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Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae


Autoři: Leon Peto aff001;  Nicola J. Fawcett aff001;  Derrick W. Crook aff001;  Tim E. A. Peto aff001;  Martin J. Llewelyn aff004;  A. Sarah Walker aff001
Působiště autorů: National Institute for Health Research (NIHR) Health Protection Research Unit on Healthcare Associated Infections and Antimicrobial Resistance, John Radcliffe Hospital, Oxford, England, United Kingdom aff001;  Nuffield Department of Medicine, University of Oxford, Oxford, England, United Kingdom aff002;  National Infection Service, Public Health England, Colindale, London, England, United Kingdom aff003;  Department of Global Health and Infection, Brighton and Sussex Medical School, Falmer, Sussex, England, United Kingdom aff004;  Department of Microbiology and Infection, Brighton and Sussex University Hospitals NHS Trust, Brighton, England, United Kingdom aff005
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pone.0222831

Souhrn

Metagenomic sequencing of fecal DNA can usefully characterise an individual’s intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer feces were spiked with carbapenemase-producing Enterobacteriaceae and incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a fecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goal.

Klíčová slova:

Staphylococcus aureus – Antimicrobial resistance – Enterobacteriaceae – DNA extraction – DNA sequencing – Vancomycin – Gene sequencing


Zdroje

1. Penders J, Stobberingh EE, Savelkoul PHM, Wolffs PFG. The human microbiome as a reservoir of antimicrobial resistance. Front Microbiol. Frontiers; 2013;4: 87. doi: 10.3389/fmicb.2013.00087 23616784

2. Ruppé E, Burdet C, Grall N, de Lastours V, Lescure FX, Andremont A, et al. Impact of antibiotics on the intestinal microbiota needs to be re-defined to optimize antibiotic usage. Clinical Microbiology and Infection. Elsevier; 2018;24: 3–5. doi: 10.1016/j.cmi.2017.09.017 28970162

3. Donskey CJ, Chowdhry TK, Hecker MT, Hoyen CK, Hanrahan JA, Hujer AM, et al. Effect of antibiotic therapy on the density of vancomycin-resistant enterococci in the stool of colonized patients. N Engl J Med. 2000;343: 1925–1932. doi: 10.1056/NEJM200012283432604 11136263

4. Donskey CJ. Antibiotic regimens and intestinal colonization with antibiotic-resistant gram-negative bacilli. Clin Infect Dis. 2nd ed. 2006;43 Suppl 2: S62–9. doi: 10.1086/504481 16894517

5. Taur Y, Xavier JB, Lipuma L, Ubeda C, Goldberg J, Gobourne A, et al. Intestinal domination and the risk of bacteremia in patients undergoing allogeneic hematopoietic stem cell transplantation. Clin Infect Dis. Oxford University Press; 2012;55: 905–914. doi: 10.1093/cid/cis580 22718773

6. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, et al. Diversity of the human intestinal microbial flora. Science. 2005;308: 1635–1638. doi: 10.1126/science.1110591 15831718

7. van Schaik W. The human gut resistome. Philos Trans R Soc Lond, B, Biol Sci. The Royal Society; 2015;370: 20140087. doi: 10.1098/rstb.2014.0087 25918444

8. Buelow E, González TDJB, Fuentes S, de Steenhuijsen Piters WAA, Lahti L, Bayjanov JR, et al. Comparative gut microbiota and resistome profiling of intensive care patients receiving selective digestive tract decontamination and healthy subjects. Microbiome. BioMed Central; 2017;5: 88. doi: 10.1186/s40168-017-0309-z 28803549

9. Andersen H, Connolly N, Bangar H, Staat M, Mortensen J, Deburger B, et al. Use of Shotgun Metagenome Sequencing To Detect Fecal Colonization with Multidrug-Resistant Bacteria in Children. Burnham CAD, editor. J Clin Microbiol. American Society for Microbiology Journals; 2016;54: 1804–1813. doi: 10.1128/JCM.02638-15 27122381

10. Lanza VF, Baquero F, Martínez JL, Ramos-Ruíz R, González-Zorn B, Andremont A, et al. In-depth resistome analysis by targeted metagenomics. Microbiome. BioMed Central; 2018;6: 11. doi: 10.1186/s40168-017-0387-y 29335005

11. Grall N, Lazarevic V, Gaïa N, Couffignal C, Laouénan C, Ilic-Habensus E, et al. Unexpected persistence of extended-spectrum β-lactamase-producing Enterobacteriaceae in the faecal microbiota of hospitalised patients treated with imipenem. Int J Antimicrob Agents. 2017;50: 81–87. doi: 10.1016/j.ijantimicag.2017.02.018 28499958

12. Sender R, Fuchs S, Milo R. Revised Estimates for the Number of Human and Bacteria Cells in the Body. PLOS Biol. Public Library of Science; 2016;14: e1002533. doi: 10.1371/journal.pbio.1002533 27541692

13. Blane B, Brodrick HJ, Gouliouris T, Ambridge KE, Kidney AD, Ludden CM, et al. Comparison of 2 chromogenic media for the detection of extended-spectrum β-lactamase producing Enterobacteriaceae stool carriage in nursing home re …—PubMed—NCBI. Diagnostic Microbiology and Infectious Disease. 2016;84: 181–183. doi: 10.1016/j.diagmicrobio.2015.11.008 26712266

14. Jett BD, Hatter KL, Huycke MM, Gilmore MS. Simplified agar plate method for quantifying viable bacteria. BioTechniques. 1997;23: 648–650. doi: 10.2144/97234bm22 9343684

15. Stoesser N, Giess A, Batty EM, Sheppard AE, Walker AS, Wilson DJ, et al. Genome sequencing of an extended series of NDM-producing Klebsiella pneumoniae isolates from neonatal infections in a Nepali hospital characterizes the extent of community- versus hospital-associated transmission in an endemic setting. Antimicrob Agents Chemother. American Society for Microbiology; 2014;58: 7347–7357. doi: 10.1128/AAC.03900-14 25267672

16. Sheppard AE, Stoesser N, Wilson DJ, Sebra R, Kasarskis A, Anson LW, et al. Nested Russian Doll-Like Genetic Mobility Drives Rapid Dissemination of the Carbapenem Resistance Gene blaKPC. Antimicrob Agents Chemother. American Society for Microbiology; 2016;60: 3767–3778. doi: 10.1128/AAC.00464-16 27067320

17. Wood DE, Salzberg SL. Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. BioMed Central; 2014;15: R46. doi: 10.1186/gb-2014-15-3-r46 24580807

18. Babraham Bioinformatics. FastQC. Available from http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

19. Bushnell B. BBMap. Available from http://sourceforge.net/projects/bbmap/

20. Truong DT, Franzosa EA, Tickle TL, Scholz M, Weingart G, Pasolli E, et al. MetaPhlAn2 for enhanced metagenomic taxonomic profiling. Nature Methods. Nature Publishing Group; 2015;12: 902–903. doi: 10.1038/nmeth.3589 26418763

21. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009;25: 1754–1760. doi: 10.1093/bioinformatics/btp324 19451168

22. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25: 2078–2079. doi: 10.1093/bioinformatics/btp352 19505943

23. Arredondo-Alonso S, Willems RJ, van Schaik W, Schürch AC. On the (im)possibility of reconstructing plasmids from whole-genome short-read sequencing data. Microbial Genomics. Microbiology Society; 2017;3. doi: 10.1099/mgen.0.000128 29177087

24. Raymond F, Boissinot M, Ouameur AA, Déraspe M, Plante P-L, Kpanou SR, et al. Culture-enriched human gut microbiomes reveal core and accessory resistance genes. Microbiome. 2019;7: 894. doi: 10.1186/s40168-019-0669-7 30953542

25. Wang CH, Koch AL. Constancy of growth on simple and complex media. J Bacteriol. American Society for Microbiology (ASM); 1978;136: 969–975. 363700

26. Gibbons RJ, Kapsimalis B. Estimates of the overall rate of growth of the intestinal microflora of hamsters, guinea pigs, and mice. J Bacteriol. American Society for Microbiology (ASM); 1967;93: 510–512. 6020422

27. Zhang T, Zhang X-X, Ye L. Plasmid metagenome reveals high levels of antibiotic resistance genes and mobile genetic elements in activated sludge. PLoS ONE. Public Library of Science; 2011;6: e26041. doi: 10.1371/journal.pone.0026041 22016806

28. Noyes NR, Weinroth ME, Parker JK, Dean CJ, Lakin SM, Raymond RA, et al. Enrichment allows identification of diverse, rare elements in metagenomic resistome-virulome sequencing. Microbiome. BioMed Central; 2017;5: 142. doi: 10.1186/s40168-017-0361-8 29041965


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2019 Číslo 11
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