AAC Accepted Manuscript Posted Online 27 April 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00120-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Two novel Salmonella genomic island 1 variants in Proteus mirabilis
2
isolates from swine farms in China
3
4
Chang-Wei Lei, An-Yun Zhang, Bi-Hui Liu, Hong-Ning Wang#, Li-Qin Yang,
5
Zhong-Bin Guan, Chang-Wen Xu, Dong-Dong Zhang, Yong-Qiang Yang
6
7
Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province;
8
Key Laboratory of Bio-resources and Eco-environment, Ministry of Education; “985
9
Project” Science Innovative Platform for Resource and environment Protection of
10
Southwestern China; College of Life science, Sichuan University, Chengdu, P. R.
11
China
12 13
#
14
NO. 29 Wangjiang Road, Chengdu, Sichuan, China, 610064.
15
Phone: +86-28-8547-1599. Fax: +86-28-8547-1599. E-mail: [email protected].
Corresponding author. Mailing address: College of Life science, Sichuan University,
16 17
Running title: Two novel SGI1 variants in P. mirabilis.
18
Keywords: Salmonella, genomic island, multidrug resistance, integron
19 20 21 1
22 23
Abstract Four different Salmonella genomic island 1 (SGI1), including two novel variants,
24
were characterized in one Salmonella enterica serovar Rissen ST1917 isolate and
25
three Proteus mirabilis isolates from swine farms in China. One novel variant was
26
derived from SGI1-B with the backbone gene S021 disrupted by a 12.72-kb
27
IS26-composite transposon containing the dfrA17-aadA5 cassettes and macrolide
28
inactivation gene cluster mphA-mrx-mphR. Another one was an integron-free SGI1
29
and contained 183-bp truncated S025 next to IS6100 and S044.
30 31
Salmonella enterica is a zoonotic pathogen and is one of the worldwide primary
32
causes of human infections. Salmonella genomic island 1 (SGI1) is an integrative
33
42.4-kb chromosomal element first identified in the multidrug resistance (MDR) S.
34
enterica serovar Typhimurium phage type DT104 clone that has been epidemic
35
among humans and domestic animals since the 1990s (1, 2). The MDR region in SGI1
36
is a complex In4-type class 1 integron (named In104) clustering five antibiotic
37
resistance genes that confer resistance to ampicillin, chloramphenicol and florfenicol,
38
streptomycin and spectinomycin, sulphonamides and tetracycline (3). SGI1 was
39
unexpectedly detected in a Proteus mirabilis clinical isolate in 2007 (4). Sequence
40
analysis showed that SGI1 in S. enterica and P. mirabilis had the same chromosomal
41
integration site corresponding to the last 18 bp of the 3’ end of the trmE (also named
42
thdF) gene (5). It has been comfirmed that SGI1 in S. enterica could be transferred by
43
conjugation with the help of IncA/C plasmid (6, 7). 2
44
Many SGI1 variants result from the homologous recombination of gene cassettes
45
within the MDR regions (3, 8-11). A few variations in SGI1 backbone are also
46
described due to the deletion, insertion and transposition (3, 9, 10, 12-15).
47
Furthermore, several novel resistance genes, including ESBL gene blaVEB-6 as well as
48
fluoroquinolone resistance genes qnrA1 and qnrB2, have been reported in SGI1 (9,
49
11), suggesting that SGI1 could act as a mobilizable element to disseminate the
50
critical resistance genes. In the present study, we characterized SGI1 among S.
51
enterica and P. mirabilis isolates from swine farms in China.
52
A total of 24 S. enterica and 61 P. mirabilis strains were isolated from samples of
53
swine stools and diseased tissues in 35 swine farms from 16 provinces in China
54
between May 2012 and February 2014. Antimicrobial susceptibility test was
55
performed by the disc diffusion method according to the CLSI guideline (16). Primers
56
used in this study are listed in Table S1. The left and right junctions of SGI1 were
57
detected in one S. Rissen and three P. mirabilis strains. The multiple locus sequence
58
typing for SGI1-containing S. Rissen strain Z4 showed that the types of the seven
59
house-keeping genes were 92 (aroC), 137 (dnaN), 8 (hemD), 524 (hisD), 206 (purE),
60
313 (sucA) and 330 (thrA) never reported to date. It was submitted to the website
61
http://mlst.warwick.ac.uk/mlst/dbs/Senterica and assigned as a new ST1917. To the
62
best of our knowledge, this is the first report of the SGI1 in S. Rissen. Three
63
SGI1-containing P. mirabilis strains belonged to different clusters by pulsed field gel
64
electrophoresis after SmaI digestion (Fig. S1). The origin, antibiotic resistance
65
profiles and cassette genes of the four SGI1-containing strains are listed in Table 1. 3
66
Four different SGI1 variants were identified through PCR-mapping and sequencing
67
(Table 2). Two known SGI1 variants, SGI1-I and SGI1-PmABB, have previously
68
been reported (10, 17). Two novel SGI1 variants, SGI1-B2 (45.93-kb) in PmSC17 and
69
SGI1-Z (24.30-kb) in PmSC42, were characterized in this study for the first time (Fig.
70
1).
71
Two gene cassettes, blaPSE-1 (1.20-kb) and dfrA17-aadA5 (1.66-kb), were detected
72
in strain PmSC17. However, the MDR region in SGI1-B2 contained only the blaPSE-1
73
cassette identical to SGI1-B (3). PCR amplicon was negative by using primers
74
S020-R and S024-outF in the PCR-mapping of SGI1-B2 backbone. Through primer
75
walking and PCR linkage, a 12.72-kb IS26-composite transposon was found inserted
76
in the backbone gene S021 never reported to date in SGI1 (Fig. 1). The transposition
77
event occurred in the region between 128-bp and 175-bp of the S021 gene, resulting
78
in a 48-bp target site duplication surrounding the IS26-composite transposon. It
79
contained the dfrA17-aadA5 cassettes and the macrolide inactivation gene
80
cluster mphA-mrx-mphR, which differed only by 4 single-base changes from the
81
corresponding regions in the Escherichia coli plasmid pEK499 (18). Therefore,
82
SGI1-B2 was derived from SGI1-B with S021 disrupted by an IS26-composite
83
transposon.
84
SGI1-Z (24.30-kb) contained backbone genes S001-S024 and 183-bp truncated
85
S025 next to IS6100 and S044. Although the strain PmSC42 harbored the
86
dfrA17-aadA5 cassettes, they were not located in SGI1. The 183-bp truncated S025
87
was exactly close to the left 14-bp inverted repeat region of IS6100. So SGI1-Z is an 4
88
integron-free SGI1 and does not contain any resistance genes. We hypothesize that an
89
IS6100-mediated transposition event might occur in the backbone gene S025 and the
90
homologous recombination happened subsequently between the two copies of IS6100
91
resulting in the loss of the integron.
92
The mobility and stability of the SGI1 have previously been confirmed in S.
93
enterica (6, 19, 20). Many SGI1s can be excised from the chromosome and the
94
resulting free circular form may be transferable with the helper plasmid (6, 20). The
95
circular extrachromosomal form was detected in all four SGI1s after two rounds of
96
PCR amplification by using the same circ1/2 primers (Fig. S2) (6), implying the
97
mobility of the SGI1. Four SGI1-containing strains were propagated lasting for 20
98
days (40 passages) in the absence of antimicrobial pressure. None SGI1-negative
99
clone was detected from the picked 827 clones (each strain picked about 200 clones)
100
in the 41st passage, suggesting that SGI1 was stable in both S. enterica and P.
101
mirabilis. Nevertheless, the homologous recombination within SGI1 integrons could
102
occur in few clones. Four SGI1-I changed to SGI1-C containing only the aadA2
103
cassette in MDR region already described (19, 20). The exchange of gene cassettes
104
between the two integrons in SGI1-B2 was detected in nine clones, generating a new
105
SGI1 MDR region containing the dfrA17 and aadA5 cassettes (Fig. S3).
106
In conclusion, four SGI1, including two novel variants, were characterized in S.
107
enterica and P. mirabilis isolates from swine farms in China. The dfrA17 and aadA5
108
cassettes, as well as mphA-mrx-mphR cluster, were reported in SGI1 for the first time.
109
The persistence of SGI1 in S. enterica and P. mirabilis might threaten public health 5
110
given that the SGI1-containing strains could spread from animal farms to humans
111
through meat consumption (5, 8, 21). Nucleotide sequence accession numbers. The complete nucleotide sequences of
112 113
four SGI1s detected in this study were submitted to GenBank and assigned accession
114
numbers KM234279 (SGI1-I in S. Rissen Z4), KP116299 (SGI1-B2 in P. mirabilis
115
PmSC17), KP057606 (SGI1-Z in P. mirabilis SC42) and KP313760 (SGI1-PmABB
116
in P. mirabilis PmXJF).
117 118
ACKNOWLEDGMENTS
119
This work was supported by “973” National Basic Research Program of China
120
(project number 2013CB127200) and Science & Technology Pillar Program in
121
Sichuan Province (grant number 2013NZ0025, 13ZC2578 and 2012GZ0001-1).
122 123
REFERENCES
124
1.
Boyd D, Peters GA, Cloeckaert A, Boumedine KS, Chaslus-Dancla E,
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Imberechts H, Mulvey MR. 2001. Complete nucleotide sequence of a
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43-kilobase genomic island associated with the multidrug resistance region of
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Salmonella enterica serovar Typhimurium DT104 and its identification in phage
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type DT120 and serovar Agona. J Bacteriol 183:5725-5732.
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Threlfall EJ. 2000. Epidemic Salmonella typhimurium DT 104--a truly
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international multiresistant clone. J Antimicrob Chemother 46:7-10.
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3. Hall RM. 2010. Salmonella genomic islands and antibiotic resistance in 6
Salmonella enterica. Future Microbiol 5:1525-1538.
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Ahmed AM, Hussein AI, Shimamoto T. 2007. Proteus mirabilis clinical isolate
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harbouring a new variant of Salmonella genomic island 1 containing the multiple
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Boyd DA, Shi X, Hu QH, Ng LK, Doublet B, Cloeckaert A, Mulvey MR.
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2008. Salmonella genomic island 1 (SGI1), variant SGI1-I, and new variant
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SGI1-O in Proteus mirabilis clinical and food isolates from China. Antimicrob
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Agents Chemother 52:340-344.
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Doublet B, Boyd D, Mulvey MR, Cloeckaert A. 2005. The Salmonella genomic
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island 1 is an integrative mobilizable element. Mol Microbiol 55:1911-1924.
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7. Douard G, Praud K, Cloeckaert A, Doublet B. 2010. The Salmonella genomic
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island 1 is specifically mobilized in trans by the IncA/C multidrug resistance
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plasmid family. PLoS One 5:e15302.
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8. Bi S, Yan H, Chen M, Zhang Z, Shi L, Wang H. 2011. New variant Salmonella
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genomic island 1-U in Proteus mirabilis clinical and food isolates from South
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China. J Antimicrob Chemother 66:1178-1179.
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9. Siebor E, Neuwirth C. 2011. The new variant of Salmonella genomic island 1
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(SGI1-V) from a Proteus mirabilis French clinical isolate harbours blaVEB-6 and
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qnrA1 in the multiple antibiotic resistance region. J Antimicrob Chemother
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66:2513-2520.
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10. Siebor E, Neuwirth C. 2013. Emergence of Salmonella genomic island 1 (SGI1) among Proteus mirabilis clinical isolates in Dijon, France. J Antimicrob 7
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Chemother 68:1750-1756.
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11. Lei CW, Zhang AY, Liu BH, Wang HN, Guan ZB, Xu CW, Xia QQ, Cheng H,
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Zhang DD. 2014. Molecular characteristics of Salmonella genomic island 1 in
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Proteus mirabilis isolates from poultry farms in China. Antimicrob Agents
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Chemother 58:7570-7572.
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12. Doublet B, Praud K, Bertrand S, Collard JM, Weill FX, Cloeckaert A. 2008.
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Novel insertion sequence- and transposon-mediated genetic rearrangements in
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genomic island SGI1 of Salmonella enterica serovar Kentucky. Antimicrob
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Agents Chemother 52:3745-3754.
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13. Doublet B, Praud K, Weill FX, Cloeckaert A. 2009. Association of
164
IS26-composite transposons and complex In4-type integrons generates novel
165
multidrug resistance loci in Salmonella genomic island 1. J Antimicrob
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Chemother 63:282-289.
167 168 169 170
14. Hamidian M, Holt KE, Hall RM. 2014. The complete sequence of Salmonella genomic island SGI1-K. J Antimicrob Chemother 70:305-306. 15. Hamidian M, Holt KE, Hall RM. 2014. The complete sequence of Salmonella genomic island SGI2. J Antimicrob Chemother. Doi: 10.1093/jac/dku407.
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16. CLSI. 2012. Performance Standards for Antimicrobial Susceptibility Testing, 22th
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Informational Supplement. CLSI document M100-S22. Clinical and Laboratory
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Standards Institute, Wayne, PA, USA.
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17. Levings RS, Lightfoot D, Partridge SR, Hall RM, Djordjevic SP. 2005. The
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genomic island SGI1, containing the multiple antibiotic resistance region of 8
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Salmonella enterica serovar Typhimurium DT104 or variants of it, is widely
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distributed in other S. enterica serovars. J Bacteriol 187:4401-4409.
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18. Woodford N, Carattoli A, Karisik E, Underwood A, Ellington MJ, Livermore
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DM. 2009. Complete nucleotide sequences of plasmids pEK204, pEK499, and
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pEK516, encoding CTX-M enzymes in three major Escherichia coli lineages from
181
the United Kingdom, all belonging to the international O25:H4-ST131 clone.
182
Antimicrob Agents Chemother 53:4472-4482.
183
19. Djordjevic SP, Cain AK, Evershed NJ, Falconer L, Levings RS, Lightfoot D,
184
Hall RM. 2009. Emergence and evolution of multiply antibiotic-resistant
185
Salmonella enterica serovar Paratyphi B D-tartrate-utilizing strains containing
186
SGI1. Antimicrob Agents Chemother 53:2319-2326.
187 188
20. Kiss J, Nagy B, Olasz F. 2012. Stability, entrapment and variant formation of Salmonella genomic island 1. PLoS One 7:e32497.
189
21. Seiffert SN, Tinguely R, Lupo A, Neuwirth C, Perreten V, Endimiani A. 2013.
190
High prevalence of extended-spectrum-cephalosporin-resistant enterobacteriaceae
191
in poultry meat in Switzerland: emergence of CMY-2- and VEB-6-possessing
192
Proteus mirabilis. Antimicrob Agents Chemother 57:6406-6408.
193
9
194 195
Tables and figure legends
196 197
Table 1. SGI1-containing strains characterized in this study.
198
Table 2. Four different SGI1 variants characterized in this study.
199 200
Fig. 1. Schematic view of the two novel SGI1 variants characterized in this study.
201
Genes and ORFs are shown as arrows and their orientations of transcription are
202
indicated by the arrowheads. DR-L and DR-R represent the 18-bp direct repeats at the
203
ends of SGI1. CS: Conserved segment; IRi and IRt: Inverted repeats defining the left
204
and right hands of integron; orf: Open reading frame.
10
205
Table 1. SGI1-containing strains characterized in this study. Province Strain
Date of isolation
Origin
Integron cassette(s)
Antibiotic resistance profile
28 October 2012
liver
aadA2, dfrA1-orfC
CHL, FFC, STR, SPT, DOX, TMP, SUL, SXT
of isolation S. Rissen Z4
Anhui
P. mirabilis SC17
Sichuan
12 April 2013
liver
dfrA17-aadA5, blaPSE-1
AMP, CHL, FFC, NAL, STR, SPT, DOX, TMP, SUL, SXT
P. mirabilis SC42
Sichuan
15 November 2013
stool
dfrA17-aadA5
CHL, FFC, NAL, CIP, STR, STP, GEN, DOX, TMP, SUL, SXT
P. mirabilis XJF
Henan
09 January 2014
stool
aacA5-aadA7
AMP, STR, STP, GEN, DOX, TMP, SUL, SXT
206
AMP, ampicillin; CHL, chloramphenicol; FFC, florfenicol; NAL, nalidixic acid; CIP, ciprofloxacin; STR, streptomycin; SPT, spectinomycin; GEN, gentamicin;
207
DOX, doxycycline; TMP, trimethoprim; SUL, sulfizoxazole; SXT, trimethoprim-sulfamethoxazole.
11
208
Table 2. Four different SGI1 variants characterized in this study. Amplification of PCR product (bp)a
SGI1 variants
Strain
209
Resistance genes in MDR region S005-S010 S020-S024 S024-S025 res-intI1
S024-S044
S. Rissen Z4
4793
3598
3579
1417
ND
P. mirabilis SC17
4793
NE
3579
1417
P. mirabilis SC42
4793
3598
NE
P. mirabilis XJF
3272
3598
3579
a
Type
Size (kb)
aadA2, floRc, tet (G), dfrA1, sul1
SGI1-I
42.48
ND
dfrA17, aadA5, blaPSE-1, sul1, mphA, mrx, mphR
SGI1-B2
45.93
NE
3399
None
SGI1-Z
24.30
1417
ND
aacCA5, aadA7, sul1
SGI1-PmABB 32.02
Primers are listed in Table S1. NE, negative. ND, not detected.
12
1
Two novel Salmonella genomic island 1 variants in Proteus mirabilis
2
isolates from swine farms in China
3
4
Chang-Wei Lei, An-Yun Zhang, Bi-Hui Liu, Hong-Ning Wang#, Li-Qin Yang,
5
Zhong-Bin Guan, Chang-Wen Xu, Dong-Dong Zhang, Yong-Qiang Yang
6
7
Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province;
8
Key Laboratory of Bio-resources and Eco-environment, Ministry of Education; “985
9
Project” Science Innovative Platform for Resource and environment Protection of
10
Southwestern China; College of Life science, Sichuan University, Chengdu, P. R.
11
China
12 13
#
14
NO. 29 Wangjiang Road, Chengdu, Sichuan, China, 610064.
15
Phone: +86-28-8547-1599. Fax: +86-28-8547-1599. E-mail: [email protected].
Corresponding author. Mailing address: College of Life science, Sichuan University,
16 17
Running title: Two novel SGI1 variants in P. mirabilis.
18
Keywords: Salmonella, genomic island, multidrug resistance, integron
19 20 21 1
22 23
Abstract Four different Salmonella genomic island 1 (SGI1), including two novel variants,
24
were characterized in one Salmonella enterica serovar Rissen ST1917 isolate and
25
three Proteus mirabilis isolates from swine farms in China. One novel variant was
26
derived from SGI1-B with the backbone gene S021 disrupted by a 12.72-kb
27
IS26-composite transposon containing the dfrA17-aadA5 cassettes and macrolide
28
inactivation gene cluster mphA-mrx-mphR. Another one was an integron-free SGI1
29
and contained 183-bp truncated S025 next to IS6100 and S044.
30 31
Salmonella enterica is a zoonotic pathogen and is one of the worldwide primary
32
causes of human infections. Salmonella genomic island 1 (SGI1) is an integrative
33
42.4-kb chromosomal element first identified in the multidrug resistance (MDR) S.
34
enterica serovar Typhimurium phage type DT104 clone that has been epidemic
35
among humans and domestic animals since the 1990s (1, 2). The MDR region in SGI1
36
is a complex In4-type class 1 integron (named In104) clustering five antibiotic
37
resistance genes that confer resistance to ampicillin, chloramphenicol and florfenicol,
38
streptomycin and spectinomycin, sulphonamides and tetracycline (3). SGI1 was
39
unexpectedly detected in a Proteus mirabilis clinical isolate in 2007 (4). Sequence
40
analysis showed that SGI1 in S. enterica and P. mirabilis had the same chromosomal
41
integration site corresponding to the last 18 bp of the 3’ end of the trmE (also named
42
thdF) gene (5). It has been comfirmed that SGI1 in S. enterica could be transferred by
43
conjugation with the help of IncA/C plasmid (6, 7). 2
44
Many SGI1 variants result from the homologous recombination of gene cassettes
45
within the MDR regions (3, 8-11). A few variations in SGI1 backbone are also
46
described due to the deletion, insertion and transposition (3, 9, 10, 12-15).
47
Furthermore, several novel resistance genes, including ESBL gene blaVEB-6 as well as
48
fluoroquinolone resistance genes qnrA1 and qnrB2, have been reported in SGI1 (9,
49
11), suggesting that SGI1 could act as a mobilizable element to disseminate the
50
critical resistance genes. In the present study, we characterized SGI1 among S.
51
enterica and P. mirabilis isolates from swine farms in China.
52
A total of 24 S. enterica and 61 P. mirabilis strains were isolated from samples of
53
swine stools and diseased tissues in 35 swine farms from 16 provinces in China
54
between May 2012 and February 2014. Antimicrobial susceptibility test was
55
performed by the disc diffusion method according to the CLSI guideline (16). Primers
56
used in this study are listed in Table S1. The left and right junctions of SGI1 were
57
detected in one S. Rissen and three P. mirabilis strains. The multiple locus sequence
58
typing for SGI1-containing S. Rissen strain Z4 showed that the types of the seven
59
house-keeping genes were 92 (aroC), 137 (dnaN), 8 (hemD), 524 (hisD), 206 (purE),
60
313 (sucA) and 330 (thrA) never reported to date. It was submitted to the website
61
http://mlst.warwick.ac.uk/mlst/dbs/Senterica and assigned as a new ST1917. To the
62
best of our knowledge, this is the first report of the SGI1 in S. Rissen. Three
63
SGI1-containing P. mirabilis strains belonged to different clusters by pulsed field gel
64
electrophoresis after SmaI digestion (Fig. S1). The origin, antibiotic resistance
65
profiles and cassette genes of the four SGI1-containing strains are listed in Table 1. 3
66
Four different SGI1 variants were identified through PCR-mapping and sequencing
67
(Table 2). Two known SGI1 variants, SGI1-I and SGI1-PmABB, have previously
68
been reported (10, 17). Two novel SGI1 variants, SGI1-B2 (45.93-kb) in PmSC17 and
69
SGI1-Z (24.30-kb) in PmSC42, were characterized in this study for the first time (Fig.
70
1).
71
Two gene cassettes, blaPSE-1 (1.20-kb) and dfrA17-aadA5 (1.66-kb), were detected
72
in strain PmSC17. However, the MDR region in SGI1-B2 contained only the blaPSE-1
73
cassette identical to SGI1-B (3). PCR amplicon was negative by using primers
74
S020-R and S024-outF in the PCR-mapping of SGI1-B2 backbone. Through primer
75
walking and PCR linkage, a 12.72-kb IS26-composite transposon was found inserted
76
in the backbone gene S021 never reported to date in SGI1 (Fig. 1). The transposition
77
event occurred in the region between 128-bp and 175-bp of the S021 gene, resulting
78
in a 48-bp target site duplication surrounding the IS26-composite transposon. It
79
contained the dfrA17-aadA5 cassettes and the macrolide inactivation gene
80
cluster mphA-mrx-mphR, which differed only by 4 single-base changes from the
81
corresponding regions in the Escherichia coli plasmid pEK499 (18). Therefore,
82
SGI1-B2 was derived from SGI1-B with S021 disrupted by an IS26-composite
83
transposon.
84
SGI1-Z (24.30-kb) contained backbone genes S001-S024 and 183-bp truncated
85
S025 next to IS6100 and S044. Although the strain PmSC42 harbored the
86
dfrA17-aadA5 cassettes, they were not located in SGI1. The 183-bp truncated S025
87
was exactly close to the left 14-bp inverted repeat region of IS6100. So SGI1-Z is an 4
88
integron-free SGI1 and does not contain any resistance genes. We hypothesize that an
89
IS6100-mediated transposition event might occur in the backbone gene S025 and the
90
homologous recombination happened subsequently between the two copies of IS6100
91
resulting in the loss of the integron.
92
The mobility and stability of the SGI1 have previously been confirmed in S.
93
enterica (6, 19, 20). Many SGI1s can be excised from the chromosome and the
94
resulting free circular form may be transferable with the helper plasmid (6, 20). The
95
circular extrachromosomal form was detected in all four SGI1s after two rounds of
96
PCR amplification by using the same circ1/2 primers (Fig. S2) (6), implying the
97
mobility of the SGI1. Four SGI1-containing strains were propagated lasting for 20
98
days (40 passages) in the absence of antimicrobial pressure. None SGI1-negative
99
clone was detected from the picked 827 clones (each strain picked about 200 clones)
100
in the 41st passage, suggesting that SGI1 was stable in both S. enterica and P.
101
mirabilis. Nevertheless, the homologous recombination within SGI1 integrons could
102
occur in few clones. Four SGI1-I changed to SGI1-C containing only the aadA2
103
cassette in MDR region already described (19, 20). The exchange of gene cassettes
104
between the two integrons in SGI1-B2 was detected in nine clones, generating a new
105
SGI1 MDR region containing the dfrA17 and aadA5 cassettes (Fig. S3).
106
In conclusion, four SGI1, including two novel variants, were characterized in S.
107
enterica and P. mirabilis isolates from swine farms in China. The dfrA17 and aadA5
108
cassettes, as well as mphA-mrx-mphR cluster, were reported in SGI1 for the first time.
109
The persistence of SGI1 in S. enterica and P. mirabilis might threaten public health 5
110
given that the SGI1-containing strains could spread from animal farms to humans
111
through meat consumption (5, 8, 21). Nucleotide sequence accession numbers. The complete nucleotide sequences of
112 113
four SGI1s detected in this study were submitted to GenBank and assigned accession
114
numbers KM234279 (SGI1-I in S. Rissen Z4), KP116299 (SGI1-B2 in P. mirabilis
115
PmSC17), KP057606 (SGI1-Z in P. mirabilis SC42) and KP313760 (SGI1-PmABB
116
in P. mirabilis PmXJF).
117 118
ACKNOWLEDGMENTS
119
This work was supported by “973” National Basic Research Program of China
120
(project number 2013CB127200) and Science & Technology Pillar Program in
121
Sichuan Province (grant number 2013NZ0025, 13ZC2578 and 2012GZ0001-1).
122 123
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Tables and figure legends
196 197
Table 1. SGI1-containing strains characterized in this study.
198
Table 2. Four different SGI1 variants characterized in this study.
199 200
Fig. 1. Schematic view of the two novel SGI1 variants characterized in this study.
201
Genes and ORFs are shown as arrows and their orientations of transcription are
202
indicated by the arrowheads. DR-L and DR-R represent the 18-bp direct repeats at the
203
ends of SGI1. CS: Conserved segment; IRi and IRt: Inverted repeats defining the left
204
and right hands of integron; orf: Open reading frame.
10
205
Table 1. SGI1-containing strains characterized in this study. Province Strain
Date of isolation
Origin
Integron cassette(s)
Antibiotic resistance profile
28 October 2012
liver
aadA2, dfrA1-orfC
CHL, FFC, STR, SPT, DOX, TMP, SUL, SXT
of isolation S. Rissen Z4
Anhui
P. mirabilis SC17
Sichuan
12 April 2013
liver
dfrA17-aadA5, blaPSE-1
AMP, CHL, FFC, NAL, STR, SPT, DOX, TMP, SUL, SXT
P. mirabilis SC42
Sichuan
15 November 2013
stool
dfrA17-aadA5
CHL, FFC, NAL, CIP, STR, STP, GEN, DOX, TMP, SUL, SXT
P. mirabilis XJF
Henan
09 January 2014
stool
aacA5-aadA7
AMP, STR, STP, GEN, DOX, TMP, SUL, SXT
206
AMP, ampicillin; CHL, chloramphenicol; FFC, florfenicol; NAL, nalidixic acid; CIP, ciprofloxacin; STR, streptomycin; SPT, spectinomycin; GEN, gentamicin;
207
DOX, doxycycline; TMP, trimethoprim; SUL, sulfizoxazole; SXT, trimethoprim-sulfamethoxazole.
11
208
Table 2. Four different SGI1 variants characterized in this study. Amplification of PCR product (bp)a
SGI1 variants
Strain
209
Resistance genes in MDR region S005-S010 S020-S024 S024-S025 res-intI1
S024-S044
S. Rissen Z4
4793
3598
3579
1417
ND
P. mirabilis SC17
4793
NE
3579
1417
P. mirabilis SC42
4793
3598
NE
P. mirabilis XJF
3272
3598
3579
a
Type
Size (kb)
aadA2, floRc, tet (G), dfrA1, sul1
SGI1-I
42.48
ND
dfrA17, aadA5, blaPSE-1, sul1, mphA, mrx, mphR
SGI1-B2
45.93
NE
3399
None
SGI1-Z
24.30
1417
ND
aacCA5, aadA7, sul1
SGI1-PmABB 32.02
Primers are listed in Table S1. NE, negative. ND, not detected.
12
