Cholera Vibrios: An Overview


The Discovery of Cholerae Causative Agent and the History of Cholera Pandemics


  • Cholera-like diseases were known from the times of antiquity. Nevertheless, the first registered epidemic outbreak of cholera emerged in India in 1817. It spread throughout the Indian sub-continent and finally was established as the first cholera pandemic in Asia.
  • In 1883 Robert Koch discovered the causative agent of cholera, later termed as classical Vibrio cholerae biotype (biovar). Later in 1906 another biovar, El Tor vibrio was isolated by E. Gotschlich on the Sinai Peninsula from the body of dead pilgrim.
  • The end of XIX and the beginning of XX century were hallmarked with six cholera pandemics. In 1923 the 6th pandemic of cholera affected the continents of southern hemisphere, North America and Europe.
  • In 1961 the seventh pandemic started from Indonesia, then spread to India and the Middle East, appeared in Africa in the 1970s and finally achieved South America at 1990s.
  • The 5th and 6th pandemics of cholera were caused by V. cholerae serogroup O1 of the biotype “classical”. Nevertheless, the 7th pandemic was produced by serogroup O1 V. cholerae of biotype El Tor.
  • Serogroup conversion of V. cholerae gave rise to novel V. cholerae serogroup O139 in 1992. It provoked the emergence of a new large epidemic in Bangladesh and India.
  • Multiple cholera cases caused by O139 strains are being registered now in Southeast Asia. Sometimes this is regarded as the start of putative eighth cholera pandemic.
  • However, O1 El Tor isolates are also detected on these territories as well as in other parts of the world. For instance, the great epidemic of cholera in 2010 in Haiti was caused by El Tor biovar.

Classification


  • Cholera vibrios belong to the order Vibrionales, family Vibrionaceae, genus Vibrio and species V. cholerae. More than 200 serogroups of Vibrio cholerae were described but only the members of O1 and recently discovered O139 serogroup were proven to cause the epidemic disease. Classical Vibrio cholerae biotype and El Tor vibrio biotype pertain to O1 serogroup.
  • Representatives of Vibrio cholerae beyond the serogroups O1 or O139 are accidental agents of moderate human diarrheal disorders being of lesser clinical relevance.
  • Another member of Vibrio genus V. рarahaemolyticus may cause diarrhea in humans; in addition, V. vulnificus engenders some individual cases of human wound infections or septicemia.

Structure and Properties of Cholera Vibrios


Morphology

  • Cholera vibrios are comma-shaped gram-negative curved rods 2-4 μm long. In old cultures or on artificial media these bacteria occur in grains, straight rods, threads or spiral forms.
  • Vibrios are non-sporeforming bacteria. Both biotypes from O1 serogroup are lack of capsule. However, they synthesize exopolysaccharide that provides microbial biofilm formation.
  • It confers also the resistance of vibrios to chlorines and bacteriophages. V. cholerae of O139 group as well as other vibrios can produce capsule.
  • Cholera vibrios are monotrichous motile microorganisms that usually carry one polar flagellum. Bacteria possess numerous pili responsible for microbial colonization.
  • Among them are mannose-sensitive hemagglutinin that ensures vibrio adherence to the chitin of marine zooplankton, and toxin-coregulated pili (TCP), which promote microbial intestinal attachment as well as reception of enterotoxin-encoding bacteriophage СТХφ.

Cultivation

  • Cholera vibrios are aerobic or facultatively anaerobic bacteria. They actively grow on basic nutrient media with increased salt concentration of 2-3% NaCl (halotolerant bacteria). The temperature range for culture is from 14 to 42°C with optimum at 37°C.
  • Vibrios can withstand alkaline pH, thereby they readily propagate at pH 8.0-9.5. Also they are resistant to bile salts.
  • In case of nutrient deprivation bacteria are capable of transforming into viable, but non-culturable organisms.
  • On solid nutrient media V. cholerae produce opaque, granular, smooth, round and convex dome-shaped colonies with a light-blue shine. In alkaline peptone broth vibrio cultures form a pellicle that contains agglomerated cholera vibrios.
  • Gelatin cultivation results in transparent granular colonies that resemble broken glass on a microscopy. The growth is followed by gelatin liquefaction.
  • Various enrichment and selective media (e.g. alkaline MPA or alkaline nutrient broth) are applied for V. cholerae cultivation. On thiosulfate-citrate-bile-sucrose (TCBS) agar with indicator bromine thymol blue the bacteria produce yellow colonies.

Biochemical properties

  • Cholera vibrios have a broad spectrum of biochemical activity. All vibrios are oxidase-positive that discerns them from Enterobacteriaceae representatives. They ferment various substrates with acid end products (glucose, maltose, sucrose, mannose, mannitol, galactose, starch, glycerol and others).
  • B. Heiberg divided vibrios into 8 groups according to their biochemical activity. Both classical Vibrio cholerae biotype and El Tor vibrio pertain to 1 Heiberg’s group and ferment sucrose and mannose whereas arabinose and lactose not.
  • Bacteria produce ammonia, indole, and reduce nitrates to nitrites. Also they secrete a number of proteases, coagulate serum and milk, liquefy gelatin.
  • Cholera vibrios render variable hemolytic and hemagglutinating properties.

Antigenic structure

  • Different vibrios share a common flagellar heat-labile H antigen.
  • Somatic lipopolysaccharide (LPS) heat-stable O-antigen is responsible for microbial antigenic specificity. More than 200 serogroups of vibrios were distinguished by O-antigen variations.
  • It was mentioned above that classical Vibrio cholerae biotype and Vibrio cholerae biotype El Tor pertain to O1 serogroup. O1 antigen contains A, B and C antigenic variations.
  • Thus, three main serotypes within O1 serogroup were established: Ogawa (AB), Inaba (AC), and Hikojima (ABC).
  • Vibrio cholerae O139 was proven to originate from El Tor vibrio. It happened after acquisition of gene cluster encoding the synthesis of novel O139 LPS antigen, which thereby substituted initial O1 antigen of El Tor.

Virulence factors

  • Numerous adherence factors of cholera vibrios play a substantial role in disease pathogenesis. Most important are toxin-coregulated pili (TCP).
  • These pili are encoded by Vibrio pathogenicity island VPI. Temperate bacteriophage VPIφ is assumed to deliver VPI genes into cholera vibrios.
  • TCP pili are responsible for microbial intestinal colonization. Moreover, TCP, expressed by cholera vibrio, act as specific receptors to bacteriophage СТХφ (cholera toxin encoding phage). СТХφ code for the production of cholerogen-enterotoxin by initially non-toxigenic bacteria.
  • LPS of the cell wall of O1 serogroup bacteria and the capsule of O139 group strains accelerate microbial intestinal colonization. Also bacterial LPS renders endotoxin activity.
  • Exopolysaccharide of V. cholerae actively participate in biofilm formation. Capsule of O139 strains protects them from phagocytosis.
  • The enzyme hemagglutinin protease facilitates microbial detachment from enterocytes thereby promoting further microbial spread along the intestinal wall.
  • Potent enterotoxin-cholerogen is the major virulence factor of cholera vibrios. Cholera toxin is a heterodimer, composed of one A subunit in combination with five В subunits with total molecular mass of 84 kDa.
  • B-subunits of toxin bind to the intestinal cells via cell membrane ganglioside receptor. Subunit A is translocated through cytoplasmic membrane into intestinal epithelial cells, undergoes thiol-dependent activation and promotes ADP-ribosylation of cell G-proteins.
  • This stimulates cellular adenylate cyclase resulting in great increase of cAMP concentration. The rise of intracellular cAMP concentration blocks active sodium chloride absorption and increases chloride and bicarbonate secretion.
  • The latter results in passive water loss with development of massive diarrhea. This is followed by marked decrease of intravascular volume, life-threatening hypoperfusion of critical organs and hypotension.
  • The cholera vibrios produce a number of invasive enzymes, e.g. hyaluronidase, collagenase, fibrinolysin, lecithinase, neuraminidase, and various proteinases.

Resistance


  • Vibrios are natural components of aquatic ecosystems. Colonization of zooplankton, plants, filamentous green algae, crustaceans and other marine inhabitants protect bacteria from unfavorable environmental conditions.
  • The El Tor vibrio biotype is characterized by relatively high resistance. It stays viable for more than 1 month in sea and river waters, up to 10 days in various foodstuffs, etc.
  • Cholera vibrios can live in feces for about a month; also they readily survive at low temperature.
  • Vibrios are sensitive to heating, UV light and desiccation (e.g, heating at 100°С immediately kills bacteria). Likewise, they are very susceptible to disinfectant treatment and acid exposure. Low concentrations of hydrochloric acid inactivate bacteria within one minute.

Pathogenesis and Clinical Findings in Cholera


  • Cholera vibrios inhabit the water of rivers, seas and oceans. Most environmental O1 strains are lack of cholerogen expression, but only toxigenic V. cholerae can cause the disease.
  • It is considered that natural strains acquire a number of virulence genes from pathogenic microbial variants, and these events can occur both in external and gastrointestinal environment.
  • The emergence of virulent V. cholerae strain results from the cascade of horizontal gene transfers that eventually convert non-pathogenic aquatic bacterium into life-threatening human pathogen.
  • Now it is generally assumed that non-toxigenic bacteria become virulent only after transduction with several temperate bacteriophages.
  • The first transduction event confers microbial cell to express toxin-coregulated pili (TCP) – receptors for cholera toxin-encoding phage СТХφ. Next cell transduction with СТХφ allows affected bacteria to produce cholera enterotoxin.
  • Moreover, phage transduction is supposed to be responsible for bacterial LPS structure changes. LPS change results in creation of new serologic variants of bacteria (e.g., vibrios of O139 serogroup), which escape from established human population immunity and provoke new large outbreaks of the infection.
  • Cholera is anthroponotic disease.It is transmitted from sick persons and carriers by fecal-oral route with infected foods or water. The causative agent is also carried by flies, or can be transmitted by contact route through contaminated hands.
  • Short incubation period of disease lasts from several hours to 5-6 days. After oral ingestion of contaminated water or food most of V. cholerae are killed by acidity of gastric juice. Thus, the infectious dose for cholera vibrios is rather high (in the range from 106 to 1011 microbial cells).
  • The rest of bacteria colonize the intestinal epithelial cells of small intestine, attach to the microvilli, and ultimately start to produce enterotoxin-cholerogen. Toxin expression is activated by gradual decrease of bile concentration along the small intestine.
  • Action of cholera enterotoxin leads to the development of disease symptoms. Patient’s stools resemble “rice water,” and contain many epithelial cells, mucus, and large number of vibrios.
  • In severe cases profuse watery diarrhea and continuous vomit results in lowering of body’s temperature, hypovolemic and hypotensive shock with lethal outcome within first 12 h of disease.
  • The total fluid loss can achieve 20-30 l per day in adults. Without adequate compensatory infusion therapy the mortality rate exceeds 20%.
  • Abortive and mild disease forms are observed in majority of cases of El Tor vibrio cholera. Patient’s carrier state rarely exceeds 1 month.
  • Post-infectious immunity is high-grade but of short duration. The immunity is both antibacterial and antitoxic; antitoxic antibodies confer most efficient protection against the disease.


Laboratory Diagnosis of Cholera


  • The specimens are collected from stool, vomit, autopsy material, water, foodstuffs, etc.
  • Microscopy is used as a preliminary test. The agglomerated gram-negative cholera vibrios resembling fish shoals appear in slide smears from stool.
  • Rapid diagnosis procedures include dark field microscopy of the stool specimens that reveal comma-like motile bacterial cells, and immunofluorescence assay.
  • Identification of cholera causative agent is performed in several steps.
  • The specimens are inoculated into alkaline peptone water and alkaline agar. After short 6-hour incubation at 37°C thin biofilm of aggregated bacteria is formed. The biofilm material is gram-stained, tested for oxidase, and examined in slide agglutination test both with O1 and O139 antisera taken in titer 1/100.
  • If the first alkaline broth cultivation results in scarce microbial growth, the material is inoculated again into alkaline peptone broth.
  • After primary examination alkaline broth culture is planted onto alkaline agar, or TCBS medium. TCBS growth reveals yellow vibrio colonies due to sucrose fermentation.
  • The vibrio culture is examined by repeat slide agglutination test and oxidase test. The latter should be positive for all vibrios.
  • To obtain the pure culture the isolate is further planted on slant alkaline agar. The final identification of culture is made by agglutination reaction with O1 and O139 sera, biochemical tests (mannose, sucrose and arabinose fermentation), positive indole test, and by susceptibility to a number of specific phages.
  • Molecular genetic methods of vibrio typing are used in specialized reference centers for epidemiological studies.
  • Classical V. cholerae and El Tor vibrios can be distinguished by the number of tests: both biotypes are sensitive to specific bacteriophages; El Tor biotype is resistant to polymyxin B, it expresses hemolysin and produces acetoin with positive Voges-Proskauer test. Classical V. cholerae has opposite traits.

Treatment and Prophylaxis of Cholera


  • The urgent treatment of cholera is based mainly on infusion replacement therapy that compensates the loss of water and electrolytes.
  • In case of adequate infusion the patient recovers from the disease. Different antibiotics, affecting gram-negative flora, can be used to facilitate convalescence. Usually oral tetracyclines are administered.
  • Antimicrobial chemoprophylaxis and vaccine prophylaxis may be used for disease prevention in family contact persons.
  • For specific prophylaxis phenol-killed vaccine and cholerogen toxoid are occasionally used now. Nevertheless, they confer only the short-term protection for 6-12 months in 50-80% of vaccinated individuals.
  • Elaboration of modern cholera vaccines is based on live microbial strains, but this work should account the possibility of attenuated vaccine bacteria to acquire virulence genes from environmental strains.
  • Non-specific prophylaxis of cholera includes the improvement of sanitation, prevention of water and foodstuffs pollution, protection of sources of water supply; proper hygienic and sanitary control measures and cholera surveillance.
  • First cases of disease should be verified and carefully registered with subsequent isolation and hospitalization of all patients, observation and laboratory testing of all contact individuals, current and final disinfection in departments for cholera patients.