Evaluation of the use of CRISPR loci for discrimination of <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Enteritidis strains recovered in Canada and comparison with other subtyping methods

Susan Nadin-Davis; Louise Pope; John Devenish; Ray Allain; Dele Ogunremi; Susan Nadin-Davis; Louise Pope; John Devenish; Ray Allain; Dele Ogunremi

doi:10.3934/microbiol.2022022

AIMS Microbiology

2022, Volume 8, Issue 3: 300-317. doi: 10.3934/microbiol.2022022

Previous Article Next Article

Research article

Evaluation of the use of CRISPR loci for discrimination of Salmonella enterica subsp. enterica serovar Enteritidis strains recovered in Canada and comparison with other subtyping methods

Ottawa Laboratory Fallowfield, Canadian Food Inspection Agency, Ottawa, ON, Canada

Received: 16 March 2022 Revised: 18 May 2022 Accepted: 29 May 2022 Published: 15 July 2022

Salmonella enterica subsp. enterica serovar Enteritidis remains one of the most important foodborne pathogens worldwide. To minimise its public health impact when outbreaks of the disease occur, timely investigation to identify and recall the contaminated food source is necessary. Central to this approach is the need for rapid and accurate identification of the bacterial subtype epidemiologically linked to the outbreak. While traditional methods of S. Enteritidis subtyping, such as pulsed field gel electrophoresis (PFGE) and phage typing (PT), have played an important role, the clonal nature of this organism has spurred efforts to improve subtyping resolution and timeliness through molecular based approaches. This study uses a cohort of 92 samples, recovered from a variety of sources, to compare these two traditional methods for S. Enteritidis subtyping with recently developed molecular techniques. These latter methods include the characterisation of two clustered regularly interspaced short palindromic repeats (CRISPR) loci, either in isolation or together with sequence analysis of virulence genes such as fimH. For comparison, another molecular technique developed in this laboratory involved the scoring of 60 informative single nucleotide polymorphisms (SNPs) distributed throughout the genome. Based on both the number of subtypes identified and Simpson's index of diversity, the CRISPR method was the least discriminatory and not significantly improved with the inclusion of fimH gene sequencing. While PT analysis identified the most subtypes, the SNP-PCR process generated the greatest index of diversity value. Combining methods consistently improved the number of subtypes identified, with the SNP/CRISPR typing scheme generating a level of diversity comparable with that of PT/PFGE. While these molecular methods, when combined, may have significant utility in real-world situations, this study suggests that CRISPR analysis alone lacks the discriminatory capability required to support investigations of foodborne disease outbreaks.
- Salmonella enterica,
- serovar,
- S. Enteritidis,
- subtyping,
- phage,
- PFGE,
- CRISPR,
- SNP-PCR
Citation: Susan Nadin-Davis, Louise Pope, John Devenish, Ray Allain, Dele Ogunremi. Evaluation of the use of CRISPR loci for discrimination of Salmonella enterica subsp. enterica serovar Enteritidis strains recovered in Canada and comparison with other subtyping methods[J]. AIMS Microbiology, 2022, 8(3): 300-317. doi: 10.3934/microbiol.2022022

Related Papers:

Abstract

Salmonella enterica subsp. enterica serovar Enteritidis remains one of the most important foodborne pathogens worldwide. To minimise its public health impact when outbreaks of the disease occur, timely investigation to identify and recall the contaminated food source is necessary. Central to this approach is the need for rapid and accurate identification of the bacterial subtype epidemiologically linked to the outbreak. While traditional methods of S. Enteritidis subtyping, such as pulsed field gel electrophoresis (PFGE) and phage typing (PT), have played an important role, the clonal nature of this organism has spurred efforts to improve subtyping resolution and timeliness through molecular based approaches. This study uses a cohort of 92 samples, recovered from a variety of sources, to compare these two traditional methods for S. Enteritidis subtyping with recently developed molecular techniques. These latter methods include the characterisation of two clustered regularly interspaced short palindromic repeats (CRISPR) loci, either in isolation or together with sequence analysis of virulence genes such as fimH. For comparison, another molecular technique developed in this laboratory involved the scoring of 60 informative single nucleotide polymorphisms (SNPs) distributed throughout the genome. Based on both the number of subtypes identified and Simpson's index of diversity, the CRISPR method was the least discriminatory and not significantly improved with the inclusion of fimH gene sequencing. While PT analysis identified the most subtypes, the SNP-PCR process generated the greatest index of diversity value. Combining methods consistently improved the number of subtypes identified, with the SNP/CRISPR typing scheme generating a level of diversity comparable with that of PT/PFGE. While these molecular methods, when combined, may have significant utility in real-world situations, this study suggests that CRISPR analysis alone lacks the discriminatory capability required to support investigations of foodborne disease outbreaks.

Acknowledgments

This work was supported by a federal government grant from the Canadian Genomics Research and Development Initiative (GRDI) programme's Food and Water project to SND and by a grant of the Ontario Ministry of Agriculture, Food and Rural Affairs, to DO.

Conflict of interest

All authors declare no conflicts of interest in this paper.

References

[1]	Carstens CK, Salazar JK, Darkoh C (2019) Multistate outbreaks of foodborne illness in the United States associated with fresh produce from 2010 to 2017. Front Microbiol 10: 2667-2667. https://doi.org/10.3389/fmicb.2019.02667
[2]	Majowicz S, Musto J, Scallan E, et al. (2010) The global burden of nontyphoidal Salmonella. Clin Infect Dis 50: 882-889. https://doi.org/10.1086/650733
[3]	Liu H, Whitehouse CA, Li B (2018) Presence and persistence of Salmonella in water: the impact on microbial quality of water and food safety. Front Public Health 6: 159-159. https://doi.org/10.3389/fpubh.2018.00159
[4]	(2018) PHACNational Enteric Surveillance Program Annual Summary 2016. Guelph, Ontario: Public Health Agency, Government of Canada.
[5]	Chai S, White P, Lathrop S, et al. (2012) Salmonella enterica serotype Enteritidis: increasing incidence of domestically acquired infections. Clin Infect Dis 54: S488-497. https://doi.org/10.1093/cid/cis231
[6]	Lane C, LeBaigue S, Esan O, et al. (2014) Salmonella enterica serovar Enteritidis, England and Wales, 1945–2011. Emerging Infect Dis 20: 1097-1104. https://doi.org/10.3201/eid2007.121850
[7]	Kozak GK, MacDonald D, Landry L, et al. (2013) Foodborne outbreaks in Canada linked to produce: 2001 through 2009. J Food Prot 76: 173-183. https://doi.org/10.4315/0362-028X.JFP-12-126
[8]	Chai SJ, Cole D, Nisler A, et al. (2017) Poultry: the most common food in outbreaks with known pathogens, United States, 1998–2012. Epidemiol Infect 145: 316-325. https://doi.org/10.1017/S0950268816002375
[9]	Middleton D, Savage R, Tighe M, et al. (2014) Risk factors for sporadic domestically acquired Salmonella serovar Enteritidis infections: a case-control study in Ontario, Canada, 2011. Epidemiol Infect 142: 1411-1421. https://doi.org/10.1017/S0950268813001945
[10]	Nesbitt A, Ravel A, Murray R, et al. (2012) Integrated surveillance and potential sources of Salmonella Enteritidis in human cases in Canada from 2003 to 2009. Epidemiol Infect 140: 1757-1772. https://doi.org/10.1017/S0950268811002548
[11]	Taylor M, Leslie M, Ritson M, et al. (2012) Investigation of the concurrent emergence of Salmonella enteritidis in humans and poultry in British Columbia, Canada, 2008-2010. Zoonoses Public Health 59: 584-892. https://doi.org/10.1111/j.1863-2378.2012.01500.x
[12]	Kuehn B (2010) Salmonella cases traced to egg producers. J Am Medl Assoc 304: 1316. https://doi.org/10.1001/jama.2010.1330
[13]	Chousalkar K, Gast R, Martelli F, et al. (2018) Review of egg-related salmonellosis and reduction strategies in United States, Australia, United Kingdom and New Zealand. Crit Rev Microbiol 44: 290-303. https://doi.org/10.1080/1040841X.2017.1368998
[14]	Tang S, Orsi RH, Luo H, et al. (2019) Assessment and comparison of molecular subtyping and characterization methods for Salmonella. Front Microbiol 10: 1591. https://doi.org/10.3389/fmicb.2019.01591
[15]	Ward LR, de Sa JDH, Rowe B (1987) A phage-typing scheme for Salmonella enteritidis. Epidemiol Infect 99: 291-294. https://doi.org/10.1017/S0950268800067765
[16]	Carrique-Mas JJ, Papadopoulou C, Evans SJ, et al. (2008) Trends in phage types and antimicrobial resistance of Salmonella enterica serovar Enteritidis isolated from animals in Great Britain from 1990 to 2005. Vet Rec 162: 541-546. https://doi.org/10.1136/vr.162.17.541
[17]	Peters TM, Berghold C, Brown D, et al. (2007) Relationship of pulsed-field profiles with key phage types of Salmonella enterica serotype Enteritidis in Europe: results of an international multi-centre study. Epidemiol Infect 135: 1274-1281. https://doi.org/10.1017/S0950268807008102
[18]	Ribot EM, Fair MA, Gautom R, et al. (2006) Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis 3: 59-67. https://doi.org/10.1089/fpd.2006.3.59
[19]	Nadon CA, Trees E, Ng LK, et al. (2013) Development and application of MLVA methods as a tool for inter-laboratory surveillance. Eurosurveillance 18: 20565. https://doi.org/10.2807/1560-7917.ES2013.18.35.20565
[20]	Bertrand S, De Lamine de Bex G, Wildemauwe C, et al. (2015) Multi Locus Variable-Number Tandem Repeat (MLVA) typing tools improved the surveillance of Salmonella Enteritidis: A 6 Years Retrospective Study. PLoS One 10: e0117950. https://doi.org/10.1371/journal.pone.0117950
[21]	Maiden MCJ, Bygraves JA, Feil E, et al. (1998) Multilocus sequence typing: A portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci 95: 3140-3145. https://doi.org/10.1073/pnas.95.6.3140
[22]	Achtman M, Wain J, Weill F-X, et al. (2012) Multilocus sequence typing as a replacement for serotyping in Salmonella enterica. PLoS Pathog 8: e1002776. https://doi.org/10.1371/journal.ppat.1002776
[23]	Yoshida C, Kruczkiewicz P, Laing C, et al. (2016) The Salmonella In Silico Typing Resource (SISTR): an open web-accessible tool for rapidly typing and subtyping draft Salmonella genome assemblies. PLoS One 11: e0147101. https://doi.org/10.1371/journal.pone.0147101
[24]	Pearce ME, Alikhan NF, Dallman TJ, et al. (2018) Comparative analysis of core genome MLST and SNP typing within a European Salmonella serovar Enteritidis outbreak. Int J Food Microbiol 274: 1-11. https://doi.org/10.1016/j.ijfoodmicro.2018.02.023
[25]	Ogunremi D, Kelly H, Dupras AA, et al. (2014) Development of a new molecular subtyping tool for Salmonella enterica serovar Enteritidis based on single nucleotide polymorphism genotyping using PCR. J Clin Microbiol 52: 4275-4285. https://doi.org/10.1128/JCM.01410-14
[26]	Shariat N, Dudley EG (2014) CRISPRs: molecular signatures used for pathogen subtyping. Appl Environ Microbiol 80: 430-439. https://doi.org/10.1128/AEM.02790-13
[27]	Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327: 167-170. https://doi.org/10.1126/science.1179555
[28]	Burmistrz M, Krakowski K, Krawczyk-Balska A (2020) RNA-targeting CRISPR-Cas systems and their applications. Int J Mol Sci 21: 1122. https://doi.org/10.3390/ijms21031122
[29]	Fricke WF, Mammel MK, McDermott PF, et al. (2011) Comparative genomics of 28 Salmonella enterica isolates: evidence for CRISPR-mediated adaptive sublineage evolution. J Bacteriol 193: 3556-3568. https://doi.org/10.1128/JB.00297-11
[30]	Fabre L, Le Hello S, Roux C, et al. (2014) CRISPR Is an optimal target for the design of specific PCR assays for Salmonella enterica serotypes Typhi and Paratyphi A. PLoS Neglected Trop Dis 8: e2671. https://doi.org/10.1371/journal.pntd.0002671
[31]	Fabre L, Zhang J, Guigon G, et al. (2012) CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections. PLoS ONE 7: e36995. https://doi.org/10.1371/journal.pone.0036995
[32]	Liu F, Barrangou R, Gerner-Smidt P, et al. (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. Appl Environ Microbiol 77: 1946-1956. https://doi.org/10.1128/AEM.02625-10
[33]	Liu F, Kariyawasam S, Jayarao BM, 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). Appl Environ Microbiol 77: 4520-4526. https://doi.org/10.1128/AEM.00468-11
[34]	Shariat N, Sandt CH, DiMarzio MJ, et al. (2013) CRISPR-MVLST subtyping of Salmonella enterica subsp. enterica serovars Typhimurium and Heidelberg and application in identifying outbreak isolates. BMC Microbiol 13: 254-254. https://doi.org/10.1186/1471-2180-13-254
[35]	Fu S, Hiley L, Octavia S, et al. (2017) Comparative genomics of Australian and international isolates of Salmonella Typhimurium: correlation of core genome evolution with CRISPR and prophage profiles. Sci Rep 7: 9733-9733. https://doi.org/10.1038/s41598-017-06079-1
[36]	Fei X, He X, Guo R, et al. (2017) Analysis of prevalence and CRISPR typing reveals persistent antimicrobial-resistant Salmonella infection across chicken breeder farm production stages. Food Control 77: 102-109. https://doi.org/10.1016/j.foodcont.2017.01.023
[37]	Li Q, Wang X, Yin K, et al. (2018) Genetic analysis and CRISPR typing of Salmonella enterica serovar Enteritidis from different sources revealed potential transmission from poultry and pig to human. Int J Food Microbiol 266: 119-125. https://doi.org/10.1016/j.ijfoodmicro.2017.11.025
[38]	Shariat N, Timme RE, Pettengill JB, et al. (2015) Characterization and evolution of Salmonella CRISPR-Cas systems. Microbiology 161: 374-386. https://doi.org/10.1099/mic.0.000005
[39]	Touchon M, Rocha EPC (2010) The small, slow and specialized CRISPR and anti-CRISPR of Escherichia and Salmonella. PLoS ONE 5: e11126. https://doi.org/10.1371/journal.pone.0011126
[40]	Nadin-Davis S, Pope L, Ogunremi D, et al. (2019) A real-time PCR regimen for testing environmental samples for Salmonella enterica subsp. enterica serovars of concern to the poultry industry, with special focus on Salmonella Enteritidis. Can J Microbiol 65: 162-173. https://doi.org/10.1139/cjm-2018-0417
[41]	Reid A Isolation and identification of Salmonella from food and environmental samples: Standard operating procedure MFHPB 20. Microbiological Methods Committee, Bureau of Microbial Hazards, Health Canada (2009).
[42]	Kumar S, Stecher G, Li M, et al. (2018) MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol 35: 1547-1549. https://doi.org/10.1093/molbev/msy096
[43]	Couvin D, Bernheim A, Toffano-Nioche C, et al. (2018) CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res 46: W246-W251. https://doi.org/10.1093/nar/gky425
[44]	PulseNetInternationalStandard Operating Procedure for PulseNet PFGE of Escherichia coli O157:H7, Escherichia coli non-O157 (STEC), Salmonella serotypes, Shigella sonnei and Shigella flexneri. (2013).
[45]	Hunter PR, Gaston MA (1988) Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J Clin Microbiol 26: 2465-2466. https://doi.org/10.1128/jcm.26.11.2465-2466.1988
[46]	Nadin-Davis S, Pope L, Chmara J, et al. (2020) An unusual Salmonella Enteritidis strain carrying a modified virulence plasmid lacking the prot6e gene represents a geographically widely distributed lineage. Front Microbiol 11: 1322. https://doi.org/10.3389/fmicb.2020.01322
[47]	Deng X, Shariat N, Driebe EM, et al. (2015) Comparative analysis of subtyping methods against a whole-genome-sequencing standard for Salmonella enterica serotype Enteritidis. J Clin Microbiol 53: 212-218. https://doi.org/10.1128/JCM.02332-14
[48]	Ogunremi D, Devenish J, Amoako K, et al. (2014) High resolution assembly and characterization of genomes of Canadian isolates of Salmonella Enteritidis. BMC Genomics 15: 713. https://doi.org/10.1186/1471-2164-15-713
[49]	Shariat N, DiMarzio MJ, Yin S, et al. (2013) The combination of CRISPR-MVLST and PFGE provides increased discriminatory power for differentiating human clinical isolates of Salmonella enterica subsp. enterica serovar Enteritidis. Food Microbiol 34: 164-173. https://doi.org/10.1016/j.fm.2012.11.012
[50]	Hiley L, Graham RMA, Jennison AV (2021) Characterisation of IncI1 plasmids associated with change of phage type in isolates of Salmonella enterica serovar Typhimurium. BMC Microbiol 21: 92-92. https://doi.org/10.1186/s12866-021-02151-z
[51]	Tankouo-Sandjong B, Kinde H, Wallace I (2012) Development of a sequence typing scheme for differentiation of Salmonella Enteritidis strains. FEMS Microbiol Lett 331: 165-175. https://doi.org/10.1111/j.1574-6968.2012.02568.x

microbiol-08-03-022-tables1.xlsx
microbiol-08-03-022-tables2.xlsx
microbiol-08-03-022-tables3.xlsx

Reader Comments

Your name:*

Email:*
© 2022 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0)