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Research article

Food Microbiology and Biotechnology (FMB)  |  Foodborne Pathogens and Food Safety

Novel Heptaplex PCR-Based Diagnostics for Enteric Fever Caused by Typhoidal Salmonella Serovars and Its Applicability in Clinical Blood Culture

Hyun-Joong Kim1, Younsik Jung2, Mi-Ju Kim2, and Hae-Yeong Kim2*

1Department of Food Engineering, Mokpo National University, Muan 58554, Republic of Korea
2Institute of Life Sciences and Resources and the Department of Food Science and Biotechnology, Kyung Hee University, Yongin 17104 Republic of Korea

Correspondence to:
Hae-Yeong Kim,      hykim@khu.ac.kr
Received: July 24, 2023; Revised: August 12, 2023; Accepted: August 14, 2023

J. Microbiol. Biotechnol. 2023; 33(11): 1457-1466

Published November 28, 2023 https://doi.org/10.4014/jmb.2307.07031

Copyright © The Korean Society for Microbiology and Biotechnology.

Abstract

Enteric fever is caused by typhoidal Salmonella serovars (Typhi, Paratyphi A, Paratyphi B, and Paratyphi C). Owing to the importance of Salmonella serovars in clinics and public hygiene, reliable diagnostics for typhoidal serovars are crucial. This study aimed to develop a novel diagnostic tool for typhoidal Salmonella serovars and evaluate the use of human blood for clinically diagnosing enteric fever. Five genes were selected to produce specific PCR results against typhoidal Salmonella serovars based on the genes of Salmonella Typhi. Heptaplex PCR, including genetic markers of generic Salmonella, Salmonella enterica subsp. enterica, and typhoidal Salmonella serovars, was developed. Typhoidal Salmonella heptaplex PCR using genomic DNAs from 200 Salmonella strains (112 serovars) provided specifically amplified PCR products for each typhoidal Salmonella serovar. These results suggest that heptaplex PCR can sufficiently discriminate between typhoidal and nontyphoidal Salmonella serovars. Heptaplex PCR was applied to Salmonella-spiked blood cultures directly and provided diagnostic results after 12- or 13.5-h blood culture. Additionally, it demonstrated diagnostic performance with colonies recovered from a 6-h blood culture. This study provides a reliable DNA-based tool for diagnosing typhoidal Salmonella serovars that may be useful in clinical microbiology and epidemiology.

Keywords

Enteric fever, typhoidal Salmonella serovar, PCR, diagnostics

Graphical Abstract


References

  1. Andino A, Hanning I. 2015. Salmonella enterica: survival, colonization, and virulence differences among serovars. Sci. World J. 2015: 520179.
    Pubmed PMC
  2. Bhan MK, Bah R., Bhatnagar S. 2005. Typhoid and paratyphoid fever. Lancet Infect. Dis. 366: 749-762.
    Pubmed
  3. Crump JA, Sjölund-Karlsson M, Gordon MA, Parry CM. 2015. Epidemiology, clinical presentation, laboratory diagnosis, antimicrobial resistance, and antimicrobial management of invasive Salmonella infections. Clin. Microbiol. Rev. 28: 901-937.
    Pubmed PMC
  4. Grimont PAD, Weill FX. 2015. Antigenic formulas of the Salmonella serovars, 9th edition. W.H.O. Collaborating Centre for Reference and Research on Salmonella. Institute Pasteur, Paris, France.
  5. Andrews JR, Ryan ET. 2015. Diagnostics for invasive Salmonella infections: current challenges and future directions. Vaccine 33: C8-C15.
    Pubmed PMC
  6. Crump JA, Luby SP, Mintz ED. 2004. The global burden of typhoid fever. Bull. World Health Organ. 82: 346-353.
  7. Gal-Mor O, Boyle EC, Grassl GA. 2014. Same species, different diseases: how and why typhoidal and non-typhoidal Salmonella enterica serovars differ. Front. Microbiol. 5: 00391.
    Pubmed PMC
  8. Gibani MM, Britto C, Pollard AJ. 2018. Typhoid and paratyphoid fever: a call to action. Curr. Opin. Infect. Dis. 31: 440-448.
    Pubmed PMC
  9. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson M, Roy SL, et al. 2011. Foodborne illness acquired in the United Statesmajor pathogens. Emerg. Infect. Dis. 17: 7-15.
    Pubmed PMC
  10. Connor BA, Schwartz E. 2005. Typhoid and paratyphoid fever in travelers. Lancet Infect. Dis. 5: 623-628.
    Pubmed
  11. Ngan GJY, Ng LM, Lin RT, Teo JW. 2010. Development of a novel multiplex PCR for the detection and differentiation of Salmonella enterica serovars Typhi and Paratyphi A. Res. Microbiol. 161: 243-248.
    Pubmed
  12. Bhutta ZA. 2006. Current concepts in the diagnosis and treatment of typhoid fever. BMJ 333: 78-82.
    Pubmed PMC
  13. Wain J, Hosoglu S. 2008. The laboratory diagnosis of enteric fever. J. Infect. Dev. Ctries. 2: 421-425.
  14. Clark AE, Kaleta EJ, Arora A, Wolk, DM. 2013. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology. Clin. Microbiol. Rev. 26: 547-603.
    Pubmed PMC
  15. Kuhns M, Zautner AE, Rabsch W, Zimmermann O, Weig M, Bader O, et al. 2012. Rapid discrimination of Salmonella enterica serovar Typhi from other serovars by MALDI-TOF mass spectrometry. PLoS One 7: e40004.
    Pubmed PMC
  16. Hatta M, Smits HL. 2007. Detection of Salmonella typhi by nested polymerase chain reaction in blood, urine, and stool samples. Am. J. Trop. Med. Hyg. 76: 139-143.
  17. Massi MN, Gotoh A, Bishnu A, Kawabata M, Shirakawa T, Gotoh A, et al. 2003. Rapid diagnosis of typhoid fever by PCR assay using one pair of primers from flagellin gene of Salmonella typhi. J. Infect. Chemother. 9: 233-237.
    Pubmed
  18. Massi MN, Shirakawa T, Gotoh A, Bishnu A, Hatta M, Kawabata M, et al. 2005. Quantitative detection of Salmonella enterica serovar Typhi from blood of suspected typhoid fever patients by real-time PCR. Int. J. Med. Microbiol. 295: 117-120.
    Pubmed
  19. Prakash P, Mishra OP, Singh AK, Gulati AK, Nath G. 2005. Evaluation of nested PCR in diagnosis of typhoid fever. J. Clin. Microbiol. 43: 431-432.
    Pubmed PMC
  20. Hirose K, Itoh KI, Nakajima H, Kurazono T, Yamaguchi M, Moriya K, et al. 2002. Selective amplification of tyv (rfbE), prt (rfbS), viaB, and fliC genes by multiplex PCR for identification of Salmonella enterica serovars Typhi and Paratyphi A. J. Clin. Microbiol. 40: 633-636.
    Pubmed PMC
  21. Ou HY, Ju CTS, Thong KL, Ahmad N, Deng Z, Barer MR, et al. 2007. Translational genomics to develop a Salmonella enterica serovar Paratyphi A multiplex polymerase chain reaction assay. J. Mol. Diagn. 9: 624-630.
    Pubmed PMC
  22. Levy H, Diallo S, Tennant SM, Livio S, Sow SO, Tapia M, et al. 2008. PCR method to identify Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B among Salmonella isolates from the blood of patients with clinical enteric fever. J. Clin. Microbiol. 46: 1861-1866.
    Pubmed PMC
  23. Woods DF, Reen FJ, Gilroy D, Buckley J, Frye JG, Boyd EF. 2008. Rapid multiplex PCR and real-time TaqMan PCR assays for detection of Salmonella enterica and the highly virulent serovars Choleraesuis and Paratyphi C. J. Clin. Microbiol. 46: 4018-4022.
    Pubmed PMC
  24. Kim HJ, Park SH, Kim HY. 2006. Genomic sequence comparison of Salmonella enterica serovar Typhimurium LT2 with Salmonella genomic sequences, and genotyping of salmonellae by using PCR. Appl. Environ. Microbiol. 72: 6142-6151.
    Pubmed PMC
  25. Kim HJ, Park SH, Lee TH, Nahm BH, Chung YH, Seo KH, et al. 2006. Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars. J. Food. Protect. 69: 1653-1661.
    Pubmed
  26. Park SH, Kim HJ, Cho WH, Kim JH, Oh MH, Kim SH, et al. 2009. Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR. FEMS Microbiol. Lett. 301: 137-146.
    Pubmed
  27. Malorny B, Hoorfar J, Bunge C, Helmuth R. 2003. Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard. Appl. Environ. Microbiol. 69: 290-296.
    Pubmed PMC
  28. Seo KH, Valentin-Bon IE, Brackett RE, Holt PS. 2004. Rapid, specific detection of Salmonella enteritidis in pooled eggs by real-time PCR. J. Food Prot. 67: 864-869.
    Pubmed
  29. Chung YH, Kim SY, Chang YH. 2003. Prevalence and antibiotic susceptibility of Salmonella isolated from Foods in Korea from 1993 to 2001. J. Food Prot. 66: 1154-1157.
    Pubmed
  30. Zhang Z, Schwartz S, Wagner L, Miller W. 2000. A greedy algorithm for aligning DNA sequences. J. Comput. Biol. 7: 203-214.
    Pubmed
  31. Al-Emran HM, Hahn A, Baum J, Espinoza LMC, Deerin J, Im J, et al. 2016. Diagnosing Salmonella enterica serovar Typhi infections by polymerase chain reaction using EDTA blood samples of febrile patients from Burkina Faso. Clin. Infect. Dis. 62: S37-41.
    Pubmed
  32. Haque A, Ahmed J, Qureshi JA. 1999. Early detection of typhoid by polymerase chain reaction. Ann. Saudi. Med. 19: 337- 340.
    Pubmed
  33. Song JH, Cho H, Park MY, Na DS, Moon HB, Pai CH. 1993. Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction. J. Clin. Microbiol. 31: 1439-1443.
    Pubmed PMC
  34. Zhou L, Pollard AJ. 2010. A fast and highly sensitive blood culture PCR method for clinical detection of Salmonella enterica serovar Typhi. Ann. Clin. Microbiol. Antimicrob. 9: 14.
    Pubmed PMC
  35. Kaye D, Palmieri M, Rocha H. 1966. Effect of bile on the action of blood against Salmonella J. Bacteriol. 91: 945-952.
    Pubmed PMC

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Article

Research article

J. Microbiol. Biotechnol. 2023; 33(11): 1457-1466

Published online November 28, 2023 https://doi.org/10.4014/jmb.2307.07031

Copyright © The Korean Society for Microbiology and Biotechnology.

Novel Heptaplex PCR-Based Diagnostics for Enteric Fever Caused by Typhoidal Salmonella Serovars and Its Applicability in Clinical Blood Culture

Hyun-Joong Kim1, Younsik Jung2, Mi-Ju Kim2, and Hae-Yeong Kim2*

1Department of Food Engineering, Mokpo National University, Muan 58554, Republic of Korea
2Institute of Life Sciences and Resources and the Department of Food Science and Biotechnology, Kyung Hee University, Yongin 17104 Republic of Korea

Correspondence to:Hae-Yeong Kim,      hykim@khu.ac.kr

Received: July 24, 2023; Revised: August 12, 2023; Accepted: August 14, 2023

Abstract

Enteric fever is caused by typhoidal Salmonella serovars (Typhi, Paratyphi A, Paratyphi B, and Paratyphi C). Owing to the importance of Salmonella serovars in clinics and public hygiene, reliable diagnostics for typhoidal serovars are crucial. This study aimed to develop a novel diagnostic tool for typhoidal Salmonella serovars and evaluate the use of human blood for clinically diagnosing enteric fever. Five genes were selected to produce specific PCR results against typhoidal Salmonella serovars based on the genes of Salmonella Typhi. Heptaplex PCR, including genetic markers of generic Salmonella, Salmonella enterica subsp. enterica, and typhoidal Salmonella serovars, was developed. Typhoidal Salmonella heptaplex PCR using genomic DNAs from 200 Salmonella strains (112 serovars) provided specifically amplified PCR products for each typhoidal Salmonella serovar. These results suggest that heptaplex PCR can sufficiently discriminate between typhoidal and nontyphoidal Salmonella serovars. Heptaplex PCR was applied to Salmonella-spiked blood cultures directly and provided diagnostic results after 12- or 13.5-h blood culture. Additionally, it demonstrated diagnostic performance with colonies recovered from a 6-h blood culture. This study provides a reliable DNA-based tool for diagnosing typhoidal Salmonella serovars that may be useful in clinical microbiology and epidemiology.

Keywords: Enteric fever, typhoidal Salmonella serovar, PCR, diagnostics

References

  1. Andino A, Hanning I. 2015. Salmonella enterica: survival, colonization, and virulence differences among serovars. Sci. World J. 2015: 520179.
    Pubmed KoreaMed
  2. Bhan MK, Bah R., Bhatnagar S. 2005. Typhoid and paratyphoid fever. Lancet Infect. Dis. 366: 749-762.
    Pubmed
  3. Crump JA, Sjölund-Karlsson M, Gordon MA, Parry CM. 2015. Epidemiology, clinical presentation, laboratory diagnosis, antimicrobial resistance, and antimicrobial management of invasive Salmonella infections. Clin. Microbiol. Rev. 28: 901-937.
    Pubmed KoreaMed
  4. Grimont PAD, Weill FX. 2015. Antigenic formulas of the Salmonella serovars, 9th edition. W.H.O. Collaborating Centre for Reference and Research on Salmonella. Institute Pasteur, Paris, France.
  5. Andrews JR, Ryan ET. 2015. Diagnostics for invasive Salmonella infections: current challenges and future directions. Vaccine 33: C8-C15.
    Pubmed KoreaMed
  6. Crump JA, Luby SP, Mintz ED. 2004. The global burden of typhoid fever. Bull. World Health Organ. 82: 346-353.
  7. Gal-Mor O, Boyle EC, Grassl GA. 2014. Same species, different diseases: how and why typhoidal and non-typhoidal Salmonella enterica serovars differ. Front. Microbiol. 5: 00391.
    Pubmed KoreaMed
  8. Gibani MM, Britto C, Pollard AJ. 2018. Typhoid and paratyphoid fever: a call to action. Curr. Opin. Infect. Dis. 31: 440-448.
    Pubmed KoreaMed
  9. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson M, Roy SL, et al. 2011. Foodborne illness acquired in the United Statesmajor pathogens. Emerg. Infect. Dis. 17: 7-15.
    Pubmed KoreaMed
  10. Connor BA, Schwartz E. 2005. Typhoid and paratyphoid fever in travelers. Lancet Infect. Dis. 5: 623-628.
    Pubmed
  11. Ngan GJY, Ng LM, Lin RT, Teo JW. 2010. Development of a novel multiplex PCR for the detection and differentiation of Salmonella enterica serovars Typhi and Paratyphi A. Res. Microbiol. 161: 243-248.
    Pubmed
  12. Bhutta ZA. 2006. Current concepts in the diagnosis and treatment of typhoid fever. BMJ 333: 78-82.
    Pubmed KoreaMed
  13. Wain J, Hosoglu S. 2008. The laboratory diagnosis of enteric fever. J. Infect. Dev. Ctries. 2: 421-425.
  14. Clark AE, Kaleta EJ, Arora A, Wolk, DM. 2013. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology. Clin. Microbiol. Rev. 26: 547-603.
    Pubmed KoreaMed
  15. Kuhns M, Zautner AE, Rabsch W, Zimmermann O, Weig M, Bader O, et al. 2012. Rapid discrimination of Salmonella enterica serovar Typhi from other serovars by MALDI-TOF mass spectrometry. PLoS One 7: e40004.
    Pubmed KoreaMed
  16. Hatta M, Smits HL. 2007. Detection of Salmonella typhi by nested polymerase chain reaction in blood, urine, and stool samples. Am. J. Trop. Med. Hyg. 76: 139-143.
  17. Massi MN, Gotoh A, Bishnu A, Kawabata M, Shirakawa T, Gotoh A, et al. 2003. Rapid diagnosis of typhoid fever by PCR assay using one pair of primers from flagellin gene of Salmonella typhi. J. Infect. Chemother. 9: 233-237.
    Pubmed
  18. Massi MN, Shirakawa T, Gotoh A, Bishnu A, Hatta M, Kawabata M, et al. 2005. Quantitative detection of Salmonella enterica serovar Typhi from blood of suspected typhoid fever patients by real-time PCR. Int. J. Med. Microbiol. 295: 117-120.
    Pubmed
  19. Prakash P, Mishra OP, Singh AK, Gulati AK, Nath G. 2005. Evaluation of nested PCR in diagnosis of typhoid fever. J. Clin. Microbiol. 43: 431-432.
    Pubmed KoreaMed
  20. Hirose K, Itoh KI, Nakajima H, Kurazono T, Yamaguchi M, Moriya K, et al. 2002. Selective amplification of tyv (rfbE), prt (rfbS), viaB, and fliC genes by multiplex PCR for identification of Salmonella enterica serovars Typhi and Paratyphi A. J. Clin. Microbiol. 40: 633-636.
    Pubmed KoreaMed
  21. Ou HY, Ju CTS, Thong KL, Ahmad N, Deng Z, Barer MR, et al. 2007. Translational genomics to develop a Salmonella enterica serovar Paratyphi A multiplex polymerase chain reaction assay. J. Mol. Diagn. 9: 624-630.
    Pubmed KoreaMed
  22. Levy H, Diallo S, Tennant SM, Livio S, Sow SO, Tapia M, et al. 2008. PCR method to identify Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B among Salmonella isolates from the blood of patients with clinical enteric fever. J. Clin. Microbiol. 46: 1861-1866.
    Pubmed KoreaMed
  23. Woods DF, Reen FJ, Gilroy D, Buckley J, Frye JG, Boyd EF. 2008. Rapid multiplex PCR and real-time TaqMan PCR assays for detection of Salmonella enterica and the highly virulent serovars Choleraesuis and Paratyphi C. J. Clin. Microbiol. 46: 4018-4022.
    Pubmed KoreaMed
  24. Kim HJ, Park SH, Kim HY. 2006. Genomic sequence comparison of Salmonella enterica serovar Typhimurium LT2 with Salmonella genomic sequences, and genotyping of salmonellae by using PCR. Appl. Environ. Microbiol. 72: 6142-6151.
    Pubmed KoreaMed
  25. Kim HJ, Park SH, Lee TH, Nahm BH, Chung YH, Seo KH, et al. 2006. Identification of Salmonella enterica serovar Typhimurium using specific PCR primers obtained by comparative genomics in Salmonella serovars. J. Food. Protect. 69: 1653-1661.
    Pubmed
  26. Park SH, Kim HJ, Cho WH, Kim JH, Oh MH, Kim SH, et al. 2009. Identification of Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium, Enteritidis and Typhi using multiplex PCR. FEMS Microbiol. Lett. 301: 137-146.
    Pubmed
  27. Malorny B, Hoorfar J, Bunge C, Helmuth R. 2003. Multicenter validation of the analytical accuracy of Salmonella PCR: towards an international standard. Appl. Environ. Microbiol. 69: 290-296.
    Pubmed KoreaMed
  28. Seo KH, Valentin-Bon IE, Brackett RE, Holt PS. 2004. Rapid, specific detection of Salmonella enteritidis in pooled eggs by real-time PCR. J. Food Prot. 67: 864-869.
    Pubmed
  29. Chung YH, Kim SY, Chang YH. 2003. Prevalence and antibiotic susceptibility of Salmonella isolated from Foods in Korea from 1993 to 2001. J. Food Prot. 66: 1154-1157.
    Pubmed
  30. Zhang Z, Schwartz S, Wagner L, Miller W. 2000. A greedy algorithm for aligning DNA sequences. J. Comput. Biol. 7: 203-214.
    Pubmed
  31. Al-Emran HM, Hahn A, Baum J, Espinoza LMC, Deerin J, Im J, et al. 2016. Diagnosing Salmonella enterica serovar Typhi infections by polymerase chain reaction using EDTA blood samples of febrile patients from Burkina Faso. Clin. Infect. Dis. 62: S37-41.
    Pubmed
  32. Haque A, Ahmed J, Qureshi JA. 1999. Early detection of typhoid by polymerase chain reaction. Ann. Saudi. Med. 19: 337- 340.
    Pubmed
  33. Song JH, Cho H, Park MY, Na DS, Moon HB, Pai CH. 1993. Detection of Salmonella typhi in the blood of patients with typhoid fever by polymerase chain reaction. J. Clin. Microbiol. 31: 1439-1443.
    Pubmed KoreaMed
  34. Zhou L, Pollard AJ. 2010. A fast and highly sensitive blood culture PCR method for clinical detection of Salmonella enterica serovar Typhi. Ann. Clin. Microbiol. Antimicrob. 9: 14.
    Pubmed KoreaMed
  35. Kaye D, Palmieri M, Rocha H. 1966. Effect of bile on the action of blood against Salmonella J. Bacteriol. 91: 945-952.
    Pubmed KoreaMed