanchet and Patey, 2011). Bacteriophages infect bacteria through lytic cycle.

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Figure 1.2 Life cycle of bacteriophages ( Bielke et al., 2012 ) .

Bacteriophages can enter either lytic or lysogenic life cycle. In lytic cycle phage DNA is inserted into host cell and many copies of daughter phages are formed before lysis of cell wall. However in lysogenic cycle phage DNA is becomes a part of bacterial DNA. Some but not all lysogenic cycles can enter lytic cycle.

Specific bacteriophages are used for specific strains (Lister et al., 2009). Mice had burnt wound infection, was infected with Pseudomonas aeruginosa. Mice were given bacteriophage therapy to cure this infection. Phages were given to mice through three different pathways, which were intramuscular, subcutaneous and intra peritoneal. Death rate was reduced by single application of phage cocktail. The pathway by which phages are given indicates efficiency of treatment (McVay et al., 2007). Lung infections are also cured by using phage therapy. Reduction in number of bacteria has decreased death rate (Morello et al., 2011). Sepsis caused by Pseudomonas aeruginosa can also be treated by this therapy.

Scientists in 1919 started to use phage therapy (d'Herelle, 1931). Many clinical trials to treat infections were done against bacteria. All clinical trials were successful although phage cocktail was also used for certain infections. Efficiency of therapy is increased by using phages and antibiotics in combination. Bacterial number is reduced to favorable extent. However, pathogenicity of bacteria can also be reduced (Sonnleitner et al., 2003). Despite the success of phage therapy scientists are reluctant to use it as major clinical practice. This may be due to antibiotics that can neutralize bacteriophage action. However, phages can also be contaminated by bacterial endotoxins and exotoxins (Pires et al., 2015). Also bacteria has developed many different systems by which bacteria can also protect itself against phages (Knezevic et al., 2013). This dilemma can be solved by using antibiotics and plant extracts in combination with bacteriophages.

Quorum sensing

Quorum sensing is management of genetic expression in response to alterations in cell density. Release of chemical signals lead to an increase in cell concentration. These chemical signal producing molecules are called auto inducers (Miller and Bassler, 2001). Chemical signals helps bacteria to communicate among themselves. By this mechanism, environmental conditions are continuously observed and changes in cell population are done if needed. However, prokaryotes and eukaryotes are difficult to identify because a bacterium can act as multicellular organism by quorum sensing mechanism (Sureshchandra, 2010). Quorum sensing helps in regulating virulence genes in Pseudomonas aeruginosa (Lee and Zhang, 2015) . However, gene expression controlled by quorum sensing helps to manage all those tasks that are beneficial when conducted by a group of bacteria (Ng and Bassler, 2009).

There are many quorum sensing systems and almost all are different in one way or another. They are operated by three main principles. One of them is auto inducers-signaling molecules. They are sensed even in low concentration and deviate or disseminate when cell mass is low. When cell density is high, there is an increased production of autoinducers (AIs). The other is identification of AIs by receptors that are present on cell sheath or in cytoplasm. The third one is regulation of gene expression for mutual behavior. Receptor mediated identification of AIs is helpful for coordination among bacterial populations (Ji et al., 1995).

There are three systems for quorum sensing in Pseudomonas aeruginosa. Two of them are Lux I/Lux R type and third one is non Lux I/Lux R. Third QS system is known as Pseudomonas quinolone system (PQS) (Bottomley et al., 2007).

Las I is target of Las R-3OC12HSL composite, a feed advancing loop that is auto inducing. Las I is homolog of rhII. RhII produces another AI, C4HSL. This attaches to RhIR, another homolog of Lux R when its concentration is high. RhIR-C4HSL triggers target genes that encode elastase, pyocyanin, and protease. Thus starting automatically second quorum sensing system (Ng and Bassler, 2009).

The use of medicines that reduce bacterial pathogenicity are more effective than to reduce bacterial growth. Drugs are based on signaling mechanism. Quorum sensing inhibitors make biofilms more vulnerable to antimicrobial agents thus reducing virulence (Rasmussen and Givskov, 2006). Infections can easily be cured. However, use of quorum sensing inhibitors is nontoxic (Kalia, 2013). Quorum sensing inhibitors work by reducing the efficiency of AHL (Acyl homoserine lactone) receptor or enzyme AHL synthase. Quorum sensing signaling molecules do not form. This is done by degradation or making copy of AHL signals (Kalia and Purohit, 2011). There are many compounds that can act as quorum sensing inhibitors but the most beneficial is to use plant extracts as quorum sensing inhibitors.

Agents used for quorum sensing inhibitors should have the following characteristics. Molecules used as inhibitors should be small so that it can help in regulating gene expression. It should be specific and nontoxic for host. It should be stable and non-degradable (Mølgaard et al., 2001).

Quorum sensing inhibitors

Usage of plant extracts as quorum sensing inhibitors is based on the resemblance between quorum signals and chemicals released by plants. Plant extract contains elements or compounds that can be used for beneficial purposes such as for curative purpose. Plant extracts can also be used as antioxidants. Some plant extracts are also used in beauty products. Raw material is used to form plant extract. The main benefit of extract is that it is nontoxic and safe for human use (Patel and Patel). Crude extract of certain parts of plant has antimicrobial properties. Many diseases are treated by it. Plant extracts are easy to obtain, cheap and one can easily make plant extract simply by dissolving it in a solvent. Plant extracts show antimicrobial characteristics due to production of secondary metabolites (Rakholiya et al., 2013). Many extracts can be used as alternative of drugs and helps to combat against antibiotic resistance.

Secondary metabolites affect gram positive bacteria more effectively than gram negative bacteria. It means that gram positive bacteria are affected by plant extracts. This distinctive character is due to cell wall structure. The cell wall of gram negative bacteria has an extra membrane containing lipopolysaccharides. This layer is called outer membrane. It has water loving surface and act as an obstacle for many plant extracts. It is not true at all. Some extracts affect gram negative bacteria more effectively than gram positive bacteria (Rakholiya et al., 2013).

Essential oils contain combination of compounds obtained as a result of secondary metabolism (Vinale et al., 2006). These oils can be used as an alternative to drugs. Commonly, bark, buds, leaves, and fruits are used to extract essential oils (EOs). For EOs extraction, plants from family Apiaceae, Rutaceae, Lamiaceae, and Myrtaceae are taken. EOs are also used to give a characteristic taste in some food industries (Roy and Chakrabarti, 2003). Infections that are caused by microorganisms in hospitals are cured by cooperative effect between two species of family Lamiaceae (Thymus maroccanus and Thymus broussonetti) (Fadli et al., 2012). However, plant extracts can also block enzymes or inactivate receptors (Teplitski et al., 2000). Many different plants are used. Most significant are garlic, ginger and cloves. Garlic has potential to prevent against infections (Bjarnsholt et al., 2008). Ginger also has anti-microbial characteristics. It can also be used for curative purposes.

Since plants also face adverse environmental conditions and are also affected by microorganisms. Like humans, plants do not have immune system that functions well by production of antibodies and by developing memory response when attacked by any antigen. With the passage of time, plants started to produce secondary metabolites as protective mechanism against microbes (Wink, 2008). Secondary metabolites can also be used as antioxidants (Rätsch, 2005). Plant extracts can be used alone or a mixture of plant extract can be used. Elements in plants have pharmacologic properties and used in drug development (Wink and Van Wyk, 2008).

Secondary metabolites in plants and their functions

Following are some major classes of plant secondary metabolites along their therapeutic applications.

Terpenes are made up of carbon ranging from C5 to C40 (Dewick, 2001). Attachment of terpenes to cell membrane and its proteins causes change in membrane permeability and effluence of cellular materials occur. This causes cell leakage or even cell death. Terpenes can also change function of membrane proteins. Terpenes can be effectively used as antimicrobial agents (Van Wyk and Wink).

Monoterpenes are present in almost all the aromatic plants. Solvent extraction is a method by which Monoterpenes can be isolated from plants. Thujone is very active and reacts readily due to presence of cyclopropane. Alkylation of proteins is the main property of thujone. It can cause many neurological disorders by alkylating proteins that have a role in signal transduction pathway. Usage of absinthe is prohibited due to this characteristic feature. Modifications in protein structure occur because monoterpenes can bind to SH group of proteins. Some monoterpenes have phenolic hydroxyl group in them or some contain aldehyde in them. These kinds of monoterpenes inhibit bacterial growth to favorable extent by attaching themselves to proteins (Chizzola, 2013).

Monoterpenes have another subdivision that contains iridoid glucosides. It has over 200 structures found mostly in all families of plants (Wink and Van Wyk, 2008). The family Gentianaceae and Menyanthaceae contains gentiopicrosides. However, taste is unpleasant. It is used to enhance appetite by improving digestion (Wink, 2015).

Aucubin and harpagoside are converted into aglycone (unstable) by β glucosidase. Functional dialdehydes are produced by opening of lactol ring. Dialdehyde show antimicrobial properties by attaching to amino acids. Rheumatism and inflammations are treated by using iridoid glucosides. Sesquiterpenes show anti-inflammatory properties by attaching to SH group of certain proteins (Van Wyk and Wink, 2015). Saponins show antimicrobial properties by changing cell membrane permeability and causes leakage of cellular metabolites. Phenolics also have antimicrobial properties and are found in many secondary metabolites of plants (Wink and Van Wyk, 2008).

Aromatic plants have myristicin, safrol, eugenol, apiole, β asarone, elemicin and estragole as secondary metabolites. These are grouped into phenylpropanoids. Phenylpropanoids show antimicrobial properties by reacting with SH group of proteins. Quinones are used to treat urinary tract infections. Phenolic compounds such as flavonoids, hidroxicinamic acids, hidroxiphenyl propens, and hidroxibenzoic acids are present in aromatic plants. Clove contains high content of eugenol about 90% and eugenol has bioactive behavior. Due to presence of eugenol, clove shows higher antimicrobial activity than other spices. High content of gallic acid also contributes to antimicrobial and anti-oxidant propertoes of clove (Cortés-Rojas et al., 2014).

Mechanism of Secondary Metabolites Activity

A large number of plants are known for their antimicrobial properties (Wink, 2010). Secondary metabolites in plants degrade bacterial cell wall by covalent and non-covalent modification of proteins and DNA bases. Proteins in cell membrane are degraded and bacterial cell burst.

Covalent modification of Proteins and DNA bases

Secondary metabolites are not present as a basic framework. It contains a lot of reactive compounds that attach to proteins and DNA (Wink and Schimmer, 2018a). Epoxide formation takes place and reaction between amino acids and DNA bases occur. Reactive compounds in extract can bind to proteins and alterations take place under environmental conditions. Proteins are most important part of cell and present everywhere such as in enzymes, and receptors etc. Any change in active site does not allow substrate binding to enzyme. Because secondary metabolites affect specific parts in organism so we cannot use them as a source of multi targeted drugs (Wink, 2007). Secondary metabolites of some plants can change DNA sequence by embedding into DNA bases and can lead to cancer (Van Wyk and Wink, 2015). DNA become stable and alterations in DNA occur causing frame shift mutations. Due to antimicrobial and cytotoxic characteristics, SM of these plants are used in drugs (Wink, 2015). But in the trade of such plants certain rules have to follow.

Non covalent modification of Proteins

Proteins being main targeted site in cell are not effectively modified by functional groups in SM. However, phenols and polyphenols can more efficiently alter protein structure due to presence of hydroxyl group (Bagetta et al., 2016). Due to hydroxyl group phenolate ions are formed. These phenolate ions react amino acids forming ionic bonds. Elasticity in protein structure is reduced. Some phenolics are glycosylated. Glycosylation in phenolics enhances hydrogen bonding and protein-phenolic synergy in maintained.

Interaction of secondary metabolites with cell membrane

All organisms have cell membrane. It is a protective covering around cell prevent leakage of cellular metabolites. Death of cell can occur if osmotic balance in cell is disturbed. This bio membrane has an assembly of proteins. SM can also attach to bio membrane proteins (Wink, 2015). Lipophilic secondary metabolites attach to membrane bilayer and change osmotic balance by enhancing membrane permeability. So lipophilic secondary metabolites demonstrate antimicrobial characteristics by bursting microbial cell membrane (Van Wyk and Wink, 2015).

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Figure 1.3 Interaction of Secondary metabolites with biomembranes

Saponins can complex membrane cholesterol; polyphenols influence 3D structure of membrane proteins (receptors, transporters, ion channels); small lipophilic terpenoids assemble in the inner lipophilic core of the biomembrane (Sun and Wink, 2014).

Secondary metabolites as antioxidants

Some active species of oxygen also affect proteins, lipids and nucleic acids. Several health related disorders occur by taking extra quantity of ROS. Secondary metabolites of plants such as phenolics, terpenoids and ascorbic acid hinder the formation of reactive oxygen species. Revelation of antioxidant activity is major aspect of secondary metabolites of plants (Su and Wink, 2015).

Combinatorial therapy between bacteriophages and plant extracts can also be used to treat bacterial infections. Due to overuse of antibiotics, combinatorial therapy is developed. Previously, both plant extracts and bacteriophages were used alone. Both have antibacterial activity (Pimchan et al., 2018). So, synergistic effect of both plant extract and bacteriophages is studied to find out how both plant extract and phages reduced bacterial growth.

In current study, plant extract was derived from Sygygium aromaticum (Clove). Bacteriophages against Pseudomonas aeruginosa were previously isolated from wastewater. The isolated phages infected almost 26 strains of Pseudomonas aeruginosa. Synergistic activity was measured by spot assay and by disc diffusion method. In the modern era, the development of new drugs is imperative and we also need to develop a system in which we do not have to use antibiotics. So, synergistic use of plant extracts and bacteriophages is considered susceptible for drug resistant strains.

Aim

The aim of the current study is to assess the synergistic antibacterial effects of plant extracts with bacteriophage as an alternative to antibiotics against Pseudomonas aeruginosa, compared with bacteriophages and plant extracts alone.

CHAPTER 2 MATERIALS AND METHODS

Bacterial Strain and Bacteriophages

A well characterized Pseudomonas aeruginosa strain PA1 (Accession number MG763232). In this study bacterial strain was cultured on LB agar, stored at 4ᵒC. An overnight bacterial culture was used for different experiments. Seven different bacteriophages GPP, Ittp, RS, RL, JPA, Sal, and Iqpl were used in research.

Table 2.1 Constituents of L-Agar

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Table 2.2 Constituents of L broth

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Table 2.3 Constituents of semisolid agar

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Table 2.4 Constituents of 30% DMSO

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Bacteriophage propagation

At first, bacteriophages were isolated by Muhammad Iqbal, latter these bacteriophages were propagated on host strain. 1ml of bacterial culture (Pseudomonas aeruginosa) and 500µl of each phage lysate was added in 100ml broth in 250ml flasks separately. Placed flasks in shaker for 24hrs at 37ºC. After overnight incubation, 500µl chloroform was added into these flasks and after half an hour the mixture was shifted to falcons and centrifuged at 4000rpm for 10min and isolated cell free lysate from mixture through usage of micro filters (0.45µm). Spot assay was performed for confirmation of bacteriophages. 900µl broth was added to first eppendorf and 990µl was added to other eppendorfs. 100µl of phage lysate was added to first eppendorf and 10µl from first dilution was taken and added to other eppendorf. Dilutions upto 10-11 were made by repeating the same procedure. 100µl from 24hrs old bacterial culture was spread on LB agar plate. Added 3-4ml of semisolid agar and gently swirled the plate. Then about 7µl spots of phage lysates were drawn on semisolid agar. Incubated the plates at 37ºC for 24hrs.

Double layer method is used for phage purification and for titer calculation. For this method, serially diluted bacteriophages were used and 100µl of bacterium was added to each dilution. Placed mixture for 5-7 min. After it, poured mixture onto LB agar plate and added 3-4ml of semisolid and swirled plates. Incubated plates at 37ºC for 24 hrs.

Bacteriophages were also purified by using this method. Isolated plaques were picked from double layer plate and purification was done.

Preparation of Clove (Syzygium aromaticum) chloroform and methanolic extracts

Clove buds were taken from local market and then dried them. About 100g of clove buds were taken and ground them. Added powdered form of cloves (50g) separately into 250ml of methanol and chloroform in 500ml flask for 72hrs and filtered through filter paper. Dried the extract in evaporator and reconstituted in 30% DMSO (Krishnan et al., 2012). 5mg of dried methanolic and chloroform extracts of clove were added in 10ml of 30% DMSO. Stored falcon at -20ºC by wrapping it with aluminum foil. The final concentration of both extracts was 500µg/ml. DMSO (30%) was used as negative control.

Antibacterial activity of plant extracts

Antibacterial activity of plant extracts was determined by using spot assay technique on LB agar for both methanol and chloroform extracts of clove. 24 hours old bacterial culture was used. Bacterial culture was spread on LB agar plate and spots of 2µl, 6µl, 10µl, 20µl were drawn on semisolid agar. Plates were placed on bench top in order to diffuse extracts into agar. Incubated at 37ºC for 24 hours. Zone of inhibition was measured.

Evaluating synergistic antibacterial effects of plant extracts and bacteriophages

L-agar and semi-solid agar were prepared and autoclaved. Then L-agar was poured in petri plates and allowed to settle down. Bacterial culture was spread by using semisolid agar onto plate. Plates were allowed to solidify. Then, spots of plant extract (4µl), bacteriophage (4µl) and bacteriophage + plant extract (2µl+2µl) were drawn. Spots were allowed to diffuse through agar by placing on bench top for a while. Incubated plates at 37ºC for 24 hours. Zone of inhibition (mm) was measured.

Filter paper discs were made using puncture. L-agar was prepared. Discs, agar, cotton buds and petri plates were autoclaved. L-agar was poured in petri plates and allowed to settle down. Overnight bacterial culture (O.D 0.5 at 600nm) was spread on agar plates with the help of cotton buds. 4µl of phage lysate, 4µl of plant extract and 4µl of mixture of plant extract and phage lysate (2µl of plant extract+2µl of phage lysate) was dispensed onto autoclaved filter paper discs and allowed to dry. After sometime, discs were placed onto agar plates. Plates were kept on bench top for some time and then placed in incubator at 37ºC for 24 hrs. Zone of inhibition was measured.

L-broth (10ml) was added to test tubes to check effectiveness of plant extracts and to check combined effect of plant extracts and bacteriophages against Pseudomonas aeruginosa. For this purpose test tubes containing 10ml of broth were autoclaved. In one test tube 200µl of bacteria PA1 (OD 0.5 at 600nm) was added (control). Equal amount (200µl) of bacteriophage (GPP, Ittp, RL, RS, Sal, JPA, and Iqpl) lysates, plant extracts (methanol and chloroform extracts of clove), mixture of bacteriophage lysate and plant extracts (100µl plant extract+ 100µl bacteriophage lysate) were added to separate test tubes. At the end, PA1 was added to all test tubes. Test tubes were placed in incubator at 37ºC. OD was checked for 24 hours at intervals of 2 hours.

CHAPTER 3 RESULTS

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Table 3.1 Susceptibility of plant extract against Pseudomonas aeruginosa based on zone diameter (mm)

Antibacterial activity of plant extracts

Both chloroform and methanol extracts (2µl each) of clove was used for assessing the antibacterial activity against Pseudomonas aeruginosa. No inhibitory effect of DMSO was found against Pseudomonas aeruginosa.

All these strains are susceptible both methanol and chloroform extracts of clove and inhibitory effect was found when small amount of plant extracts (2µl) was used. Diameter of inhibitory zone was increased by increasing amount of extract.

Table 3.2 Susceptibility of bacteriophages against Pseudomonas aeruginosa based on zone of inhibition (mm)

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Zone of inhibition (diameter mm)

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Inhibitory effect was shown by all phages against Pseudomonas aeruginosa. However, size of inhibitory zone was different for different phages against different strains of Pseudomonas aeruginosa even same phage when used against different strains showed different results. RL, RS were more susceptible against PA7 than other strains. GPP was more susceptible against PA1 and PA2 than others. JPA and Ittp were more susceptible against PA1 than other strains. Iqpl was more susceptible against PA7 and Sal was more susceptible against PA1 than other strains.

Synergism between plant extract and bacteriophages

Inhibitory effect was shown by all phages (RL, RS, GPP, JPA, Iqpl, Ittp, and Sal) against PA1, PA2, PA3 and PA7 on agar plates. Zone of inhibition (mm) is mentioned in following tables. The mixture of all the phages and plant extracts showed inhibitory effect but out of seven phages, synergistic activity of mixture of methanol extract of clove and RL and mixture of RS and methanol extract of clove showed remarkable inhibition against Pseudomonas aeruginosa.

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Table 3.3 Susceptibility of mixture of bacteriophages and plant extracts against Pseudomonas aeruginosa PA1 based on zone of inhibition (mm)

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Figure 3. 4 Combination between RL and plant extract against PA1

The effect of RL alone and in combination with plant extract was studied on agar plates. Inhibitory effect was shown by phage alone and in combination with extract. Diameter of inhibitory zone is almost equal whether RL used alone or in mixture.

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Figure 3. 5 Inhibitory effect was shown by RS alone and in combination with plant extracts.. Inhibitory effect was shown by phage alone and in combination with extract. Diameter of inhibitory zone is almost equal whether RL used alone or in mixture.

Table 3.4 Susceptibility of mixture of bacteriophages and plant extracts against Pseudomonas aeruginosa PA7 based on zone of inhibition (mm)

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Figure3.3 Combination between bacteriophages and plant extracts against PA3

a= GPP, b= GPP+ methanol extract of clove, c=GPP+ chloroform extract of clove, d=JPA+ chloroform extract of clove, e=JPA+ methanol extract of clove, f=Ittp+ methanol extract of clove, g=Ittp+ chloroform extract of clove, h= Iqpl+ methanol extract of clove, i=Iqpl+ chloroform extract of clove

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j=Sal+ methanol extract of clove, k= Sal+ chloroform extract of clove, l= Sal, m=RS+ methanol extract of clove, n=RS+ chloroform extract of clove, o=JPA, p=RS, q=Ittp, r=Iqpl.

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Figure 3.4 Combination between bacteriophages and plant extracts against PA3

Inhibitory effect is shown by bacteriophages alone and when a mixture of bacteriophages and plant extracts was used.

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Table 3.6 Susceptibility of mixture of bacteriophages and plant extracts against Pseudomonas aeruginosa PA2 based on zone of inhibition (mm)

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Figure 3.5 Combination of RL and plant extract against PA3

RL showed inhibitory effect when used alone and in combination with plant extracts. Diameter of inhibitory zone for mixture of RL and methanol extract of clove is 4.72mm and for mixture of chloroform extract of clove and RL is 4.75mm that is slightly larger than inhibitory zone of RL with diameter 4.58mm

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Table3.7 Comparative table showing the susceptibility of bacteriophages alone and in mixture with plant extracts against PA1, PA2, PA3 and PA7

Zone of inhibition (Diameter in mm)

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A = Methanol extract of clove, B = Chloroform extract of clove

Inhibitory effect is shown by all the bacteriophages. However, zone of inhibition is different for different phages. All bacteriophages showed inhibitory effect when used alone and in combination with plant extracts against different strains of Pseudomonas aeruginosa. Inhibitory zone of most phages used alone and in combination with plant extracts for one strain is almost same. It means, using phages in combination with plant extracts have no significant effect on activity of most phages. However, activity of RL phage was enhanced in presence of methanol extract of clove.

Synergistic effects of bacteriophages with plant extracts

Combination of bacteriophages and plant extract (Methanol and Chloroform extract of clove) showed susceptibility against Pseudomonas aeruginosa when used alone and in combination. However, plant extracts showed increased susceptibility when used alone.

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Figure 3.6 Synergistic effect between RL and plant extract against Pseudomonas aeruginosa

A =PA1, B = PA1+ RL, C = PA1+ Methanol extract of clove, D = PA1+ Methanol extract of clove+ RL, E =PA1+ Chloroform extract of clove, F = PA1+ Chloroform extract of clove+ RL

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Figure 3.7 Synergistic effect between RS and plant extract against Pseudomonas aeruginosa

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Figure 3.8 Synergistic effect between GPP and plant extract against Pseudomonas aeruginosa

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Figure 3.9 Synergistic effect between JPA and plant extract against Pseudomonas aeruginosa

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Figure 3.10 Synergistic effect between Iqpl and plant extract against Pseudomonas aeruginosa

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Figure 3.11 Synergistic effect between Ittp and plant extract against Pseudomonas aeruginosa

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Figure 3.12 Synergistic effect between Sal and plant extract against Pseudomonas aeruginosa

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A significant reduction in bacterial growth was observed when RL was used alone and in combination with methanol and plant extract of clove (Figure 3.6). Combination of methanol extract of clove was more effective than chloroform extract of clove (Figure 3.7). In case of RS, mixture of methanol extract was more effective than mixture of chloroform extract and phage alone. When GPP was used in combination with methanol and chloroform extract of clove, remarkable reduction in bacterial growth was observed (Figure 3.8). However, mixture of chloroform extract with GPP had greater inhibitory effect than methanol extract mixture with GPP but its activity was not maintained for 24 hours. When Iqpl was used in combination with plant extracts, phage activity was enhanced at start but it could not maintain its effect for 24 hours (Figure 3.10). The same effect was observed when Ittp was used (Figure 3.11). In case of JPA, combination of JPA with plant extracts was not as effective as JPA used alone (3.9). When Sal was used effect of mixture was almost same as that of Sal used (Figure 3.12) indicating extract had no effect on phage activity. Activity of plant extracts alone was also checked against Pseudomonas aeruginosa. Plant extracts showed increased susceptibility when used alone. To check P value t test was applied and a significant reduction in bacterial growth was observed, when bacteriophages (alone), plant extracts (alone) and mixture of bacteriophages and plant extracts were used as compared to control value. Only combination of RS and chloroform extract of clove (Figure 3.7) and combination of GPP and methanol extract of clove (Figure 3.8) did not show significant results.

CHAPTER 4 DISCUSSION

Bacteria have ability to form biofilms. Biofilm formation makes bacteria resistant to antibiotics so, many antimicrobial strategies such as using plant extracts, phage lysins or their combined use can prevent biofilm formation (Simoes, 2011). Bacteriophage and plant extracts are used to kill microorganisms. These two therapies are used separately or in combination. Combinatorial therapy can kill many infectious microorganisms, so synergistic effect between plant extract and bacteriophages is studied by other scientists but not widely explained. Phage endolysins attack at specific site in bacteria and kill them. Some bacteria have developed resistance to phage endolysins also so endolysins can be used in combination with antibiotics, plant extracts or with other endolysins to kill bacteria (Nelson, 2018). Plant secondary metabolites, antimicrobial peptides, antibodies, bacteriophages are used as an alternative to antibiotics. All these alternatives should be cost effective, toxic to bacteria and harmless to host. Because bacteria have developed resistance to most of them so combinatorial therapy can provide us more benefits than using a single therapy.

In current study, the plant extracts (Methanol extract of clove and Chloroform extract of clove), phages (RL, RS, GPP, JPA, Ittp, Iqpl, Sal) and combination of bacteriophages and plant extracts were used to check inhibitory effect.

Plant extracts showed greater inhibitory effect when used alone against Pseudomonas aeruginosa ( Dua et al., 2014 ) . Because constituents in clove extract have antimicrobial characteristics (Nzeako et al., 2006). Clove extract influenced bacterial activity by bursting of its cell membrane, by reacting to bacterial proteins and DNA bases through covalent and non-covalent bonding. Clove extract also affect lipids through oxygenation and reveals its antioxidant behavior (Gülçin et al., 2012). Reducing environment is necessary for reducing bacterial growth. Due to presence of clove extract in media pH of media changes, contributing to reducing environment. (Wong et al., 2006). So, growth of Pseudomonas aeruginosa growth in medium is affected. Media is deficit in metal ions due to chelating behavior of phenolics. Due to the presence of phenolics in extract reducing environment is maintained and bacterial growth is reduced (Dua et al., 2014).

To check inhibitory effect of clove against Pseudomonas aeruginosa clove extract was made in methanol and chloroform. Solvent is used for extraction of secondary metabolites from plant. For the extraction of flavonoids and phenols plant extract is made in methanol. Chloroform extract can extract all secondary metabolites except saponins and tannins (Dhawan and Gupta, 2017). Both methanol and chloroform extract of clove has inhibitory effect (Krishnan et al., 2012).

In current study, chloroform extract has higher antimicrobial potential than methanol extract of clove. Chloroform extract of clove showed greater inhibitory effect than methanol extract of clove. However, when a mixture of RL and methanol extract of clove was used, it had greater inhibitory effect than RL and methanol extract of clove used alone (Figure 3.6). It means, the activity of phage RL is enhanced in the presence of methanol extract of clove. Initially, RL and mixture of RL and methanol extract of clove had similar activity. When the effect was studied for 24 hours, RL activity was enhanced in the presence of methanol extract of clove. However, mixture of RL and chloroform extract of clove had greater inhibitory effect than RL used alone. However, when chloroform extract of clove was used alone, it was more effective than all. It means, in the presence of RL, chloroform extract of clove could not maintain its own activity against bacteria and bacterial growth started to increase. T test was performed to check significant difference between bacteriophage (RL) and extracts only, bacteriophage (RL) and combination of bacteriophage with extracts. P value is <0.05 so experimental data is statistically significant (Figure 3.6).

When RS phage used alone, it had inhibitory effect against bacteria. When a mixture of chloroform extract of clove and RS was used, bacterial growth was reduced at first. As time passed, bacteria started to grow in the presence of mixture of chloroform extract of clove and RS. It means, mixture could not maintain its activity for 24 hours (Figure 3.7). However, when a mixture of RS and methanol extract of clove was used, it had greater inhibitory effect than RS and methanol extract of clove used alone. Phage activity is enhanced in the presence of methanol extract of clove. Phage activity is enhanced due to presence of secondary metabolites. Effect maintained its activity for 24 hours. T test was performed to check significant difference between bacteriophage (RS) and extracts only, bacteriophage (RS) and combination of bacteriophage with extracts. P value is <0.05 except combination of RS and chloroform extract of bacteriophage (Figure 3.7).

When GPP phage was used in combination with plant extracts, a significant inhibition occurred. The inhibitory effect of mixture of GPP and methanol extract of clove was almost similar to inhibitory effect of GPP alone (Figure 3.8). However, the inhibitory effect of mixture of GPP and chloroform extract of clove was initially greater than inhibitory effect of GPP alone. But it could not maintain its activity for 24 hours. Its inhibitory effect was similar to inhibitory effect of GPP alone at the 24th hour. The mixture of GPP and plant extract did not show a marked reduction in bacterial growth than GPP used alone. T test was performed to check significant difference between bacteriophage (GPP) and extracts only, bacteriophage (GPP) and combination of bacteriophage with extracts. P value is <0.05 except combination of GPP and methanol extract of bacteriophage (Figure 3.8).

When a mixture of JPA and plant extract (methanol extract of clove and chloroform extract of clove) reduction in bacterial growth was noted. But it was not similar to that of JPA used alone. The inhibitory effect of JPA used alone is greater than that of a mixture of plant extract and phage (Figure 3.9). However, mixture of extracts and phages showed similar inhibitory activity. It means, extracts could not maintain their activity or phage activity was reduced in the presence of extract. T test was performed to check significant difference between bacteriophage (JPA) and extracts only, bacteriophage (JPA) and combination of bacteriophage with extracts. P value is <0.05 so experimental data is statistically significant (Figure 3.9).

Iqpl showed remarkable inhibitory effect against bacteria when used alone. When mixture of plant extract and Iqpl was used, Iqpl activity slightly increased (Figure 3.10). But this effect was not maintained for 24 hours. However, mixture of Iqpl with methanol and chloroform had almost equal effect on bacterial growth. Effect of mixture of Ittp and plant extract is similar to Iqpl (Figure 3.11). The mixture of phage and plant extract could not maintain its activity for 24 hours. Plant extract had no significant impact on phage activity. Plant extract could not maintain its own effect when used with phage. T test was performed to check significant difference between bacteriophage (Iqpl, Ittp) and extracts only, bacteriophage (Iqpl, Ittp) and combination of bacteriophage with extracts. P value is <0.05 so experimental data is statistically significant (Figure 3.10, Figure 3.11).

When a mixture of Sal and plant extract was used, a significant inhibition in growth of bacteria is noted. The effect of mixture of phage and plant extract was much more than plant extract used alone (Figure 3.12). But when Sal was used alone, its activity was equal to activity of mixture used. It means, inhibitory effect is due to Sal. T test was performed to check significant difference between bacteriophage (Sal) and extracts only, bacteriophage (Sal) and combination of bacteriophage with extracts. P value is <0.05 so experimental data is statistically significant (Figure 3.12).

The deficit role of plant extract in the presence of bacteriophages indicates that plant extract has lost its action to secondary metabolites. This is not true for all bacteriophages. Different bacteriophages behave differently when a mixture of plant extract and bacteriophage is used. Plant extract acts against bacteriophage only when medium is free of proteins. This vision was also supported by Chantrill and his colleagues (Chantrill et al., 1952). Plant extract could not maintain its activity for 24 hours of infection. Plant extracts can hinder viral metabolic activities in bacterial cells. This is due to antiviral action of compounds in clove extract (Lane et al., 2019). Although these compounds are present in trace amounts, their activity cannot be ignored. Inhibitory activity is indicated by all bacteriophages but effect could not be maintained for 24 hours (Pimchan et al., 2018).

However, the combinatorial therapy is still studied by scientists, the major focus is placed on combine usage of antibiotics and phages (Oechslin et al., 2016; Chaudhry et al., 2017; Tagliaferri et al., 2019), phage endolysins (Schmelcher et al., 2012) and usage of phage cocktail (Melo et al., 2016; Żaczek et al., 2016; Schooley et al., 2017) because plant extracts have slightly less antimicrobial activity than antibiotics and phage cocktail (Wolska et al., 2012). However, the current study indicated, there should be some variations. Some other compounds such as prebiotics should also be added to phages along with plant extracts to obtain better effects (Upadhyay et al., 2019).

Conclusion

Life-saving consequences are obtained by using phage therapy and it is the most outstanding advancement in scientific community. Phages are used because most of microbes have developed resistance against antibiotics. So antibiotic usage is dissatisfied. Since most of phages are also specie specific. We are in great need of therapy that can overcome such challenges. Consequently, the use of combinatorial therapy by incorporating plant extracts and phages has become an emerging area of major concern in the medical and scientific community. Most of the plants used in this therapy are those that were usually used by natives for treating infectious diseases. This can also be used as an indication that usage of plant extracts increase the antibacterial activity of phages. Synergistic activity was helpful in reducing bacterial pathogenicity. Further research is needed to explore the synergistic effect in infectious animals before testing it in clinical settings.

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