(Assignment Name)

VIBRIO CHOLERAE
Assignment report submitted to

Government College of Pharmacy, Aurangabad

By

(Mr. Chandrakant Malhare) Roll No. 12 Under Guidance: Gopal karwa Sir
Government College of Pharmacy, Aurangabad. 2013-2014.

Recent advanced techniques developed in microbiology for diagnosing some common diseases.
Contents.
Introduction History Literature view Techniques Conclusion Summary Reference

Roll No.12 Class- Pharm-D (S.Y) Sub- Pceutical Microbiology Topic Name: Vibrio Cholera

Chapter No.1
Introduction:Cholera is a diarrheal illness caused by infection with the gramnegative bacterium Vibrio cholerae. Cholera is characterized by severe, watery diarrhea, which can rapidly lead to dehydration and death in untreated patients.The clinical manifestations of cholera, the methods of arriving at a presumptive clinical and a confirmatory laboratory diagnosis, and the therapeutic approach to patients with the disease will be reviewed here. The pathogenesis of Vibrio cholerae infection is discussed separately.Vibrio vulnificus is capable of causing severe and often fatal infections in susceptible individuals. According to a recent review, this gram-negative, hemophilic bacterium was first isolated by the U.S. Centers for Disease Control (CDC) in 1964 and was initially misidentified as Vibrio parahaemolyticus. Decreased NaCl tolerance nand the ability to ferment lactose were among the characteristics first used to distinguish the as-yet unnamed V. vulnificus from V. parahaemolyticus. The bacterium was designated V. vulnificus in 1980). Sequencing of the 16S rRNA gene revealed that V. vulnificus is an outgroup from the core group of the genus Vibrio. Three biotypes (biogroups), designated 1, 2, and 3, have been established based upon characteristics, such as indole production, host specificity, serotype, and genetic subtyping (DNA sequencing of biotype 1 V. vulnificus strain YJ106 revealed that the bacterium harbors two chromosomes and a 48.5-kbPlasmid. V. vulnificus causes two distinct disease syndromes, primary septicemia and necrotizing wound infections. It is the leading cause of food-related mortality reported in the state of Florida, and the risk of foodborne disease correlates with seasonally high numbers of these bacteria during summer months. Diseases caused by V. vulnificus are among the most severe of all foodborne infections, as the case-fatality rate for primary septicemia caused by V. vulnificus has been reported at greater than 50%, and death can occur within a day or two of the onset of symptoms. However, life-threatening infections occur almost exclusively in immune compromised patients or those with iron overload resulting from liver disease or hemochromatosis. High iron levels in the host, which saturate iron binding proteins, such as transferrin, have been shown to dramatically lower the 50% lethal dose (LD50) of V. vulnificus in mice to approximately one cell. The low infectious dose of some V. vulnificus strains was confirmed in many studies, including one in which the 50% infectious dose of several V. Vibrio cholerae causes the diarrheal disease cholera and utilizes different survival strategies in aquatic environments. V. cholerae can survive as free-living or in association with zooplankton and can build biofilm and rugose colonies. The bacterium expresses cholera toxin (CT) and toxin-coregulated pilus (TCP) as the main virulence factors. These factors are co-regulated by a transcriptional regulator ToxR, which modulates expression of outer membrane proteins (OmpU) and (OmpT). The aims of this study were to disclose the role of ToxR in expression of OmpU and OmpT, biofilm and rugose colony formation as well as in association with the free-living amoeba Acanthamoeba castellanii at different temperatures.

Vibrio cholerae is a gram-negative bacterium causin severe diarrhoeal disease cholera in Asia, Africa, and America. Many millions of cholera cases occurred endemically and pandemically worldwide [1]. V. cholerae adopts several survival strategies in aquatic environments. The bacterium can survive as free-living or in association with phytoplankton/zooplankton, crustaceans, and molluscs in coastal and estuarine environments [2]. V. cholerae forms biofilms on biotic and abiotic surfaces, thereby protecting themselves with this exopolymer barrier [3], biofilm can provide protection from toxic compounds, such as antibiotics, thermal stress, and predation [4]. The bacterium has been described to switch between the smooth and rugose colony morphotypes contributing to its environmental survival. The exopolysaccharide (EPS) materials of rugose colony-forming V.

Chapter No.2
History:cholerae strains were recognized as a heavy, fibrous, electrondense, ferretin-stained layer surrounding the cells, but smooth colony V. cholera did not appear to have this EPS layer surrounding it [5]. The cell surface EPS materials confer a rugose colony morphology and resistance to osmotic and oxidative stresses. The regulation of EPS synthesis in bacteria is complex and involves multiple systems utilizing both positive and negative regulation [5]. Recent studies have shown that V. cholerae has developed a new survival strategy to grow and survive inside free-living amoeba Acanthamoeba castellanii [610]. V. cholerae expresses cholera toxin and toxin-coregulated pilus as the main virulence factors, which are coregulated by a transcriptional regulator ToxR. ToxR, however, independently of the transcriptional activators TcpP and ToxT, modulates expression of two outer membrane proteins OmpU and OmpT. Transcription of ompU is induced by ToxR, whereas transcription of ompT is repressed by ToxR [11,12]. In this paper we study the effect of ToxR regulatory protein in expression of OmpU and OmpT, biofilm and rugosity as well as survival of V. cholerae O1 with A. castellanii by constructing a toxR deletion mutant.

Cholera is a vastly underreported disease, with an estimated 3 to 5 million cases and 100,000 deaths annually. Cholera is endemic in the developing countries of Asia and Africa and has caused epidemics in Asia, the Middle East, and South and Central America . In 2009, the number of cases of cholera reported to the World Health Organization increased by 16 percent over the previous year (221,226 cases from 45 countries). Globally, a high incidence in the Americas in the early 1990s has shifted to a high incidence in Africa in the 2000s, with few cases in Asia. Recent cholera epidemics include the 2008-2009 epidemic in Zimbabwe and the 2010 epidemic following the January earthquake in Haiti. The V. cholerae strain responsible for the Haiti epidemic is nearly identical to the El Tor O1 strains predominant in southeast Asia; the ancestry is distinct from that of circulating Latin American and East African strains of V. cholerae, suggesting introduction of the strain from Asia . During the first two years of the 2010 Haiti epidemic the cumulative attack rate was 6.1 percent. The same strain was subsequently identified in cholera cases in the Dominican Republic and in Florida. (See 'Microbiology' below.) In the United States, approximately 10 laboratory-confirmed cases of cholera are reported to the Centers for Disease Control and Prevention each year. Of these, about half are acquired outside the United States; the rest are acquired via consumption of contaminated seafood. Toxigenic Vibrio cholerae infection was reported in Louisiana in October 2005, related to consumption of contaminated and improperly cooked seafood after Hurricanes Katrina and Rita.

Chapter 3 Literature review:

3.1 Diagnosis of fungal infections with a chitinase

A novel method for detecting chitin, and for diagnosing fungal infections (including yeast infections), with a chitinase or other chitin-specific binding protein. This method should allow the convenient, broad spectrum diagnosis of fungal infections in tissue samples or in body fluids. Fungal infections are a particular problem in immunocompromised hosts such as AIDS patients, where they can cause opportunistic infections. This invention overcomes difficulties experienced by prior methods of diagnosing fungal infections.

3.2 Vibrio cholerae proteins expressed during infection

Disclosed are compositions comprising in vivo expressed polynucleotides of V. cholerae. Also disclosed are methods of using such polynucleotides and the corresponding expression products to treat V. cholerae infection.

3.3 Diagnosis of multiple sclerosis with diketopiperazines

The present invention relates to the diagnosis and monitoring of diseases and conditions by quantifying markers, including degradation products of disease-associated proteins, such as diketopiperazines composed of the two N-terminal amino acids or the two C-terminal amino acids of such proteins. The methods are useful for diagnosing or monitoring various diseases, including multiple sclerosis, Alzheimer's disease and ischemia. The invention further provides binding partners specific for the markers and compositions and kits for conducting the methods of the invention.

3.4 Diagnosis of fungal infections, and a chitin-binding lectin useful in such diagnoses
A 134 kDa, calcium-independent, chitin-binding lectin called chitovibrin is secreted by marine bacteria of the genus Vibrio. The secretion of chitovibrin is inducible by chitin or chitinoligomers. Chitovibrin shows no apparent enzymatic activity, but has a strong affinity for chitin and for chito-oligomers dp9 and larger. The protein has an isoelectric pH of 3.6, shows thermal tolerance, binds chitin with an optimum at pH 6 and is active in 0-4 M NaCl. Chitovibrin is useful as a stain for fungi and other chitin-containing organisms. Chitovibrin may be used to detect the presence of chitin, particularly in diagnosing fungal infections in humans, animals, and plant materials. Fungal infections are a particular problem in immunocompromised hosts such as AIDS patients and bone marrow transplant patients, because they can cause opportunistic infections. The chitovibrin diagnostic method allows the convenient, broad spectrum diagnosis of fungal infections in tissue samples or in body fluids. Other, smaller polypeptide fragments of chitovibrin will exhibit similar chitin-binding properties, and could be used in coupling to detection systems.

3.5 Diagnosis of premalignant or malignant conditions of human secretory epithelia

A method for diagnostic or prognostic monitoring of premalignant or malignant conditions of human secretory epithelia, particularly colonic epithelia, by determining the extent of expression of a 1-3N-acetylglucosaminyltransferase is described. Associated with the expression of a wide variety of carbohydrate antigens in adenocarcinomas is the induction of 1-3Nacetylglucosaminyltransferase in epithelial cells. This enzyme is not found in normal, healthy adult colonic epithelial cells and thus indicates a novel and potentially sensitive method for screening the disease status of individual

3.6 Vaccine compositions and methods of use to protect against infectious disease

The present invention provides a novel immunogenic composition, vaccine and methods for making and using the immunogenic composition and vaccine. The immunogenic composition is capable of providing an immune response and/or a protective immunity into subjects, preferably mammals, against microorganism-associated disease. The immunogenic composition includes one or more extracellular proteins isolated from a microorganism capable of providing an immune response and/or a protective immunity into subjects against microorganism-associated disease. The isolated extracellular proteins range in molecular weight from about 10,000 Da to about 220,000 Da. Suitable microorganisms may include members of the genus Salmonella, Listeria, Pseudomonas, Staphylococcus, and Vibrio. Kits are also encompassed for detection, diagnosis and prevention of microorganism-associated disease.

3.7 IP-10 based infection diagnosis

The present invention relates to an immunological method and, more particularly, a method for measuring cell-mediated immune reactivity (CMI) in mammals based on the production of IP10.The invention further discloses an assay and a kit for measuring CMI to an antigen using whole blood or other suitable biological samples. The methods of the present invention are useful in therapeutic and diagnostic protocols for livestock and veterinary and wild life applications, thus the invention further relates to a method for diagnosing an infection in a mammal.

3.8 Diagnosis, Prevention and Treatment of Disorders Characterized by Undesirable Cell Proliferation

The present invention relates to compositions and methods for the reduction of atherosclerotic plaques and the decrease in the level of total serum cholesterol, triglycerides, serum LDL cholesterol, and serum HDL cholesterol. The present invention also relates to methods for the diagnosis, prevention and treatment of atherosclerosis and mycoplasma associated diseases.

Chapter 4
Techniques:
4.1 ToxR of Vibrio cholerae affects biofilm, rugosity and survival with Acanthamoeba castellanii
Vibrio cholerae causes the diarrheal disease cholera and utilizes different survival strategies in aquatic environments. V. cholerae can survive as free-living or in association with zooplankton and can build biofilm and rugose colonies. The bacterium expresses cholera toxin (CT) and toxin-coregulated pilus (TCP) as the main virulence factors. These factors are co-regulated by a transcriptional regulator ToxR, which modulates expression of outer membrane proteins (OmpU) and (OmpT). The aims of this study were to disclose the role of ToxR in expression of OmpU and OmpT, biofilm and rugose colony formation as well as in association with the free-living amoeba Acanthamoeba castellanii at different temperatures.

4.2 Methods for isolation and confirmation of Vibrio vulnificus from oysters and environmental sources
The gram-negative bacterium Vibrio vulnificus is a natural inhabitant of estuarine waters and poses a significant health threat humans who suffer from immune disorders, liver disease, or hemochromatosis (iron overload). V. vulnificus enters human hosts via wound infections or consumption of raw shellfish (primarily oysters), and infections frequently progress to septicemia and death in susceptible individuals. Prevalence in waters and shellfish is not correlated with fecal indicator organisms; therefore, species-specific detection and enumeration of V. vulnificus in the environment has become a priority for agencies that are responsible for shellfish safety. The many selective-differential media developed for isolation of Vibrio spp., and specifically for V. vulnificus detection, are reviewed here; however, none of the media developed to date combines the sensitivity to low numbers with the specificity necessary to inhibit growth of other organisms. Therefore, immunological and molecular protocols are needed for confirmation of the identity of the organism and are discussed in detail. Methods under development that hold promise for rapid, accurate, and sensitive detection and enumeration of the organism include multiplex and real-time PCR. Developing technologies that have proven useful for detection and investigation of other pathogens such as biosensors, spectroscopy and microarrays may provide the next generation of tools for investigation of the prevalence and ecology of V. vulnificus. 4.3 Vibrio cholerae

Vibrio cholerae is a facultative anaerobic, Gram negative, non-spore forming curved rod, about 1.04-1.06 m long. It is a facultative human pathogen found in coastal waters that causes the acute gastrointestinal disease, cholera, a major health threat in poor nations. It is widely acknowledged as one of the most important water borne pathogen of worldwide economic significance. Sea foods and water is the most common vehicle for this infection in humans. It has been isolated from wide variety of samples such as seawater, sediments, plankton, finfish and shellfishes of coastal and estuarine environments. Cholera pathogenesis is a complex process and involves synergistic action of several genes. CT is considered the most important epidemic marker among various toxins produced by V.cholerae. Detection of V.cholerae from food stuffs is problematic, since they are present at low level together with large number of competing microflora and also they may be injured by different food processing methods.

Chapter 5..
Conclusion
Our results demonstrate that ToxR does seem to play some regulatory role in the OmpT/OmpU expression shift and in the environmental survival strategies of V. cholerae such as biofilm formation, switching from smooth to rugose colony and association with the freeliving amoeba A. castellanii, suggesting a new role for this regulatory protein.ToxR does seem to play some regulatory role in the OmpT/OmpU expression shift, the changes in biofilm, rugosity and survival with A. castellanii, suggesting a new role for this regulatory protein in the environments.ToxR does seem to play some regulatory role in the OmpT/OmpU expression shift, the changes in biofilm, rugosity and survival with A. castellanii, suggesting a new role for this regulatory protein in the environments. V. vulnificus is a rare cause of disease, but is also underreported. As it is a rare entity, clinicians should have a high index of suspicion for the bacteria when patients present with gastrointestinal illness, fever or shock, with or without history of ingestion of raw seafood; or present with a wound infection after exposure to sea water. Pediatricians should also be alert as it is a potentially fatal disease, especially in immunocompromised children, so that prompt recognition would ensure immediate management, which has better prognostic implicatio A recent study showed that V. cholerae gains natural competence upon growth on chitin surfaces[8]. Natural competence enables these bacteria to take up free DNA from the environment in order to incorporate it into their genome. Blokesch and Schoolnik demonstrated that the whole O1 specific antigen cluster (size of ~32 kb) of V. cholerae O1 El Tor can be exchanged either by the O37- (size of ~23 kb) or by the O139-specific antigen cluster (size of ~42 kb) by means of chitin-induced natural competence [9]. Following this first publication, we and others demonstrated that indeed different parts of the genome can be horizontally transferred in this manner including parts of the Vibrio pathogenicity island 2 (VPI-2) and the Vibrio seventh pandemic islands (VSP-I and VSP-II), respectively (Blokesch and Schoolnik, unpublished data), the cholera toxin prophage [10] and clusters encoding metabolic pathways [6]

Chapter 6
Summary
The Cholera and Other Vibrio Illness Surveillance (COVIS) system is a national database of reported human illnesses caused by all species of Vibrio; the COVIS database is maintained by CDC. This information has been used to educate consumers about the health risks of seafood, as well as to help determine host, food, and environmental risk factors. participating health officials collect clinical data, information about underlying illness, history of seafood consumption and exposure to seawater in the 7 days before illness. Participants also conduct tracebacks of implicated seafood. Surveillance has expanded nationally, with most states reporting cases since 1997. Historically, only toxigenic V. cholerae serogroups O1 and O139 were nationally notifiable; therefore, the numbers of non-toxigenic Vibrio isolates were likely underreported. However, in January 2007, infections caused by any Vibrio species (vibriosis) became nationally notifiable, although many cases of vibriosis are undetected by clinical laboratories because vibrios are not easily identified on routine enteric media. Additionally, CDC serotypes all V. parahaemolyticus isolates received from state health departments, tests for serogroups O1, O75, O139, and O141, and determines cholera toxin production for all V. cholera isolates. This report summarizes human Vibrio infections during 2007 reported by states to CDC. Results are presented in two categories: V. cholerae isolates that produce cholera toxin. The patient infected with V. cholerae serogroup O75 had traveled domestically to Florida where he ate at an oyster bar. Health officials were unable to reach the patient to get a more detailed food history, and no traceback was undertaken. No hospitalization was required

Vibrio cholerae), and all other Vibrio isolates, including those V. cholerae isolates that do not produce cholera toxin. This report differs from previous reports, in that results for the entire country are presented together rather than including a separate section for Gulf Coast states. Additionally, results are presented by specimen type. It is important to note that isolation of some Vibrio species from a patient with illness does not necessarily indicate causation. While many Vibrio species are well-recognized pathogens, the status of V. damsela, V. furnissii, V.metschnikovii, and V. cincinnatiensis as enteric or wound pathogens is less clear.

Chapter 7
References
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9. Hlady, W.G., Klontz, K.C., 1996. The epidemiology of Vibrio infections in Florida, 19811993. J. Infect. Dis. 173, 1176 1183. 10.Hi, L., Dalsgaard, A., 2000. Evaluation of a simplified semiquantitative protocol for the estimation of Vibrio vulnificus in bathing water using cellobiose-colistin agar: a collaborative study with 13 municipal food controlling units in Denmark. J. Microbiol. Methods 41, 53 57. 11. Tauxe RV, Blake PA. Epidemic cholera in Latin America. JAMA 1992; 267:1388. 12.Swerdlow DL, Ries AA. Cholera in the Americas. Guidelines for the clinician. JAMA 1992; 267:1495