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Veterinary Microbiology 100 (2004) 247254

Detection of mutations in the gyrA gene and class I integron


from quinolone-resistant Salmonella enterica serovar
Choleraesuis isolates in Taiwan
Tzu-Ming Huang a,b , Yung-Fu Chang b , Chao-Fu Chang a,
b

a Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan


Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine,
Cornell University, Ithaca, NY 14850, USA

Received 11 November 2003; received in revised form 2 March 2004; accepted 4 March 2004

Abstract
The quinolone resistance-determining regions (QRDRs) of the gyrA gene of quinolone-resistant Salmonella enterica serovar
Choleraesuis isolates were sequenced. Four types of point mutation, Ser-83-to-Phe (TCC TTC), Ser-83-to-Tyr (TCC
TAC), Asp-87-to-Gly (GAC GGC), and Asp-87-to-Asn (GAC AAC), were found. PCR-RFLP and MAS-touch down
PCR were performed on fifty swine clinical isolates of S. enterica serovar Choleraesuis (NalR ) collected during 19972002. The
analysis indicated seven isolates with point mutations in codon 83, 13 with point mutations in codon 87, and 30 with double
mutations in both codons 83 and 87. The MICs of enrofloxacin of the isolates with a single mutation in codon 83 or 87 were
<2 g/ml, while the MICs of the isolates with double mutations in both codon 83 and 87 ranged from 2 to 64 g/ml. A class I
integron comprised of dhfr, orfF and aad2 was also identified in both human and swine S. enterica serovar Choleraesuis isolates.
These results indicate that PCR-RFLP and MAS-touchdown PCR assays can be used for surveillance of gyrA gene mutations,
which are important for fluoroquinolone resistance in Salmonella. Isolates with double mutations in gyrA codons 83 and 87 are the
major type of quinolone-resistant Salmonella isolated from swine in Taiwan. A surveillance system may be applied to the swine
industry to monitor the emergence of fluoroquinolone and/or multi-drug-resistant S. enterica serovar Choleraesuis in Taiwan.
2004 Elsevier B.V. All rights reserved.
Keywords: Salmonella enterica; Genetics; Taiwan

1. Introduction
Salmonellosis is an important zoonotic disease
of pigs. The two most common serovars causing salmonellosis in swine are S. enterica serovar
Corresponding author. Tel.: +886-2-2363-2333;
fax: +886-2-2363-2436.
E-mail address: cfchang@ntu.edu.tw (C.-F. Chang).

Choleraesuis and S. enterica serovar Typhimurium


(Schwartz, 1990, 1999). In the United States, more
than 90% of swine salmonellosis is attributed to infection by S. enterica serovar Choleraesuis (Schwartz,
1990, 1991, 1999). S. enterica serovar Choleraesuis
infection causes septicemia, enterocolitis, pneumonia
hepatitis, and occasionally meningitis, encephalitis,
and abortion in pigs (Baskerville and Dow, 1973;
Schwartz, 1999; Wilcock and Olander, 1977). S.

0378-1135/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2004.03.003

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T.-M. Huang et al. / Veterinary Microbiology 100 (2004) 247254

enterica serovar Typhimurium is the second most


frequently isolated serotype from diseased pigs. Infection in pigs is mostly asymptomatic but rarely
shows entercolitis (Schwartz, 1999) whereas human
infection is usually associated with bacteremia and
metastatic focal lesions (Cohen et al., 1987).
Integrons, which are genetic elements that lack direct or indirect repeat sequences at their ends, are
responsible for transposition of antibiotic resistance
within bacteria (Recchia and Hall, 1995). Four types
of integrons have been identified, but most clinical
isolates belong to class 1 (Naas et al., 1999; Recchia
and Hall, 1995, 1997). Class 1 integrase catalyzes
site-specific recombination between attI of the integron and a 59-base element of a mobile gene cassette
that contains various antibiotic-resistant genes and unknown open reading frames (Recchia and Hall, 1995,
1997). Integrons lack their own promoter but act as
natural expression vectors with a common promoter,
Pant , located in the conserved sequences upstream of
the inserted gene cassette (Recchia and Hall, 1995).
The presence of integrons in multi-drug-resistant
Salmonella isolates has been reported (Gebreyes and
Altier, 2002). However, the presence of integrons in
S. enterica serovar Choleraesuis isolates from Taiwan
has not been studied. Therefore, we investigated for the
presence of class 1 integron from S. enterica serovar
Choleraesuis isolates from Taiwan.
Quinolone-resistant S. enterica serovar Choleraesuis isolated from Taiwanese swine was identified in
our laboratory in 1993 (our unpublished data), and
fluoroquinolone-resistant S. enterica serovar Choleraesuis was first isolated in 1996 (Chang et al., 2002).
Increasing numbers of fluoroquinolone-resistant S. enterica serovar Choleraesuis isolates have been found
in pig farms in recent years (our unpublished data).
In Escherichia coli, topoisomerase II (DNA gyrase)
and IV are essential enzymes responsible for maintenance of topology within the bacterial cell (Piddock,
2002; Piddock et al., 1998). In most bacteria, acquired quinolone resistance is attributed to mutations
in the genes encoding DNA gyrase (GyrA, GyrB), or
DNA topoisomerase IV (ParC, ParE) (Giraud et al.,
1999; Hirose et al., 2002). Although quinolone resistance can involve a variety of different mechanisms,
mutation within gyrA resulting in amino acid substitutions in the GyrA subunit of DNA gyrase plays a
major role in quinolone resistance in Gram-negative

bacteria including Salmonella. High-level quinolone


resistance has been associated with single mutations in the quinolone resistance-determining region (QRDR) of gyrA in Salmonella, with the most
commonly described mutations being Ser-83-to-Phe
(TCC TTC), Asp-87-to-Gly (GAC GGC), and
Asp-87-to-Asn (GAC AAC) (Hakanen et al., 1999;
Hirose et al., 2002; Liebana et al., 2002; Reche et al.,
2002; Walker et al., 2001). In this study, we sequenced
the QRDR of gyrA of quinolone-resistant Salmonella
isolated from pigs. PCR-RFLP and MAS-touchdown
PCR methods were developed to detect these mutations and analyze the association between MICs and
the mutations in QRDR.

2. Materials and methods


2.1. Bacterial strains and antibiotic susceptibility
tests
Fifty S. enterica serovar Choleraesuis isolates from
several pig farms in Taiwan were collected from 1997
to 2002. These S. enterica serovar Choleraesuis isolates were isolated from diseased pigs without selection for antimicrobial resistance. Nine isolates from
human patients were collected from 1998 to 2002.
Six isolates were from Branch Office III, Center for
Disease Control, Taichung, Taiwan, and three isolates were from National Taiwan University Hospital,
Taipei, Taiwan. Disk diffusion tests (Oxoid, Ogdensburg, NY) were performed according to the NCCLS
guidelines for all isolates (NCCLS, 2002). The minimum inhibitory concentrations (MICs) for the isolates
were determined by the standard agar double dilution
procedure according to the NCCLS (2002) guidelines.
2.2. DNA preparation and PCR
All isolates were cultured overnight in LuriaBertani
broth at 37 C, bacteria from 0.5 ml medium were
pelleted, and resuspended in 200 l ddH2 O. After
boiling for 10 min, the suspension was chilled on
ice. Following centrifugation at 13,000 rpm for 3 min,
100 l supernatant was taken to a new tube and stored
at 20 C until use.
PCR-RFLP polymerase chain reaction (PCR)
was used to amplify the QRDR from gyrA. A

T.-M. Huang et al. / Veterinary Microbiology 100 (2004) 247254

313 bp DNA fragment was amplified with the primer


gyrAP1 (5 -taccgtcatagttatccacg a-3 ) and gyrAP2
(5 -gtactttacgccatgaacgt-3 ), which correspond to nucleotides 434454 and 142161 of the gyrase A
(gyrA) gene, based on the GenBank accession no.
X78977 (Griggs et al., 1996).
2.3. MAS-touchdown PCR
A multiplex allele-specific (MAS) PCR assay was
designed to detect mutations simultaneously in the
second base of Ser-83 and the first base mutation of
Asp-87 (Mokrousov et al., 2002a,b). The inner reverse
primer, gyrAP3 (5 -taccatccccacggcgattc-3 ) was selected to stop at the second base in the codon 83, and
the inner forward primer, gyrAP4 (5 -gccatacgaacga
tggtgtc-3 ) was selected to stop at the first base in the
codon 87. Any mismatch at the 3 end of the inner
primer would result in the absence of a respective amplified product. Touchdown PCR was performed as
follows: initial denaturation at 94 C for 5 min, followed by 10 cycles of 94 C for 30 s, 72 C for 30 s,
and 72 C for 1 min, the annealing temperature decreased 1 C per cycle until 62 C; followed by 25 cycles of 94 C for 30 s, 62 C for 30 s, and 72 C for
1 min; with a final elongation at 72 C for 15 min. The
amplified fragments were electrophoresed in standard
2% agarose gels and visualized under UV light as previously described (Chang et al., 1987, 1992a,b).

249

Maguire et al., 2001). The reaction consisted of denaturation of 94 C for 5 min, followed by an amplification protocol of 35 cycles of denaturation at 94 C for
30 s, annealing at 62 C for 1 min, and elongation at
72 C for 2 min, and then a final extension at 72 C for
10 min. The amplified fragment was electrophoresed
in a standard 1% agarose gel and visualized under UV
light. The PCR product was purified, ligated into pCR
2.1 and transferred into E. coli using the TA Original
cloning kit (Invitrogen, Groningen, The Netherlands).
The transformants were screened for the presence of
the required insert by the PCR described above and
subjected to sequencing.
2.5. Sequencing of PCR products and nucleotide
sequence accession number
DNA sequencing was done using an ABI model
377 automated nucleic acid sequencer at the Bioresource Center, Cornell University, New York. Homology searches were performed with NCBI, Blast
(Altschul et al., 1990). The nucleotide sequences
of class I integron of S. enterica serovar Choleraesuis have been assigned GenBank accession no.
AY551331.

3. Results

2.4. Integron detection by PCR

3.1. Sequences of quinolone resistance-determining


region of gyrA gene

Detection of class I integrons and the resistance genes located therein was performed from
all fifty isolates by polymerase chain reaction using primer IntF (5 -ggcatccaagcagcaaag-3 ) and IntR
(5 -aagcagacttgacctga-3 ) (Gebreyes and Altier, 2002;

The quinolone resistance-determining region of


gyrA gene (313 bp) was amplified and sequenced
from all 50 isolates. Fig. 1 shows the sequences of
QRDR in the gyrA gene. Four kinds of point mutation Ser-83-to-Tyr (TCC TAC), Ser-83-to-Phe

Fig. 1. Alignment of the gyrA nucleic acid sequence. There were four types of point mutation in QRDR, Ser-83-to-Phe (TCC TTC),
Ser-83-to-Tyr (TCC TAC), Asp-87-to-Gly (GAC GGC), and Asp-87-to-Asn (GAC AAC).

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T.-M. Huang et al. / Veterinary Microbiology 100 (2004) 247254

Fig. 2. (A) PCR-RFLP, digested by HinfI. Lanes 16 had mutation in codon 83. The sizes of DNA fragment were 201 and 111 bp. Lanes
79 were wild types. The sizes of DNA fragments were 111, 102, and 99 bp. (B) PCR-RFLP, digested by BanI. Lane 2 had mutation
in codon 87 (Asp-87-to-Asn, GAC AAC). The sizes of DNA fragment were 195 and 118 bp. Lanes 1 and 35 were wild types. (C)
MAS-touchdown PCR. Lanes 1 and 2 have point mutation in codon 83. Lanes 3 and 4 are wild types. Lanes 5 and 6 have point mutation
in codon 87. Lanes 7 and 8 have double mutations in both of codons 83 and 87. Lanes 9 and C were positive and negative control. The
three arrows indicate the size of PCR products are 313, 225, and 136 bp.

T.-M. Huang et al. / Veterinary Microbiology 100 (2004) 247254

251

Fig. 2. (Continued ).

(TCC TTC), Asp-87-to-Gly (GAC GGC), and


Asp-87-to-Asn (GAC AAC) were found.
3.2. Mutation detection by PCR-RFLP and
MAS-touchdown PCR
According to the sequencing data of QRDR,
a PCR-restriction fragment length polymorphism
(RFLP) assay and a multiplex allele-specifictouchdown PCR were developed to detect the mutation in the QRDR of gyrA gene. The results of QRDR
in the gyrA gene showed that there were two HinfI
cutting sites. After HinfI digestion, there were three
fragments sized 111, 102, and 99 bp, respectively.
Mutation at the first base of Ser-83 (TCC TTC
or TCC TAC) caused the loss of HinfI cutting
sites. The sizes of HinfI-digested PCR product of
QRDR of the S. enterica serovar Choleraesuis isolate with Ser-83-to-Phe or Ser-83-to-Tyr mutation
were 201 and 111 bp (Fig. 2B). The Asp-to-Gly
(GAC GGC) mutation at codon 87 created a BanI
cutting site. The sizes of BanI-digested PCR product of QRDR of the S. enterica serovar Choleraesuis

isolate with Asp-87-to-Gly mutation were 195 and


118 bp (Fig. 2C). The MAS-touchdown PCR was
used to detect mutations simultaneously in the second
base of codon 83 and the first base of codon 87. The
PCR products of the MAS-PCR of wild type S. enterica serovar Choleraesuis isolate were 313, 225, and
136 bp, respectively (Fig. 2A). The 313 bp fragment
was amplified by the primer gyrAP1 and gyrAP2 for
QRDR of gyrA gene. The 225 bp was amplified by
the primer gyrAP1 and gyrAP3 to detect the mutation
at the second base of codon 83. There was no PCR
product when the S. enterica serovar Choleraesuis
isolates had a Ser-83 mutation. The 136 bp was amplified by the primer gyrAP2 and gyrAP4 to detect
the mutation at the first base of codon 87. There
was no PCR product when the S. enterica serovar
Choleraesuis isolate had a mutation at the first base of
Asp-87 (Fig. 2A). PCR-RFLP and MAS-touchdown
PCR results indicated point mutation in Ser-83-to-Tyr
(TCC TAC) (7/50, 14%), Ser-83-to-Phe (TCC
TTC) (7/50, 14%), Ser-83-to-Tyr (TCC TAC)
and Asp-87-to-Gly (GAC GGC) (1/50, 2%),
Asp-87-to-Gly (GAC GGC) (11/50, 22%),

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T.-M. Huang et al. / Veterinary Microbiology 100 (2004) 247254

Table 1
Quinolone susceptibilities of 50 nalidixic acid-resistant Salmonella choleraesuis isolates according to the presence of the mutations at
codon 83 or 87
Mutation
Codon 83

Codon 87

Ser-83 Tyr
Ser-83 Tyr
w.t.
w.t.
Ser-83 Phe

w.t.
Asp-87
Asp-87
Asp-87
Asp-87

a
b
c

Number of
isolates

Gly
Gly
Asn
Asn

7
1
11
2
29

(14%)
(2%)
(22%)
(4%)
(58%)

MIC (g/mL)
NAa
>128
>128
>128
>128
>128

ENRb
<2
64
<1
<2
264c

Nalidixic acid.
Enrofloxacin.
MIC50 = 64 g/ml, MIC90 = 64 g/ml.

Asp-87-to-Asn (GAC AAC) (2/50, 4%) and


Ser-83-to-Phe (TCC TTC) and Asp-87-to-Asn
(GAC AAC) (29/50, 58%) (Table 1).
3.3. Quinolone susceptibility
Nalidixic acid and enrofloxacin susceptibility
of minimum inhibitory concentrations for S. enterica serovar Choleraesuis isolates was tested by
the standard agar double dilution procedure according to the NCCLS guidelines. The results are
presented in Table 1. The isolates showed resistance to nalidixic acid (>128 g/ml). The MIC of
enrofloxacin with mutation in Asp-87-to-Gly were
<1 g/ml (enrofloxacin sensitive), Ser-83-to-Tyr or
Asp-87-to-Asn were <2 g/ml (enrofloxacin sensitive), and Ser-83-to-Tyr and Asp-87-to-Gly were
64 g/ml (enrofloxacin resistant). The isolates with
mutations in both Ser-83-to-Phe and Asp-87-to-Asn
were also enrofloxacin resistant and the MIC values were ranging from 2 to 64 g/ml with MIC50 ,
64 g/ml, and MIC90 , 64 g/ml, respectively. These
findings indicate that single mutation at 83 or 87
codon in gyrA resulted in nalidixic acid resistance
whereas double mutation at both codons resulted in
enrofloxacin resistance.
3.4. Integron detection by PCR
The PCR product of primers IntF and IntR was
1.9 kb. This 1.9 kb integron existed not only in 15 out
of 50 swine isolates, but also in six out of nine human isolates. Class I integron of S. enterica serovar
Choleraesuis was highly homologous with Citrobac-

ter freundii plasmid pCTX-M3 (GenBank accession


no. AF550415). The class I integron contained three
genes, dhfr, orfF, and aadA2; the dhfr and aad2 genes
encode a sulfonamide and a streptomycin resistant protein, respectively.

4. Discussion
The emergence of quinolone and multi-drug-resistant
salmonellae was recently reported in Taiwan (Chiu
et al., 2002). Quinolone-resistant S. enterica serovar
Choleraesuis was found in human and swine isolates
in Taiwan (Chang et al., 2002; Chiu et al., 2002; Lee
et al., 2002). All mutations in the gyrA gene that are
known to confer quinolone resistance in E. coli reside
within the quinolone resistance-determining region of
the gene spanning codons 67106, which is located
in a helix-turn-helix structure close to the active site
tyrosine 122 (Piddock, 2002). The two residues of
E. coli and Salmonella most commonly associated
with quinolone resistance mutations are serine 83
and aspartate 87 (Levine et al., 1998; Piddock, 2002;
Piddock and de Jong, 1999).
Several methods are used to identify gyrA mutations including denaturing high-performance liquid
chromatography (DHPLC) (Eaves et al., 2002), a
lightCycler-based PCR-gyrA hybridization mutation
assay (GAMA) (Liebana et al., 2002; Walker et al.,
2001), or single-strand conformational polymorphism
(SSCP) (Eaves et al., 2002). However, these techniques require sophisticated and expensive equipment. Therefore, PCR-RFLP and MAS-touchdown
PCR were used to detect the gyrA mutation from S.

T.-M. Huang et al. / Veterinary Microbiology 100 (2004) 247254

enterica serovar Choleraesuis isolates. The QRDR


of the gyrA gene has two HinfI cutting sites, one is
at codon 83. PCR-RFLP with HinfI digestion was
used to detect mutation at codon 83 (Giraud et al.,
1999). Because Asp-87-Gly (GAC GGC) mutation created a BanI cutting site, PCR-RFLP with
BanI digestion was designed to detect Asp-87-Gly
mutation. When comparing single and double mutations at codons 83 and 87, all nalixidic acid resistant isolates had a substitution mutation at either
codon 83 or 87, however; only double mutations
could induce enrofloxacin resistance. The most common mutant, Ser-83-Phe and Asp-87-Asn double
mutation of S. enterica serovar Choleraesuis was enrofloxacin and ciprofloxacin resistant (Chiu et al.,
2002). In other S. enterica serovars, such as S. enterica serovar Typhi or S. enterica serovar Paratyphi A, double mutations at codons 83 and 87 contributed resistance of norfloxacin and enrofloxacin
(Hirose et al., 2002).
Mechanism of quinolone resistance in Salmonella
involving mutations in topoisomerase II and topoisomerase IV have been reported (Piddock, 2002).
However, the QRDR of gyrA gene plays a major role
in quinolone and fluoroquinolone resistance while
mutations of gyrB, parC and parE of Salmonella may
contribute to high-level resistance to fluoroquinolone
(Piddock, 2002). Therefore, the MIC value of the double mutation strains (Ser-83 Phe, Asp-87 Asn)
ranging from 2 to 64 mg/L in this study may due to
the mutation in gyrB, parE and/or parC, in additional
to the mutations in gyrA (Casin et al., 2003; Hsueh
et al., 2004). Multiple-drug-resistance may also be
due to the accumulation and export of a quinolone
across the S. enterica cell envelope (the efflux pump).
However, this was not found in S. enterica serovar
Choleraesuis isolates (Piddock, 2002). The QRDR
of the gyrA gene of nalidixic acid resistant S. enterica serovar Choleraesuis isolated from Taiwanese
swine showed point mutations located in codons 83
and 87, which were Ser-83-to-Phe, Ser-83-to-Tyr,
Asp-87-to-Gly, and Asp-87-to-Asn. These mutations
are consistent with the findings of a previous report
that showed substitutions of the gyrA gene at codons
83 and 87 of human and swine isolates in Taiwan
were Phe-83 and Asn-87, Phe-83 and Asp-87, Ser-83
and Asn-87, Ser-83 and Asp-87, Ser-83 and Gly-87,
Ser-83 and His-87 (Chiu et al., 2002).

253

In summary, one substitution mutation at codon


83 or 87 conferred resistance to quinolone while
double mutations at codons 83 and 87 were associated with resistance to fluoroquinolone in S. enterica serovar Choleraesuis isolates. PCR-RFLP and
MAS-touchdown PCR used in this study allowed a
rapid and sensitive way to identify gyrA mutations and
the presence of class I integrons in Salmonella isolates. These results warrant further study to determine
if human S. enterica serovar Choleraesuis nalidixic
resistant isolates are derived from animal sources and
what the role of using these antibiotics in animal
feeds has on the development of antibiotic resistance.

Acknowledgements
This work was supported by National Science
Council (NSC-89-2313-B-002-141), Taiwan.

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