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Subtyping Salmonella enterica serovar Derby with multilocus sequence typing (MLST) and clustered regularly interspaced short palindromic repeats (CRISPRs)
Food Control xxx (2016) 1e11
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Subtyping Salmonella enterica serovar Derby with multilocus sequence typing (MLST) and clustered regularly interspaced short palindromic repeats (CRISPRs) Huijuan Zheng 1, Yachen Hu 1, Qiuchun Li, Jing Tao, Yinqiang Cai, Yanan Wang, Jingwen Li, Zihao Zhou, Zhiming Pan*, Xinan Jiao** Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu 225009, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2016 Received in revised form 25 August 2016 Accepted 27 August 2016 Available online xxx
Salmonella Derby is one of the most prevalent serovars in pork and the most common serotype isolated from infants and toddlers. However, Salmonella Derby is also poorly understood, so we used Multilocus sequence typing (MLST) and Clustered regularly interspaced short palindromic repeats (CRISPRs) to subtype 100 Salmonella Derby isolates from pig farms, pig slaughterhouses, retail markets and humans that were collected during different years in Yangzhou, Jiangsu Province, China, in respect to the transmission of clonal groups of the serovar along the food chain. MLST analysis showed that two sequence type (ST) patterns (ST40 and ST71) were shared, and ST40 was the most common sequence type among isolates from four different sources. CRISPRs typing identified 32 different Derby CRISPR types (DCTs); ST40 and ST71 strains had 21 and 11 DCTs respectively, demonstrating the distinctiveness of the CRISPR regions among the isolates from the four sources during a seven-year period. It demonstrated that Salmonella Derby clones persisted in the same places and spread along the pork production chain. Overall, 100 spacers were analysed, including 61 for CRISPR1 (18 new) and 39 for CRISPR2 (17 new). Interestingly, we also found that the spacer arrangements were distinct between ST40 and ST71 strains, except for strain 13-S1. This analysis revealed that CRISPR genes are highly polymorphic even in the same serotype, which could be tremendously useful for bacterial subtyping during molecular epidemiological investigations. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Salmonella Derby Multilocus sequence typing Clustered regularly interspaced short palindromic repeats Molecular subtyping
1. Introduction Salmonella Derby was first isolated from humans who were infected after ingesting contaminated pork pies (Peckham & Savage, 1923). In 1946, an outbreak of gastro-enteritis among infants in a hospital was caused by Salmonella Derby; sixty eight children were involved in this outbreak and ten died (MUSHLN, 1948). Salmonella Derby is now one of the most frequent Salmonella enterica serovars found in pigs across the EU, and pork is a major source of foodborne salmonellosis in the EU and many other
* Corresponding author. Jiangsu Key Laboratory of Zoonosis, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu 225009, China. ** Corresponding author. Jiangsu Key Laboratory of Zoonosis, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu 225009, China. E-mail addresses: [email protected] (Z. Pan), [email protected] (X. Jiao). 1 These authors contributed equally to this work.
countries (Hauser et al., 2011; Kerouanton, Rose, Weill, Granier, & Denis, 2013). Salmonella Derby is the most common serotype isolated from infants and toddlers in China (Cui et al., 2009). Therefore, as one of the largest pork producing and consuming countries in the world, great attention should be paid to the prevalence of Salmonella Derby with respect to pig farms, pig slaughterhouses, retail markets and humans in China. The genetic diversity of Salmonella Derby strains has not been extensively investigated. Multilocus sequence typing (MLST), a recently developed methodology that requires minimal human input, has been used to type Salmonella strains (Kotetishvili, Stine, Kreger, Morris, & Sulakvelidze, 2002). The data available for Salmonella Derby (http://mlst.ucc.ie/mlst/mlst/dbs/Senterica/) indicate that the serovaris polyphyletic, having originated from more than one common ancestor, and it possesses several distantly related STs (Hauser et al., 2011). Clustered regularly interspaced short palindromic repeats (CRISPRs) are bacterial loci whose
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Please cite this article in press as: Zheng, H., et al., Subtyping Salmonella enterica serovar Derby with multilocus sequence typing (MLST) and clustered regularly interspaced short palindromic repeats (CRISPRs), Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.08.051
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H. Zheng et al. / Food Control xxx (2016) 1e11
dynamic nature has made them ideal targets for molecular subtyping (Shariat & Dudley, 2014). CRISPRs have been shown to be better for identification and for distinguishing between Salmonella outbreak strains/clones than PFGE (Liu, Barrangou, et al., 2011), with the additional benefits of being faster and less costly. This study was conducted to gain a better understanding of the clonality of Salmonella Derby and of the subtypes actually transmitted to humans from pigs via pork in China. For this purpose two different sequence-based approaches were applied to a set of 100 Salmonella Derby strains from pig farms, pig slaughterhouses, retail markets and humans.
2. Materials and methods 2.1. Serotyping and DNA extraction The 100 Salmonella Derby strains used for the analysis were collected in Yangzhou, Jiangsu Province, China from 2009 to 2015 (Table 1). Twenty-one Salmonella Derby strains were isolated from porcines, and 40 Salmonella Derby strains were isolated from pig slaughterhouses. Another 31 Salmonella Derby strains were isolated from pork. The remaining 8 Salmonella Derby strains were isolated from human gastroenteritis cases. All strains were serotyped according to the WhiteeKauffmanneLe Minor schemeby slide agglutination with O- and H-antigenspecific sera (SSI Diagnostika, Hiller, Denmark). Confirmed isolates were grown aerobically, overnight at 37 C in LB broth with shaking. Then genomic DNA was extracted with the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.
2.2. Multilocus sequence typing (MLST) All strains were further characterized by MLST using seven housekeeping genes aroC, dnaN, hemD, hisD, purE, sucA and thrA (Kidgell et al., 2002). The seven housekeeping gene sequences for each isolate were uploaded to the MLST database for comparison (http://mlst.ucc.ie/mlst/dbs/Senterica), which allowed us to determine the sequence type (ST). When necessary, new alleles and sequence types were submitted to the website. A minimum spanning tree was generated using BioNumerics software version 7.6 (AppliedMaths, Kortrijk, Belgium) to analyse the distribution of STs in Salmonella Derby strains from pig farms, pig slaughterhouses, retail markets and humans.
2.3. Clustered regularly interspaced short palindromic repeats (CRISPRs) CRISPR locus 1 (CRISPR1) was amplified using forward primer 0 0 0 A1 (5 -GTTGGTAAAAGAGCTGGCGA-3 ) and reverse primer A2 (5 0 GATGGACTTAGATTAGTTTC- 3 ). CRISPR locus 2 (CRISPR2) was 0 amplified using forward primer B1 (5 -CAATACCCTGATCCTTAACG0 0 0 3 ) and reverse primer B2 (5 -ATTGTTGCGATTATGTTGGT-3 ). The PCR amplicons were sequenced by Nanjing GenScript Biotech Co. (Nanjing, China). Spacers were identified for CRISPR1 and CRISPR2 using CRISPRfinder (http://crispr.u-psud.fr/Server/) (Grissa, Vergnaud, & Pourcel, 2008). Each spacer was queried against the Institut PasteurCRISPR database for Salmonella (http://www. pasteur.fr/recherche/genopole/PF8/crispr/CRISPRDB.html) to obtain the spacer and directrepeat (DR) names. For spacers or DRs that had no exact match in the DR and spacer dictionary, a new name was assigned in accordance with spacer nomenclature (Fabre et al., 2012).
Table 1 Salmonella Derby strains used for molecular analysis in this study. Strain no.
MLST (ST)
DCTs
Year of isolation
Source
09-S79 10-S57 11-S56 11-S58 11-S59 11-S60 11-S61 12-S40 12-S43 12-S44 13-S41 13-S42 13-S45 13-S46 13-S47 13-S48 13-S49 13-S50 13-S51 13-S52 13-S53 13-S54 13-S55 13-S24 13-S25 13-S28 13-S29 13-S1 13-S2 13-S3 13-S4 13-S5 14-S26 14-S27 13-S30 14-S31 14-S32 14-S33 14-S34 14-S35 14-S36 14-S37 14-S80 14-S81 14-S82 14-S83 14-S38 14-S39 14-S6 14-S7 14-S8 14-S9 14-S10 14-S11 14-S12 14-S13 14-S14 14-S15 14-S16 14-S17 14-S18 14-S19 14-S20 14-S21 14-S22 14-S23 14-S62 14-S63 14-S64 14-S65 14-S66 15-S67 15-S68 15-S69
40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 71 71 71 40 40 40 40 40 40 71 71 71 71 40 71 71 71 40 40 40 40 40 40 40 40 40 71 71 40 71 71 71 71 71 40 71 71 71 71 71 71 40 40 71 40 40 40 40 40
DCT24 DCT30 DCT26 DCT24 DCT29 DCT22 DCT24 DCT28 DCT22 DCT22 DCT24 DCT19 DCT18 DCT14 DCT16 DCT12 DCT14 DCT20 DCT17 DCT28 DCT22 DCT24 DCT22 DCT31 DCT24 DCT4 DCT5 DCT11 DCT17 DCT14 DCT14 DCT21 DCT22 DCT28 DCT4 DCT4 DCT4 DCT4 DCT28 DCT4 DCT4 DCT4 DCT27 DCT14 DCT14 DCT14 DCT18 DCT14 DCT14 DCT18 DCT15 DCT7 DCT10 DCT18 DCT2 DCT5 DCT8 DCT7 DCT6 DCT17 DCT9 DCT10 DCT1 DCT9 DCT10 DCT9 DCT28 DCT24 DCT3 DCT31 DCT32 DCT23 DCT31 DCT31
2009 2010 2011 2011 2011 2011 2011 2012 2012 2012 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2015 2015 2015
H R H R R R H S S S S S S S S S S S S S S S S S S S S R R R R R S S S S S S S S S S S H H H H H R R R R R R R R R R R R R R R R R R R R R R R F F F
Please cite this article in press as: Zheng, H., et al., Subtyping Salmonella enterica serovar Derby with multilocus sequence typing (MLST) and clustered regularly interspaced short palindromic repeats (CRISPRs), Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.08.051
H. Zheng et al. / Food Control xxx (2016) 1e11 Table 1 (continued ) Strain no.
MLST (ST)
DCTs
Year of isolation
Source
15-S70 15-S84 15-S85 15-S86 15-S87 15-S88 15-S89 15-S90 15-S91 15-S92 15-S93 15-S94 15-S95 15-S96 15-S97 15-S98 15-S99 15-S100 15-S71 15-S72 15-S73 15-S74 15-S75 15-S76 15-S77 15-S78
40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 71 71 40 40
DCT24 DCT22 DCT24 DCT24 DCT24 DCT24 DCT23 DCT24 DCT31 DCT24 DCT24 DCT24 DCT24 DCT24 DCT24 DCT24 DCT24 DCT24 DCT22 DCT13 DCT22 DCT24 DCT1 DCT1 DCT25 DCT24
2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015
F F F F F F F F F F F F F F F F F F R R R R R R R R
DCT: Derby CRISPR Type. F: Pig farm. S: Pig slaughterhouse. R: Retail market. H: human.
3. Results 3.1. Multilocus sequence typing (MLST) analysis A minimum spanning tree of all STs from the different sources was generated using BioNumerics version 7.6 (Fig. 1). Two different STs were found. ST40 (74 strains) was found in isolates from every source, and it was also the most prevalent. ST71 (26 strains) was found only in isolates from pig slaughterhouses and retail markets collected since 2013 (Table 1). The ST40 strains were found in isolates from every source in different years. ST40 differed from ST71 at all seven of the alleles profiled, so they displayed two distantly
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related STs within one serovar, despite being isolated from the same source during the same year. 3.2. Clustered regularly interspaced short palindromic repeats (CRISPRs) analysis We investigated the diversity of CRISPR spacers in a variety of Salmonella Derby strains. A total of 100 strains were analysed (Table 1). The DRs of both CRISPR loci had a conserved, 29 bp 0 0 consensus sequence 5 -CGGTTTATCCCCGCTGGCGCGGGGAACAC-3 , consistent with the result published by Fabre L et al. (Fabre et al., 2012). Sixty-one CRISPR spacers were found in the CRISPR1 region, and 39 CRISPR spacers were found in the CRISPR2 region. All spacers were 32 bp (92/100) or 33 bp (8/100) long. The 100 spacers included only five variant spacers (SNP variants). Eighteen and seventeen new CRISPR spacers were found in the CRISPR1 and CRISPR2 loci respectively, which did not match any spacers in the database (Table 2). Fig. 2 shows the differences in spacer arrangements between STs in CRISPR1 and CRISPR2. In CRISPR1, the number of spacers ranged from 11 to 61; for CRISPR2, the number of spacers ranged from 10 to 39. Interestingly, ST71 strains had more spacers than the ST40 strains, and only two spacers were common between ST40 and ST71 except in the 13-S1strain. Next, a minimum spanning tree was generated using BioNumerics software, version 7.6 (AppliedMaths, Kortrijk, Belgium) to analyse the distribution of Derby CRISPR types (DCTs) among pig farms, pig slaughterhouses, retail markets and humans. An interlinked dataset of different spacers revealed 32 DCTs among the 100 isolates, which grouped into two clusters (A-B) (Fig. 3). ST40 and ST71 strains had 21 (DCT12-DCT32) and 11 DCTs (DCT1-DCT11), respectively. An uneven distribution of the 32 DCTs was observed among isolates from the different sources. Four DCTs were found in pig farms, 14 DCTs were found in pig slaughterhouses, 24 DCTs were found in retail markets and 4 DCTs were found in humans. Some of the most prominent CRISPR types found included DCT24 (24 strains) followed by DCT14 (9 strains), DCT22 (9 strains) and DCT4 (8 strains). There is a separation between A (ST40) and B(ST71) strains, indicating that ST40 and ST71 strains have distinctly diverse CRISPR loci. Fig. 4 shows the distribution of Derby CRISPR types (DCTs) in different years. Some isolates from different years had the same CTs, such as DCT22, DCT24, DCT28and DCT31. While in some cases, isolates from the same year had different DCTs, particularly for 2013 and 2014. 4. Discussion
Fig. 1. Minimum spanning tree analysis of Salmonella Derby isolated from pig farms, pig slaughterhouses, retail markets and humans. Each circle represents one ST, and the area of the circle corresponds to the number of isolates. Different sources are represented by green, red, light blue and yellow, respectively. The number seven refers to the seven alleles that differed between ST40 and ST71. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Salmonella Derby is frequently isolated from pigs in Europe and the U.S. It ranked within the top 10 serovars identified from human salmonellosis cases in Europe in 2009 (Authority, 2015; BoleHribovsek et al., 2009; Foley, Lynne, & Nayak, 2008). Salmonella Derby is the most frequently detected Salmonella enterica serovar on pork in China (Cai et al., 2016). Therefore, this serovaris a major public health concern. This study provided an extensive characterization of Salmonella Derby isolated from different sources during different years in Jiangsu Province with the aims of understanding the transmission of Salmonella Derby from animal to human through the pork production and distribution chain, determining its clonal structure, evaluating different molecular subtyping methods for Salmonella Derby and determining the diversity among STs in the same serovar. MLST identified two Salmonella Derby clonal groups from different sources, which differed in all seven alleles profiled. ST40 was the most frequently detected genotype in this study, and Fig. 1 shows that ST40 was present in each source during all years. Therefore, we speculated that strains of this ST persist in the four
Please cite this article in press as: Zheng, H., et al., Subtyping Salmonella enterica serovar Derby with multilocus sequence typing (MLST) and clustered regularly interspaced short palindromic repeats (CRISPRs), Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.08.051
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H. Zheng et al. / Food Control xxx (2016) 1e11 Table 2 Salmonella Derby spacer name. CRISPR locus
Spacer namea (position)
Spacer DNA sequence (50 e30 )
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2
STM1var1 (1) Der1 (2) Der2 (3) Der3 (4) Der4 (5) Der5 (6) Der7 (7) Der8 (8) Der9 (9) Der10 (10) Der11 (11) Der25 (12) Der22 (13) Der23 (14) Der24 (15) Der27 (16) Der26 (17) Der13 (18) Der14 (19) Der15 (20) Der16 (21) Der17 (22) Der18 (23) Der19 (24) Der20 (25) Der22 (26) Der6 (27) Der28 (28) Der12 (29) Ago1 (30) Der55* (31) Cub1 (32) Der56* (33) Der57* (34) Cub4 (35) Cub5 (36) Der58* (37) AnkB2 (38) Der59* (39) Der60* (40) Ago6 (41) Ago7 (42) Ago8 (43) Ago9 (44) Der61* (45) Der62* (46) Der63* (47) Der64*(48) Der65*(49) Der66*(50) Der67*(51) Chol25 (52) HavB8 (53) Der68*(54) Der69*(55) Der70*(56) Der71*(57) Der72*(58) Mon42 (59) Ago2 (60) Ago3 (61) DerB1 (1) DerB2 (2) DerB3 (3) DerB4 (4) DerB5 (5) DerB8 (6) DerB9 (7) DerB10var1 (8) DerB11 (9) DerB12 (10) DerB6 (11) DerB13 (12) DerB7 (13)
TTTTCAGCCCTTGCCGACTGCGGAACGCCCCT GAGCTGGCGAACCTGATGGCGGGCGACGTGGA CCTGAATACTCGCAGCGATTTGCGCGATCTGG CATTGCGTTGTGAGCGCATTGAGTTGATTACT GGCGGCGCAGTCCAGCGCGGTAGCGGCAGAGA ATTTCGTCGTGATTTAAGTTGCAGTAGTCACG CTCCAGCTCGGCTTTTTGCTGCGCTGCGCGTT CTTCCCTTTTTTGGTTGCTGAGTCGGCCTCTC TTCTCCGGCATTTCCCCGTTGTTGAACGCAGC GGCATTTTAGATTTAGCGATCATGCTGCCAAC GATCATGCGCTGGTTAGTGCGGCGCTCAATCT GGCTGCGTCCGAACCGGTTATGTGCTGCCAGT CAGTAATCAATGCCCGATATAGAATTATCGGG CGCAGCGTTTTCGCCAGTACGGTGATATCGTT AACCCCTCAATAGACGGCGCTGATTCCGGGCG ACATGCCGAGTCGTGAGGCGACGGTTATAGAC TTCCAGTCGTCGCCGGACGTCATGTCAATCAG TTCAAAGTGAATTGCAAAAGCACAAATTGTCC CCCCTAAGCGCGGATCGTATAGTTTTTTACCC ATTGGAGATCACGTTATGAAAACAGCTATCGC AACTTTTGCTTAAAGCTTTCCATGTACTCATC TTTGTTTTTGTGCTGTCAGTGATGGCGGCGTG GTGAAGACTGCCTGTTTGCGGTGCGTGGCAAA AAGGCTCTGTCATTCCACCTTTGAGTGTTAGA ACTGGAAATACTCTGGAGTACCGGGATCAGGCG TTTGCTTCGTTATTCGATATCATATATATTCG TTTCGCTGCTTCAGCATTCCCCCTGTTAACAG CAGGTTAGATTTCTGCGCCTCCGCCATCGTCT TTTCATAATTCGCCTTATTGTTTAAATACAAA ATGGATGGCTCATATCCGGTTTCTGTTTCTGC GGTCTGACCGATGACGATTTCAAGATAAGCGA CAGCTCCAGATCGCGGGCACCGCTGGCGAACT CGCGACTGGTGGGCGTTCGCGGAGAAATTCGG GGGGCTATATGGAGCTAAACAGGATGTGGATT CGACGCAGATCACCAATCAGGCGGATATCAGC CTGATTTTGTCGAATAGTGGGCAGGACCGGAA TAACGTCCAAATAACAAATCAAAAATAAACGC TATTTAAATAAAAGTGTTCTTTATATTGGCGG GCAACCCATTACCAAAACTATTATCCCGTAAT AGCTCTTGCCCCGGAAATTAGGCTGATAGGTC TCAGGCTTGACGACAGTCTCCAGCCACTCCTG GACCGCGCGGGCTATCGTTCAGGACTATTTTT CTCTGTTACACCGTTATGCACAGACCACACAG GTTAATGCAAGCGCCATTAACAAGAAAATGAC TCGGCAATGATGTCGCTGCATATGAAGACTAC TTTCTGAGCTGGGCAAGCGCCGCCGCGCATGG TCTTCCTTAATATGGTGAATGTATGTCAATTA ACCGTCAGCCTCAAGCCCAGCTCTGGATATTT GCCTCCGGTGTAGCGTACAAAGAGCGACTTAA CGTCTCGGTGGCAAACTGTGGGAAAAAAACGG ACCATCCCAGCCGCCAGCGAGCTGCCTCATAT TGCATCGGTGTAGAGAATATCAAGTTCAACGC CGTGTGTTTGTTGTCGCAAGTGCTCGAGCCGA ACCGACACACTCTTTTACTGTCAACCAAATTT TGTTAAAACGGAATAAACTCATTAATTAACAC GGGGCTTGATTGCCTAAACAAACTTTTCTAGG GACTGGTTGAGAAGATCCGCCGCAGAGTCAGC GTGATGCTGAACAGGCTCAACGCAAACCTCAA TGTATAAAGCCGTTCTCAACGTCCTGTGGAAC TGGCGCCAGAAATATTCATGATCATCGGGATT TTCATATTCCAGAAAATGCCTGGGTGATGATC TACATCATCGGCTCAATGCGTAATTCTAAAAT CTGGGCGGCAACTTCACACGTCACGATGGTTA GTGAGTGAACGTAATGTCGAATGACGGCGAGG GACGACATGCAAAAACGCCTGGCGACGATGTT GTTCTATTTAACATAATGACCGTTACGCGCAT TATCCACTCCGGCACACATCACCGCTCGTCAA CACGTGAACCGATTTACCCGGCAGGCCGTTGA ATCATTTCAGCGGAGTATTTCAAATTGTCAGG CTTGCGGGGCGTTCAGGTCAGTAATGATGCGG AGCTTCCGCCCCCCGCCCCGGCCCGGCGTTGT GTTTGCCCCTGCTATGAAGAACATTCTCACTC TACAAGCGGCTCTGGTCGCTGATGCAAAAAGA CTGGCGCGTAACCTGCCGTTTGCCTCATATCA
Please cite this article in press as: Zheng, H., et al., Subtyping Salmonella enterica serovar Derby with multilocus sequence typing (MLST) and clustered regularly interspaced short palindromic repeats (CRISPRs), Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.08.051
H. Zheng et al. / Food Control xxx (2016) 1e11
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Table 2 (continued ) CRISPR locus
Spacer namea (position)
Spacer DNA sequence (50 e30 )
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
HilB2 (14) HilB3 (15) HilB4 (16) DerB49*(17) Ucc4var1 (18) HilB6 (19) DerB50*(20) DerB51*(21) DerB52*(22) DerB53*(23) DerB54*(24) AltB11var1 (25) DerB55*(26) DerB56*(27) DerB57*(28) Aba12 (29) DerB58*(30) DerB59*(31) DerB60*(32) DerB61*(33) DerB62*(34) DerB63*(35) StpB3 (36) DerB64*(37) DerB65*(38) CholB75var1 (39)
TGGGGAGCGTTGAGTCAAACACGACCCAGCAG ATAGAACTGCTGGGCCGGAACCTCTTCCCCTG GCAATACTGGAAAGACCCGCTCCCAGAAACGA TTGAACCAGAATATTGCCGCTGCGCCTGCGGT CGCAGCATCACAAGTTTGTATTCCTGCCAGGA CCGGGAGGCGGAATGCAGCAAAAGTAAAAACCG GGTCATTCGGCCAGCTCCCGTCCGCTCAAAAT ACCCCGTCAGGCAGCGAGTCGTAATTGAATTT GCATGAAGCTGGTAACGCTGACGCAGGACAGGC CCGTCCCACTCTATACCGTATTCGGCTGCAAG GTGTATCCGGAAGAGATAGACGGTAGTGACCA ACATATGAATAGGCATAGCCGAATGCCGGGGG ACGGAATCTCCTGATTTTGGCGCGCTGGAATC TATTTGCACCGCAGAATGCCGCGCCGCCGTTGC GACGCTAACAATTTTAAATTACTGTTTAAATT GTTGATCAATCTGGAATGATTTGTCTCACCGA TGCTCATCGATTGGTCGATCGATATAATCATT GCGAGCGCGCCAGCGCTGGCCATGATGATTTT CAATAATATTGAGTATTTTTTCTTTCGTGTTG GATAGCGTTTGATATGCGGCAGACGGCCCCTC GTTCCAACGCCTCGCGCAGTAAGGGCGGCAAC GTGAATTTGGTAATAGTGTTCATGGGTTAGCC TTCTCGGCGCGGCGCAATATCCCGGCGCACGC TCGTCCGTAATCACGGCCACCACGCCTGACGC GGTACTGATTTGGAGCTAATGCAATCAGAAGC TTCGTTGTGTTGGACACCACCGGATCGGCGCT
a
An asterisk shows a novel spacer identified in the current study.
sources and were continually propagated. Moreover, ST40 has been widely found in pigs and pork from Europe and the United States and in humans from Asia and Europe (Li et al., 2014). In this study, ST71 was only found in pig slaughterhouses and retail markets from 2013 to 2015, but it was also found in humans from Germany and Denmark (Hauser et al., 2011; Litrup et al., 2010). Cai et al. and Li et al. found only two sequence types (ST40 and ST71) of Salmonella Derby in Yangzhou, Jiangsu Province, China (Cai et al., 2016; Li et al., 2014). By searching the MLST database (http://mlst.ucc.ie/mlst/dbs/ Senterica), we found 13 different sequence types (STs) in Salmonella Derby, including ST15, ST39, ST40, ST71, ST72, ST678, ST682, ST683, ST695, ST774, ST813, ST1326 and ST1585. The serovar originates from more than one common ancestor, and it possesses several distantly related STs. Lan and coworkers' analysis of multilocus sequence typing (MLST) data showed a high level of recombination within the subspecies of S. enterica (Lan, Reeves, & Octavia, 2009). Consistent with this, several sequence types (STs) were detected in Salmonella Derby. However, for investigating outbreaks, distinguishing between closely related isolates based on MLST of housekeeping genes may be not be possible, due to their high sequence conservation (Ross & Heuzenroeder, 2005). Therefore, this method may not be suitable for closely related isolates within a serovar. Fig. 2 shows that the spacers of CRISPR1 were variable in ST40 strains, but the spacers of CRISPR2 were strictly conserved. However, strains with ST71 showed a high degree of CRISPR polymorphism in the CRISPR1 and CRISPR2 loci. The variability of CRISPRs spacer content is due to the duplication of single spacer-DR units or the deletion of single or contiguous spacer-DR units (Fabre et al., 2012). This microvariation resulted in 25 CRISPR1 types and 6 CRISPR2 types or 32 CRISPR1-CRISPR2 combined types, thus providing greater resolution than other subtyping methods, such as MLST. 13-S48, 14-S66, 15-S67 and15-S89 strains had a deletion of seventeen spacers, four spacers and one spacer adjacent to the leader array. In contrast, C. Pourcel et al. suggested that a polarity exists, in that new spacers are always added to the leader sequence of the CRISPR array (Pourcel, Salvignol, & Vergnaud, 2005). Spacer multiplication or loss occurred in 32 different CRISPR types. Tyson
GW et al. reported that spacer loss and duplication, makes CRISPR elements some of the fastest evolving loci in bacteria (Tyson GW, 2008). In addition, we found that the spacers of ST71 isolates were larger than those of ST40 in CRISPR2. The length of a CRISPR array is dependent on the number of spacers and varies dramatically among different strains (Grissa, Vergnaud, & Pourcel, 2007). Nikki Shariat et al. reported that the CRISPR spacer array is constitutively transcribed into a precursor CRISPR RNA (pre-crRNA) that is cleaved by specific Cas proteins and further processed into mature, small interfering crRNAs. These crRNAsare typically comprised of the spacer flanked on either side by portions of the DRs (Shariat & Dudley, 2014). In addition, Garneau et al suggested that mature crRNAs guide the Cas-crRNA ribonucleoprotein complex to complementary nucleic acids, typically in invading bacteriophages or plasmids (Garneau et al., 2010). The isolates obtained the new spacer through selection, possibly providing an advantage via enhanced virulence and growth. This may suggest significant differences in virulence between ST40 and ST71strains. Salmonella Derby CRISPR typing (DCT) identified 32DCTs among the 100 isolates that included 4 DCTs from pig farms, 14 DCTs from pig slaughterhouses, 24 DCTs from retail markets and 4 DCTs from humans. Therefore, CRISPR typing was shown to be better at distinguishing Salmonella Derby strains than MLST, as observed previously (Liu, Kariyawasam, et al., 2011). This result indicated that the Salmonella Derby CRISPRs types (DCTs) were more varied in pig slaughterhouses and downstream retail markets. This diversity maybe attributed to the broad origins of the pigs in slaughterhouses. The Fig. 3 B cluster DCT24 had the same CTs among isolates from all four different sources; DCT22 and DCT31 were both among isolates from pig farms, slaughterhouses and downstream retail markets. DCT14 and DCT18 showed that some isolates from pork had the same DCTs as isolates from clinical patients. Therefore, Salmonella Derby was transferred along the pork production and distribution chain from the slaughterhouse to retail markets, where people could be infected via pork contaminated with Salmonella Derby (Yi et al., 2014). Moreover, sows and piglets infected with Salmonella Derby can be asymptomatic despite its presence in
Please cite this article in press as: Zheng, H., et al., Subtyping Salmonella enterica serovar Derby with multilocus sequence typing (MLST) and clustered regularly interspaced short palindromic repeats (CRISPRs), Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.08.051
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H. Zheng et al. / Food Control xxx (2016) 1e11
Fig. 2. Graphic representation of spacers across the two CRISPR loci for a variety of Salmonella Derby strains. Repeats are not included; only spacers are represented. Each spacer is represented by a combination of a character in a particular font colour, on a particular background colour. The colour combination allows each spacer to be uniquely represented, whereby squares with similar colour schemes (combination of character colour and background colour) represent identical spacers and different colour combinations represent distinguishable spacers. The spacers are aligned, and the gaps represent the absence of a particular spacer. Above, CRISPR1; Below, CRISPR2.
Please cite this article in press as: Zheng, H., et al., Subtyping Salmonella enterica serovar Derby with multilocus sequence typing (MLST) and clustered regularly interspaced short palindromic repeats (CRISPRs), Food Control (2016), http://dx.doi.org/10.1016/j.foodcont.2016.08.051
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Fig. 2. (continued).
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Fig. 3. Minimum spanning tree analysis of Salmonella Derby isolated from pig farms, pig slaughterhouses, retail markets and humans. Each circle represents one DCT, and the area of the circle corresponds to the number of isolates. Different sources are represented by green, red, light blue and yellow, respectively. Two clusters (AeB) were identified. The numbers correspond to the different Salmonella Derby CRISPR types (DCTs). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Minimum spanning tree analysis of Salmonella Derby isolated during different years. Each circle represents one Salmonella Derby CRISPR type (DCT), and the area of the circle corresponds to the number of isolates. Different years are represented by dark green, brown, gold, bluish green, light blue, green and red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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many tissues, which can then contaminate the pork when they are slaughtered. This jeopardizes food safety (Matiasovic et al., 2014). Therefore, to prevent this serovar from entering the food chain, measures for its control should be taken at the pig farm (Hauser et al., 2011). Fig. 4 shows that some isolates from different years displayed the same DCTs or were closely related, such as DCT22, 24, 28 and 31. This demonstrated that Salmonella Derby clones persisted in the same places and spread along the pork production chain (Yi et al., 2014). The Fig. 3 A cluster shows that the diversity was greater among the ST71 strains, and the Fig. 4 demonstrates that they were only found from 2013 to 2015. This occurrence maybe due to initial isolate diversity or introduction of exotic strains. The spacer arrangements were similar among STs in the 2 CRISPR loci in Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Senftenberg (Liu, Kariyawasam, et al., 2011; Shariat et al., 2013; Yi et al., 2014). However, Fabre et al. reported that the spacer content differed greatly in different STs of Salmonella enterica serovar. This is consistent with reports by Newportas well as our findings that only two spacers are the same in the spacer arrangements of ST40 and ST71. Moreover, Fabre et al. also reported that spacer content was strongly correlated with the population structure defined by MLST (Fabre et al., 2012). All seven allele profiles differed between ST40 and ST71 and the particular spacer arrangements, we observed enormous diversity in the genomes of ST40 and ST71 strains within the same serotype. As nextgeneration sequencing has rapidly become cheaper and more amenable to standard laboratory practices, a priority is to promote whole genome sequencing (WGS) as a rapid and accurate approach to characterize microbial agents for epidemiological surveillance and outbreak detection (Aanensen et al., 2016; Deng et al., 2015). This will allow correlation of bacterial genome subtypes with a source host in order to quickly speculate on the bacterial origin (Ashton et al., 2016; Deng et al., 2015). Using whole genome sequencing, we could also systemically analyse the genome sequences of ST40 and ST71 strains and from their genetic information gain insight into the mechanism of their pathogenicity. 5. Conclusion In summary, we used MLST and Clustered regularly interspaced short palindromic repeats (CRISPR) for subtyping 100 Salmonella Derby isolates from pig farms, pig slaughterhouses, retail markets and humans in Yangzhou, Jiangsu Province, China during a sevenyear period. The results of this study illustrated CRISPRs loci evolve much faster than housekeeping genes. Therefore, CRISPRs typing provided superior discriminatory power over MLST typing for distinguishing different sources of Salmonella Derby. This study also showed that two major Salmonella Derby clones are dominant in pigs and humans in China. Some isolates from the four sources were demonstrated to be genetically similar by MLST and CRISPRs typing and were transferred along pork production chain from the pig farms to the slaughterhouses and retail markets. Contaminated pork has been identified as a vehicle for Salmonella Derby and consequently presents a risk to human health. Considering this, enough attention should be paid to prevent the further dissemination of Salmonella Derby. Acknowledgments This work was supported by the Special Fund for Agroscientific Research in the Public Interest (201403054), the National Natural Science Foundation of China (nos. 31320103907 and 31230070), the Project for Agricultural Products Quality and Safety Supervision (Risk Assessment) (18162130109236), the Program for New
Century Excellent Talents in University (NCET-12-0745), the “Six Talent Peaks Program” of Jiangsu Province (NY-028), the Yangzhou University Science and Technology Innovation Team (2016), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Aanensen, D. M., Feil, E. J., Holden, M. T., Dordel, J., Yeats, C. A., Fedosejev, A., et al. (2016). Whole-genome sequencing for routine pathogen surveillance in public health: A population snapshot of invasive Staphylococcus aureus in Europe. MBio, 7(3). Ashton, P. M., Nair, S., Peters, T. M., Bale, J. A., Powell, D. G., Painset, A., et al. (2016). Identification of Salmonella for public health surveillance using whole genome sequencing. PeerJ, 4, e1752. Authority, E. F. S. (2015). .The European Union summary report on trends and sources of zoonoses, Zionistic agents and food-borne outbreaks in 2013. MESA Journal, 13(1). l, Mariann, Davies, Robert, et al. (2009). Analysis of Bole-Hribovsek, Vojislava, Chrie the baseline survey on the prevalence of Salmonella in holdings with breeding pigs in the EU, 2008. EFSA Journal, 7(12), 1377. Cai, Y., Tao, J., Jiao, Y., Fei, X., Zhou, L., Wang, Y., et al. (2016). Phenotypic characteristics and genotypic correlation between Salmonella isolates from a slaughterhouse and retail markets in Yangzhou, China. International Journal of Food Microbiology, 222, 56e64. Cui, S., Li, J., Sun, Z., Hu, C., Jin, S., Li, F., et al. (2009). Characterization of Salmonella enterica isolates from infants and toddlers in Wuhan, China. Journal of Antimicrobial Chemotherapy, 63(1), 87e94. Deng, X., Shariat, N., Driebe, E. M., Roe, C. C., Tolar, B., Trees, E., et al. (2015). Comparative analysis of subtyping methods against a whole-genomesequencing standard for Salmonella enterica serotype Enteritidis. Journal of Clinical Microbiology, 53(1), 212e218. Fabre, L., Zhang, J., Guigon, G., Le Hello, S., Guibert, V., Accou-Demartin, M., et al. (2012). CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections. PLoS One, 7(5), e36995. Foley, S. L., Lynne, A. M., & Nayak, R. (2008). Salmonella challenges: Prevalence in swine and poultry and potential pathogenicity of such isolates. Journal of Animal Science, 86(14 Suppl), E149eE162. Garneau, J. E., Dupuis, M.-E., Villion, M., Romero, D. A., Barrangou, R., Boyaval, P., et al. (2010). The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 468(7320), 67e71. Grissa, I., Vergnaud, G., & Pourcel, C. (2007). The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats. BMC Bioinformatics, 8, 172. Grissa, I., Vergnaud, G., & Pourcel, C. (2008). CRISPRcompar: A website to compare clustered regularly interspaced short palindromic repeats. Nucleic Acids Research, 36(Web Server issue), W145eW148. Hauser, E., Hebner, F., Tietze, E., Helmuth, R., Junker, E., Prager, R., et al. (2011). Diversity of Salmonella enterica serovar Derby isolated from pig, pork and humans in Germany. International Journal of Food Microbiology, 151(2), 141e149. Kerouanton, A., Rose, V., Weill, F. X., Granier, S. A., & Denis, M. (2013). Genetic diversity and antimicrobial resistance profiles of Salmonella enterica serotype derby isolated from pigs, pork, and humans in France. Foodborne Pathogens and Disease, 10(11), 977e984. Kidgell, C., Reichard, U., Wain, J., Linz, B., Torpdahl, M., Dougan, G., et al. (2002). Salmonella typhi, the causative agent of typhoid fever, is approximately 50,000 years old. Infection, Genetics and Evolution, 2(1), 39e45. Kotetishvili, M., Stine, O. C., Kreger, A., Morris, J. G., & Sulakvelidze, A. (2002). .Multilocus sequence typing for characterization of clinical and environmental Salmonella strains. Journal of Clinical Microbiology, 40(5), 1626e1635. Lan, R., Reeves, P. R., & Octavia, S. (2009). Population structure, origins and evolution of major Salmonella enterica clones. Infection, Genetics and Evolution, 9(5), 996e1005. Li, Y. C., Pan, Z. M., Kang, X. L., Geng, S. Z., Liu, Z. Y., Cai, Y. Q., et al. (2014). Prevalence, characteristics, and antimicrobial resistance patterns of Salmonella in retail pork in Jiangsu province, eastern China. Journal of Food Protection, 77(2), 236e245. Litrup, E., Torpdahl, M., Malorny, B., Huehn, S., Christensen, H., & Nielsen, E. M. (2010). Association between phylogeny, virulence potential and serovars of Salmonella enterica. Infection, Genetics and Evolution, 10(7), 1132e1139. Liu, F., Barrangou, R., Gerner-Smidt, P., Ribot, E. M., Knabel, S. J., & Dudley, E. G. (2011). Novel virulence gene and clustered regularly interspaced short palindromic repeat (CRISPR) multilocus sequence typing scheme for subtyping of the major serovars of Salmonella enterica subsp. enterica. Applied and Environmental Microbiology, 77(6), 1946e1956. Liu, F., Kariyawasam, S., Jayarao, B. M., Barrangou, R., Gerner-Smidt, P., Ribot, E. M., et al. (2011). Subtyping Salmonella enterica serovar enteritidis isolates from different sources by using sequence typing based on virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs). Applied and Environmental Microbiology, 77(13), 4520e4526. Matiasovic, J., Stepanova, H., Kudlackova, H., Havlickova, H., Sisak, F., Rychlik, I., et al. (2014). Immune response of pigs to Salmonella enterica serovar Derby and Typhimurium infections. Veterinary Microbiology, 170(3e4), 284e290.
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H. Zheng et al. / Food Control xxx (2016) 1e11 MUSHLN, R. (1948). An outbreak of gastro-enteritis due to Salmonella derby. Journal of Hygiene (Cambridge), 46, 151. Peckham, C. F., & Savage, W. G. (1923). An outbreak of pork pie poisoning in Derby. Journal of Hygiene (London), 22(1), 69e76. Pourcel, C., Salvignol, G., & Vergnaud, G. (2005). CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology, 151(Pt 3), 653e663. Ross, I. L., & Heuzenroeder, M. W. (2005). Discrimination within phenotypically closely related definitive types of Salmonella enterica serovar typhimurium by the multiple amplification of phage locus typing technique. Journal of Clinical Microbiology, 43(4), 1604e1611. Shariat, N., DiMarzio, M. J., Yin, S., Dettinger, L., Sandt, C. H., Lute, J. R., et al. (2013).
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The combination of CRISPR-MVLST and PFGE provides increased discriminatory power for differentiating human clinical isolates of Salmonella enterica subsp. enterica serovar Enteritidis. Food Microbiology, 34(1), 164e173. Shariat, N., & Dudley, E. G. (2014). CRISPRs: Molecular signatures used for pathogen subtyping. Applied and Environmental Microbiology, 80(2), 430e439. Tyson, G. W., & Banfield, J. F. (2008). Rapidly evolving Crispers implicated in acquired resistance of microorganisms to viruses. Environmental Microbiology, 10(1), 200e207. Yi, S., Xie, J., Liu, N., Li, P., Xu, X., Li, H., et al. (2014). Emergence and prevalence of non-H2S-producing Salmonella enterica serovar Senftenberg isolates belonging to novel sequence type 1751 in China. Journal of Clinical Microbiology, 52(7), 2557e2565.
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Subtyping Salmonella enterica serovar Derby with multilocus sequence typing (MLST) and clustered regularly interspaced short palindromic repeats (CRISPRs) Huijuan Zheng 1, Yachen Hu 1, Qiuchun Li, Jing Tao, Yinqiang Cai, Yanan Wang, Jingwen Li, Zihao Zhou, Zhiming Pan*, Xinan Jiao** Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu 225009, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2016 Received in revised form 25 August 2016 Accepted 27 August 2016 Available online xxx
Salmonella Derby is one of the most prevalent serovars in pork and the most common serotype isolated from infants and toddlers. However, Salmonella Derby is also poorly understood, so we used Multilocus sequence typing (MLST) and Clustered regularly interspaced short palindromic repeats (CRISPRs) to subtype 100 Salmonella Derby isolates from pig farms, pig slaughterhouses, retail markets and humans that were collected during different years in Yangzhou, Jiangsu Province, China, in respect to the transmission of clonal groups of the serovar along the food chain. MLST analysis showed that two sequence type (ST) patterns (ST40 and ST71) were shared, and ST40 was the most common sequence type among isolates from four different sources. CRISPRs typing identified 32 different Derby CRISPR types (DCTs); ST40 and ST71 strains had 21 and 11 DCTs respectively, demonstrating the distinctiveness of the CRISPR regions among the isolates from the four sources during a seven-year period. It demonstrated that Salmonella Derby clones persisted in the same places and spread along the pork production chain. Overall, 100 spacers were analysed, including 61 for CRISPR1 (18 new) and 39 for CRISPR2 (17 new). Interestingly, we also found that the spacer arrangements were distinct between ST40 and ST71 strains, except for strain 13-S1. This analysis revealed that CRISPR genes are highly polymorphic even in the same serotype, which could be tremendously useful for bacterial subtyping during molecular epidemiological investigations. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Salmonella Derby Multilocus sequence typing Clustered regularly interspaced short palindromic repeats Molecular subtyping
1. Introduction Salmonella Derby was first isolated from humans who were infected after ingesting contaminated pork pies (Peckham & Savage, 1923). In 1946, an outbreak of gastro-enteritis among infants in a hospital was caused by Salmonella Derby; sixty eight children were involved in this outbreak and ten died (MUSHLN, 1948). Salmonella Derby is now one of the most frequent Salmonella enterica serovars found in pigs across the EU, and pork is a major source of foodborne salmonellosis in the EU and many other
* Corresponding author. Jiangsu Key Laboratory of Zoonosis, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu 225009, China. ** Corresponding author. Jiangsu Key Laboratory of Zoonosis, Yangzhou University, 48 East Wenhui Road, Yangzhou, Jiangsu 225009, China. E-mail addresses: [email protected] (Z. Pan), [email protected] (X. Jiao). 1 These authors contributed equally to this work.
countries (Hauser et al., 2011; Kerouanton, Rose, Weill, Granier, & Denis, 2013). Salmonella Derby is the most common serotype isolated from infants and toddlers in China (Cui et al., 2009). Therefore, as one of the largest pork producing and consuming countries in the world, great attention should be paid to the prevalence of Salmonella Derby with respect to pig farms, pig slaughterhouses, retail markets and humans in China. The genetic diversity of Salmonella Derby strains has not been extensively investigated. Multilocus sequence typing (MLST), a recently developed methodology that requires minimal human input, has been used to type Salmonella strains (Kotetishvili, Stine, Kreger, Morris, & Sulakvelidze, 2002). The data available for Salmonella Derby (http://mlst.ucc.ie/mlst/mlst/dbs/Senterica/) indicate that the serovaris polyphyletic, having originated from more than one common ancestor, and it possesses several distantly related STs (Hauser et al., 2011). Clustered regularly interspaced short palindromic repeats (CRISPRs) are bacterial loci whose
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dynamic nature has made them ideal targets for molecular subtyping (Shariat & Dudley, 2014). CRISPRs have been shown to be better for identification and for distinguishing between Salmonella outbreak strains/clones than PFGE (Liu, Barrangou, et al., 2011), with the additional benefits of being faster and less costly. This study was conducted to gain a better understanding of the clonality of Salmonella Derby and of the subtypes actually transmitted to humans from pigs via pork in China. For this purpose two different sequence-based approaches were applied to a set of 100 Salmonella Derby strains from pig farms, pig slaughterhouses, retail markets and humans.
2. Materials and methods 2.1. Serotyping and DNA extraction The 100 Salmonella Derby strains used for the analysis were collected in Yangzhou, Jiangsu Province, China from 2009 to 2015 (Table 1). Twenty-one Salmonella Derby strains were isolated from porcines, and 40 Salmonella Derby strains were isolated from pig slaughterhouses. Another 31 Salmonella Derby strains were isolated from pork. The remaining 8 Salmonella Derby strains were isolated from human gastroenteritis cases. All strains were serotyped according to the WhiteeKauffmanneLe Minor schemeby slide agglutination with O- and H-antigenspecific sera (SSI Diagnostika, Hiller, Denmark). Confirmed isolates were grown aerobically, overnight at 37 C in LB broth with shaking. Then genomic DNA was extracted with the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.
2.2. Multilocus sequence typing (MLST) All strains were further characterized by MLST using seven housekeeping genes aroC, dnaN, hemD, hisD, purE, sucA and thrA (Kidgell et al., 2002). The seven housekeeping gene sequences for each isolate were uploaded to the MLST database for comparison (http://mlst.ucc.ie/mlst/dbs/Senterica), which allowed us to determine the sequence type (ST). When necessary, new alleles and sequence types were submitted to the website. A minimum spanning tree was generated using BioNumerics software version 7.6 (AppliedMaths, Kortrijk, Belgium) to analyse the distribution of STs in Salmonella Derby strains from pig farms, pig slaughterhouses, retail markets and humans.
2.3. Clustered regularly interspaced short palindromic repeats (CRISPRs) CRISPR locus 1 (CRISPR1) was amplified using forward primer 0 0 0 A1 (5 -GTTGGTAAAAGAGCTGGCGA-3 ) and reverse primer A2 (5 0 GATGGACTTAGATTAGTTTC- 3 ). CRISPR locus 2 (CRISPR2) was 0 amplified using forward primer B1 (5 -CAATACCCTGATCCTTAACG0 0 0 3 ) and reverse primer B2 (5 -ATTGTTGCGATTATGTTGGT-3 ). The PCR amplicons were sequenced by Nanjing GenScript Biotech Co. (Nanjing, China). Spacers were identified for CRISPR1 and CRISPR2 using CRISPRfinder (http://crispr.u-psud.fr/Server/) (Grissa, Vergnaud, & Pourcel, 2008). Each spacer was queried against the Institut PasteurCRISPR database for Salmonella (http://www. pasteur.fr/recherche/genopole/PF8/crispr/CRISPRDB.html) to obtain the spacer and directrepeat (DR) names. For spacers or DRs that had no exact match in the DR and spacer dictionary, a new name was assigned in accordance with spacer nomenclature (Fabre et al., 2012).
Table 1 Salmonella Derby strains used for molecular analysis in this study. Strain no.
MLST (ST)
DCTs
Year of isolation
Source
09-S79 10-S57 11-S56 11-S58 11-S59 11-S60 11-S61 12-S40 12-S43 12-S44 13-S41 13-S42 13-S45 13-S46 13-S47 13-S48 13-S49 13-S50 13-S51 13-S52 13-S53 13-S54 13-S55 13-S24 13-S25 13-S28 13-S29 13-S1 13-S2 13-S3 13-S4 13-S5 14-S26 14-S27 13-S30 14-S31 14-S32 14-S33 14-S34 14-S35 14-S36 14-S37 14-S80 14-S81 14-S82 14-S83 14-S38 14-S39 14-S6 14-S7 14-S8 14-S9 14-S10 14-S11 14-S12 14-S13 14-S14 14-S15 14-S16 14-S17 14-S18 14-S19 14-S20 14-S21 14-S22 14-S23 14-S62 14-S63 14-S64 14-S65 14-S66 15-S67 15-S68 15-S69
40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 71 71 71 40 40 40 40 40 40 71 71 71 71 40 71 71 71 40 40 40 40 40 40 40 40 40 71 71 40 71 71 71 71 71 40 71 71 71 71 71 71 40 40 71 40 40 40 40 40
DCT24 DCT30 DCT26 DCT24 DCT29 DCT22 DCT24 DCT28 DCT22 DCT22 DCT24 DCT19 DCT18 DCT14 DCT16 DCT12 DCT14 DCT20 DCT17 DCT28 DCT22 DCT24 DCT22 DCT31 DCT24 DCT4 DCT5 DCT11 DCT17 DCT14 DCT14 DCT21 DCT22 DCT28 DCT4 DCT4 DCT4 DCT4 DCT28 DCT4 DCT4 DCT4 DCT27 DCT14 DCT14 DCT14 DCT18 DCT14 DCT14 DCT18 DCT15 DCT7 DCT10 DCT18 DCT2 DCT5 DCT8 DCT7 DCT6 DCT17 DCT9 DCT10 DCT1 DCT9 DCT10 DCT9 DCT28 DCT24 DCT3 DCT31 DCT32 DCT23 DCT31 DCT31
2009 2010 2011 2011 2011 2011 2011 2012 2012 2012 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2013 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2015 2015 2015
H R H R R R H S S S S S S S S S S S S S S S S S S S S R R R R R S S S S S S S S S S S H H H H H R R R R R R R R R R R R R R R R R R R R R R R F F F
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H. Zheng et al. / Food Control xxx (2016) 1e11 Table 1 (continued ) Strain no.
MLST (ST)
DCTs
Year of isolation
Source
15-S70 15-S84 15-S85 15-S86 15-S87 15-S88 15-S89 15-S90 15-S91 15-S92 15-S93 15-S94 15-S95 15-S96 15-S97 15-S98 15-S99 15-S100 15-S71 15-S72 15-S73 15-S74 15-S75 15-S76 15-S77 15-S78
40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 40 71 71 40 40
DCT24 DCT22 DCT24 DCT24 DCT24 DCT24 DCT23 DCT24 DCT31 DCT24 DCT24 DCT24 DCT24 DCT24 DCT24 DCT24 DCT24 DCT24 DCT22 DCT13 DCT22 DCT24 DCT1 DCT1 DCT25 DCT24
2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015
F F F F F F F F F F F F F F F F F F R R R R R R R R
DCT: Derby CRISPR Type. F: Pig farm. S: Pig slaughterhouse. R: Retail market. H: human.
3. Results 3.1. Multilocus sequence typing (MLST) analysis A minimum spanning tree of all STs from the different sources was generated using BioNumerics version 7.6 (Fig. 1). Two different STs were found. ST40 (74 strains) was found in isolates from every source, and it was also the most prevalent. ST71 (26 strains) was found only in isolates from pig slaughterhouses and retail markets collected since 2013 (Table 1). The ST40 strains were found in isolates from every source in different years. ST40 differed from ST71 at all seven of the alleles profiled, so they displayed two distantly
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related STs within one serovar, despite being isolated from the same source during the same year. 3.2. Clustered regularly interspaced short palindromic repeats (CRISPRs) analysis We investigated the diversity of CRISPR spacers in a variety of Salmonella Derby strains. A total of 100 strains were analysed (Table 1). The DRs of both CRISPR loci had a conserved, 29 bp 0 0 consensus sequence 5 -CGGTTTATCCCCGCTGGCGCGGGGAACAC-3 , consistent with the result published by Fabre L et al. (Fabre et al., 2012). Sixty-one CRISPR spacers were found in the CRISPR1 region, and 39 CRISPR spacers were found in the CRISPR2 region. All spacers were 32 bp (92/100) or 33 bp (8/100) long. The 100 spacers included only five variant spacers (SNP variants). Eighteen and seventeen new CRISPR spacers were found in the CRISPR1 and CRISPR2 loci respectively, which did not match any spacers in the database (Table 2). Fig. 2 shows the differences in spacer arrangements between STs in CRISPR1 and CRISPR2. In CRISPR1, the number of spacers ranged from 11 to 61; for CRISPR2, the number of spacers ranged from 10 to 39. Interestingly, ST71 strains had more spacers than the ST40 strains, and only two spacers were common between ST40 and ST71 except in the 13-S1strain. Next, a minimum spanning tree was generated using BioNumerics software, version 7.6 (AppliedMaths, Kortrijk, Belgium) to analyse the distribution of Derby CRISPR types (DCTs) among pig farms, pig slaughterhouses, retail markets and humans. An interlinked dataset of different spacers revealed 32 DCTs among the 100 isolates, which grouped into two clusters (A-B) (Fig. 3). ST40 and ST71 strains had 21 (DCT12-DCT32) and 11 DCTs (DCT1-DCT11), respectively. An uneven distribution of the 32 DCTs was observed among isolates from the different sources. Four DCTs were found in pig farms, 14 DCTs were found in pig slaughterhouses, 24 DCTs were found in retail markets and 4 DCTs were found in humans. Some of the most prominent CRISPR types found included DCT24 (24 strains) followed by DCT14 (9 strains), DCT22 (9 strains) and DCT4 (8 strains). There is a separation between A (ST40) and B(ST71) strains, indicating that ST40 and ST71 strains have distinctly diverse CRISPR loci. Fig. 4 shows the distribution of Derby CRISPR types (DCTs) in different years. Some isolates from different years had the same CTs, such as DCT22, DCT24, DCT28and DCT31. While in some cases, isolates from the same year had different DCTs, particularly for 2013 and 2014. 4. Discussion
Fig. 1. Minimum spanning tree analysis of Salmonella Derby isolated from pig farms, pig slaughterhouses, retail markets and humans. Each circle represents one ST, and the area of the circle corresponds to the number of isolates. Different sources are represented by green, red, light blue and yellow, respectively. The number seven refers to the seven alleles that differed between ST40 and ST71. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Salmonella Derby is frequently isolated from pigs in Europe and the U.S. It ranked within the top 10 serovars identified from human salmonellosis cases in Europe in 2009 (Authority, 2015; BoleHribovsek et al., 2009; Foley, Lynne, & Nayak, 2008). Salmonella Derby is the most frequently detected Salmonella enterica serovar on pork in China (Cai et al., 2016). Therefore, this serovaris a major public health concern. This study provided an extensive characterization of Salmonella Derby isolated from different sources during different years in Jiangsu Province with the aims of understanding the transmission of Salmonella Derby from animal to human through the pork production and distribution chain, determining its clonal structure, evaluating different molecular subtyping methods for Salmonella Derby and determining the diversity among STs in the same serovar. MLST identified two Salmonella Derby clonal groups from different sources, which differed in all seven alleles profiled. ST40 was the most frequently detected genotype in this study, and Fig. 1 shows that ST40 was present in each source during all years. Therefore, we speculated that strains of this ST persist in the four
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H. Zheng et al. / Food Control xxx (2016) 1e11 Table 2 Salmonella Derby spacer name. CRISPR locus
Spacer namea (position)
Spacer DNA sequence (50 e30 )
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2
STM1var1 (1) Der1 (2) Der2 (3) Der3 (4) Der4 (5) Der5 (6) Der7 (7) Der8 (8) Der9 (9) Der10 (10) Der11 (11) Der25 (12) Der22 (13) Der23 (14) Der24 (15) Der27 (16) Der26 (17) Der13 (18) Der14 (19) Der15 (20) Der16 (21) Der17 (22) Der18 (23) Der19 (24) Der20 (25) Der22 (26) Der6 (27) Der28 (28) Der12 (29) Ago1 (30) Der55* (31) Cub1 (32) Der56* (33) Der57* (34) Cub4 (35) Cub5 (36) Der58* (37) AnkB2 (38) Der59* (39) Der60* (40) Ago6 (41) Ago7 (42) Ago8 (43) Ago9 (44) Der61* (45) Der62* (46) Der63* (47) Der64*(48) Der65*(49) Der66*(50) Der67*(51) Chol25 (52) HavB8 (53) Der68*(54) Der69*(55) Der70*(56) Der71*(57) Der72*(58) Mon42 (59) Ago2 (60) Ago3 (61) DerB1 (1) DerB2 (2) DerB3 (3) DerB4 (4) DerB5 (5) DerB8 (6) DerB9 (7) DerB10var1 (8) DerB11 (9) DerB12 (10) DerB6 (11) DerB13 (12) DerB7 (13)
TTTTCAGCCCTTGCCGACTGCGGAACGCCCCT GAGCTGGCGAACCTGATGGCGGGCGACGTGGA CCTGAATACTCGCAGCGATTTGCGCGATCTGG CATTGCGTTGTGAGCGCATTGAGTTGATTACT GGCGGCGCAGTCCAGCGCGGTAGCGGCAGAGA ATTTCGTCGTGATTTAAGTTGCAGTAGTCACG CTCCAGCTCGGCTTTTTGCTGCGCTGCGCGTT CTTCCCTTTTTTGGTTGCTGAGTCGGCCTCTC TTCTCCGGCATTTCCCCGTTGTTGAACGCAGC GGCATTTTAGATTTAGCGATCATGCTGCCAAC GATCATGCGCTGGTTAGTGCGGCGCTCAATCT GGCTGCGTCCGAACCGGTTATGTGCTGCCAGT CAGTAATCAATGCCCGATATAGAATTATCGGG CGCAGCGTTTTCGCCAGTACGGTGATATCGTT AACCCCTCAATAGACGGCGCTGATTCCGGGCG ACATGCCGAGTCGTGAGGCGACGGTTATAGAC TTCCAGTCGTCGCCGGACGTCATGTCAATCAG TTCAAAGTGAATTGCAAAAGCACAAATTGTCC CCCCTAAGCGCGGATCGTATAGTTTTTTACCC ATTGGAGATCACGTTATGAAAACAGCTATCGC AACTTTTGCTTAAAGCTTTCCATGTACTCATC TTTGTTTTTGTGCTGTCAGTGATGGCGGCGTG GTGAAGACTGCCTGTTTGCGGTGCGTGGCAAA AAGGCTCTGTCATTCCACCTTTGAGTGTTAGA ACTGGAAATACTCTGGAGTACCGGGATCAGGCG TTTGCTTCGTTATTCGATATCATATATATTCG TTTCGCTGCTTCAGCATTCCCCCTGTTAACAG CAGGTTAGATTTCTGCGCCTCCGCCATCGTCT TTTCATAATTCGCCTTATTGTTTAAATACAAA ATGGATGGCTCATATCCGGTTTCTGTTTCTGC GGTCTGACCGATGACGATTTCAAGATAAGCGA CAGCTCCAGATCGCGGGCACCGCTGGCGAACT CGCGACTGGTGGGCGTTCGCGGAGAAATTCGG GGGGCTATATGGAGCTAAACAGGATGTGGATT CGACGCAGATCACCAATCAGGCGGATATCAGC CTGATTTTGTCGAATAGTGGGCAGGACCGGAA TAACGTCCAAATAACAAATCAAAAATAAACGC TATTTAAATAAAAGTGTTCTTTATATTGGCGG GCAACCCATTACCAAAACTATTATCCCGTAAT AGCTCTTGCCCCGGAAATTAGGCTGATAGGTC TCAGGCTTGACGACAGTCTCCAGCCACTCCTG GACCGCGCGGGCTATCGTTCAGGACTATTTTT CTCTGTTACACCGTTATGCACAGACCACACAG GTTAATGCAAGCGCCATTAACAAGAAAATGAC TCGGCAATGATGTCGCTGCATATGAAGACTAC TTTCTGAGCTGGGCAAGCGCCGCCGCGCATGG TCTTCCTTAATATGGTGAATGTATGTCAATTA ACCGTCAGCCTCAAGCCCAGCTCTGGATATTT GCCTCCGGTGTAGCGTACAAAGAGCGACTTAA CGTCTCGGTGGCAAACTGTGGGAAAAAAACGG ACCATCCCAGCCGCCAGCGAGCTGCCTCATAT TGCATCGGTGTAGAGAATATCAAGTTCAACGC CGTGTGTTTGTTGTCGCAAGTGCTCGAGCCGA ACCGACACACTCTTTTACTGTCAACCAAATTT TGTTAAAACGGAATAAACTCATTAATTAACAC GGGGCTTGATTGCCTAAACAAACTTTTCTAGG GACTGGTTGAGAAGATCCGCCGCAGAGTCAGC GTGATGCTGAACAGGCTCAACGCAAACCTCAA TGTATAAAGCCGTTCTCAACGTCCTGTGGAAC TGGCGCCAGAAATATTCATGATCATCGGGATT TTCATATTCCAGAAAATGCCTGGGTGATGATC TACATCATCGGCTCAATGCGTAATTCTAAAAT CTGGGCGGCAACTTCACACGTCACGATGGTTA GTGAGTGAACGTAATGTCGAATGACGGCGAGG GACGACATGCAAAAACGCCTGGCGACGATGTT GTTCTATTTAACATAATGACCGTTACGCGCAT TATCCACTCCGGCACACATCACCGCTCGTCAA CACGTGAACCGATTTACCCGGCAGGCCGTTGA ATCATTTCAGCGGAGTATTTCAAATTGTCAGG CTTGCGGGGCGTTCAGGTCAGTAATGATGCGG AGCTTCCGCCCCCCGCCCCGGCCCGGCGTTGT GTTTGCCCCTGCTATGAAGAACATTCTCACTC TACAAGCGGCTCTGGTCGCTGATGCAAAAAGA CTGGCGCGTAACCTGCCGTTTGCCTCATATCA
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Table 2 (continued ) CRISPR locus
Spacer namea (position)
Spacer DNA sequence (50 e30 )
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
HilB2 (14) HilB3 (15) HilB4 (16) DerB49*(17) Ucc4var1 (18) HilB6 (19) DerB50*(20) DerB51*(21) DerB52*(22) DerB53*(23) DerB54*(24) AltB11var1 (25) DerB55*(26) DerB56*(27) DerB57*(28) Aba12 (29) DerB58*(30) DerB59*(31) DerB60*(32) DerB61*(33) DerB62*(34) DerB63*(35) StpB3 (36) DerB64*(37) DerB65*(38) CholB75var1 (39)
TGGGGAGCGTTGAGTCAAACACGACCCAGCAG ATAGAACTGCTGGGCCGGAACCTCTTCCCCTG GCAATACTGGAAAGACCCGCTCCCAGAAACGA TTGAACCAGAATATTGCCGCTGCGCCTGCGGT CGCAGCATCACAAGTTTGTATTCCTGCCAGGA CCGGGAGGCGGAATGCAGCAAAAGTAAAAACCG GGTCATTCGGCCAGCTCCCGTCCGCTCAAAAT ACCCCGTCAGGCAGCGAGTCGTAATTGAATTT GCATGAAGCTGGTAACGCTGACGCAGGACAGGC CCGTCCCACTCTATACCGTATTCGGCTGCAAG GTGTATCCGGAAGAGATAGACGGTAGTGACCA ACATATGAATAGGCATAGCCGAATGCCGGGGG ACGGAATCTCCTGATTTTGGCGCGCTGGAATC TATTTGCACCGCAGAATGCCGCGCCGCCGTTGC GACGCTAACAATTTTAAATTACTGTTTAAATT GTTGATCAATCTGGAATGATTTGTCTCACCGA TGCTCATCGATTGGTCGATCGATATAATCATT GCGAGCGCGCCAGCGCTGGCCATGATGATTTT CAATAATATTGAGTATTTTTTCTTTCGTGTTG GATAGCGTTTGATATGCGGCAGACGGCCCCTC GTTCCAACGCCTCGCGCAGTAAGGGCGGCAAC GTGAATTTGGTAATAGTGTTCATGGGTTAGCC TTCTCGGCGCGGCGCAATATCCCGGCGCACGC TCGTCCGTAATCACGGCCACCACGCCTGACGC GGTACTGATTTGGAGCTAATGCAATCAGAAGC TTCGTTGTGTTGGACACCACCGGATCGGCGCT
a
An asterisk shows a novel spacer identified in the current study.
sources and were continually propagated. Moreover, ST40 has been widely found in pigs and pork from Europe and the United States and in humans from Asia and Europe (Li et al., 2014). In this study, ST71 was only found in pig slaughterhouses and retail markets from 2013 to 2015, but it was also found in humans from Germany and Denmark (Hauser et al., 2011; Litrup et al., 2010). Cai et al. and Li et al. found only two sequence types (ST40 and ST71) of Salmonella Derby in Yangzhou, Jiangsu Province, China (Cai et al., 2016; Li et al., 2014). By searching the MLST database (http://mlst.ucc.ie/mlst/dbs/ Senterica), we found 13 different sequence types (STs) in Salmonella Derby, including ST15, ST39, ST40, ST71, ST72, ST678, ST682, ST683, ST695, ST774, ST813, ST1326 and ST1585. The serovar originates from more than one common ancestor, and it possesses several distantly related STs. Lan and coworkers' analysis of multilocus sequence typing (MLST) data showed a high level of recombination within the subspecies of S. enterica (Lan, Reeves, & Octavia, 2009). Consistent with this, several sequence types (STs) were detected in Salmonella Derby. However, for investigating outbreaks, distinguishing between closely related isolates based on MLST of housekeeping genes may be not be possible, due to their high sequence conservation (Ross & Heuzenroeder, 2005). Therefore, this method may not be suitable for closely related isolates within a serovar. Fig. 2 shows that the spacers of CRISPR1 were variable in ST40 strains, but the spacers of CRISPR2 were strictly conserved. However, strains with ST71 showed a high degree of CRISPR polymorphism in the CRISPR1 and CRISPR2 loci. The variability of CRISPRs spacer content is due to the duplication of single spacer-DR units or the deletion of single or contiguous spacer-DR units (Fabre et al., 2012). This microvariation resulted in 25 CRISPR1 types and 6 CRISPR2 types or 32 CRISPR1-CRISPR2 combined types, thus providing greater resolution than other subtyping methods, such as MLST. 13-S48, 14-S66, 15-S67 and15-S89 strains had a deletion of seventeen spacers, four spacers and one spacer adjacent to the leader array. In contrast, C. Pourcel et al. suggested that a polarity exists, in that new spacers are always added to the leader sequence of the CRISPR array (Pourcel, Salvignol, & Vergnaud, 2005). Spacer multiplication or loss occurred in 32 different CRISPR types. Tyson
GW et al. reported that spacer loss and duplication, makes CRISPR elements some of the fastest evolving loci in bacteria (Tyson GW, 2008). In addition, we found that the spacers of ST71 isolates were larger than those of ST40 in CRISPR2. The length of a CRISPR array is dependent on the number of spacers and varies dramatically among different strains (Grissa, Vergnaud, & Pourcel, 2007). Nikki Shariat et al. reported that the CRISPR spacer array is constitutively transcribed into a precursor CRISPR RNA (pre-crRNA) that is cleaved by specific Cas proteins and further processed into mature, small interfering crRNAs. These crRNAsare typically comprised of the spacer flanked on either side by portions of the DRs (Shariat & Dudley, 2014). In addition, Garneau et al suggested that mature crRNAs guide the Cas-crRNA ribonucleoprotein complex to complementary nucleic acids, typically in invading bacteriophages or plasmids (Garneau et al., 2010). The isolates obtained the new spacer through selection, possibly providing an advantage via enhanced virulence and growth. This may suggest significant differences in virulence between ST40 and ST71strains. Salmonella Derby CRISPR typing (DCT) identified 32DCTs among the 100 isolates that included 4 DCTs from pig farms, 14 DCTs from pig slaughterhouses, 24 DCTs from retail markets and 4 DCTs from humans. Therefore, CRISPR typing was shown to be better at distinguishing Salmonella Derby strains than MLST, as observed previously (Liu, Kariyawasam, et al., 2011). This result indicated that the Salmonella Derby CRISPRs types (DCTs) were more varied in pig slaughterhouses and downstream retail markets. This diversity maybe attributed to the broad origins of the pigs in slaughterhouses. The Fig. 3 B cluster DCT24 had the same CTs among isolates from all four different sources; DCT22 and DCT31 were both among isolates from pig farms, slaughterhouses and downstream retail markets. DCT14 and DCT18 showed that some isolates from pork had the same DCTs as isolates from clinical patients. Therefore, Salmonella Derby was transferred along the pork production and distribution chain from the slaughterhouse to retail markets, where people could be infected via pork contaminated with Salmonella Derby (Yi et al., 2014). Moreover, sows and piglets infected with Salmonella Derby can be asymptomatic despite its presence in
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Fig. 2. Graphic representation of spacers across the two CRISPR loci for a variety of Salmonella Derby strains. Repeats are not included; only spacers are represented. Each spacer is represented by a combination of a character in a particular font colour, on a particular background colour. The colour combination allows each spacer to be uniquely represented, whereby squares with similar colour schemes (combination of character colour and background colour) represent identical spacers and different colour combinations represent distinguishable spacers. The spacers are aligned, and the gaps represent the absence of a particular spacer. Above, CRISPR1; Below, CRISPR2.
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Fig. 2. (continued).
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Fig. 3. Minimum spanning tree analysis of Salmonella Derby isolated from pig farms, pig slaughterhouses, retail markets and humans. Each circle represents one DCT, and the area of the circle corresponds to the number of isolates. Different sources are represented by green, red, light blue and yellow, respectively. Two clusters (AeB) were identified. The numbers correspond to the different Salmonella Derby CRISPR types (DCTs). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Minimum spanning tree analysis of Salmonella Derby isolated during different years. Each circle represents one Salmonella Derby CRISPR type (DCT), and the area of the circle corresponds to the number of isolates. Different years are represented by dark green, brown, gold, bluish green, light blue, green and red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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H. Zheng et al. / Food Control xxx (2016) 1e11
many tissues, which can then contaminate the pork when they are slaughtered. This jeopardizes food safety (Matiasovic et al., 2014). Therefore, to prevent this serovar from entering the food chain, measures for its control should be taken at the pig farm (Hauser et al., 2011). Fig. 4 shows that some isolates from different years displayed the same DCTs or were closely related, such as DCT22, 24, 28 and 31. This demonstrated that Salmonella Derby clones persisted in the same places and spread along the pork production chain (Yi et al., 2014). The Fig. 3 A cluster shows that the diversity was greater among the ST71 strains, and the Fig. 4 demonstrates that they were only found from 2013 to 2015. This occurrence maybe due to initial isolate diversity or introduction of exotic strains. The spacer arrangements were similar among STs in the 2 CRISPR loci in Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Senftenberg (Liu, Kariyawasam, et al., 2011; Shariat et al., 2013; Yi et al., 2014). However, Fabre et al. reported that the spacer content differed greatly in different STs of Salmonella enterica serovar. This is consistent with reports by Newportas well as our findings that only two spacers are the same in the spacer arrangements of ST40 and ST71. Moreover, Fabre et al. also reported that spacer content was strongly correlated with the population structure defined by MLST (Fabre et al., 2012). All seven allele profiles differed between ST40 and ST71 and the particular spacer arrangements, we observed enormous diversity in the genomes of ST40 and ST71 strains within the same serotype. As nextgeneration sequencing has rapidly become cheaper and more amenable to standard laboratory practices, a priority is to promote whole genome sequencing (WGS) as a rapid and accurate approach to characterize microbial agents for epidemiological surveillance and outbreak detection (Aanensen et al., 2016; Deng et al., 2015). This will allow correlation of bacterial genome subtypes with a source host in order to quickly speculate on the bacterial origin (Ashton et al., 2016; Deng et al., 2015). Using whole genome sequencing, we could also systemically analyse the genome sequences of ST40 and ST71 strains and from their genetic information gain insight into the mechanism of their pathogenicity. 5. Conclusion In summary, we used MLST and Clustered regularly interspaced short palindromic repeats (CRISPR) for subtyping 100 Salmonella Derby isolates from pig farms, pig slaughterhouses, retail markets and humans in Yangzhou, Jiangsu Province, China during a sevenyear period. The results of this study illustrated CRISPRs loci evolve much faster than housekeeping genes. Therefore, CRISPRs typing provided superior discriminatory power over MLST typing for distinguishing different sources of Salmonella Derby. This study also showed that two major Salmonella Derby clones are dominant in pigs and humans in China. Some isolates from the four sources were demonstrated to be genetically similar by MLST and CRISPRs typing and were transferred along pork production chain from the pig farms to the slaughterhouses and retail markets. Contaminated pork has been identified as a vehicle for Salmonella Derby and consequently presents a risk to human health. Considering this, enough attention should be paid to prevent the further dissemination of Salmonella Derby. Acknowledgments This work was supported by the Special Fund for Agroscientific Research in the Public Interest (201403054), the National Natural Science Foundation of China (nos. 31320103907 and 31230070), the Project for Agricultural Products Quality and Safety Supervision (Risk Assessment) (18162130109236), the Program for New
Century Excellent Talents in University (NCET-12-0745), the “Six Talent Peaks Program” of Jiangsu Province (NY-028), the Yangzhou University Science and Technology Innovation Team (2016), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Aanensen, D. M., Feil, E. J., Holden, M. T., Dordel, J., Yeats, C. A., Fedosejev, A., et al. (2016). Whole-genome sequencing for routine pathogen surveillance in public health: A population snapshot of invasive Staphylococcus aureus in Europe. MBio, 7(3). Ashton, P. M., Nair, S., Peters, T. M., Bale, J. A., Powell, D. G., Painset, A., et al. (2016). Identification of Salmonella for public health surveillance using whole genome sequencing. PeerJ, 4, e1752. Authority, E. F. S. (2015). .The European Union summary report on trends and sources of zoonoses, Zionistic agents and food-borne outbreaks in 2013. MESA Journal, 13(1). l, Mariann, Davies, Robert, et al. (2009). 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