A review: Use of plant extracts and their phytochemical constituents to control antibiotic resistance in S. aureus

Although the invention of antibiotics has reduced the morbidity and mortality due to infectious diseases but the inappropriate use of antibiotics both for humans and animals has led to the emergence of multidrug resistant strains of bacteria and fungi. Among the antibiotic resistant pathogens, S. aureus is the pathogen of greatest concern due to its intrinsic virulence, its ability to cause a large number of infections and to survive under various environmental conditions. Modification of antibiotic (β-lactamases), use of alternative target site (PBP2a), efflux of antibiotics (NorA) and biofilm formation are some of the mechanisms utilized by S. aureus to develop resistance against antibiotics. This has lead to the increased interest in traditional medicine that has long been used to cure various infectious diseases at different regions of the world. Plant extracts serve as an important source to combat microbial resistance because they consist of complex mixture of phytochemical constituents against which the development of resistance is very slow. In additions, phytochemical constituents present in the plant extracts also act as immunomodulatory and anti-inflammatory agents. The current study aims at stimulating the search of plant extracts and their phytochemical constituents to control microbial resistance. In future, plant extracts as well as their purified constituents could be used as potent therapeutic agents for the treatment of infections caused by S. aureus without obvious side effects.


Introduction
Discovery of penicillin in 1929 not only revolutionized the field of antibacterial therapy but also led to the discovery of large number of effective antibiotics from bacteria and fungi. However, in 1940s, penicillin-resistant strains of microbes began to appear due to its widespread use [1,2]. Similarly, bacteria have developed resistance to other commonly used antibiotics [3]. Necessity is the mother of invention; therefore, a man is in continuous search for the discovery of new remedies to control the infectious diseases and emerging resistance of microbes. Plants especially herbs have been used to cure diseases since ancient times. About 80% of the world population living in developing countries relies on herbs as well as other medicinal plants for therapeutic purposes and their uses are increasing day by day due to the side effects of allopathic medicine [4]. For the promotion and development of herbal medicine, World Health Organization has made a strategic plan which includes four areas: a) Recognition of herbal medicines as valuable resources and promotion of their use. b) Development of comprehensive program for identification, cultivation and recognition of herbal medicines as valuable products. c) Introduction of measures for the assessment of medicinal plants to ensure the quality control of plant based products in order to include the herbal medicines in the national standard or pharmacopoeia. d) Development of programs by international cooperation and coordination for the conversation of plants that serve as valuable source of traditional or modern drugs in order to prevent their extinction [5]. S. aureus as an alarm to human health In early 1970s, optimistic view of physicians that virtually all bacterial infections can be controlled by using a wide array of antimicrobial agents, was shaken due to the emergence of antibiotic resistant strains of bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis). Several factors contributed to the evolution of these antibiotic-resistant pathogens and one of these factors is the widespread use of antibiotics to control both human and animal infections [6,7]. Among the antibiotic resistant strains, S. aureus is the pathogen of greatest concern due to its intrinsic virulence, its ability to cause a large number of infections and to survive under different environmental conditions [8]. Besides the availability of effective antimicrobial agents, the mortality rate for S. aureus bacteremia remains approximately 20-40% [9]. Now, S. aureus is the leading cause of a variety of community and hospital acquired infections ranging from mild skin infections to severe infections including osteomyelitis, pneumonia, endocarditis and sepsis [10]. Molecular mechanisms of antibiotic resistance Resistance to penicillins Development of antibiotic resistance has changed the strategy to treat S. aureus associated infections In the late 1960s, more than 80% of both community and hospital acquired S. aureus strains were found to be resistant to penicillins. Rapid emergence of penicillin resistance was due to the horizontal transfer of blaZ gene that encodes β-lactamase enzyme. This enzyme hydrolyzes the β-lactam ring of penicillins and thereby rendering them ineffective [11].

Resistance to methicillin
To overcome the problems of penicillins, semisynthetic penicillinase resistant penicillin called methicillin was introduced in 1961 but its introduction was followed by a large number of cases of methicillin resistant S. aureus [12]. Resistance to penicillin is due to the presence of mecA gene that encodes penicillin binding protein 2a (PBP2a or PBP2') and is located on chromosomal island designated as staphylococcal cassette chromosome mec (SCCmec). PBP2a is a membrane bound enzyme that catalyzes the cross-linking of peptidoglycan chains [13, 14]. PBP2a has low affinity with β-lactam antibiotics and thereby allowing the S. aureus to survive in the presence of high concentrations of βlactam agents. mecA gene confers resistance not only to β-lactam agents but also to Cephalosporins [15]. Continued use of vancomycin to control MRSA infections in hospitals led to the emergence of vancomycin resistant MRSA strains. A case of an MRSA with reduced susceptibility to vancomycin was first reported in 1997 in Japan. Resistance to vancomycin is due to the mutations in the genes that are involved in the biosynthesis of cell envelope [16].

Resistance to fluoroquinolones
Another mechanism of resistance to antibiotics utilized by S. aureus is to reduce the intracellular concentration of antibiotics. Reduction in intracellular concentration of antibiotics is achieved either by decreasing the membrane permeability or by efflux of antibiotic out of the cell [17, 18]. The purpose of these efflux pumps is the elimination of intracellular metabolites that are toxic to cell. In addition, bacterial efflux pumps also play their role in the secretion of virulence determinants (hemolysins, proteases, coagulases and clumping factors) and maintenance of cell under stress conditions [19]. Some efflux pumps are specific, extruding only one antibiotic or a class of antibiotics whereas others can extrude several classes of antibiotics and are designated as multidrug resistance (MDR) efflux pumps. Until now, more than ten MDR efflux pumps have been identified in S. aureus. NorA is one of the most studied MDR efflux pump of S. aureus that is encodes by the chromosomal gene norA.

Biofilm formation
In addition to β-lactamases, PBP2a and efflux pumps, another weapon utilized by S. aureus to develop resistance to antibiotics is the formation of biofilms. Biofilm formation is used as a defense mechanism by S. aureus as it provides protection against host defense system and antibiotic therapy. Moreover, biofilm associated infections are more chronic and mostly occur in hospitals [22]. Biofilm formation in S. aureus involves three steps: initial attachment of bacteria to biotic or abiotic surface, proliferation which is preceded by the production of ECM (extracellular matrix) that consists of extracellular polysaccharides, teichoic acid and DNA released by lysed cells, and final step of biofilm production is the detachment. Detachment of biofilm is under the control of quorum sensing system Agr but the quorum sensing control of biofilm detachment in S. aureus is not yet defined.

Terpenes
Terpenes form the largest group of natural compounds and their identification is done on the basis of isoprene units they possess. Terpenoids which are the derivatives of terpenes exhibit antibacterial effects but their mechanism of antibacterial activity is not clearly defined. It is estimated that terpenoids work by disrupting the bacterial membranes [35]. Lectins Lectins are larger than polypeptides and contain mannose containing molecules. Their antimicrobial activity is due to their ability to compete with bacterial cell surface adhesion molecules for binding to host polysaccharide receptors [36, 37]. Other antimicrobial phytochemicals Besides alkaloids, phenolic compounds and lectins, many other phytochemicals have also been reported to exert antibacterial activities. There are some reports on the antibacterial activities of polyamines (spermidine), glucosides, thiosulfinates and isothiocyanates. In Brazil, acetylene compounds and flavonoids derived from plant are traditionally used for the treatment of malaria and liver disorders. Cranberry and blueberry juices have been used for therapeutic purposes for the treatment of urinary tract infections. In 1990s, researchers found that monosaccharide fructose present in cranberry and blueberry juices competitively inhibit the adsorption of pathogenic E. coli to epithelial cells of urinary tract [38].

Prevention of skin-associated infections by plant based antiseptic soaps
In order to prevent, control and reduce microbial infections, hand washing is crucial. Health care workers are potent source of transmission of MDR pathogens from one person to another. Proper hand washing and drying terminate the chain of transmission of pathogens from hands to other body parts. Hence, hand sanitizers and antiseptic soaps are being utilized for hand washing purposes. Although, chemical antiseptic soaps have played a vital role in reducing the transmission of infectious diseases but they have some adverse effects including skin irritation and microbial resistance [46]. In order to overcome the problems associated with chemical antiseptic soaps, there is a need for the development of plant based antiseptic soaps that will be less toxic and inexpensive [47]. Researchers are now in continuous search for the development and evaluation of plant based soaps that may be used as alternatives to harmful synthetic soap products. It is not surprising to find that synthetic foaming agent sodium lauryl sulfate (SLS) in soaps is replaced by  saponins [48], synthetic antibacterial agents such as Triclosan are replaced with natural antimicrobial agents [49] and synthetic antioxidant agents such as BHT (butylated hydroxytoluene) are replaced with phenolic compounds [50].

Conclusion
Discovery of antibiotics is marked as a significant scientific advancement in the field of medicine as they helped in controlling S. aureus associated infections. But an inappropriate use of antibiotics has led the evolution of S. aureus strains that are not only resistant to conventional antibiotics like penicillins but also resistant to other previously used antibiotics like fluoroquinolones.