TheScientificWorldJOURNAL (2006) 6, 931945 [626849]

Review Article
TheScientificWorldJOURNAL (2006) 6, 931–945
ISSN 1537-744X; DOI 10.1100/tsw.2006.181

©2006 with author. Published by TheScientificWorld, Ltd.; www.thescientificworld.com

931The Bacterial Microflora of Fish, Revised
B. Austin
School of Life Sciences, John Muir Building, Heriot-Watt University, Riccarton,
Edinburgh EH14 4AS, Scotland
Original review published March 4, 2002; Revised edition published August 11, 2006

This Revised Review is a revision of the review article published, Vol. 2, 2002 .
The results of numerous studies indicate that fish possess bacterial populations on or in
their skin, gills, digestive tract, and light-emitting organs. In addition, the internal organs (kidney, liver, and spleen) of healthy fish may contain bacteria, but there is debate about whether or not muscle is actually sterile. Using traditional culture-dependent techniques,
the numbers and taxonomic composition of the bacterial populations generally reflect
those of the surrounding water. More modern culture-independent approaches have permitted the recognition of previously uncultured bacteria. The role of the organisms includes the ability to degrade complex molecules (therefore exercising a potential benefit in nutrition), to produce vitamins and polymers, and to be responsible for the emission of light by the light-emitting organs of deep-sea fish. Taxa, including
Pseudomonas , may contribute to spoilage by the production of histamines in fish tissue.
KEYWORDS: bacteria, fish, microflora, methods, digestive tract, gills, skin, population size,
taxonomy, biodiversity, luminescence, degradative ability, effect of antibiotics, polymers, enzymes, spoilage

INTRODUCTION
Traditionally, studies on fish-associated microorganisms involved culture-dependent techniques of dubious sensitivity, which highlighted only the bacter ia (typically the aerobic heterotrophic bacterial
component[1]) to the exclusion of eukaryotes. Anaerobic bacteria have been comparatively
neglected[2,3,4,5] by culturists, possibly reflecting the need for more exacting techniques, although there
is increasing evidence that such orga nisms occur in large numbers especia lly within the digestive tract of
freshwater and marine fish[2]. More recently and in line with other studies of microbial biodiversity,
emphasis has been placed on mol ecular-based culture-independent techniques, which have been
generating some exciting data, a nd have revealed the presence of uncultured organisms including
anaerobes[6].
This article, which is an update d version first published in 2002[143], will synthesise the available
information on fish-associated bacteria, focusing on th e numbers, nature, and role of bacteria on or in
healthy finfish. Aspects of fish pathology will be ignored, as a wealth of information sufficient to fill
several textbooks already exists[7]. However, it is a pparent that some pathogens may be found on healthy
fish in the absence of disease. It is questionable whether such associations represent the asymptomatic
carrier state of the disease cycle, a preliminary coloni sation step prior to pathogenesis, or commensalism-
synergism. For example, Flavobacterium psychrophilum , the causal agent of coldwater disease (of

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932salmon) and rainbow trout fry syndrome, has been found in the kidney, spleen, brain, ovarian fluid,
unfertilised eggs, and milt of healthy Baltic salmon ( Salmo salar )[8].
It is apparent that fish are continuously expo sed to the microorganisms present in water and in
sediment including the contaminants in sewage/ faeces[9]. These organisms will undoubtedly influence
the microflora on external surfaces, including the gills, of fish. Similarly, the digestive tract will receive
water and food that is populated with microorganisms. Certainly, colonisation may well start at the egg
and/or larval stage, and continue with the developm ent of the fish[10]. Thus, the numbers and range of
microorganisms present in the eggs, on food, and in the water, will influence the microflora of the
developing fish. Also, it is recognised that, to some extent, it is possible to manipulate the microflora of
the developing fish by use of prebiotics, i.e., nondiges tible food ingredients that beneficially affect the
host by stimulating growth[11] and probiotics, i.e ., live microbial food supplements, which may colonise
the digestive tract for short or prolonged periods[12]. This action may have benefit for the host, such as
the moderation of fish diseases[12].
From the published literature, it may be deduced that there are three likely scenarios for the fate of
bacteria coming into contact with fish:
1. The organisms from the environment around the fish may become closely associated with and
even colonise the external surf aces of the fish. There may be accumulation of the organisms at
sites of damage, such as missing scales or abrasions[13].
2. The organisms may enter the mouth with water[10] or food and pass through and/or colonise the
digestive tract[13].
3. The organisms coming into contact with fish su rfaces may be inhibited by the resident microflora
or by natural inhibitory compounds present on or in the fish[13].
The overriding problem concerns whether or not it is possible to differentiate members of the
indigenous (fish) microflora from transients, which coul d be in the water film around fish or in water/food
within the digestive tract. This is a problem par ticularly with the culture-dependent approaches.
Unfortunately, most publications do not address this i ssue. Yet this is not so unusual insofar as similar
arguments have centred on the nature of the true microf lora of other habitats, e.g., the distinction between
microbial populations of the rhizoplane (root surface) vs. the rhizosphere (habitat around roots), and of
the phylloplane (leaf surface) vs. the phyllosphere (habitat around leaves).
It is recognised that extraneous bacteria are capab le of surviving in fish. For example, the faecal
indicator organism, Escherichia coli , has been found to survive and even multiply in the digestive tract of
rainbow trout ( Oncorhynchus mykiss ) after ingestion via contaminated food[14].
RESEARCH ACTIVITIES
Research has focused on six principal aspects of the microbiology of fish:
• The microbiology of the surface (including gills)
• The populations in the digestive tract (an area of current interest particularly involving use of
modern molecular-based culture-independent techniques)
• The possible presence of bacteria in muscle a nd the internal organs of healthy fish
• The microflora of eggs • The presence and role of bacteria associated with the light-emitting organs, particularly of deep-
sea fish
• The bacterial populations in food
As a simplification, publications have tended to emphasise either quantitative or qualitative aspects or
the supposed role of the organisms on/in fish. It is unusual for research articles to address more than one
of these aspects.

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933METHODS USED TO STUDY FISH-ASSOCIATED BACTERIA
Dilution and Spread-Plating Techniques
There has been a tendency for scientists to study fish-associated bacteria using only traditional,
comparatively simple, culture-dependent techniques, such as dilution or spread plating, incorporating
media and incubation conditions often of dubious relevance. As an example, to isolate bacteria from the
skin, the surface of the fish may be swabbed over an indeterminate area, and the inoculum spread over the
surface of nutrient-rich medium, such as tryptone soya agar (TSA[15]), with incubation at 15–25oC for 7–
14 days. A disadvantage of using swabbing techniques is that it is difficult to equate the data with a
defined unit of measurement. In addition, the re levance of the resulting data for ascertaining the
indigenous bacterial populations on the surface of fish is questionable. Yet culturing methods are still
used extensively and publications appear regularly[e.g., 16,17,18].
Criticism may also be levied at the time taken between sample collection and examination, which
may often be measured in hours or days. It is not unc ommon for whole or parts (e.g., the digestive tract)
of fish to be transported on ice, cooled or at ambien t temperature, to distant laboratories for examination.
Sections or the entire digestive tract, together with the contents, have then been homogenised, in which
case it is impossible to decide from the data if th e resulting bacteria have originated from the food
particles, lumen, and/or the wall. Some workers ha ve studied the bacterial populations of the digestive
tract by swabbing the anus and faeces[13]. Unfortunately , most scientists do not consider whether or not
there may be significant changes in the microflora during the period from collection of the fish to
microbiological examination.
As a final criticism, it is noted that the proportion of the bacterial microflora contributing to the
colony count is rarely considered in quantitative- type studies. Circumstantial evidence suggests that
populations deduced from colony counts on agar plates gr eatly underestimate the total microflora likely to
be present. Nevertheless, a study considered that at much as 50% of the microflora from the intestine of
rainbow trout produced colonies on TSA[15]; thus, a 50% error might be inferred!
Some comparisons between methods have been c onducted. For example, gentle rinsing techniques
have been evaluated and compared to excision and homogenisation with a stomacher to recover bacteria
from the surface of fish[19]. In this case, stomaching was regarded as superior for the enumeration of
total bacterial populations, but rinsing was be tter for rainbow trout than striped bass ( Morone
saxatilis )[19].
Microscopic Techniques
Microscopic techniques have found increasing use in the study of fish microflora, and include direct
microscopic counts by light[15,20,21] and electron microscopy[20,22,23,24]. These have been used to
visualise the organisms present, particularly in the digestive tract.
Automated Direct Epifluorescent Filter Technique
An automated direct epifluorescent filter technique instrument, COBRA, has been evaluated and offers
promise for enumerating bacterial populations[25].
Molecular Techniques
Molecular methods are increasingly being used. For example, numerous publications have discussed the
sequencing of 16S rRNA genes[15,26,27,28,29,30]. Also, microplate hybridisation has been successful at
identifying aeromonads in the digestive tract of freshwater fish[31].

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934QUANTITATIVE ASPECTS OF THE BACTERIAL MICROFLORA
Surface Populations
Most workers have opted for the comparatively easy approach of studying the aerobic heterotrophic
bacteria populations, with data suggesting that the num bers of bacteria on the surface of fish approximate
those of the surrounding water. Yet, in retrospect, it is apparent that the units of measurement between
water (populations ml–1) and fish surfaces (populations cm–2) are very different, and comparisons are not
especially helpful.
Scrutiny of the literature suggests that fish ha ve only low bacterial populations on the skin. For
example, Atlantic salmon ( Salmo salar ) from the U.K. were reported to possess populations of 102–103
culturable bacteria cm–2 of skin[32], whereas rainbow trout from Turkey contained bacterial populations
of 101–107 g–1[33]. However, it should be emphasised that the relevance of using weight as a unit of
measurement to estimate bacterial populations on skin is debatable. Interestingly, freshly caught mullet
(Mugil cephalus ), whiting ( Sillago ciliato ), and flathead ( Platycephalus fiscus ) from Australia were
reported to have seemingly higher populations of 4 × 103 to 8 × 104 bacteria cm–2[34]. Not surprisingly,
there are data suggesting that the bacterial population size reflects the level of water pollution, i.e., higher
counts are present on fish in polluted waters[35]. Al so, there is some evidence that the population of
aerobes exceeds that of the anaerobes[36].
Bacterial populations of skin
102 to ~104 cm–2
Overall, these low counts, which to some ex tent have been supported by scanning electron
microscopic evidence[13], indicate that only a minute ar ea of the fish surface is populated with bacteria.
However, it is conceded that the preparation fo r scanning electron microscopy may well have removed
some organisms from the skin. Thus, it could be in ferred that the surface microflora is only loosely
associated with fish skin. Coincident ally, this low level of colonisation. on fish skin corresponds well to
that of other habitats, such as the leaf surface of terrestrial plants[37].
Gills
Gill tissue has been found to harbour high bacterial populations, e.g., up to 106 bacteria g–1 of gill tissue[38].
Eyes
There is anecdotal evidence that the eyes of health y fish are devoid of bacterial colonisation[13].
Muscle and the Internal Organs
Muscle has been considered by some to be sterile [39], whereas other investigators have reported the
presence of bacteria[40]. Also, some workers have found bacteria in the kidney and liver of healthy fish,
i.e., turbot ( Scophthalmus maximus )[41].
Digestive Tract
A consensus view is that dense bacterial populations o ccur in the digestive tract (i.e., populations of up to
~108 heterotrophs g–1[42,43,44,45,46]) and ~105 anaerobes g–1[3,42,43] have been reported with numbers
appearing to be much higher than those of the surr ounding water. For example, by including contents with

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935the intestine, maximal bacterial populations of 2 × 107 colony-forming units (cfu) g–1 were recorded in the
pinfish ( Lagodon rhomboides )[47]. Moreover, counts of 1.1 × 106 to 3.6 × 108 bacteria were recorded for
the intestinal contents of deep-sea fish[45], when it was noted that more cultures were obtained at in situ
(barophilic), rather than atmospheric, pressure[45]. Also , some differences have been considered to reflect
seasonality, i.e., with maximum and minimum counts occurring in summer and winter, respectively[48]. Indeed, an effect of water temperature on the size of the microflora of pike perch ( Stizostedion
lucioperca ) has been reported[49]. In another study, seas onal variation was attributed to the monsoon
season, with maximal and minimal populations in green chromides (Etroplus suratensis ) and orange
chromides ( E. maculates ) corresponding with postmonsoon (Septe mber to December) and premonsoon
(January to April), respectively[50]. Also, the popula tion densities are likely to be influenced by the
feeding regime, with fish receiving live feeds ha ving higher populations than those with artificial
diets[46].
Differences in population size have been detected in specific regions of the digestive tract. Thus, an
estimation of aerobic heterotrophs in the digestive tract of yellowtail ( Seriola sp.) revealed counts of 2 ×
10
4 bacteria g–1, 2.5 × 105 bacteria g–1, and 6.5 × 104 to 5.9 × 106 bacteria g–1 in the pyloric caeca,
stomach, and intestine, respectively[51]. Parallel da ta were published separately[49], when the presence
of 5.5 × 103 to 5.0 × 104 cfu of aerobic heterotrophic bacteria g–1 and 1.0 × 104 to 1.0 × 107 cfu aerobic
heterotrophic bacteria g–1 were found in the stomach and intestine of pike perch, respectively. However,
higher populations were noted in the digestive tract of juvenile compared with adult farmed Dover sole
(Solea solea ), with 5.2 × 105, 8.0 × 105, and 9.8 × 106 aerobic heterotrophs g–1 recovered from the
stomach-foregut, midgut, and hindgut-rectum (of juven ile fish), respectively[4,52]. It should be
emphasised that anaerobes (7.1 × 105 anaerobic bacteria g–1) have been found in addition within the
intestines[51]. Following a familiar theme, it was ob served that there was an increase in bacterial
populations, especially of adherent organisms, along the digestive tract of herring ( Clupea harengus )
larvae[20].
Some differences have been noted according to the feeding pattern of fish. Thus, detritivorous fish
possessed higher bacterial populations than filter feeders[53]. Of course, it is likely that many organisms in the digestive tract will have been derived from the food. In this connection, it was found that there were
between 10
3 and 107 aerobic heterotrophs g–1 in commercial fish food in North America[54], whereas
comparable data from Japan indicated counts of 1.8 × 103 to 8.0 × 105 bacteria g–1[55].
Electron microscopy has substantiated the presence of high bacterial populations in the digestive
tract. In particular, scanning and transmission electron microscopy demonstrated the presence of large populations of ovoid and rod-shaped bacterial-like objects in association with the microvillous brush
borders of the enterocytes of Arctic charr ( Salvelinus alpinus )[23]. Also, bacteria were observed at and
between the tips of microvilli, and rod-shaped b acteria were seen between the microvilli of common
wolffish ( Anarhichas lupus )[22]. Evidence has pointed to endocytosis of bacteria by epithelial cells in the
pyloric caeca and midgut[23].
Bacterial populations in the digestive tract
~10
8 aerobic heterotrophs g–1
~105 anaerobic g–1
Fish Eggs and Larvae
Fish eggs may be populated by high numbers of bacteria, with 103–106 bacteria g–1 reported for salmonid
eggs[56]. There is evidence that adhesion and colonisa tion of the egg by bacteria occurs within a few
hours of fertilization[57]. Undoubtedly, these organism s and those of the food and surrounding water are
important for the establishment of a microflora in th e digestive tract of fish larvae. Incidentally, the
digestive tract of newly hatched larvae contains scant bacterial populations, but is quickly colonised[58].

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936TAXONOMY (BIODIVERSITY)
Approaches have gone from the traditional[59], through numerical taxonomy studies involving large
numbers of isolates (e.g., 197 cultures investigated in one study[60]; 473 isolates studied in another[1]),
to culture-independent molecular approaches (e.g., pa rtial sequencing of the 16S rRNA gene[15,29]). The
benefit of the latter is the recognition of organisms that may or may not be culturable by conventional
techniques[e.g., 21]. Sometimes, the phenetic approach has centered on the use of rapid systems, such as
BIOLOG or MIDI[36,61]. It is encouraging that some comparative studies have pointed to congruence between phenotypic and molecular analyses[15]. Over all, it would appear that narrow-based studies
focusing on a limited number of bacterial groups are ofte n more successful than those that attempt to be
broad-based, trying to consider all of the bacteria from fish. From the published literature, it is apparent
that there are many similarities between the bacterial populations in fish and water[33,39,40,59,62,63,
64,65,66,67,68].
Surface Microflora
The bacteria from the surface of freshwate r fish have been reported to include Acinetobacter
johnsonii [69], aeromonads (notably Aeromonas hydrophila, A. bestiarum, A. caviae, A. jandaei , A.
schubertii , and A. veronii biovar sobria[70]), Alcaligenes piechaudii, Enterobacter aerogenes,
Escherichia coli, Flavobacterium [35], Flexibacter spp., Micrococcus luteus, Moraxella spp.,
Pseudomonas fluorescens , psychrobacters[69], and Vibrio fluvialis [33,67,71]. To some extent, the
presence of aeromonads reflected whether or not the water in which the fish occurred was polluted or
clean[70]. Bacteria, typical of those in seawater, have been recovered from the surface of marine fish and
include Acinetobacter calcoaceticus, Alcaligenes faeca lis, Bacillus cereus, B. firmus, Caulobacter,
coryneforms, Cytophaga/Flexibacter, E. coli, Hyphomicrobium vulgare , Lucibacterium (Vibrio) harveyi,
Photobacterium angustum, P. logei, Prostheco microbium, Pseudomonas fluorescens, P. marina , and
Vibrio spp. (including V. albensis, V. anguillarum, V. splendidus biotype I, V. fischeri, V. ordalii , and V.
scophthalmi on the surface of turbot)[1,65,66].
As a result of a detailed numerical taxonomic study of Gram-negative, oxidase-positive bacteria
recovered from sharks, the dominance of vibr ios was noted, with representatives including V. harveyi
(=V. carchariae ), and V. alginolyticus . Other groups included Aeromonas, Photobacterium (including P.
damselae and P. damselae subsp. piscicida), Alteromonas, Plesiomonas shigelloides, Moraxella , and
Neisseria [60].
Gill Microflora
Yellow-pigmented, Gram-negative rods, especially Cytophaga spp., dominate on gills[38]. Aeromonads,
coryneforms, enterobacteria, Gram-positive cocci, p seudomonads, and vibrios have also been recovered
from the gills of healthy juvenile rainbow trout[68].
Gills of marine fish accommodate Achromobacter, Alcaligenes, Bacillus, Flavobacterium , and
Micrococcus [72] and yellow-pigmented bacteria, loosely associated with Chryseobacterium-
Flavobacterium-Flexibacter-Cytophaga [73].
Microflora in the Digestive Tract
Studies on the microflora of the digestive tract ha ve led the way in the use of culture-independent
approaches[e.g., 21]. However, the bulk of the historical data stems from culturing methods, which will
be discussed first. Ringø et al. [74] have written an excellent review on the topic.
Initially in the sac fry, only a few taxa (coryneforms and Pseudomonas ) occur within the digestive
tract[56]. It is likely that some bacteria become ing ested at the yolk-sac stage, leading to the establishment

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937of an initial intestinal microflora[57]. In addition, it has been reported that bacterial colonisation of the
digestive tract of turbot larvae coincided with the star t of feeding, when the microflora was dominated by
Aeromonas and Vibrio [75]. In an investigation of the intestinal microflora of larval sea bream
(Dicentrarchus labrax ) and sea bass ( Sparus aurata ), it was observed that when the larvae were fed with
rotifers, there was a high incidence of V. anguillarum, V. tubiashii , and nonvibrio groups[76]. However,
feeding with Artemia led to the recovery of mostly V. alginolyticus, V. proteolyticus, V. harveyi, and V.
natriegens [76]. It was concluded from these experiment s that the fluctuations in the dominant
components of the microflora reflected the bacteria in the live feed. Indeed, th e dominance of vibrios was
not recorded until the end of the larval stage[76]. Th e comparative lack of diversity in larvae continues
into older fish, and it has been suggested that the flora may be subjected to as-yet undescribed selective
effects leading to a restricted numbe r of taxa being present[59,77,78,79,80].
A comparatively wide range of taxa have been associ ated with the digestive tract of adult freshwater
fish and include Acinetobacter, Enterobacter, Escheri chia, Klebsiella, Proteus, Serratia [42],
Aeromonas [42,43,48,68,81,82], Mycoplasma [30], Clostridium[ 42] and Fusobacterium [42,74]. Isolates
have been identified by microplate hybridization as A . caviae, A. hydrophila, A. jandaei, A. sobria , and A.
veronii [31]. Alcaligenes, Eikenella [4], Bacteroides [3,83,84], Citrobacter freundii [39], Hafnia alvei [81],
Cytophaga/Flexibacter [68], Bacillus, Listeria, Propionibacterium, Staphylococcus [39], Moraxella [49],
and Pseudomonas [4,39,68,80]. In one study involving pi ke perch, it was concluded that Moraxella and
Staphylococcus were unique to the habitat when compared with the digestive tract of other fish
species[49].
Modern phenetic and molecular-based studies, including 16S rRNA sequencing have indicated
variability in the intestinal microflora of salmonids , notably rainbow trout and Atlantic salmon reflecting
the fish farm of origin[15,30], with analyses rev ealing the dominance of the gamma subclass[15,21] (i.e.,
enterics, Aeromonas , and Pseudomonas ) and beta subclass of Proteobacteria, and Gram-positive bacteria
with a low G + C-content of the DNA ( Carnobacterium )[15]. The approaches have permitted the
recognition of potentially new taxa. For example, a 16S rRNA gene sequence with similarity to
Anaerofilum pentosovorans has been detected[30].
In one detailed study, 41 culturable microbial phylotypes, and 39 sequences from 16S rRNA and 2
from 18S rRNA genes were retrieved from the diges tive and intestinal mucus of rainbow trout and
equated largely with Aeromonadaceae, Enterobacteriaceae (i.e., Buttiauxella, Enterobacter, Hafnia,
Pantoea, Plesiomonas, and Proteus ) and Pseudomonadaceae representatives. Intestinal contents contained
Arthrobacter, Bacillus, Carnobacterium, Exiguobact erium, Flavobacterium, Kokuria, Microbacterium,
Micrococcus, Rhodococcus, Sporocytophaga, and Ultramicrobacterium. Genomic DNA isolated from
intestinal contents and mucus was used to generate 104 random clones, which were mostly affiliated with
Proteobacteria (>70% of the total). Twelve seque nces were retrieved from denaturing gradient gel
electrophoresis analysis of the digestive tract or rai nbow trout, and dominant bands were mostly related to
Clostridium [29]. One of the outcomes of the study was the realization that Capnocytophaga,
Cetobacterium, Erwinia, Porphyromonas, Prevotella, Rahnella, Ralstonia, Serratia, and Veillonella were
recognised as occurring for the first time as culturable co mponents of the microflora in the digestive tract
of freshwater fish[29]. Using a parallel approach, th e digestive tract of wild and farmed salmon from
Norway and Scotland were found to be populated with Acinetobacter junii and a novel Mycoplasma
phylotype, the latter of which comprised almost all, i.e ., ~96%, of the microflora of the distal intestine of
wild salmon[30].
The digestive tract of adult marine fish has been reported to contain Aeromonas [81],
Alcaligenes [52,85], Alteromonas [20], Carnobacterium [86], Flavobacterium [52,85], Micrococcus [52],
Photobacterium [52,85], Pseudomonas [59,85], Staphylococcus [52], and Vibrio [20,52,59,62,64,80,82,85],
including V. iliopiscarius [87]. Terminal restriction fragment length polymorphism data point to a greater
diversity in the posterior compared to the an terior gut in large herbivorous fish, i.e., Kyphosus
sydneyanus [6].
Special groups, such as large (gigantobacteria) sy mbiotic bacteria, have been observed in the
digestive tract of surgeonfish from the Red Sea a nd Indo-Pacific Region[88]. Also, using a methanogen-

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938specific nested polymerase chain reaction, methanogens have been detected in the digestive tract and
faeces of flounder ( Platichthys flesus ) from the North Sea[28]. Indeed, in this study, 16S rDNA sequences
revealed 97.6–99.5% similarity to the archaea representative Methanococcoides methylutens [28].
Lactic acid bacteria, notably carnobacteria, are co mmon on/in fish, particularly in the digestive
tract[89,90,91] with investigations highlighting the presence of Lactococcus (notably L. lactis and L.
raffinolactis [90], Lactobacillus, Aerococcus -like bacteria, Leuconostoc, Pediococus, Streptococcus,
Vagococcus, and Weissella ) as part of the normal microflora[92]. To date, studies have emphasised the
taxonomy of the organisms[89], highlighting the presence of Carnobacterium [21,86,91,92,93]
particularly C. piscicola [89,91,94] and C. piscicola -like bacteria[95], and their role as putative probiotics
for use in aquaculture. Other lactic-acid bacteria pr esent in the epithelial mucosa have been equated with
Lactobacillus plantarum, Leuconostoc mesenteroides, and Streptococcus spp.[89]. In a separate
investigation, Lactobacillus, Enterococcus durans, Lactoc occus, Vagococcus, C. divergens, and C.
piscicola were recovered from freshwater fish, notably brown trout ( Salmo trutta ), and characterised
phenotypically by numerical analyses[96]. A previously undescribed species, C. inhibens , was recovered
from the intestine of Atlantic salmon, and demonstr ated antibacterial activity against fish pathogens,
notably Aeromonas salmonicida and Vibrio anguillarum [97].
Diets
Aeromonads, Bacillus , pseudomonads, and Staphylococcus dominate in diets[35,55].
Eggs
Healthy eggs are populated by Cytophaga/Flavobacterium and, to a lesser extent, Pseudomonas [56,98],
reflecting the organisms present in water[57].
Internal Organs
The liver and kidney of healthy turbot ha ve been found to be populated by mostly Pseudomonas and
Vibrio , including V. fischeri, V. harveyi, V. pelagius , and V. splendidus [41]. Similarly, Shewanella spp.
have been recovered from the intern al organs[99]. The reasons for the presence of some of these bacteria
are unclear. Moreover, it is speculative whether or not the fish are at the earliest stage of an infection
cycle.
Human Pathogens Recovered from Fish Tissue
Attention has focused on the presence of potential human pathogens in and around fish, namely Aeromonas spp., Campylobacter jejuni, Clostridium botulinum, C. perfringens, Erysipelothrix
rhusiopathiae, Edwardsiella tarda, Legionella pneumophila, Mycobacterium spp., Photobacterium
damselae, Plesiomonas shigelloides, Staphylococcus au reus, Streptococcus iniae, V. cholerae, V.
parahaemolyticus, and V. vulnificus [100]. For example, Plesiomonas shigelloides has been cultured from
the digestive tract of pike[5]. Similarly, Staphylococcus aureus and V. mimicus have been isolated
repeatedly from striped bass reared in flow-through and recirculating systems[101]. V. cholerae was
recovered from presumably healthy sharks[60]. V. vulnificus , enumerated by the most probable number
technique with serological identification, has been found in the contents of the digestive tracts of
numerous fish from the U.S. Gulf Coast[102]. In th is study, a seasonal fluctuation was recorded with
minimum and maximum numbers occurring in winter and April to October, respectively. Indeed, the
highest populations of V. vulnificus (10
8 bacterial 100 g–1) were associated with the gut contents of
bottom-feeding fish, especially those that cons umed molluscs and crustacea[102]. In contrast, the

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939plankton-feeding fish contained 105 cells of V. vulnificus 100 g–1. Overall, it was apparent from this study
that the incidence of V. vulnificus was comparatively uncommon in offshore fish, instead being restricted
to those specimens from estuaries, i.e., closer to s hore[102]. In contrast, there has not been any evidence
of Listeria monocytogenes [5], Salmonella , or Yersinia enterocolitica [36,61].
THE ROLE OF FISH BACTERIA
Although the relative numbers and types of bacteria associ ated with healthy fish are interesting, it is the
role of these bacteria that is of importance. However, the information is generally patchy. For a start, it is
relevant to inquire whether fish-associated bacteria are active metabolically or could some be inactive-
dormant-nonculturable[103]. By piecing together various data, it becomes apparent that components of
the bacterial microflora of fish have been asso ciated with numerous functions, including: (1) the
production of friction-preventing polymers (bacteria on fi sh skin, perhaps, important for the movement of
fish through the water column[104]); (2) eicosapen taenoic acid (intestinal bacteria[105]); (3) the
degradation of complex molecules, including starch (amylase production by intestinal bacteria[106,107,108]), cellulose[47,109], phospholipids (intestinal bacteria[110]), proteins[111], chitin, and collagen[52,107]; and (4) the production of neuraminidase (in Photobacterium damselae , from the
intestines of coastal fish[112]) and vitamins (e.g., vitamin B
12, which may be of value to the
host[83,113,114,115]). Moreover by use of DNA microarrays, gnotobiotic zebrafish ( Danio rerio )
revealed the presence of 212 host genes, which were regulated by the intestinal microflora[116].
Some taxa, such as Pseudomonas , have been implicated as causes of fish spoilage[117,118] by the
production of histamines[119,120], principally during storage of fish[72].
Thus, it is likely that bacteria are often beneficial by contributing to the nutritional processes of fish,
namely by degrading complex molecules in the di gestive tract[52] and by producing vitamins[83].
Luminescent Bacteria
Luminous bacteria, principally Photobacterium [121,122], including P. phosphoreum and P.
leiognathi [27], organisms related to P. phosphoreum as determined by 16S rRNA sequencing[26], and
Vibrio spp.[122], including V. fischeri [103,123,124], are responsible for the light-emitting properties of
fish from ten families and five orders[27,125,126]. In addition, obligately symbiotic luminous bacteria
that have been equated by 16S rRNA analyses as new species of Vibrio have been found in members of
the beryciform family Anomalopidae and nine fa milies in the lophiiform suborder Ceratioidei[27].
Generally, luminous bacteria are extracellular, and appear to be tightly arranged in tubules with
communication with the exterior of the light-emitti ng organ[122]. A second site for luminous bacteria has
been found in apogonid fish, Siphamia permutata and S. cephalotes [127]. The tubules release bacteria
into the digestive tract of the host and thus into the surrounding seawater, where the released organisms
are viable and culturable, and may well contri bute to the planktonic microbial populations[122].
Superficially, it would seem that this work has been largely substantiated by others who have also
recognised the presence of luminous bacteria, namely Photobacterium (P. phosphoreum ) and V. harveyi,
in the digestive tracts of some marine fish[128]. Howe ver, it should be emphasised that many fish without
light-emitting organs have also been found to po ssess luminescent bacteria in their intestines[128].
Therefore, light-emitting organs are clearly not always the source of luminous bacteria in the digestive
tract or, for that matter, in seawater.
The production of light by the light-emitting organ is a direct function of synergism or symbiosis
between luminous bacteria and fish. There is some ev idence that luminous bacteria pass from the adults to
offspring[129]. In particular, it was found that offspring from spotnape ponyfish ( Leiognathus nuchalis )
eggs, which were hatched in the absence of a dults, did not develop luminescence activity[129].
Conversely, juvenile fish developed bioluminescence w ithin 48 h of contact with adults or inoculation
with a homogenate of the adult light-emitting organs [129]. From this work, the inference was that

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940juvenile fish became infected with symbiotic lumi nous bacteria from the light-emitting organ of adult
fish, thereby gaining the ability to become bioluminescent[129].
Luminous bacteria in the intestine appear to be involved in chitin degradation, and may therefore
have a role in the digestion of complex molecu les[130]. Also, some luminous bacteria have been
attributed with the ability to produce histamine, and c ould, therefore, be involved in fish spoilage[131].
Production of Inhibitory Compounds
Some bacteria produce inhibitory compounds, particular ly in the digestive tract, and may be responsible
for controlling the colonisation of potential pathogens in fish[95,132]. For example a Vibrio sp. recovered
from the intestine of a spotnape ponyfish in Japan ese coastal waters inhibited the causal agent of
pasteurellosis/pseudotuberculosis, i.e., P. damselae subsp. piscicida [133]. Specifically, the inhibitory
compound was heat-labile and proteinaceous, with a mol ecular mass of <5 kDa, and was considered to be
possibly a bacteriocin or a bacteriocin-like substance[133].
Similarly, bacteria were isolated and found to be capable of inhibiting growth of pathogenic Vibrio
sp. from the digestive tract of halibut ( Hippoglossus hippoglossus ) larvae[134]. Here, the fraction of
pathogen inhibitors among the total number of isol ates ranged between 0–100% (first feeding) and 0–66%
(weaning). All antagonists were Gram-negative rods, most of which were fermentative, and produced
catalase and oxidase, being equated with Aeromonas and Vibrio [134].
Using a double agar layer method, 940 aerobic and anaerobic isolates obtained from the digestive
tract of river fish, water, and sediment in Japan we re examined for antagonism[84]. Some of the isolates
(i.e., Bacteroides type A and other Bacteroidaceae representativ es) from the digestive tract inhibited the
target organisms, which included A. hydrophila, A. salmonicida, E. coli , and Staphylococcus aureus . The
implication of the data was that these antagonistic bacteria may well influence the composition of the
microflora in the digestive tract by the production of inhibitory compounds[84]. In another study by the
same group, it was reported that, of 1,055 intestinal bacteria derived from 7 coastal fish in Japan, 28 isolates (2.7% of the total) inhibited the human and eel pathogen V. vulnificus [135]. Thus, marked
inhibition was displayed by 15 isolates, comprising 11 Vibrionaceae representatives, 3 coryneforms, and
1 Bacillus strain NM 12; the latter demonstrated the most profound antimicrobial activity, and was
therefore chosen for detailed study[135]. This reveal ed that one of the inhibitory compounds, which was
determined to be a heat labile siderophore of <5 kD a molecular weight, inhibited the growth of 227 out of
363 (62.5% of the total) intestinal bacterial cultur es derived from 7 fish[135]. Others have achieved this
level of success. For example, of >400 bacteria recove red from turbot, 89 inhibited the growth of the fish
pathogen V. anguillarum [136]. Similarly, of >400 isolates from the intestine and the external surface of
farmed turbot, 28% (mostly from the digestive tract) inhibited A. salmonicida, A. hydrophila , and V.
anguillarum [137].
Effect of Antimicrobial Compounds on the Microflora
When fish become exposed to antimicrobial co mpounds, there will undoubtedly be an impact on the
composition of the microflora and on antibiotic resistance patterns[20,138,139,140,141,142]. This, in
turn, may impact upon the transmission of antibiotic resistance, such as via R-factors[139], to other
bacteria, and perhaps of significance to humans.
CONCLUSIONS
Fish possess a diverse array of bacterial taxa, often reflecting the composition of the microflora of the
surrounding water. It is argued that the role of many of these fish-associated bacteria is unclear, and
future work should be directed at this aspect.

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This article should be cited as follows:
Austin, B. (2006) The bacterial microflora of fish, revised. TheScientificWorldJOURNAL 6, 931–934. DOI
10.1100/tsw.2006.181.

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