Plant Products in Dental Health

INDEX

Abbreviations

List of tables

List of figures

1. Introduction

2. Review of Literature

3. Materials and Methods

4. Results and Discussion

5. Conclusion

6. Appendices

7. Bibliography

Abbreviations

Abbildung in dieser Leseprobe nicht enthalten

List of tables

1. Common organisms forming biofilms on medical implants

2. Human infections involving bacterial/fungal biofilm

3. Various plant diseases involving biofilms

4. Details of plant selected seeds

5. Test organisms

6. Heating and cooling cycles of different Solvents during MAE

7. Classification of bacterial adhesion and biofilm formation

8. Extraction efficiency and reconstitution efficiency of seeds in various solvents

9. Percent inhibition at various concentrations of ethanolic extract of E. officinalis seeds against different human pathogens

10. Percent inhibition at various concentrations of methanolic extract of E. officinalis seeds against different human pathogens

11. Percent inhibition at various concentrations of acetone extract of E. officinalis seeds against different human pathogens

12. Percent inhibition at various concentrations of E. officinalis seeds against different Plant pathogens

13. Results of broth dilution assay of E. officinalis seed extracts against different organisms

14. Percent inhibition of S. mutans at various concentrations of different plant extracts

15. Percent inhibition of S. mutans at various concentrations of pure phytocompounds

16. Results of broth dilution assay of various seed extracts against S. mutans

17. Results of total activity of various seed extracts against different organisms

18. Time required to kill

19. Results of Disc diffusion assay of antibiotics against S. mutans

20. Result of Tissue culture plate method for biofilm formation

21. Results of crystal violet assay, tube method and viable count of ethanolic extract of E. officinalis seed against biofilm of S. mutans

22. Results of crystal violet assay, tube method and viable count of various extract of T. indica seed extract against biofilm of S. mutans

23. Results of crystal violet assay, tube method and viable count of various extracts of S. cumini seed against biofilm of S. mutans

24. Results of crystal violet assay, tube method and viable count of various antibiotics against biofilm of S. mutans

25. Comparison between effect of various plant extract/ antibiotics on planktonic and biofilm form of S. mutans

List of figures

1. Events during biofilm formation

2. Mechanisms of antibiotic resistance in biofilms

3. Emblica officinalis fruit and seeds

4. Tamarindus indica fruit and seeds

5. Syzygium cumini fruit and seeds

6. Phoneix sylvestris fruit and seeds

7. Manilkara zapota fruit and seeds

8. Correlation between extraction efficiency and average total activity (E. officinalis)

9. Correlation between extraction efficiency and average total activity (against S. mutans)

10. Effect of E. officinalis extract against S. mutans after 72h

11. Effect of T. indica extract against S. mutans after 72h

12. Effect of S. cumini extract against S. mutans after 72 h

13. Effect of P. sylvestris extract against S. mutans after 48 h.

14. Effect of Cucurmin against S. mutans after 48 h.

15. Effect of E. officinalis extract against V. cholerae after 24 h

16. Effect of E. officinalis extract against P. aeruginosa after 48 h

17. Effect of T. indica methanol extract on S. mutans biofilm

18. Effect of T. indica acetone extract on S. mutans biofilm

19. Effect of S. cumini ethanol extract on S. mutans biofilm.

20. Effect of S. cumini methanol extract on S. mutans biofilm

21. Effect of S. cumini acetone extract on S. mutans biofilm

22. Correlation between extraction efficiency and total activity (against biofilm form of S. mutans)

23. Biofilm after PBS washing

24. Destining solution after removal of it from adherent plate

25. Results of tube method

1. INTRODUCTION

1. INTRODUCTION

Over the years, many antimicrobial agents have been used for the control or elimination of bacteria in households, industry and for the treatment of common bacterial infections in humans. Over time, increasing use, and misuse of existing antimicrobial agents in medicine and in agriculture has produced strains of multiple antimicrobial resistant bacteria, making it difficult to treat most of the common microbial infections and results in high morbidity and mortality (Carlet et al., 2012). In the US alone, the extra cost for treating the drug resistant verity is around $10 billion per year (Talaro, 2008).

Continuous appearance of drug-resistant strains of pathogenic microorganisms makes it necessary to search for novel natural or synthetic antimicrobial compounds. Reduced susceptibility of biofilms to conventional antibiotics makes this challenge more daunting. Natural products have been a significant source of commercial medicines and drug leads. Plants are naturally gifted at the synthesis of bioactive compounds. Screening of crude plant extracts is the first step in the long process of discovery of novel bioactive compounds. Isolation of active principle(s) from these crude extracts followed by successful structure elucidation can provide novel lead compounds (Kothari and Seshadri, 2010). Plants synthesize several bioactive compounds such as polyphenols, terpenoids, essential oils, alkaloids, saponins, peptides and proteins, with antibacterial, antifungal, antioxidant and other properties (Kothari et al., 2010a).

Among the factors contributing to microbial resistance, is the ability of the microbes to exist in biofilm forms that allow them to withstand harsh environmental conditions and antimicrobial agents (Costerton et al., 1999). Biofilms have been associated with wide range of problems in industry, in medicine (dental plaque, clinical infections) and in agriculture . When bacteria exist in biofilm, the well-known mechanisms of antibiotic resistance, such as efflux pumps, modifying enzymes, and target mutations, do not always seem to be responsible for the protection of bacteria (Li Chen, 2011; Stewart and Costerton, 2001). Even sensitive bacteria which do not have a known genetic basis for resistance can have profoundly reduced susceptibility when they are present in a biofilm. When cells exist in a biofilm, they can become 10-1000 times more resistant to the effects of antimicrobial agents (Costerton et al., 1999; Davey and O'toole, 2000; Hiorth et al., 2007; Lewis, 2001; Mah and O'Toole, 2001; Stewart and Costerton, 2001; Scheie and Petersen, 2004). Eradication of microbial biofilms is a difficult task. Conventional antibiotics effective against planktonic pathogen may not prove equally useful against biofilms. Efforts are being made to find antimicrobial substances effective against biofilms, from plant extracts (Rukayadi and Hwang, 2006; Hossein et al., 2013).

1.1 Microbial biofilms and their occurrence

Imagine that you haven’t brushed your teeth for a while, that furry deposit on your teeth is caused by the development of a “biofilm” on the teeth surface. Initially no much attention was paid to their capacity to exist as a community. However in recent times their ability to form biofilms in various environments, and its impact on ecology, medicine, and industry has attracted considerable attention. By definition, “biofilm is a microbially derived sessile communities characterized by cells that are irreversibly attached to a substratum or interface or to each other, are embedded in a matrix of extracellular polymeric substances that they have produced, and exhibit an altered phenotype with respect to growth rate and gene expression” (Donlan and Costerton, 2002). As Andre Levchenko and Johns Hopkins has noted: “There is a perception that single-celled organisms are asocial, but that is misguided. When bacteria are under stress- which is the story of their lives they team up and form this collective called a biofilm. If you look at naturally occurring biofilms, they have very complicated architecture. They are like cities with channels for nutrients to go in, and waste to go out” (Proal, 2008, p. 1; http://mpkb.org/home/pathogenesis/microbiota/biofilm). Biofilm normally forms on both biotic (plant surfaces, human body parts) and abiotic (in aquatic environment, and on inanimate objects, e.g. ship hull, industrial pipelines, etc.) surfaces. Biofilm builds up in a wide variety of environments, ranging from the sebum that builds up in toilet bowls, and walls of swimming pools, to the constant deposition of plaque on teeth. It is this primeval tendency of making biofilms, occurring from billions of years, through which microbes have been able to colonize most habitats on earth (Talaro, 2008). In nature, biofilms generally exist as a mixed bacterial consortium, but they may also consist of a single bacterial species. In multispecies biofilm many type of positive (coaggregation, conjugation, and protection to eradication by antimicrobial agents) and negative (bacteriotoxin production, lowering of pH) interactions take place (Burmølle et al., 2006; Perumal et al., 2007). A wide majority of plant and human pathogens have been reported for their ability to form biofilm, e.g. Streptococcus mutans, Staphylococcus epidermidis, Candida albicans, Pseudomonas aeruginosa, Staphylococcus aureus, Agrobacterium tumefaciens, Xanthomonas campestris, Pseudomonas syringae, Erwinia caratovora, Aeromonas hydrophila, etc . (Burmølle et al., 2006; Canals et al., 2006; Høiby et al. 2011; Li Chen, 2011; Perumal et al., 2007; Ramey et al. 2004; Rukayadi and Hwang, 2006; Saito et al., 2012).

1.2 Why should we study biofilms instead of planktonic cells?

The first reason for studying biofilms is that in nature more than 99% of all the bacteria exist in this state (Prakash et al., 2003; Nield-Gehrig, 2005) ( http://www.educationaldimensions.com/eLearn/biofilm/introduction.php). The second reason is that in biofilm, cells are different in many ways to their free-floating planktonic counterparts and they can get many advantages in the biofilm mode (Annous et al. 2009; Costerton et al. 1999; Donlan, 2002; Vu et al., 2009) i.e.,

- Microorganisms in biofilms exhibit elevated antimicrobial tolerance and also get protected from environmental stresses such as extreme pH, oxygen, osmotic shock, heat, freezing, UV radiation, predators, etc.
- Extracellular polymeric matrix formed from the secreted exopolysaccharides (EPS) increases the binding of water resulting in decreased chance of dehydration (desiccation) of the bacterial cells, which is a common stress condition experienced by planktonic cells.
- The adherent nature of microbial cells in biofilms allows rapid exchange of nutrients, metabolites, and genetic materials.

Thus, biofilm cells are different and behave differently to planktonic cells. So if we want to be able to extrapolate results obtained in the laboratory to real life we need to study bacteria in biofilm.

Our study focused on evaluation of certain plant products/extracts for their antimicrobial activity against planktonic/biofilm forms of selected human and/or phytopathogenic microbes.

1.3 Objectives:

- To screen Emblica officinalis seed extracts for their antimicrobial property against planktonic form of selected pathogens and to determine MIC (minimum inhibitory concentration).
- To screen various extracts of Emblica officinalis, Tamarindus indica, Syzygium cumini, Phoenix sylvestris, Manilkara zapota seeds, and pure compouands (curcurmin, quercetin, and gallic acid) for their antimicrobial property against planktonic form of S. mutans.
- To evaluate the biofilm eradication potential of Emblica officinalis, Tamarindus indica, Syzygium cumini seed extracts, and their effect on viability of S. mutans biofilm.

2. REVIEW OF LITERATURE

2. REVIEW OF LITERATURE

2.1. Biofilm formation

2.2. Mechanisms of antibiotic resistance among biofilms

2.3. Problems associated with biofilm

2.3.1. Biofilm in public health

2.3.2. Biofilm in industry

2.3.3. Biofilm in agriculture

2.4. Applications of biofilm

2.5. Plant Materials

2.5.1. Embalica officinalis

2.5.2. Tamarindus indica

2.5.3. Syzygium cumini

2.5.4. Phoenix sylvestris

2.5.5. Manilkara zapota

2.6. Test Microorganisms

2.6.1. Streptococcus mutans

2.6.2. Streptococcus pyogenes

2.6.3. Staphylococcus aureus

2.6.4. Staphylococcus epidermidis

2.6.5. Pseudomonas aeruginosa

2.6.6. Vibrio cholerae

2.6.7. Salmonella paratyphi A

2.6.8. Candida albicans

2.6.9. Escherichia coli

2.6.10. Xanthomonas campestris

2.6.11. Agrobacterium tumefaceins

2.6.12. Pectobacterium caratovorum

2.6.13. Pseudomonas syringae

2.7. Extraction of Plant material

2.7.1. Microwave assisted extraction (MAE)

2.8. Antimicrobial susceptibility testing

2.8.1. Antibacterial susceptibility testing

2.8.1.1 Broth dilution assay

2.8.2. Antifungal susceptibility testing

2.9. Time required to kill

2.10. Methods for the study of biofilm

2.10.1 Tissue culture plate (TCP) method

2.11. The ‘Eagle effect’

2.1. Biofilm formation

Biofilm formation is a multistage process (Fig 1). The initial step in biofilm formation involves reversible attachment of planktonic (freely moving individual cell) bacteria to a surface (colonization) by using adhesins (Donlan and Costerton, 2002; De Beer and Stoodley, 2006; Høiby et al. 2011). For example, polysaccharide adhesin (PS/A) of S. epidermidis initiates adhesion on naked or coated polymer surface (expression is controlled by the inter-cellular adhesion operon (Ica) (Li Chen, 2011; Tojo et al., 1988; Zhang et al., 2003). In Streptococcus pyogenes, various cell surface molecules such as proteins and lipoteichoic acid are important for adherence on cultured human cells (Nobbs, 2009). The adhesin SpaP (PAc) in Streptococcus mutans is important for adhesion on teeth surfaces, and its expression is enhanced by sucrose or preexisting biofilm (Li Chen, 2011). In Vibrios, lateral flagella provide mechanism for attachment on surfaces (Atlas and Bartha, 1998). In P. aeruginosa, one of the virulence determinants, alginate plays important role in the adherence of the organism on trachael epithelium (Anwer et al., 1992; Marcus et al., 1989). In S. aureus, SasC protein factor plays important role in colonization during infection (Schroeder et al., 2009). The adhesion process is also affected by physiological state of the organism; in some organisms attachment is high in log phase, while in others attachment is high in stationary phase (Fletcher, 1999). The bacteria are still susceptible to antibiotics at this stage.

Abbildung in dieser Leseprobe nicht enthalten

Figure 1: Events during biofilm formation

(Anwer, 1992; Annous, 2009; Høiby, 2011; Proal, 2008; Scheie and Petersen, 2004)

The next step in biofilm formation is turning the initial reversible binding of organisms to a surface into irreversible binding, followed by multiplication of the bacteria resulting in microcolony formation, after which production of a polymer matrix around the microcolony converts it into a mature biofilm (Annous et al. 2009; De Beer and Stoodley, 2006; Høiby, 2011). Mature biofilm architecture varies from flat homogeneous layer of cells, to organized mushroom-like or tower-like structures (Folkesson et al., 2008; Høiby et al., 2011). This maturation stage is controlled by quorum sensing (QS) systems, such as N-acyl-homoserine lactone (AHL) and 4-quinolone systems (in gram-negatives), AgrD peptide systems (in gram-positives), AI2/LuxS system (in both gram-negatives and gram-positives), and farnesol systems (in fungi) (Høiby et al., 2011; Li Chen, 2011).The subsequent biofilm develoment involves focal dissolution, liberating bacterial cells (erosion), that can then spread to other locations where new biofilms can be formed from these liberated bacteria. This liberation process may be triggered by bacteriophage activity within the biofilm. The mature biofilm matrix may contain water-filled channel like structures and thereby resemble primitive, multicellular organisms (Annous et al., 2009).

2.2. Mechanisms of antibiotic resistance among biofilms

Many mechanisms (Fig 2) have been proposed for antibiotic resistance in biofilms. One of the proposed resistance mechanisms is based on the possibility of slow or incomplete penetration of the antibiotics into the biofilm (Donlan and Costerton, 2002), due to EPS matrix in which microorganisms are embedded in biofilm (Mah and O’Toole, 2001; Stewart and Costerton, 2001). A well-known disinfectant chlorine was able to reach only upto 20% of that in bulk media within a mixed species biofilm of P. aeruginosa and Klebsiella pneumoniae (De Beer et al., 1994). Al-Fattani and Dauglas (2004) investigated penetration of antifungal drugs through Candida biofilms, and concluded that poor antifungal penetration is not a major drug resistance mechanism for Candida biofilms. They found that mixed species biofilm of bacteria (S. epidermidis) and yeast (C. albicans) allowed slower penetration of drugs than single species biofilm of C. albicans. Many researchers reported that penetration of aminoglycosides is retarded in P. aeruginosa biofilm, it is due to binding of aminoglycoside with alginate (polysaccharide) (Donlan and Costerton, 2002; Stewart and Costerton, 2001).

Abbildung in dieser Leseprobe nicht enthalten

Figure 2: Mechanisms of antibiotic resistance in biofilms

(Mah and O'Toole, 2001; Stewart and Costerton, 2001)

Another resistance mechanism focuses on altered chemical microenvironment within the biofilm. Microscale gradient formation in nutrient concentrations is a well-known feature of biofilms. Oxygen can be completely consumed in the surface layers of a biofilm, which ultimately lead to anaerobic environment in the deeper layers. Local accumulation of acidic waste products might lead to pH differences greater than 1 between the bulk fluid and the biofilm interior. Physiological heterogeneity and gradient formation is very well recorded within the biofilms. All the cells present in the biofilm are not in the same physiological or metabolic state (Joshi et al., 2010). Combined with this heterogeneity in microenvironment, slower growth rate of the microbes in biofilm than its planktonic stage, antibiotic action may be antagonized (Donlan and Costerton, 2002; Stewart and Costerton, 2001). Most antibiotics are best effective against actively growing cells. There can be significant differences in the metabolic and/or growth rates of biofilm bacteria compared to their planktonic counterparts. Welch et al. (2012) derived the specific growth rate of S. mutans bacterial biofilm. They found the specific growth rate of S. mutans in biofilm mode of growth was 0.70 h⁻¹, compared to1.09 h⁻¹ in planktonic growth. Growth related effect of antibiotics in mixed species biofilms of P. aeruginosa, Escherichia coli and S. epidermidis were reported by Mah and O’Toole (2001). They observed increase in sensitivity to tobramycin or ciprofloxacin with increasing growth rate in both- planktonic and biofilm mode. This indicates that slow growth rate of organisms in biofilm may offer them protection from antimicrobial agents (Folsom, 2010; Mah and O’Toole, 2001).

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