Molecular Mechanisms of Inhibition of Streptococcus Species by Phytochemicals

This review paper summarizes the antibacterial effects of phytochemicals of various medicinal plants against pathogenic and cariogenic streptococcal species. The information suggests that these phytochemicals have potential as alternatives to the classical antibiotics currently used for the treatment of streptococcal infections. The phytochemicals demonstrate direct bactericidal or bacteriostatic effects, such as: (i) prevention of bacterial adherence to mucosal surfaces of the pharynx, skin, and teeth surface; (ii) inhibition of glycolytic enzymes and pH drop; (iii) reduction of biofilm and plaque formation; and (iv) cell surface hydrophobicity. Collectively, findings from numerous studies suggest that phytochemicals could be used as drugs for elimination of infections with minimal side effects.


Introduction
The aim of this review is to summarize the current knowledge of the antimicrobial activity of naturally occurring molecules isolated from plants against Streptococcus species, focusing on their mechanisms of action. This review will highlight the phytochemicals that could be used as alternatives or enhancements to current antibiotic treatments for Streptococcus species. The scope of the review is limited to inhibitory effects of phytochemicals, mainly polyphenols, against Streptococcus species and where possible, their mechanisms of action against the major virulence factors will be discussed. Due to their major implications on human health, this review has largely focused on four Streptococcus species: (i) S. mutans (ii) S. pyogenes (iii) S. agalactiae and (iv) S. pneumoniae. To explain the potential mechanisms of inhibition of the phytochemicals, S. mutans has been used as the major example.

Adhesion, Plaque, and Biofilm Formation of Streptococcal Species
To cause disease, a bacterial pathogen needs to meet several basic requirements. First, it must be able to adhere to the tissue surface and compete with the normal microbiota present on that surface [5,34,35]. Subsequently, for sustainable attachment, biofilms are developed and this may lead to invasion of the host tissue [6]. To establish biofilm, planktonic bacteria attaches to either inert or coated surfaces and this can be mediated by electrostatic contacts or bacterial surface adhesins [36]. Attachment is followed by proliferation of the primary colonizers and their co-aggregation with other planktonic bacteria, production of exopolysaccharide which stabilizes the architecture, leading to the maturation of the biofilm [36]. Sessile bacteria then could detach and form biofilms at different site [36][37][38]. Biofilm formation is not an attribute only specific to a few species, but a general ability of all microorganisms. Biofilm formation pathways are species specific, diverse, and dependent on environmental factors [39]. Although diverse, there are common features among all biofilms: (i) cells in the biofilm are glued together by an extracellular matrix made of exopolysaccharides, proteins, and occasionally nucleic acids; (ii) biofilm formation is initiated by environmental and bacterial signals; and (iii) biofilm offers bacteria protection from antibiotics and environmental stresses including immunological responses of the host [39]. Bacterial biofilms can build up on abiotic (plastic, glass, metal, etc.) or biotic (plants, animals, and humans) surfaces [34,38,40]. Mammalian-tissue colonizing species of Streptococcus live within biofilm in the natural environment [6,41,42].
Bacteria increase the expression of their outer cell surface adhesins when environmental conditions allow promoting cell-cell and cell-surface interaction [6,43]. Streptococci owe their success in colonization to their wide range of proteins expressed on their surfaces [5,6]. Surface adhesins facilitate interrelation with salivary, serum, extracellular matrix elements, host cells and other microbes [5,6]. Many of these adhesins are anchored to the cell wall peptidoglycan via their C-terminus or to the cell membrane via their N-terminal lipid (lipoproteins), and other adhesins remain surface localized through non-covalent interactions with other proteins or polysaccharides on the cell surface [6,44].
Most bacterial pathogens, including streptococci, have long filamentous structures known as pilli or fimbriae that are also involved in adhesion and biofilm formation [34]. In Gram-positive bacteria, hydrophobic components can be found: (i) covalently bound to cell wall, such as streptococcal M and F proteins, (ii) in the cytoplasmic membrane (e.g., lipoteichoic acid (LTA) of S. pyogenes or sialic acid of S. agalactiae) or (iii) located on the surface, like pilli or fimbriae [6,44,45]. Aside from adherence, biofilms are of significant importance as approximately 65% of human bacterial infections involve biofilms [45] including Streptococcus species (e.g., S. mutans, S. pyogenes, S. agalactiae, and S. pneumoniae) [34,40,41,46]. Clinically, biofilms are important because they reduce susceptibility of the bacteria to antimicrobials, prospering resistant bacteria leading to persistent infections [47,48].
The primary cause of dental caries is dental plaque which is a complex biofilm [41]. Broad spectrum of saliva proteins contribute to and initiate adhesion and dental biofilm formation [41,49,50]. Adhesion of S. pyogenes to various host cells is facilitated by the capsule and several factors embedded in the cell wall including M protein, LTA, and F protein [6,25,51]. M protein not only helps bacteria to attach to the host tissue but also inhibits opsonization by binding to host complement regulators and to fibrinogen [52]. A recent study has demonstrated that S. pyogenes pilus promotes pharyngeal cell adhesion and biofilm formation [53]. Altering surface hydrophobicity by sub-minimum inhibitory concentration of penicillin and rifampin reduces the adhesion of S. pyogenes to epithelial cells suggesting that surface-associated LTA will determine the surface hydrophobicity content of S. pyogenes, which consequently affects the bacteria's interaction with mammalian host cells [54][55][56].
S. agalactiae produces several virulence factors such as adhesins [6]. These surface proteins and LTA of S. agalactiae bacterial cell wall contribute to the adhesion process mediating the invasion of eukaryotic cells [30]. Non-encapsulated S. agalactiae strains show increased adherence to eukaryotic cells [30]. In vitro studies have shown that S. agalactiae adheres to vaginal, buccal, endothelial and pulmonary epithelial cells [30]. Many clinical isolates of S. mutans, S. pyogenes, and S. agalactiae have been reported to be hydrophobic while their avirulent counterpart strains lacked this feature [57][58][59][60]. Studies have shown that S. pneumoniae adheres to abiotic surfaces, e.g., polystyrene or glass, and forms three-dimensional biofilm structures that are about 25 micrometers deep [34]. This three-dimensional structure enables the bacteria to survive for long periods within the bacterial community [34].

Proton-Extrusion and Glycolysis of Streptococcal Species
Vital to the survival of bacteria is the regulation of the cytoplasmic pH as cellular activity requires a specific pH range [61]. Cytoplasmic pH is modulated by environmental pH, production, or consumption of internal protons, and transferring acids and bases across the plasma membrane [62]. The function of F-adenosine triphosphatase (F-ATPase) in streptococci is to regulate internal pH by pumping protons out of the cell [62,63]. The physiological role of streptococcal F 0 F 1 -ATPase is to alkalinize the cytoplasmic pH in the acidic pH range and to establish a proton reserve for a variety of secondary transport systems [64][65][66]. Streptococci are deficient in respiratory chains and are unable to produce a large proton gradient across the membrane, however, they make up for this lack by utilizing a range of basic transport systems [66]. For example, synthesizing a cytochrome-like respiratory chain, formation of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate by coupling the nicotinamide adenine dinucleotide hydrogen (NADH) oxidation with phosphorylation reaction [66][67][68]. Generally, ATPase in streptococci does not function as ATP synthase because of lack of a functional electron transport system; thus, it functions as hydrolase for proton movements coupled to ATP hydrolysis that are used for the generation of the proton gradient [66]. Streptococci utilize the glycolytic pathway to metabolize glucose to lactic acid [4,66]. Glucose is taken up, phosphorylated to glucose-6-phosphate through the phosphoenolpyruvate-dependent phosphotransferase system, and then converted to pyruvate, and eventually to lactic acid [66,69].
S. pneumoniae and oral streptococci could adapt to different environments and this capability is facilitated by ATPase regulating the intracellular concentration of solutes, including protons, and maintaining the pH homeostasis by proton extrusion [66,70]. Adherence is dependent: (i) on the synthesis of extracellular polysaccharides (mostly glucans) from the disaccharide sucrose through glucosyltransferases (GTFs) for S. mutans, and (ii) bacteria's ability to produce acid by glycolysis and its tolerance to the produced acid [71]. S. mutans has the properties of acid production from sugar metabolism causing a drop in pH in dental plaque [72]. Low pH values in the plaque matrix leads to demineralization of tooth enamel, selection of acid-tolerant streptococci and eventually dental caries [72]. The glucans synthesized by GTFs promote the binding and accumulation of S. mutans and other bacteria on the tooth surface and contribute to the formation of biofilms [72][73][74][75]. S. mutans increases the proton-translocation, and F-ATPase activity when the environment's pH drops, thereby this bacterium could withstand acidification influences [66,76,77]. F-ATPase transfers protons out of cells with the assistance of ATP hydrolysis to maintain its intracellular pH (e.g., more alkaline than the extracellular environment) [76]. F-ATPase enzyme is composed of two domains; (i) F 1 , the cytoplasmic catalytic domain; and (ii) F 0 , the proton-conducting membrane domain [67,78]. S. mutans does not produce catalase or cytochromes (thus a heme-based electron transport system) and so does not have oxidative phosphorylation linked to trans-membrane electron transport [66,79].

Glucan Synthesis, Aggregation and Quorum Sensing of Streptococcal Species
Glucans interact with surface-associated glucan binding proteins of S. mutans to initiate colonization, cell-cell aggregation and the firm adherence of its cells to tooth surfaces [72,80]. S. mutans produces three types of GTFs: GTFB, GTFC, GTFD, and each of these enzymes are composed of two functional domains: (i) an amino-terminal catalytic domain (CAT); and (ii) a carboxyl-terminal glucan-binding domain (GBD) [81]. GTFB and GTFC, located on the cell surface, are encoded by gtfB and gtfC genes and GTFD is encoded by the gtfD gene [82]. Therefore, one of the strategies to control biofilm formation and dental caries is to inhibit the activity of GTFs: (i) GTFB (which synthesizes a polymer of mostly insoluble α1, 3-linked glucan); (ii) GTFC (which synthesizes a mixture of insoluble α-1,3-linked glucan and soluble α-1,6-linked glucan); and/or (iii) GTFD (which synthesizes water-soluble glucans rich in α-1,6-glucosidic linkages) [83,84].
Many streptococci use quorum-sensing systems to regulate several physiological properties, including the ability to incorporate foreign deoxyribonucleic acid (DNA), tolerate acid, form biofilm, and become virulent [85][86][87][88]. Quorum sensing, a strategy of cell-to-cell communication in a biofilm community, regulates unnecessary over-population and nutrient competition [89,90]. Bacterial activities including virulence gene expression within biofilms is regulated by the occurrence of quorum sensing [91]. This topic as well has comprehensively been discussed in review articles [87,92,93].

Treatment of Streptococcal Infection
Penicillin or one of its derivatives (e.g., amoxicillin and ampicillin) are the recommended antibiotic treatment for non-allergic patients diagnosed with S. pyogenes and S. agalactiae infections [27]. For allergic individuals, azithromycin and clarithromycin are recommended and in fact, azithromycin is prescribed more commonly than penicillin [94]. For severe S. pyogenes infections like necrotizing fasciitis and toxic shock syndrome, a combination of penicillin and clindamycin are prescribed [95]. S. pyogenes and S. agalactiae are not resistant to penicillin, but over time they have become resistant to clindamycin, tetracycline, vancomycin and macrolides (e.g., erythromycin, azithromycin and clarithromycin) [27]. Clarithromycin, clindamycin and vancomycin resistance among S. pyogenes and S. agalactiae strains are most concerning [27].

Antibiotic Resistance and Emerging Threats
Antimicrobial resistance is compromising the treatment of invasive infections including severe streptococcal infections [27]. This threat becomes significant in vulnerable patients (e.g., individuals undergoing chemotherapy, dialysis and organ transplants) due to infection-related complications [27]. This puts healthcare providers in the position to use antibiotics that may be more toxic to the patient, and frequently more expensive, leading to an increased risk of long-term disability and lower survival rates [27].
According to Frieden, director of the U.S. Center for Disease Control and Prevention (CDC), antimicrobial resistance is a serious health threat in the 21st century [27]. Infections caused by resistant bacteria are now on the rise and their resistance to multiple types and classes of antibiotics is worrisome [96]. The decrease in the rate of pathogen susceptibility to antibiotics has made it much more difficult to combat the infectious diseases [27]. The CDC's 2013 report has prioritized drug-resistant S. pneumoniae as a serious threat, and erythromycin-resistant S. pyogenes and clindamycin-resistant S. agalactiae as concerning threats [27].

Possible Alternatives for Classical Antibiotics
Plants produce diverse secondary metabolites or phytochemicals, most of which are isoprenoids and polyphenols and their oxygen-substituted derivatives such as tannins that could be raw materials for future drugs [97]. Herbs and spices contain useful medicinal compounds including antibacterial chemicals, and researchers have found that many of these compounds inhibit the growth of pathogenic bacteria [97]. Accordingly, experimental observations have shown that herbal preparations are active against many of the pathogens ( Table 2).
From the period of 1981 to 2006, 109 new antibacterial drugs were approved for treatment of infectious diseases of which 69% originated from natural products, and 21% of antifungal drugs were natural derivatives or compounds mimicking natural products [98]. Various medicinal plants have recently been tested for their antimicrobial activity and all have proven that phytochemicals, particularly polyphenols, exhibit significant antibacterial activity against Streptococcus species (Table 3).

Anti-Streptococcal Attributes of Phytochemicals
Many fruits and plants have shown to possess anti-streptococcal effects (Table 3). Folklore medicinal plants have long been used for the treatment of S. pyogenes infections ( Table 2) including pharyngitis. For example cashew plant (Anacardium occidentale), stickwort (Agrimonia eupatoria), mountain daisy (Arnica montana), bayberry (Myrica cerifera), soft leafed honeysuckle (Lonicera japonica), cuajilote (Parmentiera aculeate) or baobab (Adansonia digitata) [99][100][101][102][103][104], (Table 2). Particularly more attention has been given to anti-streptococcal effects of phytochemicals against S. mutans due to its cariogenic properties. A wide range of commercial and freshly prepared polyphenolic rich extracts (70% propanone) of various teas including green and black tea, lemon, cinnamon, hibiscus, peppermint, grape seed, sloe berry skin, cocoa, blackberry, pomegranate skin, blackcurrant, hawthorn berry skin, red and white wine was tested for their anti-streptococcal activity against oral streptococci (various strains of S. mutans, S. oralis, S. gordonii, S. salivarius, S. sanguis) [105]. All the tested products exhibited their minimum inhibitory effect at concentrations ranging 0.25-32 mg/mL against Streptococcus species [105]. Red grape seed propanone extract was most potent against S. mutans and Agro tea extract least effective with minimum inhibitory concentration of 0.5 mg/mL and 32 mg/mL respectively [105]. Phytochemicals, although very limited, also have been shown to hinder the growth of S. agalactiae [106][107][108][109][110][111][112]. Aqueous, ethanolic and chloroform extracts of bael, Indian gooseberry, moringa, neem, Chinese mahogany exert their minimum inhibitory effects at concentrations ranging from 0.15 mg/mL to 10 mg/mL against S. agalactiae, chloroform extract of Chinese mahogany being the most active one [111]. In a study by Nguelefack et al. ethyl acetate bark extract of Distemonanthus benthamianus at Minimum Bactericidal Concentration (MBC) of 4096 µg/mL was effective against S. agalactiae and its phytochemical profile was indicative of presence of flavonoids and phenolics and absence of sterols, triterpenes and alkaloids [113]. Moderate inhibitory effect of wild Asparagus racemosus ethanol extract at concentration of 500 µg/disc was also reported for S. agalactiae [114].

Phytochemicals with Inhibitory Activities against Adhesion, Plaque, and Biofilm Formation
Phytochemical-rich extracts and their associated pure compounds have repeatedly shown inhibitory effects against adhesion, plaque, and biofilm formation of streptococcal species (Tables 4  and 5). High molecular weight non-dialysable materials extracted from cranberry juice (NDM) exhibit adhesion reduction activity in a dose-dependent manner at concentrations of 66-1330 µg/mL against S. sobrinus [115]. In another study, the ethanolic extract of Helichrysum italicum at concentrations of 15-31 µg/mL inhibited the sucrose-dependent adherence of S. mutans cells to a glass surface by 90% to 93% [116]. Cranberry juice powder (25%) at 500 µg/mL concentration inhibited the biofilm formations of S. sobrinus and S. sanguinis significantly [117]. In the same study, cranberry juice powder decreased the cell surface hydrophobicity of S. mutans and S. sobrinus 6715 by more than 40% [117]. Table 2. Folklore medicine used for Streptococcal diseases or diseases with similar clinical Presentations.
Adhesion of S. mutans to the tooth surface was hindered after treatment with UA at 256 µg/mL [176]. Sub-MIC dose of UA also affected the adhesion consequently hindering the biofilm formation [176]. UA moreover eradicated the biofilm cells at concentrations of 500-2000 µg/mL [176]. Polyphenolics-rich tea extract at concentrations as low as 1-4 mg/mL prevented the attachment of S. mutans to collagen coated hydroxyapatite beads [181].
In another study, the effect of cocoa polyphenol fractions on S. mutans biofilm reduction in the absence and presence of sucrose were measured. At 35 µM concentration and after 4 h, biofilm mass was reduced to 68% in the absence of sucrose and to 44% in the presence of sucrose [165]. Biofilm of S. mutans on saliva coated hydroxyapatite surface was preformed and then treated (60 s) with purified proanthocyanidin (PAC)-containing fraction of cranberry (various degree of polymerization) [182]. At concentrations of 100 µM (single or combined fractions in 1:1 ratio), confocal 3D images show distorted architecture and deficient biofilm accumulation suggestive of reduced biomass and thickness of adherent bacteria and EPS [182]. Expressions of 119 genes of S. mutans within biofilm were altered post exposure to PAC-rich fractions of cranberry [182]. The expression of genes particularly related to adhesion, acid stress tolerance, glycolysis and other cellular activities during biofilm development were downregulated [182]. Structure activity relationship analysis revealed that PAC oligomers with more than eight epicatechin units exhibit higher anti-adhesion effects up to 85% against S. mutans however the increase in potency is not proportional [182]. This not only is associated with degree of polymerization but may also be associated with number and location of A-type linkages in the oligomers, and type of interflavan bonds [182].
The anti-adhesive properties of root extract of Pelargonium sidoides have been studied against S. pyogenes attachment to human epithelial type 2 (HEp-2) cells [164]. Results have shown that after pre-treatment of S. pyogenes with methanol insoluble and methanol soluble fractions of the extracts of Pelargonium sidoides at concentrations of 30 µg/mL, adhesion of the pathogen to HEp-2 cells was inhibited up to 30% to 35% [183]. To characterize the anti-adhesive constituents of these fractions, comparative chemical studies were performed. The study revealed that the proanthocyanidins content of the fraction was of prodelphinidin nature, and inhibition of the adhesion was in a specific rather than non-specific manner [164,183]. Successful inhibition of adhesion and hydrophobic interactions could reduce and or prevent sore throat caused by S. pyogenes [164]. It has been suggested that polymeric flavonoids or other large molecule polyphenols may exhibit higher anti-adhesion effects against streptococci [180]. Coffee high molecular weight fraction nearly completely (91%) hindered the adhesion of S. mutans [184].
Similarly, a study on the binding activity of S. pneumoniae and S. agalactiae to different molecular size fractions (F1, F2, F3) of Vaccinium family polyphenols found that binding was highest to wild cranberry (Vaccinium oxycoccos) [112]. S. pneumoniae cells bound mostly to cranberry juice low-molecular size fraction (F1) and S. agalactiae cells to high-molecular size fraction (F3) [112]. S. pneumoniae bound to F1 of bilberry and cranberry juices and S. agalactiae attached most actively to F2 and F3 of berry and juice preparations belonging to Vaccinium species [112]. Phytochemical analysis has shown that F2 and F3 fractions contain polyphenol macromolecular complexes, including proanthocyanidins and polyhydroxy flavonoids [112]. At sub-MIC level of 2 mg/mL red grape marc extract, composed of 20% polyphenols and 3% anthocyanin, inhibited the adherence of S. mutans and Fusobacterium nucleatum cells to glass surface [166]. Morin, a flavonol, reduced biofilm biomass of S. pyogenes at concentrations exceeding 225 µM up to 65% [175]. Epigallocatechin gallate (EGCG) of Camellia sinensis has various physiological effects on S. mutans UA159 (Figure 1) and has been proven to inhibit the enzymatic activity of glucosyltransferases, F 1 F 0 -ATPase, lactate dehydrogenase, biofilm formation and growth [153]. fractions contain polyphenol macromolecular complexes, including proanthocyanidins and polyhydroxy flavonoids [112]. At sub-MIC level of 2 mg/mL red grape marc extract, composed of 20% polyphenols and 3% anthocyanin, inhibited the adherence of S. mutans and Fusobacterium nucleatum cells to glass surface [166]. Morin, a flavonol, reduced biofilm biomass of S. pyogenes at concentrations exceeding 225 µM up to 65% [175]. Epigallocatechin gallate (EGCG) of Camellia sinensis has various physiological effects on S. mutans UA159 ( Figure 1) and has been proven to inhibit the enzymatic activity of glucosyltransferases, F1F0-ATPase, lactate dehydrogenase, biofilm formation and growth [153].

Phytochemicals with Inhibitory Activities against F-ATPase and Glycolytic pH-drop
Phytochemical-rich extracts not only possess anti-adhesion, anti-plaque and anti-biofilm attributes, but also have demonstrated inhibitory effects on streptococcal species F-ATPase and glycolytic pH-drop activities (Table 6, Figure 2). Plants and fruits have been studied for their anti-streptococcal effects and fruits such as cranberry (V. macrocarpon), cocoa (Theobroma cacao), babchi (Psoralea corylifolia), mangosteen (Garcinia mangostana) and grape (Vitis vinifera) have shown inhibitory effects on F0-ATPase

Phytochemicals with Inhibitory Activities against F-ATPase and Glycolytic pH-drop
Phytochemical-rich extracts not only possess anti-adhesion, anti-plaque and anti-biofilm attributes, but also have demonstrated inhibitory effects on streptococcal species F-ATPase and glycolytic pH-drop activities (Table 6, Figure 2). Plants and fruits have been studied for their anti-streptococcal effects and fruits such as cranberry (V. macrocarpon), cocoa (Theobroma cacao), babchi (Psoralea corylifolia), mangosteen (Garcinia mangostana) and grape (Vitis vinifera) have shown inhibitory effects on F 0 -ATPase and F 1 -ATPase, glucosyltransferases (GTFB and GTFC) and acid production activities of S. mutans [80,84,165,185]. The lack of inhibitory activity of monophenolic compounds suggest that the inhibition of F 1 -F 0 -ATPase by phenolics require two or more phenolic structures [186]. The flavones have also been shown to interact with other ATPases, such as Ca 2+ -ATPase [187] and Na + /K + -ATPase [188], in addition to their inhibitory effects on F 1 -F 0 -ATPase [189]. Glycolysis of S. mutans is inhibited by α-mangostin leading to indirect inhibition of respiration by α-mangostin [190]. Glucan production by GTFs and F-ATPase is inhibited by α-mangostin suggesting that S. mutants can be eliminated selectively [190]. and F1-ATPase, glucosyltransferases (GTFB and GTFC) and acid production activities of S. mutans [80,84,165,185]. The lack of inhibitory activity of monophenolic compounds suggest that the inhibition of F1-F0-ATPase by phenolics require two or more phenolic structures [186]. The flavones have also been shown to interact with other ATPases, such as Ca 2+ -ATPase [187] and Na + /K + -ATPase [188], in addition to their inhibitory effects on F1-F0-ATPase [189]. Glycolysis of S. mutans is inhibited by α-mangostin leading to indirect inhibition of respiration by α-mangostin [190]. Glucan production by GTFs and F-ATPase is inhibited by α-mangostin suggesting that S. mutants can be eliminated selectively [190].

Conclusions and Prospects
Each class of classical antibacterial agents (antibiotics) usually targets different sites and processes of pathogenic bacteria. Major antimicrobial actions include disruption of membrane structure, inhibition of protein synthesis, and inhibition of production of folate coenzymes, nucleic acids, and peptidoglycans. Natural antimicrobials like their synthetic counterparts (antibiotics) target different molecules and processes to inhibit the colonization and viability of the bacteria or to inactivate bacterial toxins and or modulate the molecules and processes pre-requisite for bacteria's metabolic pathways or reduce the rate of protein synthesis. It is worth noting that natural antimicrobial products not necessarily have to be bactericidal to suppress such processes and activities. It is plausible that a compound is likely to be efficient bacterial growth inhibitor if it can deteriorate the cytoplasmic pH, increase the permeability of plasma membrane, prevent extracellular and intracellular microbial enzyme production, interrupt bacterial metabolic pathways, or disrupt plaque and biofilm formation. As observed, there is considerable amount of scientific evidence that phytochemicals exert significant multiple anti-streptococcal effects and apart from their bactericidal effects, their main bacteriostatic strategy is the anti-adhesiveness attribute.
The efficacy of natural products as antimicrobials with fewer or no side effects is likely to depend on the structure of the compound that interacts with the toxin or pathogen and not with molecules of the host meaning that their effect is specific. This approach has become the rationale for natural drug design studies as a new field of research. Attempts have been made to understand certain features relating to phytochemical structure and the associated antibacterial activity. High molecular weight and complex phytochemicals exert greater inhibitory effects such as pentamer polyphenolic fraction

Conclusions and Prospects
Each class of classical antibacterial agents (antibiotics) usually targets different sites and processes of pathogenic bacteria. Major antimicrobial actions include disruption of membrane structure, inhibition of protein synthesis, and inhibition of production of folate coenzymes, nucleic acids, and peptidoglycans. Natural antimicrobials like their synthetic counterparts (antibiotics) target different molecules and processes to inhibit the colonization and viability of the bacteria or to inactivate bacterial toxins and or modulate the molecules and processes pre-requisite for bacteria's metabolic pathways or reduce the rate of protein synthesis. It is worth noting that natural antimicrobial products not necessarily have to be bactericidal to suppress such processes and activities. It is plausible that a compound is likely to be efficient bacterial growth inhibitor if it can deteriorate the cytoplasmic pH, increase the permeability of plasma membrane, prevent extracellular and intracellular microbial enzyme production, interrupt bacterial metabolic pathways, or disrupt plaque and biofilm formation. As observed, there is considerable amount of scientific evidence that phytochemicals exert significant multiple anti-streptococcal effects and apart from their bactericidal effects, their main bacteriostatic strategy is the anti-adhesiveness attribute.
The efficacy of natural products as antimicrobials with fewer or no side effects is likely to depend on the structure of the compound that interacts with the toxin or pathogen and not with molecules of the host meaning that their effect is specific. This approach has become the rationale for natural drug design studies as a new field of research. Attempts have been made to understand certain features relating to phytochemical structure and the associated antibacterial activity. High molecular weight and complex phytochemicals exert greater inhibitory effects such as pentamer polyphenolic fraction of cocoa, high molecular weight non-dialyzable material of cranberry and F2 or F3 fractions of crowberry and bilberry. The side effects of the current antimicrobials and the spread of drug-resistant microorganisms have become a significant concern and a threat to successful therapy of microbial diseases. Therefore, there is an urgent demand for the discovery of safe natural compounds with diverse chemical structures and mechanisms of action satisfying both the consumer and the healthcare providers as potential useful therapeutic tools of the post-antibiotic era. Intensive research on such plants could lead to the incorporation of the most potent chemically defined extracts into nutraceuticals or natural health products and becoming a solution to this global concern of evolution of drug-resistant microorganisms.