Vibrio cholerae

Mobashar Hussain Urf Turabe Fazil1 & Durg V Singh†1 Infectious Disease Biology, Institute of Life Sciences, Nalco Square, Bhubaneswar–751023, Orissa, India Author for correspondence: Tel.: +91 674 230 2754 n Fax: +91 674 230 0728 n [email protected]

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Vibrio cholerae is the causative agent of the diarrheal disease cholera. Although antibiotic therapy shortens the duration of diarrhea, excessive use has contributed to the emergence of antibiotic resistance in V. cholerae. Mobile genetic elements have been shown to be largely responsible for the shift of drug resistance genes in bacteria, including some V. cholerae strains. Quorum sensing communication systems are used for interaction among bacteria and for sensing environmental signals. Sequence analysis of the ctxB gene of toxigenic V. cholerae strains demonstrated its presence in multiple cholera toxin genotypes. Moreover, bacteriophage that lyse the bacterium have been reported to modulate epidemics by decreasing the required infectious dose of the bacterium. In this article, we will briefly discuss the disease, its clinical manifestation, antimicrobial resistance and the novel approaches to locate drug targets to treat cholera.

Vibrio cholerae is the causative agent of the diarrheal disease cholera, which can be transmitted after consuming contaminated water and food. The most distinct salient feature of cholera is its epidemiologic behavior, which tends to cause explosive outbreaks. Toxigenic serogroups of V. cholerae O1 and O139 have been reported to cause epidemic cholera, whereas non-O1 and non-O139 V. cholerae are known to cause sporadic diarrhea and extraintestinal infections [1] . Mobile genetic elements such as plasmids, phages, transposons and integrons play a crucial role in the horizontal transfer of genes in bacterial populations. Integrative conjugative elements (ICEs) share a set of genes performing specific functions, such as excision, transfer, and integration, but differ in genes that are not essential for transmissibility [2] . In V. cholerae, antibiotic resistance determinants have been found to be associated with Class I integrons and the SXT element. With a rise in multidrug-resistant (MDR) V. cholerae strains it has become necessary to make a concerted effort to understand and devise mechanisms to counter the increasing repertoire of drug resistance in this organism. Herein, we provide an overview of cholera, its clinical manifestation, antimicrobial resistance and finally discuss the emerging insights into novel approaches to find drug targets and phage therapy to treat the disease. Cholera gravis

Cholera gravis, caused by toxigenic strains of V. cholerae, has been reported to infect populations throughout the world, and is endemic in regions of Asia and Africa. Approximately 3–5 million cholera cases have been reported per 10.2217/FMB.11.93 © 2011 Future Medicine Ltd

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Vibrio cholerae infection, novel drug targets and phage therapy

annum, with a mortality rate of 2–3% that could increase if the disease is not treated in time [101] . The severity of the infection depends on several factors including local intestinal immunity, size of the inoculums, adequacy of the stomach gastric acid barrier, and the patient’s blood group. A high infectious dose (108 bacteria) caused severe cholera in healthy volunteers when compared with a much lower dose (105 bacteria) when administered with antacids [3] . The bacteria that survive after passing the stomach acid barrier reach the intestine where they adhere, colonize and produce cholera toxin (CT), ultimately causing symptoms of cholera. The organism survives better in aquatic environments, which serve as reservoirs for transmission of the disease. V. cholerae with biofilm shed in human stools appears to be another efficient strategy for the spread of the disease and survival of pathogen in aquatic environment [4] . A large number of asymptomatic human carriers harbor V. cholerae. This is primarily because of the ability of the bacterium to avoid clearance and persist in the gut via prolonged colonization. A recent study has demonstrated the role of secreted accessory toxins in the colonization at the carrier stage, and this has been linked to the spread of the disease [5] . Clinical manifestations

The most distinctive feature of cholera is the painless purging of voluminous stools resembling rice water with a fishy odor. The rate of purging may quickly reach up to 500– 1000 ml/h, leading to acute dehydration [6] and is characterized by the absence of, or low volume, peripheral pulse, undetectable blood Future Microbiol. (2011) 6(10), 1199–1208

Keywords antibiotic resistance biofilm n drug target n quorum sensing n V. cholerae n vaccine n n

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pressure, poor skin turgor, sunken eyes and wrinkled skin. The patients first become restless and extremely thirsty, but later become apathetic and may lose consciousness as shock progresses [7] . The rapid fluid loss may further the risk of death within a few hours of onset of the disease. Most deaths occur during the first day of the establishment of the disease; however, the patient would survive if rehydration fluids are provided in time and in sufficient quantities. Prevention, treatment & vaccine

Cholera can be prevented by achieving a clean drinking water supply and improving sanitation. Community or mass chemoprophylaxis has no effect on the spread of cholera; rather it leads to the emergence of MDR [102] . The key to therapy is provision of adequate rehydration either by intravenous infusion of fluid or by oral rehydration therapy [8] . The disease may continue usually for 1–5 days in the absence of antimicrobial therapy. Doxycycline is the drug of choice, but tetracycline and other antibiotics (e.g., erythromycin, co-trimoxazole, ciprofloxacin and azithromycin) are also used for treatment [9] . Efficient protection against cholera infection depends on the composition of a vaccine containing biotypes of V. cholerae. It is known that classical biotype of V. cholerae provides selective protection against classical biotype strains and the El Tor strains provide protection against El Tor strains [10] . Naturally acquired immunity has been reported to last for at least 3 years; however, longer immunity would be possible depending on the individual host genetic makeup. Antigenic components of the pathogen that contribute to the protective immunity include CT, lipopolysaccharide, flagella, fimbriae and outer membrane proteins. Although cholera is a toxin-mediated disease, the predominant protective immune mechanism appears to be antibacterial rather than antitoxic [11] . Antibodies against lipopolysaccharide have been found in immunized individuals with V. cholerae O1 or from convalescent patients. The role of lipopolysaccharide in protective immunity has been demonstrated [12] , but the contribution of other components of this bacterium in defense has not been clearly identified. The vaccine Dukoral® (SBL Vaccine, Sweden), which consists of nonvirulent cells of toxigenic V. cholerae of both biotypes and an inactive CT B subunit (CTB), is one of the vaccines recommended and registered by the WHO for vaccination. The vaccine is given in two separate doses within 1–6 weeks and stimulates antibacterial 1200

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and antitoxic immunity [13] . Both live and killed vaccines of cholera have undergone field trials [103] . Parenteral whole cell killed vaccine, oral whole cell killed with CT B subunit and recombinant B subunits were popular. Live oral cholera vaccines containing the CVD103HgR strain were earlier used in endemic areas, but later discontinued [14] . Two oral cholera vaccines (Dukoral and Shanchol [Shantha Biotech Ltd, India]) are the ones promoted by the WHO for cholera vaccine initiatives [14] . However, there exist reports on certain adverse effects against this cholera vaccine as well [14] . A study evaluating the effects of oral cholera vaccine (whole cell killed with CTB subunit) on HIV-infected patients revealed that the vaccine increased viral load in the plasma of patients [15] . Furthermore, thiomersal, a complex salt-containing mercury, which is used as stabilizer in certain vaccine preparations has been reported to cause adverse reactions [16] . The most important drawback, however, would be the cost of the vaccine. The present cost is beyond the consumer capacity for prophylaxis programs in endemic regions because of very low income per capita in these regions [17] . Shanchol, the newly licensed lowcost vaccine, developed for use in choleraaffected countries, can join Dukoral on the WHO’s list of prequalified cholera vaccines to be used in developing countries. Antibiotic resistance & SXT

Antibiotic resistance and cholera infections are complementary to each other. Antibiotic susceptibility tests of O1 strains isolated from the 1979 outbreak in Bangladesh demonstrated resistance to tetracycline, in addition to other antibiotics; ampicillin, kanamycin, streptomycin and cotrimoxazole (trimethoprim–sulfamethoxazole). The subsequent follow-up study of the epidemiological assessment of the outbreak indicated that the O1 isolates were not resistant to tetracycline, streptomycin, chloramphenicol, amoxicillin or nalidixic acid. All the classical V. cholerae strains isolated in Bangladesh between 1988 and 1989 were resistant to tetracycline, whereas strains belonging to the El Tor biotype were sensitive to the drug [18] . Almost after a decade, tetracycline-resistant El Tor strains reemerged in Bangladesh in 1991 and in Tehran in 1998 [18,19] . In October 1995, emergence of nalidixic acid-resistant O1 V. cholerae El Tor strains were reported in Southern India [20] . Moreover, 80–100% of V. cholerae O1 isolated in Kenya, South Sudan, Peru and Guinea-Bissau and 65–90% of isolates from Somalia, isolated future science group

Vibrio cholerae infection, novel drug targets & phage therapy

between March 1994 and December 1996, were not resistant to tetracycline [18] . On the other hand, all the O1 isolates from Tanzania and Rwanda were resistant to this drug [18] . A number of V. cholerae O1 strains isolated between 1992 and 1997, and in 2011 in Kolkata showed resistance to tetracycline, as well as ampicillin, chloramphenicol, co-trimoxazole, neomycin, streptomycin, and emerging resistance to nalidixic acid [21,22] . Antibiotic susceptibility tests of V. cholerae O1 strains isolated from Kottayam, Alleppey and Trivandrum, Southern India, in 2000 and from East Delhi between 2004 and 2006 showed resistance to nalidixic acid, and/ or neomycin and/or streptomycin, respectively, and sensitivity to tetracycline suggested existence of different R-types of V. cholerae strains in different locations [23,24] . Continuous surveillance of the outbreak of cholera in Kerala, Southern India, showed that the emergence of nalidixic acid-resistant strains of V. cholerae O1 in 2002. After the extensive cholera outbreaks caused by V. cholerae O139, V. cholerae O1 El Tor strains re-emerged in 1994 as the predominant cause of cholera in the Indian continent. In contrast to El Tor strains isolated before the O139 outbreak, the re-emerged El Tor strains, such as the initial O139 isolates, were resistant to furazolidone, sulfamethoxazole, trimethoprim, chloramphenicol and streptomycin [25] . The corresponding genes were found to be located in an ICE. The SXT is a 62 kb conjugative, self-transmissible integrating element encoding resistance to sulfamethoxazole, trimethoprim, chloramphenicol and streptomycin [26] . This element was first detected in the newly emerged O139 serogroup of V. cholerae in 1992 [26] . Since 1994, V. cholerae O1 strains isolated from India, Bangladesh, Mozombique, and Laos were found to contain the SXT [2,27,28] . In El Tor V. cholerae strains, the resistance genes are located in the SXT element. Variations were noted in recent V. cholerae O139 isolates from India, which contain SXT but demonstrated varying resistance to streptomycin but sensitivity to sulfamethoxazole and trimethoprim [22,29,30] . Bacterial mobile genetic elements (MGEs), such as bacteriophages, transposons, conjugative plasmids, integrons and ICEs harboring drug-resistance genes have been shown to be largely responsible for the shift of drug resistance genes in bacteria including V. cholerae O1 and O139 serogroups [2,26,27,31–33] . Integrons are gene-capturing systems incorporating exogenous open reading frames by future science group

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site-specific recombination. This is mediated by a tyrosine recombinase, converting the open reading frames to functional genes by ensuring their correct expression [34] . The recruitment of exogenous genes is the most efficient means by which bacterial species can survive various environmental challenges, including exposure to antimicrobial compounds. However, with the discovery of superintegrons, and the thousands of gene cassettes associated with integrons in the genomes of environmental bacterial species, the importance of these elements clearly extends beyond the phenomenon of antibiotic resistance. Experimental and phenotypic data suggest that superintegrons could be the ancestor of both mobile integrons and the resistance gene cassette observed in the genome of bacterial strains of clinical significance [34] . Integrative conjugative elements integrate into the host genome in a site-specific manner like bacteriophages, disseminate to new hosts via conjugative transfer like conjugative plasmids [2] , and confer resistance to antibiotics and heavy metals. The first ICE discovered in V. cholerae O139 strains was the SXT element [26] . The SXT possessed by V. cholerae O1 strains (designated ICEVchInd1) differed from those of V. cholerae O139, which possess a trimethoprim resistance determinant distinct from other antibiotic resistance genes [2] . Subsequently SXT has been detected with or without drugresistance genes among V. cholerae isolated from Mexico, Laos, Vietnam, Mozambique, South Africa [2] and China [35] . Interestingly, factors promoting the spread of SXT among bacteria have not been clearly established. However, the SOS response has been shown to induce horizontal spread of SXT in a manner similar to the lytic life cycle of the bacteriophage-g [36] . SXT elements show considerable variation with respect to drug resistance genes, suggesting genetic rearrangement in these elements. V. cholerae isolated before the emergence of the O139 serogroup rarely possessed SXT [2] , indicating that SXT may have not been an integral part of the V. cholerae genome. Hence, the origin and acquisition of SXT by V. cholerae may provide insight into the evolution of SXT and acquisition of drug resistance. Although antibiotic therapy shortens the duration of diarrhea [37] , use of antibiotics has contributed to the emergence of resistance in V. cholerae. V. cholerae strains resistant to multiple antibiotics have been found [38] . The resistance pattern is dependent on the source of isolation and the geographical area. Knowledge-based www.futuremedicine.com

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treatment of local antibiotic-resistant strains was carried out earlier for prophylaxis [38] . Furazolidone has been widely used for treatment of cholera. In cases where furazolidone-resistant strains were reported, erythromycin and azithromycin were provided to treat V. cholerae infection [38] . Similarly, ciprofloxacin was used in the treatment of the disease where tetracycline proved ineffective [39] . However, studies on treatment of cholera patients and carriers of vibrios with different antimicrobials showed that bacterial relapses occurred in subjects treated with chloramphenicol, tetracycline and combinations of chloramphenicol and tetracycline [39] . The rate of secondary infection was very high with MDR strains [39] . It is known that major parts of antibiotics used for the treatment of humans are not metabolized, which then added to the environment, and pour antibiotics and their residues in aquatic niches [40] . There is increasing concern over such abuse of antibiotics that could lead to toxicity in the environment and promote rapid development of resistance, disrupting public health [40] . Novel avenues for drug targeting

The antibiotic resistance in bacteria broadly occurred through: mutations in genes, efflux pumps and horizontal gene transfer. All three mechanisms have been found to play a role in the acquisition of drug resistance and emergence of MDR in V. cholerae strains. Cumulative mutations in the quinolone targets leads to high rate of flouroquinolone resistance in V. cholerae strains in endemic areas [41] . At least six multidrug efflux proteins that keep a variety of structurally and functionally distinct antimicrobials at bay have been reported in non-O1 V. cholerae strains [39] . Although environmental V. cholerae usually do not contain plasmids, transfer of the MDR phenotype from V. cholerae to other Enterobacteriaceae members has been shown by conjugation [2] . Integrons are one of the principal apparatus for incarceration and propagation of antimicrobial resistance genes among Gramnegative bacteria [31] . Later V. cholerae O139 strains possessing the SXT element devoid of antibiotic resistance genes were reported to have caused cholera outbreaks [2] . Variable antibiotic resistance, including MDR, has been reported among V. cholerae strains [42] . Therefore, there is a need to search for novel avenues for drug targets and new designs for vaccines to prevent spread of the disease. Thus, various new methods have been looked upon to either design vaccines, and 1202


new methods of drug targeting or intervention in the causation and spread of the disease [43] . Therefore, we will discuss approaches that need to be considered for the exploitation of new targets and therapy that could be of utmost interest in the prevention and spread of cholera. Targeting bacterial signal transduction pathways

Survival of bacteria relies on the integration of multicellular responses and acclimatization to changes that occur in the environment through cell–cell communication in the process known as quorum sensing (QS). Interestingly QS in V. cholerae is accomplished through the synthesis, release and subsequent detection of signaling molecules called auto-inducers, which control virulence gene expression and biofilm formation [44,45] . At least three distinct sensory pathways are known to effect the regulation of virulence gene expression, biofilm formation and protease production [46] . QS system-1 is composed of the cholera autoinducer-1 (acyl homoserine lactone) and a two-component sensor kinase CqsS. System 2 is composed of autoinducer (AI)-2 (a furanosyl borate diester AI), the periplasmic binding protein LuxP and the two-component sensor kinase LuxQ [47] . The sensory information from both of these systems is conveyed through a phosphorelay mechanism mediated by a phosphorelay protein, LuxU. Recently the third system consisting of the histidine kinase response regulator pair of VarS and VarA has been described [48] . Since bacterial signal transduction systems including QS have no counterpart in the human host, components of this machinery are considered for development of drugs and/or vaccines against this human pathogen. QS proteins in other enteropathogenic bacteria have been successfully used to evaluate the effectiveness of microbial inhibitors. Matrix production and QS constitute the major pathways for biofilm formation. c-diGMP, a cyclic dimer of two GMP molecules, is an intracellular signaling molecule that has diguanylate cyclase activity and phosphodiesterase activity. The levels of c-di-GMP depend on the relative concentration of these two enzymes [49] . c-di-GMP induces biofilm formation through activation of the expression of the vpsT gene, essentially by up-regulating genes that promote biofilm formation in V. cholerae. The biofilms and conditionally viable environmental cells (CVECs) have an important role to play in the transmission and survival of V. cholerae. future science group


A short-lived hyperinfectious state of V. cholerae, which after passage through human host, transiently gains explosive behavior that causes cholera outbreaks. Huq et al. describe two modes of survival: one biofilms and other viable but nonculturable state in the environment for V. cholerae [50] . The infectious potential of V. cholerae increases in biofilms primarily because of the dose that may be as high as 1 × 109 cells/clump [50] . The bacterium in the biofilm may well be protected against acidic pH and various antibiotics in the gut. Faruque et al. argue the presence of CVECs in V. cholerae and demonstrated that CVECs were partially dormant cells that revive to viable bacteria both under in vitro and in vivo conditions [51] . They further emphasized that the clumps of CVECs akin to biofilms are responsible for the hyperinfectious state in V. cholerae. The new therapeutics against V. cholerae should encompass QS effectors because of their important role in biofilm formation and virulence gene regulation [44,45] . The objective of targeting virulence genes and their regulators would be to decrease the quantitated dose of toxins released by the bacteria, thus reducing the overall severity of infection. However, targeting biofilm has its own advantages. Since concentration of nutrients tends to be high at surfaces, the attachment of bacteria to the surface is a major step in establishing infection. Apart from chemical stress, bacterial biofilms improve survival during osmosis. The most salient feature of biofilms in disease would be their role in surface attachment that decreases the volume of water required to deliver an infectious dose. Biofilms also form a physical barrier against the proper dispersal and diffusion of antibiotics. A small nontoxic molecule, LED209, which inhibits QseC (a histidine kinase) autophosphorylation and consequently inhibits QseCmediated activation of virulence gene expression, has been identified and employed against E. coli, S. typhimurium and F. tularensis [52] . Acyl homoserine lactones (AHLs) constitute the signaling molecule of the QS1 in V. cholerae. A novel antibacterial was indeed designed based on the structure of AHL autoinducer and its receptors present in both cytoplasm and membrane in V. harveyi [53] . AHL analogs, such as N-acyl cyclopentylamine are known that can interrupt signaling in the Lux QS system [54] . Hydrolysis of the AHL signal by a cylase enzyme is known in other bacteria. When nucleoside analogs screened for their ability to perturb AI-2-based QS, p-methoxy phenyl propionamide furanosyl future science group

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derivatives was found to show inhibition of QS [55] . It was presumed that LuxPQ proteins act as sensor for the phosphorelay, because the most probable component was inhibited by the phenyl propionamide derivative. The activity of LuxS, the enzyme that catalyzes the production of AI-2, was blocked by S-anhydroribosyll-homocysteine and S-homoribosyl-l-cysteine through a signaling circuit involving AI-2 [56] . Hemagglutinin/protease regulatory protein (HapR) is the terminal effector in QS pathways of V. cholerae. HapR brings about changes in the expression levels of various proteins regulating biofilm and virulence. HapR has a dimeric, two-domain, helical structure that is conserved in the TetR family of transcriptional regulators [57] . The N-terminal DNA-binding domain contains a helix–turn–helix DNAbinding motif and C-terminal domain containing a solvent-accessible channel connected to an amphipathic cavity. Cinnamaldehyde and furanone derivatives have been found to disrupt AI-2-based QS in Vibrio spp. by decreasing the DNA-binding activity of the response regulator LuxR (a HapR homolog) [58,59] . Another potential target is the LuxO response regulator protein, which acts in two different signaling cascades, culminating in expression/repression of HapR. Response regulators typically consist of a receiver domain and an ATPase domain separate from DNA-binding domain. Although ATP-binding domains remain the pet for the pharmaceutical industry, the other two domains provide ample opportunity to disrupt sensing circuits through the signaling system in V. cholerae. Bearing in mind the aforementioned observations, novel approaches to treat V. cholerae should include QS pathways, not only to check antibiotic resistance, but also because it could lead to discovery of a common target against diverse toxigenic serogroups of V. cholerae. Targeting toxins & vaccines

Cholera toxin is a hexameric A-B5 type toxin. The toxicity is attributed to enzymatic activity of the A-subunit that catalyzes ADP ribosylation of the a-subunit of GTP-binding protein, GS, activating adenylate cyclase and elevating cAMP, leading to hypersecretion of Cl- ions and water, ultimately causing profuse diarrhea. CT A subunit consists of two polypeptide chains, CTA1 and CTA2. CTA1 confers CT-mediated toxicity, whereas CTA2 acts as a linker between CTA1 and CTB subunit. The five B-subunits of the toxin bind to receptor monosialoganglioside (GM1) present on the www.futuremedicine.com

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surface of the intestinal epithelial cells. The B subunit is considered a molecular recognition unit and delivery vehicle for A-subunit. When administered orally CTB induces immunogenicity at mucosal surfaces [60,61] . This event is possible because of the binding of the CTB subunit to the eukaryotic cell surface via GM1, which elicits a mucosal immune response against the pathogen when coupled chemically with other antigens. One of the several approaches would be to find inhibitors of the toxin receptor that can be administered as a therapeutic agent for the treatment of infection. Although the action of CT is conserved among classical and El Tor strains, the CTB sequence differs among the two biotypes, which is the basis for ctxB genotyping. Based on nonrandom base variations, three types of ctxB genes ensuing in a change in the deduced amino acid sequence positions at 39, 46 and 68 have been described [62] . Genotype 1 is found in classical biotype strains worldwide and in US Gulf Coast strains, genotype 2 in El Tor biotype strains from Australia, and genotype 3 in El Tor biotype strains from the seventh pandemic and the Latin American epidemics. New ctxB variants showing an additional polymorphism at amino acid positions 28 and 34 have recently been described in V. cholerae O139 strains [63] . Structure-based drug design against CT is one of the impervious methods that have had success in finding inhibitor(s) and vaccine design initiatives against cholera. Previous studies indicated that amino acid sequence diversity in ctxB, not involved in receptor binding, can be one of the reasons for epitope variation of CTB [64] . The results of our study on natural CTB subunit variants indicated subtle variations in binding to various carbohydrate ligands of the receptor [65] . Mutation analysis of CTB revealed variations in immunoreactivity, hemolysis and GM1 binding ability for the toxin subunit [66,67] . Therefore, there is need to consider these differences to reassess their contribution in developing antitoxin titers in vivo and for the development of synthetic or natural vaccines against cholera. Targeting transcription regulators & virulence genes

The progress in diagnosis and treatment of the disease, including the discovery of new drugs and proposed avenues for drug targeting has lead researchers to find answers to counter the increasing incidence of MDR strains causing cholera. For effective evaluation of drugs, one 1204


needs to scan large amounts of genomic data, analyze its physicostructure and find drug target sites useful in controlling cholera infection. This tedious task, in the recent past, has been largely relieved owing to exploitation of computational methods [68] . There is considerable argument regarding the use of in silico homology modeling in drug discovery initiatives. Structural genomics along with substantial dynamic simulations could provide a practical and cost-effective alternative for generating convincingly accurate models for drug discovery. A combination of methods has been successful in identifying a suitable molecules using docking simulation, binding accuracy and ligand–receptor interaction, mutagenesis experiments and rationalizing structure–activity relationship data. Apart from the aforementioned targets, there are other prominent pathways and the possibility of exploiting these for targeting drugs and vaccines. In a review on antisecretory agents, Thiagarajah et al. reported that there are two potential drug targets to look for: enkephalinase inhibitors (e.g., racecadotril) and CFTR inhibitors (e.g., thiazolidinone included glycine hydrazine class of CFTR inhibitors) for the treatment of cholera [69] . Yamaichi and colleagues [70] evaluated another class of inhibitors exploiting the second chromosomal replication initiator RctB. A bactericidal replication initiation inhibitor, vibrepin, was identified that represents a new class of antibiotic that specifically targets a particular family of microorganisms. The authors argue that the genes mediating resistance to these compounds will not arise and transfer from nonvibrios to vibrios. Virstatin is another potential inhibitor designed to act against this pathogen by inhibiting the transcriptional regulator ToxT, thus preventing expression of CT and the toxin coregulated pilus [71] . The genomic target database puts three locus tags VC0015, VC2730 and VC2770 for V. cholerae-specific drug targets in unique metabolic pathways [104] . The crystal structure of novel bacterial topoisomerase inhibitor of Staphylococcus aureus DNA gyrase that presents a unique method of inhibition, dodging fluoroquinolone resistance was a respite to this important drug target [72] . This could be a healthy development considering the frequent use of flouroquinolones in the treatment of V. cholerae. Moreover, the VC0015 region that represents DNA gyrase with topoisomerase activity can be exploited as a possible drug target. However, a recent computational study on potential drug targets in V. cholerae indicate future science group


that there are approximately 36 protein-coding genes that can be considered for drug target initiatives [73] . Phage therapy

Phage therapy for cholera was established as a helpful tool for the treatment of cholera patients because phage are able to kill large number of bacteria, thus reducing the burden of the pathogenic toxin and, ultimately, transmission of the disease [74] . The first report of phage therapy that used high doses of anticholera phage (100–200 phage per vibrio) showed the killing of cholera bacterium but the phage were not able to complete many cycles of replication and amplification [74] . When bacteriophage therapy was conducted on ill patients in a hospital and compared with tetracycline treatment and with fluid replacement alone as a control, it was noted that very high-dose phage therapy was comparable to tetracycline in reducing the excretion of vibrios in stools; this reduction, however, did not translate into overall clinical improvement (i.e., shorter duration of diarrhea and more rapid recovery) [74,75] . Several problems were noted that complicated the evaluation of phage therapy in cholera; first was the discovery of the diversity of serotypes of vibrios and the varying susceptibility of these bacteria to the phage stocks employed; second was the rapid transit of ingests phage through gastrointestinal track

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of cholera patients, a fact that may have precluded the second round of phage infection essential in low multiplicity of infection therapy [74] . Several reports have shown the utilization of bacteriophages in the treatment of severe cases of cholera [75] . Considering the need for such prophylactic action at that time, the cholera bacteriophage therapy could be argued successful. The impetus for considering bacteriophage-mediated regulation would be the fact that phage-resistant strains rarely occur in a natural habitat. Conclusion

Little is known about the efficiency of novel drugs and available drug targets in V. cholerae. Apart from conventional antibiotics and vaccines that could be obsolete in near future, we are at dire straits without adequate machinery and knowledge regarding infectious disease processes. The present climatic conditions and population dynamics of V. cholerae demand a rapid breakthrough in drug discovery research against cholera. Elaboration of the proposed sites for drug discovery like QS effectors, toxins and bacteriophage therapy could give deeper insights into methodologies to subdue this pathogen. It would be more than useful to employ unconventional approaches such as computational biology, in silico target identification, homology modeling and structure-based drug design to pursue

Executive summary Cholera, clinical manifestation & antibiotic resistance Vibrio cholerae is the causative agent of the deadly disease cholera. The most distinctive feature of cholera is the painless purging of watery stool with a fishy odor. n The key to the therapy includes either intravenous injection or oral rehydration therapy. n Multidrug resistant V. cholerae is on the rise. n Resistance mechanisms adapted by the bacteria comprise of mutations, horizontal gene transfer and efflux pumps. n n

Novel avenues of drug targeting, vaccines & phage therapy Bacteriophage therapy was earlier used in cholera prophylaxis in endemic areas but with the advent and rise of antibiotic use, it lost its effectiveness against the pathogen. This therapy requires a serious reconsideration for use against this disease. n Although vaccines and drugs targeting the cholera toxin are available, the contentious issue would be the amino acid variations in ctxB subunit to bind GM1 receptor. n Quorum sensing pathways provide a unique opportunity to exploit the pathogen at unconventional junctures targeting cellular communication. n This will provide avenues to search for new inhibitor molecules, which target signaling cascades, to treat cholera. n

Conclusion Elaboration of the proposed sites for drug discovery like quorum sensing effectors, toxins and bacteriophage therapy could give deeper insights into methodologies to subdue this pathogen. n It would be more than useful to employ unconventional approaches, such as computational biology, in silico target identification, homology modeling and structure-based drug design to pursue the course of antibacterial drug discovery at a pace that could keep up with the rapid transitions in multidrug-resistant phenotypes of this bacterium. n

Future perspective Many more avenues need to be explored to find suitable inhibitors or therapy to control cholera infection. The use of unorthodox technologies, such as computational biology should be explored for this purpose.

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antibacterial drug discovery that could keep up with the rapid transitions of MDR phenotypes of this bacterium. Future perspective

In the backdrop of the emergence of MDR V. cholerae and as well as looking for newer drugs, various new approaches need to be assessed to design and evaluate vaccines or to find novel target inhibitors involved in QS or toxin binding to receptors and phage therapy as a possible remedy to cure and prevent the spread of cholera. Many more avenues need to be explored to find suitable inhibitors or therapy to control cholera infection. The use of unorthodox technologies, such as computational biology could be explored and used for this purpose. Bibliography Papers of special note have been highlighted as: n of interest nn of considerable interest 1.

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Khan WA, Saha D, Rahman A, Salam MA, Bogaerts J, Bennish ML. Comparison of single-dose azithromycin and 12-dose, 3-day

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This research was supported by the Department of Science and Technology, New Delhi grant (SP/SO/ HS-51/2002) to DV Singh and funds contributed by the Department of Biotechnology, New Delhi, to the Institute of Life Sciences, Bhubaneswar, India. Senior Research Fellowship awarded by the Indian Council of Medical Research, New Delhi and the Institute of Life Sciences, Bhubaneswar, to MHUT Fazil is gratefully acknowledged. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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