Università degli Studi di Sassari
SCUOLA DI DOTTORATO DI RICERCA
Scienze dei Sistemi Agrari e Forestali
e delle Produzioni Alimentari
Indirizzo Scienze e Tecnologie Zootecniche
Ciclo XXIII
Microbial ecology of the intestinal tract of gilthead sea
bream (Sparus aurata Linnaeus, 1758)
Dr. Rosanna Floris
Direttore della Scuola: Prof. Giuseppe Pulina
Referente di Indirizzo: Prof. Nicolò Macciotta
Docente Guida: Prof. Giuseppe Pulina
Tutor: Nicola Fois
Anno accademico 2009-2010
1
To Paolo, Gabriele and Letizia
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
1
ACKNOWLEDGEMENTS
I am grateful to my husband Paolo, my children Gabriele and Letizia for
their patience when I was worried and tired from my experiments and I
did not have time to stay and play with them. They have encouraged me
during these three years giving me a great deal of peace of mind.
I sincerely wish to thank Dr. Silvana Manca for her technical support in
the laboratory and for her encouragement.
I also thank Dr. Elisabetta Daga for her scientific advice and Dr. Ilaria
Duprè for her suggestions on sequence analysis.
I thank Dr. Fabrizio Chessa and Sig. Marco Trentadue for technical help.
I want to express my gratefulness to Massimo Pes and Riccardo Di Salvo
for their friendship, advice and encouragement.
I wish to thank Prof. Nicolò Macciotta and Prof. Corrado Dimauro for
their clear and precious lectures on statistics and for their humanity.
I wish to express a special thought to my mother and my father (not alive
any more) because they taught me to work with enthusiasm and this has
always allowed me to face up to all the difficulties of life.
Last but not least I have to thank the AGRIS Department of Animal
Production (DIRPA) where I work and Dr. Nicola Fois for giving me the
chance to improve my scientific knowledge by taking this PhD,
and I wish to thank Dr. Roberta Comunian, Dr. Antonio Paba and all the
colleagues in the microbiology laboratory of DIRPA for their kind support
and encouragement during this research.
I also wish to thank the “La Maricoltura Alghero s.r.l” and the
“Cooperativa Pescatori Tortoli”.
This research has been funded by AGRIS-DIRPA Servizio Risorse Ittiche.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus,
1758).-Tesi di Dottorato in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di
Sassari.
2
INDEX
ABSTRACT
pag. 6
CHAPTER 1:
SPARUS AURATA LINNAEUS, 1758
pag. 7
1.1 Biology of gilthead seabream (Sparus aurata, L.)
pag. 8
1.2 Types of rearing
pag. 9
1.3 Productions
pag. 11
CHAPTER 2:
FISH GUT
pag. 14
2.1 Anatomy and general physiology of the teleost‟ gut
pag. 15
2.2 Digestive metabolism
pag. 19
2.3 Role of the digestive tract for osmoregulation and endocrine control
pag. 21
in teleosts
2.4 Endocrine and nervous functions of the gut
pag. 24
2.5 Immune functions of the gut
pag. 25
CHAPTER 3:
FISH INTESTINAL MICROBIOTA
pag. 27
3.1 Overview
pag. 28
3.2 Microflora composition of the gut
pag. 30
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
3
3.3 Role of intestinal bacteria for nutrition
pag. 32
3.4 Probiotics
pag. 35
3.4.1 Probiotics in aquaculture
pag. 37
3.5 Pathogens
pag. 39
CHAPTER 4:
METHODS FOR STUDYING BACTERIAL FLORA
pag. 42
4.1 Conventional techniques
pag. 43
4.2 Molecular techniques
pag. 44
4.2.1. Methods for direct detection of bacteria in fish products
pag. 49
CHAPTER 5:
AIM
pag. 53
CHAPTER 6:
MATERIALS AND METHODS
pag. 56
6.1 Fish farms and sampling
pag. 57
6.2 Microbiological analyses
pag.62
6.2.1 Sampling and processing
pag. 62
6.2.2 Microbial quantitative analyses
pag. 64
6.2.2.1 Culture media and growth conditions
pag. 64
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
4
6.2.3 Microbial qualitative analyses
pag. 64
6.2.3.1 Basic phenotipic tests
pag. 64
6.2.3.2 Genetic analyses
pag. 65
6.2.3.2.1 Bacterial “type” strains
pag. 65
6.2.3.2.2 Cell lysis and DNA estraction
pag. 66
6.2.3.2.3 Quantification of DNA samples
pag. 67
6.2.3.2.4 Amplification of the 16S rRNA gene
pag. 67
6.2.3.2.5 Amplified ribosomal DNA restriction analysis (ARDRA)
pag. 68
6.2.3.2.6 Purification of PCR products for 16S rRNA gene sequencing
pag. 69
6.2.3.2.7 Estimation of purified PCR products and sequence analyses
pag. 70
6.3 Statistical analyses
pag. 71
CHAPTER 7
RESULTS
pag. 72
7.1 Microbiological quantitative analyses
pag. 73
7.1.1. Enumeration of intestinal microflora
pag. 73
7.2 Microbiological qualitative analyses
pag. 76
7.2.1 Basic phenotypic tests
pag. 76
7.2.1.1 Bacterial growth on selective culture media
pag. 78
7.2.2 Genetic analyses
pag. 80
7.2.2.1 Amplification of 16S rRNA gene
pag. 81
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
5
7.2.2.2 Amplified ribosomal DNA restriction analysis (ARDRA)
pag. 82
7.2.2.2.1 Analysis of ARDRA profiles
pag. 85
7.2.2.3 Comparison of ARDRA profiles of the bacterial isolates with the
pag. 88
“type” strains
7.2.2.4 Intestinal microbial ecology
pag. 89
7.2.2.4.1 Identification of bacteria by sequence analysis
pag. 89
7.2.2.4.2 Intestinal microflora of gilthead sea bream reared in the “ La
pag. 90
Maricoltura Alghero s.r.l “ facility and the Tortoli lagoon
7.2.2.4.3 Intestinal microbial community of Sparus aurata
pag. 93
DISCUSSION
pag. 95
CONCLUSIONS
pag. 105
LIST OF FIGURES
pag. 108
LIST OF TABLES
pag. 111
LITERATURE CITED
pag. 112
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
6
ABSTRACT
The study of fish gut microflora is important because it reflects the bacterial
composition of the rearing environment and the dietary regimen of ingested food;
moreover, these microbiota play a role in the health and the quality of adult fish. The
aim of this research was to study the microbial ecology of the gut of two groups of
gilthead sea bream (Sparus aurata L.) reared in off-shore floating cages and in a lagoon,
located in Sardinian coast, in order to quantify the heterotrophic bacteria and to identify
at genus and species level the dominant bacterial communities of the intestinal tract by
means of the ARDRA technique and sequencing of the 16S rRNA gene. This study
aimed to test the microbiological quality of fish and intestinal microbial biodiversity in
order to detect a possible link with the rearing system. The results showed a
significantly higher bacterial load in the gilthead sea bream farmed in the lagoon than in
the fish from the off-shore cages and highlighted a different bacterial qualitative
composition of the gut microflora in the two groups of fish, although the presence of the
Pseudomonas spp. was observed in all the fish studied. A greater microbial diversity at
species level was observed in the sea bream reared in the lagoon.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
7
CHAPTER 1
SPARUS AURATA LINNAEUS, 1758
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
8
1.1 Biology of gilthead sea bream (Sparus aurata, L.)
Sparus aurata L. (Teleostei: Sparidae) inhabits the Eastern Atlantic coasts from Great
Britain to Senegal, the Mediterranean and the Black Sea (rare) and is considered one of
the most important marine fish in fishery and aquaculture. It is naturally found in both
marine and brackishwater environments such as coastal lagoons and estuarine areas, on
rocky and seagrass (Posidonia oceanica) meadows, but it is also frequently caught on
sandy grounds especially during the initial stages of its life cycle. The body is oblong
and the mouth has 4-6 canines in front of both jaws with behind them and at back 2-4
rows of teeth. The colour of the body is silvery gray with large dark patch at origin of
lateral line, overlapping the upper part of the opercle and underlined by a reddish area.
The head presents a golden curved bar, bordered by two dark zones, especially in adults
(Bauchot and Hureau, 1986). The natural reproductive cycle starts in the open sea where
the fish is born during October-December, while the juveniles of gilthead seabream
migrate in early spring towards protected coastal waters, where they can find trophic
resources and milder temperatures. Gilthead sea bream is a protandrous hermaphrodite
and ovaries develop asynchronically releasing the reproductive cells by mass spawning
daily and for a period of 3 to 4 months. Sexual maturity develops in males at 2 years of
age (20-30 cm) and at size of over 30 cm they become females, which at 2-3 years (3340 cm) produce 20 to 80 thousands spherical and transparent eggs with a diameter of
less than 1mm and a single oil droplet (Arabaci et al., 2010). This fish species does not
spawn in Black Sea (Bauchot and Hureau, 1986). Gilthead sea bream are mainly
carnivorous (shellfish including mussels and oysters), occasionally herbivorous.
Arabaci et al., (2010) in a study on breeding stocks of gilthead sea bream aimed at
analysing its behaviour and morphology in the hatchery and in the wild, showed that a
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
9
social behaviour exists although it is considered also a solitary fish species. In hatchery
they observed the presence of one leader candidate, normally a male with dark vertical
bands and not necessarily of large size. If two leader candidates occur they struggle for
life till one wins and the the losing one, having been rejected by the group, dies from
injuries or starvation. Each individual in the stock obeys the winning leader. As regards
the wild gilthead sea bream, the same authors observed that they form schools of
different number, size and phenotypes and it is not clear whether they belong to
different populations or not.
1.2 Types of rearing
Gilthead seabream are traditionally cultured extensively in coastal lagoons and saltwater
ponds and have been reared intensively systems both in ponds and in cages since the
1980s. Sparus aurata is farmed in coastal ponds and lagoons using extensive and semiintensive methods which differ on fish farming density and food supply (FAO, 2010).
The extensive system is based on naturally migrationing juveniles being collected in
fishing traps. The productive cycle of juveniles and adult fish in the extensive system
starts with reproduction in the estuary or pond in autumn, the development of yolk sac
fry followed by larval stage at 7-10 days, the post larvae of 14-18 mm and their
migration to the open sea or pond in February-April for extensive fattening. The
extensive method of rearing provides a limited source of natural juveniles, therefore,
modern commercial extensive production structures use both wild-caught and hatcheryreared juveniles and a high proportion of sea bream fingerlings come today from
unselected broodstocks (Arabaci et al., 2010). Thus, the gilthead sea breams‟ juveniles
are produced in the hatcheries and when they reach the weight of 2-3 g are normally
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
10
seeded into the lagoons in April-May achieving the commercial size of 350g in 20
months.
As regards semi-intensive systems, they consist of seeding lagoons with juveniles prefattened in an intensive systems and in this case it is possible to increase the natural
food source by fertilization of farming area. Other types of semi-intensive farming are
based on the provision of artificial feed and supplemental oxygen and this practice is
performed in limited areas of the lagoons.
The production cycle of juveniles of Sparus aurata in intensive system is quite different
from that found in nature. Indeed the broodstock, which is a group of various aged
individuals from 1-year-old males to 5 year-old females is kept in tanks equipped with a
water heating/cooling system under computerized control of temperature and light
conditions. The breeders, originating from a farm or from the wild, are fed a specific
artificial diet commonly represented by fish meal. As soon as (mainly in an artificial
way), spawning starts, selected breeders are transferred to the spawning tanks.
Afterwards, the yolk sac fries represents the first step of the larval phase and after 7-10
days they become larvae. Gilthead seabream larvae generally deplete their yolk sacs
after 3-4 days of endogenous feeding. At this stage, the eyes are pigmented and the
mouth developed for praying on living organisms such as rotifers (e.g. Brachionus
plicatilis). After 10-11 days, Artemia salina nauplii integrates the diet of the larvae until
metamorphosis occurs (32-35 days post hatching). The 45-day old fish constitute the
prefattened juveniles of 5 g which is transferred into a section of the hatchery equipped
with larger tanks (10-25 m3) where the weaning rearing system takes place. This step
consists of a intensive rearing system where feed is given at 2-hour intervals from 8.00
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
11
to 20. 00 using artificial feeds composed of 150-300 μm particles (FAO, 2010). As
regards the rearing of gilthead seabream in intensive systems, tanks at densities ranging
from 15-45 kg/m³ with a massive oxygen support and temperatures from 18-26°C, are
used. The intensive grow-out normally follows different phases starting from
reproduction, larval rearing as described above and pre-fattening during which small
gilthead seabream (5 g) reach initial commercial size (350-400 g) in about one year.
This takes place in land-based installations with rectangular tanks that vary in size (2003000 m³) according to fish size and the demands of production. Grow-out may also
occur in sea cages, either in sheltered or semi-exposed sites (floating cages) or totally
exposed sites (semi-submersible or submersible cages). Intensive systems may use
juveniles purchased from separate hatcheries, but large production units normally rear
their own. The fattening system normally used in the Mediterranean basin is represented
by the sea cages technique characterized by densities of 10-15 kg/m³ (lower than in
tanks) and consisting of a simple and economical method for fattening with no energy
costs for pumping, aeration etc. Feeds are artificial and distributed by automatic feeders
or by hand for larger fish at intervals during the day. The negative side of this system is
represented by the longer rearing period (about 16 months ) before the fish are of
market size.
1.3 Productions
Fishery in EU has shown a decline during the last two decades from 9 million in 1989 to
7 million t in 2005, while aquaculture has been increasing from that time on ward
(Melotti and Roncarati, 2009). According to EU statistics and FEAP reports (2008)
finfish aquaculture production in 2006 reached a quantity of 1,415,632 t. Considering
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
12
the EU member states, the four largest fish producers were the United Kingdom
(145,739 t), Greece (83,000 t), Italy (71,900 t), Spain (65,515 t) and France (49,900 t).
Other European countries which contribute to aquaculture productions are Norway
(47.8%) and Turkey (6.1%).
Farming of eurhyaline species as Sparus aurata started developing in the mid-1980s
and a large scale production of gilthead seabream juveniles was achieved in 1988-1989
in the Mediterranean basin particularly in Spain, Italy and Greece where artificial
breeding, hatchery production and farming have become well established. In 2008, the
largest producer of Sparus aurata was Greece with 49,000 t (47 %) followed by Turkey
with 17,000t, Spain with 21,100t, Italy with 9.200t (Roncarati and Melotti, 2007; FEAP,
2008) and other producers such as Croatia, Cyprus, Egypt, France, Malta, Morocco,
Portugal, Tunisia, Israel and Kuwait (FAO, 2010). After the crisis of the recent years,
Greece remains the main producer of sea bream and seabass while Turkey and Spain
productions have been growing more than the Italian one which has shown a lower
growth rate over the last ten years (Melotti and Roncarati, 2009).
Extensive farming still remains a traditional activity in some regions but with a low
impact on the market. According to lagoon productivity, total production by extensive
system with fish density generally not exceeding 0.0025Kg/m³ ranges from 30-150Kg
/ha/yr. The production in semi-intensive systems with a density of about 1 kg/m³ ranges
from 500-2400 kg/ha/yr. Annual production of Sparus aurata in intensive systems, both
in ponds and in cages with a fish density of 10-15 kg/m³, have increased regularly until
2008 reaching a peak of 128,943 t (Arabaci et al., 2010). However, the rapid
development of production led to prices falling by approximately 60 % between 1990
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
13
and 2000 and are still decreasing. Until the 1990s, most of sea bream production
originated from land based farms, while from that time onwards, with the development
of mariculture techniques, this fish has been produced using floating cages in sheltered
areas, as well as submersible or floating cages in open sea area (Roncarati and Melotti,
2007). As regards Sardinian production of Sparus aurata in semi and intensive systems
estimated in 2008, it reached the value of 1,385 t (Viale, 2009).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
14
CHAPTER 2
FISH GUT
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
15
2.1 Anatomy and general physiology of teleost’ gut
A detailed description of fish gut, which represents the tract between the mouth and the
anus, was provided by Smith (2009). This anatomic section is generally divided into the
head gut, the foregut, the midgut and the hindgut with some peculiarities according to
the dietary regimen of the fish (Fig. 1). The head gut comprised the mouth and the gill
(branchial, pharyngeal) cavities. The foregut begins behind the gills and includes the
oesophagus, the stomach, and the pylorus. In fish, such as the cyprinus, which lack both
a stomach and pylorus, the foregut consists of the oesophagus and an intestine anterior
to the opening of the bile duct. The midgut includes the intestine behind the pylorus and
often includes a variable number of pyloric caecae (pyloric appendages), although
pyloric caecae are always absent in fish which lack stomachs. The midgut is always the
longest portion of the gut and may be coiled into intricated loops (often characteristic
for each species). The beginning of the hindgut is marked by an increase in diameter of
the gut which ends with the anus. The gut of teleosts forms very early during embryonic
development and its length may change during development. Indeed the larvae of most
fish are carnivorous and have a short, simple, agastric gastrointestinal tract that
apparently has limited digestive capabilities (Buddington et al., 1997). Acquisition of
adult gastrointestinal tract characteristics coincides with the transition to the adult diet,
which for many fish occurs at metamorphosis. The morphology and physiology of the
adult fish‟s digestive tract vary a lot from fish to fish and different factors determine the
final structure of the gut: the phylogeny of a species and feeding habits. Based on the
nature of the food ingested, three broad categories are distinguished: herbivores and
detritophags, omnivores which consume small invertebrates, and carnivores which
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
16
consume fish and bigger invertebrates (Kapoor et al., 1975). In teleosts the gut lengths
are 0.2-2.5, 0.6-8.0 and 0.8-15.0 times body length in carnivores, omnivores, and
herbivores, respectively. Thus, the longest guts are found in herbivores (Smith, 2009)
but not all herbivores have long guts in fact the gut lengths of some herbivores are
shorter than those of some carnivores and this can be due to the fact that they have to
adapt to variable conditions of life represented by new foods sometimes ingested with
indigestible material (e.g. mud). The mouth of fish exhibits a variety of adaptations for
capturing, holding and sorting food. Most the Cyprinidae like common carp have
pharyngeal teeth placed in front of the oesophagus which are used as the primary
chewing apparatus although other groups of fish also show a abrading or triturating
ability with some part of the gill bars. Many fish which chew their food have some
capcity to secrete mucus which is only partly comparable to saliva. The oesophagus, is a
short, distensible, muscular passageway between the mouth and the stomach so that
large objects can be swallowed, but not all fish have a stomach. In most fishes where a
stomach is present, it may vary in shape, size and structure according to the diet of the
various species. Fish stomachs can be straight with an enlarged lumen, a U-shaped
stomach as in Salmo, a Y-shaped stomach as in Alosa, Anguilla, the cod, and ocean
perch particularly suited for holding large prey. One possible explanation for the loss of
stomachs in some species of fish is that they live in a chloride-poor environment and
providing large amounts of chloride ion for operating a stomach is bioenergetically
disadvantageous. More over, fish which eat mud or other small particles more or less
continuously only need a small stomach. The function of stomachs is probably to
produce hydrochloric acid and the enzyme pepsin. The transport of food from the
stomach into the midgut is controlled by a muscular sphincter, the pylorus which can be
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
17
absent in some species. The midgut of fish is more developed in carnivores and can
include accessory structures that increase the absorbent surface area (Buddington and
Diamond, 1987). These include pyloric caecae, which are blind finger-like diverticula
attached to the anterior part of the midgut, have a taxonomic significance and are
always absent in fish that lack a stomach. The midgut represents the major absorptive
surface area of the body and is composed of distinct epithelial, absorptive and secretory
cells. It is mildly alkaline and contains enzymes from the pancreas and the intestinal
wall, as well as bile from the liver. These enzymes attack all three classes of foods proteins, lipids, and carbohydrates. The demarcation between midgut and hindgut is
often minimal in terms of gross anatomy and fisiology even if they are differentiated
histologically.
Liver and pancreas represented the secretory glands which produce digestive secretions.
The liver is the primary organ for synthesis, detoxification, and storage for many
nutrients and it produces bile which is secreted into the intestine, usually via the gall
bladder and whose primary function is to emulsify fats into small globules
(chilomicrons) for absorption or to make hydrolysis by lipases easier (Lovel, 1998). The
pancreatic tissue in teleost fishes is quite diffuse and consists of acini (ramified tubules)
scattered
along
the
intestinal
surface,
and
within
the
liver
and
spleen.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
18
Head gut
Foregut
Midgut
Hindgut
Figure 1. Representative scheme of the anatomy of the digestive tract of Teleost fish on
the basis of different dietary regimen (from Smith, 2009).
a. Rainbow trout (carnivore)
b. Cat fish (omnivores, animal sources of food)
c. Carp (omnivores, plant sources of food)
d. Milk fish (microphagous planktovore)
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
19
2.2 Digestive metabolism
Digestion represents all the physical and chemical activities through which ingested
materials pass through the gut and are reduced to molecules for absorption and passage
into the blood stream. In this process the proteins are hydrolyzed to amino acids,
digestible carbohydrates to simple sugars, and lipids to fatty acids and glycerol (Smith,
2009). Digestion begins in the stomach where most of the protein digestion occurs even
if it also continues in the intestine. The parietal cells of the stomach secrets hydrochloric
acid, enzymes and mucus to protect this organ. Another type of cells the chief cells
produce pepsinogen which is hydrolyzed to pepsin, a proteolytic and predominant
gastric enzyme which is active at pH 1.5 to 3.0. Thus pepsin and hydrochloric acid
partially hydrolyze proteins into shorter chain polypeptides. Minerals and mineralized
tissue are solubilized in the acid stomach but no fat or carbohydrate breakdown occur in
the stomach (Lovel, 1998). The pyloric sphincter placed in the posterior end of the
stomach holds food until it is fluid for passing into the anterior intestine. Considering
the pH immediately below the pylorus, it changes drastically becoming alkaline (from 7
to 9), reaching a maximum level of 8.6 in the upper intestine, and decreasing to the
neutrality in the hindgut (Page et al., 1976). Pancreas delivers to the upper midgut
through the bile duct different substances like bicarbonate buffering compounds and the
zymogens as trypsinogen, chimotrypsinogen which are the precursors of enzymes which
digest proteins as trypsin which is predominant, chimotrypsin, carboxypeptidases and
aminopeptidases), carbohydrates (α-amilase, maltase in sea bream and ayu), lipids
(lipase), chitin (chitinase), and nucleotides. Moreover, a lipolytic activity was found in
extracts of intestine, liver, spleen, bile, pyloric caecae, stomach and pancreas of
different fish species and cellulase activity was reported in intestinal extracts from some
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
20
fish, but this probably came from intestinal bacteria (Luczkovich et al., 1993; Bairagi et
al., 2002). There were reports of chitinolytic activity in some fish which eat crustaceans
and this was from bacterial activity (Kihara et al., 2001). Digestive enzyme activity in
fish generally indicates a strong correlations with diet in fact herbivores possess higher
levels of carbohydrases (e.g α- amilase) than are found in carnivorous fish which show
higher protease activities (e.g. pepsin and trypsin) (Hidalgo et al., 1999; Fernandez et
al., 2001). On the other hand, this is not always true because a study carried out by
German et al., (2004) on different enzyme activities such as α- amylase, lipase, trypsin
and aminopeptidase in herbivorous and carnivorous prickleback fishes belonging to the
family of Stichacidae showed that despite dietary differences these sister taxa displayed
the most similar digestive enzyme activities. The results support the hypothesis that
phylogeny influences digestive enzyme activities in these fish. In addition, another
study performed on the same species confirmed that activity of α- amilase follows a
pattern influenced more by phylogeny than by diet and indicated that no significant
differences in pepsin and trypsin activities were present between related herbivorous
and carnivorous fish species (Chan et al., 2004). However, fish with relatively broad
diets can modulate digestive enzyme activities in response to changes in dietary
composition (Fernandez et al., 2001) and in salinity in euryhaline teleosts (Psochiou et
al., 2007). Absorption of soluble food takes places predominantly in the midgut and
probably to some degree in the hindgut and species with relative short intestine, such as
the carnivorous S.aurata present significant nutrient absorbtion in their posterior
intestines (Ferraris and Ahearn, 1984).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
21
2.3 Role of the digestive tract for osmoregulation and endocrine control in teleosts
It has estimated that 95% of teleost species are stenohaline which means that live in
either fresh water or sea water. The remaining 5% are euryhaline which have the
capacity to withstand large changes in environmental salinity (McCormick, 2001). The
renal physiologist from the last century, Smith (1930) outlined „„The physiological
problem of maintaining the osmotic pressure and salt composition of the blood against
the stress of fresh or salt water was faced by the vertebrates since the time of their
evolution‟‟. The capacity to regulate the concentration of plasma ions is a necessity for
fish which move between fresh water and seawater as part of their life cycle. The
survival of teleost fish in a dehydrating, marine environment is possible tanks the
uptake of ingested seawater across the intestine and elimination of excess NaCl by the
gills (Veillette et al., 2005). Indeed, marine teleosts have extracellular fluids less
concentrated than their environment, and this could determine a continual water loss. In
order to compensate with this, they drink significant amounts of seawater, a solution
having a pH of about 8.5 by absorbing water and salts across the gut and secreting
excess mono-valent ions across the gills and divalent ions through the kidney
(McCormick, 2001). Moreover, a too high a salt content in the intestine might be not
suitable for the activity of some enzymes reducing the rate of digestion. In this regard,
the stomach (in eels, the oesophagus), having a pH of 4 or lower in most fish, dilutes the
incoming seawater and in this way the final osmoregulatory product of the gut is a rectal
fluid composed of magnesium and other divalent ions having about the same total
concentration as blood. Thus, digestion and osmoregulation are so inter-related that
problems in one system could disrupt the functions of the other. Various physiological
studies were performed on the role of intestine during the osmoregulation in fishes.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
22
Veillette et al., 2005 showed that the transfer of salmon from freshwater to seawater
causes an significant increase of both fluid uptake and Na+/K+ ATPase activity as well
as high physiological cortisol levels in plasma. These authors identified pyloric ceca as
a major site of osmoregulation in chinook salmon. According to McCormick (2001) the
neuroendocrine system takes part to the osmoregulatory adaptations of the teleost fish.
It is interesting as this author described the endocrine control of osmoregulation in some
teleost fish as represented in Fig. 2. This physiological process is complex and involved
mainly the chloride cells situated in the gills where secretion and uptake of salt occur.
These cells have a different morphology in seawater and freshwater and generally are
characterized by numerous mitochondria and a tabular system that is continuous with
the basolateral membrane. The major transporters implicated in salt secretion in the gills
are placed in the chloride cells and include Na+/K+-ATPase (the sodium pump) and
Na+, K+, 2Cl- cotransporter (NKCC) and an apical Cl- channel (Fig. 2). In this process,
the hormone control of osmoregulation is performed by growth hormone (GH) and
insulin-like growth factor I (IGF-I) which give a support to cortisol for regulating salt
secretion in teleosts. In particular, GH causes general cell proliferation of the gill
creating more undifferentiated cells (stem cells) that can then be acted on by cortisol
which promotes the differentiation and the increase of the chloride cells which
determine the ion secretion in seawater environment. A study of this physiological
process made by Shrimpton and McCormick (1998) showed that GH treatments caused
an increase of the number of the cortisol receptors located in chloride cells of the gills in
Atlantic salmon. The same authors found that the number of these receptors is
correlated with the capacity of cortisol to stimulate Na+, K+ ATPase of the gills. In
addition, McCormick (2001) in his article affirms that the GH-IGF I axis may have
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
23
other target tissue like renal epithelia, hepatic receptors and the gut even if this
physiological mechanism vary a lot among the different Teleosts. On the other hand,
prolactin promotes the development of fresh water chloride cells which work for ion
uptaking. In conclusion, as McCormick (2001) states “growth hormone promotes the
acclimation to seawater, prolactin promotes acclimation to fresh water, and cortisol
interacts with both of these hormones thus having a dual osmoregulatory function. It
should be noted that only a small number of teleosts were examined and that we still
know little or nothing about the complex hormonal control of osmoregulation in the vast
majority of fish”.
Figure 2. Endocrine control of osmoregulation in teleost fish: morphology and transport
mechanism of gill chloride cells in seawater and fresh water (from McCormick, 2001).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
24
2.4 Endocrine and nervous functions of the gut
Intestine and associated organs produce hormones which regulate digestion and
metabolic processes in fish. Aldman and Holmgren (1995) described the response of the
trout gall bladder to the passage of nutrients in the digestive tract. They observed that
both fat and amino acids increased the tonus and the frequency of contractions of the
gall bladder, possibly by the release of cholecystokinin. As far as the distribution of
peptide hormone-like substances in the gastrointestinal tract (GIT) is concerned, a study
by means of immunofluorescence technique showed that somatostatin producing cells
were present in the glandular epithelium of the stomach of fish or in the pyloric
appendage of different species while few somatostatin-immunoreactive cells were
scattered in the epithelium of the pancreatic ducts of other species of fish (Langer et al.,
1979). In addition, these studies showed that another hormone, the glucagon was
produced by cells located in the epithelium of the upper mid-gut near the stomach or in
the intestine and that the pancreatic polypeptide was produced by cells located in
various places of the gut in different species of fish. Moreover, another
immunohistochemical study showed that diverse neuropeptides such as bombesin, the
so called gastrin releasing peptides, the enkephalin, gastrin/cholecystokinin,
neuropeptide Y, neurotensin, substance P, vasoactive intestinal polypeptide and
somatostatin have a wide distribution in the gut nerves of fish (Bjenning and Holmgren,
1988). The physiological actions of these peptides were investigated by the same
authors and can be generally summarized as follows: the neurotensis exerts an
excitatory effect on stomach, the somatostatin, substance P affect the gut motility and
the vasoactive intestinal polypeptyde reduces gastric secretion by decreasing the ion
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
25
selectivity of tight junctions in combination with serotonin which is secreted by cells
associated with the intestine as well.
2.5 Immune functions of the gut
The intestine constitutes a surface exposed to pathogens, allergens, and toxins and has
to prevent the invasion by pathogens and noxious components present in the diet and
environment. Therefore, the enteric immune system of fish possess a barrier represented
by non-specific and specific defense mechanisms. Fish are more dependent on nonspecific immune functions such as the tight junctions which link the enterocytes and
provide a physical barrier while the mucous secreted by goblet cells reduces the ability
of bacteria to adhere to the enterocytes and protects against physical and chemical
damage (Landolt, 1989). Phagocytic cells are also present in the mucosa and provide
another means of non specific immunity. However, studies performed on the enteric
immune system of rainbow trout indicated a variety of eosinophilic granule cells which
were considered immunomodulatory agents (Powell et al., 1991, 1993). The highest
densities of intraepithelial macrophages capable of antigen presentation were found in
the distal intestine. As regards a specific enteric immune activity, two distinc cell types
corresponding with the B and T lymphocytes of mammals were found in the gut of carp
(Rombout et al., 1993). Although mucosal accumulations of lymphoid cells, the socalled Peyer‟s patches, have not been described in fish, lymphocytes were found to be
distributed throughout the intestinal epithelium and underlying the tissue layers (Hansen
and Olafsen, 1999). A study performed on plasma and bile fluid of the antartic teleost
fish Trematomus bernacchii evidenced the presence of antibodies specific for a
nematode parasites and and immunoglobulins IgM-like were detected in different
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
26
tissues like liver, gall bladder, common bile duct and anterior intestine (Abelli et al.,
2005).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
27
CHAPTER 3
FISH INTESTINAL MICROBIOTA
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
28
3.1 Overview
Different studies have been performed on the indigenous microflora of fish in
aquaculture. They include descriptions of the quality of farmed fish during ice storage
(Tejada and Huidobro, 2002; Papadopoulos et al., 2003), the relationship between
environment and fish microflora (Horsley, 1973; Sugita et al., 1989), the study of the
bacterial microflora associated with fish farms (Allen et al., 1983) and the nutritional
role of the intestinal flora (Goodrich and Morita, 1977; Sugita et al., 1991). The
microbiology of the intestinal tract of marine and freshwater fish has been investigated
by different researchers and most of them have aimed to determine the origin of the
organism responsible for the spoilage of freshly caught fish. These surveys have
demonstrated that the quality and the quantity of bacteria are a reflection of different
factors: the aqueous environment (temperature, salinity, etc.) (Sugita et al., 1989),
seasonal variation (Al-Harbi and Uddin, 2004; Pujalte et al. 2003a), diet, the different
regions and the anatomy of gastrointestinal tract (Austin and Al-Zahrani, 1988; Ringo et
al., 1998; Ringo and Olsen, 1999; Ringo et al., 2006; Heikkinem et al., 2006), the
stages of fish development (Campbell and Buswell, 1983), type of rearing facility, fish
species (Cahill, 1990) and in general the way of life of the host fish species (Izvekova et
al., 2007). As a consequence, the study of the gut microflora is considered important in
aquaculture because it reflects both the bacterial composition of the rearing environment
(water) and the dietary regimen of ingested food. Intestinal microflora has been
considered an important component of the digestive tract in animals including fish
during larval development when the feeding regimes are represented by rotifers and
Artemia. Furthermore, at this stage, in order to osmoregulate, marine fish larvae start
“drinking” before the yolk sac is consumed (Reitan et al., 1998) and bacteria enter the
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
29
digestive tract before active feeding commences (Hansen and Olafsen, 1999). In this
way, bacteria present in the environment and successively in the live feed are the initial
intestinal colonisers which can prevent the establishement of pathogenic bacteria as
Vibrio anguillarum which dominates the larval intestine (Grisez et al., 1997). The
intestinal microflora must adapt to various conditions of nutrient composition, pH,
anaerobiosis, concentration of bile salts and digestive enzymes, the hosts‟ immune
system, and the presence of other members of intestinal community. According to
Sugita et al., (1988), the development of the gut microflora in Carassius auratus has
three stages: the transitory (accidental) microflora, which is scarce, does not remain for
long in the intestine, and occurs also in the water, food, and on the surface of fish eggs;
the permanent indigenous microflora recorded at all stages of fish ontogeny; and the
“adult” microflora, which first appears within approximately two months after hatching.
These microbiota play a role in the health and the quality of adult fish and constitute a
protection barrier against disease since the intestine is one of the major routes of
infection of certain pathogen bacteria such as Salmonella spp. or Escherichia coli.
These bacteria present in the intestine could contaminate the edible portions of fish and
cause human disease. On the basis of the literature cited, by studying the gut microflora
is possible to have information on the quality of the fishery product and storage life as
well by monitoring the bacterial contents of fish organs.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
30
3.2 Microflora composition of the gut
The colonization of the digestive tract by bacteria depends on different endogenous
factors such as pH, intestinal peristalsis, the contents of bile acids, digestive enzymes,
and the immune response of the host to bacterial invasion, as well as the presence of
autochthonous bacteria and their antibacterial activities (Fuller, 1989). Thus, the
survival of bacteria in the gut depends on their capacity to resist to antibacterial
mechanisms (both chemical and physical) which operate in the gut. Different studies on
the composition of intestinal bacteria have indicated that it is similar to that of
integuments, gills and bolus and is mostly represented by Gram negative both aerobic or
facultatively anaerobic (Cahill, 1990). Gram negative aerobes were found in a greater
number of species and occur with equal frequencies in freshwater and marine fish.
Gram negative aerobes were characteristic mainly of predatory and benthophagous fish
(Izvekova et al., 2007). On the other hand, there are data showing that the intestines of
fish (especially of herbivorous species) contain both facultative and obligate anaerobes
and the population levels of obligate anaerobes present in salmonids are lower than
those of facultative anaerobes (Ringo et al., 1995). The relevant findings on fish
intestinal microbiota show that they include the genera Pseudomonas, Aeromonas and
Enterobacteriaceae, Plesiomonas, Carnobacterium, Flavobacterium and obligate
anaerobic bacteria of the genera Bacteroides, Fusobacterium, Eubacterium in fresh
water-reared fish and Vibrio and Pseudomonas in fish from sea water (Cahill, 1990;
Hansen and Olafsen, 1999; Ringo and Olsen, 1999; Huber et al., 2004). A recent study
performed on Salmo salar and sea trout Salmo trutta trutta juveniles (0+ years old)
from the same environmental conditions showed that the predominant microbiota in the
intestinal tract of sea trout were Enterobacteriaceae (52%), Aeromonas (22%) and
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
31
Pseudomonas (14%) (Skrodenyte-Arbaciauskiene et al., 2008). A total of 38 different
bacterial species were identified in the intestines of fish and are as follows: Aeromonas
caviae, A. hydrophila, A. jandaei, A. sobria, A. veronii, A. punctata, Vibrio
anguillarum, V. tubiashii, V. anginolyticus, V. proteolyticus, V. harveyi, V. natriegens,
V. cholerae, Photobacterium damselae, Plesiomonas shigelloides, Providencia stuartii,
Serratia liquefaciens, Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae,
Pseudomonas
fluorescens,
Shewanella
putrefaciens,
Enterococcus
durans,
Corynebacterium afermentas, C. urealyticum, Bacillus circulans, B. cereus, B. pumilus,
Carnobacterium piscicola, C. divergens and Curtobacterium pusillum, Lactobacillus
acidophilus, L. casei, L. paracasei subsp.paracasei, L. plantarum, L. brevis, and L.
fermentum (Sugita et al., 1996a; Gonzalez et al., 1999; Al-Harbi and Uddin, 2004).
Most of them are considered potential pathogens for the host and represent the
etiological agents of pathogenesis in fish especially when diverse factors of stress
(temperature, salinity, rearing conditions etc.) occur with the consequence of
determining an increase in bacterial number and a dismicrobism in the host. On the
other hand other bacterial species like lactic acid bacteria (LAB), represent the group of
probiotics which are occasionally present in fish intestine in low number and are
considered beneficial for living organisms (Ringo and Gatesoupe, 1998; Itoi et al.,
2008). Other bacterial genera typical of homeothermal animals such as Bifidobacteria,
Bacteroides, Eubacterium are either absent or only occasionally present in fish (Isolauri
et al., 2004).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
32
3.3 Role of intestinal bacteria for nutrition
The role of gut microbiota in fish nutrition has been studied in various fish species of
teleosts and considered important since they metabolize different substrates furnishing
cell substances or micronutrients such as essential fatty acids, vitamins, minerals or
enzymes useful for the host. Most of the studies on fermentative bacterial activity are
referred to herbivorous fish (Luczkovic and Stellwag, 1993; Clements and Choat, 1997;
Mountfort et al., 2002), omnivorous (Kihara and Sakata, 2002) and detritivorous
teleosts (Kihara and Sakata, 1997) but various studies have also been performed on
carnivorous fish (Kihara et al., 1995; Kihara and Sakata, 2001; Mahious et al., 2006;
Burr et al., 2010). These studies have focused on bacterial fermentative activity present
in the intestinal contents coming mainly from the hindgut (posterior intestine, distal
end) of various teleost species. The intestinal bacteria involved in fermentative
activities, the substrates used by these microbiota, the products of their metabolic
activities and the fish species where the studies referred to, are presented in Tab. 1. The
bacteria present in the gut are represented by different species not always identified at
taxonomic level whose metabolism is heterofermentative, in fact monosaccharides like
glucose, mannitol, glucuronic acid, galactose, fructose, raffinose, gentiobiose etc. and
polysaccharides like starch, glycogen, sulfated galactans, laminarin and cellulose,
hemicellulose are transformed into different compounds like acetic acid, acetate,
propionate, butyric acid, isobutyrate, butyrate, butyric acid, formate, valerate,
isovalerate, sulfate and CO2. As regards the short-chain fatty acids (SCFAs) produced
in the digestive tract of fish, the most abundant one is acetate, followed by propionate
and butyrate. Different studies showed that these compounds, present at a high level in
herbivorous marine species, constitute an important source of energy and biosynthesis
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
33
for these fish (Seeto et al., 1996; Fidopiastis et al., 2006). Furthermore, butyrate has
been demonstrated to have an antibiotic effect in combating Salmonella in poultry (Van
Immerseel et al., 2006). Interestingly, other studies have found that intestinal bacteria
are able to utilize skim milk, proteins and some aminoacids for producing iso-butyric
acid, iso-valeric acid and CO2 (Tab. 1).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
.
34
Table 1. Mayor intestinal bacterial species involved in fermentative activities: substrates and products of their metabolism, host fish species and diet.
(C=carnivorous; H=herbivorous; O=Omnivorous; D=detritivorous)
Bacterial species
Substrates
Products
Host fish species
Diet
References
Fusobacteria bacterium
mannanoligosaccharide,
galactooligosaccharide, inulin
acetic acid, acetate, CO2
Hybrid striped bass (Morone chrysops x Morone
saxatilis)
C
Burr et al., 2010
Mesophilic sulfate reducers
acetate
HS-, HCO3-,CO2
Kyphosus sydneyanus, Odax pullus, Aplodactylus
H
arctidens
Mountfort et al., 2002
Methanogens
acetate
methane
Kyphosus sydneyanus, Odax pullus, Aplodactylus
H
arctidens
Mountfort et al., 2002
Enterovibrio sp., Faecalibacterium sp.
glucose, lactose, acetate,
Zebraperch (Hermosilla azurea)
H
Fidopiastis et al., 2006
Desulfovibrio sp.
sulfated galactans
(polysaccharides)
Zebraperch (Hermosilla azurea)
H
Fidopiastis et al., 2006
Bacteroides sp.
starch, laminarin (polysaccaride),
hemicellulose
Zebraperch (Hermosilla azurea)
H
Fidopiastis et al., 2006
Vibrio pelagius, Vibrio spp.
glucose, peptone, inulin,
oligofructose
Turbot larvae (Scophthalmus maximus); Arctic
charr (Salvelinus alpinus);Turbot larvae (Psetta
maxima)
C&H
Ringo et al., 1992a; Ringo et al., 1992b; Mahious et al., 2006
Common carp (Cyprinus carpio), rainbow trout
(Oncorhynchus mykiss)
O&C
Kihara and Sakata, 2001; Kihara and Sakata, 2002
Unknown bacteria
chitin, glucose, lactosucrose,
raffinose, gentiobiose, soybeanoligosaccarides (stachyose,
sucrose)
acetate, propionate, valerate ,
butyrate, isobutyrato, formate,
lactate
acetate, propionate, valerate ,
butyrate, isobutyrate, sulfate
acetate, propionate, valerate,
isobutyrate
eicosapentaenoic acid (20:5 n-3)
(EPA) Fatty acids (16:1n-7,
18:1n-9; 20:1, 22:1)
butyric acid, butyrate,
propionate, CO2,
Unknown bacteria
skim-milk,
carboxymethylcellulose, chitin,
proteins, valine, leucine,
isoleucine, glucose, lactosucrose
iso-butyric acid, iso-valeric acid, Pinfish (Lagodon rhomboides), Red seabream
CO2
(Pagrus major)
H&C
Luczkovich and Stellwag, 1993, Kihara et al., 1995
Unknown bacteria
α-starch
propionate, acetate, n-butyrate
Tilapia (Oreochromis niloticus)
D
Kihara and Sakata, 1997
Unknown bacteria
mannitolo, glucose, glucurinic
acid, galactose, fructose
acetate, propionate
Kyphosus sydneyanus, Odax pullus, Aplodactylus
arctidens, herring cale (Odax cyanomelas), sea
H
carp(Crinodus lophodon)
Mountfort et al., 2002; Seeto et al.,1996
Unknown bacteria
starch, glycogen
acetate, propionate, isobutyrate,
butyrate, isovalerate, valerate
Kyphosus spp.
Clements and Choat, 1997
H
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
35
Moreover, various studies tell of the beneficial capacity of gut bacteria of synthesizing
enzymes and vitamins such as vitamin B12, available to the fish by direct absorption
from the gastrointestinal tract (Limsuwan and Lovel, 1981; Sugita et al., 1991; Izvekova
et al., 2007) and by furnishing cell micronutrients such as minerals and essential fatty
acids (Ringo et al., 1992a, 1992b). Interesting studies have demonstrated that some
gastrointestinal bacteria added to a lipid diet can enhance the growth of catfish
(Ictalurus punctatus) thanks to their ability to sintetyze biotin (Robinson and Lovel,
1978; Lovel and Buston, 1984). Moreover, microbial components of intestinal liquor
from turbot larvae (Scophthalmus maximus) and Arctic charr (Salvelinus alpinus),
identified as belonging to Vibrio spp., have been found to produce eicosapentaenoic
acid (EPA; 20:5 n-3) (Ringo et al., 1992a, 1992b). EPA production by intestinal
microorganisms present in some fish represents an important aspect in aquaculture,
because of the beneficial effects of these lipids for the host fish and, being a source of
PUFA, for food trade. In any case, we can presume that these lipid substances together
with other products of microbial metabolism as outlined above can have a role in the
probiotic effect attributed to fish intestinal bacteria which will be described in the
following paragraph.
3.4 Probiotics
A precise definition of probiotics was made by Fuller (1989). This author considered
them “a live microbial feed supplement which beneficially affects the host animal by
improving its intestinal microbial balance”. This action is performed in different modes:
a) by suppressing pathogen growth by producing antibacterial compounds, and
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
36
competing for nutrients or adhesion sites; b) by stimulating immunological response by
increasing antibody levels and macrophage activity. Different species of bacteria, which
were proved to have a beneficial action on animals, are mainly of intestinal origin:
Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus delbrueckii ssp. delbrueckii,
Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus
lactis, Lactobacillus salivarius, Lactobacillus plantarum, Streptococcus thermophilus,
Carnobacterium, Enterococcus faecium, Enterococcus faecalis, Bifidobacterium spp.
and Escherichia coli. The only two species which do not originate from the intestine are
represented by Lactobacillus bulgaricus and Streptococcus. thermophilus, which have a
dairy origin and together with most of the above mentioned bacteria are called lactic
acid bacteria (LAB) because they produce lactic acid as a major or the sole product of
fermentative metabolism. They are Gram positive, containing both rods and cocci.
Various studies have demonstrated that they are part of the normal intestinal microbiota
in fish (Ringo et al., 1995) and have positive effects on the health and well-being of
hydrobionts by producing harmless proteins called bacteriocins against fish pathogens,
stimulating the gut‟s immune system with an increase in intestinal T cells and
acidophilic granulocytes and by improving growth performances (Ringo et al., 1998;
Carnevali et al., 2006; Rollo et al., 2006; Abelli et al., 2009). In the above studies these
bacteria were experimented as feed supplements for rearing larvae using rotifers and
Artemia as living vectors or for culturing juveniles of teleosts. Carnevali et al., 2006
considered the effect of the bacteria on sea bass gut colonization, growth performance
and the level of the stress hormone cortisol. The results of these studies indicated that
the addition of LAB to a live diet determined a high gut colonization by these bacteria
together with significantly lower cortisol level than that found in the fish fed on a diet
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
37
with no LAB addition. In this regard, Rollo et al., 2006, studied the effect of probiotics
on sea bream fry under pH stress and they found that the tolerance to acute stress was
higher in fry under probiotic treatment. Considering the growth performances (standard
length and body weight) of European seabass juveniles, Carnevali et al., 2006 carried
out a study on the expression of two antagonist genes involved in muscular growth after
the addition of LAB to the diet. The results showed that in the fish fed on a diet with
LAB added, the genes involved in muscular growth were transcribed at a higher level
than in the control group and this was in agreement with an increase of the body weight
of the treated sea bass.
3.4.1 Probiotics in aquaculture
The intensification of aquaculture and globalization of the seafood trade have led to
remarkable developments in the aquaculture industry. However, most modern
aquaculture practices such as discontinuous culture cycles, disinfection or cleaning of
ponds or tanks together with high fish densities in rearing plants, have increased the
exposure of the fish to elevated stressful conditions (Wang et al., 2008). Physiological
stress is one of the primary factors of fish disease and mortality in aquaculture. The
development of microbial communities under intensive rearing conditions is affected by
both deterministic and stochastic factors. Deterministic factors include salinity,
temperature, oxygen concentration, and quantity and quality of the feed while stochastic
factors are represented by chance, favoring organisms in a certain place at a certain time
and prolifering if the conditions are suitable (Verschuere et al., 2000). The idea that
both environmental conditions and chance influence the emergence of microbial
communities opens up opportunities for the concept of probiotics as biological
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
38
conditioning and control agents. Instead of allowing spontaneous primary colonization
of the rearing water by bacteria accidentally present, generally probiotics can be applied
in the feed or added to the culture tank or pond as preventive agents against infection by
pathogenic bacteria, although nutritional effects are also often attributed to probiotics. A
probiotic organism should be non-pathogenic and biochemically and physiologically
well-characterized, should be normal inhabitants of the new environment, and capable
of surviving and growing under the biotic and abiotic conditions such as the pH,
digestive enzymes, bile salts and immune response of the site of application; in addition
it should maintain its viability and activity throughout product manufacture and storage
(Hansen and Olafsen, 1999). Probiotic preparations are added in different ways as liquid
fresh cells, frozen cultures, powders or spore in the genus Bacillus. All these “formula”
have to guarantee a high level of viable bacteria (Wang et al., 2008). Generally,
probiotics has constituted part of the autochthonous intestinal microflora of different
fish species and produced inhibitory substances against pathogenic bacterial species
(Olsson et al., 1992; Sugita et al., 1996b). Most probiotics proposed as control agents in
aquaculture belong to LAB as stated above, and different studies have focused on their
effect on fish larvae. In fact, during the development of fish eggs a large number of
microorganisms colonize the eggs‟ surface and the nutrients released during hatching
allow to proliferate the opportunistic bacteria which in certain cases cause problems for
the larvae (Hansen and Olafsen, 1999) especially in intensive aquaculture during the
first feeding of larvae when large amounts of nutrients are added to the live food
cultures. The use of probiotics is therefore designed to find the optimal mix of bacteria
that has a positive effect on the development of larvae so limiting the proliferation of
the opportunistic harmful bacteria. An interesting study was made on turbot (S.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
39
maximus) larvae using as food source Brachionus plicatilis which served as a vector for
introducing the LAB into the larvae (Gatesoupe, 1991). This study showed a significant
increase in the mean weight of turbot larvae fed rotifers compared to the control. In
addition, other bacterial genera such as Vibrio (Vibrio alginolyticus etc.), Bacillus,
Pseudomonas, Aeromonas of different origin (intestine, eggs, larvae, fish mucus,
seawater, Rotifer culture, Artemia culture etc.) proved to behave as probiotics
(Onarheim et al., 1994; Verschuere et al., 2000). An in vivo study of the effect of
probiotic strains on gilthead sea bream larvae (Sparus aurata) was performed by
Makridis et al., 2005. The addition of these bacteria belonging to the genera Cytophaga,
Roseobacter, Ruergeria, Paracoccus, Aeromonas and Shewanella improved the survival
of gilthead sea bream larvae.
In the light of these considerations, probiotics in intensive rearing of marine organisms
has a great potential to respond to the harmful environmental effects due to the use of
antibiotics and the expense associated with vaccinations. The use of host specific strains
used in a prophylaxis program can be an alternative to the antibiotics and a promising
approach in aquaculture.
3.5 Pathogens
Bacteria that cause disease in marine fish are called pathogens and are represented
mainly by a group of Gram-positive and Gram-negative ubiquitous or opportunistic
microorganisms. This means that they are usually widespread in the aquatic
environment or form part of the normal flora of healthy individuals and under
environmental stressors (temperature, oxygen concentration, pH, osmotic strength etc.)
can multiply and invade the host tissue causing diseases as a consequence of the
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
40
weakened defenses of fish. In this way chemical or abrasive forces may damage the
integrity of the mucus layer and favour bacterial access to host epithelial surfaces.
Virulence determinants of pathogenic bacteria may be “regulated” by environmental
factors (Hansen and Olafsen, 1999). A few bacteria are obligate pathogens and depend
on a living host for their propagation e.g., Renibacterium salmoninarum and
Mycobacterium spp. (Hansen and Olafsen, 1999). Bacterial diseases are the major cause
of economic loss affecting fish farms. The most common bacterial fish pathogens are
represented by different species of genera Vibrio, Aeromonas and Pseudomonas.
Vibrio vulnificus is indigenous to estuarine environments and can be a human pathogen
implicated in septicemia generally originating from ingestion of raw oysters harboring
the bacterium (Brauns et al., 1991). Vibrio harveyi is a waterborne bacterium commonly
found in tropical marine environment. Some strains are pathogenic to aquatic fauna,
invertebrates and a variety of finfish such as Trachurus spp. (mackerel), Carcharhinus
spp. (sharks), Squalus spp. (dogfish), and other strains may be considered opportunistic
pathogens and dangerous in immuno compromised hosts. Many of these fish are farmed
and the presence of V. harveyi could have a dramatic economic impact (Oakey et al.,
2003). Furthermore, other studies have recognized numerous strains of V. harveyi as the
most significant pathogens in aquaculture being the etiological agent of different types
of diseases in marine vertebrates and invertebrates (Austin and Zhang, 2006) such as
prawn with a mortality of 100% in larval stages, mollusks and corals (Cano-Gomez et
al., 2009). A primary pathogen for several cultured fish species, such as Atlantic
salmon, rainbow trout, turbot, cod, gilthead sea bream and eel is represented by Vibrio
anguillarum which can cause many mortalities among fish larvae (Balebona et al.,
1998).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
41
In addition, members of the genus Aeromonas are known to be important waterborne
pathogens of animals and humans. They can cause furunculosis, fin rot in fish,
septicaemia, gastrointestinal infections in humans and are commonly found in the
microflora of trout (Lee et al., 2002).
Another opportunistic pathogen of fish species is Pseudomonas anguilliseptica which
was originally described as the etiological agent of “red spot disease” in the Japanese
eel, Anguilla japonica but which has been isolated from various cultured and wild fish
such as European eel, Anguilla Anguilla (L.), Atlantic salmon, Salmo salar (L.), sea
trout, Salmo trutta (L.), rainbow trout Oncorhyncus mykiss (Walbaum), turbot , Psetta
maxima (L.), seabass (Dicentrarchus labrax), black spot sea bream, Pagellus bogaraveo
(Brunnich), farmed cod, Gadus morhua (L.) and gilthead sea bream, Sparus aurata (L.)
(Balboa et al., 2007). In juvenile and adult farmed gilthead sea bream in the
Mediterranean area, P. anguilliseptica has been reported to cause the so called “winter
disease”, a pathology which develops with the decrease of the water temperature below
11-12°C and is characterized by hemorrhagic septicemia associated with keratitis
presenting an average mortality rate of 30% (Doménech et al., 1997). Another pathogen
which causes losses in aquaculture is Photobacterium damselae, (formely Vibrio
damselae) the causative agent of pasteurellosis. This disease has affected cultured fish
species in Europe such as gilthead sea bream, sea bass (Dicentrarchus labrax), turbot
and yellowtail (Seriola quinqueradiata) (Osorio et al., 1999).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
42
CHAPTER 4
METHODS FOR STUDYING BACTERIAL FLORA
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
43
4.1 Conventional techniques
The study of a bacterial community is a hard and complex work which is often based on
a polyphasic study which takes time and requires the use of different techniques both
phenotypic (conventional) and genotypic (molecular) which are various in their level of
resolution. The choice of one rather than another depends on various factors: the ease of
application, time of processing, cost, number of samples to screen, discrimination
power. The ideal bacterial typing for identification purposes in aquaculture should have
a high level of resolution, high reproducibility, ease of performance, speed and low cost
(Cano-Gomez et al., 2009).
Most studies in fish have used only conventional bacteriological techniques to
investigate the intestinal microflora (González et al., 1999) and often involve the
cultivation and the isolation of bacteria using a variety of selective and non-selective
media, incubated under a variety of conditions. After isolation, bacteria can be
characterized at genus, species or strain level using a battery of tests which comprise
morphology, biochemical profiles specific for a bacterial genus or species such as the
commercial kits API 20E and Biolog GN. In this way, Gram-negative bacterial isolates
are identified on the basis of sugar utilization or by the presence of specific enzymatic
activities and the resistance to some antibiotic (Alsina and Blanch, 1994). A
biochemical method used for differentiating bacterial genera and species of the family
Vibrionaceae is the study of their cellular fatty acid composition (FAME) (Lambert et
al., 1983). Furthermore, serological methods have been used for identification of V.
harvey in aquaculture by means of an enzyme-linked immunosorbent assay (ELISA)
based on the use of polyclonal antibodies against live cells (Phianphak et al., 2005) and
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
44
the profile of antimicrobial substances has been used in epidemiological studies to
characterize pathogenic strains of the above Vibrio species (Musa et al., 2008).
However, these methods are often laborious, time consuming and not reliable for
distinguishing genera, species or strains. This is due to the fact that many bacteria are
morphologically and biochemically similar and often the phenotypic traits are instable
being linked to extrachromosomal mobile genetic elements (plasmids) which can be lost
after various cultivations on synthetic media. In addition, various genetic recombinant
events such as point mutations, chromosomal rearrangements, duplication, infection by
bacteriophages, and horizontal gene transfer might be responsible for changing
phenotypes among bacteria (Cano-Gomez et al., 2009). Furthermore, bacteria identified
using these methods can represent only a small fraction of the natural microbial
communities (Pond et al., 2006) many of them being fastidious to cultivate.
4.2 Molecular techniques
The use of molecular techniques for characterizing the microflora colonizing a specific
biological niche can be considered fundamental for research purposes. Only about 20%
of naturally occurring bacteria have been characterized and selective and enriched
media and growth conditions are not sufficient to mimic the natural habitat where
microbes live. Furthermore, many microorganisms are bound to tissue and sediment
particles and it is not possible to detect them by conventional microscopy (Muyzer et
al., 1993). A comparison of conventional and molecular techniques used for studying
the intestinal microflora of rainbow trout was performed by Pond et al., 2006. In
general, molecular methods offer new opportunities for the analysis of the genome and
generally possess a higher discriminatory power and a better reproducibility than
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
45
phenotypic tests. The advent of genotypic fingerprinting methods has led to the rapid
identification of large number of bacterial communities present in environmental
samples and in the intestine of fish (Huber et al., 2004; Pond et al., 2006). These
techniques are various, and have their own peculiarities as described in Tab. 2.
A technique which is considered an advanced phenotypic tool, standing between
conventional and molecular methods with a good reproducibility, is represented by
sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of wholecell protein electrophoresis. This technique has been used for studying the diversity of
microbial populations and the similarity of strains at species level (Tryfinopoulou et al.,
2002; Tryfinopoulou et al., 2007) and for characterizing the immmunoglobulines and
the antibacterial proteins which take part in the immune defences of various fish organs
(Abelli et al., 2005; Ruangstri et al., 2010). This methodology is labour intensive, it
consists of the analysis of protein extracts separated electrophoretically on
polyacrylamide gels and the protein patterns can be analysed by specific commercial
software.
However, the studies carried out for identifying bacteria at species and at strain level,
mainly focus on the analysis of the genomic DNA. DNA-DNA hybridisation (DDH)
with nucleic acid probes is normally used for species delineation with both good power
of resolution and reproducibility. The technique consists of a complex and expensive
procedure by which a bacterial DNA is put on a specific membrane or on polystyrene
microplates and hybridizes with a known DNA probe previously labelled with a
fluorescent or radioactive compound included in commercial kits. The result of the
hybridization is detected by indirect enzymatic activities (Willems et al., 2001) and the
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
46
criterion for establishing two strains as belonging to the same species is a DNA-DNA
similarity of more than 70%.
A more recent technology used for identifying and typing bacteria based on genomic
DNA-DNA similarity, is provided by DNA microarrays in which bacterial genomes are
fragmented randomly, spotted on a glass slide and hybridized with reference strains to
test genomes at species and strain level (Cho and Tiedje, 2001). This is an expensive
and technically demanding technique and hybridization profiles are used in statistical
procedures with the possibility of creating a database. Furthermore, a great number of
genetic studies have considered the use of whole-genome fingerprinting techniques as
an alternative method for species identification. A labour intensive but valid and
powerful technique is represented by amplified fragment length polymorphism (AFLP)
which is based on size separation patterns of fragments amplified with two primer sets
after initial restriction cutting of genomic DNA (Cano-Gomez et al., 2009). Another
fingerprinting technique with high power of resolution for strain typing and with a good
reproducibility is the pulsed field gel electrophoresis (PFGE) where chromosomal DNA
is digested with endonucleases and the products of digestion are resolved in an agarose
gel made run in an electric pulsed field (Mannu et al., 1999). Due to the cost and the
labour intensive protocols, the above mentioned genetic analyses are considered not
suitable for the routine identification of a large number of isolates and other techniques
are taken into account for species classification. Repetitive extragenic palindromic
elements PCR (REP-PCR) which amplify repetitive, highly conserved DNA sequences
of the chromosomal DNA is quite a fast method with a reasonable level of
reproducibility which can differentiate bacterial isolates at species and strain level
(Gomez-Gil et al., 2004). A similar technique of PCR fingerprinting commonly used
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
47
because it is easy to perform, rapid, and not expensive is represented by random
amplified polymorphic DNA (RAPD), which uses one primer of arbitrary nucleotide
sequence for randomly amplifying the genomic DNA. This method is used for
epidemiological investigation and as a tool for calculating genetic similarities among
bacterial strains from different environments although it suffers from poor
reproducibility (Mannu et al., 1999; Pujalte et al., 2003; Musa et al., 2008). In addition,
the identification of a bacterial species is possible by means of assays based on PCR
amplification of single genes and the successive study of their sequences which are
compared with the public databases of GeneBank. The analysis of the 16S or the 23S
RNAs genes, which codes for the ribosomal RNAs is of great importance because the
genes are highly conserved and essential to the viability of bacterial cells; in addition
they contain short variable sequences useful to determine microbial diversity, giving
information at the level of family, genus and species (Kita-Tsukamoto et al., 2006).
Thus, the use of PCR and sequencing of ribosomal genes are performed in the
assessment of the natural phylogenetic relatedness between isolated and uncultured
prokaryotes. A technique used for analysing ribosomal genes for taxonomic purposes is
represented by amplified ribosomal DNA restriction analysis (ARDRA) which consists
of an amplification of the ribosomal gene and its digestion with restriction enzymes
(Cano-Gomez et al., 2009). A description of this methodology is made in the materials
and methods of this dissertation.
Another technique used for species identification and epidemiological studies, based on
the detection of sequence differences within or flanking ribosomal RNA genes, is the
ribotyping or ribosomal RNA Restriction Fragment Length Polymorphism Analysis
(rRNA-RFLP). Ribotyping fingerprints have been performed for studying genotypic
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
48
diversity of Vibrio isolates from seawater and skin of turbot (Scophthalmus maximus)
(Montes et al., 2006). The methodology is recognized to be very reproducible but quite
complex and time-consuming. In fact chromosomal DNA is digested with at least two
endonucleases and restriction fragments are separated electrophoretically in agarose
gels with a successive transfer of DNA fragments to a nylon hybridization membrane
(southern blots); this membrane is successively hybridized using a digoxigenin-labeled
probe complementary to 16S and 23S rRNA of Escherichia coli. Ribotype membranes
are scanned and analyzed with software packages so that electronic fingerprints can be
included in a database and compared with a commercially available standard platform
(Pujalte et al., 2003).
A useful technique used for identifying very close bacterial species in biological
samples is the denaturing gradient gel electrophoresis (DGGE) technique which is based
on electrophoretic separation of PCR-amplified 16S rDNA regions by means of
polyacrylamide gels containing a linearly increasing gradient of denaturants; indeed, in
denaturing gradient gel electrophoresis, DNA fragments of the same length but with
different base-pair sequences can be separated on the basis of the different
electrophoretic mobility of the partially melted DNA molecule which is decreased
compared with that of the completely helical form of the molecule. In this way, the
fragments which differ among the samples can be successively sequenced or after a
blotting can be hybridized with specific oligonucleotide probes for identification
purposes (Muyzer et al., 1993).
New molecular identification markers are represented by other conserved genes such as
rec A, toxR (transmembrane transcriptor regulator) used for discriminating Vibrio spp.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
49
in products from aquaculture (Kim et al., 1999; Conejero and Hedreyda, 2003;
Thompson et al., 2004) etc. whose variable sequences, have been proven to give
information for species delineation. In this regard, a more complete molecular technique
with a high resolution and a good reproducibility is represented by multilocus sequence
analysis (MLSA), which is based on the analysis of diverse genes like rpoA, rec A,
pyrH for taxonomic purposes (Thompson et al., 2005). The MLSA approach has a webbased database and this allows for online strain and species identification rather than the
exchange of data among laboratories. However, it is expensive, time consuming and
requires experience.
All the above mentioned DNA fingerprinting techniques give different information
according to their resolution power and all require a careful standardization of the
methodology. In addition the use of known “type” strains from different collections is
mandatory in order to compare a large number of patterns under study.
To conclude it is not easy to find a molecular method which can give complete genetic
information and only the use of different techniques can lead to a robust taxonomic
identification.
4.2.1. Methods for direct detection of bacteria in fish products
The study of DNA from bacterial populations in complex biological samples has
prompted the demand for direct detection methods. In this regard, the in situ
hybridization
(FISH)
technique
provides
satisfactory
information
by
using
oligonucleotide probes which target visualize the 16S rRNA of fixed cells by
epifluorescence microscopy (Webster and Negri, 2006). This method is time-consuming
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
50
and not suitable for routine purposes. Another more rapid, sensitive and reliable method
is Real-Time PCR amplification in which SYBR Green I fluorescent dye binds to the
minor grooves of the amplified DNA during the primer annealing and extension steps of
each PCR cycle. Accumulation of amplified DNA is measured by determining the
increase in fluorescence which is recorded by using the SYBR Green I optic channel set
at a wavelength of 495 nm. This technique has been applied for detection and
quantification of specific Vibrio pathogens in aquaculture products and for an accurate
survey of bacterial dynamics in the ecosystems of fish farms (Goarant and Merien,
2006; Panicker et al. 2004). The use of all these methods should always be supported by
initial validation studies using isolates from respective systems.
Molecular methods have also proved useful for direct detection of virulence genes in
aquacultural products intended for the food chain (market). These studies show up the
presence of potentially pathogenic bacterial strains in aquacultural samples and are
important in initiating disease control measures. In this regard, Austin and Zhang (2006)
described the pathogenicity mechanisms of Vibrio harveyi, a serious pathogen of marine
fish and invertebrates.
In addition, it seems that, as reported by Cano-Gomez et al., 2009, pathogenic traits are
borne by mobile elements present in bacteria and non-virulent strains can become
virulent after gene duplication, plasmid uptake, lateral gene transfer from pathogenic
bacterial species or bacteriophage-mediated transfer of a toxin gene(s) or a gene(s)
controlling toxin production. The complex mechanism of pathogenity is often
determined by various environmental causes represented by close contact between
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
51
bacterial strains in mixed biofilms and animal guts and in general by host-pathogen
interaction.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
52
Table 2. Mayor molecular techniques used for studying bacteria: name, type of analysis, information obtained, advantages and disadvantages.
Technique
Type of analysis and information
Advantages
Disadvantages
References
Sodium dodecyl sulphate polyacrylamide Analysis of whole-cell protein extracts; study
gel electrophoresis (SDS-PAGE)
of similarities at species and strain level
High resolution power, high reproducibility
Time consuming
Tryfinopoulou et al., 2007; Ruangstry et al., 2010
DNA-DNA hybridisation (DDH)
Analysis of whole genome; species delineation
High resolution power, high reproducibility
Technically demanding; expensive
Willem et al., 2001; Gomez-Gil et al., 2004
DNA Microarrays
Analysis of whole genome; species delineation
Hybridization profiles are statistically used
Technically demanding; expensive
Cho and Tiedje, 2001
and strain typing
Amplified fragment lenght
polymorphism (AFLP)
Genomic fingerprinting; species delineation
High resolution power, high reproducibility
Technically demanding; time consuming Cano-Gomez et al., 2009
Pulsed field gel electrophoresis (PFGE)
Genomic fingerprinting; strain typing
High resolution power, high reproducibility
Technically demanding; time consuming Mannu et al., 1999
Repetitive extragenic palindromic
elements PCR (REP-PCR)
Genomic fingerprinting; species delineation;
strain typing
Easy to perform; rapid; low cost
Moderate reproducibility
Gomez-Gil et al., 2004
Random amplified polymorphic DNA
(RAPD)
Genomic fingerprinting; species delineation;
strain typing
Easy to perform; rapid; low cost
Poor reproducibility
Pujalte et al., 2003; Musa et al., 2008
Amplified ribosomal DNA restriction
analysis (ARDRA)
Ribosomal gene restriction analysis; family,
genus; species delineation
High reproducibility; easy to perform, rapid Poor resolution power for close species
Kita-Tsukamoto et al., 2006; Cano-Gomez et al., 2009
Ribosomal restriction fragments
Ribotype after southern hybridization
High resolution power, high reproducibility
Pujalte et al., 2003; Montes et al., 2006
length polymorphism (RFLP)
of digested genomic DNA with ribosomal probes;
Technically demanding;
time consuming; expensive
species delineation and epidemiological studies
Denaturing gradient gel electrophoresis
(DGGE)
Analysis of amplified 16S rDNA variable regions; High resolution power, high reproducibility,
species delineation
separation of different base-pair sequences
Technically demanding; time consuming Muyzer et al., 1993
Multilocus sequence analysis (MLSA)
Cluster analysis of sequences from different
phylogenetic genes; species delineation.
High resolution power; great number of loci Technically demanding, expensive;
analyzed; high reproducibility.
time consuming
Conejero and Hedreyda, 2003;Thompson et al., 2005
Real time PCR
Fluorescence measure of amplified DNA;
Rapid, sensitive, quantification of PCR
Goarant and Merien., 2006; Panicker et al., 2004
species delineation
product
Expensive
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
53
CHAPTER 5
AIM
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
54
The importance of eurhyaline fish farming has been developing since the mid-1980s for
food trade and, from that time on, large scale fish production has been achieved by
intensive rearing systems. The aim of fish farming practices is to obtain products in
large quantities as well as of good quality respecting the environment and animal
welfare. The intensification of modern aquaculture practices, such as discontinuous
culture cycles, disinfection or cleaning of ponds or tanks, together with high fish
densities in rearing plants or in natural aquatic environments have led to a strict control
of bacteria associated with fish, which are the major cause of economic losses to fish
farms. In this contex, the study of microbiota present in the intestine of fish is of great
importance because these bacteria reflect different factors such as the diet, general
rearing conditions, aqueous parameters and the general the way of life of the host
species. Various studies concerning the indigenous gut microflora of fresh water fish
have been carried out while, not many studies have been focused on eurhyaline teleost
species cultured on Mediterranean coasts and specifically on the microflora present in
the intestine of Sparus aurata.
In addition the strict European rules on the microbiological criteria for food safety (Reg.
EC N°. 2073/2005) impose the necessity of gueranteeing the quality of the productions
destinated to food market with respect to animal welfare and of the environment. In the
light of these considerations, the knowledge of the number as well as of the composition
of the bacterial communities of a reared fish, proves to be foundamental for the quality
which is by far mostly influenced by the microbiological activity.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
55
The specific aim of this research was to study the microbial ecology of the midgut and
hindgut of gilthead sea breams grown in two different geographical locations along the
Sardinian coast and reared by two different systems of farming: an intensive system and
an extensive one in order to quantify the heterotrophic bacteria with conventional
methods and to identify at genus and species level the dominant bacterial communities
by means of the analysis of the16S ribosomal (rRNA) gene.
This study was performed to test the microbiological quality of fish and rearing
conditions and the molecular analyses of the bacterial gut communities were also aimed
at detecting their biodiversity at species level in order to detect a possible link with the
rearing system of Sparus aurata.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
56
CHAPTER 6
MATERIALS AND METHODS
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
57
6.1 Fish farms and sampling
The sites were the samplings were carried out are represented in Fig. 3. Thirty gilthead
sea bream (mean weight 325±61g) starved for 48 hours were caught in OctoberNovember 2008 from a single floating cage belonging to the off shore fish farming
facility “La Maricoltura Alghero ” s.r.l. (MA), a 2.15 ha area which is sited in Alghero
Bay (north western Sardinia, Italy: Lat 40°33,730‟N, Long 08°16,140‟E) at about 2
miles from the cost and on a 38 m average water depth (Fig. 4).
Figure 3. Sampling sites.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
58
Figure 4. Floating cages of “La Maricoltura Alghero” s.r.l..
The rearing facility of “La Maricoltura Alghero” was constituted of 9 floating cages
“REFA TLC (Tension Leg Cages), five of 800 m3 where gilthead sea bream specimens
(Sparus aurata Linnaeus, 1758) are reared with an intensive system and four of 2,500
m3 (Fig. 4).
According to Brambilla et al., 2007, fish density ranged from 0.4 to 20 Kg m-3 and daily
feed ratio varied from 40 to 190 kg cage-1 with a daily average of 98 kg cage-1. Gilthead
sea bream were fed with a complete feed for marine fish (PERLA PLUS 2.0,
SKRETTING) whose composition was as follows: crude protein (45.0%), crude fat
(18.0%), ash (7.6%), crude fibre (2.4%), phosphorus (1.1%). The mean estimated
production in 2008 was 80 t/year according to fishermen and the average temperature of
the seawater measured in the Autumn 2008 was 18-20°C.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
59
Some phases of fishing at “La Maricoltura Alghero” are represented in Fig. 5 (a) and
(b).
Figure 5 (a). Fishing at “La Maricoltura Alghero”s.r.l.
Figure 5 (b). Fish put into ice after capturing.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
60
Thirty sea bream (mean weight 349±41g) were captured in January-February 2009
from the Tortoli lagoon (T) (eastern Sardinia, Italy: Lat 39°56‟ 854‟‟N, Long
9°41‟160‟‟) an area of 250 hectars which has been fished by the fishermen and called
”Cooperativa Pescatori Tortoli” since 1944 where Sparus aurata is reared with an
extensive system (Fig. 6). The lagoon has a depth which ranges from 3 to 4 m and
possesses two entries from the sea where two capturing systems called “lavorieri” are
present, one situated in the north and the other placed in the south.
Fig. 6 shows the capturing system “lavoriero” where the gilthead seabream samples
were captured. The mean water average chemical-physical parameters were as follows:
temperature 12.6 (°C), salinity 30 (‰), dissolved oxygen 102 (%) and pH 8.2 and the
waters were classified as mesotrophic (Cannas et al., 1998). According to the
“Cooperativa Pescatori di Tortoli” the mean total productivity of the lagoon proved to
be 58.4 t/year plus 108 t/year of mussels.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
61
Figure 6. Tortoli lagoon: “lavoriero” where gilthead seabreams were
captured.
Figure 7. Fish put into ice after capturing
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
62
6.2 Microbiological analyses
6.2.1 Sampling and processing
After capture, a total of 60 fish from the two fish farms were stored immediately in ice
(Figs. 5 (b) and 7) and transported inside a refrigerated bag to Bonassai laboratory
within 6-8 h. Following a series of preliminary studies to optimise methodology, a
sampling and processing protocol was defined. Each time ten gilthead sea bream were
analyzed at 7 day-intervals. On arrival at the laboratory, gilthead sea bream were
weighed, measured and underwent intestine extraction. For bacteriological analyses of
the gut, the fish belly was sterilised by flame and the peritoneal cavity was aseptically
opened with a sterile blade. The intestine (mean weight 5.22±2g for (MA) fish and
3.4±1 for (T) fish) between the pyloric caeca (midgut) and the anus (hindgut) of each
fish was removed (Fig. 8), weighed aseptically, diluted (10% w/v) in peptone saline
solution (0.85% NaCl, 0.1 g peptone), transferred to a stomacher bag and homogenised
for 30 seconds in plastic bags by Stomacher® 400 at room temperature (Fig. 9). One ml
of homogenate was used for serial dilutions. One ml of each dilution prepared was
placed on the bottom of the petri dish and successively 20 ml of molten agar media was
poured onto duplicate and mixed with the inoculum.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
63
Figure 8. Removal of intestine from a seabream for microbiological analyses.
(a)
(b)
Figure 9. Phases of preparation of sea bream intestinal homogenate: (a) Stomacher®
400; (b) serial dilutions.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
64
6.2.2 Microbiological quantitative analyses
6.2.2.1 Culture media and growth conditions
The total count of aerobic heterotrophic bacteria was determined on Plate Count Agar
(PCA; Microbiol) incubated at 30°C for 48-72h and on Nutrient Agar (NA; Microbiol)
after incubation at 28°C for 72h. The enumeration of Enterobacteriaceae, Coliforms and
Escherichia coli was performed on Violet Red Bile Glucose Agar (VRBGA; Microbiol)
and Violet Red Bile Agar Mug (VRBA-MUG; Microbiol) put at 30°C for 24h,
respectively. The cultivation and the count of probiotics were made on de Man-RogosaSharpe Agar (MRS; Microbiol) incubated at 28°C for 7 days. Bacterial counts were
expressed as colony forming units per gram (CFU g-1).
6.2.3 Microbiological qualitative analyses
6.2.3.1 Basic phenotypic tests
A total of 200 colonies coming from intestinal samples from 8 fish captured at (MA)
facility and from 10 gilthead seabream collected from the (T) farm, were isolated
randomly from Nutrient Agar plates and streaked on fresh media four times to obtain
pure culture. The purified isolates were stored at -80°C in a 40% (v/v) glycerol-Nutrient
broth solution. A polyphasic approach was carried out in order to identify bacterial
isolates at genus and species level. For this purposes, after reactivation on Nutrient Agar
medium, basic conventional microbiological tests such as cell shape and motility (by
phase-contrast microscope), Gram staining and catalase reaction (gas production from
H2O2) were performed after 24-72h of incubation. Afterwards, all the isolates were
cultivated on thiosulphate-citrate-bile salts-sucrose (TCBS, Microbiol) agar and
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
65
incubated at 28°C and at 40°C for 24-48h as indicated by Reid et al. (2009) in order to
identify presumptively the members of the Vibrio genus. TCBS medium was utilised for
differentiating sucrose fermenters Vibrio which produced yellow colonies and nonfermenters that yielded green colonies. The colonies which grew well on TCBS agar
presenting the characteristics according to the technical sheet, were tentatively
considered to belong to the Vibrio genus. In addition, all the same isolates were
cultivated on Pseudomonas agar F medium (Liofilchem) at 37°C for 24h for
identification of presumptive Pseudomonas and the strains which grew on
Pseudomonas agar F medium and appeared surrounded by a yellow to greenish-yellow
zone resulting from fluorescent pyoverdin production, were considered to belong to the
Pseudomonas genus; if pyiocianin (a typical Pseudomonas soluble pigment) was also
synthesized, a bright green colour was produced which fluoresces under UV light.
6.2.3.2 Genetic analyses
In order to identify the bacterial communities characterizing the microflora of gilthead
sea bream at species level, the polyfasic study of the bacterial colonies was further
carried out by means of molecular techniques. A total of 200 purified bacterial colonies
coming from the intestines of (MA) fish and (T) sea bream were reactivated on Nutrient
Agar medium for collecting the cells to be processed for DNA extraction and PCR
(Polymerase Chain Reaction) analyses.
6.2.3.2.1 Bacterial “type” strains
For comparative identification purposes, different “type” strains belonging to the
Official Spanish Bacterial Collection of the University of Valencia were utilised. The
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
66
“type” strains were the following: Vibrio vulnificus (Farmer 1980) CECT (Colección
Espa ola de Cultivos Tipo, Valencia) 529, V. aestuarianus, CECT 625, Aeromonas
hydrophyla subsp. Hydrophila, CECT 839 and Pseudomonas anguilliseptica, CECT
899.
Before genetic analyses, the reference bacterial strains were reactivated according to the
recommended instructions and the grown culture was processed as above said for the
genetic analyses of intestinal isolates.
6.2.3.2.2 Cell lysis and DNA extraction
Bacterial cell preparation for DNA extraction to be used for PCR amplification was
carried out as described by Lemanceau et al. (1995). Purified strains were reactivated by
culturing for 24-72h at 28°C on Nutrient Agar medium. A loopful of cells of each
isolate were transferred to a 1.5 ml Eppendorf tube containing 500 μl Millipore-Q
water. The mixture was vortexed and, after centrifugation at 10,000 rpm for 10 min at
4°C, the supernatant was discarded. This procedure was repeated for three times. The
washed pelleted cells were stored at -20°C until DNA extraction. For DNA extraction
the washed cells were resuspended in 100 μl of sterile Millipore-Q water; then, 100 μl
of 10 mM Tris-HCL (pH 8.3) and 13 μl of proteinase K (Sigma Chimie, St. Quentin
Fallavier, France) (1μg/ml in sterile Millipore-Q water) were added to each sample,
mixed well and incubated overnight at 55°C. Thereafter, the proteinase K was
inactivated by incubating the cell suspension for 10 min at 100°C. After proteinase K
digestion, the treated cells were submitted to three cycles of 1 min in liquid nitrogen and
2 min in boiling water to ensure maximum lysis as indicated by Mhamdi et al. (2002).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
67
6.2.3.2.3 Quantification of DNA samples
The cell suspensions were used for quantifying the released DNA. An aliquot of 3 μl of
cell lysates was analysed by means of Nano Drop 2000 Thermo Scientific (Euroclone
S.p.a) in order to determine the quantity and the quality of extracted DNA. The
absorbances at 260 nm and 280 nm were estimated and their ratio was calculated in
order to test the purity of DNA. The absorbance reading at 260 nm represents the
quantity of DNA which was expressed as ng/μl.
6.2.3.2.4 Amplification of the 16S RNA gene
Universal primers designed to anneal to the conserved region of the 16S rRNA gene
were selected from those cited in the literature (Marchesi et al., 1998). These primers
were designed to amplify approximately 1,300 bp of Escherichia coli 16S rRNA gene
corresponding to nucleotides 63-1387. The sequences were as follows: forward primer
63f (5‟-CAG GCC TAA CAC ATG CAA GTC-3‟) and reverse primer 1387r (5‟-GGG
CGG WGT GTA CAA GGC-3‟).
The reaction mixture (Euro Taq kit, Life Science Division, Italy) and PCR conditions
were as indicated by Pond et al. (2006) with some modifications for magnesium
chloride concentration, primers quantity and annealing temperature. The PCR mixture
contained
31μl
sterile
molecular
grade
water,
5μl
reaction
buffer,
5μl
deoxyribonunucleotide triphosphate (dNTPs, 200 μM), 1.5 μl magnesium chloride (1.5
mM), 1 μl each primer (50 pmoli/μl) (Sigma Genosys), 0.5 μl Taq polymerase (Euro
Taq kit, Life Science Division, Italy) and 5 μl of cell lysates (from 25 to 100 ng DNA
template) to give a total reaction of 50 μl. The PCR was performed in a Peltier
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
68
thermocycler (Euroclone, 96 Universal Peq STAR). PCR conditions were as follows: 30
cycles of denaturation at 94 °C for 1 min, annealing at 58°C for 1 min and elongation at
72°C for 2 min, with a final elongation at 72 °C for 10 min. PCR reactions were carried
out using equipment autoclaved at 121 °C for 15 min and under Vertical 700 Laminar
flow Cupboard (ASALAIR).
In order to set up the right quantity of DNA for PCR amplification, different aliquots of
DNA were used for amplification trials in preliminary experiments. Subsequently, 10 μl
aliquots of the amplified 16S rRNA products were electrophoresed in a 1% (w/v)
agarose (Eppendorf, Italia) -Tris-acetate EDTA (Sambrook et al., 1989) gel at 90 V for
1 h. In order to visualise the amplified bands under UV light, the molten Tris-acetate
EDTA buffer agarose solution was mixed with 1.5 μl SYBER Safe DNA gel stain ®
(Oregon, USA) and poured in the stamp. A 10 μl aliquot (1 μl Marker III (Roche) 56421,220 base pairs (bp), 3 μl loading buffer and 8 μl sterile water) were used inside the
gel for identification of the band with a specific molecular weight. The amplification
products were photographed under UV light and gel images recorded using a digital
camera (KodaK Digital Science 1D LE 3.0).
6.2.3.2.5 Amplified ribosomal DNA restriction analysis (ARDRA)
The PCR products obtained from bacterial isolates were digested with the restriction
enzymes HaeIII and HhaI (BioLabs, New England). These endonuclease cut DNA
molecules in different ways in the presence of the nucleobases guanine and cytosine.
According to the manufacturer‟s instructions, the digestion mixture contained 10 l of
the PCR amplification product, 4 l of the specific recommended buffer for each
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
69
enzyme, 2 l of the enzyme (10,000 U/ml) and 4 l of sterile milli-Q water. The
digestion mixtures were incubated at 37°C for 3 h in a water bath and the fragments
obtained were resolved on 2% (w/v) agarose (Eppendorf, Italia) gel in Tris-acetate
EDTA added with 2.0 l SYBER Safe DNA gel stain ® (Oregon, USA) at 80 V for 2 h.
Restriction digests were run with a 1 Kb Plus DNA ladder Marker Size (100-12,000 bp)
and 100 bp DNA Ladder (Invitrogen) (100-2,072 bp).
6.2.3.2.6 Purification of PCR products for 16S rRNA gene sequencing
A total of 11 isolates representing the most numerous different ARDRA groups were
furtherly processed and examined for DNA sequencing. For these strains, DNA
products obtained after the amplification of the 16S RNA gene were purified with a
commercial kit (Amicon ® Ultra -0.5ml 30K Centrifugal Filter Devices; Millipore,
USA) in order to remove salts, components of PCR mixture and residuals of cell
lysates. The procedure was as follows: an aliquot of about 35 l of amplification
products was added to the Amicon Ultra filter device previously inserted into a
microcentrifuge tube provided, after the addition of 400 l sterile molecular grade
water. The capped filter assembled device was placed into the centrifuge rotor and spun
at 3000 rpm x 15 min. Successively, the Amicon Ultra filter device was separated from
the microcentrifuge tube and inserted into another cleaned tube. After the addition of 20
l of sterile molecular grade water, the Amicon Ultra filter device was placed upside
down in the clean micro centrifuge tube and spun at 3000 rpm x 2 min in order to
recover the concentrated purified solute containing the DNA to be sequenced.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
70
6.2.3.2.7 Estimation of purified PCR products and sequence analyses
An accurate estimate of the quantity of purified DNA products was necessary for
sequence analysis. For these purposes an aliquot of 10 l of Amicon ultra filter purified
PCR products was run on 1% agarose gel using Low DNA Mass Ladder (Invitrogen)
100-2,000 bp and stained by SYBER Safe DNA gel stain ® (Oregon, USA) as
described above. The quantity of purified DNA fragments was estimated on the basis of
the intensity of the band compared to the DNA marker. After quantification, PCR
products were sent to the commercial facility service (BMR Genomics s.r.l., Università
di Padova, Spin-off ufficiale, Italy). The concentrations of DNA to be sequenced, as
well as those of the primer used for sequencing were according to the BMR Genomics
instructions. Fifty ng of purified DNA was mixed with 6.4 pmoli of primer 63f and,
after evaporating water at 65°C, the pellet was used for sequencing. The partial 16S
rDNA sequences obtained were edited with the software Chromas version 1.43 (Griffin
University, Brisbane, Qld, Australia) and the results of the sequencing were submitted
for homology searches by BLAST (Basic Logical Alignment Search Tool; Altschul et
al., 1990) after unreliable sequences at the 3‟ and 5‟ ends were removed. The data-base
used for sequence pairing was the NCBI (National Center for Biotechnology
Information) http://www.ncbi.nml.nih.gov, a large database containing sequences of
different organisms. The identities were determined on the highest score basis.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
71
6.3 Statistical analyses
Data of microbial counts were manipulated using the One-Way ANOVA test in order to
compare the number of the different bacterial groups estimated on the intestines of the
gilthead sea bream from the two fish farms.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
72
CHAPTER 7
RESULTS
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
73
7.1 Microbiological quantitative analyses
7.1.1. Enumeration of intestinal microflora
The microbiological quantitative analyses were performed by means of conventional
methods performed on 60 intestinal samples from Sparus aurata. A graphic
representation of the observed bacterial colonies grown on PCA and VRBGA medium
are shown in Figs. 10 (a) and (b). The results of the the mean counts of total aerobic
heterotrophic bacteria estimated on PCA, Enterobacteriaceae counted on VRBGA and
Coliforms enumerated on VRBA-MUG, expressed as colony forming units per gram
(CFU g-1) are presented in Tab. 3 and in Fig. 11. The number of bacteria grown on NA
medium are not indicated because they proved to be almost the same as those counted
on PCA. A large variability in the number of colonies was observed both inside and
between each group of fish and this was evident from the broad range of CFU g-1
quantified among all the samples. In any case higher values of microbial load were
recorded in the sea bream farmed by extensive system (Fig. 11).
Moreover, it was also noted that an intestinal sample from one sea bream reared in the
Tortoli lagoon did not show any colony growth on any of the media under the
conditions employed.
Considering the counts of probiotics cultivated on MRS medium, a complete absence of
any culturable microflora was found when cultivating intestinal samples from (MA)
facility and only 6 out of 30 fish captured from the (T) farm showed the presence in
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
74
their intestine of bacteria able to grow on this medium, counting up to 6 colonies per
plate.
(a)
(b)
Figure 10. Bacterial colonies from sea bream intestinal samples grown on PCA medium
(a) and on VRBA-MUG (b).
As regards the comparisons of the number of different bacterial groups detected in the
intestinal samples of the two groups of sea bream by ANOVA, they differ significantly
in the two farms both for total aerobic heterotrophics counted on PCA (P≤0.01) and for
coliforms enumerated on VRBA -MUG (P≤0.05) while, considering the number of
Enterobacteriaceae estimated in the two groups of fish, the results of the counts
performed on VRBGA proved different but with a P=0.06 (Tab. 3).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
75
Table 3. Counts (average CFU g-1 ± SE and min-max values) of different microbial groups
estimated on intestinal samples of gilthead sea bream captured from the “La Maricoltura
Alghero” (MA) and the Tortoli lagoon (T)
Farms
Bacterial groups
Total
heterotrophics
Enterobacteriaceae
Min-max
*:
Coliforms
Min-max
Min-max
MA
125 ±20
10-495
73.8±31
0-935
40.9±12.5
0-370
T
1521±428
0-8550
408.5±173
0-4250
187±65
0-1405
P*
P≤0.01
P=0.06
P≤0.05
ANOVA One-Way .
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
76
10,000
1,000
CFU g-1
T
MA
100
10
1
Total heterotrophics
Enterobacteriaceae
Total coliforms
Figure 11. Mean values of different bacterial groups detected in the gut of gilthead sea
bream reared in the Tortoli lagoon (T) and in the “La Maricoltura Alghero” (MA) farm.
7.2 Microbiological qualitative analyses
7.2.1 Basic phenotypic tests
The qualitative characterisation of the isolates from intestines of fish from the (MA)
farm and from the (T) lagoon, was carried out in a polyphasic study. The microscopic
observations of the bacteria indicated that the dominant microflora which colonizes the
intestine of Sparus aurata from both the groups was constituted by Gram negative
microorganisms, motile and non-motile (Fig. 12).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
77
Figure 12. Gram negative bacteria isolated from the intestine of Sparus aurata L.
observed by the phase-contrast microscope.
In particular, the Gram stain and the study of the morphology of the bacteria composing
the intestinal microflora of (MA) sea bream, indicated that 76 out of 90 isolates were
Gram negative and had a rod-like shape except for 9 strains which appeared to be
coccobacilli. The other 14 isolates proved Gram positive and among them 5 isolates
were rods, 6 had a coccal cell morphology while 2 colonies were coccoid rods and one
spore forming rod-shaped colony was observed in this group.
As regards the bacteria isolated from sea bream captured in the lagoon, microscopic
observations highlighted the fact that 77 out of 100 isolates were Gram negative isolates
which comprised 75 rod-shaped bacteria and only 2 coccal shaped ones. The other 23
colonies proved to be Gram positive including 17 rods and 6 cocci and one spore
forming rod-shaped isolate was also found inside this group.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
78
Considering the catalase test, all the screened bacterial isolates coming from both
groups of fish proved to posses this enzyme.
7.2.1.1 Bacterial growth on selective culture media
All the Gram negative isolates, being the dominant group, were successively studied. In
order to make a preliminary differentiation of the colonies they were streaked on
differential media TCBS and Pseudomonas agar F which were used for tentatively
differentiating Vibrio and Pseudomonas genera, respectively. On the basis of the
growth, the diameter and the colour of the colonies and pigment production, a
preliminary identification of the isolates was made.
Regarding the bacterial colonies isolated from (MA) fish, the group of microorganism
which grew well on TCBS agar and appeared yellow (able to ferment sucrose) or green
(not able to ferment sucrose) were considered to be presumptive Vibrio spp. (Figs. 13
(a) and (b)). This group of bacteria represented 24% of strains and inside this group
19% were able to grow up at both 28°C and 40 °C (thermophilic) and 5% could grow
only at 28°C (mesophilic).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
79
.
(a)
(b)
Figure 13. Bacterial strains grown on TCBS agar: (a) sucrose fermenter strain; (b) non
sucrose fermenter strain.
The group of Gram negative bacteria isolates from (MA) gilthead sea bream which
grew well on selective Pseudomonas agar F medium and showed colonies surrounded
by a yellow to greenish zone resulting from fluorescence of the pigments produced,
were tentatively considered to belong to Pseudomonas genera (Fig. 14) and constituted
10% of Gram negative isolates from this group of fish.
top
bottom
(a)
(b)
Figure 14. Presumptive Pseudomonas spp. strains grown on Pseudomonas agar F. (a):
an isolate observed under U.V. light showing pyocianin production; (b): pigment
producers isolates (bottom) and non pigment producing strains (top) observed at visible
light.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
80
As a matter of fact, 5 strains merged in the Pseudomonas group, grew very well also on
TCBS agar medium and produced green-blue colonies.
Considering the bacterial isolates from fish reared in the (T) lagoon, we found one
group of presumptive Vibrio representing 27% of the strains which formed both yellow
(sucrose fermenters) and green colonies (non sucrose fermenters) and inside this group
21% of strains were mesophylic bacteria so able to grow at 28°C and not at 40°C and
6% of colonies proved to be thermophylic able to grow up at both 28°C and 40 °C.
The growth trial on selective media Pseudomonas agar F allowed for the formation of a
second group which merged the presumptive Pseudomons spp. representing 7% of
Gram negative bacteria and one isolate of the group was able to grow also on TCBS
agar medium producing blue green colonies.
7.2.2 Genetic analyses
The polyphasic study of the microflora of the intestine of Sparus aurata was further
carried out by the analysis of the 16S rRNA gene in order to identify the strains at
species level firstly by comparison of ARDRA profiles of intestinal bacteria under study
with the “type” bacterial strains used and successively by sequence analyses. After an
initial phase spent on setting up the protocols of DNA extraction and DNA
amplification, the DNA was extracted from all the studied isolates and quantified by
Nanodrop before PCR analysis. A quantity of DNA ranging from 62 ng/µl to 7000
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
81
ng/µl was detected in the cell lysed samples and a quantity from 25 to 100 ng was
established for performing DNA amplification.
7.2.2.1 Amplification of 16S RNA gene
Amplification of the 16S rRNA gene by means of 63f and 1387r primers was performed
and a single band of approximately 1,350 bp corresponding to nucleotides 63-1387 of
Escherichia coli 16S rRNA gene, was produced (Fig. 15). Amplification products were
obtained by all the “type” strains and by most of the analyzed isolates, however some
isolates both from (MA) and (T) seabream did not produce any amplification PCR
products.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
82
1
2
3
4
5
6
7
8
9
10
1375bp947bp564bp-
Figure 15. Products of amplification of the 16S rRNA gene from DNA of intestinal
bacteria run on 1% agarose gel. 1: Marker III (564- 21,226 bp)(Roche); 2-6: (MA)
isolates; 7-10 “type” strains of Spanish collection: CECT 625 (Vibrio aestuarianus),
CECT 529 (Vibrio vulnificus); CECT 839 (Aeromonas hydrofila); CECT 899
(Pseudomonas anguilliseptica).
7.2.2.2 Amplified ribosomal DNA restriction analysis (ARDRA)
The digestion of the amplicons of the 16S rRNA gene was performed by means of
HaeIII and HhaI restriction enzymes using the ARDRA (Amplified Ribosomal DNA
Restriction Analysis) technique in order to detect the differences present in the16S
rRNA gene base composition among all the strains. This analysis allowed them to be
grouped in-to different clusters of ribotypes representing the various philogenetic
groups present in the intestinal microflora.
Examples of ARDRA profiles from isolates from (MA) and (T) sea bream obtained
after digestion with Hae III and Hha I restriction enzymes are shown in Figs. 16, 17, 18
and 19.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
83
1
2
3
4
5
6
7
8 9
10
11 12
13
14
15 16
-2,000bp
1000bp650bp-
-600bp
100bp-
Figure 16. ARDRA profiles from (MA) bacterial isolates run on 2% agarose gel
obtained after digestion with HaeIII restriction enzyme. 1: 1 Kb Plus DNA ladder
Marker Size (100-12,000 bp) (Invitrogen); 10: 100 bp DNA Ladder (1002,072bp)(Invitrogen); 2-9, 11-12, 15-16: different ribotypes; 13: CECT 625 (Vibrio
aestuarianus); 14: CECT 529 (Vibrio vulnificus).
1
G
2
3
4
G
5
6
7
8
9
10
11 12
G
13 14 15
-2,000bp
1000bp650bp-
-600bp
100bp-
Figure 17. ARDRA profiles from (MA) bacterial isolates run on 2% agarose gel
obtained after digestion with Hha I restriction enzyme. 1: 1 Kb Plus DNA ladder
Marker Size (100-12,000 bp) (Invitrogen); 8: 100 bp DNA Ladder (1002,072bp)(Invitrogen); 2-7, 9,10,15: different ribotypes; 11: CECT 625 (Vibrio
aestuarianus); 12: CECT 529 (Vibrio vulnificus); 13: CECT 839 (Aeromonas
hydrofila); 14 CECT 899 (Pseudomonas anguilliseptica). The lanes indicated with G
represent the ribotypes G
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
84
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
12,000bp-2,072bp
1000bp650bp-
100bp-
Figure 18. ARDRA profiles from (T) isolates run on 2% agarose gel obtained after
digestion with Hae III restriction enzyme. 1: 1 Kb Plus DNA ladder Marker Size (10012,000 bp) (Invitrogen); 9: 100 bp DNA Ladder (100-2,072bp)(Invitrogen); 2-8,10-16:
different ribotypes.
1
2
3
4
5
6
7 8
9
10 11 12 13 14 15
2,000bp-
600bp-
100bp-
Figure 19. ARDRA profiles from (T) bacterial isolates run on 2% agarose gel obtained
after digestion with Hha I restriction enzyme. 1: 1 Kb Plus DNA ladder Marker Size
(100-12,000 bp) (Invitrogen); 8: 100 bp DNA Ladder (100-2,072bp)(Invitrogen); 2-7, 915: different ribotypes.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
85
7.2.2.2.1 Analysis of the ARDRA profiles
A total of 111 ARDRA profiles were obtained, analysed visually and the groupings of
different restriction profiles were made on the basis of the number and the molecular
weight of the different bands (fragments) determined with the aid of the DNA markers
indicated. The number of bands produced after the digestion by HaeIII and HhaI
restriction enzymes ranges from 2 to 10 and their molecular weight from 80 to about
1000bp (Figs. 16, 17, 18 and 19). In this way, a specific “ribotype” for each enzyme per
each isolate was established and in the end, a unique “ribotype” was defined for each
strain combining the analyses of the ARDRA profiles produced by the two enzymes.
The criterion was as follows: the bacterial strains which had an identical profile after
digestion with both enzymes were considered the same “ribotype” and when they
produced different ARDRA profiles with at least one enzyme, they were designated as
belonging to a diverse cluster of ribotypes. The various “ribotypes” so delineated,
marked in italics, were named with upper case letters for indicating (MA) bacterial
isolates, with lower case letters for targeting (T) strains and when the same “ribotype”
was found on both farms, it was named with upper case letters for labelling both (MA)
and (T) strains. In this way, different ARDRA biotypes, which represented the various
philogenetic types composing the heterotrophic microflora of Sparus aurata, were
identified for each group of fish.
A total of 19 different “ribotypes” out of 63 isolates were found out in (MA) fish and 17
“ribotypes” out of 48 analysed strains coming from (T) intestinal samples proved
different (Tab. 4). The biodiversity indexes which determined the ratio between the
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
86
different “ribotypes” over the total number of isolates were calculated for each group of
sea bream and was 0.3 for (MA) biotypes and 0.35 for (T) ones (Tab. 4).
As regards all the ribotypes obtained in this study, a dominant biotype which
represented the most numerous microbial group identified on both fish farms was the
cluster named G. Indeed, ribotype G merged 14 isolates (22%) from the (MA) farm
coming from 7 out of 8 analysed fish and 15 bacterial strains (31%) from 5 out of 10 (T)
fish. Considering other ribotypes in common with the two farms, ribotype B was shown
by 9 strains (14%) from 2 (MA) fish and 1 (T) isolate (2%) while ribotype N was
produced by 7 isolates (11%) from 4 (MA) fish and 1 (T) strain (2%) (Tab. 4). The
other ribotypes observed in this study were not in common and therefore were
considered characteristic for each farm.
As regards the dominant ARDRA profiles found on the (MA) farm, a numerous group
was identified as ribotype A which included 9 isolates (14%) from 4 fish while the
ribotypes which characterised (T) sea bream were biotype e merging 6 bacteria (13%)
from 1 fish and biotype p which comprised 6 bacterial strains (13%) from 4 fish (Tab.
4).
Furthermore, it was interesting to detected a bacterial diversity within each fish, in fact
a number varying from 3 to 7 of different ARDRA biotypes were observed per fish in
(MA) sea bream where a mean of 8 isolates per fish were tested, and a number ranging
from 1 to 5 of different ribotypes were found in the gut microflora of single (T) sea
bream for which a mean number of 5 isolates per fish were analysed.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
87
Table 4. Different “ribotypes” found in the gut microflora of gilthead sea bream reared
in the MA and T farms: number of isolates (N°), percentage (%) and number of fish they
were isolated from.
Farms
Ribotypes
La Maricoltura Alghero s.r.l
Tortoli lagoon
A
a
B
b
C
c
D
d
E
e
F
f
G
g
H
h
I
i
L
l
M
m
N
n
O
o
P
p
Q
R
S
T
U
Total
Biodiversity index
Total different
Isolates (N°) % Fish (N°) Isolates (N°)
9
14 4
1
9
14 2
1
2
1
2 1
1
1
2 1
3
1
2 1
6
1
2 1
2
14
22 7
15
2
2
3 2
2
1
2 1
1
1
2 1
1
4
6 3
1
7
11 4
1
1
2
3 2
2
4
6 3
6
1
2 1
2
3 2
1
2 1
1
2 1
1
2 1
63
48
19/63
17/48
19
17
%
Fish (N°)
2
2
4
1
1
2
2
1
6
1
13 1
4 1
31 5
4 2
4
2
2
1
2
1
2
2
2
1
1
1
4
1
13 4
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
88
7.2.2.3 Comparison of ARDRA profiles of the bacterial isolates with the “type”
strains
In order to proceed with the taxonomic identification of the isolates, four “type” strains
belonging to the CECT Spanish Collection were analysed in the same way as the
isolates under study for comparative purposes.
The ARDRA HhaI profiles from ribotypes G, which proved to be the dominant
microbial philogenetic group of the intestinal microflora of Sparus aurata coming from
both fish farms, presented the same ARDRA HhaI profiles as Pseudomonas
anguilliseptica CECT 899 (Fig. 17: lanes 2, 5, 14). On the other hand, considering the
ARDRA HaeIII profiles of these strains, only 1 band out of 3 differs slightly in
molecular size from the Pseudomonas anguilliseptica “type” strain.
Furthermore, two other isolates from fish reared on the (T) farm (ribotypes a and m)
showed the same ARDRA HaeIII profiles as the “type” strain CECT 839 (Aeromonas
hydrofila).
These results were considered important because they were consistent with the
taxonomic identification at genus level made successively by sequence analysis which
ascribed ribotype G to Pseudomonas genus and ribotypes a and m to Aeromonas spp.
demonstrating the validity of ARDRA studies for taxonomic identification at genus
level.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
89
7.2.2.4 Intestinal microbial ecology
7.2.2.4.1 Identification of bacteria by sequence analysis
Intestinal bacterial isolates studied using the ARDRA technique were finally identified
by sequence analysis. The analysis of the sequence of the 16S RNA gene was
performed on one representative biotype from each dominant ARDRA groups. In some
cases, more than one strain from the same ARDRA group was studied by sequence
analysis.
An example of the partial sequence of the 16S rRNA gene of a P. fragi strain isolated
from a (T) gilthead sea bream‟s gut is shown in Fig. 20.
Figure 20. Partial sequence of the 16S rRNA gene of a P. fragi strain isolated from
the intestine of a (T) gilthead sea bream.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
90
Tab. 5 lists the intestinal bacteria identified by ARDRA and sequence analyses in the
present work. Different bacterial species were found in the two groups of sea bream
studied. As for the G biotype dominant on both farms, it was ascribed to Pseudomonas
spp. Indeed, the representative strain of the ARDRA G group isolated from (MA) fish
presented a 100% similarity with P. fluorescens while the ribotypes G from the
“Tortoli” farm were classified as P. fragi species with a 99% homology by BLAST
program. The other ARDRA biotypes found on both farms were represented by ribotype
B which was identified as Rainbow trout intestinal bacterium T115 in the NCBI
database with an identity of 100% and by ribotype N classified as Psychrobacter sp.
with 99% identity (Tab. 5). Considering the dominant biotypes characteristic of each
fish farm, ribotype A, representative of the (MA) farm, was classified as Myroides
profundi at 100% identity, while ribotypes e and p characteristic of (T) sea bream, were
affiliated to the species Sphingomonas paucimobilis and Arctic soil bacterium,
respectively. Other different bacterial species identified by sequence analysis in this
study as composing the gilthead sea bream‟s intestinal microflora are presented in Tab.
5.
7.2.2.4.2 Intestinal microflora of gilthead sea bream reared in the “La Maricoltura
Alghero s.r.l” facility and the Tortoli lagoon
Thus, the present study has so far shown that the dominant bacterial species which
colonise the gut of Sparus aurata reared on the (MA) and (T) farms were as shown in
Fig. 21. Considering the percentage distribution of the different microbial groups
identified in the present study, a great biodiversity was found in the intestinal tract of
gilthead sea bream reared in the Tortoli lagoon, in fact 8 different bacterial species were
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
91
detected in the intestinal microflora of these fish while 5 different taxonomic groups
were found among the isolates from “La Maricoltura Alghero” (Fig. 20).
Table 5 Intestinal bacteria of Sparus aurata identified using sequence analysis of the 16S
rRNA gene: ARDRA biotypes, number of sequenced bases (N°), species name, percentage of
identity in NCBI data base (%).
ARDRAgroup
Sequenced bases(N°)
Biotypes
Identification
Identity(%)
Species name
A
618
Myroides profundi
100
a
420
Aeromonas salmonicida subsp.
99
salmonicida
B
480
Rainbow trout intestinal bacterium T115
100
D
639
Chryseobacterium sp.
100
e
394
Sphingomonas paucimobilis
99
G
535
Pseudomonas fluorescens (MA)*
100
G
653
P. fragi (T)*
99
h
618
Leucobacter sp.
100
m
379
Aeromonas molluscorum
100
N
520
Psychrobacter sp.
99
p
600
Arctic soil bacterium A1T3
99
*Letters in brackets indicates the farm where the bacterial strain where isolated. (MA): “La Maricoltura
Alghero”; (T): Tortoli lagoon.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
92
14%
36%
14%
2%
(a)
22%
11%
Rainbow trout intestinal bacterium or Providencia rettgeri (Enterobacteriaceae)
Myroides profundi (Flavobacteriaceae)
Cryseobacterium sp. (Flavobacteriaceae)
Pseudomonas fluorescens (Pseudomonadaceae)
Psychrobacter sp.(Moraxellaceae)
Other GRAM -
2% 2%
31%
13%
4%
13%
2%
(b)
2%
31%
Aeromonas salmonicida subsp.salmonicida (Vibrionaceae)
Aeromonas molluscorum (Vibrionaceae)
Sphingomonas paucimobilis (Sphingomonadaceae)
Leucobacter sp.(Microbacteriaceae)
Artic soil bacterium A1T3
Rainbow trout intestinal bacterium (Enterobacteriaceae)
Pseudomonas fragi
Psychrobacter sp.(Moraxellaceae)
Other GRAM-
Figure 21. Percentage distribution of bacterial species present in the gut of Sparus
aurata reared in the “La Maricoltura Alghero” (a) and the Tortoli lagoon (b).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
93
7.2.2.4.3 Intestinal microbial community of Sparus aurata
From the polyphasic survey of the present study performed on 111 strains, the bacterial
qualitative composition of the gut of Sparus aurata is represented by the diverse
phylogenetic groups shown in Tab. 6. The Pseudomonadaceae (29/111= 26% of the
total) comprised the species Pseudomonas fluorescens (MA fish) and P. fragii (T fish),
the Enterobacteriaceae (9/111= 9% of the total) were represented by Rainbow trout
intestinal bacterium T115 species, followed by Moraxellaceae (8/111= 7% of the total)
with the Psychrobacter genus, by Flavobacteriaceae (10/111= 9% of the total) with the
species Myroides profundi and the Chryseobacterium sp. (MA sea bream), by
Sphingomonadaceae (6/111= 5% of the total) with Sphingomonas paucimobilis (T fish)
and by an unclassified philogenetic group typical of cold environments identified as
Arctic soil bacterium (6/111= 5% of the total) by Blast search, peculiar of (T) gilthead
seabream. As regards the other taxonomic groups found in the intestinal microflora of
Sparus aurata, the Aeromonadaceae (2/111= 2% of the total) with the species
Aeromonas salmonicida and A. molluscorum were characteristic only of (T) gilthead sea
bream. Furthermore, 2 isolates from (T) fish (2% of the total) were ascribed to the
Microbacteriaceae group represented by the Gram-positive Leucobacter sp. However,
up to now a total of 38 out of 111 isolates (31%) could not be assigned to any genus or
bacterial species and this study is in progress.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
94
Table 6. Major bacterial philogenetic groups composing the microbial community of
the gut of Sparus aurata, closest relative (from Blast search), number of the total
isolates (N°/Total), percentage of the total (%).
Philogenetic group
Closest relative
N°/Total
%
Pseudomonadaceae
Pseudomonas fluorescens
29/111
26
10/111
9
Pseudomonas fragii
Enterobacteriaceae
Rainbow trout intestinal
bacterium
Moraxellaceae
Psychrobacter sp.
8/111
7
Flavobacteriaceae
Myroides profundi
10/111
9
Chryseobacterium sp.
Sphingomonadaceae
Sphingomonas paucimobilis
6/111
5
Unclassified bacteria
Arctic soil bacterium
6/111
5
Aeromonadaceae
Aeromonas salmonicida subp.
2/111
2
2/111
2
73/111
65
salmonicida
Aeromonas molluscorum
Microbacteriaceae (high GC
Leucobacter sp.
Gram positives)
Total
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
95
DISCUSSION
In the present research, a total of 60 gilthead seabream reared in off-shore floating cages
(intensive system) and grown in a lagoon (extensive system) were sampled for studying
the gut bacterial load and the qualitative bacterial composition. Considering the number
of different microbial groups analysed, higher values were detected in the fish reared in
the extensive system than in the sea bream grown in the floating cages, although this
bacterial load proved low if compared with studies performed on other fish species like
hybrid tilapia (Al-Harbi et al., 2004), Atlantic cod (Gadus morhua L.) (Ringo et al.,
2006), Atlantic salmon (Salmo salar) (Ringo et al., 2008), sea trout (Salmo trutta trutta)
(Skrodenyte-Arbaciauskiene et al., 2008) and rainbow trout (Oncorhynchus mykiss)
(Pond et al., 2006). In general, typical bacterial counts estimated on fish gut (106-108
CFU g-1) are lower than those reported for humans and terrestrial animals (approx. 1011
CFU g-1) and this reflects the higher number of anaerobes in the intestine of endotherms
compared to that of fish (Kim et al., 2007).
According to the literature mentioned in the introduction, the substantial differences in
the intestinal microbial load detected in the two groups of gilthead sea bream analysed
in the present study can be generally attributed to the different type of rearing systems
including dietary regimen and so on as well as the different ages of the fish; indeed the
gilthead sea bream grown in the lagoon ought to be older than the fish reared in floating
cages. As regards the bacterial counts of various microbial groups, the ANOVA showed
significant differences between the two groups of fish except for the number of
Enterobacteriaceae which presented a P=0.06 although their mean value was clearly
higher in the fish captured in the lagoon compared to the sea bream reared in floating
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
96
cages. In this regard, however, an important aspect to be considered is represented by
the day-to-day fluctuations and the inter-individual differences which may occur in fish
(Ringo et al., 1995). Indeed, it is important to know that the present study showed a
great variability in the fish of the same group (one T fish did not contain any type of
bacteria) probably linked to a different ethology of the fish living in a large and variable
environment such as a lagoon.
In any case, the relative low number of bacteria associated with the digestive tract,
registered especially in the gilthead sea bream harvested in “floating” cages,
demonstrated a good quality of fish, good hygienic conditions inside the cages as well
as a suitable rearing density and a balanced diet for the sea bream.
As regards the counts of probiotics, only up to 6 colonies per plate were recovered in 6
(T) fish and a complete absence of any culturable microflora was found on MRS agar
when cultivating intestinal samples from (MA) sea bream; these results were in
agreement with previous studies on intestinal microbial communities analysed on
rainbow trout (Oncorhynchus mykiss) where no studied bacteria were able to grow on
MRS (Kim et al., 2007).
Throughout the present study, the characterisation of gut microflora was carried out by
a polyphasic study. The basic phenotypic observations indicated that Gram negative
aerobes with a rod morphology were present in high numbers and occurred with equal
frequencies in (MA) and (T) fish. Indeed, the dominance of the Gram negative aerobes
was described as characteristic mainly of predatory and benthophagous fish (Izvekova
et al., 2007) and for this reason we focused our study on this type of bacteria. Since
various microbiological studies showed that the Vibrio-Pseudomonas group dominated
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
97
the intestine of marine fish, we tried to use a fast methodology of tentatively
discriminating these bacterial genera on a large number of strains. The use of TCBS
medium proposed by Reid et al., 2009 and by other researchers as a crude method for
tentatively identifying the Vibrio spp. from other bacteria, was demonstrated to be
unsuccessful; indeed, the results achieved in the present study, showed that members of
various bacterial species were able to grow on TCBS, while no Vibrio spp. were found
among the strains identified by sequence analysis, although the bacteria studied in the
present work grew well on this medium. On the other hand, the species Vibrio harveyi
was isolated from internal organs (kidney and liver) of gilthead sea bream (Sparus
aurata) and in a greater number from European sea bass (Dicentrarchus labrax)
cultured on Spanish fish farms but almost exclusively on warm months (June to
November) (Pujalte et al., 2003a). In any case, the absence of Vibrio as a dominant
group was observed in the bacterial composition of other fish species such as freshwater
salmon, sea trout (Skrodenyte-Arbaciauskiene et al., 2008) and rainbow trout ( Pond et
al., 2006; Kim et al., 2007).
Despite this fact, the general screening performed on the intestinal isolates by means of
selective-differential medium for Pseudomonas, gave information on the presence of
this genus in the gut of the gilthead sea bream analysed although just a part of this
bacterial species was detected by means of this medium. Indeed, the following genetic
investigations highlighted a more consistent presence of Pseudomonas spp. in the two
groups of fish than was detected by the use of selective medium.
As far the genotypic studies were concerned, they were performed on 111 bacterial
isolates. PCR-amplification of the 16S rRNA gene from the bacterial strains and the
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
98
analyses of the restriction fragments were useful in identifying the dominant species
constituting the indigenous gut microflora of Sparus aurata. The use of the forward
primer 63f and reverse primer 1387r design by Marchesi et al., 1998, combined with
improved PCR, ARDRA studies and sequencing technology, widened the knowledge of
the microbial ecosystem of the gilthead sea bream considered, allowing a broad range of
bacterial species both within and between the two groups of fish to be highlighted.
Interestingly, a total of 32 different biotypes were observed by the use of two restriction
enzymes among the 111 intestinal strains screened and 11 distinct taxonomic bacterial
groups, representing the species of the most numerous ARDRA groups, were identified
by sequencing of the partial 16S rRNA gene, sharing a 99% minimum sequence
similarity with the closest known species in GeneBank. As a matter of fact, the ARDRA
technique using two restriction enzymes was discriminative for the identification at
genus but not at species level because the G biotypes were identified both as P.
fluorescens in the gut microflora of (MA) fish and as P. fragi in the intestine of (T) sea
bream. Indeed they proved to have a high homology by BLAST search. This confirms
the fact that 16S rDNA is not variable enough for the identification of some species or
that the use of two restriction enzymes is not sufficient for differentiating between these
phylogenetically close Pseudomonas species. Therefore, the combination of different
techniques is fundamental for taxonomic purposes and the use of other restriction
enzymes combined with sequence analysis of a greater number of intestinal isolates is
necessary for a more complete conclusion.
In any case, the present study showed that the gut microflora of Sparus aurata is quite
complex being constituted of various phylogenetic dominant groups such as
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
99
Pseudomonadaceae
(26%),
Enterobacteriaceae
(9%),
Moraxellaceae
(7%),
Flavobacteriaceae (9%) found also in the gut of other fish species such as rainbow trout
(Kim et al., 2007), Atlantic cod (Ringo et al., 2006) and Atlantic salmon (Ringo et al.,
2008).
Considering the Pseudomonas spp. which proved predominant in the intestinal tract of
S. aurata, it is well-documented that they are ubiquitous bacteria in nature in both
animal and plant products. Pseudomonas spp. represent a heterogeneous phylogenetic
microbial group which was found to be a component of the edible part of fish and
responsible of the spoilage of gilthead sea bream Sparus aurata from Mediterranean
Sea waters (Tryfinopoulou et al., 2002). Since the same bacterial species were identified
in the gut of the gilthead sea bream used in the current study, we can confirm that a
possible contamination of the edible portion from gastrointestinal sources can occur in
fish products, corroborating other studies which affirm that the composition of intestinal
microflora is similar to that of integuments, gills and bolus (Cahill, 1990). Furthermore
it is interesting to find that various biovars of P. fragi and P. fluorescence were
described as the principal aerobic Gram-negative spoilage microflora on meat and
certain meat products at chill temperatures (Molin and Ternström, 1982).
Despite this fact, however, it is also true that the members of the Pseudomonas group
such as P. fluorescence merge species of environmental origin which were found to be
abundant in the soil, water, plant and snow algae of the arctic-alpine-tundra as well as in
the alpine-tundra soils of the Colordo Front Range (Männistö and Häggblom, 2006;
Molin and Ternström, 1986). In these studies some authors also highlighted the
presence of protease, lipase and cellulase in Pseudomonas strains (Tryfinopoulou et al.,
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
100
2002; Männistö and Häggblom, 2006) and therefore a possible role in the nutrition of
fish can be assigned to these bacteria other than that of taking part in the storage
processes.
Another bacterial species found in common in the two groups of gilthead sea bream was
identified as the Rainbow trout intestinal bacterium T115 by DNA homology of the 16S
ribosomal RNA gene as observed in the gut of rainbow trout by Kim et al., 2007.
The results of the present study showed that Psychrobacter sp. of the Moraxellaceae
group was present in significant numbers (11%) in the fish coming from floating cages.
It is interesting to note that this bacterial genus, commonly isolated from cold
environments including soil, sea-ice and the skin and gills of fish (Scholes and Shewan,
1964), was found in another study carried out on Atlantic cod fed different diets and
proved dominant in the foregut and in the midgut of fish fed bioprocessed soybean meal
(Ringo et al., 2006) and in the intestine of Atlantic salmon (Ringo et al., 2008).
On the other hand, clear differences were observed when comparing the microflora of
the gilthead seabream from the two farms. Regarding the group of Flavobacteriaceae
which included the species Myroides profundi (14%) and Chryseobacterium sp. (2%),
representative of the (MA) farm, the first species was found to be a quite numerous
component of the intestinal microflora of the sea bream studied. Indeed, the Myroides
genus is typical of aquatic environments and Dang et al., 2009 isolated from Chinese
deep-sea sediments a Myroides strain which was found to possess protease and DNase
activities. The same study indicated other strains of sedimentary origin, ascribed to
Pseudomonas and Psychrobacter spp. which also contained amylases and lipases.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
101
Considering the presence of Chryseobacterium sp., as typical of sea bream reared in
floating cages, the occurrence of this genus was also noted in the hindgut of Atlantic
cod especially in fish fed soybean meal (Ringo et al., 2006), although the genus
Chryseobacterium was primarily isolated in soil, water or in gills, skin, kidney and in
muscle lesions from diseased aquatic animals (Bernardet et al., 2005).
Thus, on the basis of the results of the qualitative microbial analysis of the (MA) fish
and considering the similar results described by Ringo et al. 2006, we can state and
confirm that gut microflora is markedly influenced by feeding and therefore can give
information both on the dietary regimen and the rearing farm as reported in literature
(Ringo and Olsen, 1999; Dimitroglou et al., 2010).
By using 16S rRNA genes to study intestinal microflora of gilthead sea bream, we were
able to find other bacterial species peculiar to the fish captured in the Tortoli lagoon and
some of them had not previously been reported in the intestinal tract of fish. It was
interesting to identify a group of strains classified as Arctic soil bacterium A1T3 (13%)
by Blast search which proved quite numerous in the gut of these sea bream (4 fish). As
a matter of fact, Arctic soil bacteria are described in literature as they represent a large
group of psychrotolerant species related to the Pseudomonas genus and commonly
found in Arctic coastal environments (coastal lagoons and the surface of marine
macroalgae). Various studies have highlighted the ability of some strains ascribed to
this philogenetic group to degrade contaminating compounds such as polychlorinated
biphenils (PCBs) (Master and Mohn, 1998; Master et al., 2008) and mercury (Poulain et
al., 2007) in marine environments. According to these investigations, since these
microorganisms can grow on contaminated substrates and are found to be able to
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
102
remove these pollutants from the environment, they could be considered useful for
bioremediation processes and represent one means by which these sites may be restored
to their original condition. Furthermore, in the current polyphasic study, it was
interesting to identify the Sphingomonas paucimobilis species (13%) in the intestinal
microflora of the gilthead sea bream from the Tortoli lagoon. A recent study described
this species as an agent of clinical endophthalmitis (Wook et al., 2008), yet, diverse
papers have descrbed the ability of some strains isolated from environmental samples
(soil, or waste water) to be able to biodegrade lignin-related biphenyl compounds (Peng
et al., 2002) and to transform toxic triphenylmethane dyes such as Malachite Green
used in the textile industries and in aquaculture into non-toxic substances (Ayed et al.,
2009). The ability of bacteria present in the intestine of an aquacultural fish specimen
such as Sparus aurata to degrade Malachite Green dye is of great importance, because
this substance is used for treating several illnesses in reared fish caused by a protozoan
(ictioftiriasi), a fungus (saprolegnosi) (Foster and Wooddbury, 1936) and for controlling
kidney disease (Clifton-Hadley and Alderman, 1987). In addition, other studies have
reported that a S. paucimobilis strain is one of the microorganisms most able to utilize
potentially hazardous high-molecular-weight polycyclic aromatic hydrocarbon (PAH)
such as fluoranthene as a sole carbon source and as energy for growth (Mueller et al.,
1990). The same research has shown catabolic activities of this strain towards other
PAHs as 2,3-dimethylnaphthalene, anthracene, fluoranthene, fluorene, naphthalene, and
phenancene and other papers have demonstrated that some strains of S. paucimobilis
possess degradative systems active against naphthalene, antracene and phenanthrene
(Story et al., 2001).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
103
As regards the other bacterial species Aeromonas salmonicida subsp. salmonicida and
Aeromonas molluscorum, identified in (T) fish in low numbers, the literature indicates
that Aeromonas spp. including Aeromonas salmonicida strains are ubiquitous in fish,
being commonly found among the microflora of trout (Lee et al., 2002; Pond et al.,
2006; Kim et al., 2007), in the intestinal microbial community of carp (Namba et al.,
2007) and in the gut of the salmon (Skrodenyte-Arbaciauskiene et al., 2008).
Additionally, high GC Gram positive bacteria belonging to Leucobacter sp. were found
among the non dominant bacteria of two (T) gilthead sea bream. In this regard,
members of the genus Leucobacter have been described in literature and in particular
two new species including L. luti and L. alluvii. By Blast search this research showed
that both these species are taxonomically close to to the strains identified in this study
which showed a 97 % of 16S rDNA homology with L. luti and a 96 % 16S rDNA
similarity with L. alluvii. These novel species were reported to be able to grow in the
presence of a certain concentration of chromium, in fact they were isolated from diverse
environments such as activated sludge and sediments of a river receiving chromium
contaminated water in Central Portugal (Morais et al. 2006). Furthermore, the same
authors investigated other new species L. aridicollis and L. chromiireducens which were
found to be able to reduce the carcinogenetic Cr (VI) to the non harmful form Cr (III)
(Morais et al., 2004). It was interesting to find that the Leucobacter sp. strains identified
in the present study had a of 96% 16S rDNA homology with the species L. aridicollis
and L. komagatae and of 95% with L. chromiireducens .
However, Morais et al., 2004 concluded that the ability to transform the harmful Cr
(VI) into Cr (III) does not seem to be acquired by lateral gene transfer from other
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
104
chromium-resistant organisms that colonize the environment, and can be considered a
common feature of the strains of the genus Leucobacter, since another strain of L.
komagatae not isolated from a chromium enriched environment also showed the same
degree of chromium resistance.
In any case, the presence of high GC Gram-positive bacteria belonging to the
Microbacteriaceae group has also been indicated for the intestine of rainbow trout (Kim
et al., 2007).
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
105
CONCLUSIONS
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
106
The present research evidenced that the bacterial species of the Pseudomonas genus are
characteristic in S. aurata and generally present in fish as reported in other studies on
the microflora of the gut of other fish specimens and in researches on the bacteria
responsible of spoilage of food products. Other bacterial species identified in this study
as Psychrobacter sp. and Chryseobacterium sp., seem to be indicators of the type of diet
of the gilthead sea bream. Moreover, some other bacterial species such as the group of
Arctic soil bacteria, the species Sphingomonas paucimobilis and Leucobacter sp.,
identified in the intestinal microflora of Sparus aurata reared in the Tortoli lagoon, had
not previously been reported in the intestinal tract of fish while they have been isolated
from environmental samples. On the basis of these data, it is evident the there is a close
link between the intestinal microflora and the rearing system of Sparus aurata.
The microbial biodiversity observed in the gut microflora of the two groups of fish is
very interesting and futher studies on strain “typing” are necessary in order to know
better the genetic biodiversity of these microbial populations.
In the light of the results presented here, a general conclusion can be drawn: the
biodiversity highlighted in this study, the general low quantity of bacteria found, the
presence of bacterial species of environmental origin or deriving from diet, which do
not represent pathogen bacteria in the rearing conditions of the sea bream under study,
together with the good state of health of the fish, indicate a microbial balance in the
intestinal microflora composition of these fish which reflects good rearing conditions
(rearing density, dietary regimen etc.) and a general well-being of the animals.
The presence in some fish of bacterial species described as potential natural
bioremediators is very interesting in the light of further studies aimed at investigating
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
107
the degradative properties of these bacteria on diverse substrates in order to find out a
possible beneficial role both for fish welfare and for natural biodegradation strategies
for biotechnological applications.
Furthermore, the fact that most of the bacteria identified are reported to carry enzymes
like amylases, proteases, and lipases, encourages further studies aimed at studying the
biochemical properties of these microbial species in order to assess their potential
nutritional role.
This is an initial study on the intestinal microbial ecology of S. aurata reared on the
Mediterranean coast. Further analyses are being conducted on a larger number of
isolates from other fish from the same sites and from different marine areas in order to
go into the intestinal microbial ecology of Sparus aurata specimen in more depth. This
will help to evaluate the ecological significance of the strains as bioindicators.
Finally, this study represents a step in the knowledge of S. aurata gut biology.
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
108
LIST OF FIGURES
Figure 1. Representative scheme of the anatomy of the digestive tract of Teleost fish on
the basis of different dietary regimen (Smith, 2009).
pag. 18
Figure 2. Endocrine control of osmoregulation in teleost fish: morphology and transport
mechanism of gill chloride cells in seawater and fresh water (McCormick, 2001).
.
pag. 23
Figure 3. Sampling sites.
pag. 57
Figure 4. Floating cages of “La Maricoltura Alghero” s.r.l..
pag. 58
Figure 5 (a). Fishing at “La Maricoltura Alghero”s.r.l..
pag. 59
Figure 5 (b). Fish put into ice after capturing.
pag. 59
Figure 6. Tortoli lagoon: “lavoriero” where gilthead seabreams were captured. pag.61
Figure 7. Fish put into ice after capturing.
pag. 61
Figure 8. Removal of intestine from a seabream for microbiological analyses.
pag. 63
Figure 9. Phases of preparation of sea bream intestinal homogenate: (a) Stomacher®
400; (b) serial dilutions.
pag. 63
Figura 10. Bacterial colonies from sea bream intestinal samples grown on PCA
medium (a) and on VRBA-MUG (b).
pag. 74
Figure 11. Mean values of different bacterial groups detected in the gut of gilthead sea
bream reared in Tortoli lagoon (T) and in “La Maricoltura Alghero” (MA) farm. pag. 76
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
109
Figure 12. Gram negative bacteria isolated from the intestine of Sparus aurata L.
observed by the phase-contrast microscope.
pag. 77
Figure 13. Bacterial strains grown on TCBS agar: (a) sucrose fermenter strain; (b) non
sucrose fermenter strain.
pag. 79
Figure 14. Presumptive Pseudomonas spp. strains grown on Pseudomonas agar F. (a):
an isolate observed under U.V. light showing pyocianin production; (b): pigment
producers isolates (bottom) and non pigment producing strains (top) observed at visible
light.
pag. 79
Figure 15. Products of amplification of the 16S rRNA gene from DNA of intestinal
bacteria run on 1% agarose gel. 1: Marker III (564- 21,226 bp)(Roche); 2-6: (MA)
isolates; 7-10 “type” strains of Spanish collection: CECT 625 (Vibrio aestuarianus),
CECT 529 (Vibrio vulnificus); CECT 839 (Aeromonas hydrofila); CECT 899
(Pseudomonas anguilliseptica).
pag. 82
Figure 16. ARDRA profiles from (MA) bacterial isolates run on 2% agarose gel
obtained after digestion with HaeIII restriction enzyme. 1: 1 Kb Plus DNA ladder
Marker Size (100-12,000 bp) (Invitrogen); 10: 100 bp DNA Ladder (1002,072bp)(Invitrogen); 2-9, 11-12, 15-16: different ribotypes; 13: CECT 625 (Vibrio
aestuarianus); 14: CECT 529 (Vibrio vulnificus).
pag. 83
Figure 17. ARDRA profiles from (MA) bacterial isolates run on 2% agarose gel
obtained after digestion with Hha I restriction enzyme. 1: 1 Kb Plus DNA ladder
Marker Size (100-12,000 bp) (Invitrogen); 8: 100 bp DNA Ladder (1002,072bp)(Invitrogen); 2-7, 9,10,15: different ribotypes; 11: CECT 625 (Vibrio
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
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aestuarianus); 12: CECT 529 (Vibrio vulnificus); 13: CECT 839 (Aeromonas
hydrofila); 14 CECT 899 (Pseudomonas anguilliseptica). The lanes indicated with G
represent the rybotypes G.
pag. 83
Figure 18. ARDRA profiles from (T) isolates run on 2% agarose gel obtained after
digestion with Hae III restriction enzyme. 1: 1 Kb Plus DNA ladder Marker Size (10012,000 bp) (Invitrogen); 9: 100 bp DNA Ladder (100-2,072bp)(Invitrogen); 2-8,10-16:
different ribotypes.
pag. 84
Figure 19. ARDRA profiles from (T) bacterial isolates run on 2% agarose gel obtained
after digestion with Hha I restriction enzyme. 1: 1 Kb Plus DNA ladder Marker Size
(100-12,000 bp) (Invitrogen); 8: 100 bp DNA Ladder (100-2,072bp)(Invitrogen); 2-7, 915: different ribotypes.
pag. 84
Figure 20. Partial sequence of the 16S rRNA gene of a P. fragi strain isolated from the
intestine of a (T) gilthead sea bream.
pag. 89
Figure 21. Percentage distribution of bacterial species present in the gut of Sparus
aurata reared in the “La Maricoltura Alghero” (a) and the Tortoli lagoon (b).
pag. 92
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
111
LIST OF TABLES
Table 1. Mayor intestinal bacterial species involved in fermentative activities: substrates
and products of their metabolism, host fish species and diet.
pag. 34
Table 2. Mayor molecular techniques used for studying bacteria: name, type of
analysis, information obtained, advantages and disadvantages.
pag. 52
Table 3. Counts (average CFU g-1 ± SE and min-max values) of different microbial
groups estimated on intestinal samples of gilthead sea bream captured from the “La
Maricoltura Alghero” (MA) and the Tortoli lagoon (T).
pag. 75
Table 4. Different “ribotypes” found in the gut microflora of gilthead sea bream reared
in the MA and T farms: number of isolates (N°), percentage (%) and number of fish they
were isolated from.
pag. 87
Table 5. Intestinal bacteria of Sparus aurata identified using sequence analysis of the
16S rRNA gene: ARDRA biotypes, number of sequenced bases (N°), species name,
percentage of identity in NCBI data base (%).
pag. 91
Table 6. Major bacterial philogenetic groups composing the microbial community of the
gut of Sparus aurata, closest relative (from Blast search), number of the total isolates
(N°/Total), percentage of the total (%).
pag.94
Rosanna Floris- Microbial ecology of the intestinal tract of gilthead sea bream (Sparus aurata Linnaeus, 1758).-Tesi di Dottorato
in Scienze e Tecnologie Zootecniche- XXIII Ciclo, Università degli Studi di Sassari.
112
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