Title: Detection of antibiograms, calss I integron and antibiotic resistance genes for Salmonella
Choleraesuis strains isolated from swine in southern Taiwan
JEN-CHIEH PANG1, TSAI-HSIN CHIU1, WEN-ZHE HWANG1, MING-HUEI LIAO2, GAN-NAN CHANG2
AND HAU-YANG TSEN1*.
1
2
*
Department of Food Science, National Chung-Hsing University, Taichung, Taiwan, R.O.C.
Department of Veterinary Medicine, National Ping-tung University, Ping-tung, Taiwan, R.O.C.
Corresponding author:
Hau-Yang Tsen, Professor
Department of Food Science,
National Chung-Hsing University,
Taichung, Taiwan, R.O.C.
Abstract
Antibiotic susceptibilities for 95 Salmonella Choleraesuis strains collected from the swine field in Southern
Taiwan were analyzed. A total of 17 antibiotics were tested. Results showed that 43 antibiograms were obtained.
All these 95 strains were multidrug resistant strains. PCR was used to screen the genes encode for the resistance
to ampicillin (PSE group), streptomycin-spectinomycin (aadA), kanamycin (aphA), sulfadiazine (sulI),
trimethoprim (dfrA12), chloramphenicol (cat, cmlA) and the presence of class I integron. Results showed that
32.6% of the 95 strains of Salmonella Choleraesuis have the class I integron. The antibiotic resistance genes in
integron cassette were further analyzed by PCR mapping. Five types of patterns including dfrA12-aadA2,
oxa1-aadA, dfrA12-aad2-su1l, pse and dfrA12 were found. Our data demonstrated that the problem of antibiotic
resistance for Salmonella Choleraesuis need to be paid with attention, since this Salmonella species has recently
emerged as the major species for swine infection in Taiwan. .
Key words: Salmonella Choleraesuis, integron, antibiogram.
INTRODUCTION
Antibiotics have been used in the therapeutic, subtherapeuitic and growth promotion for domestic animals, such
as swine, in many countries (Aarestrup, et al., 2001). Many antibiotics used for human are also used as feed
additives in Taiwan, for example, avoparcin, virginiamycin, kanamycin and enrofloxacin, that analog to
vancomycin, kanamycin, gentamicin ciprofloxacin, ofloxacin, quinupristin, and dalfopristin. Certain antibiotics
are critical for human use because there is no other drug available for the treatment of human infected by
multi-drug resistant pathogens, or because alternative therapies are less effective or are associated with increased
side effects. There are now an increasing number of reports suggesting that critical forms of antibiotic resistance
arise in human pathogens as a result of using certain antibiotics in food-producing animals, and that such
resistance can be transmitted to humans via the food chain (Molbak, et al., 1999; Smith, et al., 1999; Stobberingh,
et al., 1999; Soltani, et al., 2000; McDonald, et al., 2001). In 2000, the Council of Agriculture in Taiwan
prohibited the use of several antimicrobial agents, such as avoparcin, kanamycin, kitasamycin, lasalocid,
spiramycin, salinomycin, and streptomycin, which had been widely used as growth promoters or prophylactic
agents in animal husbandry in Taiwan during the past 2 to 3 decades (Hsueh, et al., 2002). In Taiwan, recent
reports have shown the widespread emergence of reduced-susceptibility and full-resistance to fluoroquinolones
among human isolates of Salmonella spp (Ho, et al., 1999; Chiu, et al., 2002). In recent years, S. Choleraesuis has
become the most frequent isolated Salmonella serotype from swine. The hosts of this Salmonella spp. could be
swine and humans (Bäumler, et al., 1998). Thus, regular surveillance of S. Choleraesuis in domestic animals
could be helpful for human health.
An efficient route of acquisition and vertical and horizontal dissemination of resistance determination is
through mobile elements including plasmids, transposons, and gene cassettes in integrons (Stokes, et al., 1989;
Recchia, et al., 1995). Recent advances in molecular characterization of antibiotic resistance mechanisms
highlighted the existence of integrons (Hall, et al., 1995), those integrons contain many different antibiotics gene
cassettes (Mazel, et al., 1998; Fluit, et al., 1999). A class 1 integron has the following formal arrangement
(Bennett, et al., 1999):
IntI attI (r59b)n qacEΔ1 sul1 orf5 ofr6.
Where r59b represents a resistance gene cassette and n indicates the number of gene cassettes integrated at the
locus. The set of gene cassettes are currently known to accommodate genes that confer the resistance of many
antibiotics.
To elucidate the relationship of antibiotics resistance between the phenotypes and the genotypes, in this study,
we assayed the antibiotics resistance susceptibility for S. Choleraesuis isolates from swine fields in Southern
Taiwan and used the PCR to detect their the resistance genes.
MATERIAL AND METHODS
Strains
Salmonella Choleraesuis strains were obtained from swine fields in southern Taiwan. They were isolated
during the period of 2000 to 2002. 67 of these 95 strains were isolated from diarrhea swine and 28 isolated strains
from systemic infection swine by the Department of Veterinary Medicine, National Pingtung University,
Pingtung, Taiwan. Reference strains that have been identified to have integrons map were obtained from the
National Laboratory of Food and Drug, Department of Health, Taipei. Those strains were S. Typhimurium U302
from chicken, S. Typhimurium DT008 from egg yolk, S. Typhimurium from beef, S. Schwarzengrund from
chicken. All the isolated strains were grown on Tryptic Soy broth (TSB) and were stored frozen at -80℃ for
future transfer.
Antibiotic susceptibility testing
The 95 of S. Choleraesuis isolates were subcultured onto Mueller-Hinton agar (BD) for disk diffusion.
Antibiotic susceptibilities of these isolates were determined according to the guidelines of National Committee
for Clinical Laboratory Standards (NCCLS). 17 of antimicrobial agents were used. The antibiotic and its conc.
per disc were contained ampicillin (10μg), amoxicillin-clavulanic acid (20 and 10μg), gentamicin (10μg),
cephalothin (30 μ g), chloramphenicol (30 μ g), erythromycin (15 μ g), tetracycline (30 μ g),
sulfamethoxaeole-trimethopmin (1.25 and 23.75μg), norfloxacin (10μg), ciprofloxacin (5μg), ofloxacin (5μ
g), nalidixic acid (30μg), sulfonamides (300μg), strepmycin (10μg), carbencillin (100μg), kanamycin (30μ
g), and stretionmycin (10μg). Determination of the susceptibility were according to the NCCLS standard
breakpoint for gram-negative enteric organisms. Escherichia coli ATCC 25922, the control strain, was routinely
used.
PCR amplification and sequence
Bacteria cells were grown on TSB overnight at 37℃, then transfered 100l culture to 3ml TSB, and
incubated for 8h. The cells were collected and lysed by heating at 100℃ for 10 min. 10l of cell dilution
(approximately 105 CFU/10l) was mixed with PCR buffer (10mM Tris-HCl, pH 8.8; 1.5mM MgCl2; 50mM
KCl; and 0.1% Triton X-100), 200 pmol each of dATP, dGTP, dCTP, dTTP (Boeheringer Mannheim, Germany),
50 pmol each of the PCR primer, and 0.2 U ProTaq. The PCR conditions were denaturation at 94℃ for 5 min
followed by 35 cycles of 94℃, 30s; 60℃, 30s; 72℃, 30s, and a single final extension of 10 min at 72℃. PCR
amplification specific for pse group (AmpicillinR), dfrA12 (Sulfamethoxaeole- trimethopmin or TrimethoprimR),
oxa1 (AmpicillinR), aadA1a (streptomycinR and spectinomycinR), aphA (kanamycinR), cmlA (chloramphenicol
R
), sul, QS (SulfonamideR), aadB (Gentamycin R), cat (Chloramphenicol R) (Table 1) genes were carried out.
The amplified products were analyzed by electrophoresis on 2% agarose gel.
Integron analysis
The integrons in Salmonella strains were detected using primers 5’CS-3’CS, and intI1F-intI1R (Table 1).
These primers were specific for integron conserved segments and class I integronase gene. For the primers
5’CS-3’CS, DNA from target bacteria was amplified during 35 cycles of 94℃, 30 s; 55℃, 30 s; 72℃, 2 min.
PCR conditions for primers intI1F-intI1R was the same as those for the analysis of antibiotic resistance genes.
The amplified product was examined by electrophoresis on 2% agarose gel.
RESULTS
Antibiotic susceptibility test
Results from the antimicrobial susceptibility test are shown in Table 2. The total strains could be grouped into
43 antibiotic resistant patterns. All the 95 strains were multi-drug resistant strains. They were resistant to two to
fifteen antimicrobial agents. For example, strains in pattern No. 38 were resistant to 15 kinds of antimicrobial
agents. All of the isolates were resistant to nalidixic acid and sulfonamides (100%), which have been grouped to
quinolones and folate pathway inhibitors. More than 80% of the strains were resistant to ampicillin (89.5%),
erythromycin (80.0%), tetracycline (93.7%), kanamycin (80.0%), and carbenicillin (87.4%). All of the 95 isolates
were susceptible to amoxicillin-clavulanic acid, which was -lactam and -lactamase inhibtor combination.
About 21.1 % to 24.2% isolates were fluoroquinolone- resistance (norfloxacin 21.1%, ciprofloxacin 21.1%, and
24.2%) (Table 3). The result also presented that S. Choleraesuis isolates from systemic infection were more
antibiotics resistant than the diarrhea isolates. For example, the fluoroquinolone- resistant strains were only found
in systemic infection.
PCR amplification of the antibiotic resistance gene
Nine PCR primer pairs specific for antibiotic resistant genes were used for screening of the 95 S. Choleraesuis
isolates. The results showed that 30 of 95 isolates (31.6%) have dfrA12 gene, 1 of 95 isolates (1.1%) have pse
gene, 29 of isolates (30.5%) have aadA1a gene, 82 of 95 isolates (86.3%) have aphA gene, 1 of 95 isolates
(1.1%) have oxa1 gene, 29 of 95 isolates (30.5%) have cmlA gene, 26 of 95 isolates (27.4%) have qs gene, were
PCR positive while aadB and cat genes were not found. The sizes of all the PCR products were as expected
(Table 1).
Integron analysis
Results from the determination of the class 1 integron in these 95 S. Choleraesuis isolates showed that 31
strains (32.6%) were with the class 1 integron. Most of the strains from systemic infection swine harbored the
integrons. As 5’CS and 3’CS primers were used to amplify the integrons of 31 strains. PCR products obtained
showed that the sizes of integrons were in between 650 to 2100 bp. Integronase gene also have been detected
with int1F and int1R primer pair. All of the 31 strains with class I integron generated positive reactions. They all
generated a 280 bp product. Further PCR analysis for antibiotic resistant genes was then performed for these 31
strains which harbored integrons. Five patterns, including dfrA12-aadA1a, oxa1-aadA1a, dfr12-aadA1a-sul, pse
and dfrA12 patterns were found (Table 4). These integrons preferentially harbored one or two of dfrA12, aadA2,
and pse gene cassettes. The 1500 bp integron size was found to encode for the streptomycin-spectinomycin and
-lactams resistance, i.e., with the oxa1- aadA1a gene cassettes. Other four types of gene cassettes belong to an
integron of 2100 bp size, which encode for the resistance to trimethoprim- sulfonamides and -lactams.
DISCUSSION
Antibiotics are often used in domestic animals for therapy and prophylaxis of bacterial infections and for
promotion of their growth. The use of antibiotics for domestic animals may cause problems in the therapy of
infections due to the antibiotic resistance of bacterial pathogens which infected animals or humans (Aarestrup, et
al., 1999). In this study, we incestigated the antibiotic resistant patterns for S. Choleraesuis isolates from swine.
The antibiotics surveyed were those commonly used for clinical therapy and animal treatment. High percentage
of these strains were found to be antimicrobial resistant and multi-drug resistant. Because of the emergence of
multiple resistant bacteria which may cause infection in humans, some antimicrobial agents have now become
last line-drugs in treatment of the bacteria infection.
Chiu, et al. (2002) reported that approximately 90% of S. Choleraesuis isolates from humans was found to be
resistant
to
at
least
one
of
the
following
antibiotics,
ie,
ampicillin,
chloramphenicol,
and
trimethoprim-sulfamethoxazle. Also, the rate of floroquinolone resistant strains was 60 percent. In this study, we
found that for S. Choleraesuis isolates from swine, the rate of ampicillin resistant strains was about 90%,
chloramphenicol resistant strains was 76%, trimethoprim-sulfamethoxazle resistant strains was 33% and
floroquinolone resistant strains was about 21%. The lower rates of resistant strains for isolated from swine might
be due to the samples from different sources. For example, human isolates were generally obtained from patients
with blood and other organs infection, and swine isolates were obtained from swine withdiarrhea (about 70%)
and systemic infection (30%). However, almost all the isolates from systemic infection were resistant to those
three antimicrobials. Ampicillin, chloramphenicol, and sulfa drugs have been recognized as standard first-line
drug for the therapy of Salmonella infection. If human infections were caused by Salmonella with
multidrug-resistance, all of these antibiotics will be ineffective, and treatment of Salminella infection will be
difficult. In Taiwan, those antimicrobial agents are no longer the drugs of choice for treatment of serious
nontyphoidal Salmonella infection. Cephalosporins and floroquinolone are now the preferred drugs for treatment
of invasive Salmonella infection (Yang, et al., 1998). However, emergence of floroquinolone resistance would
change the policy for treatment of S. Choleraesuis infection on humans or swine in Taiwan (Chiu, et al., 2002;
Aarestrup, et al., 2003).
As for the detection of antibiotic resistance genes by PCR, positive PCR results did not coincide exactly with
the phenotype. This could be due to the following reasons. First, the phenotype of antimicrobial resistance was
due to the expression of several different resistance genes. For example, resistance to ampicillin may be due to
oxa1, oxa2, and pse genes or others. Second, that resistant gene was unexpressed, such genes may be
pseudo-genes, which may have not complete regulator region including promoter.
By using PCR mapping with primers specific for integrons, the major finding of this study was that class 1
integron carrying the dfr12- aadA1a was widely distributed in S. Choleraesuis isolates from swine in Taiwan. If S.
Choleraesuis strains were resistant to streptomycin, spectinomycin, and sulfamethoxaeole-trimethopmin, the class
1 integron carrying the gene cassettes of this type could be found.
Surveillance and monitoring of antimicrobial-drug resistance of S. Choleraesuis from animals, including the
work of screening for class 1 integrons, are necessary steps in study of epidemiology and gene evolution of
antimicrobial resistance.
ACKNOWLEDGMENTS
This project is supported by the Nation Science Council, Taipei, Taiwan. The project No. is NSC
91-2313-B-005-074.
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TABLE 1. PCR primers used in the identification of integrons and antibiotic resistant gene.
primers
sequence
target
5’CS
3’CS
intI1F
GGC ATC CAA GCA GCA AGC
intI1R
PSE G1
TCC ACG CAT CGT CAG GC
ACC GTA TTG AGC CTG ATT TA
PSE group (PSE-1,
PSE G2
ATT GAA GCC TGT GTT TGA GC
PSE-4,CARB-3 , )
dfr12-F
dfr12-B
oxa1-F
oxa1-R
ACT CGG AAT CAG TAC GCA
dfrA12
ant-3’-1aF
ant-3’-1aB
aphA-1L
aphA-1R
cat-F
cat-B
aadB-F
GTG GAT GGC GGC CTG AAG CC
aadB-B
cmlA-F
cmlA-B
sul1-F
sul1-R
QS-1
QS-2
CTG TTA CAA CGG ACT GGC CGC
specific antibiotic
resistant
accession no.
M73819
Levesque, et al.,
1995.
integrase I
-
M73819
Orman, et al.,
2002
oxa1
aadA1a
ATT GCC CAG TCG GCA GCG
aphA
321
trimethoprim
AF175203
ampicillin
AJ009819
streptomycin,
spectinomycin
kanamycin
M10241
U63147
Guerra, et al.,
2001.
462
Sandvang, et al.,
1998.
708
Sandvang, et al.,
1998.
527
Ng, et al.,1999
489
GAG AAA ACT CAC CGA GGC AG
CCT GCC ACT CAT CGC AGT
cat
chloramphenicol
U46780
CCA CCG TTG ATA TAT CCC
GAG CGA AAT CTG CCG CTC TGG
TGTCATTTACGGCATACTCG
aadB
cmlA
gentamycin
chloramphenicol
ATCAGGCATCCC ATT CCC AT
CCT CGA TGA GAG CCG GCG GC
sul
GCA AGG CGG AAA CCC GCG CC
ATG AAA GGC TGG CTT TTT CTTG
TGA GTG CAT AAC CAC CAG CC
280
Bert, et al., 2002.
ampicillin
ATT CGA CCC CAA GTT TCC
TTA TGC CTC TTC CGA CCA TC
－
-
GTG TAC GGA ATT ACA GCT
AGC AGC GCC AGT GCA TCA
product size
(bp)
integron
AAG CAG ACT TGA CCT GAT
CCC TCC CGC ACG ATG ATC
reference
qac/sul1
sulfonamide
AF078527
M64556
Guerra, et al.,
2001.
632
Frana et. al.,
2001
310
Guerra et al.,
2001.
435
Sandvang et al.,
1998.
437
Boyd et al., 2002
850
TABLE 2. Antiograms of Salmonella Choleraesuis isolates from swine in southern Taiwan.
Type
Antibiogarm
No. of strain
Strain
1
AmpGmCfCETSxtNaSuSpSCbK
1
SCV02
2
AmpGmCfCETSxtNaSuSpSCb
1
SCV03
3
AmpGmETNaSuCbK
1
SCV04
4
AmpCETNaSuCbK
7
SCV05, SCV06, SCV08,
SCV15, SCV19, SCV46, SCV47
5
AmpGmCETNaSuCbK
11
SCV07, SCV09, SCV28,
SCV38, SCV39, SCV40,
SCV44, SCV50, SCV52,
SCV58, SCV59
6
AmpGmCETSxtNaSuSCbK
1
SCV10
7
AmpGmCETSxtNaSuCbK
3
SCV11, SCV32, SCV71
8
ENaSu
3
SCV12, SCV18, SCV66
9
AmpCTNaSuCbK
1
SCV13
10
AmpETNaSuSpCbK
1
SCV14
11
GmETNaSu
1
SCV16
12
AmpGmCfETNaSuCbK
2
SCV17, SCV65
13
AmpGmCETNaSuSCbK
8
SCV20, SCV24, SCV30,
SCV31, SCV36, SCV49,
SCV57, SCV64
14
AmpCTNaSuCb
1
SCV22
15
AmpGmCfCETNaSuCbK
2
SCV23, SCV69
16
AmpGmCfCETSxtNaSuSpS
1
SCV25
17
ETNaSu
2
SCV26, SCV27
18
AmpTNaSuCbK
1
SCV29
19
AmpCETNaSu
1
SCV33
20
GmCNaSuS
1
SCV34
21
AmpTNaSuSCbK
1
SCV35
22
AmpGmCfCETNaSuSCbK
2
SCV37, SCV42
23
AmpCETNaSuCb
1
SCV41
24
ETNaSuS
2
SCV43, SCV53
Type
Antibiogarm
No. of strain
Strain
25
AmpETNaSuCbK
3
SCV45, SCV55, SCV70
26
AmpGmENaSu
1
SCV54
27
AmpGmETNaSuSCbK
1
SCV60
28
CTNaSuS
1
SCV61
29
AmpGmCETNaSu
1
SCV62
30
AmpGmCETNaSuCbK
1
SCV63
31
AmpGmCTNaSuS
1
SCV67
32
GmCETNaSuCbK
1
SCV68
33
AmpCfETSxtNaSu
1
SCV72
34
AmpGmCETSxtNaSuSCb
2
SCV1a, SCV 17a
35
AmpGmCTSxtNorCipOflNaSuSpSCbK
7
SCV 2a, SCV 3a, SCV 4a, SCV
6a, SCV 8a, SCV 11a, SCV 14a
36
TSxtNorCipOflNaSu
1
SCV 5a
37
AmpGmCETSxtNorCipOflNaSuSpSCbK
11
SCV 9a, SCV 10a, SCV 12a,
SCV 13a, SCV 15a, SCV 18a
SCV 22a, SCV 23a, SCV 24a,
SCV 26a, SCV 27a
38
AmpGmCfCETSxtNorCipOflNaSuSpSCb
1
SCV 16a
39
AmpCETSxtNaSuSpSCbK
2
SCV 19a, SC
40
AmpGmCETSxtOflNaSuSpSCb
1
SCV 20a
41
AmpGmCETSxtNaSuSpSCbK
1
SCV 21a
42
AmpGmCETSxtNaSuSpSCb
1
SCV 25a
43
AmpCTSxtNaSuSpSCbK
1
E55
Amp:ampicillin AMC: amoxicillin-clavulanic acid Gm:gentamicin Cf:cephalothion C:chloramphenicol
E:erythromycin T:tetracycline Sxt:sulfamethoxaeole-trimethopmin Nor:norfloxacin Cip:ciprofloxacin
Ofl:ofloxacin Na:nalidixic acid Su:sulfonamides Sp:strepmycin S:stretionmycin Cb:carbencillin K:kanamycin
TABLE 3 . Phenotypes of antimicrobial resistance for S. Choleraesuis isolates
Clsaa and/or antimicrobial
% Resistance strains
(n=95)
Penicillins
Ampicillin
89.5%
Amoxicillin-clavulanic acid
0%
Carbenicillin
87.4%
Aminoglycosides
Gentamicin
67.4%
Streptomycin
70.6%
Kanamycin
80%
Cephalosporin I
Cephalothin
7.4%
Phenicols
Chloramphenicol
75.8%
Tetracycline
93.7%
Sulfonamides and potentiated sulfonamides
Trimethoprim-sulfamethoxazole
32.6%
Sulfonamides
100%
Quinolones and fluoroquinolones
Nalidixic acid
100%
Ciprofloxacin
21.1%
Norfloxacin
21.1%
Ofloxacin
24.2%
Macrolides
Erythromycin
80%
Aminocyclitols
spectinomycin
31.6%
TABLE 4. Genotypic and phenotypic characteristics for antibiotic resistance of Salmonella Choleraesuis isolates.
AMP
Strain
SCV02, SCV03, SCV24a
SCV62
SCV1a
TMP
STR
KAN
CHL
GEN
SUL
qacE/
oxa1
blaPSE
dfrA12
aadA1a
aphA
cat
cmlA
aadB
sul1
sul
－
－
＋
＋
＋
－
＋
－
－
－
＋
－
＋
＋
＋
－
－
－
－
－
－
＋
＋
＋
＋
－
＋
－
＋
＋
integron
pattern
integron
size
dfrA12-aadA2
~2100 bp
oxa1-aadA2
~1500 bp
dfrA12-aadA2-sul1
-2100 bp
pse
SC, E55
SCV2a, SCV3a, SCV4a, SCV5a,
－
－
＋
－
＋
－
＋
－
＋
＋
dfrA12
-2100 bp
－
＋
＋
＋
－
＋
－
＋
＋
dfrA12-aadA2-sul1
-2100 bp
－
－
＋
＋
－
－
＋
－
－
－
dfrA12-aadA2
-2100 bp
－
－
＋
＋
－
－
＋
－
＋
＋
dfrA12-aadA2-sul1
-2100 bp
－
SCV6a, SCV9a, SCV10a,
SCV11a , SCV12a, SCV13a,
SCV14a, SCV15a, SCV18a,
SCV19a , SCV21a, SCV22a,
SCV23a, SCV26a, SCV27a
SCV8a ,SCV16a,
SCV17a , SCV20a, SCV25a
a.
Amp: ampicillin 10μg per disk, Neo: Neomycin 30μg per disk, Chl: Chloramphenicol 30μg per disk, Kan: Kanamycin 30μg per disk, Str: Streptomycin 10μg per disk,
Sul: Sulfonamide 250μg per disk, Cef: Cefoperazone 75μg per disk, Sxt: Trimrthoprim-sulfamethoxazole, Nor: Norfloxacin 10μg per disk, Tet: Tetracyclin 30μg per disk.
1
2
3
4
5
6
7
8
9
10
11
M a b
c
d
e f
g h
i
jM k
l
m n
o p
q
12
13
14
15
16
17
18
19
20
Fig. 1 PCR amplification of the integrons and gene cassettes for S. Choleraesuis
21
22
23
24
25
26
isolates. Lane M: 100 bp ladder marker; lane a-d: products of 5’CS/3’CS primer pair
of SCV2, SCV62, SCV1a, and SCV2a; lane e-g: gene cassettes of dfrA12, aadA1, and
dfrA12-aadA1 of SCV2; lane h-j: gene cassettes of oxa-1, aadA1, and oxa-1-aadA1 of
SCV62; lane k-m: gene cassettes of dfrA12, aadA1,and sul1 of SCV1a; lane n-p: gene
cassettes of dfrA12, aadA1,and sul1 of SCV2a; lane q: gene cassettes of dfrA12 of
E55.