CRISPR-Cas influences the acquisition of antibiotic resistance in Klebsiella pneumoniae


Autoři: Natalie A. Mackow aff001;  Juntao Shen aff002;  Mutayyaba Adnan aff001;  Aisha S. Khan aff001;  Bettina C. Fries aff001;  Elizabeth Diago-Navarro aff001
Působiště autorů: Department of Medicine, Infectious Disease Division, Stony Brook University, Stony Brook, New York, United States of America aff001;  School of Life Science and Biotechnology, Dalian University of Technology, Dalian, PR China aff002;  Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, United States of America aff003
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225131

Souhrn

In the US Carbapenem resistance in Klebsiella pneumoniae (Kp) is primarily attributed to the presence of the genes blaKPC-2 and blaKPC-3, which are transmitted via plasmids. Carbapenem-resistant Kp (CR-Kp) infections are associated with hospital outbreaks. They are difficult to treat, and associated with high mortality rates prompting studies of how resistance is obtained. In this study, we determined the presence of CRISPR-Cas in 304 clinical Kp strains. The CRISPR-Cas system has been found to prevent the spread of plasmids and bacteriophages, and therefore limits the horizontal gene transfer mediated by these mobile genetic elements. Here, we hypothesized that only those Kp strains that lack CRISPR-Cas can acquire CR plasmids, while those strains that have CRISPR-Cas are protected from gaining these plasmids and thus maintain sensitivity to antimicrobials. Our results show that CRISPR-Cas is absent in most clinical Kp strains including the clinically important ST258 clone. ST258 strains that continue to be sensitive to carbapenems also lack CRISPR-Cas. Interestingly, CRISPR-Cas positive strains, all non-ST258, exhibit lower resistance rates to antimicrobials than CRISPR-Cas negative strains. Importantly, we demonstrate that the presence of CRISPR-Cas appears to inhibit the acquisition of blaKPC plasmids in 7 Kp strains. Furthermore, we show that strains that are unable to acquire blaKPC plasmids contain CRISPR spacer sequences highly identical to those found in previously published multidrug-resistance-containing plasmids. Lastly, to our knowledge this is the first paper demonstrating that resistance to blaKPC plasmid invasion in a CRISPR-containing Kp strain can be reversed by deleting the CRISPR-cas cassette.

Klíčová slova:

Antibiotic resistance – Antibiotics – CRISPR – Plasmids – Polymerase chain reaction – Repeated sequences – Sequence analysis – Sequence databases


Zdroje

1. Centers for Disease C, Prevention. Vital signs: carbapenem-resistant Enterobacteriaceae. MMWR Morb Mortal Wkly Rep. 2013;62(9):165–70. 23466435.

2. Hauck C, Cober E, Richter SS, Perez F, Salata RA, Kalayjian RC, et al. Spectrum of excess mortality due to carbapenem-resistant Klebsiella pneumoniae infections. Clin Microbiol Infect. 2016;22(6):513–9. doi: 10.1016/j.cmi.2016.01.023 26850824.

3. Chen L, Mathema B, Chavda KD, DeLeo FR, Bonomo RA, Kreiswirth BN. Carbapenemase-producing Klebsiella pneumoniae: molecular and genetic decoding. Trends Microbiol. 2014;22(12):686–96. doi: 10.1016/j.tim.2014.09.003 25304194.

4. Diago-Navarro E, Chen L, Passet V, Burack S, Ulacia-Hernando A, Kodiyanplakkal RP, et al. Carbapenem-resistant Klebsiella pneumoniae exhibit variability in capsular polysaccharide and capsule associated virulence traits. J Infect Dis. 2014;210(5):803–13. Epub 2014/03/19. doi: 10.1093/infdis/jiu157 24634498.

5. Hirsch EB, Tam VH. Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrug-resistant infection. J Antimicrob Chemother. 2010;65(6):1119–25. doi: 10.1093/jac/dkq108 20378670.

6. Neuner EA, Yeh JY, Hall GS, Sekeres J, Endimiani A, Bonomo RA, et al. Treatment and outcomes in carbapenem-resistant Klebsiella pneumoniae bloodstream infections. Diagn Microbiol Infect Dis. 2011;69(4):357–62. Epub 2011/03/15. doi: 10.1016/j.diagmicrobio.2010.10.013 21396529.

7. Satlin MJ, Chen L, Patel G, Gomez-Simmonds A, Weston G, Kim AC, et al. Multicenter Clinical and Molecular Epidemiological Analysis of Bacteremia Due to Carbapenem-Resistant Enterobacteriaceae (CRE) in the CRE Epicenter of the United States. Antimicrob Agents Chemother. 2017;61(4). doi: 10.1128/AAC.02349-16 28167547.

8. Chen L, Chavda KD, DeLeo FR, Bryant KA, Jacobs MR, Bonomo RA, et al. Genome Sequence of a Klebsiella pneumoniae Sequence Type 258 Isolate with Prophage-Encoded K. pneumoniae Carbapenemase. Genome Announc. 2015;3(3). doi: 10.1128/genomeA.00659-15 26089425.

9. Munoz-Price LS, Poirel L, Bonomo RA, Schwaber MJ, Daikos GL, Cormican M, et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis. 2013;13(9):785–96. Epub 2013/08/24. doi: 10.1016/S1473-3099(13)70190-7 23969216.

10. Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle JW, Steward CD, et al. Novel carbapenem-hydrolyzing beta-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother. 2001;45(4):1151–61. doi: 10.1128/AAC.45.4.1151-1161.2001 11257029.

11. Bowers JR, Kitchel B, Driebe EM, MacCannell DR, Roe C, Lemmer D, et al. Genomic Analysis of the Emergence and Rapid Global Dissemination of the Clonal Group 258 Klebsiella pneumoniae Pandemic. PLoS One. 2015;10(7):e0133727. doi: 10.1371/journal.pone.0133727 26196384.

12. Marsh JW, Krauland MG, Nelson JS, Schlackman JL, Brooks AM, Pasculle AW, et al. Genomic Epidemiology of an Endoscope-Associated Outbreak of Klebsiella pneumoniae Carbapenemase (KPC)-Producing K. pneumoniae. PLoS One. 2015;10(12):e0144310. doi: 10.1371/journal.pone.0144310 26637170.

13. Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005;60(2):174–82. doi: 10.1007/s00239-004-0046-3 15791728.

14. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005;151(Pt 8):2551–61. doi: 10.1099/mic.0.28048-0 16079334.

15. Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology. 2005;151(Pt 3):653–63. doi: 10.1099/mic.0.27437-0 15758212.

16. Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2007;35(Web Server issue):W52–7. doi: 10.1093/nar/gkm360 17537822.

17. Hargreaves KR, Flores CO, Lawley TD, Clokie MR. Abundant and diverse clustered regularly interspaced short palindromic repeat spacers in Clostridium difficile strains and prophages target multiple phage types within this pathogen. MBio. 2014;5(5):e01045–13. doi: 10.1128/mBio.01045-13 25161187.

18. Barrangou R, Marraffini LA. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity. Mol Cell. 2014;54(2):234–44. doi: 10.1016/j.molcel.2014.03.011 24766887.

19. Marraffini LA, Sontheimer EJ. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science. 2008;322(5909):1843–5. doi: 10.1126/science.1165771 19095942.

20. Palmer KL, Gilmore MS. Multidrug-resistant enterococci lack CRISPR-cas. MBio. 2010;1(4). doi: 10.1128/mBio.00227-10 21060735.

21. Burley KM, Sedgley CM. CRISPR-Cas, a prokaryotic adaptive immune system, in endodontic, oral, and multidrug-resistant hospital-acquired Enterococcus faecalis. J Endod. 2012;38(11):1511–5. doi: 10.1016/j.joen.2012.07.004 23063226.

22. Price VJ, Huo W, Sharifi A, Palmer KL. CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis. mSphere. 2016;1(3). doi: 10.1128/mSphere.00064-16 27303749.

23. Ostria-Hernandez ML, Sanchez-Vallejo CJ, Ibarra JA, Castro-Escarpulli G. Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae. BMC Res Notes. 2015;8:332. doi: 10.1186/s13104-015-1285-7 26238567.

24. Shen J, Lv L, Wang X, Xiu Z, Chen G. Comparative analysis of CRISPR-Cas systems in Klebsiella genomes. J Basic Microbiol. 2017;57(4):325–36. doi: 10.1002/jobm.201600589 28156004.

25. Diancourt L, Passet V, Verhoef J, Grimont PA, Brisse S. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol. 2005;43(8):4178–82. doi: 10.1128/JCM.43.8.4178-4182.2005 16081970.

26. Diago-Navarro E, Calatayud-Baselga I, Sun D, Khairallah C, Mann I, Ulacia-Hernando A, et al. Antibody-Based Immunotherapy To Treat and Prevent Infection with Hypervirulent Klebsiella pneumoniae. Clin Vaccine Immunol. 2017;24(1). doi: 10.1128/CVI.00456-16 27795303.

27. Porwollik S, Santiviago CA, Cheng P, Long F, Desai P, Fredlund J, et al. Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium. PLoS One. 2014;9(7):e99820. doi: 10.1371/journal.pone.0099820 25007190.

28. Coordinators NR. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2013;41(Database issue):D8–D20. doi: 10.1093/nar/gks1189 23193264.

29. Park SO, Liu J, Furuya EY, Larson EL. Carbapenem-Resistant Klebsiella pneumoniae Infection in Three New York City Hospitals Trended Downwards From 2006 to 2014. Open Forum Infect Dis. 2016;3(4):ofw222. doi: 10.1093/ofid/ofw222 27942542.

30. Li HY, Kao CY, Lin WH, Zheng PX, Yan JJ, Wang MC, et al. Characterization of CRISPR-Cas Systems in Clinical Klebsiella pneumoniae Isolates Uncovers Its Potential Association With Antibiotic Susceptibility. Front Microbiol. 2018;9:1595. doi: 10.3389/fmicb.2018.01595 30061876.

31. Wyres KL, Wick RR, Judd LM, Froumine R, Tokolyi A, Gorrie CL, et al. Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones of Klebsiella pneumoniae. PLoS Genet. 2019;15(4):e1008114. doi: 10.1371/journal.pgen.1008114 30986243.

32. Huang W, Wang G, Sebra R, Zhuge J, Yin C, Aguero-Rosenfeld ME, et al. Emergence and Evolution of Multidrug-Resistant Klebsiella pneumoniae with both blaKPC and blaCTX-M Integrated in the Chromosome. Antimicrob Agents Chemother. 2017;61(7). doi: 10.1128/AAC.00076-17 28438939.

33. Aydin S, Personne Y, Newire E, Laverick R, Russell O, Roberts AP, et al. Presence of Type I-F CRISPR/Cas systems is associated with antimicrobial susceptibility in Escherichia coli. J Antimicrob Chemother. 2017;72(8):2213–8. doi: 10.1093/jac/dkx137 28535195.

34. Touchon M, Charpentier S, Pognard D, Picard B, Arlet G, Rocha EP, et al. Antibiotic resistance plasmids spread among natural isolates of Escherichia coli in spite of CRISPR elements. Microbiology. 2012;158(12):2997–3004. doi: 10.1099/mic.0.060814-0 28206908.

35. Deleo FR, Chen L, Porcella SF, Martens CA, Kobayashi SD, Porter AR, et al. Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 2014;111(13):4988–93. Epub 2014/03/19. doi: 10.1073/pnas.1321364111 24639510.

36. Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health. Proc Natl Acad Sci U S A. 2015;112(27):E3574–81. doi: 10.1073/pnas.1501049112 26100894.


Článek vyšel v časopise

PLOS One


2019 Číslo 11