Vol. 2, No. 2, September 2009
J. Ris. Kim.
MULTIDRUG RESISTANCE (MDR) OF V. Parahaemolyticus
Marlina
Faculty of Pharmacy,University of Andalas
e-mail: marlina_adly@yahoo.com
ABSTRACT
A total of 97 V. parahaemolyticus isolate from Padang were examined for their resistance to 15
antibiotics. V. parahaemolyticus isolated behaved as resistant to sulfamethoxazole (100%),
rifampin (95%) and tetracycline (75%) and sensitive to norfloxacin (96%). Ampicillin still sensitive
for V. parahaemolyticus isolated from human stools. All of isolates were sensitive to namely
chloramphenicol and floroquinolones (ciprofloxacin and norfloxacin agents). RAPD-PCR profiling
with three primers (OPAR3, OPAR4 and OPAR8) produced four major clusters (R1, R2, R3 and
R4), 7 minor clusters (I, II, III, IV, V, VI and VII) and three single isolates.
Keywords: V. parahaemolyticus, MDR, RAPD
INTRODUCTION
Food safety is an increasingly important public
health issue. Governments all over the world
are intensifying their efforts to improve food
safety. These efforts are in response to
an increasing number of food safety problems
and rising consumer concerns. Vibrio
parahaemolyticus is one of a prevalent foodborne pathogen in many Asian countries where
marine foods are frequently consumed. It is an
important food-poisoning pathogen in coastal
countries, especially in Japan and Taiwan
(Wong et al., 2000; Feldhusen, 2000; Lipp et
al., 2002; Tantillo et al., 2004).
Food can be considered an excellent way
for introducing pathogenic microorganisms
in
general
population
and
in
immunocompromised people, and therefore it
may transfer antibiotic-resistant bacteria to the
intestinal tract of consumers very efficiently.
There is an increasing demand for fish and fish
products around the world (Feldhusen, 2000).
However, there is substantial evidence that fish
and seafood are high on the list of foods
associated with outbreaks of foodborne
diseases (Huss, 1997).
V. parahaemolyticus has been recognized as an
agent of gastroenteritis associated with
112
consumption of seafood and considered to be
one of the causes of diarrhoeal disease in
humans. Usually diarrhea caused by V.
parahaemolyticus is self-limiting with duration
of 2–5 days but it can persist for 2 weeks or
longer (Nishibuchi, 2004). Antimicrobial
therapy is not required except in severe cases
long-lasting
V.
parahaemolyticus
and
infections. Tetracycline and cotrimoxazole are
normally considered the drug of choice for
diarrhoeal infection, but ampicillin or
amoxicillin are also recommended.
In general, the majority of Vibro spp. are
resistant to a large number of beta lactam
antibiotics particularly for B-lactam penicillin
groups (ampicillin and amoxicillin) (Jacoby
and Munos-price, 2005; Wang et al., 2006;
Tjaniadi et al., 2003; Lesmana et al., 2001) and
susceptible for tetracycline, chloramphenicol
and nalidixic acid (Wang et al., 2006). In
addition, majority of V. parahaemolyticus are
resistant to trimethoprim and sulphonamides
(Radu et al., 1998). The available information
on antimicrobial susceptibilities of V.
parahaemolyticus differ somewhat from
different countries (Wong et al., 2000;
Ottaviani et al., 2001; and Lesmana et al.,
2001) an but resistance has been reported to be
increasing particularly to fluroquinolones
(Zhao and Drlica, 2001). There is growing
ISSN : 1978-628X
J. Ris. Kim.
scientific evidence that the use of antibiotics in
veterinary particularly in developed countries
leads to the development of resistant
pathogenic bacteria that can reach humans
though food chain. In Indonesia, there are few
reports on the antimicroibial susceptibility of
V. parahaemolyticus isolated from humans
(Lesmana et al., 2002 and Tjaniadi et al.,
2003) but no documented reports yet on the
susceptibilities of V. parahaemolyticus strains
isolated from food and environments. To
readdress this situation, we investigated the
antimicrobial
susceptibilites
of
V.
parahaemolyticus isolated from shellfish,
hospital wastewater and human stools in
Padang, West Sumatera, Indonesia.
MATERIAL AND METHODS
Antibiotic Susceptibility Testing
A total 97 isolates of V. parahaemolyticus
isolated from shellfish (B. violacae, C.
moltkiana and F. ater), hospital wastewater
and human stools in Padang, West Sumatera,
Indonesia
were
used
for
antibiotic
susceptibility testing. Fifteen antibiotics were
used in this experiment: ampicillin (10 μg),
ceftriaxone (30 μg), ceftadizime (30 μg),
cefuroxime (30 μg), chloramphenicol (30 μg),
ciprofloxacin (30 μg), erythromycin (15 μg),
gentamycin (10 μg), kanamycin (30 μg),
nalidixic acid (30μg), norfloxacin (30 μg),
rifampin (5 μg), streptomycin (10 μg),
sulfametoxazole (25 μg) and tetracycline (30
μg).
The antibiotic sensitivity testing was carried
out based on the standard Kirby-Bauer
diffusion method (Bauer et al., 1966). The
method indicated susceptibility of the
organism to the antibiotic by clear zone of
inhibited growth around the reservoir (the
discs), the diameter of this zone being
proportional to the degree of susceptibility.
The results were analysed by hierarchic
numerical methods using the software package
BioNumeric version 4.6 (Applied Maths,
Kortrijk,
Begium).
Resemblance
was
computated
with
Pearson
correlation
coefficient and agglomerative clustering was
ISSN : 1978-628X
Vol. 2, No. 2, September 2009
performed with the
linkage (UPGMA).
unweighted
average
RAPD-PCR Amplification
The DNA of the V. parahaemolyticus strains
were extracted by mini-preparation method of
Ausubel et al., (1987). The RAPD assay was
conducted in a final volume of 25 l
containing 2.5 l 10x reaction buffer, 1.6 l,
25 mM MgCl2, 0.5 l, 10 mM each dATP,
dCTP, dGTP and dTTP (Bioron), 0.5 M
primer, 0.2l. 5 U/unit of Taq polymerase and
genomic DNA. Amplifications were carried
out in the thermal cycler (Perkin Elmer Cetus
2400) for 35 cycles of 1 minute at 94ºC, 1
minute at 36ºC and 2 minutes at 72ºC. A final
elongation step at 72ºC for 5 minutes was
included. Amplification products were
fractionated by electrophoresis through 1.5%
agarose gel stained with ethidium bromide.
1kb DNA ladder (Bioron) was used as DNA
size marker.
Results
A total of 97 V. parahaemolyticus isolates
were examined for their resistance to fifteen
antibiotics. Sixteen isolates were from clinical
samples (human stools), twenty isolates were
from the hospital wastewater, and sixty-one
isolates from shellfish which were collected
from two different locations (Lake Singkarak
and Lake Maninjau). Correlation of resistance
pattern among three different sample types
(clinical samples, hospital wastewater, and
shellfish samples) was investigated in present
study. The diffusion susceptibility results were
analysed by hierarchic numerical methods to
cluster isolates and to group antimicrobials
according to similarity profile.
The highest prevalence of resistance in the V.
parahaemolyticus from shellfish isolates were
to the antimicrobials, rifampin (100% of all B.
violacae isolates), sulfametoxazole (100% for
B. violacae and F. ater isolates) and
erythromycin (100% for C. moltkiana and B.
violacae). None of the V. parahaemolyticus
from shellfish isolates from B. violacae were
found to be resistant to norfloxacin.
113
Vol. 2, No. 2, September 2009
Similarly, the highest prevalence of resistance
in V. parahaemolyticus from human stools
isolates was to rifampin (all of human stools
isolates), followed by sulfametoxazole (95%)
and kanamycin (75%). Sulfametoxazole
resistance was common in all types of V.
parahaemolyticus isolates examined, followed
by rifampin and streptomycin. Human stools
isolates were found to be completely
susceptible to 5 of the examined
antimicrobials. The highest prevalence of
resistance among V. parahaemolyticus from
hospital wastewater isolates was also to
rifampicin (all of hospital wastewater isolates),
followed
by
sulfametoxazole
(95%),
kanamycin and erythromycin (75%). All
hospital wastewater isolates were found to be
susceptible to 7 of the antimicrobials tested. In
contrast, most of the hospital wastewater and
human stools isolates were sensitive to second
generation
cephalosporin
(ceftadizime,
ceftriaxone and cefuroxime). Overall the
highest prevalence of resistance in all of the 97
isolates tested was found to the antimicrobial
rifampin, followed by sulfametoxazole,
streptomycin and kanamycin. All 97 V.
parahaemolyticus isolates were found to be
susceptible to ciprofloxacin, norfloxacin and
ceftriaxone (Table 4.5). Overall, V.
parahaemolyticus isolated are resistant to
sulfametoxazole (100%) and rifampin (96%).
Norfloxacin was the most active agent against
V. parahaemolyticus (3% resistant isolates),
followed by ciprofloxacin (22% resistant
isolates) and chloramphenicol (25% resistant
isolates). V. parahaemolyticus isolated from
human stools still sensitive to ampicillin.
V. parahaemolyticus isolated from shellfish (F.
ater) were distributed in cluster C1 (VpFar 3.1
and VpFar 3.2), two isolates could be found in
cluster C2 (VpFar4.1 and VpFar6.1), two
isolates in cluster C5 (VpFar5.1 and
VpFar5.2), one isolates in cluster C6
(VpFar1.2) and two isolates in cluster C12.
V. parahaemolyticus isolated from C.
moltkiana were mainly distributed in cluster
C6, C11 and C12 (7 isolates). Two isolates
were in cluster C3 and C10. One isolates each
in cluster C4, cluster C5 and C7. Three
isolates were in cluster C8 and C9.
114
J. Ris. Kim.
Ten of twenty V. parahaemolyticus isolated
from hospital wastewater were distributed in
cluster C5, other isolates were distributed in
cluster C2 (5 isolates), C8 (2 isolates) and C1,
C6 and C7 (1 isolates each) respectively.
V. parahaemolyticus isolated from human
stools samples mainly could be found in
cluster C9 (6 isolates), 2 isolates in cluster C4,
C5 and C8 respectively and 3 isolates in cluster
C7.
Cluster C1 comprised of 2 isolates from F. ater
and one isolates from hospital wastewater were
resistant to ceftadizime, erythromycin,
tetracycline and sulfametoxazole and sensitive
to ampicillin, cefuroxime, ceftriaxone and
norfloxacin. Cluster C2 consisted of hospital
wastewater and F. ater were resistant only to
rifampin and sulfametoxazole.
All isolates in cluster C3 were resistant to
ampicillin, ceftadizime, cefuroxime, rifampin,
erythromycin, tetracycline, streptomycin and
sulfametoxazole. Cluster C4 include only
shellfish isolates showed resistance to
ampicillin, ciprofloxacin, chloramphenicol and
tetracycline. Isolates in cluster C5 displayed
resistance to smaller number of antibiotics (12
isolates in cluster C5 resistant only to 7
antibiotics).
The RAPD technique is mainly used to assess
the genetic relatedness and to discriminate very
closely related species/strains. Nine OPAR
primers were screened, but 7 of the primers did
not produce any polymorphic bands or did not
amplify clear products. Only clearly visible
bands with sizes between 250 to 1300-bp were
computed. All bands were polymorphic. From
nine RAPD primers previously evaluated, three
were selected because they produced more
polymorphic and reliable amplification
patterns. Cluster analysis was carried out using
the Unweighted Pair Group Method Using
Arithmetic Averages (UPGMA) provided by
RAPD Distance version 4.0 and was calculated
using the Jaccard's coefficient (Cruz and
Carneiro (2003) appraise the genetic variability
between and within the formed clusters.
According to the dendrogram (Figure 1), V.
parahaemolyticus isolates were divided into
two clusters and at a similarity level of 20%.
The isolates were subdivided into seven subclusters, namely group I, II, III, IV, V, VI and
VII.
ISSN : 1978-628X
J. Ris. Kim.
Discussion
All V. parahaemolyticus isolates displayed
resistance towards rifampin (94.6% for
Corbicula moltkiana; 100% for Batissa
violacae; 90% for Faunus ater; 100% for
hospital waste water and 100% stools samples.
This seems to be characteristic of the V.
parahaemolyticus isolates and not related to
clinical or shellfish since more than 90% of the
isolates had the same behavior. V.
parahaemolyticus
studied
behaved
as
susceptible to ampicillin, chloramphenicol and
tetracycline (29.4%) and cefuroxime (18.2%)
for stools isolates. This low number human
stools isolates resistance towards ampicillin
and tetracycline is very interesting. This is
because these antimicrobials were usually used
for diarrhea treatment in the hospital Padang,.
Therefore the result indicate that these
antibiotics are still effective for diarrhea
treatment in Padang hospital even though
many studies elsewhere had shown high
resistance toward these antibiotics (Tjaniadi et
al., 2003).
V. parahaemolyticus isolates from shellfish,
hospital wastewater and human stools in
Padang, Indonesia were tested for their
resistance to 15 antibiotics. All V.
parahaemolyticus isolates displayed resistance
towards rifampin (94.6% for Corbicula
moltkiana; 100% for Batissa violacae; 90% for
Faunus ater; 100% for hospital waste water
and 100% stools samples). This seems to be
characteristic of the V. parahaemolyticus
isolates and not related to clinical or shellfish
since more than 90% of the isolates had the
same behavior. These results did not agree
with Li et al., 1999, that V. parahaemolyticus
isolated from Hong Kong were susceptible to
this antibiotic.
With
a
few
exceptions,
the
V.
parahaemolyticus
studied
behaved
as
susceptible to ampicillin, chloramphenicol and
tetracycline (29.4%) and cefuroxime (18.2%)
for stools isolates. This low number of human
stools isolates resistant towards ampicillin and
tetracycline is very interesting. This is because
these antimicrobials were usually used for
diarrhea treatment in the hospital Padang.
Therefore the result indicate that these
ISSN : 1978-628X
Vol. 2, No. 2, September 2009
antibiotics are still effective for diarrhea
treatment in Padang hospital even though
many studies elsewhere had shown high
resistance toward these antibiotics (Tjaniadi et
al., 2003).
β-lactam antibiotics are widely used in the
clinical field. β-lactamases are the production
of the major resistance mechanism toward
these antibiotics in Gram negative bacteria
(Jacoby and Munos-price, 2005). Although
ampicillin susceptibility reached 100% in
cluster C8 and C9, this was true only for
human stools and shellfish (raw B. violacae
and cooked C. moltkiana) isolates.
In Indonesia, tetracycline and co-trimoxazole
had been the first line antibiotic to diarrhoeal
treatment, sulfamethoxazole was used as the
second
line in this situations where V.
parahaemolyticus is found resistant. In this
study, most of the isolates from Padang were
resistant to sulfametoxazole (100%) and 65%
isolates were resistance to tetracycline. These
findings do not agree with Tjaniadi et al.
(2003) who found that V. parahaemolyticus
isolated from Jakarta still susceptible to
trimethoprim-sulfamethoxazole. From these
results, it was evident that V. parahaemolyticus
appears to be increasingly resistant to
suflamethoxazole.
Comparing the resistance profile of V.
parahaemolyticus, 86.6% of
B. violacae
isolates showed resistance to ampicillin, and
93.3% of them were resistant to gentamycin,
while resistance was found in 88.8% of human
stools isolates to sulfametoxazole. Variety of
profiles, involving resistance to tetracycline
and chloramphenicol, together with other
antimicrobial agents have been described by
Radu et al. (2003) who detected multiple
resistance of Aeromonas in retail fish in
Malaysia, but correlation with drug resistance
was not shown. The presence of resistance to
these antibiotics in food and clinical samples
makes their use in either clinical therapy or on
farms a concern. The resistance to
chloramphenicol,
sulfamethoxazole
and
tetracycline has been reported to be acquired
and encoded by plasmids or transposons
(Casas et al., 2005).
The RAPD technique is mainly used to assess
the genetic relatedness and to discriminate very
115
Vol. 2, No. 2, September 2009
J. Ris. Kim.
closely related species/strains. The great
phenotypic and molecular diversity of Vibrio
including of V. parahaemolyticus isolated
from raw shellfish has been demonstrated in
other studies (Warner, 1999; Sarkar et al.,
2003; Lu et al., 2006). Because this method is
simple and sensitive, it can become an
important tool for the characterization of
strains of V. parahaemolyticus. In this study,
RAPD assay was used to type 73 isolates of V.
parahaemolyticus isolated from shellfish (B.
violacae, C. moltkiana and F. ater), hospital
waste water and human stools samples. DNA
fingerprinting and RAPD–PCR are powerful
methods that are often used in taxonomic and
epidemiological studies (Gomes et al., 1995).
One of the advantages in using the RAPD
technique over other genomic fingerprinting
methods is that unlike ribotyping and RFLP
(Restriction Fragment Length Polymorphism)
analyses, RAPD uses the entire genome
(Janssen et al., 1996).
raw C. moltkiana (VpCmr6.1) and VpCmr2.2.
(single isolate) which had the lowest similarity,
were different in the cluster traits. In group R1
there were nine isolates with different types of
characteristics; most of them had hospital
wastewater isolates (VpE6.1; VpE5.1, VpE7.2;
VpE6.2 and VpE8.1), but there were also
genotypes from shellfish (VpFar3.1 and
VpBvr1.2). Isolates from Human stools was
also located in this group (VpC1.1).
The variations in the banding patterns
indicated genomic variability among the
isolates were very high. Such a high variability
among the V. parahaemolyticus strains can be
explained by the fact that the RAPD method
derives information from the whole bacterial
genome where regions with higher variability
are present.
The third group (R3) were comprised of only
isolates from shellfish. The clustering among
isolates were closer indicating similarity in
their genetic background were higher. The
high similarity between C. moltkiana and B.
violacae clones in the cluster R3 was
somewhat unexpected, since the first was
isolated from Singkarak Lake and the second
from Maninjau Lake. This suggested that some
Maninjau strains of V. parahaemolyticus could
possibly be introduced into in Singkarak
waters or vice versa. According Sudheesh et
al. (2002) RAPD assay is very useful for
typing and differentiating environmental
marine vibrios, which are relatively difficult to
identify. The genetic variation observed in
these bacteria should be further probed
employing
latest
molecular
biological
approaches.
In most cases, the clusters were not in
agreement with the type’s samples traits,
sometimes not even with the origin or location
of the samples. For example, in cluster R1, V.
parahaemolyticus from raw Faunus ater
(VpFar3.1) in spite of its name did not come
from the same location and showed high
similarity to a V. parahaemolyticus isolate
from raw Batissa violacae (VpBvr1.1). The
genotypes of V. parahaemolyticus isolate from
116
In the second group (R2), fourteen isolates
could be found. Isolates originated from
Singkarak Lake shellfish samples (raw and
cooked C, moltkiana and raw B. violacae)
were belonged to this group. The other six
isolates were originated from human stools
sample from M. Jamil Hospital and wastewater
from the same hospital. Two isolates from
cooked C, moltkiana (VpCmp3.1) and human
stools sample (VpC8.1) showed the same
similarity level (56%).
ISSN : 1978-628X
J. Ris. Kim.
Vol. 2, No. 2, September 2009
Pearson correlation
ibu marlina
100
86.6
75.5
72
87.5
80.4
63.2
C12
100
85.3
100
59.9
85.3
Rl
S
K
Cn
Te
C
E
Rd
NA
Nor
Cip
Cro
Caz
Cxm
Amp
100
95
90
85
80
75
70
65
60
55
50
45
35
30
25
20
15
Similarity
40
ibu marlina
Cmr8.1
raw Corbicula moltkiana
Cmr9.1
raw Corbicula moltkiana
Cmp2.1
cooked Corbicula moltkiana
Cmr10.2
raw Corbicula moltkiana
C7.1
Human Stool
Far2.2
raw Faunus ater
Far4.2
raw Faunus ater
Cmr10.1
raw Corbicula moltkiana
Cmr2.2
raw Corbicula moltkiana
Cmr11.1
raw Corbicula moltkiana
Cmp2.2
cooked Corbicula moltkiana
Cmp4.1
cooked Corbicula moltkiana
Cmp5.1
cooked Corbicula moltkiana
Cmr3.2
raw Corbicula moltkiana
Bvp1.4
cooked Batissa violacae
Bvr3.2
raw Batissa violacae
Cmp1.1
cooked Corbicula moltkiana
Cmp7.1
cooked Corbicula moltkiana
Cmp9.2
cooked Corbicula moltkiana
Bvp1.1
cooked Batissa violacae
Cmp1.2
cooked Corbicula moltkiana
Cmr2.1
raw Corbicula moltkiana
Cmr4.1
raw Corbicula moltkiana
Bvr1.1
raw Batissa violacae
Bvr5.1
raw Batissa violacae
C11.2
Human Stool
C8.1
Human Stool
Bvr1.2
raw Batissa violacae
C12.1
Human Stool
C11.1
Human Stool
Bvr5.2
raw Batissa violacae
C9.1
Human Stool
Cmp5.2
cooked Corbicula moltkiana
Cmp8.2
cooked Corbicula moltkiana
Bvr4.1
raw Batissa violacae
Cmp6.1
cooked Corbicula moltkiana
C3.1
Human Stool
E1.2
Hospital waste water
E11.2
Hospital waste water
Cmp7.2
cooked Corbicula moltkiana
C6.2
Human Stool
Cmp8.1
cooked Corbicula moltkiana
Cmp6.2
cooked Corbicula moltkiana
C5.1
Human Stool
Bvp1.2
cooked Batissa violacae
C10.2
Human Stool
Bvr6.1
raw Batissa violacae
Bvr2.1
raw Batissa violacae
Bvr2.2
raw Batissa violacae
Cmr6.1
raw Corbicula moltkiana
C4.1
Human Stool
C6.1
Human Stool
E13.1
Hospital waste water
Cmp9.1
cooked Corbicula moltkiana
Cmr7.2
raw Corbicula moltkiana
Far1.2
raw Faunus ater
Cmr3.1
raw Corbicula moltkiana
Cmr5.2
raw Corbicula moltkiana
Cmr1.2
raw Corbicula moltkiana
Cmr6.2
raw Corbicula moltkiana
Cmp3.1
cooked Corbicula moltkiana
Cmr7.1
raw Corbicula moltkiana
E2.1
Hospital waste water
E10.1
Hospital waste water
E9.1
Hospital waste water
C1.1
Human Stool
C2.1
Human Stool
Far5.1
raw Faunus ater
Far5.2
raw Faunus ater
E12.1
Hospital waste water
E7.2
Hospital waste water
E8.2
Hospital waste water
Far1.1
raw Faunus ater
E6.1
Hospital waste water
E7.1
Hospital waste water
Cmr1.1
raw Corbicula moltkiana
E3.1
Hospital waste water
E5.2
Hospital waste water
E5.1
Hospital waste water
Far2.1
raw Faunus ater
Bvp1.3
cooked Batissa violacae
C10.1
Human Stool
C9.2
Human Stool
Bvr3.1
raw Batissa violacae
Cmr4.2
raw Corbicula moltkiana
Cmr10.3
raw Corbicula moltkiana
Cmr5.1
raw Corbicula moltkiana
E6.2
Hospital waste water
Far6.1
raw Faunus ater
E4.1
Hospital waste water
Far4.1
raw Faunus ater
E11.1
Hospital waste water
E2.2
Hospital waste water
E8.1
Hospital waste water
E1.1
Hospital waste water
Far3.2
raw Faunus ater
Far3.1
raw Faunus ater
79.1
100
53.1
68.4
86.6
C1
85.3
1
85.3
68.6
C1
85.3
45.3
100
87.5
0
82.7
80.4
73.2
87.3
67.2
100
86.6
40.6
60.1
81.5
C9
87.3
66.1
59.7
87.3
37.3
80.3
53.2
C8
85.3
77.6
62.4
53.9
73.9
47.4
C
33.1
73.9
56.5
7
70
53.3
85.3
44.3
71.7
C6
86.6
59.5
57.6
70
100
87.5
100
80.4
41.4
71.8
28.8
68.9
86.6
79.4
61.5
55.7
100
76.4
26.1
61.7
44.7
85.3
C5
76.8
24.1
99.3
98.7
98.3
C
22
C
46.7
11.4
C
2
31.2
4
99.3
70.7
59.5
1
98.7
97.3
98.3
28
C
Figure 1.1 Dendrogram showing the clustering of antibiotics patterns for V. parahaemolyticus isolates from
shellfish, hospital waste water and human stools using BioNumeric Software Version 4.6 (Applied Maths,
Kortrijk, Belgium), computed with Pearson correlation coefficient and agglomerative clustering with
UPGMA. Black box: resistant response, and white box: sensitive response
ISSN : 1978-628X
117
Vol. 2, No. 2, September 2009
J. Ris. Kim.
Figure 2. Dendrogram showing the clustering of RAPD patterns for V. parahaemolyticus isolates from
shellfish, hospital waste water and human stools using primer Gold Oligo PAR 3, Gold Oligo PAR 4 and
Gold Oligo PAR 8 using the RAPD Distance Software Version 4.0
REFERENCES
1. D. Ottaviani, I. Bacchiocchi, L. Masini, F.
Leoni, A. Carraturo, M. Giammarioli, and
G. Sbaraglia, Antimicrobial susceptibility
of potentially halophilic vibrios isolated
from seafood, International Journal of
Antimicrobial Agents 18: 135-140, (2001).
2. A. Cespedes, and E. Larson, Knowledge,
attitude and practices regarding antibiotic
use among Latinos in the United States:
Review and Recommendations, American
Journal of Infection Control 34: 495-502,
(2006).
3. M.
Lesmana,
D.
Subekti,
C.H.
Simanjuntak, P. Tjaniadi, J. R. Campbell,
and B. A. Ofoyo, Vibrio parahaemolyticus
associated with cholera-like diarrhea
118
among patients in North Jakarta,
Indonesia, Diagnostic Microbiology and
Infectious Disease, 39: 71-75, (2001).
4. S. Lu, B. Liu, B. Zhou, And R. E. Levin,
Incidence and Enumeration of Vibrio
parahaemolyticus in Shellfish from two
retail Sources and the Genetic Diversity of
isolates as Determined by RAPD-PCR
Analysis, Food Biotechnology, 20: 193209, (2006).
5. M. Nishibuchi, Vibrio parahaemolyticus.
In International handbook of foodborne
pathogens, ed. M.D. Milliots and J. W.
Bier, United States: Marcel Dekker, Inc. P,
2004, 237-252.
6. L. Poirel, M. R. Martinez, H. Mammeri, A.
Liard, and P. Nordmann, Origin of
Plasmid-Mediated Quinolone Resistance
ISSN : 1978-628X
J. Ris. Kim.
Determinant QnrA, Antimicrobial Agents
and Chemotherapy, 49: 3523-3525,
(2005).
7. S. Radu, N. Elhadi, Z. Hassan, G. Rusul, S.
Lihan, N. Fifadara, Yuherman and E.
Purwati, Characterization of Vibrio
vulnificus isolated from cockles (Anadara
granosa): antimicrobial resistance, plasmid
profiles and random amplification
of polymorphic DNA analysis, FEMS
Microbiology Letters, 165: 139–143,
(1998).
8. S. Radu, N. Ahmad, F. H. Ling, and A.
Reezal,
Prevalence
and
resistance
to antibiotics for Aeromonas species from
retail fish in Malaysia, International of
Journal Food Microbiology, 81: 261–266,
(2003).
9. B. Sarkar, N. R. Chowdhury, G. B. Nair,
M. Nishibuchi, S. Yamasaki, Y. Takeda, S.
K. Gupta, S. K. Bhattacharya, and
Ramamurthy, Molecular characterization
of Vibrio parahaemolyticus of similar
serovars isolated from sewage and clinical
cases of diarrhea in Calcutta, India,
World Journal of Microbiology and
Biotechnology, 19: 771-776, (2003).
ISSN : 1978-628X
Vol. 2, No. 2, September 2009
10. S. Schwarz, and E. Chaslus-Dancla, Use of
antimicrobials in veterinary medicine and
mechanisms of resistance, Veterinary
Residue, 32: 201–225, (2001).
11. H. Sörum, and T.M. L’Abèe-Lund,.
Antibiotic resistance in food-related
bacteria – a result of interfering with the
global web of bacterial genetics,
International
Journal
of
Food
Microbiology, 78: 43–56, (2002).
12. P. Tjaniadi, M. Lesmana, D. Subekti, N.
Machpud, S. Komalarini, W. Santoso,
C. H. Simanjuntak, N. Punjabi, J. R.
Campbell, W. K. Alexander, H. J.
Beecham, A. L. Corwin, and B. A. Oyofo,
Antimicrobial Resistance of Bacterial
Pathogens Associated with Diarrheal
Patients in Indonesia, American Journal
of Tropical Medicine and Hygiene,
68: 666-670, (2003).
13. X. Zhao, and D. Drlica, Restricting
the Selection of Antibiotic-Resistant
Mutants: A General Strategy Derived
from Fluoroquinolone Studies, Clinical
Infectious Diseases, 33: S147-S156,
(2001).
119