In Vitro Antimicrobial Activity of Tea Tree Oil on Microorganisms in Association with Oral Infections

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In vitro antimicrobial activity of tea tree oil on microorganisms in association with oral infections

 

Chapter 1. Introduction

The human oral cavity is a site that harbours more than 700 different bacterial species, which have been detected microbiologically (Larsen and Fiehn, 2017)(Karygianni et al., 2016). These bacteria naturally form different biofilms on different oral sites, and dental infections are associated etiologically with a change in these healthy oral biofilms, turning them into pathological biofilms. Biofilms present on tooth surfaces may cause dental caries, while supra- and subgingival biofilms, along and under the gingival margin, may cause periodontal diseases (Larsen and Fiehn, 2017). Although these are both bacterial diseases, dental caries and periodontal infections are not strictly infectious diseases because they result from complex interactions between commensal microbiota, host susceptibility and environmental factors, such as diet and smoking (Chinsembu, 2016).

Dental infections have been controlled by specific and non-specific control measures. Non-specific measures aims to disrupt the dental plaque or biofilm mechanically. On the other hand, specific measures include the use of chemicals to inhibit the causative agents of dental infections These measures included some non-conventional therapeutics which proved effective like enzymes, detergents, probiotics, and prebiotics (Allaker and Ian Douglas, 2015).

Around 250,000 of natural products are proved to have some level of biological activity (Demain, 2013). Hundreds of natural agents were examined for their antimicrobial effects on microorganisms of medical importance. Fortunately, many of these agents had dramatic impact on the drug industry in dentistry and they were already incorporated into medicinal preparations and mouthwashes (Jeon et al., 2011)(Karygianni et al., 2016)(Chinsembu, 2016).

With the increase in the incidence of antibiotic resistance by bacterial dental biofilms, efforts were directed to nature and its elements for therapy and cure. Essential oils (EOs), plant-derived compounds like polyphenols and animal extracts were all potential leads for antimicrobial testing (Maróti et al., 2011)(Kouidhi, Al Qurashi and Chaieb, 2015).

1.1 Microbiology of oral infections

Dental caries is considered the most common poly-microbial disease worldwide, and it is characterised by the demineralisation of a teeth without concurrent inflammation of the surrounding tissues. The development of caries was thought to only be due to a few Gram-positive bacterial species present in a biofilm, known as the specific biofilm/plaque hypothesis (Larsen and Fiehn, 2017). Based on 16S ribosomal Ribonucleic acid (rRNA) sequences, oral Streptococci are classified into four groups: the mutans, the salivarius, the mitis, and the anginosus groups (Kawamura et al., 1995). Strongly acidogenic and aciduric Streptococci, such as Streptococcus mutans and Streptococcus sobrinus, together with some Lactobacillus species, play a key role (Chinsembu, 2016). S. mutans is one of the mutans streptococci group and the most common type reported in epidemiological studies to be associated with dental caries. In addition, it is the first species to colonise infants, especially in caries active individuals (Loesche, 1986).

In dental caries biofilm formation the predominant initial colonisers are primarily from the Streptococcus mitis group, followed by Gram-positive rods, especially Actinomyces species (Larsen and Fiehn, 2017)(Costalonga and Herzberg, 2014). In total, 25.5% of bacteria found in large amounts in caries active individuals can be attributed to S. mitis, while S. mutans was found to represent 1.2% of bacteria isolated from the same patients. This indicates that S. mutans is related more with initial carious lesions (Costalonga and Herzberg, 2014), and therefore dental caries can be thought of as a two-stage process. Oral Streptococci causes the initial lesion, which then progresses due to the effect of Lactobacilli or Actinomyces species (Larsen and Fiehn, 2017)(Loesche, 1986)(Costalonga and Herzberg, 2014). Glucosyltransferase enzymes found in saliva produce glucans that form the extracellular polyscharide of dental plaque biofilms, and glucans are binding sites for cariogenic bacteria. If dental biofilms are not mechanically removed and/or are exposed to dietary sucrose, these bacteria will synthesise organic acids that dissolve the hard surface of a tooth (Jeon et al., 2011).

Interestingly, caries can be detected in the absence of S. mutans and other acidogenic/aciduric species, and contrarily, these may be present without the development of caries. This has led to the formulation of the ecological biofilm/plaque hypothesis. This hypothesis proposes that whether caries develop or not is determined by the existing ecological balance within the biofilm present on the tooth surface, and caries develop when there is a shift in the balance of the resident microorganisms (Larsen and Fiehn, 2017).

S. mutans and S. mitis have been found to be implicated in extra-oral infections that lead to cardiovascular diseases. S. mutans, for example, was found to be the most commonly isolated species from patients with heart valve abnormalities and from aneurysmal wall specimens. S. mutans has also been reported to cause infections in pregnant women, patients with inflammatory bowel disease, and patients with infective endocarditis (Han and Wang, 2013).

Enterococci are the most commonly retrieved species from root filled teeth with apical periodontitis, although their numbers are decreased in untreated necrotic teeth (Molander et al., 1998). Vancomycin-resistant enterococci (VRE) have had a significant impact on the field of medical therapeutics (Karygianni et al., 2016), and an endocarditis-associated Enterococcus faecalis virulence gene has been recovered from resistant strains in root canals (Karygianni et al., 2014)(Al-Ahmad et al., 2014).

There has been controversy concerning the presence of Staphylococcus aureus as a component of oral flora (Robertson and Smith, 2009)(Smith, Bagg and Jackson, 2001). However, it was found that S. aureus causes a range of perioral infections, such as angular cheilitis, oral mucositis in older patients and highly dependent patients receiving intravenous nutrition, and severe dental abscesses in children (Robertson and Smith, 2009)(Smith, Bagg and Jackson, 2001). Methicillin-Susceptible S. aureus (MSSA) isolates were reported by laboratories to be present in 26% of angular cheilitis cases. S. aureus is known to produce exotoxins, which may have a role in the redness and burning sensation observed in oral mucositis (McCormack et al., 2015). Viridans Streptococci and Staphylococci are two of the most common bacteria to be cultured from head and neck space infections, accounting for 28.9% and 8.9% of all isolates, respectively (Rega, Aziz and Ziccardi, 2006).

S.aureus has been found in association with acute dentoalveolar infections, and denture-induced stomatitis is another condition from which S. aureus has been cultured. The involvement of orally originating S. aureus in extraoral infections and systemic diseases has also been reported (Smith, Bagg and Jackson, 2001)(Han and Wang, 2013), and the most common systemic disease caused by oral S. aureus is prosthetic joint infection, with 8 out of 23 cases of late prosthetic joint infection found to be due to oral S. aureus (Smith, Bagg and Jackson, 2001).

In one study 50% of all periodontal lesions were shown to contain staphylococci species (Rams, Feik and Slots, 1990)(Smith, Bagg and Jackson, 2001). The high recovery rates of S. aureus from plaque in individuals with poor oral hygiene and periodontal disease is similar to their levels in the same patients’ trachea, i.e. patients with respiratory tract infections had high recovery rates of S. aureus from their plaque samples (Han and Wang, 2013).

Methicillin-resistant S. aureus (MRSA) colonises the oropharynx and resists eradication by regular antibiotics (Han and Wang, 2013)(Smith, Bagg and Jackson, 2001). Two cases have been reported of MRSA cross-infections in dental clinics in which they have re-colonised other body sites. In addition, MRSA may be involved in denture stomatitis because these bacteria demonstrated a preference for denture surfaces (Smith, Bagg and Jackson, 2001).

Similar to MRSA, MSSA was found in the saliva of patients with white sponge nevus (an oral lesion). Antibiotic therapy, resulted in some improvement in the lesion indicating a role for S. aureus infection in the disease process of white sponge nevus (Marrelli et al., 2012).

1.2 Antibiotic resistance of selected microorganisms

In the UK, amoxicillin, penicillin, metronidazole and erythromycin are the most commonly prescribed antibiotics in the management of acute dental abscesses. With the exception of metronidazole, an increase in antibiotic resistance in microbes recovered from acute dental abscesses has been reported. Reduced susceptibility to penicillin is more prevalent in the mitis group of streptococci than in the anginosus group, while resistance to macrolides appears to have a higher prevalence in Viridans Streptococci group and anaerobic Streptococci (Robertson and Smith, 2009). It is well known in literature that S.aureus is a virulent pathogen. It has developed resistance to different antibiotics e.g.Methicillin, Vancomycin, Daptomycin and many others (Gardete and Tomasz, 2014)(Petrovic-Jeremic et al., 2008)(Bayer, Schneider and Sahl, 2012)(Hiramatsu et al., 2014).

S.mutans was found to have moderate resistance to fluoride. Two special genes were found to code for chloride channels and were responsible for its fluoride resistance (Men et al., 2016). Also, S.mutans genome has at least 11 genomic islands that are acquired by horizontal gene transfer. Changes in these genes may lead to high susceptibility to Trimethoprim (Chattoraj et al., 2009). Moreover, S.mutans and E.faecalis had 45% and 25% frequency of Clindamycin resistance, respectively (Loyola-Rodriguez et al., 2014).

78% of E.faecalis samples were resistant to at least one out of ten of the commonly used antibiotics (Fernandes et al., 2015).  In a study testing five herbal extracts against multidrug resistant strains including Streptococcus mutans, Staphylococcus aureus,and Enterococcus faecalis,  E.faecalis was the most resistant of the three  (Khan et al., 2009). Phylogenetic analysis of a larger set of E. faecalis strains showed a high rate of recombination and horizontal gene transfer (Werner et al., 2013). This may justify high resistance rate of E.faecalis.

S.mitis has a higher resistance rate than other viridans Streptococci. Around 60 % of the tested isolates of S.mitis were resitant to Penicillin. Since long, it has been known that Streptococci resistance to Penicillin is due to an altered penicllin binding protein (Potgieter, Koornhof and Chalkley, 1992). Also, it has shown double rate of resistance to Cephalosporin when compared to viridans Streptococci group (Chun, Huh and Lee, 2015). Additionally, mitis group Streptococci developed high level Daptomycin resistance in vitro and in vivo (Garcia-de-la-Maria et al., 2013). S.mitis was able to receive genetic material from Veillonella species and to develop resistance to Tetracycline (Hannan et al., 2010). Interestingly, S.mitis has relatively low resistance to fluoride (Men et al., 2016).

Amongst all the available antibacterial agents, chlorhexidine (CHX) is a broad spectrum antimicrobial that is most effective against both Gram-positive and Gram-negative microorganisms (Jain et al., 2015)(Salam et al., 2015). In dentistry, CHX, at a 0.2% concentration, has become the standard international concentration for plaque control. However, it has been found to have a number of adverse side effects. These might include an alteration in taste, staining of teeth, restorative material or the dorsum of the tongue, as well as supragingival calculus formation (Jain et al., 2015).

In addition to the previous drawbacks of Antibiotics, they may also have undesirable side effects, such as vomiting, diarrhoea, allergy, and Blood thinning or clotting. Due to growing antibiotic resistance and side effects, researchers shifted their attention to folk or traditional medicine to look for antimicrobial alternatives (Karygianni et al., 2016).

1.2 Natural products as antimicrobials

Research in natural products and their antimicrobial effects is an important cornerstone for the industry of drug discovery (Brown, Lister and May-Dracka, 2014). Moreover, the potential use of natural compounds as biofilm preventive agents in dentistry have long been documented. They showed effectiveness to inhibit the growth of oral pathogens, to reduce the development of biofilms and dental plaque, to decrease the adhesion of bacteria to surfaces, and to lessen the symptoms of oral diseases (Palombo, 2011). Several reviews qualitatively summarized the antimicrobial activity of medicinal herbs against in vitro, ex vivo and in vivo formed multispecies oral biofilms (Karygianni et al., 2016). In traditional medicine, animal extracts and minerals as well as plants are used in antimicrobial preparations (Jain et al., 2015)(Cheng et al., 2015).

Natural antimicrobial agents, based on their chemical composition, can be divided into 3 categories : (i) phenolic compounds, which are made from simple sugars, containing benzene rings, hydrogen and oxygen; (ii) terpenoids, which are made from mevalonic acid and composed almost entirely of carbon and hydrogen; and (iii) alkaloids, which are nitrogen-containing compounds (Chinou, 2008). These chemical groups have been associated with biological effects of natural products, which may include antimicrobial, antioxidant, and anticancer activities (Newman and Cragg, 2007).  Most of the studies found that the effective compounds in natural products belong to polyphenol compounds. A polyphenol is any substance that contains at least 1 aromatic ring with 1 or more hydroxyl groups (other substituents can be present) (Yoo et al., 2012). Polyphenol compounds are derived from different kinds of natural products, and in vitro experiments indicated that some of them could be effective in killing bacteria or inhibiting biofilms (Ferrazzano et al., 2011)(Cheng et al., 2015).

Several Crude or total plant extracts were proved to be antibacterial against S.mutans and other oral pathogens (Palombo, 2011).  The extracts exhibited a bactericidal effect on planktonic S.mutans (Lee, 2013). We have reason to believe that natural products have antibacterial activity against oral microorganisms due to their ability to:1) Inhibit the Bacterial Growth, 2)inhibit acid production, 3)inhibition Extracellular matrix formation,thus preventing biofilm formation. A summary of relevant literature of some previously-evaluated natural agents against the four microorganisms tested in this study is shown in table.1

Natural Product Biological activity

(antimicrobial activity) and Oral Organisms

Refernces
Grape extract It has high bactericidal activity against planktonic s.mutans (Thimothe et al., 2007)(Karygianni et al., 2016)
Theobromacacao (Cacao)  / cacao bean husk extracts It showed anticariogenic action and  decreased biofilm formation of S.mutans (Percival et al., 2006)(Karygianni et al., 2016)(Jeon et al., 2011)
Punica granatum (Pomegranate) It is effective in eradicating oral microorganisms like Streptococcus mitis (Menezes, Cordeiro and Viana, 2006)(de Oliveira et al., 2013)(Karygianni et al., 2016)
Allium sativum (Garlic) It prevents biofilm formation  of s.mutans (Kim et al., 2011) (Bakri and Douglas, 2005)(Karygianni et al., 2016)
Garlic in Crudeform It has maximum inhibitory activity against s.mutans (Jain et al., 2015)
Allium sativum, Garlic juice It exhibited impressive inhibition of Streptococcus mutans (Francis Xavier and Vijayalakshmi, 2007)(Salam et al., 2015)
Curcuma xanthorrhiza (Javanese Ginger) It works against planktonic and biofilm formation of S. mutans (Rukayadi and Hwang, 2006)(Kim et al., 2008)(Karygianni et al., 2016)
gingier rhizomes organic solvent form

 

It had the maximum inhibitory activity against S. mutans
Ginger Ethanolic extract It possess antimicrobial potential against S.mutans and E.faecalis (Jain et al., 2015)
MESWAK (SALVADORA PERSICA [SP]) It inhibits the growth and acid production of Streptococcus mutans (Giriraju and Yunus, 2013)
Enterococcus faecalis is the most sensitive microorganism affected by the use of S.persica miswak. SP showed strong antimicrobial effects on the growth of

Enterococcus faecalis , Streptococcus sp. and Staphylococcus aureus.

 (Al-Bagieh and Weinberg, 1988)
S. persica miswak extracts had antimicrobial effects on Streptococcus mutans and E. faecalis. (Almas, Al-Bagieh and Akpata, 1997) (Al lafi and Ababneh, 1995)
SP has antimicrobial activity on S. aureus, Streptococcus mutans, Streptococcus pyogenes, E. faecalis, (Almas, 1999)
BANYAN (FICUS RELIGIOSA) or

Neem stick extract

It inhibits the growth of Streptococcus mutans, Streptococcus mitis, Enterococcus faecalis and  shows significant reductions in bacterial adhesion. in vitro (Al-Bayati and Sulaiman, 2008)
Green tea and oolong
tea (Camellia sinensis)
It shows antibacterial activity against S. mutans (Prashant et al., 2007)
It has inhibitory activities of green tea
extracts on cariogenic and periodontopathic bacteria including S. mutans. Also, oral rinsing of green tea solution without sugar for a short time could strongly inhibit salivary
and plaque numbers of S. mutans.
(Vanka et al., 2001)
Thymol and eugenol It inhibits  the growth of mutans streptococci (Salam et al., 2015)
Propolis (Bee glue) It shows significant antibacterial
activity against S. mutans  in vitro
(Sakanaka et al., 1989) (Jeon et al., 2011)
  an olive leaf extract and mastic gum extracts They have antimicrobial activity against Streptococcus mutans , Enterococcus faecalis ATCC 29212, and S. aureus
Rosemary It has antimicrobial activity against Streptococcus mutans. Its antimicrobial potentiality was higher than chlorhexidine mouthrinse
clove and clove bud oil It shows potent antimicrobial activity against five dental organisms  including Streptococcus mutans and Staphylococcus aureus (Araghizadeh, Kohanteb and Fani, 2013)(Awadalla et al., 2011)(Cheng et al., 2015)
Emblica officinalis or amalaka, oval, amla, amlaki

*aqueous extract of amla

It has the potential
to prevent dental caries by inhibiting the virulence
factors of Streptococcus mutans. Also, the antibacterial efficiency of Amla was found higher than the chlorhexidine and
more effective mouthwash.
(Shapiro, Meier and Guggenheim, 1994) (Shapiro and Guggenheim, 1995)(Jeon et al., 2011)
  It has the maximum inhibitory effect against Streptococcus mutans (Jeon et al., 2011)
Myristica fragrans, known as ‘nutmeg’, It shows inhibitory activity of 3.9 µg/ml against Streptococcus mutans. (Karygianni et al., 2014)
Peppermint essential oil (Mentha piperita)

 

Active against Staphylococcus
aureus
(Nalina and Rahim, 2007) (Dalirsani et al., 2011)(Salam et al., 2015)
peppermint
and spearmint essential oils
They inhibited the growth of Methicillin-resistant Staphylococcus aureus (MRSA) (Cai, 1996)(Salam et al., 2015)
The crude herbal mixture  of 10%
Qurecus aegilops L. (ground of oak bark), 20%
Salvadora persica L.(ground of miswak), 20%
Cinnamomum zeilanicum (ground of Cinnamon
bark), 10% Mentha spicata L. (leaves of mint), 5%
Syzygium aromaticum (dried flower buds of
clove), 30% glycerine oil and 5% Matricaria
chamomilla L. (flowers of camomile)
This mixture possesses  strong antibacterial activity against range
of studied bacteria like Streptococcus
mutans, Staphylococcus aureus.
(Hasan et al., 2012)
Oil of lavender (Lavandula angustifolia Mill.) It shows strong antiseptic activity against MRSA and  vancomycin -resistant strains of Enterococcus species Genus (VRE) (Jain et al., 2015)
thyme oil It has high inhibiting activity on bacterial strains like Staphylococcus aureus (Chung et al., 2006)(Salam et al., 2015)
It demonstrates strong antibacterial activity on multidrug resistant clinical strains of Staphylococcus, Enterococcus (Iscan et al., 2002)
oregano and savory oils They have high activity against antibiotic-resistant bacteria e.g. Enterococcus faecalis and

Staphylococcus aureus MRSA genera

(Imai et al., 2001) (Edris, 2007)
Manuka Honey oil which is derived from Leptospermum scoparium and  is known as red tea tree, It shows bactericidal properties against Enterococcus faecalis and   Staphylococcus aureus (Al-Taee et al., 2012)(Salam et al., 2015)

Table 1. Summary of previous work about antimicrobial agents against the four test microorganisms.

1.3 Essential oils as antibacterial agents

Aromatic plants were commonly used in embalming by Ancient Egyptians. They were believed to stop bacterial growth and prevent decay, an effect largely attributed to a great extent to their essential oils. Essential Oils (EO) are secondary metabolites of those aromatic plants. EO can also be considered an important adjuvant therapy due to its antibacterial, antimycotic, antiviral and anti-inflammatory (sage and thyme oils), anaesthetic (clove and lavender oils) properties as well as antioxidant activity (Edris, 2007). Essential oils show bactericidal activity against oral and dental pathogenic microorganisms and can be incorporated into rinses or mouth washes for pre-procedural infection control (Yengopal, 2004).

Thymol, eucalyptol and menthol are the constituents most active against oral pathogens, such as Streptococcus mutans, S. milleri, and Candida albicans. Due to their antigingivitis effects, they are often included in mouth rinses (Cecchini et al., 2012) (Bernardes et al., 2010) (Sienkiewicz, Kowalczyk and Wasiela, 2012).

Many authors report a synergistic activity between antibiotics and essential oils (Reichling et al., 2009) (Sienkiewicz, Kowalczyk and Wasiela, 2012) (Seow et al., 2014). A mixture of Thymol together with some biologically-active compounds contained in oil from Thymus magnus (Nakai) increases the activity of Norfloxacin antibiotic against resistant strains of Staphylococcus aureus (Babu et al., 2012).

The problem in using plant extracts and essential oils when compared to synthetic molecules is that it is a time-consuming process to extract, purify and distil the crude form of the plant. Also, special apparatus is required to isolate the active molecules from the raw plant. The inconsistent source material, the ambiguity in the isolation process and the cost of extraction are all obstacles facing the use of natural agents. Finally, every natural extract holds some side effects e.g. ginger and garlic can increase a tendency to bleed. Also, some plants have strong smells and flavours. Allergy and toxicity are the two most serious reactions caused by natural agents like Neem and Aloe Vera (Costalonga and Herzberg, 2014) (Jain et al., 2015).

1.4 Tea tree oil (Melaleuca alternifolia)

Tea tree oil (TTO) comes from the Australian indigenous tree, Melaleuca alternifolia (Carson and Riley, 1993). It is produced by steam distillation of freshly harvested leaves and terminal branchlets with water. The condensed aqueous distillate is separated from the oil, but produces a very low yield (Altman, 1989).

According to the literature, the essential oil derived from tea tree (M. alternifolia) is the strongest aroma therapeutic antibacterial medicine (Reichling et al., 2009). The scientific basis for the use of M. alternifolia oil as an antimicrobial agent has only been partially verified. TTO had first known for its antimicrobial activity in 1937 when Penfold and Morrison reported that a 2.5% solution of Melasol (or tea tree oil) killed haemolytic Streptococci in 30 seconds.  Also, TTO is seen to have a rapid cidal effect against Staphylococcus aureus using a 1 in 60 dilution of Melasol (TTO) (Carson and Riley, 1993).

TTO has various chemotypes that differ between M. alternifolia plants, some of these varieties are suitable for medicinal use while some are not (Merry, Williams and Home, 1990). TTO is reported to contain more than 100 components, the main ingredients of TTO are Terpinen-4-ol, Gamma-Terpinene, 1,8-Cineole, alpha-Terpinene, other terpene hydrocarbons like monoterpenes and their associated alcohols (Carson and Riley, 1993)(Tighe, Gao and Tseng, 2013).

Most TTO is extracted from M. alternifolia plant species, although other Melaleuca species can produce a therapeutic oil. The suitability of TTO for medicinal use is dependent upon the percentage content of Terpinen-4-ol. M. alternifolia is one of the richest plants in Terpinen-4-ol content (Merry, Williams and Home, 1990). In addition, P-cymene is one of the degradation products of TTO that is produced after the terpinen-4-ol is oxidized (Brophy et al., 1989). It is found in TTO at concentrations of between 1-12% (Altman, 1988) (Brophy et al., 1989) (Williams and Home, 1989) (Carson and Riley, 1993). For TTO to be of beneficial effect in medicine, the International Organization for Standardization (ISO) guidelines recommend that the concentrations of TTO and terpinen-4-ol are more than 30%, while that of 1, 8-cineole is less than 15% (Lee et al., 2013).

Previous work has shown the antimicrobial effect and anti-inflammatory properties of TTO in vitro (Hammer et al., 2003).TTO has proved very effective in treating bacterial and viral infections of the respiratory system. For example, TTO had Anti Mycoplasma pneumoniae Activity (Reichling et al., 2009).  Following investigations into the bactericidal properties of tea tree essential oil against bacteria, it was seen to be active against Gram-positive bacteria (Staphylococcus aureus and Streptococcus viridans). TTO has been shown to inhibit the growth and adhesion of Streptococcus mutans (Salam et al., 2015) (Groppo et al., 2002). TTO is also used as root canal irrigant, but was found less effective compared to Ethylenediaminetetraacetic acid (EDTA, a known root canal irrigant) and Sodium hypochlorite (NaClO) (Sadr Lahijani et al., 2006)(Salam et al., 2015). A more recent study showed that TTO was similar to sodium hypochlorite and chlorhexidine in the inhibition of bacterial growth of E.faecalis (Kamath et al., 2013). Based on other previous studies TTO can be used as an alternative to CHX as a mouthwash (Groppo et al., 2002) (Chandrdas et al., 2014) (Salam et al., 2015). The antibacterial activity of some skin-wash formulas containing TTO and others having pure TTO was evaluated against Staphylococcus aureus. All formulations showed antibacterial activity, but the efficacy of TTO appeared to be dependent on the formulation and the concentration tested (Messager et al., 2005). Therefore, topical preparations containing TTO can be considered in regimens for eradication of methicillin-resistant Staphylococcus aureus in hospitals (Dryden, Dailly and Crouch, 2004) (Edris, 2007). TTO showed a high antibacterial activity against S. aureus in vitro and in vivo (Caelli et al., 2000) (Dryden, Dailly and Crouch, 2004) (Reichling et al., 2009).

Reports suggest that repeated use of formulations containing tea tree essential oil (TTO) does not lead to dermatological problems, nor affect the original protective bacterial flora of the skin (Carson and Riley, 1995). One study reported that when the concentration of TTO was reduced to 5%, the irritation was diminished. No skin irritation was observed when the concentration was decreased to 2.5%. In another study, TTO presented dose-dependent inhibitory effects against the growth of S. aureus (Lee et al., 2013).

Tea tree and lavender oils demonstrate not only strong antibacterial activity but also have an immunostimulatory effect. After inhalation of essential oils, no bacteria were found in the sputum, while alleviation of clinical symptoms and increased granulocyte chemiluminescence was observed, as well as a reduction in the incidence of infection in patients with bacterial respiratory infections [32].

The application of strong vapours from eucalyptus oil and tea tree oil to prevent respiratory system diseases as colds, flus, pneumonia, tuberculosis, and opportunistic infections in individuals with cystic fibrosis was reported. The tested essential oils are active against bacteria caused by opportunistic infections as Staphylococcus aureus. Another study described the use of some essential oils in preventing, treating and curing infections of the human respiratory system by pathogens causing severe acute respiratory syndrome (SARS). The authors recommend the use of vapours of oils Melaleuca alternifolia to reduce the risks of infection in public places (Sienkiewicz, Kowalczyk and Wasiela, 2012).

1.5 TTO mechanism of action

The lipophilic nature of M. alternifolia oil enables it to penetrate skin and potentiates its antiseptic action. On the other hand, it can increase the possibility of toxicity due to dermal absorption (Carson and Riley, 1993). The enhanced antimicrobial activity of terpinen-4-ol is thought to be as a result of its favourable hydrophobic hydrophilic character, in that it possesses sufficient hydrophilicity to diffuse through surrounding water to the bacterial cytoplasmic membrane and sufficient hydrophobicity to then diffuse through the bacterial cytoplasmic membrane (Carson, Mee and Riley, 2002). Furthermore, non-oxygenated components of TTO, such as gamma-terpinene, reduce the aqueous solubility of terpinen-4-ol, thereby reducing the concentration at the bacterial cell surface and reducing the level of bactericidal activity shown by the TTO complete formulation (Cox, Mann and Markham, 2001).

Although it is widely accepted that TTO is a nonspecific membrane active biocide, it has been suggested that other mechanisms such as bacterial enzyme antagonism or blockage of membrane bound proteins may exist due to the large number of its components (Carson, Mee and Riley, 2002).

1.6 TTO toxicity

“Oil of Melaleuca: terpinen- 4-ol type (tea tree oil)” has been the international standard to regulate the composition of tea tree oil since 1996 (Jain et al., 2015). Prior to this there was an Australian standard which specified the 1,8-cineole content of tea tree oil to not exceed 15%, while that of  terpinen-4-ol content must exceed 30% (Carson and Riley, 2001).

There are numerous reports in the literature of positive patch tests to tea tree oil (de Groot, 1996) (De Groot and Willem Weyland, 1992). Unfortunately, there is no sound data about the exact composition of TTO in safety and toxicity studies.  However, some effort has been made to identify the tea tree oil components responsible for allergic reactions. Those found that oxidation products are the likely allergens (Hausen, 1999) (Harkenthal, Hausen and Reichling, 2000). Since oxidized tea tree oil appears to be a more potent allergen than fresh tea tree oil, adverse reactions may be minimized by reducing exposure to aged oil (Carson, 2001).

TTO can be toxic if ingested, as evidenced by experimental studies in rats and from cases of human poisoning. A case of an adult poisoned after drinking approximately half a tea cup of TTO, corresponding to a dose of approximately 0.5–1.0 ml/kg body weight, was reported (Carson and Riley, 1995). In another incident, a 60-year-old man who swallowed one and a half teaspoonfuls of TTO as a cure for a cold presented with a red rash covering his feet, knees, upper body and arms including his palms and elbows. Apart from these reports, there are no data on the systemic toxicity of TTO in humans. However, the available knowledge clearly demonstrates the toxicity of TTO following oral exposure and the ingestion of TTO should not be recommended (Elliott, 1993).

Contact allergy occurred in response to 100% pure TTO as well as lower concentrations of TTO in various formulations. In a study of 725 consecutive patients presenting to a patch test clinic and tested with 5%, 1% and 0.1% TTO, only one patient had a positive patch test to 5% and 1% oil and No patient reacted to 0.1% oil (Lisi et al., 2000)(Hammer et al., 2006). In another series, 550 patients were tested with 100% pure oil. Allergic reactions were recorded in 13 (2.4%) of the patients (Coutts, Shaw and Orton, 2002). In addition, these patients also reacted to one or more of the components D-limonene, aterpinene, aromadendrene, terpinen-4-ol and a-phellandrene at 1%, 5% or 10%. Newly distilled TTO appears to have a relatively low sensitising capacity, whereas TTO kept for prolonged periods is a moderate to strong sensitizer and has a significantly increased peroxide value (Hausen, 1999)(Hammer et al., 2006). This same study also suggests that the most important allergens formed could be terpinolene, a-terpinene, ascardiole and 1, 2, 4-trihydroxymethane (Hausen, 1999) (Hausen, 2004) (Hammer et al., 2006).

The acute oral toxicity of tea tree oil in rats has been determined. This was similar to the oral toxicity of other common essential oils such as eucalyptus oil and indicates that tea tree oil should not be administered orally (Altman, 1990).

Furthermore, treatment of the fibroblast cells with either TTO or terpinen-4-ol did not result in a statistically significant reduction in viability. Also, statistical analysis of the results also revealed that increasing the concentration of both TTO and terpinen-4-ol had no significant effect on fibroblast viability (Carson, Mee and Riley, 2002). Moreover, comparison of the toxicity of terpinen-4-ol and TTO against human fibroblasts revealed that neither agent, at the concentrations tested, were toxic over the 24-h test period. This finding confirms the results of a previous in vitro study which reported that TTO was only slightly toxic in very low concentrations (Söderberg, Johansson and Gref, 1996). The results of both patch testing and clinical trials indicate that TTO is associated with negligible skin irritancy (Aspres and Freeman, 2003) (Dryden, Dailly and Crouch, 2004) (Veien, Rosner and Skovgaard, 2004) (Loughlin et al., 2008).

1.6 Rationale

Antibiotic resistance develops due to several factors like poor penetration of the antimicrobial through biofilm, bacterial stress response strategy, low rates of bacterial growth and/or metabolism, multi-drug resistant efflux systems, interbacterial quorum sensing , and an alteration in the protein composition of the bacterial cell membrane in response to antibiotics (Kouidhi, Al Qurashi and Chaieb, 2015). Searching for novel and effective antibiotics is a domain that will always be needed. A study that examined Susceptibility tests on 207 isolates of nine species of alpha-haemolytic streptococci, including Streptococcus mutans, Streptococcus salivarius, Streptococcus oralis and Streptococcus mitis, found that only S. mutans was universally susceptible to penicillin. Four blood culture isolates of S. mitis were resistant to penicillin with Minimum inhibitory concentrations of 16–32 mg/L; they were also resistant to the aminoglycosides gentamicin, kanamycin and tobramycin (Potgieter et al., 1992). S. oralis and S. mitis showed the highest penicillin resistance amongst the alpha-haemolytic streptococci. As with penicillins, the alpha-haemolytic streptococci show high resistance to cephalosporins. Antibiotic profiling of alpha-haemolytic streptococci isolated from the oropharynx of healthy Greek children showed 23% of isolates were resistant to tetracycline the majority of isolates were S. mitis. Also, another study found two high-level tetracycline-resistant isolates of S. mitis (Konig, Reinert and Hakenbeck, 1998).

While there are many studies that showed the antimicrobial effect of TTO against s.mutans, s.aureus and E.faecalis, there is scant research about TTO effect on s.mitis. Additionally, there is a paucity of literature that compares the antimicrobial efficacy of TTO against this array of oral microorganisms. Moreover, There are no previous studies that examined the time kill curves of TTO on bacteria except the ones for s.aureus and e.faecalis (Jay et al , 2000). Furthermore, TTO can be incorporated in preparation with known antibiotics to boost their action or reduce their overall needed dose. The combination therapy was tested  with TTO and proved effective (Forrer et al., 2013)(Hammer, Carson and Riley, 2011).

1.7 Aim

The purpose of this study is to investigate the antibacterial effect of TTO and to determine its Minimum Inhibitory Concentration (MIC) with S.mutans, S.aureus, E.feacalis, and S.mitis. Also, this study aims to compare time-kill and survival assays of TTO against these four microorganisms in association with oral infections.

Chapter 2. Materials and Methods

2.1 Natural reagents

The Tea Tree Oil (TTO) was purchased from a local health food store (100% pure essential oil of Melaleuca alternifolia leaf and Limonene, Dr.Organic Ltd, Swansea, UK). This oil has more than 38% terpinen-4-ol as specified by the supplier. Sterile liquid Paraffin Wax was used as an emulsifier for TTO because it was the best available miscible ingredient used after several trials with other agents see Appendix A.

2.2 Bacterial strains and growth conditions

Four standard cultures were used in this study. Two of them were National Collection of Type Cultures (NCTC) strains of Streptococci mutans  and Streptococci mitis with NCTC 10449 and NCTC 12261 codes respectively. The other two reference strains were Staphylococci aureus M77, X-80, and Enterococci faecalis 328, X-80 recovered from glycerol-frozen stocks in the Manchester University Collection of Bacteria (MUCOB). Using plastic bacterial loops, S.aureus and E.feacalis were cultured aerobically on Chocolate agar (Oxoid Ltd,Basingstoke, Hampshire, UK) at 37⁰C for 24 hours . S.mitis and S.mutans were cultured aerobically on Columbia Blood Agar (Oxoid Ltd, Basingstoke, Hampshire, UK) in a 5% CO2 incubator at 37⁰C for 24 and 48 hours ,respectively. All expirements were performed on from fresh culture plates of the four microorganisms. Pictures of the agar plates are provided in Appendix.B.

2.3 Preparation of inocula

Colonies from each 24-hour old pure culture was emulsified in saline to obtain a turbidity equivalent to McFarland 0.5 standard . This dilution will yield 1.5X108 Colony Forming Unit (CFU) per millilitre (ml). Then, the suspensions were used as inocula for all subsequent expirements.

2.4 In vitro antibacterial assay

Bacterial lawns were prepared from the McFarland suspensions of S.aureus, E.feacalis and S.mitis on the surface of Muller Hinton Agar plates (Oxoid Ltd, Basingstoke, Hampshire, UK) using sterile cotton swabs . S.mutans was inocluated onto Muller Hinton Blood Agar plate (Oxoid Ltd, Basingstoke, Hampshire, UK).  Four wells of 6 mm diameter were bored into every plate using a sterile cork-borer. Two wells were filled with fifty microlitres of two different concentrations of TTO (100% and 50% v/v) using a micropipette (Labozone, model no.OP100). Same volumes of Corsodyl (Chlorhexidine Digluconate 0.2% w/v)(CHX) and normal saline were used as positive and negative controls, respectively. Plates of S.aureus, E.feacalis and S.mitis were incubated at 37°C for 24 hour. S.mutans plate was incubated at 37°C in 5% CO2 for 48 hours.  If the bacterial growth was semi-confluent , zones of inhibition were measured in millimitres (mm) using a measuring ruler. All tests were performed in triplicate and the mean values were calculated.

2.5 Minimum inhibitory concentration determination

Minimum Inhibitory Concentrations (MICs) of Essential Oils (EOs) were determined by modified broth micro-dilution method from Wiegand et al. Of each bacterial suspension , 10 ( L) was added to an equal volume of different concentrations of TTO ( 100%, 75%, 50%, and 25% v/v) in 0.5 ml Eppendorf tubes and mixed using a vortex (IKA® Lab Dancer) . Then, 20 L of each mixture was subcultured on Chocolate agar and incubated at 37°C for 24 hour for Plates of S.aureus, E.feacalis and S.mitis. S.mutans plate was incubated at 37°C in 5% CO2 for 48 hours. The concentration at which there was no single colony of bacteria after incubation was taken as MIC. The MIC is defined as the lowest concentration of antimicrobial agent that completely inhibits the growth of the organism. Each experiment was repeated twice.

2.6 Time kill assays

TTO in 100% and 50% concentrations and the positive control were assayed against the four tested organisms. Using a micropipette (Labozone,model no.OP1000)  100 L of each standardised bacterial suspensions of the four organisms was mixed in plastic bijou tubes with 100 L of one of the antimicrobial agent in each tube. The tubes were continuously shaken using a vortex. Aliquots were removed from the inoculum mixture after timed intervals of incubation (at 0 minutes, 5 minutes, 10 minutes, 15minutes , 30 minutes and 60 minutes) . At each sample time, numbers of viable cells were determined by the plate count technique which involved plating 20 L of each sample on a chocolate agar using a micropipette. The plates were incubated in the suitable conditions. After incubation, the colonies were counted and the mean values in (CFU/mL) for each test and controls were determined. The experiments were performed in duplicate. Data were analysed as killing curves by plotting the log10 (CFU/mL) versus time (minutes). Two different concentrations of TTO were considered bactericidal when a 3 log10 decrease in CFU/mL was reached compared to the initial inocula.

2.7 Cell viability assays

Cell viability counts reveal the extent to which treated cells are able to survive and reproduce to form colonies when removed from the presence of tea tree oil and re-cultured in a nutrient medium. 200 L of 24-hour Cultures of bacteria with 50% TTO were centrifuged (for 3minutes at 6000 revolution per minute (rpm), washed and resuspended in 200 L of saline to give the standard concentration of cells. This was repeated twice. Then, the suspension  is incubated and the number of viable cells is determined by the pour plate method using a streak plate method reported in earlier work (Palombo, 2011)

 2.8 Statistical analyses

Calculation of means and Statistical analyses used in this study have been carried out using Microsoft® Excel (2010) where a comparison of means of antimicrobial activity was performed. One way ANOVA was used to compare the means of antimicrobial activity to know the significance of results at p value of 0.05.

Chapter 3. Results

3.1 In vitro antibacterial assay

The antimicrobial effect of TTO was evaluated against four oral pathogenic bacteria. These bacteria demonstrated varying susceptibility patterns to TTO. The antimicrobial activity and inhibition zones are summarized in table.2. This data shows that TTO, in full and half concentrations, had the maximum antimicrobial effect against S.aureus. However, it falls short than CHX in its antibacterial effect against the other 3 organisms. S.mitis had moderate sucibtibility towards TTO ,while S.mutans and E.Faecalis had the weakest sucebtibility. No antibacterial eefect was seen with Saline, which was used as a negative control.

Table 2. Zones of Inhibition showing the antimicrobial Activity of 100%TTO, 50% TTO ,Chlorhexidine and Saline

3.2 Minimum inhibitory concentration

The MIC values of TTO against s.mutans and s.mitis was 50% v/v . While its MIC value against s.aureus and e.faecalis was 75% v/v. There was no significant difference in MICs between the four pathogens (p value= 0.49).

3.3 Time kill assays

TTO induced a bactericidal effect in all tested organisms, but the onset of the bactericidal activity was dependent on the concentration of the antimicrobial and differed between the different species. S.mitis experienced the most rapid killing during the first five minutes with the two test concentrations of TTO and the positive control (Figures.1,2,3) . The slowest organism to be killed by TTO was E.faecalis as shown in Figure.1 and 2. It demonstrated 15 minutes and 30 minutes intervals when treated with by 100% TTO and 50% TTO, respectively. With 100 % TTO , both s.aureus and s.mutans were completely killed after 10 minutes (Figure.1). Unlike 100% TTO, 50% TTO needed 15 minutes and 30 mintues to completely kill s.mutans and s.aureus respectively (Figure.2).  Though there seems to be a discrepancy in the time TTO needs to kill the four organisms, there was actually no significant difference in its time-killing ability when the four organisms were compared using One-way ANOVA (p-value of 0.45) . Positive control, CHX, was effective in killing all organisms in less than 5 minutes.

Figure 1: Time-Kill curves for 100% Tea Tree Oil.

Figure 2: Time-Kill curves for 50% Tea Tree Oil

Figure 3: Time-Kill Curves for o.2% Chlorhexidine.

3.4 Viability assays

TTO had a bactericidal effect on all the four tested organisms because there was no growth of viable cells after 24 hour incubation, washing and resuspension in saline. Therefore, none of the four organisms remained viable when incubated with 50% v/v TTO.  Results of individual cell lines are not reported.

Chapter 4. Discussion

This study aimed to examine the differences in the activity of TTO against selected oral microorganisms. It had the greatest antibacterial activity against S.aureus . This agrees with previous published work about the effectiveness of TTO in killing S.aureus (Cox et al,2000).

In this study , 50% v/v TTO was able to kill all the four microorganisms in less than 15 minutes. This finding agrees with a previous study in which TTO demonstrated a relatively short killing time (less than 60 min) for multidrug-resistant organisms, including MRSA, glycopeptide-resistant Enterococci (May, 2000). MRSA showed the highest resistance and the longest time for eradication. It was concluded that the antimicrobial activity of TTO is attributed to its high content of terpenen-4-ol (Edris, 2007). Generally, TTO was relatively quick in killing the four microorganisms. This fact supports its use in formulations to be used everyday like pastes or mouthwashes.

The strains tested in this study were not as susceptible to TTO as others have described in previous studies using similar methods.This might be due to several facts. In this study , an unusual solvent (liquid paraffin) of TTO was used. It was tested against the four microorganisms but it showed no antibacterial activity. Previous reports of TTO mentioned smaller percentages of the oil was tested and proved effective. Hence, the selection of the solvent affected the dilution factor.

In our results, slight variation in MICs between the four organisms was noted. These findings might be greatly affected by the technique used in making the different dilutions for MIC testing. The viscosity of TTO and the paraffin made it even harder.

To discuss the mode of action of TTO based on our findings , other natural antibiotics should be considered. Natural compounds targeting bacterial viability are typically aiming at bacterial eradication via of several modalities. They may disrupt the cell wall biosynthesis and/or cell membrane permeability, bind to surface-adsorbed components, inhibit protein synthesis or nucleic acid metabolism, and/or inhibit the enzyme activity by oxidizing bacterial proteins (Jeon et al., 2011). Also, the mechanisms by which EOs can inhibit microorganisms involve different modes of action, and in part may be due to their hydrophobicity. As a result, EOs get partitioned into the lipid bilayer of the cell membrane leading to leakage of vital cell contents (Burt and Reinders, 2003)(Kim, Marshall and Wei, 1995)(Juven et al., 1994)(Edris, 2007).

TTO is thought to affect the cytoskeletal makeup of a bacterial cell. For example, when M. pneumonia was treated with 0.006% TTO in ethanol (1%) for 12 h, the cells lost their typical ‘pear-shaped’ appearance and became rounded.Therefore, M. pneumoniae cells lost their virulence. On the other hand, the integrity of the cell membrane was not impaired by TTO (Reichling et al., 2009). This confirm the theory of hydrophobicity and the merging of TTO molecule with the bacterial cell membrane that leaves no damage to the membrane.

The biological effect of TTO on cell structures, such as cytoplasm, cytoplasmic membrane, and cell wall was studied using electron microscopy. After 12 h exposure to a subminimum inhibitory concentration of 0.12% TTO, no irregularities were noted in cell shape, cell wall or cytoplasmic membrane. However, an interruption in bacterial cell division was seen. Also, lamellar like membrane rods were seen in the bacterial cytoplasm.  These mesosome-like membrane structures can reflect the internal cell damage. All these findings suggest a permanent detremintal effect on the bacterial cell at 0.12% TTO.  After 12 h of incubation of the bacterial cells with the subminimum inhibitory concentration of 0.25% TTO, dramatic cellular alterations became visible on electron microscopic image. Similar to 0.12% TTO, cell division was affected. A complete inhibition of the bacterial cell division occurred. Also, new fibrous electron-dense structures were observed, and  the lamellar-like membrane rods, tht was seen with 0.12% TTO, were no longer visible.

Another effort in studying the mechanism of action of TTO involved measuring the cellular respiration, detecting  the leakage of intracellular cations, and examining the change in the permeability of the bacterial cell membrane. It was found that the inhibitory concentrations of TTO, that killed Echerechia coli and staphylococcus aureus, were able to inhibit cell respiration, alter the permeability of the cell membrane, and stimulate the leakage of intracellular potassium ions (Cox et al., 2001).

Moreover, TTO and its components were found to change the permeability and fluidity of the plasma membranse of 3 different yeasts (Hammer, 2004). Based on these results, it was assumed that the essential oils may have antimicrobial activity by influencing bacterial and fungal targets involved in cytoplasmic and cell wall metabolism. Components of TTO are the actual players in its mechanism of action. They, especially monoterpenes, were found to disturb the structure of the membrane-embedded proteins, inhibit cell respiration, partition into the cell membrane, and alter the ion transport processes (Reichling et al., 2009).

A significant inhibition of respiratory oxygen consumption in cultures of  three organisms (Escherechia coli, S.aureus, and candida albicans) resulted upon exposure to tea tree oil. The inhibitory effects of tea tree oil were consistent with effects related to the partitioning of its monoterpene components into cell membranes.

There was a decline in viability and the inhibition of respiration was accompanied by increased cell membrane permeability evident by  fluorescent  dye. However, cells with damaged or permeabilised cell membranes do not exclude the stain. Therefore, staining of cells indicates cytoplasmic membrane (bacteria) and plasma membrane (yeast) damage. In the case of Staph.aureus, monitoring K+ efflux may be a more sensitive indicator of membrane damage than  fluorescent  staining.

The ability of tea tree oil to inhibit respiration and increase membrane permeability in microbial cells suggests that its lethal actions are primarily the result of inhibition of membrane-located metabolic events and a loss of chemiosmotic control.( Sean et al, 2001)

Now , the possibility that tea tree oil directly inhibits a specific respiratory enzyme or metabolic event cannot be eliminated.

However, our findings also reveal that minimum inhibitory levels of tea tree oil altered cell membrane structure. Increased uptake of the nucleic acid stain propidium iodide, to which the cell membrane is normally impermeable, was observed. Also, leakage of potassium ions commenced immediately upon adding tea tree oil to suspensions containing E. coli and within 5min for Staph. aureus cells.

Toxic effects on membrane structure and function have generally been used to explain the antimicrobial action of essential oils and their monoterpenoid components (Andrews etal. 1980; Uribe etal. 1985; Knobloch etal. 1988).

 4.2 Limitations

This preliminary study examined the effects of TTO in vitro. It has some considerable limitations. First, the tested microorganisms are not clinical isolates from human saliva. Second, the strains are being tested in the planktonic phase while they present clinically in biofilms. Single or multiple species biofilms can demonstrate the real effect of an agent against the microorganisms. Also, a biofilm study might take into account the effect of the environmental factors on the formation and survival of biofilms. Moreover, an ideal antimicrobial agent for clinical application should only affect oral pathogens without disturbing normal oral streptococci (Sbordone and Bortolaia, 2003). Therefore, susceptibility assay of normal oral streptococci for the herbal extracts should have been investigated (Lee, 2013).

Conveniently, a commercial TTO formulation was used instead of the pure oil that could be extracted from M. alternifolia plant. Also, sterile paraffin was used because it was the best available material to dissolve TTO. However, previous studied successfully used Tween 80 (Hammer, Carson and Riley, 2011). Finally, examining the mechanism of action of TTO on the specific strains used was not done.

 4.3 Future Applications

Although TTO is popular and is being extensively studied, no comprehensive investigation was carried on to look into the potential of some microorganisms to develop resistance against it. One study examined the frequency of resistance of S.aureus to TTO (Ferrini et al., 2006). Another study attempted to induce resistance of MRSA to TTO and found a sub population of the organsims was resistant to TTO though not previously exposed to this antimicrobial (Nelson, 2000). This suggests that if the organism was exposed to TTO ,a selection of the resistant strains will happen and inevitable resistance to TTO will eventually emerge. In addition, a study was done to know the frequency of resistance of single-step mutants of Gram positive bacteria to TTO. It concluded that there is very low frequency of resistance to TTO is found in staphylococci and Enterococci species (Hammer, Carson and Riley, 2011).

Microbiological culture csn  selectively detects viable organisms after they have been acted upon by an  antibiotic. However, because only a small percentage of species can be cultured, this strategy underestimates microbial diversity. There are several sensitive and reliable assays to screen for antibiotic agents. Polymerase Chain reaction (PCR) has many applications in biomedical research. It can be used to screen the genomic Deoxyribonucleaic acid DNA for evidence of damage or mutation.

To overcome this limitation of old antimicrobial testing techniques , alternative PCR-based strategies have been developed. A review has shoen two complementary strategies (Cangelosi and Meschke, 2014). One strategy, termed viability PCR, correlates viability with cell envelope impermeability. In viability PCR, microbes in samples are incubated with a membrane-impermeative reagent such as propidium monoazide (PMA).  If cells were viable, a strong PCR signal will be seen. In nonviable cells, damaged membrane and free DNA will not be protected from PMA and hence no amplification will occur. The second strategy, termed “molecular viability testing” (MVT), correlates viability with the ability to rapidly synthesize a macromolecule (a species-specific rRNA precursor) (Cangelosi and Meschke, 2014) . All these techniques are promising but will cost a lot of grants.

To establish its safety for various clinical situtions, in vivo efficacy and safety studies of TTO are required. Some in vivo and clinical trials are being held and they are reported some positive findings (Ernst and Huntley, 2004).

Microbiological culture can selectively detect viable organisms after they have been acted upon by an antibiotic. However, because only a small percentage of species can be cultured, this strategy underestimates microbial diversity (1–6). To overcome this limitation, alternative PCR-based strategies have been developed. A review has shown two complementary strategies (Gerard A. Cangelosi, John S. Meschke,2014). One strategy, termed viability PCR, correlates viability with cell envelope impermeability (9, 10). In viability PCR, microbes in samples are incubated with a membrane-permeable reagent such as propidium monoazide (PMA).  If cells were viable, a strong PCR signal will be seen. In nonviable cells, damaged membrane and free DNA will not be protected from PMA and hence no amplification will occur. The second strategy, termed “molecular viability testing” (MVT), correlates viability with the ability to rapidly synthesize a macromolecule (a species-specific rRNA precursor) (12–14) (Gerard A. Cangelosi, John S. Meschke,2014). All these techniques are promising but will require funding to further investigate.

Conclusion

TTO has long been known for its antimicrobial activity against a large scale of microorganisms. This was a preliminary laboratory study to evaluate the antimicrobial ability of TTO against S.mutans ,S.aureus, E.feacalis and S.mitis. Although TTO, in its two concentrations, was  less potent than chlorhexidine (CHX), it is still relatively effective in inhibiting the growth of these oral bacteria in vitro. TTO was able to kill all the tested organisms in less than 15 minutes. It had bactericidal effect on all the tested organisms which makes it a powerful antimicrobial agent. Due to the limitations of this study , more work is needed to test TTO against the same organisms in vivo.  The testing of  TTO’s  antimicrobial activity using molecular techniques will confirm our findings using the conventional methods of antimicrobial testing.

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