The antimicrobial possibilities of green tea

Green tea is a popular drink, especially in Asian countries, although its popularity continues to spread across the globe. The health benefits of green tea, derived from the leaves of the Camellia sinensis plant, have been studied for many years. Fairly recently, researchers have begun to look at the possibility of using green tea in antimicrobial therapy, and the potential prevention of infections. The particular properties of catechins found in the tea have shown promise for having antimicrobial effects. There are four main catechins (polyphenols) found in green tea: (-)-epicatechin (EC), (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin (EGC), and (-)-epigallocatechin-3-gallate (EGCG). Three of these, ECG, EGC, and EGCG have been shown to have antimicrobial effects against a variety of organisms. These catechins have exhibited a variety of antimicrobial mechanisms. The results of studies on the antimicrobial effects of green tea have shown that the potential for preventive and therapeutic purposes is present. Further data collection on studies performed with human consumption during the course of infections, and studies on the occurrence of infections in populations that consume regular amounts of green tea will be necessary to complete the picture of its antimicrobial possibilities.


INTRODUCTION
Tea is a very popular drink world-wide. It is produced from the plant Camellia sinensis, which is grown in at least 30 countries, and grows best in certain tropical and subtropical regions (Gupta et al., 2014). Tea is mainly produced as four varieties; white, green, Oolong, and black. White tea is made from very young tea leaves or buds; green tea is made from mature unfermented leaves; Oolong tea from partially fermented leaves; and black tea from fully fermented leaves (Jigisha et al., 2012;Gupta et al., 2014). Most of the black tea produced is consumed in the United States, Oolong tea is most popular in China and Taiwan, and green tea is most popular in China, Japan, and Korea (Cabrera et al., 2006). Among the health benefits that have been studied using green tea are: as an antioxidant, anti-inflammatory, anticarcinogenic, in cardiovascular health, oral health, and as an antimicrobial. Antioxidant effects come from the ability of green tea to limit the amount of free radicals by binding to reactive oxygen species (ROS) (Serafini et al., 2011;Jigisha et al., 2012;Gupta et al., 2014). Anti-inflammatory effects may be a result of increased production of IL-10, an anti-inflammatory cytokine. Inflammation is involved in, among other conditions, arthritis, cardiovascular disease, aging, and cancer (Serafini et al., 2011;Jigisha et al., 2012). The anticarcinogenic effects of green tea have been seen in many types of cancer, and the mechanisms may include inhibiting angiogenesis and cell growth, and inducing apoptosis in cancer cells (Serafini et al., 2011;Jigisha et al., 2012;Subramani and Natesh, 2013). Cardiovascular effects include the antioxidant and anti-inflammatory effects, and consumption of green tea has been shown to inhibit atherosclerosis, reduce lipid levels overall, and improve the ratio of LDL to HDL (Serafini et al., 2011;Jigisha et al., 2012). The effects for oral health are related to both teeth and gums. The main cause of dental caries is the bacteria Streptococcus mutans. Green tea has a direct antimicrobial effect on this bacteria, plus it seems to inhibit the attachment of the bacteria to oral surfaces. In addition, green tea is a natural source of fluoride (Jigisha et al., 2012;Gupta et al., 2014). Green tea has been shown to have antimicrobial effects against a variety of gram positive and gram negative bacteria (e.g., Escherichia coli, Salmonella spp., Staphylococcus aureus, Enterococcus spp.), some fungi (e.g., Candida albicans), and a variety of viruses (e.g., HIV, herpes simplex, influenza) (Jigisha et al., 2012;Steinmann et al., 2013). These antimicrobial effects will be discussed in more detail later in this paper.

GREEN TEA COMPOSITION
The medically important components of green tea are the polyphenols, most importantly the flavonoids. The main flavonoids in tea are the catechins, making up 30-40% of the water-soluble solids in green tea (Wang and Ho, 2009;Roowi et al., 2010). The different types of tea vary in the amount of catechins that they contain, with green tea containing the most, then Oolong tea, then black tea. The initial steaming process in the production of green tea destroys the enzyme polyphenol oxidase, thus protecting the polyphenol content. There are four main catechins in tea. (-)-epicatechin (EC), (-)-epicatechin-3-gallate (ECG), (-)-epigallocatechin (EGC), and (-)-epigallocatechin-3gallate (EGCG). In green tea, EGCG is the most abundant, representing approximately 59% of the total catechins. EGC is next, making up approximately 19%. Then ECG, at 13.6%; and EC, at 6.4% (Cabrera et al., 2006;Jigisha et al., 2012). In addition to the type of tea, the amount of catechins can be affected by: where on the plant the leaves are harvested, leaf processing, geographical location, growing conditions, and tea preparation (Fernandez et al., 2002;Lin et al., 2003;Cabrera et al., 2006).
In order for any of the components of green tea to be a health benefit they have to have bioavailability This is most commonly assessed by measuring plasma, urine, and perhaps (in directed studies) tissue levels of the components after ingestion; usually by taking samples at regularly timed intervals. Various studies conducted using consumption of green tea beverage (Henning et al., 2004;Stalmach et al., 2009;Clifford et al., 2013), green tea extract (Lee et al., 1995;Yang et al., 1998;Henning et al., 2004), or specific catechins (Chow et al., 2001;Van Amelsvoort et al., 2001), have shown that ECG, EGCG, and metabolites of EC and EGC can be detected in blood plasma; and metabolites of EC and EGC only can be detected in urine. The peak concentration in blood plasma occurs at approximately 2 h after ingestion; and the peak concentration in urine occurs approximately 4-6 h after consumption. Some studies were performed using various dosages of the catechins, and show that the bioavailability of the catechins is directly proportional to the amount consumed (Nakagawa et al., 1997;Yang et al., 1998;Chow et al., 2001Chow et al., , 2003Ullmann et al., 2003). Table 1 shows a summary of the results of these various bioavailability studies, giving data specifically for EGCG and EGC. The most abundant catechins in green tea, EGCG and EGC, also occur in the greatest concentration in the body after consumption (EGCG in plasma, and EGC in both plasma and urine).

KEY CONCEPT 2 | Bioavailability
Bioavailability is a measure of how much of a substance can be found in blood, tissues, urine, etc., after being introduced into the body. Enough of the substance must be present at the site of an infection in order to be useful in fighting the microorganism(s) involved.
In a previous study (Reygaert and Jusufi, 2013), it was projected that using a brewed preparation of Japanese green tea (containing approximately 20 mg of EGC per gram of tea) would result in approximately 150 mg of ECG per cup of tea. The amount of EGC excreted in the urine would be approximately 3.5 mg per cup of tea.

ANTIMICROBIAL PROPERTIES OF GREEN TEA
In addition to the direct antimicrobial effects of catechins discussed below (damage to the bacterial cell membrane, inhibition of fatty acid synthesis, inhibition of enzyme activity, etc.), there are also some effects that may contribute to the total antimicrobial effect in infected individuals. These effects include inhibition of inflammation (particularly inflammation caused by oxidative stress, such as vascular), more specifically, by increasing the synthesis of nitric oxide (Yamakuchi et al., 2008), inhibiting angiotensin II and IL-6-induced C-reactive protein expression (Li et al., 2012), suppression of IL-6 and RANKL production in infected osteoblast-like cells (Ishida et al., 2007), inhibiting IL-8 production (Hirao et al., 2010), and inhibiting hyaluronidase activity (activated by chronic inflammation) by inhibition of IL-12 (Adcocks et al., 2002).

DAMAGE TO THE BACTERIAL CELL MEMBRANE
Many of the direct effects of tea catechins are a result of the catechins binding to the bacterial lipid bilayer cell membrane which then causes damage to the membrane (Sirk et al., 2008(Sirk et al., , 2009). This damage can then lead to a variety of related antimicrobial effects. Researchers studying Escherichia coli found that when exposed to green tea polyphenols, the bacterial response was changed regulation of 17 individual genes. Nine genes were upregulated and eight were downregulated. One of the major outcomes of this change in regulation was damage to the bacterial cell membrane (Cho et al., 2007). Tea catechins have less effect on gram negative bacterial cell membranes due to the fact that the LPS outer membrane of gram negative bacteria is negatively ∼3000 μg (4-6 h) Yang et al., 1998 charged (Ikigai et al., 1993). Bacterial cell membrane damage inhibits the ability of the bacteria to bind to host cells (Sharma et al., 2012), and inhibits the ability of the bacteria to bind to each other to form biofilms, which are significant in pathogenesis (Blanco et al., 2005). Bacterial membrane damage also results in an inability of the bacteria to be able to secrete toxins (Sugita-Konishi et al., 1999;Shah et al., 2008). Directly related to the homeostasis of the bacterial cell membrane is fatty acid synthesis.

INHIBITION OF FATTY ACID SYNTHESIS
Fatty acids in bacteria have important functions; as a component of phospholipid cell membranes (and mycolic acid in cell walls of mycobacteria), and as an excellent energy source. Researchers have recently begun to look at the potential of targeting fatty acid biosynthesis for antimicrobial drug development (Wang and Ma, 2013). Researchers have found that green tea components (especially EGCG) inhibit specific reductases (FabG, FabI) in bacterial type II fatty acid synthesis (Zhang and Rock, 2004;Li et al., 2006). Inhibition of fatty acid synthesis by green tea has also been found to inhibit bacterial production of toxic metabolites (Sakanaka and Okada, 2004).

INHIBITION OF OTHER ENZYME ACTIVITY
In addition to the enzymes involved in fatty acid synthesis, research with green tea has been shown to have effects on other essential bacterial enzymes. Researchers found that green tea catechins have an inhibitory effect on protein tyrosine phosphatase and cysteine proteinases in certain anaerobic oral bacteria (Okamoto et al., 2003(Okamoto et al., , 2004. Researchers have also found that green tea catechins have the ability to interfere with DNA replication by interacting with, and thereby inhibiting the function of DNA gyrase (Grandišar et al., 2007). Studies on antifolate activity in microorganisms have shown that green tea polyphenols can inhibit the enzyme dihydrofolate reductase in bacteria and yeast, effectively blocking the ability of the microorganisms to synthesize folate (Navarro-Martinez et al., 2005. More recently is has been discovered that bioflavonoids (including those from green tea) could inhibit the activity of bacterial ATP synthase, reducing the ability of the microorganisms to produce enough energy (Chinnam et al., 2010).

OTHER INHIBITORY EFFECTS
Researchers have found that green tea catechins have inhibitory effects on many other bacterial functions. One such effect is inhibiting synthesis of PBP2 in methicillin-resistant

KEY CONCEPT 3 | Resistant
Microorganisms have a variety of methods that they use to overcome antimicrobial agents (antimicrobial-resistance). There are four main mechanisms that are used: inhibiting uptake of a drug, increased excretion of a drug (e.g., efflux pumps), structural or functional modification of the microorganism drug target, or inactivating the drug directly. This antimicrobial-resistance may be acquired from another similar organism, or a result of a mutation in one or more of the microorganism's own genes.

KEY CONCEPT 4 | MRSA
Methicillin-resistant Staphylococcus aureus (MRSA) have acquired a gene that allows production of an altered penicillin-binding protein (PBP), PBP2 . PBPs are transpeptidases that are used in the synthesis of the peptidoglycan layer of the bacterial cell wall. PBP2 has a greatly reduced affinity for most of the β-lactam drugs, making these drugs ineffective against MRSA. This makes the treatment of infections with MRSA much more difficult and costly.
of resistance to β-lactam drugs (Yam et al., 1998). Another effect on bacteria is the inhibition by green tea catechins on the conjugative transfer of the R plasmid in Escherichia coli (Zhao et al., 2001a). This could lead to decreased sharing of antimicrobial genes between bacteria. Research on staphylococci has shown the ability of green tea catechins to inhibit the activity of efflux pumps. In this particular study the efflux pump was Tet(K) which is involved in resistance to tetracycline (Sudano Roccaro et al., 2004). This same type of activity could very well-occur with other efflux pumps, which could enhance the ability of certain drugs to have an antimicrobial effect. A study performed with Helicobacter pylori found that green tea catechins blocked the ability of the bacteria to bind to the toll-like receptor-4 (TLR-4) on gastric epithelial cells . This would lead to a drastic reduction in that organism's ability to cause gastric disease. There are also inhibitory effects found with viruses. Research with HIV-1 has shown that green tea EGCG binds to the CD4 T-cell receptor, blocking the binding of the virus. This ability to block viral cell binding has been proposed for use in therapy for HIV-1 infection (Williamson et al., 2006).

SYNERGY OF GREEN TEA WITH ANTIMICROBIALS
Researchers have taken the concept of green tea catechins as having antimicrobial effects and expanded on this by performing studies that would determine if the green tea catechins might work synergistically with known antimicrobial agents. Many of these studies have focused on Staphylococcus aureus (particularly MRSA), Staphylococcus epidermidis, and Escherichia coli, among others. Examples of those studies have found that green tea catechins work synergistically with tetracycline against S. aureus and S. epidermidis (Sudano Roccaro et al., 2004;Fanaki et al., 2008); with penicillin against S. epidermidis (Haghjoo et al., 2013); with penicillin, oxacillin, ampicillin/sulbactam, and imipenem against MRSA (Hu et al., 2001(Hu et al., , 2002Zhao et al., 2001b;Stapleton et al., 2006;Aboulmagd et al., 2011); and with chloramphenicol, ciprofloxacin, and cefotaxime against isolates of E. coli having various levels of resistance, including extended-spectrum betalactamase (ESBL) producers (Fanaki et al., 2008;Cui et al., 2012a;Passat, 2012).

KEY CONCEPT 5 | ESBL
Extended-spectrum β-lactamase (ESBL) producing organisms are most often members of the gram negative Enterobacteriaceae family. Betalactamases are enzymes produced by the organism that are able to hydrolyze β-lactam drugs, rendering these drugs inactive. Like MRSA, the ESBLs have an increased resistance to penicillins and cephalosporins, but are sensitive to carbapenems and β-lactamase inhibitors (drugs that inhibit the activity of β-lactamases and are used together with a β-lactam drug, such as ampicillin/sulbactam). Many ESBL producers are also multidrugresistant organisms (MRSOs), making treatment of these infections very difficult.

GREEN TEA CATECHINS ANTIMICROBIAL SCOPE
A large variety of research has been performed assessing whether or not green tea has antimicrobial activities. A summary of examples of this research is included in Table 2. The research results shown do suggest that green tea may be effective against many organisms. The research is pretty well-balanced between gram positive and gram negative organisms, with a few viruses, fungi and a parasite also represented. The main gram positive organism represented is S. aureus. That is not surprising considering the ongoing menace of MRSA. The most commonly studied gram negative organism is E. coli. These results represent potentially useful information for battling the emerging threat of ESBL and Carbapenem-resistant Enterobacteriaceae (CRE) organisms. In a previous study, green tea extract was shown to be effective against E. coli strains isolated from UTIs. Many of these isolates (59/80) were multi-drug resistant (MDROs), and six of the strains were ESBLs (Reygaert and Jusufi, 2013). The results shown in Table 2, along with the possible synergistic activities mentioned earlier, suggest that green tea could be extremely useful as an antimicrobial agent. There are certain things to keep in mind when viewing the results in Table 2. First, is the type of green tea and the preparation used. The research may have used green tea from various locations around the globe. For the experiments, the preparation may have included freshly brewed green tea, green tea extracts (water, alcohol), or purified extracts of certain of the green tea catechins. There are also various other factors that may affect the composition of the tea (refer to the section on green tea composition). The second thing to pay attention to is the type of result reported. There are four basic types of results shown: growth in colony-forming units (CFUs), or plaque-forming units (PFUs) for viruses; minimal inhibitory concentration (MIC); the size of zones of inhibition; and the optical density (O.D.) of growth in liquid cultures. The percentage of increase or decrease of any of these parameters may also be shown. In addition, for viruses, other growth or detection measures made be shown. Overall, the results indicate that green tea has great potential as an antimicrobial agent.

KEY CONCEPT 6 | CRE
Carbapenem-resistant Enterobacteriaceae (CREs) are similar to ESBLs. These organisms are resistant to all β-lactam drugs, and are not inactivated by β-lactamase inhibitors. The most common ESBL-producing organisms are Escherichia coli and Klebsiella pneumoniae. Treatment options are extremely limited for infections involving these organisms.

DISCUSSION
The data discussed shows that green tea catechins EGCG and EGC are bioavailable in plasma (both) and urine (EGC only) at potentially therapeutic levels and in a timely manner (see Table 1). Antimicrobial studies using various green tea extracts have shown that there is a huge potential for the use of green tea in antimicrobial therapy (see Table 2). In a previous study, the amount of EGC from brewed Japanese green tea excreted in the urine was calculated to be approximately 3.5 mg per cup of tea. Since the results of the study showed that most of the bacterial strains tested were susceptible to EGC levels of ≤0.72 mg, one cup of tea should be able to control growth of the bacteria for up to 6 h or even longer (Reygaert and Jusufi, 2013). While the research results that have been discussed suggest that green tea may be effective against many organisms, there are certain issues that need to be addressed concerning these results. The first issue is how the results are displayed. The difficulty of assessing these types of results, and how these results relate to each other among the different research groups, can be confusing. This brings us to the second major issue. Various agencies, such as the Clinical and Laboratories Standards Institute (CLSI) in the United States, have strict protocols for the determination of antimicrobial susceptibility. Most of the research results that have been performed using green tea have not necessarily followed any of these types of guidelines. Therefore, the results of these experiments may differ from what the results would have been if strict protocols had been used. In the future, it will be most helpful if researchers would take the time to use these protocols. That will make comparing the results, and assessing if the results   are meaningful so much easier. In addition (and most importantly), in order for the results to be able to be translated into clinical use, the protocols will need to be followed. That having been addressed, it is still very encouraging to look at the research performed and see that green tea could very possibly be an effective antimicrobial agent, especially against multidrugresistant strains, and in particular, MRSA and ESBL producing organisms. Hopefully, in the future, researchers will be able to study the effects of green tea on infections in humans. This type of research is a critical part of determining the antimicrobial capabilities of green tea. It might possibly be incorporated into research with other antimicrobial compounds. Perhaps naturopathic practitioners could begin to collect data on patients using green tea. With emerging multidrug-resistant organisms and the lack of effective new antimicrobial drugs being produced, we cannot afford to ignore the potential of green tea.