Vibrios, that comma shaped pest. – A Survey of the the Marine Microbes

Vibrios – A Survey of the Marine Microbes

©1998 Jonathan Lowrie

Introduction

Just mentioning the word “Vibrio” around veteran aquarists can cause quite a stir.  These small comma shaped bacteria, when discussed as a genus, refer to  gram-negative, motile, facultatively anaerobic curved rods.  They can also spell disaster for marine aquarists, as Vibrios are pathogeneic to many marine organisms.  There are tentatively over 175 species of Vibrios, and possibly many more when  sub-strains are taken into account.  Some Vibrios release enterotoxins that affect intestinal tissue, while others release exotoxins that are so fast acting that   mortality can result within hours after exposure.  As such, certain species pose a risk not only to various marine animals, but also to humans who are in contact with them.  The following is an overview of some of the more well known Vibrios in the marine environment, their identification, effect, and treatment for both marine animals and humans.

General Description

Vibrios belong to the family Vibronacae, a group composed of curved gram-negative, catalase and oxidase producing bacteria that include three genera: Vibrio, Aeromonas, and Plesiomonas.  Gram-negative rods, as a group, are considered important because of the public health risks typically associated with them. The gram negative rods can be further divided into: aerobic rods and cocci, facultative anaerobic rods, and anaerobic straight, curved, and helical rods - typically motile with polar flagella.  The term "vibrioid" is used to describe the typical curvature in their shape. Vibrio belong to the facultative aerobe group, as they can grow under both aerobic and anaerobic conditions. They are fermentative to a variety of substrates, can reduce nitrate to nitrite, and many are capable of digesting chitin and cellulose

The following chart lists common facultative anaerobic gram-negative rods that are pathogenic to man (and many higher animals), exclusive of Vibrio

Escherichia coli	Opportunistic and occasionally lethal infections
Shigella dysenteriae	dysentery
Salmonella typhi	Typhoid fever
Salmonella sp.	Food poisoning
Klebsiella pneumoniae	Pneumonia
Yersinia pestis	Bubonic plague
Pasteurella multocida	Animal bite infections
Haemophilus influenzae	Meningitis, pediatric disease

Location

Vibrios are found worldwide, primarily in water, and are often found in marine and brackish environments such as coastal waters and estuaries. They are found most often in nutrient rich, warm, polluted and relatively unflushed areas.  Vibrios also prefer alkaline environments with a pH of 8.5-9.0. Most also grow best in salinites that range from 5-20 ppt. Both pathogenic and non-pathogenic strains are commonly isolated from bays, river outlets, and sewage treatment sites, as well as some species occurring in oceanic waters.  As such, many  are halophilic, meaning “loving salt . Most marine Vibrios can grow in sea water with ammonium and glycerol alone as nutrients. This environment is also ideal for the shellfish so commonly associated with  Vibrio infections.  Those species common to open ocean and low nutrient water tend to be smaller and coccoid in shape.

Much work needs to be done is isolating various sources for Vibrios in the environment, as they are periodically found associated with various micorenvironments such as sediments, zooplankton, shellfish, worms, plants, and the intestinal tracts of marine vertebrates.  Vibrios are also found normally in the intestinal tract of animals feeding on zooplankton, and in sediments enriched with the feces of animals who have fed on zooplankton. Many other Vibrios exist in the marine environment, some completely unknown and/or unspeciated, and may be more or less pathogenic to fish and humans.

Occurrence of Some Common Vibrios

	Open ocean	Estuaries	Mollusks	sediment	Fish	Crust aceans	Corals	Fresh water
aesturaianus		X
alginolyticus	X	X	X	X	X	X	X	X
anguillarum	X	X	X		X	X		X
campbelli			X
carchariae	X		X		X	X		X
Charcharia							X
cholerae	X	X			X			X
cincinnatiensis
damsela	X		X		X
diazotrophicus		X
fischeri	X	X			X	X
fluvialis		X
furnissii
harevyi			X		X
hollisae
marinus	X	X	X			X		X
Mediterranei							X
metschinikovii	X	X	X			X		X
mimicus
ordalli	X	X	X		X	X
orientalis			X
parahaemolyticus		X	X	X	X	X	X
pelagia	X		X
splendidis			X			X
vulnificus		X	X	X	X	X	X

Biology

Vibrios have a fermentative and respiratory metabolism.  They are typically flagellated, contributing to their nature as highly pathogenic. Generally, Vibrios are of high tissue virulence, although not all have high epidemic virulence. In other words, they are generally not highly contagious. Hemolysins, exotoxins, and enterotoxins are commonly produced by Vibrios as part of their pathogenic response in affected animals.  At least some species also produce their own antimicrobial agents effective against other bacteria. These compounds are vibriocin and microcin. Interestingly, V. cholerae and other Vibrio spp. from estuarine and other marine environments also produce tetradotoxin, a potent neuromuscular toxin previously associated only with pufferfish. 

Some Vibrios are called "swarers," and have lateral, rather than polar, flagella that aide in their attachment to substrates.

Commercial Hazards

Vibrio is not only a human public health hazard, but it’s an economic hazard since it can infect shellfish and become a common economic problem when harvests are contaminated with Vibrios.  Many of the recent "seafood scares" in public media have been based on oysters contaminated with V. vulnificus. Also, many commercially important food fish and eels are susceptible to Vibrio infections, thus depleting the numbers available for fishermen.  Caught fish typically have a normal intestinal flora of slightly less than 50% Vibrio spp., with bottom dwelling, sediment-associated and commercially available flatfish (flounder, halibut, sole) having up to 100%  of the normal intestinal flora composed of Vibrio spp.. V. marinus is but one such common component of fish gut flora. V. fischeri, also a gut flora species,  is now considered to be part of a newer taxa of luminescent bacteria, Photobacterium spp . 

Animal Hazards

Vibrio spp. affect other terrestrial animals than humans, as well.  V. parahaemolyticus , most commonly associated with muddy sediments and zooplankton, can cause acute enteritis in avian species like mynahs, canaries, parakeets, and finches. Water fowl and game are also at risk, as V. metchinokovii can produce a subacute disease characterized by hemorrhagic enteritis.  The same symptoms can be caused by a Vibrio sp. in masupials and monotremes.  Swine and other peccaries can contract  a severe intestinal malady from V. coli.  Vi. chloerae  can affect primates, and has been isolated in frogs and birds. One of the common factors in many cases of Vibrio  disease is the source.  Contaminated water seems to be a prevalent source for Vibrio infection.  Most of the terrestrial animals that succumb to the numerous Vibrio infections have encountered, drank from, or bathed in water ripe with Vibrios.  Most of the birds and land mammals that contract Vibrio do so via a  common watering hole.  It is possible that original freshwater contamination of many of these sites was from the bird droppings of an infected avian.

Human Hazards

As a human public health hazard, Vibrio ranks very high, causing a number of primarily enteric (intestinal) infections. Vibrios affect humans primarily through the ingestion or contact with infected shellfish (raw oysters, clams, and crustaceans). Vibrio cholerae, the agent that causes cholera in humans, is common in third-world nations with less than ideal sanitary conditions and poor sewage management practices. The disease is caused by drinking water contaminated with the V. cholerae bacterium, and it can cause severe dehydration from diarrhea. Vibrio parahaemolyticus is a bacteria which causes an infection of the intestinal system.  The disease is characterized by watery diarrhea and abdominal cramps.  The main transmission of this Vibrio to humans is through eating raw oysters.  Vibrio vulnificus is another deadly Vibrio to humans.  It is also one associated with shell fish poisoning, and the ingestion of this bacteria by healthy individuals can lead to gastroenteritis and death.  Death by diarrhea is not a pleasant thought, and it is no wonder that so much media attention has been focused on this species in recent years.

Eleven  Vibrio species are known to be pathogenic to humans and are found in the marine environment. There are likely to be other human pathogenic species yet to be identified, as well as unreported cases of either misidentified Vbrio species, or of known but unnamed species. The Centers for Disease Control and Prevention also have evidence of disease being caused by those working with fish and marine mammals. Workes are reported as getting Vibrio associated skin lesions  just from handling fish, shellfish or pinnepeds.      Humans are at risk of Vibrio caused zoonosis from animal sources, as well. 

    Vibrios Pathogenic to Humans

	Disease caused
V. alginolyticus	Septicemia Enterocolitis Conjunctivitis Stump/skin ulcers
V. carchariae	Gastroenteritis
V. cholerae	Cholera
V. damsela	Skin lesions
V. fluvialis	Gastroenteritis
V. furnissi	Gastroenteritis
V. hollisae	Gastroenteritis
V. marinus	Skin lesions
V. metschinikovii	Gastroenteritis (?)
V. mimicus	Gastroenteritis
V. parahaemolyticus	Gastroenteritis, Septicemia, Wound infection
V. vulnificus	Gastroenteritis, Septicemia, Wound infection, Meningitis, Pneumonia, Keratitis

Marine Animal Hazards

There are also a number of Vibrios that are pathogenic to fish species.  These include V. fischeri, V. anguillarum, V. damsela, V. ordelli, V.alginolyticus, V. vulnificus and V. carcharriae, among others.  In fish, the term vibriosis describes a number of symptoms and synonyms, including “red pest,” "red sore," "red spot," "red disease," saltwater furunculosis, boil disease, Hitra disease, and ulcer disease caused by V anguillarum. Vibriois, a type of septicemia can cause skin discoloration, lesions, mouth and fin infections, gastric infections, exopthalmia, gut and organ distention and swelling, hemorrhagic lesions, and local necrosis. Poor water conditions, overcrowding in aquaculture facilities, heavy nutrient loading of coastal waters, and high temperature can all lead to increased outbreaks and infection. The treatment for Vibrio infections in fish populations is generally acoomplished with the use of sulfa antibiotics.

Vibrio angulliformes  and V. damsela are a hazard for American eels.  At various stages, these infections reach epidemic proportions within the eel population.  The infected eels cannot be used for human consumption, and this is an economic hardship.  Other commercial fishes are also affected.  Common skin lesions show up on fish in the infections resulting from  Vibrio parahaemolyticus.

Interestingly, the shellfish infected with Vibrios do not usually show signs of infection, as Vibrio are typically non-pathogenic to them.  For example, it is not easy to tell if a contaminated oyster has active disease, as it will show no signs of illness without histological examination.  Most invertebrates, especially those adapted for filter feeding and bacterial capture, seem largely immune to Vibrio virulence except in opportunistic infections resulting from stressful conditions..  However, V. anguillarum, V. alginolyticus, V. parahaemolyticus, V. pelagia, and V splendidus can cause Vibriosis in oysters and shellfish with an exotoxin that inhibits larval swimming. In adult shellfish, the effects of Vibrio pathogenicty are typically a loss of digestive function , loss of motility, and velum abnormalities. Mass mortalites are more frequent during the warmer summer months. Cytolysis and/or hemolysis may occur in advanced stages. Even so, it has been repeatedly mentioned in such reports that these cases are ones in which  Vibrio pathogens in shellfish are regarded as opportunistic  since such occurrences typically occur in highly stressed hatcheries.

V. anguillarum plays a very clear role in fish and eel disease, but the problem with determinations of many ubiquitously associated Vibrios is that they are common to both healthy and diseased fish and marine life. Thus, their role in pathogenicity, if any, is often quite loosely fit. Furthermore, there are two Vibrios now classified as Photobacterium spp., that are associated with bioluminescent organs. A mutualistic relationship existss between sea urchins and V. diazotrophicus, as well as between other Vibrio spp. and shellfish.

Vibrios Pathogenic to Marine Life

	Fish	Mollusks	Crustaceans	Cnidarians
Type AK-1				X
V. alginolyticus	X ulcer disease	X	X
V. anguillarum	X vibriosis
V. campbelli		X
V. carchariae	X vasculitis
V. cholerae	X Petechial hemorrahage
V. damsela	X Skin lesions	X
V. fischeri	X
V. harveyi	X	X		Briareum sp.?
V. metschnikovii		X
V. ordalli	X vibriosis
V. orientalis		X
V. parahaemolyticus	X Ulcer disease
V. splendidis		X
V. vulnificus	X Skin lesions

Susceptibility and Determination.

The three basic tests used to isolate the genus from other bacteria are growth: on TCBS nutrient medium, anaerobic growth, and susceptibility to the 0/129 compound. Agent 0/129 (2,4 diamin, -6, 7 diisopropyl pteridine) is a compound used in determinative assays of Vibrio, and is a vibriostatic agent to which many Vibrios are susceptible.  Approximately 50% of species are susceptible to the antibiotics chloramphenicol and polymyxin-B. Tetracycline is also commonly used along with terramycin is sensitivity assays.  Many Vibrios are susceptible to one or more these agents. Most are resistant to heavy metals. Various substrates and agars can be used, along with fermentation tests, etc. in the speciation of Vibrios in basic determinative bacterial plating assays.

General Determination of Vibrios

Exclusive of Strains

Algino lyticus	Anguil larum	Car chariae	Cholerae	Damsela	Fluvialis	Metsch nikovii	Ordalli	Para haemo lyticus	Vulni ficus
R S	S S	R S	S S	S S			S S	R S	S S	0/129 10µg 150µg
+	-	+	-	-			-	V	-	Swarming
- + +/V	+ - -	- + +	- + +	+ - -	+ - -	V V -	- - -	- + +	- + +/-/V	Amino acids Arg Lys Orn
A - a - a/+	A - a - a	A - a a a	A -/+ a - a/+ -	Ag - ag - -	- + +	V - + -	A - - - a	A - a - - V	A A A A -	Fermentation Glu Lac Man Sal Suc Ara
-	-	+	-	+			-	V	V/-	urease
+	+	+	+	-	-	V	-	-/+	-/+	Indole
- + + + +	V + + v -	- + + + -	V/- + v - -	- + v - -	V -	- V -	- + + - -	- + + + -	- + +/- - -	%NaCl growth 0 3 6 8 10
+	-	-	+	-			-	+	+/-	42oC
+			+		+	-		+	+	NO3>NO2
+			+		+	-		+	+	oxidase

The, synergistic combination on sulfonamides and trimethoprim is a commonly used antibiotic treatment of Vibrio infections.  Polymyxin B is another antibiotic commonly used for its effect on many species  of Vibrio. Increased oxygen tension can also be used for a bacteriostatic effect. Some are also effected by hydrostatic pressure - in other words, they are less likely to grow at deep depths, as has been shown for V. alginolyticus and V. marinus.

General Antibiotic Susceptibility for Vibrios

	Dosage	Route	Target	Species affected
Aminoglycosides				V. vulnificus
Ampicillin				V. vulnificus
Chlorampenicol				Vibrios. V. vulnificus, parahaemolyticus
Cotrimoxazole				V. vulnificus
Erythromycin				V. parahaemolyticus
Furanace	2 mg/l	Bath	Trout	Vibrios
Gentamycin				V. parahaemolyticus
Halquinol	25 mg/l	Bath	Turbot	Vibrios
Kanamycin				V. parahaemolyticus
Piromidic acid	10-40 mg/kg	Oral	Salmon, eels	Vibrios
Polymyxin B				Vibrios
Sulfadoxine - TMP	30 mg.kg	Oral	Ayu	Vibrios
Sulfamethoxazole - TMP	25 mg/l	Bath	Turbot	Vibrios
Teracycline				Vibrios, V. vulnificus, parahaemolyticus, cholerae

Vibrios on the Reef and in Aquaria

As mentioned, Vibrios are ubiquitous and important components of marine environments. Their occurrence in oceanic and reef environments is much less common, but they still exist.  Vibrios are found in all types of sediments and on  substrates, especially those enriched with organic material.  Soft lagoonal sediments will find higher numbers of Vibrio spp than coarse reef sediments.  Vibrios play a role in denitrification and nutrient regeneration, as they are capable of fermentation and reduction.   Vibrios are also extremely common components of coral mucus. Their pathogenicity to invertebrates is unreported except as opportunistic species capable of potential pathogenicity.  One species has  found to induce coral bleaching in extenuating circumstances Some, like V. vulnificus, may be nutrient limited; in the case of V. vulnificus, iron appears to be essential for the cytolysin activity. Under nutrient limitation, Vibrio readjust their metabolism toward long term stasis, adhering tightly to substrates until conditions are more suitable for their growth and reproduction.  Thus, it is unlikely that one can “get rid” of Vibrios in a marine environment. 

Corals are known to both culture and utilize both bacteria and bacterial byproducts that are grown on and near their mucus. Most Vibrios associated with coral mucus are swarmers, and seem especially suited to their carbohydrate rich medium. Several species are part of the normal bacterial flora of coral mucus and can comprise well in excess of 50% of total microbial populations in both soft and stony corals.  The two Vibrio species persistently found associated with coral mucus are V. parahaemolyticus and V. alginolyticus. At least V. parahaemolyticus shows the potential to be pathogenic to coral tissue, especially under stress, although their presence seems to indicate that both are non-pathogenic and normal flora associated with corals.  V. campbelli and V. haloplanktis have been found as normal non-pathogenic strains on healthy colonies of Briareum abestinum. Decomposing coral tissue (and any decomposing organic matter) seems to be quickly settled by Vibrios, and various unidentified strains, along with V. fluvialis, V. furnissi, and V haloplanktis have been identified in such cases.

To further illustarte the innateness of reef invertebrates and Vibrios, the toxin of V. cholerae peptides from V. alginolyticus, and small molecules of V. cholerae present in bacterial film, induce the settlement of various invertebrate larvae.  Corals, especially, seem likely to settle on bacterial films dominated by those species common to their normal microbial flora, specifically V. alginolyticus.. Gorgonians and other soft corals produce a number of antimicrobial compounds that show a wide range of effects on various bacteria, with specificty leant towards those bacteria which are potentially non-native and pathogenic to their area and surface composition.  Perhaps not coincidentally, they have been found to have a very low activity against Vibrios.  Similarly, scleractinian corals also produce a wide range of antimicrobial substances that help reduce surface fouling that can lead to pathogenicity and stress.  V. parahaemolyticus and V. algnolyticus are often initial colonizers in such cases, as might be expected by their abundance and regular presence on coral and substrate surfaces. As with gorgonian studies, stony corals show little activity towards V. parahaemolyticus, despite most corals showing significant and often specific activity towards various other bacterial species.  Only Tubastraea sp. shows any significant activity, and this is against against V. alginolyticus. Given their propensity to have these bacteria dwelling on their surface, if pathogenicity was a significant threat, it is likely that substances would be produced to show antibacterial action towards them; yet, this does not occur in species studied thus far over the past 40 years to the very recent studies   The discovery several years ago of an unnamed Vibrio (type AK-1) as an agent of coral bleaching was the first record of Vibrio pathogenicity in corals.  Brown (1997)  has confirmed that the circumstances of its occurrence were unusual.

Conclusion

Vibrios are a unique bacterial constituent of marine and estuarine environments.  They have become quite infamous because of their tendency towards virulence and pathogenicity in humans and ornamental fish.  Certainly, they can be quite dangerous, and care should be excercised when working with aquariums, specifically corals and substrates where these microbes tend to exist prolifically.  Cuts and openings in the skin should be protected, with hand washing using a true antibacterial soap performed after handling or working in aquarium water.  Any cuts or abrasions that occur from working in an aquarium should be immediately attended to with antibiotic applications.  Not only are Vibrios a potential danger in aquaria, but there are many other genera which have equally dangerous potentials for infection and disease.  Nonetheless, it should be apparent that the thought of Vibrios in the aquarium should not be met with trepidation, but understanding.  These bacteria play a number of important roles in marine habitats, from denitrification to providing settlement cues and acting as trophical resources for many organisms.  Their presence is normal to the environment, and, hopefully, understanding these bacteria will allow them to be met with less fear and a greater appreciation of their role and presence both in the wild and in marine aquaria.

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