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MICROBIAL ECOLOGY
@Springer-VerlagNew York Inc. 1990
Marian M. Cahill
Department of Microbiology,University of Queensland, St Lucia, Queensland 4067, Australia
Abstract. Bacterial floras isolated from eggs, skin, gills, and intestines
have been described for a limited number of fish species. Generally, the
range of bacterial genera isolated is related to the aquatic habitat of the
fish and varies with factors such as the salinity o f the habitat and the
bacterial load in the water. In many investigations, identification of isolates
to the genus level only makes it difficult to determine the precise relation-
ships o f aquatic and fish microfloras. Bacteria recovered from the skin and
gills may be transient rather than resident on the fish surfaces. Microfloras
o f fish intestines appear to vary with the complexity of the fish digestive
system. The genera present in the gut generally seem to be those from the
environment or diet which can survive and multiply in the intestinal tract,
although there is evidence for a distinct intestinal microflora in some species.
While obligate anaerobes have been recovered from carp and tilapia in-
testines, low ambient temperatures may prevent colonization by anaerobes
in species such as rainbow trout.
Introduction
Various aspects o f the normal microbial flora associated with fish have been
studied. These include the changes in flora during storage [36], the effects of
catching methods or handling on microflora which might lead to deterioration
[ 12, 15], the relationship between environmental and fish microfloras [ 17, 41,
47-50], and the establishment of bases for monitoring changes in fish farms
[1-3, 25].
In attempting to define the bacterial groups normally associated with various
fish species, many researchers have come across problems with taxonomy.
Shewan [37] stated that when he began to study the bacteriology of fresh and
spoiling fish he found that it was difficult to identify m a n y isolates even to the
genus level. Most papers written before 1980 only refer to the genera of bacteria
isolated, but recently, with the use of numerical taxonomy and determination
o f % G + C, identification to the species level and the recognition of new phena
is occurring. For this review, I have used the identifications given by the authors
of the articles quoted, although some of these identifications are now out of
date. The genus Achromobacter, for example, is now Moraxella-Acinetobacter.
Differentiation o f the Flavobacterium-Cytophaga group has been rationalized,
and there has been much revision o f the species within the genus Vibrio. Most
22 M. M. Cahill
Skin Microflora
Gill Microflora
Both marine and freshwater fishes have been shown to support quite high
populations of a range of bacterial genera on their gills. Studies reviewed by
Shewan [36] recorded Pseudomonas, Achromobacter, Flavobacter, and Vibrio
species, in descending order of frequency, on gills of marine fish from the North
Sea and Norwegian waters, whereas only Bacillus and Micrococcus were isolated
from gills of fish from warmer waters off India. These differences probably
reflect differences in environmental temperatures, with more psychrophiles and
fewer mesophiles in the cold North Sea waters. Results of more recent studies
are shown in Table 3.
Horsley's study [17] of bacterial genera on the gills of Atlantic salmon mi-
grating up the Dee River in Aberdeenshire, Scotland showed that the relative
numbers o f the different genera changed with changes in the environment of
the fish, from marine to freshwater. Moraxella comprised 32% of the flora of
fish from marine sampling sites but only 8% of that from upland sites. Also,
Vibrio spp. were isolated only from fish caught at marine sites, whereas Aero-
rnonas spp. were present in highest numbers on gills offish taken from upstream
Waters. From all of the sampling stations the major components of the flora
of the fish were similar to those present in the water, which supports the
hypothesis that the external flora of fish is a reflection of their environment.
The Atlantic salmon can be caught in marine, estuarine, and freshwater en-
vironments and so allows comparison of the flora of one fish species with that
of different aquatic environments.
26 M. M. Cahill
Intestinal Microflora
The microbiology of the intestinal tract of marine and freshwater fish has been
investigated by many researchers. There is evidence that dense microbial pop-
ulations occur within the intestinal contents, with numbers of bacteria much
higher than those in the surrounding water, indicating that the intestines provide
favorable ecological niches for these organisms (see [7], p. 26). Table 4 sum-
marizes the results of a number of these investigations.
The method of sampling of intestinal bacteria varied in these surveys. The
use of anal swabs by Colwell [ 12] has been criticized (see [7], p. 37) as it probably
does not give a representative sample of gut flora. In some cases [16, 24, 30,
41-43, 46] only the intestinal contents were sampled, while in others the in-
testinal tract plus contents were homogenized and used for culturing [19, 26,
38, 45, 46]. Austin and A1-Zahrani [6] distinguished between the flora of the
gut contents and that intimately associated with the wall of the gastrointestinal
tract, and noted that scanning electron microscopy showed only sparse micro-
bial colonization of the wall. In this study, there was a progressive decline in
the numbers of aerobic heterotrophic bacteria along the digestive tract. An-
aerobes were detected only in the upper intestine and in the intestinal contents.
However, Trust and Sparrow [46] found that numbers of bacteria in fresh-
water salmonids increased between the stomach and the posterior portion of
the intestine. They suggested that these numbers must represent active mul-
tiplication in the tract as they could not be accounted for by ingestion. The
numbers detected in this survey are probably an artificially low estimate since
the methods used did not allow for the isolation and growth of strict anaerobes,
species sensitive to oxygen, nutritionally fastidious species, or those requiring
low growth temperatures (below 20~ Also, the counts obtained were based
on the total tissue weight in each sample, while the bacteria actually populate
only the epithelial surface of the tract, and the rest of the tissue of the tract is
Table 4. Intestinal microflora o f fish"
to
Achro- Flavo- Coryne- oo
Pseudo- mobac- bacte- bacte- Aero- Micro-
Fish species Location monas Vibrio ter rium rium Bacillus monas coccus
Various (see Table 2) Puget Sound, U S A 48.1 13.9 17.7 2.5 1.3 5.1 5.1
Flatfish (Kareius bicoloratus) Fish market, J a p a n + + + +
Bluefish (Pomatomus saltatrix) Long Island Sound, 22.4 28.4 18.2 1.6 0.9 1.4
USA
Striped bass (Morone saxatilis) H u d s o n River, U S A 23 10 5 2 0.5 33 1
(estuarine)
Long Island Sound, 11 27 18 1 0 21 3
U S A (marine)
Plaice (Pleuronectus platessa) Scotland + + + + + +
Salmonids Freshwater lakes, + + + + + + +
Canada
R a i n b o w T r o u t (Salmo gairdnerO Fish farm, Scotland 12 4 12 8 16 12
14
Plaice (Pleuronectus platessa) + Gilmour et al. [ 16]
Salmonids + + Trust and Sparrow
[46]
R a i n b o w Trout (Salmo gairdneri) 2O Austin and Al-Zahrani
[6]
Dace, field gudgeon, c o m m o n minnow, + + Sugita et al. [41 ]
stone moroko, carp, crucian carp,
and brackish goby
Goldfish (Carassius auratus), + Sugita et al. [42]
1-year-old
R a i n b o w trout (Salrno gairdnerO + + Nieto et al. [26]
Carp (Cyprinus carpio) Sugita et al. [43]
Salmonids + Trust and Sparrow
[46]
Grass carp (Ctenopharyngodon + Trust et al. [45]
idella)
Tilapia zillii Sakata et al. [30]
Tilapia zillii Sakata et al. [30]
Tilapia (Sarotherodon niloticus) Sakata and Koreeda
[29]
bo
30 M.M. Cahill
sterile. There was no significant difference in the bacterial flora offish o f different
species, sex, breeding status, weight, or geographical source.
The predominant species were Enterobacter, Aeromonas, and Acinetobacter
in these freshwater fishes. This contrasts with those in digestive tracts o f marine
fishes, where Vibrio, Pseudomonas, Achromobacter, Corynebacterium, Flavo-
bacterium, and Micrococcus predominated [12, 24].
showed fluctuations. G r a m - p o s i t i v e cocci from these fish did not show the
same correlation with those from the water supplying the ponds, but handling
and c o n t a m i n a t e d feed m a y have affected the n u m b e r s o f staphylococci detected
in the fish [26].
Farming practices for some fish [e.g., rainbow trout (Sahno gairdneri)] require
a constant flow o f fresh water through the fish ponds. W h e r e large n u m b e r s o f
fish are being raised, the addition o f fish feed (some o f which is not utilized)
and fish feces to the water can have a detrimental effect on the microbial quality
o f the effluent water from the fish farm. A study in Finland [25] showed that
effluents from two fish farms had elevated n u m b e r s o f total coliforms and fecal
coliforms, and on one farm, effluent had m o r e fecal streptococci than the
influent. The majority o f coliforms were identified as Enterobacter and Citro-
bacter, and Aeromonas hydrophila was quite c o m m o n . High concentrations o f
fecal streptococci and total coliforms were detected in fish feces. Fecal strep-
tococci and coliforms appeared to enter the farms via influent water and feed,
and seemed to multiply in the fish intestines and possibly in the sediment. A
quantitative study o f the intestinal microflora o f the farmed fish was not done,
although such a study could have indicated m o r e accurately the relationship
between intestinal bacteria and those in the effluent.
Austin [4] also c o m p a r e d the microbial floras o f the inflow and outflow from
four rainbow trout farms. T h e r e was no consistent increase in coliforms or in
Aeromonas hydrophila in the effluents f r o m these farms, although Enterobacter
aerogenes increased from 0 (inflow) to 5% (outflow) on two farms, and there
was a similar increase in Escherichia coil on another two farms. In these cases,
increases were considered due to increasing eutrophication and were not linked
to multiplication within the fish intestinal tract.
While the microorganisms present in water can influence the microbial flora
associated with fish, the contribution o f bacteria shed from fish to water is also
worth consideration, especially where dense populations offish are maintained,
as in aquaculture.
Total viable counts and determinations o f the varieties o f bacteria in the
water o f a tank used for rearing carp were m a d e before, during, and after the
addition o f the fish [43]. Total viable counts increased after the introduction
of carp, and the diversity and p r e d o m i n a n c e o f bacterial groups also changed.
Before the introduction o f fish, Pseudomonas and the Flavobacterium-Cytoph-
aga group were most n u m e r o u s in the water, and Enterobacteriaceae, the Vibrio-
Aeromonas group, and Bacteroidaceae were not detected. T h e Vibrio-Aero-
rnonas group b e c a m e p r e d o m i n a n t after the fish had been added. As the only
bacteria present in the pelleted diet o f the fish were Bacillus and Clostridium,
it appeared that the Vibrio-Aeromonas and Bacteroidaceae in the water had
originated from the fish feces. T h e presence o f these organisms in the water
seemed d e p e n d e n t on a continual input from fish excreta, as they disappeared
once fish were removed.
has been cultured extensively in Japan and elsewhere. It was considered a good
subject for a study o f intestinal flora because o f its ability to adapt to fresh and
sea water, and its o m n i v o r o u s diet, including phytoplankton, algae, and some-
times water plants. Its intestine is five to seven times its b o d y length and has
an a b u n d a n t c o m m e n s a l flora [30]. Counts and identification o f viable hetero-
trophic aerobic bacteria were m a d e from h o m o g e n i z e d sections o f intestines
offish kept in fresh or sea water, and the variety o f these bacteria was c o m p a r e d
with that o f the corresponding water.
T h e n u m b e r o f genera isolated from the fish intestines was m u c h m o r e
restricted than that isolated from the surrounding water. Aeromonas p r e d o m -
inated in the intestines o f freshwater Tilapia, whereas Flavobacterium and
Pseudomonas p r e d o m i n a t e d in freshwater. In marine fish, Vibrio and Aero-
monas f o r m e d the greater percentage o f the intestinal flora, but Pseudomonas
was most frequently isolated from the sea water. H o w e v e r , where fish had been
kept for a long time (181 days) in sea water, there were m o r e Aeromonas than
Pseudomonas in the water, indicating that fish feces m a y be a source of Aero-
monas in water.
In a further study o f the microbial flora of Tilapia intestines, separate groups
o f fish were reared in freshwater and in 25, 50, 75, or 100% sea water [40].
T h e bacteria o f the stomach, fore-intestine, and postintestine (sic) were isolated.
Bacteroides types A and B, aerobic gram-negative rods, coryneforms, and strep-
tococci were isolated from all three regions. Bacilli, aerobic gram-positive cocci,
and yeasts were found only in the stomach and fore-intestine regions. Where
the fish were reared in 75 and 100% sea water, n u m b e r s o f obligate anaerobes
decreased a n d / o r disappeared, and aerobic and facultatively anaerobic gram-
negative rods predominated. Bacteroides type A was found to grow in NaCl
concentrations o f 0 to 3%, Bacteroides type B in 0 to 2%, and Vibrio-Aeromonas
in 0 to 4%. Therefore, sensitivity to increased salinity is a possible reason why
the bacterial flora in the gastrointestinal tract o f Tilapia nilotica changes during
the process o f the fish's adaptation to sea water.
The bacterial flora o f the gut o f two marine fish has been investigated in an
attempt to clarify the relationship between these bacteria and the bacterial flora
of their diets, and to d e t e r m i n e the effect o f the degree o f specialization o f the
digestive tracts on their floras [34]. Bacteria were isolated from the gastric and
intestinal contents o f two fish species, red sea bream and file-fish, at various
time intervals after the fish were fed in the laboratory. Bacteria were also isolated
from the fishes' e n v i r o n m e n t and from various diets (fishmeat, fishmeat sup-
plemented with chitin or starch, and shellfish). The file-fish has a relatively
undeveloped stomach in comparison with that o f the red sea bream. T h e
stomach and intestinal contents o f the file-fish closely reflected the bacterial
flora of their diet, whereas in red sea bream, the d o m i n a n t flora always consisted
o f one or both o f two groups o f vibrios (distinct from those present in the
diets). These two groups o f vibrios were both resistant to 2% bile and low p H
(5.5) and were considered to be m e m b e r s o f an indigenous flora o f the digestive
Bacterial Flora of Fishes 33
tract o f sea bream. This work implied that the bacterial flora of fish with
relatively undeveloped digestive tracts reflected that o f the fishes' food, whereas
fish with more specialized tracts have a distinctive gut microflora. In the red
sea bream, the composition of the bacterial floras of the stomach and intestine
changed with time after feeding. Half an hour after ingestion most o f the bacteria
isolated from the stomachs resembled those of the fishmeat diet, but after 6
hours, vibrios resistant to bile and low pH predominated.
Further work comparing representative strains of these indigenous vibrios
from the red sea bream with other isolates from the stomach and intestine
(peritrichous rods, Achromobacter species, and cocci) showed that the vibrios
were able to survive in the presence of gastric juice at pH 4, and were able to
grow, although at a reduced rate, at pH 5, while most o f the other isolates were
inhibited by these conditions [35].
N e w m a n et al. [24] sampled the microbial flora o f the bluefish (Pomatomus
saltatrix) intestine to see whether the microorganisms present were dependent
on the type o f food most recently ingested. The bacteria isolated were similar
to the flora o f some other marine fish (e.g., skate, sole, and cod) which may be
prey to bluefish. Whether these fish actually were prey was not determined,
and no observations o f the flora of prey fish from the same geographic location
was available, so this conclusion was rather conjectural. The gut flora merely
seems typical o f that o f most marine fish.
Medicated feeds o f the types used in the treatment o f fish diseases contain
antimicrobial agents, such as oxolinic acid, oxytetracycline, sulphafurazole,
erythromycin, and penicillin G. Administration o f these to rainbow trout caused
changes in the population composition of the gastrointestinal flora [6]. Organ-
isms sensitive to these compounds appeared to be inhibited and were succeeded
by a resistant microflora. This suggests that the presence of the normal micro-
flora o f the gut may prevent its colonization by other bacteria. Antimicrobial
treatment also resulted in higher populations of bacteria in the lower intestine,
but the reason for this was not clear.
Most investigators offish intestinal microflora have used methods which would
isolate only aerobic or facultatively anaerobic bacteria. Trust et al. [45] isolated
obligate anaerobes from intestines of grass carp and goldfish, showing that fish
can have a resident nonpathogenic anaerobic microflora. The number of an-
aerobes in the gastrointestinal tract plus contents ranged from 6.6 x 1 0 4 to 1.6
• 109 g-l, depending on the medium used and the region of the tract sampled.
The highest count was from the hindgut of grass carp. Strict anaerobes isolated
from the different fish are shown in Table 5 [45]. Other strict anaerobes, mostly
gram-negative rods, but also some gram-positive cocci, which were isolated
had not previously been described.
34 M . M . Cahill
Fish Bacteria
Grass carp Actinomyces
Bacteroides
Clostridium
Eubacterium
Fusobacterium
Pepgostreptococcus
Goldfish Bacteroides melaninogenicus
Bacteroides thetaiotaomicron
Bacteroides sp.
Rainbow trout Bacteroides melaninogenicus
Clostridium
Fusobacterium
From Trust et al. [45]
Masu salmon Oncorhynchus masou and chum salmon O. keta were cultured
from the egg to the fry stage to determine when the normal intestinal microflora
Bacterial Flora of Fishes 35
was established [50]. In sac fry and advanced fry the bacterial counts from the
whole digestive tract ranged from nil to 104 g-J, whereas in fingerlings similar
counts yielded 103 to 107 bacteria g-~. In sac fry, coryneforms and Pseudomonas
were isolated from the digestive tract, and these organisms appeared to be
derived from bacterial flora of the water and the fishes' diet. In later stages
(i.e., in advanced fry and fingerlings), the intestinal flora consisted mainly of
Aerornonas. Therefore, it seems that the normal intestinal microflora of sal-
monids is established when the yolk is absorbed and the digestive tract is
activated at the advanced fry stage.
An investigation of the aerobic and anaerobic heterotrophic intestinal flora
of goldfish Carassius auratus demonstrated that the intestinal microflora be-
came relatively stable at about 67 days after hatching, and consisted of Aero-
rnonas hydrophila, Pseudomonas, Clostridium, and Bacteroides type A. Other
transient bacteria appeared in intestinal samples, including Plesiomonas shi-
gelloides, Enterobacteriaceae, and Moraxella. These transients were also de-
tected in fish diets and fish eggs, and in water or sediment, but did not become
established in the intestines. Bacteroides type A was first detected at about 44
days and became the predominant organism in the intestinal contents of the
adult fish. Thus, the permanent intestinal microflora consisted of bacteria which
were also present in the surroundings but which were able to persist and grow
in the environment provided by the intestinal tract [42].
Another aspect o f the intestinal microflora o f fish is their role in fish nutrition.
Microbial breakdown of substances, such as cellulose and chitin, in the gut
could make nutrients available for absorption. Cellulase activity was found to
occur in the stomachs of 17 of 62 fish species examined and was apparently
due to the production o f this enzyme by gut microflora [39].
Chitin-decomposing bacteria were isolated from the digestive tracts of ayu
(Plecoglossus altivelis), carp (Cyprinus carpio), and rainbow trout (Salmo gaird-
neri), in relatively high numbers and frequencies (up to 4.4 x 107 g-~ wet weight
of whole digestive tract, and 3 to 91% of the total count). Most of these chitin-
decomposers were identified as Aeromonas species from freshwater fish, and
mainly Vibrio from marine fish [20]. Direct evidence o f a nutritional role for
these bacteria was not given.
Commercial fish feeds contain food materials held together by various bind-
ers to form pellets or particles suitable for fish of different sizes to eat. In a
study o f farmed plaice [16], bacteria isolated from the intestines of fish which
had eaten feeds bound with different binders were tested for their ability to use
each binder as a sole carbon source. Samples of intestinal contents were in-
cubated in modified ZoBell's medium for 4 days before being plated out, and
the bacteria which grew were identified. Because of the method used, relative
numbers o f the genera from the intestines could not be determined. The iden-
tification methods used did not distinguish between the Pseudomonas and
Achromobacter/Alcaligenes/Agrobacterium species which were isolated. Other
bacterial groups were Vibrio/anaerogenic Aeromonas, Enterobacteriaceae, Mor-
36 M.M. Cahill
fish should not contain populations of potential human pathogens which are
larger than the threshold values for these organisms. Threshold values for
viruses (as demonstrated by the exposure of fish to phage T2) were quite low
(102 per fish).
The spleen, liver, and kidneys should be sterile in healthy fish [26], and the
presence of bacteria in these organs could indicate breakdown of immunological
defense mechanisms as the result of stress. Stress factors include poor water
quality, temperature changes, nutritional deficiencies, overcrowding, trauma,
parasitism, or primary viral infections [14]. There are reports of isolations of
bacteria from internal organs of apparently healthy fish [18, 26, 46]. Such
organisms may have been particularly resistant to the host's defense mecha-
nisms. Presumably, they would eventually either be destroyed, or survive to
initiate an infection.
Some breakdown of the fish's natural defenses could also explain the occur-
rence of bacteria in organs such as the liver and kidney. The immune response
in fish is significantly affected by temperature [ 13]. When the immune response
is suppressed by lower temperatures, fish have no immune protection against
the pathogenic mechanisms of bacteria in their environment, whether these
organisms are obligate pathogens or environmental organisms, such as Aero-
m o n a s hydrophila.
Conclusions
Acknowledgment. This work was supported by funding from the Mayne bequest fund of the
University of Queensland.
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