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Research article
Prevalence and Characteristics of Salmonella spp. Isolated from Raw Chicken Meat in the Republic of Korea
1Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju 28159, Republic of Korea
2Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, Center for Food and Bioconvergence, Research Institute for Agricultural and Life Science, Seoul National University, Seoul 08826, Republic of Korea
J. Microbiol. Biotechnol. 2022; 32(10): 1307-1314
Published October 28, 2022 https://doi.org/10.4014/jmb.2207.07031
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract
Introduction
Many foodborne illness outbreaks of harmful bacteria in foods are reported each year, and these human health-threatening incidences have had various patterns [1]. Across the globe, outbreaks of food poisoning tend to occur in groups and become larger while the key pathogenic organisms causing food poisoning include
The average number of individuals in a
In addition, according to data from the Korea Meteorological Administration, the year 2018 was one of the hottest ever, with a total of 27.8 heat wave days that had a daily maximum apparent temperature of 33°C or higher [5]. In terms of temperature-induced proliferation of
Based on this background, we aimed to isolate
Materials and Methods
Sampling
From February to April 2018, 113 samples of chicken and by-products sold in department stores, large discount stores and traditional markets in Chungcheong Province were collected. After purchase, the samples were kept in a cooling box for refrigeration while being transported to the laboratory for testing.
Isolation and Identification of Salmonella spp.
In accordance with the testing method of the Korean Food Code laid out by the MFDS, a 25 g sample and 225 ml of Buffered Peptone Water (Oxoid, UK) were mixed thoroughly and enriched in the incubator at 37°C for around 24 h. The enriched culture solution was added to two enrichment media, 1 ml to 10 ml of teterathionate medium (Biomeriux Inc., Spain) and 0.1 ml to 10 ml of Rappaport-Vassiliadis medium (Oxid, UK), which underwent secondary enrichment at 37°C (tetrathionate) and 42°C (RV medium) for 20-24 h. The secondary enrichment culture solution was smeared on the selective media of XLD agar (Oxoid) and Brilliant green sulfa agar (Remel, UK), and cultured at 37°C for 18-24 h, and then a typical colony was selected and subcultured in the nutrient medium, which was identified by Vitek MS (Biomeriux Inc., France).
Pathogenic Gene Analysis Using Polymerase Chain Reaction (PCR)
Genes subject to genetic characterization of
For
For
-
Table 1 . Primers/probe and PCR conditions used in the present study.
Target gene Sequence (5'-3') Size (bp) PCR cycling conditions spvC F: AATGAACTACGAAGTGGGCG
R: TCAAACGATAAAACGGTTCCTC
P: FAM-ATGGTGGCGAAATGCAGAGACAGGC-BHQ1112 50°C, 2 m→95°C, 10 m→95°C, 15 s→60°C, 1 m: 40 cycles sefA F: GGCTTCGGTATCTGGTGGTGTA
R: GGTCATTAATATTGGCCCTGAATA
P:Cy5-CCACTGTCCCGTTCGTTGATGGACA-BHQ298 50°C, 2 m→95°C, 10 m→95°C, 15 s→60°C, 1 m: 40 cycles hin F: TCCATGAGAAAAGCGACTAAAAT
R: AGCCGACTAATCTGTTCCTGTTC572 95°C, 3 m→95°C, 30 s→57°C, 30 s→72°C, 1 m: 30 cycles→72°C, 2 m
For
For
Serology Testing
Tests were conducted in accordance with the method provided by the MFDS [4] to verify serotypes of isolated strains. Difco Antisera by somatic (O) antigen (A, B, C, D, E, Vi) and by flagellar (H) antigens (a, b, c, d, e, h, i, k, r, y, z) were used to perform slide and tube agglutination tests for identification of serotypes.
Pulsed-Field Gel Electrophoresis (PFGE)
PFGE analysis of
A 1 mm-thick slice was cut from the washed plug and reacted at 37°C for 2 h using 40 U/μl XbaI (Roche, Switzerland). Electrophoresis was performed with the plug gel treated with the restriction enzyme using the electrophoresis equipment at 14°C for 18 h under an initial time of 2.16 s, final time of 63.8 s, a voltage gradient of 6 V/cm, and an included angle of 120°.
Multilocus Sequence Typing (MLST)
With MLST, the sequence type was identified by analyzing the sequences of seven house-keeping genes (
-
Table 2 . PCR and sequencing primer for MLST used in this study.
Gene Primer sequence (5'-3') Product size (bp) thrA F: GTCACGGTGATCGATCCGGT
R: CACGATATTGATATTAGCCCG852 purE F: GACACCTCAAAAGCAGCGT'
R: AGACGGCGATACCCAGCGG635 sucA F: CGCGCTCAAACAGACCTAC
R: GACGTGGAAAATCGGCGCC793 hisD F: GAAACGTTCCATTCCGCGC
R: GCGGATTCCGGCGACCAG788 aroC F: CCTGGCACCTCGCGCTATAC
R: CCACACACGGATCGTGGCG826 hemD F: GAAGCGTTAGTGAGCCGTCTGCG
R: ATCAGCGACCTTAATATCTTGCCA666 dnaN F: ATGAAATTTACCGTTGAACGTGA
R: AATTTCTCATTCGAGAGGATTGC833
Sequences were assembled and analyzed using Lasergene 7.2.1 software (DNAStar). Sequence type (ST) numbers were assigned by submitting the sequences and strain information to the
Results
Prevalence of Salmonella spp. from Raw Chicken Meat
Among the 113 samples of chicken purchased in retail stores in Chungcheong Province, 24 chicken samples (21.2%) were determined to be positive for
Distribution of Salmonella Serotypes
The identified
-
Table 3 . Serotypes of
Salmonella spp. isolated from raw chicken meat.Sample No. Source of isolates Somatic antigens Flagellar antigens Serovar Group I Group II O-antigen H phase 1 H phase 2 1 Meat C Group O:8 (C2-C3) 6, 8 r 1, 2 S . Bsilla2 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis3 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis4 Gizzard D Group O:9 (D1) 9, 12 g, m - S . Enteritidis5 Meat C Group O:7 (C1) 61,2, 7 g, m, s [1, 2, 7] S . Montevideo6 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis7 Gizzard D Group O:9 (D1) 9, 12 g, m - S . Enteritidis8 Meat C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow9 Meat C Group O:8 (C2-C3) 6, 8 r 1, 2 S . Bsilla10 Meat C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow11 Meat C Group O:7 (C1) 61, 7 r 1, 2 S . Virchow12 Meat C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow13 Meat C Group O:7 (C1) 61,2, 7, 14 y 1, 5 S . Bareilly14 Heart D Group O:9 (D1) 1, 9, 12 g, m - S . Enteritidis15 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis16 Feet C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow17 Gizzard C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow18 Meat E Group O:1,3,19 (E4) 1, 3, 19 g, s ,t - S . Dessau19 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis20 Meat C Group O:8 (C2-C3) 8, 20 z4, z24 - S . Albany21 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis22 Meat C Group O:7 (C1) 61,2, 7, 14 g, m, s [1, 2, 7] S . Montevideo23 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis24 Gizzard D Group O:9 (D1) 9, 12 g, m - S . Enteritidis
PCR Targeted to Pathogenic Genes
The results from gene detection of the 24 isolates of
-
Table 4 . Pathogenic gene-targeted PCR results of
Salmonella serovars.Sample No. Serovar Serological type Real-time PCR PCR O-antigen Group H Phase 1 H Phase 2 his invA stn sefA spvC hin 1 S . BsillaC r 1, 2 + + + - - + 2 S . EnteritidisD g, m - + + + + + - 3 S . EnteritidisD g, m - + + + + + - 4 S . EnteritidisD g, m - + + + + + - 5 S . MontevideoC g, m, s [1, 2, 7] + + + - - - 6 S . EnteritidisD g, m - + + + + + - 7 S . EnteritidisD g, m - + + + + + - 8 S . VirchowC r 1, 2 + + + - - + 9 S . BsillaC r 1, 2 + + + - - + 10 S . VirchowC r 1, 2 + + + - - + 11 S . VirchowC r 1, 2 + + + - - + 12 S . VirchowC r 1, 2 + + + - - + 13 S . BareillyC y 1, 5 + + + - - + 14 S . EnteritidisD g, m - + + + + + - 15 S . EnteritidisD g, m - + + + + + - 16 S . VirchowC r 1, 2 + + + - - + 17 S . VirchowC r 1, 2 + + + - - + 18 S . DessauE g, s ,t - + + + - - - 19 S . EnteritidisD g, m - + + + + - / - - 20 S . AlbanyC z4, z24 - + + + - - - 21 S . EnteritidisD g, m - + + + + + - 22 S . MontevideoC g, m, s [1, 2, 7] + + + - - - 23 S . EnteritidisD g, m - + + + + + - 24 S . EnteritidisD g, m - + + + + + -
Comparison of Isolates of Salmonella Bacteria Using PFGE
The PFGE results on the 24
-
Fig. 1. Relatedness of
Salmonella spp. isolated from raw chicken meat by PFGE analysis with XbaI.
MLST Analysis
Six Sequence Types (STs) from
-
Table 5 . ST definitions based on allele type for each of seven loci sequenced and assigned by the
Salmonella enterica database.ST Allele type No. isolates % of total thrA purE sucA hisD aroC hemD dnaN 11 11 6 6 7 5 3 2 11 45.8 16 14 8 10 10 6 10 7 8 33.3 4 4 34 13 13 43 16 41 2 8.3 203 17 68 12 12 81 36 69 1 4.2 14 13 7 8 8 7 8 6 1 4.2 292 48 104 9 78 104 54 100 1 4.2
After being classified through the PubMLST program to verify the diversity of clones, 6 STs belonged to ST11 and 16. Of the 6 STs, ST11 (11 strains) and ST16 (8 strains) were the most common types. The rest of the strains were ST4 (2 strains), and ST203, 14, 292 (1 strain). In addition, in most cases, ST11, ST16, ST4, ST203, ST14, and ST292 appeared in isolates of bacterial species in 2001 but did not appear in those thereafter.
Discussion
In this study,
The serotypes of
With respect to genotypes of isolated
In this study, the serotype of
The Sequence Type determined by the MLST can be used as an important clue for traceback investigation particularly when multiple outbreaks of foodborne illness derived from the same
Along with the rapid growth of the global food trade, the consumption of food or ingredients has become highly dependent on importation, and the possibility of food poisoning sources from imported food is increasing. In the case of an outbreak suspected to be caused by an imported food source, a traceback investigation is conducted by authorities in the importing and exporting countries and the investigating country requests the gene sequence data of isolated pathogens from the suspected source, which becomes important scientific evidence for the investigation. So, it is crucial to monitor the prevalence and gene sequence profile of pathogens isolated from domestic products through sustainable national surveillance programs in response to a foodborne illness outbreak investigation as well as to protect the health of people and the agricultural industry.
In this study, we attempted to investigate the various characteristics of
Acknowledgment
This research was supported by a grant (18161MFDS033) from the Ministry of Food and Drug Safety in 2018.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB
Article
Research article
J. Microbiol. Biotechnol. 2022; 32(10): 1307-1314
Published online October 28, 2022 https://doi.org/10.4014/jmb.2207.07031
Copyright © The Korean Society for Microbiology and Biotechnology.
Prevalence and Characteristics of Salmonella spp. Isolated from Raw Chicken Meat in the Republic of Korea
Youngho Koh1,2, Yunyoung Bae1, Yu-Si Lee1, Dong-Hyun Kang2*, and Soon Han Kim1*
1Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju 28159, Republic of Korea
2Department of Food and Animal Biotechnology, Department of Agricultural Biotechnology, Center for Food and Bioconvergence, Research Institute for Agricultural and Life Science, Seoul National University, Seoul 08826, Republic of Korea
Correspondence to:Dong-Hyun Kang, kang7820@snu.ac.kr
Soon Han Kim, lambndog@korea.kr
Abstract
In this study, we sought to investigate the various characteristics of Salmonella spp. isolated from raw chicken meats available in Korean markets. The data collected, such as food source of isolation, sampling information, serotype, virulence, and genetic profile including sequence type, were registered in the database for further comparative analysis of the strains isolated from the traceback investigation samples. To characterize serotype, virulence and gene sequences, we examined 113 domestically distributed chicken meat samples for contamination with Salmonella spp. Phylogenetic analysis was conducted on 24 strains (21.2%) of Salmonella isolated from 113 commercially available chicken meats and by-products, using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Serotyping of the isolated Salmonella spp. revealed S. Enteritidis in 11 strains (45.8%), S. Virchow in 6 strains (25%), S. Montevideo in 2 strains (8.3%), S. Bsilla in 2 strains (8.3%), S. Bareilly in 1 strain (4.2%), S. Dessau in 1 strain (4.2%), and S. Albany in 1 strain (4.2%). The genetic correlation indicated that 24 isolated strains were classified into 18 clusters with a genetic similarity of 64.4-100% between them. Eleven isolated S. Enteritidis strains were classified into 9 genotypes with a sequence identity of 74.4%, whereas the most distantly related S. Virchow was divided into five genotypes with 85.9% identity. Here, the MLST analysis indicated that the major Sequence Type (ST) of the Salmonella spp. isolated from domestic chicken sold in Chungcheong Province belongs to the ST 11 and 16, which differs from the genotype of Salmonella isolated from imported chicken. The differential sequence characteristics can be a genetic marker for identifying causative bacteria for epidemiological investigations of food poisoning.
Keywords: Salmonella, serotype, sequence type, chicken, foodborne pathogen
Introduction
Many foodborne illness outbreaks of harmful bacteria in foods are reported each year, and these human health-threatening incidences have had various patterns [1]. Across the globe, outbreaks of food poisoning tend to occur in groups and become larger while the key pathogenic organisms causing food poisoning include
The average number of individuals in a
In addition, according to data from the Korea Meteorological Administration, the year 2018 was one of the hottest ever, with a total of 27.8 heat wave days that had a daily maximum apparent temperature of 33°C or higher [5]. In terms of temperature-induced proliferation of
Based on this background, we aimed to isolate
Materials and Methods
Sampling
From February to April 2018, 113 samples of chicken and by-products sold in department stores, large discount stores and traditional markets in Chungcheong Province were collected. After purchase, the samples were kept in a cooling box for refrigeration while being transported to the laboratory for testing.
Isolation and Identification of Salmonella spp.
In accordance with the testing method of the Korean Food Code laid out by the MFDS, a 25 g sample and 225 ml of Buffered Peptone Water (Oxoid, UK) were mixed thoroughly and enriched in the incubator at 37°C for around 24 h. The enriched culture solution was added to two enrichment media, 1 ml to 10 ml of teterathionate medium (Biomeriux Inc., Spain) and 0.1 ml to 10 ml of Rappaport-Vassiliadis medium (Oxid, UK), which underwent secondary enrichment at 37°C (tetrathionate) and 42°C (RV medium) for 20-24 h. The secondary enrichment culture solution was smeared on the selective media of XLD agar (Oxoid) and Brilliant green sulfa agar (Remel, UK), and cultured at 37°C for 18-24 h, and then a typical colony was selected and subcultured in the nutrient medium, which was identified by Vitek MS (Biomeriux Inc., France).
Pathogenic Gene Analysis Using Polymerase Chain Reaction (PCR)
Genes subject to genetic characterization of
For
For
-
Table 1 . Primers/probe and PCR conditions used in the present study..
Target gene Sequence (5'-3') Size (bp) PCR cycling conditions spvC F: AATGAACTACGAAGTGGGCG
R: TCAAACGATAAAACGGTTCCTC
P: FAM-ATGGTGGCGAAATGCAGAGACAGGC-BHQ1112 50°C, 2 m→95°C, 10 m→95°C, 15 s→60°C, 1 m: 40 cycles sefA F: GGCTTCGGTATCTGGTGGTGTA
R: GGTCATTAATATTGGCCCTGAATA
P:Cy5-CCACTGTCCCGTTCGTTGATGGACA-BHQ298 50°C, 2 m→95°C, 10 m→95°C, 15 s→60°C, 1 m: 40 cycles hin F: TCCATGAGAAAAGCGACTAAAAT
R: AGCCGACTAATCTGTTCCTGTTC572 95°C, 3 m→95°C, 30 s→57°C, 30 s→72°C, 1 m: 30 cycles→72°C, 2 m
For
For
Serology Testing
Tests were conducted in accordance with the method provided by the MFDS [4] to verify serotypes of isolated strains. Difco Antisera by somatic (O) antigen (A, B, C, D, E, Vi) and by flagellar (H) antigens (a, b, c, d, e, h, i, k, r, y, z) were used to perform slide and tube agglutination tests for identification of serotypes.
Pulsed-Field Gel Electrophoresis (PFGE)
PFGE analysis of
A 1 mm-thick slice was cut from the washed plug and reacted at 37°C for 2 h using 40 U/μl XbaI (Roche, Switzerland). Electrophoresis was performed with the plug gel treated with the restriction enzyme using the electrophoresis equipment at 14°C for 18 h under an initial time of 2.16 s, final time of 63.8 s, a voltage gradient of 6 V/cm, and an included angle of 120°.
Multilocus Sequence Typing (MLST)
With MLST, the sequence type was identified by analyzing the sequences of seven house-keeping genes (
-
Table 2 . PCR and sequencing primer for MLST used in this study..
Gene Primer sequence (5'-3') Product size (bp) thrA F: GTCACGGTGATCGATCCGGT
R: CACGATATTGATATTAGCCCG852 purE F: GACACCTCAAAAGCAGCGT'
R: AGACGGCGATACCCAGCGG635 sucA F: CGCGCTCAAACAGACCTAC
R: GACGTGGAAAATCGGCGCC793 hisD F: GAAACGTTCCATTCCGCGC
R: GCGGATTCCGGCGACCAG788 aroC F: CCTGGCACCTCGCGCTATAC
R: CCACACACGGATCGTGGCG826 hemD F: GAAGCGTTAGTGAGCCGTCTGCG
R: ATCAGCGACCTTAATATCTTGCCA666 dnaN F: ATGAAATTTACCGTTGAACGTGA
R: AATTTCTCATTCGAGAGGATTGC833
Sequences were assembled and analyzed using Lasergene 7.2.1 software (DNAStar). Sequence type (ST) numbers were assigned by submitting the sequences and strain information to the
Results
Prevalence of Salmonella spp. from Raw Chicken Meat
Among the 113 samples of chicken purchased in retail stores in Chungcheong Province, 24 chicken samples (21.2%) were determined to be positive for
Distribution of Salmonella Serotypes
The identified
-
Table 3 . Serotypes of
Salmonella spp. isolated from raw chicken meat..Sample No. Source of isolates Somatic antigens Flagellar antigens Serovar Group I Group II O-antigen H phase 1 H phase 2 1 Meat C Group O:8 (C2-C3) 6, 8 r 1, 2 S . Bsilla2 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis3 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis4 Gizzard D Group O:9 (D1) 9, 12 g, m - S . Enteritidis5 Meat C Group O:7 (C1) 61,2, 7 g, m, s [1, 2, 7] S . Montevideo6 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis7 Gizzard D Group O:9 (D1) 9, 12 g, m - S . Enteritidis8 Meat C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow9 Meat C Group O:8 (C2-C3) 6, 8 r 1, 2 S . Bsilla10 Meat C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow11 Meat C Group O:7 (C1) 61, 7 r 1, 2 S . Virchow12 Meat C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow13 Meat C Group O:7 (C1) 61,2, 7, 14 y 1, 5 S . Bareilly14 Heart D Group O:9 (D1) 1, 9, 12 g, m - S . Enteritidis15 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis16 Feet C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow17 Gizzard C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow18 Meat E Group O:1,3,19 (E4) 1, 3, 19 g, s ,t - S . Dessau19 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis20 Meat C Group O:8 (C2-C3) 8, 20 z4, z24 - S . Albany21 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis22 Meat C Group O:7 (C1) 61,2, 7, 14 g, m, s [1, 2, 7] S . Montevideo23 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis24 Gizzard D Group O:9 (D1) 9, 12 g, m - S . Enteritidis
PCR Targeted to Pathogenic Genes
The results from gene detection of the 24 isolates of
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Table 4 . Pathogenic gene-targeted PCR results of
Salmonella serovars..Sample No. Serovar Serological type Real-time PCR PCR O-antigen Group H Phase 1 H Phase 2 his invA stn sefA spvC hin 1 S . BsillaC r 1, 2 + + + - - + 2 S . EnteritidisD g, m - + + + + + - 3 S . EnteritidisD g, m - + + + + + - 4 S . EnteritidisD g, m - + + + + + - 5 S . MontevideoC g, m, s [1, 2, 7] + + + - - - 6 S . EnteritidisD g, m - + + + + + - 7 S . EnteritidisD g, m - + + + + + - 8 S . VirchowC r 1, 2 + + + - - + 9 S . BsillaC r 1, 2 + + + - - + 10 S . VirchowC r 1, 2 + + + - - + 11 S . VirchowC r 1, 2 + + + - - + 12 S . VirchowC r 1, 2 + + + - - + 13 S . BareillyC y 1, 5 + + + - - + 14 S . EnteritidisD g, m - + + + + + - 15 S . EnteritidisD g, m - + + + + + - 16 S . VirchowC r 1, 2 + + + - - + 17 S . VirchowC r 1, 2 + + + - - + 18 S . DessauE g, s ,t - + + + - - - 19 S . EnteritidisD g, m - + + + + - / - - 20 S . AlbanyC z4, z24 - + + + - - - 21 S . EnteritidisD g, m - + + + + + - 22 S . MontevideoC g, m, s [1, 2, 7] + + + - - - 23 S . EnteritidisD g, m - + + + + + - 24 S . EnteritidisD g, m - + + + + + -
Comparison of Isolates of Salmonella Bacteria Using PFGE
The PFGE results on the 24
-
Figure 1. Relatedness of
Salmonella spp. isolated from raw chicken meat by PFGE analysis with XbaI.
MLST Analysis
Six Sequence Types (STs) from
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Table 5 . ST definitions based on allele type for each of seven loci sequenced and assigned by the
Salmonella enterica database..ST Allele type No. isolates % of total thrA purE sucA hisD aroC hemD dnaN 11 11 6 6 7 5 3 2 11 45.8 16 14 8 10 10 6 10 7 8 33.3 4 4 34 13 13 43 16 41 2 8.3 203 17 68 12 12 81 36 69 1 4.2 14 13 7 8 8 7 8 6 1 4.2 292 48 104 9 78 104 54 100 1 4.2
After being classified through the PubMLST program to verify the diversity of clones, 6 STs belonged to ST11 and 16. Of the 6 STs, ST11 (11 strains) and ST16 (8 strains) were the most common types. The rest of the strains were ST4 (2 strains), and ST203, 14, 292 (1 strain). In addition, in most cases, ST11, ST16, ST4, ST203, ST14, and ST292 appeared in isolates of bacterial species in 2001 but did not appear in those thereafter.
Discussion
In this study,
The serotypes of
With respect to genotypes of isolated
In this study, the serotype of
The Sequence Type determined by the MLST can be used as an important clue for traceback investigation particularly when multiple outbreaks of foodborne illness derived from the same
Along with the rapid growth of the global food trade, the consumption of food or ingredients has become highly dependent on importation, and the possibility of food poisoning sources from imported food is increasing. In the case of an outbreak suspected to be caused by an imported food source, a traceback investigation is conducted by authorities in the importing and exporting countries and the investigating country requests the gene sequence data of isolated pathogens from the suspected source, which becomes important scientific evidence for the investigation. So, it is crucial to monitor the prevalence and gene sequence profile of pathogens isolated from domestic products through sustainable national surveillance programs in response to a foodborne illness outbreak investigation as well as to protect the health of people and the agricultural industry.
In this study, we attempted to investigate the various characteristics of
Acknowledgment
This research was supported by a grant (18161MFDS033) from the Ministry of Food and Drug Safety in 2018.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.
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Table 1 . Primers/probe and PCR conditions used in the present study..
Target gene Sequence (5'-3') Size (bp) PCR cycling conditions spvC F: AATGAACTACGAAGTGGGCG
R: TCAAACGATAAAACGGTTCCTC
P: FAM-ATGGTGGCGAAATGCAGAGACAGGC-BHQ1112 50°C, 2 m→95°C, 10 m→95°C, 15 s→60°C, 1 m: 40 cycles sefA F: GGCTTCGGTATCTGGTGGTGTA
R: GGTCATTAATATTGGCCCTGAATA
P:Cy5-CCACTGTCCCGTTCGTTGATGGACA-BHQ298 50°C, 2 m→95°C, 10 m→95°C, 15 s→60°C, 1 m: 40 cycles hin F: TCCATGAGAAAAGCGACTAAAAT
R: AGCCGACTAATCTGTTCCTGTTC572 95°C, 3 m→95°C, 30 s→57°C, 30 s→72°C, 1 m: 30 cycles→72°C, 2 m
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Table 2 . PCR and sequencing primer for MLST used in this study..
Gene Primer sequence (5'-3') Product size (bp) thrA F: GTCACGGTGATCGATCCGGT
R: CACGATATTGATATTAGCCCG852 purE F: GACACCTCAAAAGCAGCGT'
R: AGACGGCGATACCCAGCGG635 sucA F: CGCGCTCAAACAGACCTAC
R: GACGTGGAAAATCGGCGCC793 hisD F: GAAACGTTCCATTCCGCGC
R: GCGGATTCCGGCGACCAG788 aroC F: CCTGGCACCTCGCGCTATAC
R: CCACACACGGATCGTGGCG826 hemD F: GAAGCGTTAGTGAGCCGTCTGCG
R: ATCAGCGACCTTAATATCTTGCCA666 dnaN F: ATGAAATTTACCGTTGAACGTGA
R: AATTTCTCATTCGAGAGGATTGC833
-
Table 3 . Serotypes of
Salmonella spp. isolated from raw chicken meat..Sample No. Source of isolates Somatic antigens Flagellar antigens Serovar Group I Group II O-antigen H phase 1 H phase 2 1 Meat C Group O:8 (C2-C3) 6, 8 r 1, 2 S . Bsilla2 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis3 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis4 Gizzard D Group O:9 (D1) 9, 12 g, m - S . Enteritidis5 Meat C Group O:7 (C1) 61,2, 7 g, m, s [1, 2, 7] S . Montevideo6 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis7 Gizzard D Group O:9 (D1) 9, 12 g, m - S . Enteritidis8 Meat C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow9 Meat C Group O:8 (C2-C3) 6, 8 r 1, 2 S . Bsilla10 Meat C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow11 Meat C Group O:7 (C1) 61, 7 r 1, 2 S . Virchow12 Meat C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow13 Meat C Group O:7 (C1) 61,2, 7, 14 y 1, 5 S . Bareilly14 Heart D Group O:9 (D1) 1, 9, 12 g, m - S . Enteritidis15 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis16 Feet C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow17 Gizzard C Group O:7 (C1) 61,2, 7 r 1, 2 S . Virchow18 Meat E Group O:1,3,19 (E4) 1, 3, 19 g, s ,t - S . Dessau19 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis20 Meat C Group O:8 (C2-C3) 8, 20 z4, z24 - S . Albany21 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis22 Meat C Group O:7 (C1) 61,2, 7, 14 g, m, s [1, 2, 7] S . Montevideo23 Meat D Group O:9 (D1) 9, 12 g, m - S . Enteritidis24 Gizzard D Group O:9 (D1) 9, 12 g, m - S . Enteritidis
-
Table 4 . Pathogenic gene-targeted PCR results of
Salmonella serovars..Sample No. Serovar Serological type Real-time PCR PCR O-antigen Group H Phase 1 H Phase 2 his invA stn sefA spvC hin 1 S . BsillaC r 1, 2 + + + - - + 2 S . EnteritidisD g, m - + + + + + - 3 S . EnteritidisD g, m - + + + + + - 4 S . EnteritidisD g, m - + + + + + - 5 S . MontevideoC g, m, s [1, 2, 7] + + + - - - 6 S . EnteritidisD g, m - + + + + + - 7 S . EnteritidisD g, m - + + + + + - 8 S . VirchowC r 1, 2 + + + - - + 9 S . BsillaC r 1, 2 + + + - - + 10 S . VirchowC r 1, 2 + + + - - + 11 S . VirchowC r 1, 2 + + + - - + 12 S . VirchowC r 1, 2 + + + - - + 13 S . BareillyC y 1, 5 + + + - - + 14 S . EnteritidisD g, m - + + + + + - 15 S . EnteritidisD g, m - + + + + + - 16 S . VirchowC r 1, 2 + + + - - + 17 S . VirchowC r 1, 2 + + + - - + 18 S . DessauE g, s ,t - + + + - - - 19 S . EnteritidisD g, m - + + + + - / - - 20 S . AlbanyC z4, z24 - + + + - - - 21 S . EnteritidisD g, m - + + + + + - 22 S . MontevideoC g, m, s [1, 2, 7] + + + - - - 23 S . EnteritidisD g, m - + + + + + - 24 S . EnteritidisD g, m - + + + + + -
-
Table 5 . ST definitions based on allele type for each of seven loci sequenced and assigned by the
Salmonella enterica database..ST Allele type No. isolates % of total thrA purE sucA hisD aroC hemD dnaN 11 11 6 6 7 5 3 2 11 45.8 16 14 8 10 10 6 10 7 8 33.3 4 4 34 13 13 43 16 41 2 8.3 203 17 68 12 12 81 36 69 1 4.2 14 13 7 8 8 7 8 6 1 4.2 292 48 104 9 78 104 54 100 1 4.2
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