Herbal Compounds-An Alternative for Multi-Drug Resistant Vibrio Cholerae

Corresponding Author: Hotam Singh Chaudhary Department of Biotechnology, Madhav Institute of Technology and Science Gwalior, RGPV, India Email: hotamsingh@gmail.com Abstract: Vibrio cholerae is a causative agent of cholerae, many people dies every year, especially in developing countries around the world. The outbreaks of cholera are responsible for approximately 120,000 deaths annually. Cholera is a self limiting illness; however antibiotics are used as a part of treatment regimen. But at present, the treatment against cholera has become very critical issue worldwide, because most of the strain developed multidrug resistance. Efflux pumps, spontaneous chromosomal mutation, conjugative plasmids, SXT elements and integrons are discussed as an antibiotics resistant mechanism. Now at present the demand is to find an alternative and promising strategy and development of novel therapeutics. The present chapter is mainly focus on the treatment, strategies and developing resistance against these antibiotics. Later section mainly focused on the utility of natural remedies against V. cholerae infection.


Introduction
V. cholerae, a member of the family Vibrionaceae is a facultative anaerobic, Gram-negative, non-spore-forming curved rod, about 1.4-2.6 mm long, capable of respiratory and fermentative metabolism; it is well defined on the basis of biochemical tests and DNA homology studies (Baumann et al., 1984). Discovery of V. Cholerae is credited to Filipo Pacini, who first time describes V. cholerae and also made microscopic slide first time. V. Cholerae is classified by the heat-stable surface somatic "O" antigen, present in the outer polysaccharide layer. This classification was firstly described by Gardner and Venkatraman (1935). Presently the organism is classified into 206 "O" serogroups (Shimada et al., 1994;Yamai et al., 1997). Until recently, epidemic cholera was exclusively associated with V. Cholerae strains of the O1 and O139 serogroups. The O1 serogroup exists as two biotypes, classical and El Tor; antigenic factors allow further differentiation into two major serotypes-Ogawa and Inaba. The cholera was originated in the India subcontinent, it has been prevalent in the Ganga Delta from ancient times (Sack et al., 2004). The first cholera pandemic occurred in India in Bengal region starting in 1817 through 1824. The second pandemic lasted from 1827 to 1835 and the affected countries were United States and Europe due to the results of advancement in transportation and global trade and increase human migration. The third pandemic began in 1839 and persisted until 1856, extended from North America to south America for the first time specially Brazil. During fourth pandemic from 1863 to 1875 cholera hit the sub-Saharan African region. During 1881-1896 and 1899-1923 the fifth and sixth pandemic occurred. Seventh pandemic originated in 1961 in Indonesia and is marked by the emergence of new strain named E1 Tor which still persistent in developing countries (Aberth, 2011). The pandemic stages emerged because of the resistance to antibiotic. The strains of V. cholerae became multi-drug antibiotic resistant.

Pathogenicity for Human and Virulence Factor
The genes that enable a strain to infect and cause disease are called virulence genes and the proteins they encode are called virulence factors. Many of the virulence genes in V. Cholerae are located in so-called Pathogenicity islands (PAIs). In V. cholerae these are called 'V. cholerae Pathogenicity Islands' (VPI-I and VPI-II) and 'Vibrio Seventh Pandemic islands' (VSP-I and VSP-II). These PAIs have been identified by analysis of the G+C content along the genome. The PAIs usually have a lower G+C content than the surrounding DNA. This indicates that these sequences have been acquired by horizontal gene-transfer, by mechanism similar to those whereby drug-resistance genes are being exchanged. Analysis of the flanking regions indicate that transduction by temperate bacteriophages are a likely source (Dziejman et al., 2002;Karaolis et al., 1998). In order to cause cholera the strain must carry the temperate phage CTXφ, encoding the Cholera Toxin (CT). The existence of cholera enterotoxin was first given by Robert Koch in 1884 and demonstrated 75 year later by Dutta et al. (1959) working independently. Structural analysis of toxin showed it to consist of a subunit and 5 smaller identical B subunit (Finkelstein and LoSpalluto, 1969). The A subunit has a specific enzymatic function and acts intracellularly. It raises the cellular level of cAMP and thereby altering the net absorptive tendency of small intestine to one of net secretion. The B subunit binds the toxin to eukaryotic cell receptor ganglioside GM1. In toxigenic V. Cholerae O1 and O139 has a dynamic 4.5kb core region, called as a virulence cassette but not found in non-toxigenic strains (Trucksis et al., 1993). This virulence cassette carry atleast six genes including ctxAB (encoding the A and B subunits of CT), zot (encoding zonula occludens toxin (Fasano et al., 1991)), cep (encoding core-encoded pilin (Pearson et al., 1993)), ace (encoding accessory cholera enterotoxin (Trucksis et al., 1993)) and orfU (encoding a product of unknown function (Trucksis et al., 1993)). The two major virulence factors in V. cholerae are Cholera Toxin (CT) and Toxin Co-regulated Pili (TCP). TCP is a type IV pilus required for intestinal colonization (Rhine and Taylor, 1994). They cause the bacteria to aggregate in crypts of the small intestine but they not involved in adhesion of epithelial cell. CTXφ phage also gets attached to TCP. The genes for TCP formation (tcpA-F) are located in VPI-1 Pathogenicity Island and the CT gens (ctxAB) are located on CTXφ phage and they both are under control of transcriptional activator ToxT.

Treatment
Earlier lytic bacteriophages were used for the treating the cholera when the molecular biology and antibiotics not discovered in India in late 1920s and early 1930s Summers, 1993. Oral Rehydration Salt (ORS) is primarily recommended for the treatment of cholera. In severe cases antibiotics are used to treat cholerae either in combination with ORS. Many antibiotics used against V. cholerae such as doxycycline, cotrimoxazole, erythromycin, tetracycline, chloramphenicol, furazolidone, etc. Doxycycline is used as first line treatment in adults. Whereas azithromycin is recommended as first-line treatment for children and pregnant women. Tetracycline, chloramphenicol these antibiotics inhibit the protein synthesis in bacteria (Franklin and Snow, 2005). Ciprofloxacin (1g orally in 1dose) and doxycycline (300 mg in 1dose) are the antibiotics choose for adult except pregnant women.
The reason behind the increasing drug resistant is undoubtedly the immense amount of antibacterial drug used in the treatment of humans and animals worldwide. It's now became necessary to minimize the unnecessary use of antibacterial drugs, in order to control the resistance. However now the need is to find a renewed antibiotics and new drug targets. It became resistant through various mechanism like exporting drugs through efflux pump, chromosomal mutation or developing genetic resistant via the exchange of conjugative plasmids, conjugative transposons, integrons or self transmissible chromosomally integrating SXT elements (Kitaoka et al., 2011).

Bacterial Efflux Pumps
Efflux pump used by V. cholerae to export a broad range of antibiotics, detergents, dyes that are structurally and chemically unrelated (Paulsen et al., 1996). VcaM, a V. cholerae ABC (ATP-binding cassette) multidrug resistant pump is a ATP-driven pump. It confers resistant to structurally divergent drugs (e.g., tetracycline, norfloxacin, ciprofloxacin and doxorubicin). V. Cholerae uses an array of MATE (multidrug and toxic compound extrusion)-family efflux systems, namely VcmB, VcmD, VcmH, VcmN, VcmA and VcrM (Begum et al., 2005;Huda et al., 2003). MFS transporters in V. cholerae include the V. cholerae efflux systems (Colmer et al., 1998) that confer resistance to bile (deoxycholate), antibiotics (e.g., chloramphenicol and nalidixic acid) and the proton gradient-uncoupling agent carbonyl cyanide mchlorophenylhydrazone (Colmer et al., 1998;Woolley et al., 2005). Recently shown that the classical O395 strain carries the MFS efflux protein EmrD-3, which confers resistance to linezolid, rifampicin, erythromycin and chloramphenicol when expressed in a drughypersensitive Escherichia coli strain (Smith et al., 2008). Collectively these results show that the efflux pump is exclusively employed in drug resistant.

Spontaneous Mutations
Mutation in bacterial chromosomes can also be a reason for antibacterial drug resistant. It has found that mutation cause resistant to the cell wall biosynthesis inhibitor alafosfalin and to the DNA replication inhibitor family of quinolones in V. cholerae (Allen et al., 1979;Gellert et al., 1977;Goss et al., 1965;Sugino et al., 1977). From comprehensive study it was found that during 1980s 0 cholera epidemic in the United Republic of Tanzania, the rate of mutation in V. cholerae genes is higher than the E. coli genes. This facilitates the resistance to antibiotics such as alafosfalin . Chromosomal mutation in genes gyrA and parC which encodes subunits of DNA Gyrase and topoisomerase IV, respectively, a resistant against quinolones is developed in V. cholerae. There are various reports documented the multi-drug resistance in V. cholerae including the antibiotics tetracycline, erythromycin, chloramphenicol, quinolones, streptomycin and cotrimoxazole (Abera et al., 2010;Das et al., 2008;Islam et al., 2009;Karki et al., 2010;Ngandjio et al., 2009;Ranjbar et al., 2010;Roychowdhury et al., 2008;Kumar et al., 2014;2012).

SXT Elements and Integrons
The SXT elements was first described in V. cholerae serogroup O139 based on its ability to harbor genes which provide the host bacterium with resistant to sulfamethoxazle, trimethoprim and streptomycine (Waldor et al., 1996). Beaber et al. (2004) has found that the horizontal dissemination of SXT-encoded antibiotic resistant genes is regulated by bacterial SOS response. Further research demonstrated that stress alleviates the SXT-encoded repressor setR, which in turn activates excision and conjucation of the elements. Ciprofloxacin act as an inducing molecule that can promote horizontal transfer of SXT elements. These results suggest that the antimicrobial agents can promote the spread of antibiotic resistant genes. SXT elements have capacity to mobilize conjugative plasmids and genomic islands in trans (Daccord et al., 2010;Hochhut et al., 2000), providing an alternative mechanism for antibiotic resistant gene transfer. All V. cholerae isolates facilitates the large chromosomal integrons that provide them the capacity to rapidly transfer gene cassettes containing antibiotic resistant genes (Mazel, 2006).

Conjugative Plasmids
Many V. cholerae strains are identified which developed resistant against tetracycline antibiotics, an oral drug often given to patient during rehydration therapy (Greenough et al., 1964). First reported tetracycline resistant strain (exhibiting resistant to tetracycline, streptomycin and chloramphenicol) was isolated in the Astrakhan region of the USSR circa 1970 (Kitaoka et al., 2011). This resistance was transferable to E.coli K-12 and thses strains carry a single plasmid. Similarly during cholera outbreak in Bangladesh in 1970 it was caused by the strain which carried a multi drug resistant plasmid transferable through conjugation with other bacteria, including E.coli (Glass et al., 1980). This plasmid showed resistance to a no. of antibiotics in addition to tetracycline including ampicillin, kanamycin, streptomycin, gentamicin and trimethopim.
These are the method by which the strains of V. cholerae became resistant to antibiotics. Now the main target of present day is to find an alternative for these diseases.
Herbal Plants-good antimicrobial Activity: There are many herbal plants presents in the nature which has high medicinal plant. These plant can be use as a source of antimicrobial compounds which can be use to kill the Pathogenicity of various pathogenic bacteria. There are various plant reported which showed antimicrobial activity. In historical times, traditional therapeutics used to treat the infection caused by V. cholerae from various medicinal plants. The active compounds present in natural compound can be used to treat V. cholerae by various pharmacologic mechanisms. Some compound shows direct antimicrobial activity against V. cholerae and some inhibit the binding of CT to GM receptors at epithelial cell surface. On this mechanism many herbal compounds identified against V. cholerae.
• Neem: This is the most important and ancient medicinal plant of India who's each part has some medicinal value. Neem oil suppresses growth of several species of pathogenic bacteria such as S. aureus, S. typhosa (Chaurasia and Jain, 1978) V. cholerae (Kunin, 1993) • Green Tea: there are many compounds found in green tea which show antimicrobial property. Extensive research on catechin showed that it inhibits the growth of vibrio cholarae (Borris, 1996). • Allium cepa: Abdul Hannan et al. (2010) showed that Allium cepa has antimicrobial property and it inhibit the growth of V. cholerae. They found that the antimicrobial activity of purple type of allium cepa extract was better as compared to yellow Allium cepa axtract • Indian Species: Praveen Singh et al. (2013) Kalirajan et al. (2012) studied that the antimicrobial activity of methanol and aqueous extract of herbal plant cocculus hirsutus using E. coli, V. cholerae, staphylococcus aureus, etc. they found that the aquous and methanol extract of plant is more effective against V. cholerae and staphylococcus aureus • Psidium guajava: Also known as "goiabeira" found to have antimicrobial property. Rahim et al. (2010) observed the antimicrobial activity of psidium guajava taking its leaf and bark against V. cholerae and suggested that nature of its bioactive component is nonprotiec • Garlic Extract: Researchers found that galactan polysaccharide a bioactive compound present in garlic extract as a mojor anti-choleric component. Politi et al. (2006) repoted the inhibitory property of galactan against B-subunit of CT In this context many more researches has been done and reported many nautral compounds against vibrio cholarea. Polyphenol extract of apples shows good anticholeric properties. They inhibit the enzymatic activity of a subunit of CT (Saito et al., 2002). The therapeutics component present in apple is chlorogenic acid, phloridzin, phloretin, caffeic acid and p-coumaric acid, monomeric Catechins, procynidine. Similar study conducted by Hör et al. (1995) proanthocyanidines extracted from Guazumaulimfolia, a medicinal plant present in Mexico, can provide in vivo inhibitory properties against cholera toxin. Oi et al. (2002) reported the pharmacological properties of rhubarb galloyl tannin (RG-tannin), an active compound isolated from Rhei rhizome (Rheum palmatum), against CT including ADP-ribosylation and fluid accumulation. Studies conducted on animal models (rabbit and mouse) indicated the heterologus polyphenol gallate inhibit fluid accumulation induced by CT. Chatterjee et al. (2010) reported the bioactive component present in red chilli showed an inhibitory effect against V. cholerae. Further study found that the bioactive compound capsaisin present in red chilli act as antimicrobial agent against many pathogenic bacteria V. cholerae, bacillus sps. Etc.
Similarly some more compounds found from in-silico and in-vitro studies which are inhibiting the growth of V. cholerae. A study in mice reveals that the aqueous and etholic extract of leaves of spondias mombin and Senna occidentalis and stem sap of Musa sapeintum against two epidemic strain of V. cholerae O1(BA O1 and CVC O1) could be a good alternatives in the treatment of cholera (Shittu et al., 2014). They found in the in vivo studies that the intestinal sample of mice showed mild loss of villi at lower dosages regime and at higher dosages no lesion were observed compare to control groups. This study also suggests that the aqueous extract of spondias mombin and Senna occidentalis can be an alternative for the treatment of epidemic Cholerae.

Conclusion
Since antibiotic is widely used for the regime of cholera, the number of pathogenic strain of V. cholerae resistant is increasing, as summarized in Table1. V. cholerae is an environment organism; it means it has ability to acquire resistance genes from intimate contact with intrinsically resistant environment bacteria (Martinez, 2008) through mobilizable genetic elements. V. cholerae can share these resistant genes with other bacteria (Mekalanos et al., 1997;Sedas, 2007) in human gut. To prevent this resistance it is require limiting the use of antibiotics to cholera patient.
The milestone of cholera treatment is to develop a vaccine for the children. Vaccine will prevent the cholera effectively in comparison to other treatment. However, in spite of some recent advances in understanding of host-pathogen interactions, molecular mechanism underlying pathogenesis of V. cholerae, still such a vaccine yet not developed (Provenzano et al., 2006).
Another promising approach is to discover the therapeutics by selecting new targets. The drugs that disable the bacterium by inhibiting their virulence mechanism (Hung et al., 2005). Transcriptional activator ToxT, ToxR and TcpP required for the synthesis of cholera toxin and TCP represents a promising target. Because the expression of cholera toxin and TCP is control by transcriptional activator ToxT, if a drug inhibit this target both the virulence factor of V. cholerae will not be expressed.
The most promising and significant approach will be the natural compounds. There are many compounds present in the herbal plants which have high medicinal value and these could be used as therapeutics alternatives for these resistant bacteria (Chomnawang et al., 2009). Recent studies found that there are some herbal plants which have some bioactive compound that can inhibit the growth of V. cholerae. Arjun extract of plant Terminalia arjuna inhibited that growth of V. cholerae (Fakruddin et al., 2011). This study reveals that Terminalia arjuna would be a good antibacterial drugs in the treatment of V. cholerae infection, provided if found effective and non-toxic through in vivo studies. Now many of herbal compounds have been found till date, but to find the most potential and drug-like property still remains. From this entire compound to found the most potent compound we can use in-silico method. Computer aided method is an easy and a preliminary steps to screen the novel therapeutics agents and the discipline is an emerging strategy as it reduces many complexities of drug discovery process. By using in-silico method we can found a potential drug for V. cholerae and their effectiveness can also be checked by in-vivo studies. This can be a better alternative to found an alternative therapeutics against V. cholerae infection. In-silico reduces the complexity and time as compared to traditional method of drug designing. However until these approaches will successfully implemented till we have to fall back on three key principles in managing this potentially deadly source: Clean water supplies, contaminant of cholera patient to stop transmission and use of oral rehydration therapy with antibiotics.