In this article we will discuss about:- 1. Introduction to Vibrio 2. The Organisms of Vibrio and their Characteristics 3. Pathogenesis and Clinical Features 4. Isolation and Identification 5. Association with Foods.
- Introduction to Vibrio
- The Organisms of Vibrio and their Characteristics
- Pathogenesis and Clinical Features of Vibrio
- Isolation and Identification of Vibrio
- Vibrio’s Association with Foods
1. Introduction to Vibrio:
Historically, cholera has been one of the diseases most feared by mankind. It is endemic to the Indian subcontinent where it is estimated to have killed more than 20 million people this Century. During the 19th Century there were a number of pandemics of ‘Asiatic cholera’ spread from the Indian subcontinent throughout Europe and the Americas.
It spread inexorably across Europe at a rate of about eight kilometres a day reaching England in 1831, where it thrived in the appalling overcrowded, insanitary conditions of the burgeoning towns and cities.
The approach of a second outbreak in 1848 prompted Parliament to establish the Central Board of Health which began the long task of improving sewerage and water supply systems. Similar apprehension of an approaching cholera outbreak in 1866 inspired the foundation of a similar Board in New York in the United States.
Pacini (1854) is credited with the first description of the etiological agent of cholera when he observed large numbers of curved bacilli in clinical specimens from cholera patients in Florence.
Pacini findings were not however generally accepted because of the widespread occurrence of similar but harmless vibrios in the environment. It was Robert Koch who firmly established the causal link between Vibrio cholerae and cholera when working in Egypt in 1886.
Koch isolated what is now known as the classical V. cholerae biotype which was responsible for most outbreaks of cholera until 1961. The El Tor biotype, first isolated in 1906 by Gotschlich from pilgrims bound for Mecca at the El Tor quarantine station in Sinai, Egypt, is responsible for the current (7th) pandemic.
This started in Celebes in Indonesia in 1961, reached Africa in 1970 and the Americas in 1991. Of the 594694 cases reported to the WHO in 1991, 391 220 were in South and Central America.
It was recognized in the 1930s that both biotypes are agglutinated by a single antiserum designated O1. Other strains of V. cholerae do not react with this antiserum and are termed non-agglutinable, or more correctly non-O1 strains, though some do produce cholera toxin.
In 1992 a new serotype, O139, was associated with epidemic cholera in India and Bangladesh and has also been isolated from cholera patients in Thailand
A number of other species of Vibrio have been recognized as pathogens causing wound and ear infections, septicaemia as well as gastrointestinal upsets (Table 7.9). In particular, V. parahaemolyticus, which was first shown to be an enteropathogen in 1951, is responsible for 50-70% of outbreaks of foodborne gastroenteritis in Japan.
V. fluvialis has been isolated -from sporadic cases of diarrhoea in some countries, particularly those with warm climates, although its exact role is uncertain since other enteropathogens were often present in the stool samples. V. mimicus, which produces diarrhoea, is distinguishable from V. cholerae only by its ability to produce acid from sucrose and acetoin from glucose.
V. vulnificus does not usually cause diarrhoea but severe extra-intestinal infections such as a life-threatening septicaemia. Patients normally have some underlying disease and have eaten seafood, particularly oysters about a week before the onset of illness.
2. The Organisms of Vibrio and their Characteristics:
Vibrios are Gram-negative pleomorphic (curved or straight), short rods which are motile with (normally) sheathed, polar flagella. Catalase and oxidase-positive cells are facultatively anaerobic and capable of both fermentative and respiratory metabolism. Sodium chloride stimulates the growth of all species and is an obligate requirement for some.
The optimum level for the growth of clinically important species is 1-3%. V. parahaemolyticus grows optimally at 3% NaCl but will grow at levels between 0.5% and 8%. The minimum aw for growth of V. parahaemolyticus varies between 0.937 and 0.986 depending on the solute used.
Growth of enteropathogenic vibrios occurs optimally at around 37 °C and has been demonstrated over the range 5-43 °C, although ≈ 10 °C is regarded as a more usual minimum in natural environments.
When conditions are favourable, vibrios can grow extremely rapidly; generation times of as little as 11 min and 9 min have been recorded for V. parahaemolyticus and the non-pathogenic marine vibrio, V. natrigens respectively.
V. parahaemolyticus is generally less robust at extremes of temperatures than V. cholerae. Numbers decline slowly at chill temperatures below its growth minimum and under frozen conditions a 2-log reduction has been observed after 8 days at — 18 °C. The D49 for V. parahaemolyticus in clam slurry is 0.7 min compared with a D49 for V. cholerae of 8.15 min measured in crab slurry.
Other studies have recorded higher ID values for V. parahaemolyticus, for instance 5 min at 60 °C produced only 4- 5 log reductions in peptone/3% NaCl. Pre-growth of the organism in the presence of salt is known to increase heat resistance.
V. parahaemolyticus will grow best at pH values slightly above neutrality (7.5-8.5) and this ability of vibrios to grow in alkaline conditions up to a pH of 11.0 is exploited in procedures for their isolation. Vibrios are generally viewed as acid sensitive although growth of V. parahaemolyticus has been demonstrated down to pH 4.5-5.0.
The natural habitat of vibrios is the marine and estuarine environment. V. cholerae can be isolated from temperate, sub-tropical, and tropical waters throughout the world, but seem to disappear from temperate waters during the colder months.
Long-term survival may be enhanced by attachment to the surfaces of plants and marine animals and a viable but non-culturable form has also been described where the organism cannot be isolated from the environment using cultural techniques even though it is still present in an infective form (see also Campylobacter).
V. parahaemolyticus is primarily associated with coastal inshore waters rather than the open sea. It cannot be isolated when the water temperature is below 15 °C and cannot survive pressures encountered in deeper waters.
The survival of the organism through winter months when water temperatures drop below 15 °C has been attributed to its persistence in sediments from where it may be recovered even when water temperatures are below 10 °C.
Most environmental isolates of both V. cholerae and V. parahaemolyticus are nonpathogenic. The majority of the V. cholerae are non-O1 serotypes and even those that are not tend to be non-toxigenic. Similarly 99% of environmental strains of V. parahaemolyticus are non-pathogenic.
3. Pathogenesis and Clinical Features of Vibrio:
Cholera usually has an incubation period of between one and three days and can vary from mild, self-limiting diarrhoea to a severe, life-threatening disorder.
The infectious dose in normal healthy individuals is large when the organism is ingested without food or buffer, of the order of 1010 cells, but is considerably reduced if consumed with food which protects the bacteria from stomach acidity.
Studies conducted in Bangladesh indicate that 103-104 cells may be a more typical infectious dose. Individuals with low stomach acidity (hypochlorohydric) are more liable to catch cholera.
Cholera is a non-invasive infection where the organism colonizes the intestinal lumen and produces a potent enterotoxin. In severe cases, the hyper-secretion of sodium, potassium, chloride, and bicarbonate induced by the enterotoxin results in a profuse, pale, watery diarrhoea containing flakes of mucus, described as rice water stools.
The diarrhoea, which can be up to 201 day -1 and contains up to 108 vibrios ml-1, is accompanied by vomiting, but without any nausea or fever.
Unless the massive losses of fluid and electrolyte are replaced, there is a fall in blood volume and pressure, an increase in blood viscosity, renal failure, and circulatory collapse. In fatal cases death occurs within a few days. In untreated outbreaks the death rate is about 30-50% but can be reduced to less than 1 % with prompt treatment by intravenous or oral rehydration using an electrolyte g-1 lucose solution.
The reported incubation period for V. parahaemolyticus food poisoning varies from 2 h to 4 days though it is usually 9-25 h.
Illness persists for up to 8 days and is characterized by a profuse watery diarrhoea free from blood or mucus, abdominal pain, vomiting and fever. V. parahaemolyticus is more entero-invasive than V. cholerae, and penetrates the intestinal epithelium to reach the lamina propria. A dysenteric syndrome has also been reported from a number of countries including Japan.
Pathogenicity of V. parahaemolyticus strains is strongly linked to their ability to produce a 22 kDa, thermo-stable, extracellular haemolysin. When tested on a medium known as Wagatsuma’s agar, the haemolysin can lyse fresh human or rabbit blood cells but not those of horse blood, a phenomenon known as the Kanagawa reaction. The haemolysin has also been shown to have enterotoxic, cytotoxic and cardio-toxic activity.
Most (96.5%) strains from patients with V. parahaemolyticus food poisoning produce the haemolysin and are designated Kanagawa-positive (Ka+) while 99% of environmental isolates are Ka —. Volunteer feeding studies have found that ingestion of 107-1010 Ka- cells has no effect whereas 105-107 Ka+ cells produce illness. A number of other virulence factors have been described but have been less intensively studied.
V.vulnificus is a highly invasive organism that causes a primary septicaemia with a high fatality rate (≈50%). Most of the cases identified occurred in people with preexisting liver disease, diabetes or alcoholism. Otherwise healthy individuals are rarely affected and, when they are, illness is usually confined to a gastroenteritis.
In foodborne cases, the symptoms of malaise followed by fever, chills and prostration appear 16-48 h after consumption of the contaminated food, usually seafood’s, particularly oysters. Unlike other vibrio infections, V. vulnificus infections require treatment with antibiotics such as tetracycline.
4. Isolation and Identification of Vibrio:
The enrichment media used for vibrios exploit their greater tolerance for alkaline conditions. In alkaline peptone water (pH 8.6-9.0) the incubation period must be limited to 8 h to prevent overgrowth of the vibrios by other organisms. Tellurite/ bile salt broth (pH 9.0-9.2) is a more selective enrichment medium and can be incubated overnight.
The most commonly used selective and differential agar used for vibrios is thiosulfate/citrate/bile salt/sucrose agar (TCBS).
The medium was originally designed for the isolation of V. parahaemolyticus but other enteropathogenic vibrios grow well on it, with the exception of V. hollisae. V. parahaemolyticus, V. mimicus, and V. vulnificus can be distinguished from V. cholerae on TCBS by their inability to ferment sucrose which results in the production of green colonies. V. cholerae produces yellow colonies. Individual species can then be differentiated on the basis of further biochemical tests.
V. cholerae is divided into the serogroups O1 and non-O1. O1 strains can be further classified into the classical (non-haemolytic) or E1 Tor (haemolytic) biotypes each of which can be subdivided by serotypirtg into one of three groups: Ogawa, Inaba, or Hikojima. Clinical strains of V. parahaemolyticus can be serotyped for epidemiological purposes using a scheme based on 11 thermo-stable O antigens and 65 thermolabile K (capsular) antigens
5. Vibrio’s Association with Foods:
Cholera is regarded primarily as a waterborne infection, though food which has been in contact with contaminated water can often serve as the vehicle. Consequently a large number of different foods have been implicated in outbreaks particularly products such as washed fruits and vegetables which are consumed without cooking.
Foods coming from a contaminated environment may also carry the organism, for example sea-foods and frog’s legs. In the current pandemic in South and Central America, an uncooked fish marinade in lime or lemon juice, ceviche has been associated with some cases.
V. parahaemolyticus food poisoning is invariably associated with fish and shellfish. Occasional outbreaks have been reported in the United States and Europe, but in Japan it is the commonest cause of food poisoning. This has been linked with the national culinary habit of consuming raw or partially cooked fish, although illness can also result from cross-contamination of cooked products in the kitchen.
Though the organism is only likely to be part of the natural flora of fish caught in coastal waters during the warmer months, it can readily spread to deep-water species through contact in the fish market and it will multiply rapidly if the product is inadequately chilled.