Revista de Biología Tropical ISSN Impreso: 0034-7744 ISSN electrónico: 2215-2075

Resistance of Clostridioides difficile spores (Clostridiales: Peptostreptococcaceae) to sodium dichloroisocyanurate


bacterial endospores
sporicidal agent
exosporium proteins
endosporas bacterianas
agentes esporicidas
proteínas de exosporio

How to Cite

González-Carballo, G. C., & Rodríguez, C. (2021). Resistance of Clostridioides difficile spores (Clostridiales: Peptostreptococcaceae) to sodium dichloroisocyanurate. Revista De Biología Tropical, 69(2), 755–762.


Clostridioides difficile is a significant cause of diarrhea in hospitals and the community. This bacterial pathogen is transmitted through the ingestion of endospores, which are challenging to eliminate due to intrinsic resistance to a variety of chemical disinfection agents. The well-characterized laboratory strain CD630 displays low virulence, has not caused outbreaks, and is highly susceptible to disinfectants. Nonetheless, a closely related strain termed NAPCR1 caused outbreaks in Costa Rica and later became endemic in many hospitals from this country. This strain causes disease through unusual mechanisms and is genotypically distinct from CD630. Consequently, its epidemic potential could be influenced by as yet unknown spore phenotypes, such as increased resistance to disinfectants. Objective: To determine whether the NAPCR1 strain is more resistant to a conventional and highly effective C. difficile sporicidal agent than strain CD630 and to identify potential explanatory mechanisms at the genomic level. Methods: We used an in vitro dilution-neutralization method to calculate the sporicidal activity of sodium dichloroisocyanurate (DCC) against purified spores from three subtypes of NAPCR1 isolates (LIBA-2945, LIBA-5761, and LIBA-6276), CD630, and a representative of the highly virulent and epidemic NAP1 strain (LIBA-5758). This phenotypic characterization was complemented with a genomics-steered search of polymorphisms in 15 spore- or sporulation-related genes. Results: Whereas DCC at a final concentration of 0.1 % (w/v) eradicated CD630 endospores with high efficacy (log10 reduction factor (LFR) ≥ 5), it only partially inactivated NAPCR1 (average LFR range: = 1.77-3.37) and NAP1 endospores (average LRF = 3.58). As hypothesized, the three NAPCR1 subtypes tested were more resistant to DCC than strain CD630 (ANOVA, P < 0.05), with LIBA-5761 showing the highest level of DCC resistance overall (ANOVA, P < 0.05). All three NAPCR1 isolates showed large deletions in bclA1. Besides, isolates LIBA-5761 and LIBA-6276 had deletions in bclA2. Conclusions: Our in vitro tests revealed a differential resistance of spores from the C. difficile NAPCR1 strain to DCC. They highlight the importance of continuously evaluating the efficacy of deployed disinfection agents against circulating strains and hint to a potential role of structural proteins from the exosporium in resistance to disinfectants in C. difficile.


Balsells, E., Filipescu, T., Kyaw, M.H., Wiuff, C., Campbell, H., & Nair, H. (2016). Infection prevention and control of Clostridium difficile: a global review of guidelines, strategies and recommendations. Journal of Global Health, 6(2), 020410.

Barbut, F. (2015). How to eradicate Clostridium difficile from the environment. Journal of Hospital Infection, 89, 287–295.

Cortezzo, D.E., Koziol-Dube, K., Setlow, B., & Setlow, P. (2004). Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress. Journal of Applied Microbiology, 97, 838–852.

Dawson, L.F., Valiente, E., Donahue, E.D., Birchenough, G., & Wren, B.W. (2011). Hypervirulent Clostridium difficile PCR-Ribotypes exhibit resistance to widely used disinfectants. PLoS ONE, 6(10), e25754.

Dubberke, E.R., Carling, P., Carrico, R., Donskey, C.J., Loo, V.G., McDonald, L.C., Maragakis, L.L., Sandora, T.J., Weber, D.J., Yokoe, D.S., & Gerding, D.N. (2014). Strategies to prevent Clostridium difficile infections in acute care hospitals: 2014 update. Infection Control & Hospital Epidemiology, 35(6), 628–645.

Durovic, A., Widmer, A.F., & Tschudin-Sutter, S. (2018). New insights into transmission of Clostridium difficile infection - a narrative review. Clinical Microbiology and Infection, 24, 483–492.

Fraise, A.P., Wilkinson, M.A.C., Bradley, C.R., Paton, S., Walker, J., Maillard, J., Wesgate, R.L., Hoffman, P., Coia, J., Woodall, C., Fry, C., & Wilcox, M. (2015). Development of a sporicidal test method for Clostridium difficile. Journal of Hospital Infection, 89, 2–15.

Gallandat, K., Stack, D., String, G., & Lantagne, D. (2019). Residual maintenance using sodium hypochlorite, sodium dichloroisocyanurate and chlorine dioxide in laboratory waters of varying turbidity. Water, 11(1309), 1–14.

Ghose, C. (2013). Clostridium difficile infection in the twenty-first century. Emerging Microbes and Infections, 2, 1–8.

Gil, F., Lagos-Moraga, S., Calderón-Romero, P., Pizarro-Guajardo, M., & Paredes-Sabja, D. (2017). Updates on Clostridium difficile spore biology. Anaerobe, 45, 3–9.

Guerrero-Araya, E., Meneses, C., Castro-Nallar, E., Guzmán, A.M., Álvarez-Lobos, M., Quesada-Gómez, C., Paredes-Sabja, D., & Rodríguez, C. (2020). Origin, genomic diversity and microevolution of the Clostridium difficile B1/NAP1/RT027/ST01 strain in Costa Rica, Chile, Honduras and Mexico. Microbial Genomics, 6(5), e000355.

Khan, F.Y., & Elzouki, A.N. (2014). Clostridium difficile infection: a review of the literature. Asian Pacific Journal of Tropical Medicine, 7(1), S6–S13.

Kouhsari, E., Abbasian, S., Sedighi, M., Yaseri, H.F., Nazari, S., Bialvaei, A.Z., Dahim, P., Mirzaei, E.Z., & Rahbar, M. (2018). Clostridium difficile infection: a review. Reviews in Medical Microbiology, 29, 103–109.

Leggett, M.J., McDonnell, G., Denyer, S.P., Setlow, P., & Maillard, J.Y. (2012). Bacterial spore structures and their protective role in biocide resistance. Journal of Applied Microbiology, 113, 485–498.

Loo, V.G. (2015). Environmental interventions to control Clostridium difficile. Infectious Disease Clinics, 29, 83–91.

López-Ureña, D., Quesada-Gómez, C., Montoya-Ramírez, M., Gamboa-Coronado, M.D.M., Somogyi, T., Rodríguez, C., & Rodríguez-Cavallini, E. (2016). Predominance and high antibiotic resistance of the emerging Clostridium difficile genotypes NAPCR1 and NAP9 in a Costa Rican hospital over a 2-year period without outbreaks. Emerging Microbes and Infections, 5(1), 1–5.

Martin, J., Monaghan, T.M., & Wilcox, M.H. (2016). Clostridium difficile infection: epidemiology, diagnosis and understanding transmission. Nature Reviews: Gastroenterology & Hepatology, 13, 206–216.

Murillo, T., Ramírez-Vargas, G., Riedel, T., Overmann, J., Andersen, J.M., Guzmán-Verri, C., Chaves-Olarte, E., & Rodríguez, C. (2018). Two groups of cocirculating, epidemic Clostridiodes difficile strains microdiversify through different mechanisms. Genome Biology and Evolution, 10(3), 982–998.

O’Connor, J.R., Johnson, S., & Gerding, D.N. (2009). Clostridium difficile infection caused by the epidemic B1/NAP1/027 strain. Gastroenterology, 136, 1913–1924.

Paredes-Sabja, D., Shen, A., & Sorg, J.A. (2014). Clostridium difficile spore biology: sporulation, germination, and spore structural proteins. Trends in Microbiology, 22(7), 406–416.

Phetcharaburanin, J., Hong, H.A., Colenutt, C., Bianconi, I., Sempere, L., Permpoonpattana, P., Smith, K., Dembek, M., Tan, S., Brisson, M.C., Brisson, A.R., Fairweather, N.F., & Cutting, S.M. (2014). The spore-associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium difficile. Molecular Microbiology, 92(5), 1025–1038.

Pizarro-Guajardo, M., Calderón-Romero, P., Castro-Córdoba, P., Mora-Uribe, P., & Paredes-Sabja, D. (2016). Ultrastructural variability of the exosporium layer of Clostridium difficile spores. Applied and Environmental Microbiology, 82(7), 2202–2209.

Pizarro-Guajardo, M., Olguín-Araneda, V., Barra-Carrasco, J., Brito-Silva, C., Sarker, M., & Paredes-Sabja, D. (2014). Characterization of the collagen-like exosporium protein, BclA1, of Clostridium difficile spores. Anaerobe, 25, 18–30.

Quesada-Gómez, C., Gamboa-Coronado, M.D.M., Rodríguez-Cavallini, E., Du, T., Mulvey, M.R., Villalobos-Zúñiga, M., Boza-Cordero, R., & Rodríguez, C. (2010). Emergence of Clostridium difficile NAP1 in Latin America. Journal of Clinical Microbiology, 48(2), 669–670.

Quesada-Gómez, C., López-Ureña, D., Acuña-Amador, L., Villalobos-Zuñiga, M., Du, T., Freire, R., Guzmán-Verri, C., Gamboa-Coronado, M.D.M., Lawley, T.D., Moreno, E., Mulvey, M.R., Brito, G.A., Rodríguez-Cavallini, E., Rodríguez, C., & Chaves-Olarte, E. (2015). Emergence of an outbreak-associated Clostridium difficile variant with increased virulence. Journal of Clinical Microbiology, 53(4), 1216–1226.

Rineh, A., Kelso, M.J., Vatansever, F., Tegos, G.P., & Hamblin, M.R. (2014). Clostridium difficile infection: molecular pathogenesis and novel therapeutics. Expert Review of Anti-infective Therapy, 12(1), 131–150.

Roberts, A.P., & Mullany, P. (2016). Clostridium difficile Methods and Protocols. Humana Press.

Turner, N.A., & Anderson, D.J. (2020). Hospital infection control: Clostridoides difficile. Clinics in Colon and Rectal Surgery, 33, 98–108.

Ungurs, M., Wand, M., Vassey, M., OBrien, S., Dixon, D., Walker, J., & Sutton, J.M. (2011). The effectiveness of sodium dichloroisocyanurate treatments against Clostridium difficile spores contaminating stainless steel. American Journal of Infection Control, 39(3), 199–205.

Vohra, P., & Poxton, I.R. (2011). Efficacy of decontaminants and disinfectants against Clostridium difficile. Journal of Medical Microbiology, 60, 1218–1224.



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