MLS 513 Vibrio Cholerae PDF

MLS 513 Vibrio Cholerae Dr SC Chollom  Introduction  Vibrios are highly motile, gram-negative, curved or comma-shaped rods with a single polar flagellum. Of the vibrios that are clinically significant to humans, Vibrio cholerae O group 1, the agent of cholera, is the most important. Vibrio...

MLS 513 Vibrio Cholerae Dr SC Chollom  Introduction  Vibrios are highly motile, gram-negative, curved or comma-shaped rods with a single polar flagellum. Of the vibrios that are clinically significant to humans, Vibrio cholerae O group 1, the agent of cholera, is the most important. Vibrio cholerae was first isolated in pure culture by Robert Koch in 1883, although it had been seen by other investigators, including Pacini, who is credited with describing it first in Florence, Italy, in 1854.  Cholera is a life-threatening secretory diarrhea induced by an enterotoxin secreted by V cholerae. Cholera and the cholera enterotoxin are increasingly recognized as the prototypes for a wide variety of non-invasive diarrheal diseases, collectively known as the enterotoxic enteropathies; of these, diarrhea due to enterotoxigenic strains of Escherichia coli is the most important.  Cholera remains a major epidemic disease. There have been seven great pandemics. The latest, which started in 1961, invaded the Western Hemisphere (for the first time this century) with a massive outbreak in Peru in 1991. There have since been more than a million cases in Central and South America as well as a few imported cases in the U.S. and Canada. V cholerae serogroup O139, which arose in October of 1992 in India and Bangladesh, may become the cause of the 8th great pandemic of cholera.  Other vibrios may also be clinically significant in humans, and some are known to cause diseases in domestic animals. Nonpathogenic vibrios are widely distributed in the environment, particularly in estuarine waters and seafoods. For this reason, isolation of a vibrio from a patient with diarrheal disease does not necessarily indicate an etiologic relationship.  Structure, Classification and Antigenic Types  The cholera vibrios are Gram-negative, slightly curved rods whose motility depends on a single polar flagellum. Their nutritional requirements are simple. Fresh isolates are prototrophic (i.e., they grow in media containing an inorganic nitrogen source, a utilizable carbohydrate, and appropriate minerals). In adequate media, they grow rapidly with a generation time of less than 30 minutes. Although they reach higher population densities when grown with vigorous aeration, they can also grow anaerobically. Vibrios are sensitive to low pH and die rapidly in solutions below pH 6; however, they are quite tolerant of alkaline conditions. This tolerance has been exploited in the choice of media used for their isolation and diagnosis.  Until 1992, the vibrios that caused epidemic cholera were subdivided into two biotypes:  classical and El Tor.  Classical V cholerae was first isolated by Koch in 1883. Subsequently, in the early 1900s, some vibrios resembling V cholerae were isolated from Mecca-bound pilgrims at the quarantine station at El Tor, in the Sinai peninsula, that had been established to try to control cholera associated with pilgrimages to Mecca. These vibrios resembled classical V cholerae in many ways but caused lysis of goat or sheep erythrocytes in a test known as the Greig test. Because the pilgrims from whom they were isolated did not have cholera, these hemolytic El Tor vibrios were regarded as relatively insignificant except for the possibility of confusion with true cholera vibrios. In the 1930s, similar hemolytic vibrios were associated with relatively restricted outbreaks of diarrheal disease, called paracholera, in the Celebes. In 1961, cholera caused by El Tor vibrios erupted in Hong Kong and spread virtually worldwide. Although in the course of this pandemic most V cholerae biotype El Tor strains lost their hemolytic activity, a number of ancillary tests differentiate them from vibrios of the classical biotype.  Structure, Classification and Antigenic Types  The operational serology of the cholera vibrios which belong in O antigen group 1 is relatively simple. Both biotypes (El Tor and classical) contain two major serotypes, Inaba and Ogawa. These serotypes are differentiated in agglutination and vibriocidal antibody tests on the basis of their dominant heat-stable lipopolysaccharide somatic antigens. The cholera group has a common antigen, A, and the serotypes are differentiated by the type-specific antigens, B (Ogawa) and C (Inaba). An additional serotype, Hikojima, which has both specific antigens, is rare. V cholerae O139 appears to have been derived from the pandemic El Tor biotype but has lost the characteristic O1 somatic antigen; it has gained the ability to produce a polysaccharide capsule; it produces the same cholera enterotoxin; and it seems to have retained the epidemic potential of O1 strains.  Other antigenic components of the vibrios, such as outer membrane protein antigens, have not been extensively studied. The cholera vibrios also have common flagellar antigens. Cross-reactions with Brucella and Citrobacter species have been reported. Because of DNA relatedness and other similarities, other vibrios formerly called “nonagglutinable” are now classified as V cholerae. The term nonagglutinable is a misnomer because it implies that these vibrios are not agglutinable; in fact, they are not agglutinable in antisera against the O antigen group 1 cholera vibrios, but they are agglutinable in their own specific antisera. More than 139 serotypes are now recognized. Some strains of non-O group 1 V cholerae cause diarrheal disease by means of an enterotoxin related to the cholera enterotoxin and, perhaps, by other mechanisms, but these strains have not been associated with devastating outbreaks like those caused by the true cholera vibrios. Recently, vibrio strains that agglutinate in some O group 1 cholera diagnostic antisera but not in others have been isolated from environmental sources  Structure, classification and Antigenic Types  Humans apparently are the only natural host for the cholera vibrios. Cholera is acquired by the ingestion of water or food contaminated with the feces of an infected individual.  Previously, the disease swept the world in six great pandemics and later receded into its ancestral home in the Indo-Pakistani subcontinent. In 1961, the El Tor biotype (a subset distinguished by physiologic characteristics) of V cholerae, not previously implicated in widespread epidemics, emerged from the Celebes (now Sulawesi), causing the seventh great cholera pandemic.  In the course of their migration, the El Tor biotype cholera vibrios virtually replaced V cholerae of the classic biotype that formerly was responsible for the annual cholera epidemics in India and East Pakistan (now Bangladesh).  The pandemic that began in 1961 is now heavily seeded in Southeast Asia and in Africa. It has also invaded Europe, North America, and Japan, where the outbreaks have been relatively restricted and self-limited because of more highly developed sanitation. Several new cases were reported in Texas in 1981 and sporadic cases have since been reported in Louisiana and other Gulf Coast areas. This now endemic focus appears to be due to a clone which is unique from the pandemic strain.  In 1991, the pandemic strain hit Peru with massive force and has since spread through most of the Western Hemisphere, causing more than a million cases. Fortunately, mortality has been less than 1 percent because of the effectiveness of oral rehydration therapy. The vibrios surprised us again, in 1992, with the emergence of O139 in India and Bangladesh. For a while it appeared that O139 would replace O1 (both classical and El Tor) but it has exhibited quiescent periods when O1 reemerges.  Clinical Manifestation  Following an incubation period of 6 to 48 hours, cholera begins with the abrupt onset of watery diarrhea  The initial stool may exceed 1 L, and several liters of fluid may be secreted within hours, leading to hypovolemic shock.  Vomiting usually accompanies the diarrheal episodes. Muscle cramps may occur as water and electrolytes are lost from body tissues. Loss of skin turgor, scaphoid abdomen, and weak pulse are characteristic of cholera. Various degrees of fluid and electrolyte loss are observed, including mild and subclinical cases.  The disease runs its course in 2 to 7 days; the outcome depends upon the extent of water and electrolyte loss and the adequacy of water and electrolyte repletion therapy. Death can occur from hypovolemic shock, metabolic acidosis, and uremia resulting from acute tubular necrosis.  Diagnosis  Rapid bacteriologic diagnosis offers relatively little clinical advantage to the patient with secretory diarrhea, because essentially the same treatment (fluid and electrolyte replacement) is employed regardless of etiology. Nevertheless, rapid identification of the agent can profoundly affect the subsequent course of a potential epidemic outbreak.  Because of their rapid growth and characteristic colonial morphology, V cholerae can be easily isolated and identified in the bacteriology laboratory, provided, first, that the presence of cholera is suspected and, second, that suitable specific diagnostic antisera are available. The vibrios are completely inhibited or grow somewhat poorly on usual enteric diagnostic media (MacConkey agar or eosin-methylene blue agar).  An effective selective medium is thiosulfate-citrate-bile salts-sucrose (TCBS) agar, on which the sucrose-fermenting cholera vibrios produce a distinctive yellow colony. However, the usefulness of this medium is limited because serologic testing of colonies grown on it occasionally proves difficult, and different lots vary in their productivity.  This medium is also useful in isolating V parahaemolyticus. They can also be isolated from stool samples or rectal swabs from cholera cases on simple meat extract (nutrient) agar or bile salts agar at slightly alkaline pH values. Following observation of characteristic colonial morphology with a stereoscopic microscope using transmitted oblique illumination, microorganisms can be confirmed as cholera vibrios by a rapid slide agglutination test with specific antiserum.  Classic and El Tor biotypes can be differentiated at the same time by performing a direct slide hemagglutination test with chicken erythrocytes: all freshly isolated agar-grown El Tor vibrios exhibit hemagglutination; all freshly isolated classic vibrios do not. In practice, this can be accomplished with material from patients as early as 6 hours after streaking the specimen in which the cholera vibrios usually predominate.  However, to detect carriers (asymptomatically infected individuals) and to isolate cholera vibrios from food and water, enrichment procedures and selective media are recommended. Enrichment can be accomplished by inoculating alkaline (pH 8.5) peptone broth with the specimen and then streaking for isolation after an approximate 6-hour incubation period; this process both enables the rapidly growing vibrios to multiply and suppresses much of the commensal microflora.  Diagnosis  Diagnosis can be made retrospectively by confirming significant rises in specific serum antibody titers in convalescents. For this purpose, conventional agglutination tests, tests for rises in complement-dependent vibriocidal antibody, or tests for rises in antitoxic antibody can be employed. Convenient microversions of these tests have been developed. Passive hemagglutination tests and enzyme-linked immunosorption assays (ELISAs) have also been proposed.  Cultures that resemble V cholerae but fail to agglutinate in diagnostic antisera (nonagglutinable or non-O group 1 vibrios) present more of a problem and require additional tests such as oxidase, decarboxylases, inhibition by the vibriostatic pteridine compound 0/129, and the “string test.” The string test demonstrates the property, shared by most vibrios and relatively few other genera, of forming a mucus-like string when colony material is emulsified in 0.5 percent aqueous sodium deoxycholate solution. Additional tests for enteropathogenicity and toxigenesis may be useful. Genetically based tests such as PCR are increasingly being used in specialized laboratories.  Control  Treatment of cholera consists essentially of replacing fluid and electrolytes. Formerly, this was accomplished intravenously, using costly sterile pyrogen-free intravenous solutions. The patient's fluid losses were conveniently measured by the use of buckets, graduated in half- liter volumes, kept underneath an appropriate hole in an army-type cot on which the patient was resting. Antibiotics such as tetracycline, to which the vibrios are generally sensitive, are useful adjuncts in treatment. They shorten the period of infection with the cholera vibrios, thus reducing the continuous source of cholera enterotoxin; this results in a substantial saving of replacement fluids and a markedly briefer hospitalization. Note, however, that fluid and electrolyte replacement is all-important; patients who are adequately rehydrated and maintained will virtually always survive, and antibiotic treatment alone is not sufficient.  Recently it has been recognized that almost all cholera patients and others with similar severe secretory diarrheal disease can be maintained by fluids given orally if the solutions contain a usable energy source such as glucose. Because of this discovery, packets containing appropriate salts are distributed by such organizations as WHO and UNICEF to cholera-afflicted areas, where they are dissolved in water as needed. One such formulation, called ORS for oral rehydration salts, contains NaCl, 3.5 g; KCl,1.5 g; NaHCO3, 2.5 g (or trisodium citrate, 2.9 g); and glucose, 20.0 g. This mixture is dissolved in 1 L of water and taken orally in increments. Flavoring may be added. Improved versions of ORS, including rice-based formulations that reduce stool output and can be made at home, have been recommended. Unfortunately, this technique, which will save countless millions of lives in developing countries, has not yet been widely accepted by practicing physicians in developed countries.  The possibility of pharmacologic intervention (e.g., a pill that will stop choleraic diarrhea after it has started), has been considered. Two drugs, chlorpromazine and nicotinic acid, have been effective in experimental animals, although the precise mechanism of action has yet to be defined.  Control  Like smallpox and typhoid, cholera—under natural circumstances—appears to affect only humans; therefore, V cholerae as an etiologic entity could conceivably disappear with the last human infection. Nevertheless, the spectrum of cholera-like diarrheal diseases probably will persist for some time.  Cholera is essentially a disease associated with poor sanitation. The simple application of sanitary principles—protecting drinking water and food from contamination with human feces—would go a long way toward controlling the disease. However, at present, this is not feasible in the underdeveloped areas that are afflicted with epidemic cholera or are considered to be cholera receptive. Meanwhile, development of a vaccine that would effectively prevent colonization and manifestations of cholera would be extremely helpful. As indicated above, such vaccines are presently being tested. Antibiotic or chemotherapeutic prophylaxis is feasible and may be indicated under certain circumstances. It also should be mentioned that the incidence of cholera is significantly higher in formula-fed than in breast-fed babies.  Present information indicates that V parahaemolyticus enteritis could be almost completely prevented by applying appropriate procedures to prevent multiplication of the organisms in contaminated seafood, such as keeping it refrigerated continually.   Other vibrios may be clinically significant also. These include non-O group 1 V cholerae. Vibrio parahaemolyticus, a halophilic Other (salt-loving) vibrio associated with enteritis is acquired by ingestion of raw or improperly cooked seafoods. Another halophilic vibrio, which ferments lactose and for this reason was called the L + vibrio, has recently been identified as V vulnificus. It has been associated with wound infections as well as fatal septicemias.  Other groups of vibrios, previously referred to as group F and EF-6, have recently been classified into species: V fluvialis, V Vibrio  hollisae, V furnissia, and V damsela. Vibrio mimicus is a recently described sucrose-negative species. Vibrio fetus, a group of anaerobic to microaerophilic spirally curved rods associated with venereally transmitted infertility and abortion in domestic animals, is now called Campylobacter jejuni and is considered to belong in the family Spirillaceae rather than Infections in the family Vibrionaceae. Campylobacter jejuni has been associated with dysentery-like gastroenteritis, duodenal and gastric ulcers, as well as with other types of infection, including bacteremic and central nervous system infections in humans.  Another vibrio-like organism, Helicobacter pylori (formerly known as C pylori) causes gastritis and predisposes to duodenal ulcers and gastric cancer. Although some similarities in habitat and other properties occur, members of the family Vibrionaceae are separated taxonomically from members of the family Enterobacteriaceae. The oxidase test (vibrios are usually oxidase positive) is particularly useful.  Other vibrios exist, and some of these may be responsible for diseases in fish and other lower animals. As vibrios are widely distributed in the environment, particularly in estuarine waters and in seafoods, reports of their isolation from patients with diarrheal disease do not necessarily always imply an etiologic relationship.  Cholera-like vibrios have been reported in Maryland's Chesapeake Bay but have not been associated with any human cases despite more than 15 years of extensive surveillance. These vibrios are probably nonpathogenic nonagglutinable (non-O group 1) vibrios, or the atypical O group 1 vibrios mentioned above, which do not contain the genes for toxin production, do not colonize, and are avirulent.  Other Vibrio Infections  Relatively little is known about the epidemiology of nonagglutinable vibrios. When sought, these vibrios have been found widely in brackish surface waters (sewers, marshes, bogs, and coastal areas), and are generally more numerous in warmer months. They appear to be free-living aquatic organisms; whether particular subsets are potential pathogens is not yet clear.  Strains isolated from humans with diarrheal disease more frequently give positive responses in assays for enterotoxins or enteropathogenicity, but the pathogenic mechanism of other isolates associated with shellfish remains undefined.An epidemiologic pattern is more evident with V parahaemolyticus, which is clearly part of the normal flora of coastal and estuarine waters throughout the world.  Although originally recognized in Japan, V parahaemolyticus enteritis has been reported virtually worldwide within the last decade. Its reported frequency varies widely, partly because of inherent differences in distribution and partly because many laboratories do not use the appropriate culture medium (TCBS) to isolate these organisms. Two types of clinical syndromes, both usually self-limited, have been observed.  The most common is a watery diarrhea, perhaps with associated abdominal cramps, nausea, vomiting, and fever, with a modal incubation period of 15 hours. A dysenteric syndrome with a short incubation period of 2 1/2 hours also has been described. In Japan, about 24 percent of reported cases of food poisoning are attributed to V parahaemolyticus.  The disease occurs primarily during summer, possibly reflecting the increased presence of the organism in the marine environment during those months, as well as the enhanced opportunity for it to multiply in unrefrigerated foods. It appears to be transmitted exclusively by food, primarily raw or improperly prepared seafood. As growth of this organism is inhibited at temperatures below 15° C, rapid cooling and refrigeration of seafoods that are eaten raw would vastly reduce the incidence of disease.  The organisms are killed by heating to 65° C for 10 minutes; therefore, properly handled cooked seafood should present no problem. The role played in virulence and pathogenesis by the thermostable direct hemolysin, which is responsible for the positive Kanagawa phenomenon (a hemolytic reaction around colonies growing on a particular blood agar medium), is not yet fully defined. This hemolysin is clearly associated with pathogenicity, but whether it is merely an associated marker or intimately involved in the disease process awaits further research. Be this as it may, only strains that possess the Kanagawa hemolysin are considered pathogenic. In laboratory studies, the isolated hemolysin has been reported to be cytotoxic, cardiotoxic, and lethal.