Laboratory diagnosis of different Vibrio species from a practical point of view for UG and PG students.

1. VIBRIO – Practical Notes
 Vibrios are
 Gram-negative,
 Asporogenous,
 Straight curved, or comma-shaped rods.
 Motile (Darting) by means of sheathed monotrichous or multitrichous polar flagella.
 Halophilic
 Predominately oxidase-positive.
 Sodium chloride stimulates the growth of all vibrios an is required by most.
 The minimal concentration of optimum growth varies from 0.029 to 4.1 percent NaCl.
 Facultatively anaerobic.
 Vibrios are ubiquitous, in aquatic environments.
 They are found in the water column, in sediments, and associated with plants,
invertebrates, and vertebrate animals within those, ecosystems.
 Twelve species are considered human pathogens
 V. alginolyticus
 V. cholerae,
 V. cincinnatiensis,
 V. damsela (P damselae),
 V. fluvialis,
 V. furnissii,
 V. harveyi (synony V. carchariae),
 V. hollisae,
 V. metschnikovii,
 V. mimicus
 V. parahaemolyticus, and
 V. vulnificus.
 These species are the most frequently isolated by clinical laboratories.
 HABITAT
 Vibrios are ubiquitous aquatic organisms whose range is determined by salinity,
temperature, and nutrient availability.
 In temperate climates, vibrios peak in the water column during the summer months and
may be very abundant.
 The seasonal distribution of human infections generally correlates with this increase.
 When water temperature drops, so does the concentration of vibrios and they may
become limited to sediments.
 Vibrios that require lower concentrations of salt, such as V. cholerae and V. mimicus,
can be found in fresh-water lakes and rivers as well as estuarine and marine
environments.
 Fecal contamination of drinking water is important in the epidemiology of cholera.
 Infection of humans does act as an amplification cycle that is important in the epidemic
spread of V. cholerae.
 Colony Characteristics
 Vibrios can produce a large variety of colony morphologies on nonselective media.
 Most produce smooth, convex, creamy colonies.
 Acapsular isolates tend to produce translucent colonies
 Encapsulated strains are opaquer and may even be somewhat mucoid for heavily
encapsulated V. vulnificus strains.

2.  Vibrios can also switch between the common smooth morphology and a wrinkled, or
rugose, colony morphology that is adherent to the agar and difficult to emulsify.
 In V. cholerae, this phenotype is associated with the production of an
exopolysaccharide and increased resistance to chlorine, ultraviolet (UV) light, and
other environmental stresses.
 V. parahaemolyticus, V. harveyi, and V. alginolyticus may swarm on solid media due
to the production of lateral flagella.
 Swarming occurs most frequently at room temperature.
 CULTURE
 Samples for vibrios should be processed expeditiously and never refrigerated.
 Fecal specimens should be placed in Cary–Blair transport media.
 Enrichment broths such as alkaline peptone water, incubated for 6–8 h, can be useful
for increasing recovery of V. cholerae from formed stools, food samples, and water
samples.
 A second enrichment step is often used for food and water samples.
 Vibrios grow rapidly on most nonselective media containing 0.5–1.0 percent NaCl.
 Most vibrios will not grow in nutrient agar without added salt
 The range of salt concentrations that support growth is an important test for identifying
the many species of vibrios.
 The most commonly used selective media is thiosulfate-citrate-bile saltssucrose
(TCBS) agar.
 In addition to selecting for vibrios, TCBS differentiates strains by their ability to
ferment sucrose.
 YELLOW COLONIES (ferment sucrose)
 V. cholerae,
 V. alginolyticus, and
 V. fluvialis.
 GREEN COLONIES (nonsucrose fermenting)
 V. vulnificus
 V. mimicus
 V. parahaemolyticus
 P. damselae
 V. hollisae
 V. metschnikovii.
 Other selective media include
 Taurocholate tellurite gelatin agar (TTGA) (or Monsur’s agar)
 Polymyxin-mannose-tellurite agar
 Thiosulfate-chlorideiodide agar.
 Vibrios will also grow well on blood agar and usually as clear lactose negative colonies
on MacConkey agar after overnight incubation.
 Blood agar plates from stool cultures should be examined for oxidase positive colonies
to increase recovery of vibrios and Aeromonas spp.
 Growth Characteristics on BAP
 Blood agar is particularly useful for isolation, recognition, and identification of vibrios
as it allows one to test individual colonies for oxidase production.
 V. cholerae and V. vulnificus smooth, creamy, buffcolored colonies.
 V. metschnikovii, and V. alginolyticus flat grayish glistening colonies and
 V. alginolyticus swarming growth.

3.  -hemolytic on T-soy agar with five percent sheep blood
 V. mimicus,
 V. hollisae,
 V. fluvialis,
 P. damsela,
 V. metschnikovii
 Classical biotype V. cholerae O1 are nonhemolytic
 Most strains of eltor biotype, serogroup O139, and non-O1, non-O139 V. cholerae
are -hemolytic.
 V. metschnikovii produces a double zone of hemolysis.
 Alpha-hemolytic or nonhemolytic species include V. parahaemolyticus, V.
alginolyticus, and V. vulnificus.
 Although not b-hemolytic on sheep-blood agar, V. parahaemolyticus is b-hemolytic
on Wagatsuma agar, which contains human blood. This is known as the KANAGAWA
PHENOMENON and is associated with virulence.

4. LABORATORY ISOLATION AND IDENTIFICATION
 The first step in laboratory identification of vibrios is to assign the isolate to the
correct genus.
 This is becoming more challenging because of
 Changes in phenotype,
 Discovery of more Vibrio spp. with unusual characteristics
 Many laboratories no longer have access to conventional biochemicals.

5.  It is important for the laboratory to proactively look for vibrios in clinical
specimens, particularly when there is a history of
 Consumption of raw or undercooked shellfish,
 Exposure of wounds to seawater, or
 Foreign travel.
 Vibrios, on nonselective media
 Similar in appearance to enterics, Aeromonas spp., and Plesiomonas shigelloides
 May be overgrown by other bacteria, particularly in stool cultures.
 Selective agar TCBS
 Can increase recovery of vibrios.
 Growth and colony morphology on TCBS agar is helpful in identifying vibrios.
 Not all vibrios will grow on TCBS
 There is significant lot-to-lot variation in selectivity.
 The oxidase test
 particularly critical for separating vibrios, Aeromonas spp., and Plesiomonas
shigelloides fromenteric gram-negative rods.
 Vibrios (except V. metschnikovii and V. gazogenes), aeromonads, and P.
shigelloides are oxidase-positive.
 Triple sugar iron (agar) (TSI)
 Can be useful to separate vibrios from Enterobacteriaceae and oxidase-positive
nonfermentors.
 Vibrios ferment sugars but do not produce gas from glucose (except some strains
of V. furnissii and P. damsela).
 Susceptibility to the vibriostatic compound 2,4-diamino-6,7-diisopropyl-
pteridine phosphate (O/129) used to be a key test for identifying vibrios.
 Unfortunately, in recent years, resistance to O/129 has become widespread in V.
cholerae.
 The string test and growth in 6 percent NaCl remains valuable for identifying
vibrios. The string test is performed by mixing a large loop of growth from a
noninhibitory agar with a drop of 0.5 percent sodium deoxycholate on a glass
microscope slide.
 The deoxycholate lyses vibrios, releasing the cellular contents, which causes the
solution to become viscous.
 When the loop is slowly lifted off the slide, a viscous ‘string’ is visible between the
slide and the loop-like cheese on a particularly gooey pizza.
 Some strains of V. parahaemolyticus, V. cincinnatiensis, and P. damsela give
variable results.
 Most Aeromonas spp., P. shigelloides, and enterics are string-test-negative.
 The salt requirement is tested by growth in nutrient broth with 0 percent or 6 percent
NaCl added.
 Vibrios can grow at 6 percent NaCl, while aeromonads, P. shigelloides, and
enterics cannot.
 The halophilic vibrios cannot grow at 0 percent NaCl.
 Salt broths must be inoculated lightly so that they are not visibly turbid before
incubation, and the inoculum should be fresh from a nonselective medium.
 The tubes should be examined at 24 h, but negative tubes may be held for up to 7
days.

6.  For definitive, reliable identification, classical tube biochemicals should be used and
supplemented to a final concentration of 1 percent NaCl.
 Tests should be incubated in ambient air at 35–37C and read daily for up to 3 days.
 V. cholerae and V. mimicus can be differentiated from other vibrios by their ability to
grow in nutrient broth without added salt.
 V. cholerae is sucrose-positive and V. mimicus is sucrose-negative.
 Different concentrations of salt from one to eight percent can help differentiate species.
 Lysine decarboxylase (LDC), ornithine decarboxylase (ODC), and arginine
dihydrolase (ADH) are key tests for dividing vibrios into groups.
 V. hollisae is the only clinical species that is negative for all three tests.
 P. damselae, V. fluvialis, and V. furnissii are ADH-positive.
 Other critical tests are
 Indole,
 Voges–Proskauer,
 Simmon’s citrate
 fermentation of arabinose.
 Fermentation of myoinositol is key for identification of V. cincinnatiensis

9. Some Important Biochemical Identification Features of Non Cholera Vibrios
Organism Colony
colour on
TCBS
O
129
VP NaCl Conc.
Survival
Sugar
fermentation
V.
parahemolyticus
GREEN R
V. mimicus GREEN 0%
V. vulnificus Lactose/
Cellobiose
V. fluvialis YELLOW -VE Cellobios
(+ve)
V. anginolyticus YELLOW +VE Cellobios
(-ve)

10. API 20/E- V. cholerae identification

11. BD CRYSTAL ID System
Molecular detaction Genes targeted are
 16S rDNA
 toxR
 vcg
 tcpA
 tdh
 tdh2x

13. Vibriocholerae oxidase test in a positive reaction, the bacterial growth
becomes dark purple within 10 seconds. Positive and negative controls should
be tested at the same time.

14. V. cholerae on MAC agar.
V. cholerae O-129 susceptibility test.

15. A V. parahemolyticus.

17. IDENTYTHE PROBABLE CAUSATIVE ORGANISM WITH
JUSTIFICATION?