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Volume 8, Issue 1

Characterization of the CRISPR1-Cas array and its subtyping potential in from Malaysia Open Access

Abstract

is a gram-positive bacterium and a common cause of hospital-associated infections. Three major CRISPR loci have been discovered in this species, namely, CRISPR1-, CRISPR2 and CRISPR3-. We developed novel primers which target the CRISPR1- loci in and tested these primers on 26 isolates isolated from diverse settings from Segamat, Malaysia. Half of the isolates were found to carry the CRISPR1- locus, and the CRISPR1 array was successfully amplified in 12 out of 13 isolates that contained the gene. Characterization of the CRISPR array shows that CRISPR1- shares similar array length and typical repeat sequences with CRISPR2 but differs significantly in terms of spacer identities and terminal repeat (TR) sequences. Most CRISPR spacers encode for chromosomal DNA sequences. Genotype characterization based on ancestral spacer (AS) and TR sequences indicates that with the same CRISPR1-AS genotype do not always harbour the same CRISPR2-AS genotypes and vice versa. A combined CRISPR1- and CRISPR2 typing offers comparable discriminatory power to MLST, suggesting its potential to be used in short-term strain identification and epidemiological surveillance at a lower sequencing cost. Our study provides a genetic reference for future studies in Southeast Asia.

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Keyword(s): CRISPR-Cas , Enterococcus faecalis , epidemiology , MLST , repeat-spacer and typing
Funding
This study was supported by the:
  • Ministry of Higher Education, Malaysia (Award FRGS/1/2019/SKK01/MUSM/01/1)
    • Principal Award Recipient: SadequrRahman
© 2026 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2026-01-30
2026-03-07

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References

  1. Palmer KL, Gilmore MS. Multidrug-resistant enterococci lack CRISPR-cas. mBio 2010; 1:e00227-10 [View Article] [PubMed]
     [Google Scholar]
  2. Daniel DS, Lee SM, Dykes GA, Rahman S. Public health risks of multiple-drug-resistant Enterococcus spp. in Southeast Asia. Appl Environ Microbiol 2015; 81:6090–6097 [View Article] [PubMed]
     [Google Scholar]
  3. Dos Santos BA, de Oliveira J da S, Parmanhani-da-Silva BM, Ribeiro RL, Teixeira LM et al. CRISPR elements and their association with antimicrobial resistance and virulence genes among vancomycin-resistant and vancomycin-susceptible enterococci recovered from human and food sources. Infect Genet Evol 2020; 80:104183 [View Article] [PubMed]
     [Google Scholar]
  4. Gholizadeh P, Aghazadeh M, Ghotaslou R, Rezaee MA, Pirzadeh T et al. Role of CRISPR-Cas system on antibiotic resistance patterns of Enterococcus faecalis. Ann Clin Microbiol Antimicrob 2021; 20:49 [View Article] [PubMed]
     [Google Scholar]
  5. Hullahalli K, Rodrigues M, Schmidt BD, Li X, Bhardwaj P et al. Comparative analysis of the orphan CRISPR2 locus in 242 Enterococcus faecalis strains. PLoS One 2015; 10:e0138890 [View Article] [PubMed]
     [Google Scholar]
  6. Maikova A, Severinov K, Soutourina O. New insights into functions and possible applications of Clostridium difficile CRISPR-Cas system. Front Microbiol 2018; 9:1740 [View Article] [PubMed]
     [Google Scholar]
  7. Ishino Y, Krupovic M, Forterre P. History of CRISPR-Cas from encounter with a mysterious repeated sequence to genome editing technology. J Bacteriol 2018; 200:e00580-17 [View Article] [PubMed]
     [Google Scholar]
  8. Koonin EV, Makarova KS. Origins and evolution of CRISPR-Cas systems. Philos Trans R Soc Lond B Biol Sci 2019; 374:20180087 [View Article] [PubMed]
     [Google Scholar]
  9. Horvath P, Romero DA, Coûté-Monvoisin A-C, Richards M, Deveau H et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J Bacteriol 2008; 190:1401–1412 [View Article] [PubMed]
     [Google Scholar]
  10. Bhaya D, Davison M, Barrangou R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 2011; 45:273–297 [View Article] [PubMed]
     [Google Scholar]
  11. Rath D, Amlinger L, Rath A, Lundgren M. The CRISPR-Cas immune system: biology, mechanisms and applications. Biochimie 2015; 117:119–128 [View Article] [PubMed]
     [Google Scholar]
  12. Lier C, Baticle E, Horvath P, Haguenoer E, Valentin A-S et al. Analysis of the type II-A CRISPR-Cas system of Streptococcus agalactiae reveals distinctive features according to genetic lineages. Front Genet 2015; 6:214 [View Article] [PubMed]
     [Google Scholar]
  13. Shariat N, Dudley EG. CRISPRs: molecular signatures used for pathogen subtyping. Appl Environ Microbiol 2014; 80:430–439 [View Article] [PubMed]
     [Google Scholar]
  14. Bourgogne A, Garsin DA, Qin X, Singh KV, Sillanpaa J et al. Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF. Genome Biol 2008; 9:R110 [View Article] [PubMed]
     [Google Scholar]
  15. Katyal I, Chaban B, Ng B, Hill JE. CRISPRs of Enterococcus faecalis and E. hirae isolates from pig feces have species-specific repeats but share some common spacer sequences. Microb Ecol 2013; 66:182–188 [View Article] [PubMed]
     [Google Scholar]
  16. Lyons C, Raustad N, Bustos MA, Shiaris M. Incidence of type II CRISPR1-Cas systems in Enterococcus is species-dependent. PLoS One 2015; 10:e0143544 [View Article] [PubMed]
     [Google Scholar]
  17. Jackson CR, Fedorka-Cray PJ, Barrett JB. Use of a genus- and species-specific multiplex PCR for identification of enterococci. J Clin Microbiol 2004; 42:3558–3565 [View Article] [PubMed]
     [Google Scholar]
  18. Clinical and Laboratory Standards Institute CLSI Supplement M100: Performance Standards for Antimicrobial Susceptibility Testing, 28th Ed Wayne: Clinical and Laboratory Standards Institute; 2018
     [Google Scholar]
  19. Couvin D, Bernheim A, Toffano-Nioche C, Touchon M, Michalik J et al. CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res 2018; 46:W246–W251 [View Article] [PubMed]
     [Google Scholar]
  20. Gawryszewska I, Malinowska K, Kuch A, Chrobak-Chmiel D, Trokenheim LL et al. Distribution of antimicrobial resistance determinants, virulence-associated factors and clustered regularly interspaced palindromic repeats loci in isolates of Enterococcus faecalis from various settings and genetic lineages. Pathog Dis 2017; 75:ftx021 [View Article] [PubMed]
     [Google Scholar]
  21. IBM Corp IBM SPSS Statistics for Windows, version 26.0. Armonk, NY: IBM Corporation; 2019
  22. Ruiz-Garbajosa P, Bonten MJM, Robinson DA, Top J, Nallapareddy SR et al. Multilocus sequence typing scheme for Enterococcus faecalis reveals hospital-adapted genetic complexes in a background of high rates of recombination. J Clin Microbiol 2006; 44:2220–2228 [View Article] [PubMed]
     [Google Scholar]
  23. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 2018; 35:1547–1549 [View Article] [PubMed]
     [Google Scholar]
  24. Carriço JA, Silva-Costa C, Melo-Cristino J, Pinto FR, de Lencastre H et al. Illustration of a common framework for relating multiple typing methods by application to macrolide-resistant Streptococcus pyogenes. J Clin Microbiol 2006; 44:2524–2532 [View Article] [PubMed]
     [Google Scholar]
  25. Kumar S, Stecher G, Suleski M, Sanderford M, Sharma S et al. MEGA12: molecular evolutionary genetic analysis version 12 for adaptive and green computing. Mol Biol Evol 2024; 41:msae263 [View Article] [PubMed]
     [Google Scholar]
  26. Letunic I, Bork P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res 2024; 52:W78–W82 [View Article] [PubMed]
     [Google Scholar]
  27. Hunter PR, Gaston MA. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J Clin Microbiol 1988; 26:2465–2466 [View Article] [PubMed]
     [Google Scholar]
  28. Chylinski K, Makarova KS, Charpentier E, Koonin EV. Classification and evolution of type II CRISPR-Cas systems. Nucleic Acids Res 2014; 42:6091–6105 [View Article] [PubMed]
     [Google Scholar]
  29. Horvath P, Coûté-Monvoisin AC, Romero DA, Boyaval P, Fremaux C et al. Comparative analysis of CRISPR loci in lactic acid bacteria genomes. Int J Food Microbiol 2009; 131:62–70 [View Article] [PubMed]
     [Google Scholar]
  30. Shmakov SA, Sitnik V, Makarova KS, Wolf YI, Severinov KV et al. The CRISPR spacer space is dominated by sequences from species-specific mobilomes. mBio 2017; 8:e01397-17 [View Article] [PubMed]
     [Google Scholar]
  31. Heussler GE, O’Toole GA. Friendly fire: biological functions and consequences of chromosomal targeting by CRISPR-Cas systems. J Bacteriol 2016; 198:1481–1486 [View Article] [PubMed]
     [Google Scholar]
  32. Wimmer F, Beisel CL. CRISPR-Cas systems and the paradox of self-targeting spacers. Front Microbiol 2019; 10:3078 [View Article] [PubMed]
     [Google Scholar]
/content/journal/acmi/10.1099/acmi.0.001070.v3
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Characterization of the CRISPR1-Cas array and its subtyping potential in Enterococcus faecalis from Malaysia
Access Microbiology 8, 001070.v3 (2026); https://doi.org/10.1099/acmi.0.001070.v3
/content/journal/acmi/10.1099/acmi.0.001070.v3
/content/journal/acmi/10.1099/acmi.0.001070.v3
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