RESEARCH ARTICLE

Characterization of antibiotic resistance in Vibrio spp. isolated from

farmed marine shrimps (Penaeus monodon)
Sirikorn Kitiyodom1, Sirintip Khemtong1, Janenuj Wongtavatchai2 & Rungtip Chuanchuen1
1

Department of Veterinary Public Health, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand; and 2Department of Veterinary
Medicine, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand

Received 19 August 2009; revised 29 November

2009; accepted 26 January 2010.
Final version published online 4 March 2010.
DOI:10.1111/j.1574-6941.2010.00846.x
Editor: Riks Laanbroek

Abstract
A total of 83 Vibrio isolates from farmed marine shrimps (Penaeus monodon) were
tested for the presence of class 1, 2 and 3 integrons, SXT constin and tetracycline
resistance-encoding genes. Mutations in the quinolone resistance-determining
regions (QRDRs) of the gyrA and parC genes were determined in fluoroquinoloneresistant Vibrio strains (n = 17). Five isolates were found to carry class 1 integrons,
of which only one contained the partial rumA gene in the variable region. All the
Vibrio strains were devoid of class 2 and 3 integrons. Seven isolates harbored SXT
constin. None of the Vibrio isolates were positive to the tet(K), tet(L), tet(M),
tet(O) and tet(S) genes. Ten fluoroquinolone-resistant Vibrio strains carried a
point mutation G-248-T in the gyrA QRDR, leading to a Ser-83-Ile substitution in
GyrA, but none of these strains had mutations in the QRDR of the parC gene.

MICROBIOLOGY ECOLOGY

Keywords
ICE; integrons; shrimps; Thailand; Vibrio.

Introduction
Over the past decade, shrimp farming has grown significantly in many parts of the world in response to increased
global-market demand. The fast-growing marine shrimp
culture industry has increased the need for intensive farming
practices to maximize production; however, the shrimp
culture industry has been beset by devastating diseases
mostly due to bacterial infection. Bacteria of the genus
Vibrio are ubiquitous in marine environments where
shrimps are farmed and occur naturally (Roque et al., 2001;
Vaseeharan & Ramasamy, 2003). Vibrio spp., for example
Vibrio parahaemolyticus, Vibrio cholerae, Vibrio fluvalis and
Vibrio vulnificus are generally considered opportunistic
pathogens, causing disease when shrimps are stressed, and
have been known to be one of the most frequent pathogens
contributing to major losses in marine shrimp culture. The
bacterium has also emerged as the causative agent of
foodborne, waterborne and zoonotic diseases (Vaseeharan
& Ramasamy, 2003; Sapkota et al., 2008).
Antimicrobial agents have been applied to the shrimp
feed and water in large quantities primarily to treat and
prevent diseases in farmed shrimps. Consequently, antimicrobial agents persist in sediment and aquatic environments,
leading to deteriorated environmental conditions and conFEMS Microbiol Ecol 72 (2010) 219227

ferring antimicrobial resistance to the sediment bacteria. Of

particular concern is the indiscriminate use of antibiotics
leading to the development of multiple-antibiotic-resistant
Vibrio spp. that have adversely affected antibiotic therapy of
Vibriosis in shrimps and humans (Zanetti et al., 2001). In
this case, shrimps could serve as delivery vehicles of antimicrobial resistance to Vibrio from aquatic environments to
humans and from one country to another.
Thailand has been one of the worlds largest culturedshrimp suppliers since 1991 and this aquaculture sector has
become a major contributor of export revenues (Rosenbery,
1995). However, shrimp farming in the country has faced
problems with Vibrio outbreaks (Dalsgaard et al., 1995).
Large portions of antibiotics have been used in farmed
shrimp production, of which the commonly used antibiotics
include tetracycline, fluoroquinolones and sulfonamides,
and antimicrobial-resistant Vibrio isolates have been isolated from farmed marine shrimps (Zanetti et al., 2001).
Despite the rapid expansion of the shrimp-farming sector,
only limited data are available on the molecular mechanisms
of antimicrobial resistance in Vibrio from farmed marine
shrimps.
The aim of this study was to characterize the genetic basis
underlying the antibiotic resistance phenotypes in Vibrio
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Correspondence: Rungtip Chuanchuen,

Department of Veterinary Public Health,
Faculty of Veterinary Science, Chulalongkorn
University, Bangkok 10330, Thailand. Tel.:
166 2 218 9578; fax: 166 2 218 9577;
e-mail: rchuanchuen@yahoo.com

220

spp. (i.e. V. parahaemolyticus, V. cholerae, V. fluvalis and

V. vulnificus) isolated from farmed marine shrimp, with particular attention to class 1, 2 and 3 integrons and SXT constin.
We also determined the presence of tetracycline-resistanceencoding genes and mutations in the quinolone resistancedetermining regions (QRDRs) of the gyrA and parC genes.

Materials and methods

Bacterial strains

Antimicrobial susceptibility testing

All of the Vibrio isolates were tested for minimum inhibitory
concentrations using a serial twofold agar dilution technique
in accordance with the Clinical and Laboratory Standards
Institute (CLSI, formerly NCCLS) (NCCLS, 2002). The
antibiotics and breakpoints used were as follows: ampicillin
(32 mg mL1), chloramphenicol (32 mg mL1), ciprofloxacin
(4 mg mL1), erythromycin (8 mg mL1), enrofloxacin
(4 mg mL1), kanamycin (64 mg mL1), streptomycin
(64 mg mL1), sulfamethoxazole (76 mg mL1), tetracycline
(16 mg mL1) and trimethoprim (4 mg mL1). All antibiotics
were purchased from Sigma-Aldrich (St. Louis, MO).

PCR amplification and DNA sequencing

The DNA template for PCR testing was prepared as
described previously (Ahmed et al., 2005). Amplification
reactions were carried out in a final volume of 25 mL using
PCR Master Mix (Fermentass, Mainz, Germany) according
to the manufacturers instructions. All the primers used in
this study are listed in Table 1.
Class 1 integrase (intI1) was detected using primers int1F
and int1R. All intI-positive strains were further tested for the
inserted-gene cassette region using the 5 0 -CS and 3 0 -CS
primer pair. The presence of intI2 and intI3 genes was
examined using the primer pairs int2F/int2R and int3F/
int3R, respectively.
The presence of SXT constin was investigated by detection
of the SXT integrase (intSXT) gene using primers int1SXT-F
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and int1SXT-R. All intSXT-positive isolates were examined

for the resistance genes typically associated with SXTMO10
and R391. The floR gene encoding for florphenicol resistance
was detected using primers FloR-F and FloR-R. Trimethoprim-resistance genes were determined using primers
dfrA1-F and dfrA1-R for dfrA1 and primers dfrA18-F and
dfrA18-R for dfrA18. The sulfonamide-resistance gene sulII
was tested using the primer pair sul2-F and sul2-R. Streptomycin-resistance genes were determined using primers strAF and strA-R for strA and primers strB-F and strB-R for strB.
The tetA gene encoding tetracycline resistance was detected
using primers tetA-F and tetA-R. The organization of the
resistance genes as shown in SXTMO10 was determined using
PCR amplification with various combinations of primers
TMP3/FloR-F, StrBF/Sul2R and sul2F/RUMA. The genes
clustered in the rumAB operon were amplified using primers
LEFTF3 and RUMA. PCR primers TMP3 and TMP4 were
used to define the extent of the region flanking dfrA18. The
regions around hot spots for insertion of additional DNA
sequences s073-traF, s043-traL and traA-s054 were analyzed
using the primer pairs YL6/TraF-R, HS1F/HS1R and
HotS2F/HotS2R, respectively. Three conserved regions traI,
traC and setR were amplified using the primer pairs orf171/
traI-R, traC-F/traC-B and setRpF/setRpR, respectively. The
unique features of R391 between the attL-xis and s026-s027
regions were analyzed using the primer pairs VisLF/VisLR3
and S026F/S027R.
The presence of tetracycline-resistance-encoding genes
tet(K), tet(L), tet(M), tet(O) and tet(S) was detected using
specific primer pairs. Campylobacter coli CAC041 was used
as a positive control for tetO (Ekkapobyotin et al., 2008).
The positive control strains for tet(K), tet(L), tet(M) and
tet(S) genes were Escherichia coli with the relevant gene
obtained previously (Bryan et al., 2004).
Mutations in the QRDRs of the gyrA and parC genes were
determined by PCR amplification and DNA sequencing.
The gyrA and parC genes were amplified using the primer
pairs VGYR-2/VGYR-4 and VGYR-3/VGYR-4, respectively.
Two Vibrio isolates susceptible to ciprocloxacin and enrofloxacin were included as controls.
Amplified DNA was purified from agarose gels using the
Nucleospin Gel extraction kit (Nucleospins, Gutenberg,
France) and submitted for sequencing at Macrogen Inc.
(Seoul, South Korea). The nucleotide sequence was analyzed
by comparisons with the gene sequences in the GenBank
database using BLAST software.

Results
Antibiotic resistance phenotypes
Among 83 Vibrio isolates tested, 89% were found to be
resistant to at least one antibiotic and 26% were
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A total of 83 Vibrio spp. comprising of 26 V. parahaemolyticus, 18 V. cholerae, 23 V. fluvalis and 16 V. vulnificus, were
obtained from a strain collection of the Department of
Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University. The isolates were collected from marine shrimps (Penaeus monodon) farmed in Thailand during
20012003. All the strains were isolated using the standard
method as described in the FDA Bacteriological Analytical
Manual (1998) (Elliot et al., 1998) and the species were
identified by API 20E (bioMe rieux, Craponne, France). All
of the bacterial strains were stored as 20% glycerol stocks at
 80 1C.

S. Kitiyodom et al.

221

Resistance in Vibrio spp. from shrimps

Table 1. Primers used in this study

Primer

Region

Sequence of primers (5 0 3 0 )

Reference

Int1F
Int1R
Int2F
Int2R
Int3F
Int3R
5 0 -CS
3 0 -CS
tetK-I
tetK-II
tetL-I
tetL-II
tetM-I
tetM-II
tetO-I
tetO-II
tetS-FW
tetS-RV
int1 SXT-F
int1 SXT-R
TMP3
TMP4
YL6
TraF-R
FloR-F
FloR-2
LEFTF3
RUMA
traC-F
traC-R
orf171
traI-R
setRpF
setRpR
VisLF
VisLR3
S026F
S027R
HS1F
HS1R
HotS2F
HotS2R
dfrA1-F
dfrA1-R
dfrA18-F
dfrA18-R
sul2-F
sul2-R
strA-F
strA-R
strB-F
strB-R
tetA-F
tetA-R
VGYR-2
VGYR-3
VGYR-4

intI1

CCTGCACGGTTCGAATG
TCGTTTGTTCGCCCAGC
GGCAGACAGTTGCAAGACAA
AAGCGATTTTCTGCGTGTTT
CCGGTTCAGTCTTTCCTCAA
GAGGCGTGTACTTGCCTCAT
GGCATCCAAGCAGCAAG
AAGCAGACTTGACCTGA
CAATACCTACGATATCTA
TTGAGCTGTCTTGGTTCA
TGGTCCTATCTTCTACTCATTC
TTCCGATTTCGGCAGTAC
GGTGAACATCATGACACGC
CTTGTTCGAGTTCCAATGC
AGCGTCAAAGGGGAATCACTATCC
CGGCGGGGTTGGCAAATA
ATCAAGATATTAAGGAC
TTCTCTATGTGGTAATC
GCTGGATAGGTTAAGGGCGG
CTCTATGGGCACTGTCCACATTG
CATGCTGTTTCTCGACGGTG
GATCCGATCTGTTTGTTCAG
TGTGGAACGGCTTTCTGACG
GGGGTTCTCATTGTCAGCTC
TTATCTCCCTGTCGTTCCAGCG
CCTATGAGCACACGGGGAGC
GGTGCCATCTCCTCCAAAGTGC
CGAGCAATCCCCACATCAAG
TTGACGTGTTTTCACCAACG
GGCACGACCTTTTTTCTCCC
GCAAGTCCTGATCCGCTATC
CCAGGCATCTCATATGCGT
ACGGCGGAGATGTTTTTGT
GTGCGCCAATGCTCAGTT
GAGTACAAATTCCGTTTTAG
GCATTCTCCTGAAAATCAATG
GAGCAATGGGCGAGAGTTCC
TCAGCGACAACCGGAGAATG
GGCTATTCCACCGGTGGTG
TGCCGATCACTAGCCCCAAC
TTCCAGCAATCAGCGCCG
CAGTTGTCCTATGTGGACTCGG
CAATGGCTGTTGGTTGGAC
CCGGCTCGATGTCTATTGT
TGGGTAAGACACTAGTCATGGG
ACTGCCGTTTTCGATAATGTGG
GCGCAGGCGCGTAAGCTGAT
CGAAGCGCAGCCGCAATTC
TGGCAGGAGGAACAGGAGG
AGGTCGATCAGACCCGTGC
GCGGACACCTTTTCCAGCCT
TCCGCCATCTGTGCAATGCG
GCTGTCGGATCGTTTCGG
CATTCCGAGCATGAGTGCC
AA(C/T)GA(C/T)TGAA(C/T)AA(A/G)(G/C)C
AA(A/G)TA(C/T)CA(C/T)CC(A/C/G/T)CA(C/T)G
TC(A/C/G/T)GT(A/G)TA(A/C/G/T)C(G/T)CAT((A/C/G/T)GC

Chuanchuen et al. (2007)

intI2
intI3
Variable region
tet(K)
tet(L)
tet(M)
tet(O)

intSXT
tnpB-s013
s073-traF
floR
rumBA
traC
traI
setR
attL-xis
s026-s027
s043-traL
traA-s054
dfrA1
dfrA18
sul2
strA
strB
tetA
gyrA
parC

Ekkapobyotin et al. (2008)

Levesque et al. (1995)
Klare et al. (2007)
Werner et al. (2003)
Werner et al. (2003)
Klare et al. (2007)
Charpentier et al. (1993); Gevers et al. (2003)
Hochhut et al. (2001b)
Hochhut et al. (2001b)
Hochhut et al. (2001b)
Iwanaga et al. (2004)
Hochhut et al. (2000)
Burrus et al. (2006)
Hochhut et al. (2001b)
Bani et al. (2007)
Bani et al. (2007)
Bani et al. (2007)
Burrus & Waldor (2004); Hochhut et al. (2001b)
Bani et al. (2007)
Toma et al. (2005)
Burrus et al. (2006)
Chuanchuen et al. (2008)
Hochhut et al. (2001b)
Chuanchuen & Padungtod (2009)
Chuanchuen & Padungtod (2009)
Chuanchuen & Padungtod (2009)
Chuanchuen et al. (2008)
Okuda et al. (1999)
Okuda et al. (1999)

Used in combination with primer VGYR-4.

FEMS Microbiol Ecol 72 (2010) 219227

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tet(S)

Ekkapobyotin et al. (2008)

222

S. Kitiyodom et al.

multiresistant (resistant to at least three different classes of

antibiotics). The resistance rates to ampicillin, ciprofloxacin,
enrofloxacin, erythromycin, streptomycin, sulfamethoxazole, tetracycline and trimethoprim were 61%, 20%, 14%,
42%, 7%, 28%, 19% and 20%, respectively. All of the Vibrio
isolates were susceptible to chloramphenicol and kanamycin. Analysis of the antimicrobial resistance profiles revealed
28 resistance patterns, of which the most frequent resistance
pattern was AMP (22%) (Table 2).

Class 1, 2 and 3 integrons

All the Vibrio strains were screened for the presence of class
1, 2 and 3 integrons. While none of the isolates were positive
to the intl2 and intl3 genes, five isolates i.e. V. cholerae (one)
and V. fluvalis (four) were found to harbor the intl1 gene
(Table 2). Out of the five intl1-positive strains, only the
V. cholerae isolate yielded an 500-bp CS-PCR product,
which, upon sequence analysis, revealed the presence of the

Table 2. Phenotypes and genotypes of the antimicrobial-resistant Vibrio isolates (n = 83)

Strain

Resistance pattern

Class 1 integrons

ICE

GyrA mutationw

V. cholerae

Vch16
Vch32
Vch33
Vch45
Vch76
Vch77
Vch78
Vch106
Vch125
Vch127
Vch143
Vch157
Vch179
Vch197
Vfl1
Vfl3
Vfl6
Vfl7
Vfl11
Vfl13
Vfl21
Vfl49
Vfl57
Vfl63
Vfl72
Vfl73
Vfl75
Vfl81
Vfl132
Vfl153
Vfl159
Vfl164
Vfl186
Vfl192
Vfl286
Vpa14
Vpa19
Vpa58
Vpa59
Vpa62
Vpa64
Vpa83
Vpa94
Vpa103
Vpa104
Vpa177

AMP
AMP, SUL, TMP
SUL
CIP, ENR, TMP
AMP
AMP, CIP, ERY, ENR
ERY
AMP, ERY, SUL, TMP
AMP, SUL, TET
AMP, ERY, SUL
STR, SUL, TMP
AMP, CIP
AMP
ERY, STR, SUL
AMP, CIP, ENR, ERY, STR, SUL, TET, TMP
AMP, CIP, ENR, ERY, SUL, TET, TMP
AMP, ERY, SUL, TET, TMP
AMP, ERY
AMP
ERY
AMP, SUL, TMP
AMP
AMP, ERY
AMP, SUL, TMP
AMP, ERY, TET
AMP
AMP, ERY
AMP, TET
CIP, ERY, SUL, TET, TMP
AMP
CIP, ENR, ERY
CIP, ERY, STR
TET
AMP
ERY
AMP
AMP, ERY, STR, SUL, TET
AMP, ERY, SUL, TMP
AMP
AMP, ERY
AMP
AMP, TET
AMP, ERY
ERY
AMP
AMP



1















1


1


1
1
























1(ICEVchTha2)




1(ICEVchTha3)

1(ICEVchTha1)







1(ICEVflTha1)


1(ICEVflTha2)














1(ICEVpaTha1)















1


























1
1













V. fluvalis

V. parahaemolyticus

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Species

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Resistance in Vibrio spp. from shrimps

Table 2. Continued.
Strain

Resistance pattern

Class 1 integrons

ICE

GyrA mutationw

V. vulnificus

Vpa178
Vpa184
Vpa191
Vpa234
Vpa235
Vpa237
Vpa245
Vpa250
Vpa251
Vpa265
Vpa270
Vpa279
Vpa281
Vpa282
Vvu5
Vvu17
Vvu25
Vvu34
Vvu43
Vvu44
Vvu46
Vvu61
Vvu67
Vvu109
Vvu183
Vvu217
Vvu223
Vvu267
Vvu268

AMP, CIP, STR

AMP, TET
ERY
ERY
SUL
ERY
AMP, STR, SUL, TET, TMP
SUL
CIP, ERY
AMP
ERY
AMP, CIP, STR
AMP, CIP
AMP, CIP, ERY, ENR, SUL, TMP
AMP, ERY, SUL, TMP
AMP, CIP, ERY, ENR, SUL, TMP
AMP, CIP, ENR
ERY
ERY, TET
AMP, CIP, ENR, TET
AMP
AMP
AMP, CIP, ERY, SUL, TMP
AMP, ERY
TET
TET
SUL, TMP
AMP, ERY
AMP



























































1(ICEVvuTha1)



1







1



1
1

1
1



1







Positive for the ICE (name of the ICE found). (ICE, integrating conjugative element.)
w

Positive for a point mutation G-248-T.

AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; ERY, erythromycin; ENR, enrofloxacin; KAN, kanamycin; STR, streptomycin; SMX,
sulfamethoxazole; TET, tetracycline; TRI, trimethoprim.

partial rumA gene. The other integrons were empty integrons with 5 0 and 3 0 conserved segments, but without gene
cassettes.

Analysis of the integrating conjugative

elements (ICEs)
Of the 83 Vibrio strains, seven Vibrio isolates including three
V. cholerae, two V. fluvalis and one isolate of each V. vulnificus
and V. parahaemolyticus were positive for the intSXT integrase
gene. The similarity of the ICEs in these isolates to the SXT/
R391 family elements was assessed (Table 2 and Fig. 1). The
ICEs named ICEVflTha1, ICEVchTha1 and ICEVpaTha1,
found in V. fluvalis Vfl1, V. cholerae Vch143 and V. parahaemolyticus Vpa19, carried the resistance genes typically associated with the SXTMO10 or R391 including the genes
encoding resistance to florphenicol (floR), streptomycin (strA
and strB) and sulfonamides (sul2). Because PCR amplification
of the rumAB operon in these strains was not successful, the
organization of the resistance genes was determined instead.
FEMS Microbiol Ecol 72 (2010) 219227

PCR products obtained with primer sets TMP3/FloR-F, StrBF/

Sul2R and sul2F/RUMA were 2800 bp, 2300 bp and 3900 bp,
respectively, and confirmed the genetic organization floR-strBstrA-sul2 as shown in the SXTMO10. All these three ICEs were
negative to the dfrA18 gene reported for SXTMO10. Therefore,
PCR with primers TMP3-TMP4 was performed and a 1300-bp
amplicon consisting of tnpB3 and s013 was obtained. For
analysis of three hot spots, DNA sequencing revealed the
presence of orf37-orf38 (840 bp) that separated s043 and traL
in ICEVchTha1, ICEVflTha1 and ICEVpaTha1. In
ICEVflTha1, the intergenic region from traA to s054 contained
s052-s053 that is present in SXTMO10, but the same conserved
regions in ICEVchTha1 and ICEVpaTha1 harbored a 1049 bp
putative gene of an ORF encoding a hypothetical protein.
Four ICEs, designated ICEVchTha2, ICEVflTha2,
ICEVchTha3 and ICEVvuTha1, identified in V. cholerae
V45, V. fluvalis V49, V. cholerae V125 and V. vulnificus
V268, failed to yield a PCR amplicon with the primers
designed to detect the resistance genes floR, strA, strB, sul2,
dfrA1 and dfrA18. PCR amplification of the rumAB operon
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Species

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S. Kitiyodom et al.

orf 45-orf55

(a)

merRTPCA

R391
Common
scaffold

intl

rumBA

s026-s027

traI

s043-traL traA-s054

traC

s073-traF

setR

SXTMO10

(b)
NI

ICEVf l Tha1
(Vfl1)

s026-s027

ICEVpaTha1
(Vpa19)

s026-s027

Unknown

ICEVchTha1
(Vch143)

s026-s027

Unknown

NI
NI

rumBA

s026-s027
s026 s027

ICEVf l Tha2
(Vfl49)

rumBA

s026-s027

ICEVchTha3
(Vch125)

rumBA

ICEVvuTha1
(Vvu268)

rumBA

NI
NI

NA

NI

NA

NI

Fig. 1. Genetic organization of SXTMO10 and R391 and seven new ICEs. (a) Linear map (not to scale) of the SXT/R391 common scaffold (the gray
arrows). Specific insertions for R391 (the black arrow) and SXTMO10 (the white arrow) are shown above and below the common scaffold, respectively. (b)
Linear maps of the seven ICEs identified in Vibrio isolates from farmed marine shrimps in the present study. The dashed line in SXTMO10 indicates the
region deleted in ICEVflTha1, ICEVchTha1 and ICEVpaTha1. The black triangles indicate the three hot spots. NA, not amplified; NI, no insertion.

revealed an 800-bp amplicon in all four ICEs, suggesting

the integrity of the rumAB operon. The gene cluster s052s053 was detected in the traA-s054 intergenic regions of
ICEVchTha2, ICEVflTha2 and ICEVchTha3. In ICEVvuTha1, orf45-orf46 (763 bp) and orf37-orf38 were found in
the traA-s054 and s043-traL intergenic region, respectively.
No insertion of additional DNA sequence was detected in
the intergenic region from s073 to traF in all the ICEs
identified in this study. All these seven ICEs were positive
to three conserved genes traI, traC and setR. PCR amplification of the attL-xis region revealed the 450 bp in all ICEs,
except ICEVvuTha1. All ICEs, except ICEVchTha3 and
ICEVvuTha1, yielded an 400-bp amplicon with PCR
amplification of the s026-s027 region. ICEVvuTha1 yielded
an 2300-bp product with PCR amplification of the attL-xis
region.

Tetracycline-resistance genes and mutations in

gyrA and parC
All the Vibrio isolates were determined for the presence of
the tet(K), tet(L), tet(M), tet(O) and tet(S) genes and none of
them were found to contain the tet genes tested.
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All the Vibrio isolates resistant to ciprofloxacin and/or

enrofloxacin were examined for mutation(s) within the
QRDRs of gyrA and parC genes. From DNA sequence
analysis, 10 of these 17 strains (i.e. four V. parahaemolyticus,
three V. vulnificus, two V. fluvalis and a V. cholerae strain)
carried a point mutation G-248-T in the QRDR of gyrA,
leading to a Ser-83-Ile substitution in GyrA. Nine Vibrio
isolates with mutations in gyrA additionally harbored a
silent mutation C-363-T in the same gene. None of the
strains had mutations in the QRDR of the parC gene. No
mutations were detected in the QRDRs of gyrA and parC of
the fluoroquinolone-sensitive controls.

Discussion
One of the main findings in this study was the wide spread
of antimicrobial resistance among Vibrio spp. isolated from
farmed marine shrimps. Resistance to ampicillin and erythromycin was prevalent, which was in agreement with the
previous studies (Roque et al., 2001; Akinbowale et al.,
2006). This is not surprising because both antibiotics are
naturally produced and dispersed in the environment, and
thus readily select for the resistance determinants or resistant bacterial strains (Rosser & Young, 1999; Bani et al.,
FEMS Microbiol Ecol 72 (2010) 219227

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ICEVchTha2
(Vch45)

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FEMS Microbiol Ecol 72 (2010) 219227

Taviani et al., 2008). Because the antibiotic resistance genes

in the ICEs were different and not always present, it suggests
that these resistance genes are not intrinsic components of
the SXT-related ICEs (Hochhut et al., 2001b). Instead, they
are transmissible and could be inserted themselves and
maintained in bacterial populations upon selective pressure.
The presence of antibiotic resistance genes detected in the
ICEs in this study did not support the total resistance
phenotypes observed and all four strains carrying the intact
rumAB operon were resistant to antibiotics, indicating the
existence of non-SXT-related resistance mechanisms.
Further studies are required to elucidate such resistance
mechanisms. Moreover, the inserted sequences in the three
hotspots among the ICEs in this study varied and insertion
between s073-traF in all ICEs was absent, which was
consistent with a previous study in environmental isolates
(Taviani et al., 2008). Variations of sequences inserted into
the antibiotic resistance gene clusters and the hotspots may
occur as a result of adaptation to environmental changes
(Iwanaga et al., 2004).
Some traits of ICEVvuTha1 appeared to be more closely
related to R391 than SXTMO10. These ICEs yielded the
2300-bp attL-xis fragment and contained the cluster of
orf37-orf38 genes in the s043-traL region corresponding to
R391 (Burrus et al., 2006). The cluster of orf45-orf46 genes
that was part of the inserted sequences between traA-s054 in
R391 is present in the same region of ICEVvuTha1. Loss of
the other genes in the same cluster in ICEVvuTha1 might be
a result of deletion during genetic recombination. The
R391-kanamycin resistance-encoding gene carried by a
transposon is usually inserted between s026 and s027 in
R391, but is absent in ICEVvuTha1. This feature has been
observed previously in ICEVchMex1 from a human isolate
of V. cholerae (Burrus et al., 2006). In addition, the orf37orf38 sequence was identified in the s043-traL region of
ICEVflTha1, ICEVpaTha1 and ICEVchTha1, and this was
demonstrated previously in SXTLAOS and R391 (Iwanaga
et al., 2004). The presence of sequences belonging to both
SXTMO10 and R391 in one ICE suggested that they may be
the recombinant ICEs (Hochhut et al., 2001a; Burrus &
Waldor, 2004) and supported the idea that SXTMO10 and
R391 are descended from the same ancestor that was
genetically reorganized and acquired different inserted
sequences (Burrus & Waldor, 2004; Taviani et al., 2008).
Several Vibrio strains were resistant to tetracycline, but
none of the five tetracycline resistance genes [tet(K-S)] were
detected, indicating the presence of other tetracycline resistance mechanisms. Several tet genes [e.g. tet(A), tet(B) and
tet(D) genes encoding active efflux pumps] have been
identified previously in Vibrio spp. from a maricultural
environment (Dang et al., 2006, 2007). Further studies are
required to elucidate the mechanisms underlying tetracycline resistance in the isolates in our collection.
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2007). The resistance rate to tetracycline is rather high,

which was consistent with former reports (Tendencia & de
la Pena, 2001; Vaseeharan et al., 2005). Antibiotics in this
family, particularly oxytetracycline, are commonly used in
shrimp farming and this could be an explanation the
resistance dissemination observed. The enrofloxacin-resistance rate is higher than that reported previously in Australia and Mexico (Roque et al., 2001; Akinbowale et al.,
2006), but much less than that observed in the isolates from
India (Vaseeharan et al., 2005). Such differences in the
resistance phenotypes are likely due to differences in antimicrobial use in different geographical areas.
Only one Vibrio isolate harbored class 1 integrons containing an incomplete rumA gene cassette that could not
confer any benefits to the host. The rumAB operon resides in
SXT elements in Vibrio spp., but it is unclear how this partial
rumA gene was inserted into the integrons. It is possible that
this partial gene may have been captured through an
aberrant recombination event during gene transfer (Yu
et al., 2003). Another four class 1 integrons were found to
lack gene cassettes integrated into the variable regions. Such
empty integrons have been identified previously in bacteria
isolated from marine aquaculture sites (Rosser & Young,
1999; Schmidt et al., 2001). It has been suggested that the
empty integrons may be integrons that have not acquired
gene cassette inserts or that gene cassettes may be excised
from the insert region in the absence of sustained antimicrobial pressures (Schmidt et al., 2001); regardless, their empty
variable regions are available for integration of the new
emerging gene cassette(s). In addition, none of the Vibrio
strains in this study were positive to class 2 and 3 integrons.
To date, class 2 integrons have been identified only in
V. cholerae isolates (Ahmed et al., 2006; Opintan et al., 2008),
but class 3 integrons have never been found in Vibrio spp.
PCR and sequencing analysis of the SXT constin revealed
that seven ICEs identified in this study are different from
SXTMO10. PCR with primers TMP3 and TMP4 in
ICEVflTha1, ICEVpaTha1 and ICEVchTha1 yielded an
1300-bp amplicon, suggesting the deletion of sequences
that flank dfrA18 as reported previously in SXTLAOS
(Iwanaga et al., 2004). The other four ICEs i.e. ICEVchTha2,
ICEVflTha2, ICEVchTha3 and ICEVvuTha1, were characterized by the absence of the antibiotic resistance genes
typically inserted into the rumAB operon of SXTMO10. ICEs
devoid of antibiotic resistance genes have already been
found in human and environmental Vibrio spp. (Taviani
et al., 2008). These empty SXT constins are available for
acquisition of not only antibiotic resistance determinants
but also virulent genes that could be transferred to other
strains (Iwanaga et al., 2004). It has been suggested that the
circulation and maintenance of ICEs with no antibiotic
resistance determinants may confer advantages unrelated to
antibiotic resistance to their hosts (Burrus et al., 2006;

226

Acknowledgements
This work was supported by the MAG Window II grant,
MRG-WII515S054, cofunded by the Thailand Research
Fund and the Chulalongkorn University (through the 90th
anniversary of Chulalongkorn University fund). It was
supported in part by Chulalongkorn University-Veterinary
Science Research Fund RG20/2552.

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evaluate the risk of transfer of resistant bacteria/genes to
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S. Kitiyodom et al.

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