Sensitizing Clostridium difficile Spores With Germinants on Skin and Environmental Surfaces Represents a New Strategy for Reducing Spores via Ambient Mechanisms

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Michelle Marie Nerandzic
Curtis J. Donskey


Background: Clostridium difficile is a leading cause of healthcare-associated infections worldwide. Prevention of C. difficile transmission is challenging because spores are not killed by alcohol-based hand sanitizers or many commonly used disinfectants. One strategy to control spores is to induce germination, thereby rendering the spores more susceptible to benign disinfection measures and ambient stressors.

Methods/Results: C. difficile spores germinated on skin after a single application of cholic acid-class bile salts and co-germinants; for 4 C. difficile strains, recovery of viable spores from skin was reduced by ~0.3 log10CFU to 2 log10CFU after 2 hours and ~1 log10CFU to >2.5 log 10CFU after 24 hours. The addition of taurocholic acid to 70% and 30% ethanol significantly enhanced reduction of viable spores on skin and on surfaces. Desiccation, and to a lesser extent the presence of oxygen, were identified as the stressors responsible for reductions of germinated spores on skin and surfaces. Additionally, germinated spores became susceptible to killing by pH 1.5 hydrochloric acid, suggesting that germinated spores that remain viable on skin and surfaces might be killed by gastric acid after ingestion. Antibiotic-treated mice did not become colonized after exposure to germinated spores, whereas 100% of mice became colonized after exposure to the same quantity of dormant spores.

Conclusions: Germination could provide a new approach to reduce C. difficile spores on skin and in the environment and to render surviving spores less capable of causing infection. Our findings suggest that it may be feasible to develop alcohol-based hand sanitizers containing germinants that reduce spores on hands.


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1. Loo VG, Poirier L, Miller MA, Oughton M, Libman MD, Michaud S, Bourgault AM, Nguyen T, Frenette C, Kelly M, Vibien A, Brassard P, Fenn S, Dewar K, Hudson TJ, Horn R, Rene P, Monczak Y, Dascal A. A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med. 2005;353(23):2442-9. PubMed PMID: 16322602. doi: 10.1056/NEJMoa051639

2. Poxton IR, McCoubrey J, Blair G. The pathogenicity of Clostridium difficile. Clin Microbiol Infect. 2001;7(8):421-7. PubMed PMID: 11591205.

3. Lessa FC, Mu Y, Bamberg WM, Beldavs ZG, Dumyati GK, Dunn JR, Farley MM, Holzbauer SM, Meek JI, Phipps EC, Wilson LE, Winston LG, Cohen JA, Limbago BM, Fridkin SK, Gerding DN, McDonald LC. Burden of Clostridium difficile infection in the United States. N Engl J Med. 2015;372(9):825-34. PubMed PMID: 25714160. doi: 10.1056/NEJMoa1408913

4. Weiner LM, Fridkin SK, Aponte-Torres Z, Avery L, Coffin N, Dudeck MA, Edwards JR, Jernigan JA, Konnor R, Soe MM, Peterson K, McDonald LC. Vital Signs: Preventing Antibiotic-Resistant Infections in Hospitals - United States, 2014. MMWR Morb Mortal Wkly Rep. 2016;65(9):235-41. PubMed PMID: 26963489. doi: 10.15585/mmwr.mm6509e1

5. Oughton MT, Loo VG, Dendukuri N, Fenn S, Libman MD. Hand hygiene with soap and water is superior to alcohol rub and antiseptic wipes for removal of Clostridium difficile. Infect Control Hosp Epidemiol. 2009;30(10):939-44. PubMed PMID: 19715426. doi: 10.1086/605322

6. Edmonds SL, Zapka C, Kasper D, Gerber R, McCormack R, Macinga D, Johnson S, Sambol S, Fricker C, Arbogast J, Gerding DN. Effectiveness of hand hygiene for removal of Clostridium difficile spores from hands. Infect Control Hosp Epidemiol. 2013;34(3):302-5. PubMed PMID: 23388366. doi: 10.1086/669521

7. Rutala WA, Weber DJ. New disinfection and sterilization methods. Emerg Infect Dis. 2001;7(2):348-53. PubMed PMID: 11294738. Pubmed Central PMCID: 2631727. doi: 10.3201/eid0702.700348

8. Riggs MM, Sethi AK, Zabarsky TF, Eckstein EC, Jump RL, Donskey CJ. Asymptomatic carriers are a potential source for transmission of epidemic and nonepidemic Clostridium difficile strains among long-term care facility residents. Clin Infect Dis. 2007;45(8):992-8. PubMed PMID: 17879913. 10.1086/521854

9. Sethi AK, Al-Nassir WN, Nerandzic MM, Bobulsky GS, Donskey CJ. Persistence of skin contamination and environmental shedding of Clostridium difficile during and after treatment of C. difficile infection. Infect Control Hosp Epidemiol. 2010;31(1):21-7. PubMed PMID: 19929371. doi: 10.1086/649016

10. Setlow P. I will survive: DNA protection in bacterial spores. Trends Microbiol. 2007;15(4):172-80. PubMed PMID: 17336071.

11. Leggett MJ, McDonnell G, Denyer SP, Setlow P, Maillard JY. Bacterial spore structures and their protective role in biocide resistance. J Appl Microbiol. 2012;113(3):485-98. PubMed PMID: 22574673. doi: 10.1111/j.1365-2672.2012.05336.x

12. Gould GW. Symposium on bacterial spores: IV. Germination and the problem of dormancy. J Appl Bacteriol. 1970;33(1):34-49. PubMed PMID: 4246071.

13. Sorg JA, Sonenshein AL. Bile salts and glycine as cogerminants for Clostridium difficile spores. J Bacteriol. 2008;190(7):2505-12. PubMed PMID: 18245298. Pubmed Central PMCID: 2293200. doi: 10.1128/JB.01765-07

14. Akhtar S, Paredes-Sabja D, Torres JA, Sarker MR. Strategy to inactivate Clostridium perfringens spores in meat products. Food Microbiol. 2009;26(3):272-7. PubMed PMID: 19269568. doi: 10.1016/

15. Durban E, Goodnow R, Grecz N. Changes in resistance to radiation and heat during sporulation and germination of Clostridium botulinum 33A. J Bacteriol. 1970;102(2):590-2. PubMed PMID: 4911549. Pubmed Central PMCID: 247591.

16. Wheeldon LJ, Worthington T, Lambert PA, Hilton AC, Lowden CJ, Elliott TS. Antimicrobial efficacy of copper surfaces against spores and vegetative cells of Clostridium difficile: the germination theory. J Antimicrob Chemother. 2008;62(3):522-5. PubMed PMID: 18544601. doi: 10.1093/jac/dkn219

17. Nerandzic MM, Donskey CJ. A Quaternary Ammonium Disinfectant Containing Germinants Reduces Clostridium difficile Spores on Surfaces by Inducing Susceptibility to Environmental Stressors. Open Forum Infect Dis. 2016;3(4):ofw196. PubMed PMID: 28066792. Pubmed Central PMCID: 5198585. doi: 10.1093/ofid/ofw196

18. Nerandzic MM, Donskey CJ. Effective and reduced-cost modified selective medium for isolation of Clostridium difficile. J Clin Microbiol. 2009;47(2):397-400. PubMed PMID: 19073869. Pubmed Central PMCID: 2643678. doi: 10.1128/JCM.01591-08

19. Perez J, Springthorpe VS, Sattar SA. Clospore: a liquid medium for producing high titers of semi-purified spores of Clostridium difficile. J AOAC Int. 2011;94(2):618-26. PubMed PMID: 21563698.

20. American Society for Testing and Materials. Standard Guide for Evaluation of the Effectiveness of Hand Hygiene Topical Antimicrobial Products using ex vivo Porcine Skin (Method E 2897-12). West Conshohocken, PA: American Society for Testing and Materials. 2012.

21. Kochan TJ, Somers MJ, Kaiser AM, Shoshiev MS, Hagan AK, Hastie JL, Giordano NP, Smith AD, Schubert AM, Carlson PE, Jr., Hanna PC. Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores. PLoS Pathog. 2017;13(7):e1006443. PubMed PMID: 28704538. Pubmed Central PMCID: 5509370. doi: 10.1371/journal.ppat.1006443

22. American Society for Testing and Materials. Standard Quantitative Carrier Test Method to Evaluate the Bactericidal, Fungicidal, Mycobactericidal, and Sporicidal Potencies of Liquid Chemicals. (Method E 2111-12). West Conshohocken, PA: American Society for Testing and Materials. 2012.

23. Hilburn J, Hammond BS, Fendler EJ, Groziak PA. Use of alcohol hand sanitizer as an infection control strategy in an acute care facility. Am J Infect Control. 2003;31[2]:109-116.

24. Girou E, Loyeau S, Legrand P, Oppein F, Brun-Buisson C. Efficacy of handrubbing with alcohol based solution versus standard handwashing with antiseptic soap: randomised clinical trial. BMJ. 2002;325(7360):362. PubMed PMID: 12183307. Pubmed Central PMCID: 117885.

25. Voss A, Widmer AF. No time for handwashing!? Handwashing versus alcoholic rub: can we afford 100% compliance? Infect Control Hosp Epidemiol. 2004;18[3]:205-208.

26. Kampf G, Kramer A. Epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs. Clin Microbiol Rev. 2004;17(4):863-93, table of contents. PubMed PMID: 15489352. Pubmed Central PMCID: 523567. doi: 10.1128/CMR.17.4.863-893.2004

27. Pultz NJ, Stiefel U, Donskey CJ. Effects of daptomycin, linezolid, and vancomycin on establishment of intestinal colonization with vancomycin-resistant enterococci and extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae in mice. Antimicrob Agents Chemother. 2005;49(8):3513-6. PubMed PMID: 16048971. Pubmed Central PMCID: 1196280. doi: 10.1128/AAC.49.8.3513-3516.2005

28. Pultz NJ, Stiefel U, Subramanyan S, Helfand MS, Donskey CJ. Mechanisms by which anaerobic microbiota inhibit the establishment in mice of intestinal colonization by vancomycin-resistant Enterococcus. J Infect Dis. 2005;191(6):949-56. PubMed PMID: 15717271. doi: 10.1086/428090

29. Francis MB, Allen CA, Sorg JA. Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination. J Bacteriol. 2015;197(14):2276-83. PubMed PMID: 25917906. Pubmed Central PMCID: 4524186. doi: 10.1128/JB.02575-14

30. De Sordi L, Butt MA, Pye H, Kohoutova D, Mosse CA, Yahioglu G, Stamati I, Deonarain M, Battah S, Ready D, Allan E, Mullany P, Lovat LB. Development of photodynamic antimicrobial chemotherapy [PACT] for Clostridium difficile. PLoS One. 2015;10[8]:e0135039.

31. Ghosh S, Setlow P. Isolation and characterization of superdormant spores of Bacillus species. J Bacteriol. 2009;191(6):1787-97. PubMed PMID: 19136594. Pubmed Central PMCID: 2648361. doi: 10.1128/JB.01668-08

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