Antimicrobial resistance (AMR) and plant-derived antimicrobials (PDAms) as an alternative drug line to control infections

Infectious diseases caused by antimicrobial-resistant microbes (ARMs) and the treatment are the serious problems in the field of medical science today world over. The development of alternative drug line to treat such infectious diseases is urgently required. Researches on ARMs revealed the presence of membrane proteins responsible for effusing the antibiotics from the bacterial cells. Such proteins have successfully been treated by plant-derived antimicrobials (PDAms) synergistically along with the commercially available antibiotics. Such synergistic action usually inhibits the efflux pump. The enhanced activity of plant-derived antimicrobials is being researched and is considered as the future treatment strategy to cure the incurable infections. The present paper reviews the advancement made in the researches on antimicrobial resistance along with the discovery and the development of more active PDAms.


Introduction
Increasing antimicrobial resistance (AMR) among microbes caused the emergence of new resistant phenotypes and further caused the development of new antimicrobial compounds (Goossens 2013). Infectious diseases caused by antimicrobial-resistant microbes (ARM) have been frequently reported since last few years (Vila and Pal 2010). About 440,000 new cases of multidrug-resistant tuberculosis (MDR-TB) are recorded annually, causing approximately 150,000 deaths all over the world. Recently, a joint meeting of medical societies, the first ever in India was held to tackle the challenges of antimicrobial resistance in developing world (Ghafur 2013). As a result of this conference ''Chennai declaration'' came into existence, initiating efforts through a national policy to control the rising trend of AMR in India and abroad (Ghafur et al. 2012).
Multidrug-resistant (MDR) microbes are resistant to three or more antibiotics (Styers et al. 2006), however; strains of Mycobacterium tuberculosis, resistant to virtually all classes of antimicrobials have also been identified in the Kwa Zulu Natal Province of South Africa (Gandhi et al. 2006), a typical example of Extremely Drug-Resistant Tuberculosis (XDR TB) reported in 64 countries to date (World Health Organization 2011). The global emergence of MDRs is increasingly limiting the effectivity of the existing antibiotic drugs (Hancock 2005) for e.g. methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci spp. (Norrby et al. 2005). The development of resistance among the microbes is the result of continuous selection pressure of antibiotics and their surroundings causing genetic alterations (Bush 2004) which, are transferred to the next generation and reach out to the wider range of other geographical regions through the transfer of genetic information exchange between microbes (Amábile-Cuevas 2003) ( Table 1 presents the examples of some of the common MDRs). In this review, attempt has been made to understand specific issues such as factors causing resistance, the role of developing world with a quick overview of plant-derived antimicrobials (PDA m ) and synergistic compounds as an alternative drug line.

Factors causing AMR
Microbes comprise 50 % of total living biomass and are well-survived life forms on earth. There exists a sharp distinction between microbes as pathogenic and non-pathogenic although; one-way exchange of genetic elements (Amábile-Cuevas 2003) may confer the pathogenic characters to the non-pathogenic microbe. Pathogenic microbes cause infectious diseases in humans and animals and are treated with antibiotics. Antibiotics also known as antimicrobials are chemical substances, toxic for most of the life forms. Irrational and deliberate use of antibiotics, migration of infected individuals to other communities (Memish et al. 2003), prolonged use of medical health care systems in hospitals, hunger and malnutrition are some of the main causes of the development of resistance against antibiotics in the microbes (Byarugaba 2004;Vila and Pal 2010). Antimicrobial use in veterinary practices especially as food additives is one of the causes of development of AMRs in zoonotics that may spread to humans (Memish et al. 2003) through the food chain. In this connection, reports of Schlegelova et al. (2008) suggest, least chances of spreading of a resistant strain through the dairy products, however; improperly processed raw meat is strongly discouraged for human consumption in developed nations (Threlfall 2002).

Molecular understanding of AMR
Microbes attain resistance very rapidly against most of the currently available antibiotics because of the adaptability feature conferred by plasmids. Table 1 presents the examples of such plasmids carrying integron and gene cassettes in most common MDRs which on transfer, widespread the resistance (Kumarasamy et al. 2010). Gram-negative (Kumarasamy et al. 2010) and Gram-positive bacteria (Grohman et al. 2003) both exhibit conjugative transfer of plasmids, a natural way of horizontal gene transfer for e.g. the horizontal transfer of plasmid in between Vibrio fluvialis and Vibrio cholerae conferring resistance to V. fluvialis (Rajpara et al. 2009). Recent cases of AMR development include Pseudomonas aeruginosa and Acinetobacter baumannii resistant to nearly all antibiotics including the carbanems (Huang and Hsueh 2008). Antibiotic inactivation (degradation of antibiotics by the microbial enzymes e.g. transferase and b-lactamase) causes resistance in microbes (Wright 2005;Jacoby and Munoz-Price 2005), more than 1,000 such b-lactamases are identified till date (Bush and Fisher 2011). Different antibiotics have different mode of actions, therefore, their use is largely dependent on variety of traits other than resistance (Amábile-Cuevas 2010) which either undergo rapid enzymatic degradation or actively effused by the resistant bacteria. Efflux pump in MDRs was first described by Roberts (1996) for tetracycline and macrolide antibiotics. In general, efflux pumps act through membrane proteins of substrate specificity, effuse the antibiotics from the bacterial cell, resulting in a low intracellular ineffective concentration of the drug (Gibbons 2004;Thorrold et al. 2007) altering the permeability of membrane. In a study, staphylococcal accessory regulator (sarA) was reported to contribute promising role, imparting resistance in S. aureus (Riordan et al. 2006). In addition, Kuete et al. (2011) reported two efflux pumps viz., AcerAB-TolC (Enterobacteriaceae) and MexAB-OprM (Pseudomonas aeruginosa) imparting resistance in Gram-negative bacteria against natural products. AMR is a genetically-modified manifestation, linked to the point mutation in bacterial non-chromosomal DNA. As in case of MRSA, the resistance to methicillin is associated with acquisition of a mobile genetic element, SCCmec, which contains mecA-resistant gene (Okuma et al. 2002). Analytical procedure followed on Escherichia coli showed reversible function of class 1 integron integrase gene machinery under selective pressure (Díaz-Mejía et al. 2008). Similar results were also observed by Hsu et al. (2006) whereby E. coli MDR was found associated with the class 1 integron gene. Detailed mechanism of development of AMR among microbes has been extensively reviewed by Byarugaba (2010).

Developing world: the factory of MDRs
Developing world especially the countries of South East Asia, Western and Central Africa, India and Pakistan are the most vulnerable for various infectious pandemic diseases. Byarugaba (2004) comprehensively reviewed and reported the AMR in developing countries. Several factors are associated with the AMR development including nosocomial infections, unsafe disposal of biomedical waste, inappropriately used antibiotics, self drug abuse, shortfall of antibiotic course and lack of mass awareness of infectious diseases and personal hygiene (Okeke et al. 2005a, b). In addition to these, lack of surveillance data, providing information of microbial infections common to a geographic location and the invasive microbial species have been suggested as the major causes of MDRs development in developing countries (Okeke et al. 2005a, b;Giske and Cornaglia 2010;Kartikeyan et al. 2010;Lalitha et al. 2013). Giske and Cornaglia (2010) emphasized on the surveillance practices especially the monitoring and sampling techniques of invasive microbial isolates. Surveillance of resistance in many developing countries is suboptimal (Okeke et al. 2005b) and unable to present the real picture of infectious diseases and the medication. Recent reports of Lalitha et al. (2013) showed the feasibility of proper surveillance of resistance by carrying experimental surveillance study on the school children in different geographic locations of Indian subcontinent. In India for Salmonella typhi, MDR has become a norm in strains. This widespread resistant bacterium is associated with contaminated water supply in developing countries and through food products such as contaminated meat in developed countries (Threlfall 2002). Remarkable report of Kumarasamy et al. (2010) provides sufficient evidences in support of the positive role of developing world in the development of ARMs. Resistance to carbapenem conferred by plasmid encoded New Delhi metallo-b-lactamase-1 (bla NDM-1 ) is a worldwide health problem, especially in UK, (Kumarasamy et al. 2010) having the roots in India and Pakistan. The selective pressure on the bacterial cells is associated with the adaptations causing resistance among microbes for multiple antimicrobials for e.g. genes encoding NDM-1, OXA-23 and OXA-51 enzymes (hydrolyzing specific antibiotics) were observed in three different isolates of Acinetobacter baumannii in India (Kartikeyan et al. 2010). Alterations in gene structure were reported in A. baumannii as a result of selection pressure of antibiotics (Kartikeyan et al. 2010). The literature suggest, substandard surveillance of resistance, nonprescribed antibiotic usage causes huge selection pressure resulting in the development of AMR in developing countries and their suburbs (Byarugaba 2004;Okeke et al. 2005b;Kumarasamy et al. 2010). Figure 1 shows a schematic diagram showing the development of MDR microbe in community.

Plants derived antimicrobial (PDA m ): a ray of hope
Antimicrobial resistance is rapidly increasing along with the development of classical antibiotics consequently, there is an urgent need to develop a different drug line to treat and control MDR bacterial infections. Medicinal values of plants were known to earlier traditional medical practitioners (Emeka et al. 2012). PDA m substances are plantoriginated secondary metabolites and have great concern because of their antibiotic activity without conferring resistance (Baris et al. 2006;Palaniappan and Holley 2010).
PDA m s are classified as antimicrobial on the basis of dose ranging from 100 to 1,000 lg ml -1 for the minimum inhibitory concentration (MIC) susceptibility test performed on bacteria (Tegos et al. 2002).  (Table 3). Target specific plant's secondary metabolites having potential to treat and control the infections are being screened out globally for e.g. Coumarins having specificity on Staphylococcus aureus and ineffective on Gram-negative bacteria (Lewis and Ausubel 2006). The literature such as Cowan (1999); Lewis and Ausubel (2006) and González-Lomothe et al. (2009) provides comprehensive information on the major secondary metabolites of plant origin. Precise mechanistic approach of PDA m and their activity on microbes has been discussed by Lewis and Ausubel (2006). In general, PDA m s (mostly secondary metabolites) are phenol derivatives, sufficiently able to control microbes by reducing pH, increasing membrane permeability, altering efflux pumping. Examples mentioned in Table 2 followed by recent studies of (Machado et al. 2003;Ram et al. 2004;McGaw et al. 2008;Renisheya et al. 2011;Ahmed et al. 2012;Emeka et al. 2012;Upadhyaya 2013) and the references there in, suggest the antimicrobial potential of various local and exotic plant species, although very few reports have suggested the mechanism of their actions. The affectivity of PDA m s largely depends upon the extraction methods (Das et al. 2010 (2) (4) (5) Fig. 1 Illustrative sketch of the development of MDR microbes. The sketch is divided into various segments: (1) Bacterial infection was treated with calculated amount of antimicrobial drug (X) followed by complete cure, in the same time prolonged use of drug (X) put selective pressure causing point mutation (D). (2) Second infection (in a community only) was treated with same drug (X) with a higher dose, a delayed response was displayed because of mutant bacterial strain, (3) Third time infection (in a community only) trigger the resistance, in particular microbe for a particular drug (X); therefore, synergistic compounds (Y) were administered along with (X) may be for clinical trials, the successful treatment, leading to the production of new antimicrobial drug (Z), (4) Since the earlier bacteria attained resistance in due course of time for the drug (X) transferred the resistant gene into another strain of same species of bacteria resistant to the drug (Z) which was introduced in this community from the other one, gene cassettes got recombined on the plasmid to confer multi-drug resistant status to the new introduced bacteria. Infection caused by both these bacteria might be having same symptoms which would be treated with the newly developed drug (Z) keeping the resistance against (X) in consideration. (5) Infection could not be cured because the drug was applied to cure the (X) drug-resistant bacteria however; another bacteria having resistance against (Z) remained as such activity of all the extracts, however; the target specificity of plant extracts could not be established because of uncertain mechanism of plant-derived antimicrobial compounds. A generalized mechanism of PDA m s on microbes suggests the effects of efflux pumping on MDRs: increasing permeability and reduce selection pressure (Lewis and Ausubel 2006). Antimicrobial peptides (AMPs) are also produced by plants against the infections also called as defensins. Plant defensins are small basic peptides, having characteristic 3D folding pattern, stabilized by eight disulfide linked cysteines (Thomma et al. 2002). AMPs have antimicrobial properties too ) and have been suggested as an alternative approach to improve treatment outcome (Brouwer et al. 2011), for e.g. IbAMP1, a plant originated disulfide linked b-sheet antimicrobial peptide (Wang et al. 2009).

Synergistic actions of PDA m s
The AMR is conferred by several factors which have already been reviewed in previous sections. Plasmid encoded resistance facilitate bacterial cells to develop resistance of various degrees. For instance, unlike Grampositive, MDR Gram-negative bacterial species have developed a sophisticated permeability barrier as outer membrane comprised of hydrophilic lipopolysaccharide restricting the entry of hydrophobic (quinones and alkaloids) and amphipathic antibiotic compounds (Lewis and Ausubel 2006). The biased effect of PDA m s on Grampositive and -negative species has been a key to the discovery of the synergistic compounds of plant origin (Lewis 2001). Plant antimicrobials act well in combinations with other amphipathic compounds. In addition to this, resistance in MDRs conferred by efflux pumping can be treated with the synergistic combinations of antimicrobial with an efflux pump inhibitor (EPI) and altering outer membrane permeability of MDR bacteria providing an effective drug (Savage 2001;Gibbons 2004;Baskaran et al. 2009). Studies of Chusri et al. (2009) reported another example of synergistic effect of plant-derived phenolics such as Ellagic acid (a derivative of Gallic acid) a non-antimicrobial, administered as EPI in combination with classical antibiotic to control Acinetobacter baumannii. Another example belongs to the well-studied plant Berberis fremontii and its amphipathic cation berberine inhibits the NorA MDR pump of Staphylococcus aureus when applied in combination with 5 0 -MHC (5 0 -methoxyhydnocarpin, an amphipathic weak acid) a real inhibitor of the pump enhancing the activity of berberine (Stermitz et al. 2000). Similar nonantimicrobial compounds known to enhance effectivity of antimicrobials have been discussed by Lewis (2001).
Detailed mechanism of PDA m s on MDR S. aureus has been discussed in the review by Gibbons (2004). Wang et al. (2009) defined that the role of AMP plant defensin Ib-AMP1 isolated from plant Impatiens balsamina have a prime target, intercellular components, forming small channels that permit the transit of ions or protons across the bacterial membrane, the same activity was also observed in the linear analogs of this peptide.

Future studies
Researches on the AMR and alternating drug system are endless and a lot of scope is there in the field of ethnopharmacology. Scientists are working on the development of Chemical structure given in front of corresponding group of antimicrobials is not to be considered as generalized one, the references are in correspondence with bacteria safe and effective antimicrobials all over the world. Future studies may involve the development of new plant-derived synergistic compounds capable of enhancing the activity of PDA m s. A lot of research potential is also there to answer the questions for e.g. mechanism of resistance in different bacterial species, development of XDRs and their control.

Conclusion
AMR is a worldwide problem. Research literatures suggest that the substandard living in major parts of developing world is one of the major causes of the development of resistance among bacteria. The developed world is also vulnerable of getting widespread infections for e.g. USA is surrounded by the developing countries having high rates of resistance development. Nosocomial, water borne, health care systems and food products especially meats are some of the most common means of widespread of resistant gene globally. Thanks to the modern molecular approaches for making better understanding of the pathways of resistance development and its remedy. Pharmacologists are developing new antibiotic drugs to treat and control various infections, however; the chances of the development of resistance are equal to the emergence of new drugs. In addition, research suggest that the combinations of PDA m s and the synergistic compounds work efficiently on resistant strains ensuring no further resistance development. Moreover; concerted efforts have been solicited by the world community because poor countries are worst affected by the antimicrobial resistance and the developed countries are no longer safe (Diáz-Granados et al. 2008). In this regard, PDA m s in combination with plant-derived synergistic compounds may be the cost-effective approach to deal with global antimicrobial resistance.