Advanced Studies in Biology, Vol. 4, 2012, no. 9, 407 – 418
The First Isolation of Campylobacter jejuni
from Caspian Sea's Water
Masood Ghane, Fahimeh Ghorbani Moein* and Shirin Massoudian
Corresponding author: Fahimeh Ghorbani Moein
Department of microbiology, Tonekabon branch, Islamic Azad University,
Tonekabon, Iran.
e-mail address: ghorbani_fahimeh@yahoo.com
Abstract
The major purpose of this study was isolation, identification and characterization
of Campylobacter spp. from Caspian Sea’s water in north of Iran. Sampling from
the water was conducted within 4 seasons. Campylobacter spp. was isolated using
standard method then identified by Phenotyping tests. Finally, the identification of
strain was verified by PCR method. After conducting of the Phenotyping methods
and their confirmation with molecular technique, seven strains of Campylobacter
jejuni were identified. With regard to the obtained results, prevalence of this
bacterium in the coastal waters of the Caspian Sea’s was evaluated as 2.66
percent. The result obtained from this study is, in fact, regarded as the first report
from the isolation of Campylobacter jejuni from Caspian Sea. Knowledge of the
state of the entry and time span of the survival of the Campylobacter in the water
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M. Ghane, F. Ghorbani Moein and Sh. Massoudian
environments to control the water quality and prevention from the disease is of
importance.
Keywords: Campylobacter, Isolation, Caspian Sea, Iran
1. Introduction
Campylobacter’s are gram negative bacteria, which are seen in curve and Sshaped [1]. These bacteria have single and polar flagella in one or both end of the
cell [2]. Campylobacter’s are microaerophilic, non proteolytic, non sacharolytic
and non lipolytic, therefore to energy obtain from oxidation of amino acid or
tricarboxilic acid [3, 4]. These bacteria commonly live in intestinal track of warm
blood animals [4, 5].
Campylobacter’s are one of the most common causes of diarrhea in world wide.
According to world health organization estimated 2-35% of diarrhea induced by
campylobacter’s. So that, annually 400-500 million peoples in the word, which
infected for these bacteria [6]. Disease in human being occurs with consumption
of contaminated water and animal products viz., meat and milk [7, 8]. Studies
showed that 95% of Campylobacteriosis in human induced by C.jejuni, 4% by C.
coli and 1% by other species [9, 10].
Waters play important role in transmission of Campylobacter to animal and
human. These bacteria through of urine and feces of animal and birds into the
water [11]. Several studies showed that Campylobacter jejune isolated from
surface water [12-14], rivers [15, 16], ground water [17], rivers sediments [18],
lakes [19] lagoons [20], sea waters [21-23], sea sands [24] as well as
contaminated water and soil to sewage [20]. This is why reported existence of
Campylobacter jejune in untreated drinking water [15, 17]. Hence, surface water
appropriate source to transmission of this bacteria to Human beings [14].
Isolation of C. jejuni from Caspian Sea
409
Therefore, based on foregoing evidence Campylobacter infections are considered
a worldwide problem of public healthy, causing considerable suffering and no
more information is available concerning to isolation and frequency of occurrence
of this bacterium from Caspian sea, in order to achieve minimum information
concerning to the same.
2. Materials and methods
2.1 . Sample collection
In total, 263 water samples were obtained from south coastal Caspian Sea in one
year. The water samples were collected in 500 ml sterile bottles (falcon tube),
from one deep on surface sediment and transported to the laboratory at ambient
temperature and stored at 4°C. In order to keep the temperature low, all test tubes
containing the water samples were placed in the full-of-ice flask. Sampling time
was from 8 a.m. to 2 p.m., and the maximum time of transferring the samples to
laboratory was one hour. The environmental features, including atmospheric
condition, pH, temperature and salinity of water have been taken into
consideration.
2.2. Sample processing and isolation
at first, the tubes containing water sample were centrifuged in 4000 rpm (ALC,
Italy) within 10 minutes; then, supernatant was discarded and 1 to 2ml of it was
used in order to isolate the bacterium. In the next step, water sample was
transferred to the brain heart infusion broth (Merck-Germany) by the 0.45
micrometer filter. The tubes were incubated at 37°C for 48 h under
microaerophilic conditions.
After this period a one loop was taken from the bacterial suspension and the
spread culture was carried out on the CAMP medium (Merck-Germany), enriched
by the defibrinated blood of sheep. All cultured plates were placed in an incubator
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M. Ghane, F. Ghorbani Moein and Sh. Massoudian
at temperature of 37OC for 72 hours and under microaerophilic conditions in order
to isolate the Campylobacter spp.
2.3. Identification and biotyping of Campylobacter spp
Campylobacter identification was performed by subjecting all the suspected
colonies to microscopic examination, gram staining, glucose fermentation,
oxidase and catalase test. The isolates were characterized by using standard
Campylobacter phenotypic identification tests recommended by Atabay & Corry
[25]. At the end, the PCR method was carried out in order to confirm the
Phenotyping results.
2.4. DNA extraction and PCR method
DNA was extracted from suspected colony using phenol chloroform method. The
purity of the extracted DNA was analyzed based on absorbance of the extracted
DNA at 260 and 280 nm wavelengths by biophotometer (Eppendorf-Germany).
specific Primers produced by TAG Copenhagen (Denmark) were used to amplify
16s rRNA for campylobacter gene. The sequences of forward and reverse primers
were
5'-GGTTAAGTCCCGCAACGAGCCGC-3'
and
5'-
GGCTGATCTACGATTACTAGCGAT-3', respectively. Each reaction was
performed in a total volume of 25 µl contained 14.5 µl of molecular biology-grade
water (sigma aldrich company ltd.), 2.5 µl of 10×PCR buffer (cinagen-Iran), 1 µl
of 10 pmol each forward and reverse PCR primers, 1 µl of a 10 mM dNTPs
(cinagen-Iran), 0.5 µl of smar taq polymerase (cinagen-Iran), 1 µl of 50mm
MgCl2 (cinagen-Iran) and 5 µl of DNA template. Non-template control (NTC)
tube contained the same PCR reagents as above but had 5 µl of water substituted
for template. PCR amplification conditions on thermocycler (Biorad-Germany)
were as follows: 94°C for 4 min, followed by 30 cycles of 94°C for 60 s, 52°C for
45 s and 72°C for 45 s, with a final extension at 72°C for 5 min and storage at
4°C.
Isolation of C. jejuni from Caspian Sea
411
All PCR products were run on a 1.5% (w/v) agarose gels with a 1kb DNA ladder
(Fermntas-Russia). Aliquots of PCR products were electrophoresed at 75 V for 40
min; DNA was visualized using ethidium bromide and photographed after UV
transillumination with Uvidoc (England).
3. Results
Sampling from the Caspian Sea’s water was conducted within 4 seasons. Average
water salinity of the Caspian Sea was 12.85 (ppt) in the various seasons. The
minimum and maximum water temperature in the various seasons was reported 7
and 24oC, respectively. Also, water pH was ranged from 5 to 8 (Table 1).
After conducting of the Phenotyping methods and their confirmation with
molecular technique, seven strains of Campylobacter jejuni were identified. With
regard to the obtained results, prevalence of this bacterium in the coastal waters of
the Caspian Sea’s was evaluated as 2.66 percent (table 2).
Of 7 isolated strains, 2 strains in spring and 5 strains in summer were identified,
respectively. It is required to mention that the isolation of bacterium was not
performed in the autumn and winter seasons. In all cases which strains of
Campylobacter jejuni were isolated, the sea had the calm state so that the isolation
of bacterium was not successful in the stormy and wavy state.
4. Discussion
As mentioned above, C. jejuni cause of zoonotic infection, transmitted water and
foods [26]. These bacteria identified from surface water, water cannel, swage and
sea water [22, 27]. Amongst, intestinal tract pathogens include, Escherichia,
Salmonella, Vibrio, and Campylobacter jejuni is also transmitted by swage. This
species, despite that they are transferred to water through the birds’ feces, but the
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M. Ghane, F. Ghorbani Moein and Sh. Massoudian
swage is the main source of transferring the pathogen to rivers and surface water
[28-30].
Isolation and identification of C. jejuni from Caspian Sea was not reported. The
results obtained from present study achieved during 4 season from 263 samples
were collected of which 7 strain of C. jejuni isolated. This is evidently could
concluded that there exist this bacteria in coastal water of Caspian Sea.
In addition, within the studies conducted by Jose et al (1990) on the presence of
the Campylobacter in the sea waters of Spain, 25 strains (13%) were isolated out
of 192 collected Samples [31]. Within another research under the topic of the
identification of the Campylobacter’s in the water resources of the Africa’s west
conducted by deer guard in 2004, they could report the prevalence of the
Campylobacter jejuni in these waters up to 13.6% in this research [14]. In a
research carried out in Israel in 1999, it was specified that, out of 115 collected
samples, 52 strains (45%) from the sample of the swimming beaches of this
country are contaminated with Campylobacter [24].
The main way of the entry of these bacteria in to the Caspian Sea’s water can be
thought the house hold sewages and surface waters pouring into the sea.
Within the studies under the title of identifying Campylobacter’s from the
environmental samples in the north of Iran carried out by Ghane et al., 2010 they
reached this conclusion that one of the main sources of Campylobacter in this
geographical region in surface waters and rivers. Of 65 samples of the surface
waters and 27 samples of the sewage, they identified 22 strains (33.84%) and 2
strains (7.4%) Campylobacter, respectively which were the highest prevalence
related to the species of the Campylobacter jejuni [32].
The existence of this bacterium in the sea water can be attributed to the presence
of the seagulls as well because this bacterium doesn’t create a specified disease in
the gulls. But can enter in to sea water through feces [33, 34].
The existence of the environmental contamination and swage assists noticeably
the survival of the species of this bacterium so that they are found more greatly in
Isolation of C. jejuni from Caspian Sea
413
the contaminated environments. This is because of providing the nutrients for the
microorganisms. This affair shows that there exists a direct relationship between
the sewage. Infections and growth of the Campylobacters in the waters, including
the seawater [35].
The results obtained from this study is, in fact, regarded as the first report from
the isolation of campylobacter jejuni from Caspian sea. Knowledge of the state of
the entry and time span of the survival of the campylobacter in the water
environments to control the water quality and prevention from the disease is of
importance.
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Fig. 1. Agarose gel (1.5%) analysis of a PCR diagnostic test. Lane M: size marker
1 kb, Lane 1: positive control, Lane 2-4: positive sample, Lane 5: Negative
control.
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Table 1. Temperature, pH and Salinity of Caspian Sea in 4 season sampling
seasons
Temperature
pH
Salinity
Fall
7-11
6/5-7
12/80
Winter
5-11
5-6/5
12/85
Spring
7-18
6/5-8
12/95
Summer
13-24
7
13/00
Table 2. Frequency of occurrence of C.jejuni isolated from Caspian sea
seasons No.
sample
collected
Isolated
number
(%)
Fall
41
0
Winter
61
0
Spring
67
Summer
94
Total
263
2
(0.76%)
5 (1.9%)
7
(2.66%)
Received: May, 2012