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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(2): 755-762, April-June 2021 (Published June 30, 2021)

Resistance of Clostridioides difficile spores from the NAP

CR1

strain

(Clostridiales: Peptostreptococcaceae) to sodium dichloroisocyanurate

Gian Carlo González-Carballo

; https://orcid.org/0000-0002-2188-4461

César Rodríguez

1,2

*; https://orcid.org/0000-0001-5599-0652

1. Facultad de Microbiología, Universidad de Costa Rica, Ciudad Universitaria Rodrigo Facio, 11501-2060, San Pedro

de Montes de Oca, San José, Costa Rica; ggcarballo8@gmail.com

2. Centro de Investigación en Enfermedades Tropicales (CIET), Universidad de Costa Rica, Ciudad Universitaria

Rodrigo Facio, 11501-2060, San Pedro de Montes de Oca, San José, Costa Rica; cesar.rodriguezsanchez@ucr.ac.cr

(*Correspondence)

Received 10-VI-2020. Corrected 31-V-2021. Accepted 18-VI-2021.

ABSTRACT

Introduction: 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 NAP

CR1

caused outbreaks in Costa Rica and later became endemic in many hospi-

tals 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 NAP

CR1

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 NAP

CR1

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

(log

reduction factor (LFR) ≥ 5), it only partially inactivated NAP

CR1

(average LFR range: = 1.77-3.37) and

NAP1 endospores (average LRF = 3.58). As hypothesized, the three NAP

CR1

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 NAP

CR1

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 NAP

CR1

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.

Key words: bacterial endospores; disinfection; sporicidal agent; exosporium proteins.

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. https://doi.

org/10.15517/RBT.V69I2.42255

https://doi.org/10.15517/RBT.V69I2.42255

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Revista de Biología Tropical, ISSN: 2215-2075 Vol. 69(2): 755-762, April-June 2021 (Published June 30, 2021)

Clostridioides difficile is a Gram-positive,

spore-forming, anaerobic bacterium that may

cause diarrhea in susceptible individuals. It col-

onizes and proliferates in the gastrointestinal

tract of humans in response to structural and

functional imbalances in the residing micro-

biota elicited by antibiotic therapy (Martin et

al., 2016). Its main virulence factors are tox-

ins TcdB and TcdA, from the family of large

clostridial toxins (Khan & Elzouki, 2014).

Additional known virulence factors in this bac-

terium are cysteine proteases, S-layer proteins,

and a binary toxin with ADP-ribosyltransferase

activity (Kouhsari et al., 2018).

C. difficile infection (CDI) has increased in

terms of severity and incidence in the last two

decades (Khan & Elzouki, 2014) and become

one of the most critical healthcare-associated

diseases in developed and developing countries

(Khan & Elzouki, 2014).

C. difficile is a highly heterogeneous spe-

cies, and so, different strains have emerged and

predominated in different places of the world.

Of all contemporary strains, the NAP/RT27/

ST01 strain (MLST Clade 2) has received the

most attention because it includes epidemic

and hypervirulent isolates whose infections

are more severe and linked to higher mortality

rates (O’Connor et al., 2009). Furthermore, it

is highly resistant to disinfectants due to the

activity of efflux pumps and ABC transport-

ers (Dawson et al., 2011). This genotype has

circulated in Costa Rica for more than a decade

(Guerrero-Araya et al., 2020). It occasionally

predominates, yet it has been outnumbered by

other lineages (López-Ureña et al., 2016), such

as the NAP9 (MLST Clade 4) and the NAP

CR1

(MLST Clade 1) strains.

The NAP

CR1

/RT012/ST54 strain was

detected for the first time during a C. difficile

outbreak (Quesada-Gómez et al., 2010) and

became endemic in Costa Rican hospitals

(López-Ureña et al., 2016). Despite its very

small genomic distance to the non-epidemic

reference strain CD630 within the C. difficile

MLST Clade 1, the NAP

CR1

strain shows

increased virulence and causes disease by

atypical mechanisms (Quesada-Gómez et al.,

2015). Subsequent genomic studies have sub-

divided it into at least three clusters (I, II,

and III) owing to a differential representation

of mobile genetic elements in its pangenome

(Murillo et al., 2018).

C. difficile infection (CDI) is transmitted

through the ingestion of endospores, which

are characterized by a distinct multilayer ultra-

structure and intrinsic resistance to harsh envi-

ronmental conditions (Paredes-Sabja et al.,

2014). The coat layer of endospores is prin-

cipally responsible for this trait, as it contains

highly impermeable disulfide and dityrosine

cross-links and detoxifying enzymes, includ-

ing superoxide dismutase and peroxiredoxin

chitinase (CotE). Moreover, the lipids found in

the inner membrane of the endospore are sig-

nificantly compressed by the spore cortex and

consequently show low permeability (Leggett

et al., 2012; Gil et al., 2017).

CDI patients excrete endospores that can

remain viable in the environment for weeks or

months until they are ingested by susceptible

hosts (Martin et al., 2016). These developmen-

tal forms can contaminate bathrooms, wards,

beds, and cleaning and medical equipment at

the hospital level. Besides, healthcare staff may

act as vectors due to poor hygiene practices,

such as inadequate handwash or lack thereof

(Durovic et al., 2018).

Household bleach (5.25 % sodium hypo-

chlorite) or chlorine-releasing agents, such as

sodium dichloroisocyanurate (DCC) (1 000-5

000 ppm), have proven to be more effective

than neutral detergents and quaternary ammo-

nium compounds for environmental decon-

tamination of rooms of patients with CDI and

high-touch surfaces (Dubberke et al., 2014;

Loo, 2015; Turner & Anderson, 2020). When

dissolved in water, DCC reaches a pH between

6-7 and increases the availability of free chlo-

rine in the form of hypochlorous acid (Gallan-

dat et al., 2019). This latter compound, as other

oxidizing agents, damages the inner membrane

of endospores and blocks their germination

(Cortezzo et al., 2004).

Given the importance of spores in CDI

spreading (Rineh et al., 2014), we hypothesized

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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(2): 755-762, April-June 2021 (Published June 30, 2021)

that the epidemic potential of the NAP

CR1

explained, at least in part, by a greater resis-

tance of its endospores to disinfectants, which

has the potential to play important roles in its

transmission dynamics. This study was per-

formed to assess this notion and to pinpoint

potential explanatory mechanisms based on

the availability of whole-genome sequences

(WGS) for various NAP

CR1

isolates.

MATERIALS AND METHODS

Bacterial strains: This study was done

with isolates LIBA-2945 (Cluster I), LIBA-

5761 (Cluster II), and LIBA-6276 (Cluster

III), which represent each of the three clusters

into which the NAP

CR1

strain can be classi-

fied (Murillo et al., 2018). Strain CD630 and a

NAP1 isolate (LIBA-5758) were also studied

as a proxy for C. difficile strains with low

(Dawson et al., 2011) and high (Ghose, 2013)

resistance to disinfectants, respectively.

Preparation of endospores: Bacterial

strains were grown at 37 ºC under anaerobic

conditions on trypticase soy agar plates supple-

mented with 0.5 % (w/v) yeast extract. After 5

days of incubation, plates were scratched with

sterile loops, and the biomass recovered was

suspended in sterile 1X phosphate buffer solu-

tion (PBS) containing 1 % bovine serum albu-

min (BSA, Sigma). These suspensions were

homogenized through vigorous vortexing and

loaded onto an equal volume of a 100 % (w/v)

solution of the non-ionic density gradient medi-

um Histodenz™ (Sigma-Aldrich) to reach a

final concentration of 50 % (w/v). Endospores

were separated from vegetative cells through

centrifugation (10 000 rcf x 10 min). Pel-

lets containing the structures of interest were

washed thrice with distilled water to remove

Histodenz

remnants (5 000 rcf x 5 min), and

the resulting materials were resuspended in

1X PBS supplemented with 1 % BSA to avoid

agglutination (Pizarro-Guajardo et al., 2016).

The purity and abundance of viable endospores

in these preparations were checked by light

microscopy and a plate count method. Brain

heart infusion agar plates supplemented with

0.01 % (w/v) sodium taurocholate to induce

germination were used in this verification step

(Roberts & Mullany, 2016). Endospore suspen-

sions were standardized to contain 10

endo-

spores/mL and stored at 4 ºC until use.

Sporicidal assays: The sporicidal activity

of 0.1 % (w/v) DCC was determined using a

dilution-neutralization method (Vohra & Pox-

ton, 2011). Briefly, a work solution containing

1 000 ppm of DCC prepared from a stock of

10 000 ppm. Later, 100 mL of spores were added

to 800 mL of DCC and 100 mL of 1 % BSA to

simulate the presence of organic matter in the

clinical environment. After 5 min of exposure

at room temperature, the activity of DCC was

stopped by mixing 100 mL of the mixture with

100 mL of sterile distilled water and 800 mL of

0.5 % sodium thiosulfate as a neutralization

agent. Three serial, decimal dilutions of the

neutralized mixture were made using 0.85 %

sterile physiological saline solution and there-

after 100 µL of each dilution were spread onto

Brucella agar plates supplemented with 0.01

% (w/v) sodium taurocholate. After incubation

under anaerobiosis at 37 ºC for 72 h, colony

counts were recorded and exploited to calcu-

late log

reduction factors (LRF) (Fraise et

al., 2015). A disinfectant agent for clinical use

should reach an LRF equal to or greater than 5

to be considered effective (Fraise et al., 2015),

which corresponds to a 99.999 % reduction of

the initial microbial load. All experiments were

performed in triplicate. Differences between

disinfectants and strains were examined using

one-way ANOVA tests followed by Tukey and

Bonferroni post-hoc tests.

Bioinformatic analysis of sporulation

genes: Bowtie 2 was used to map trimmed

Illumina reads obtained for the LIBA isolates

(PRJEB5034, European Nucleotide Archive)

to a high-quality, close genome sequence of

CD630 (GCF_000009205.2). The resulting

bam archives were examined for structural

variations in the sequence of genes encoding

crucial spore- or sporulation-related proteins

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(Table 1). Protein sequences were aligned using

MUSCLE and the resulting alignments were

visualized with Geneious R10.

TABLE 1

Genes for spore- or sporulation-related proteins

scrutinized for structural variants

Gene (locus) (Possible) Localization

spoIVA (CD2629)

Basement layer

sipL (CD3567)

Basement layer

cotA (CD1613)

Exosporium

cotB (CD1511)

Exosporium

cotCB (CD0598)

Coat and exosporium

cotD (CD2401)

Exosporium

cotE (CD1433)

Exosporium

cotF (CD0597)

Exosporium

cotJB2 (CD2400)

cotG (CD1567)

Exosporium

sodA (CD1631)

Coat

bclA1 (CD0332)

Exosporium

bclA2 (CD3230)

Exosporium

bclA3 (CD3349)

Exosporium

cdeC (CD1067)

Exosporium

: as yet unconfirmed;

ND: not determined.

RESULTS

Sporicidal effect of disinfectants: As

indicated in Fig. 1, DCC fully inactivated spores

from strain CD630 (LRF ≥ 5) and induced

undistinguishable reductions of intermediate

magnitude for the NAP

CR1

isolates LIBA-2945

(LRF = 3.64 ± 0.63) and LIBA-6276 (LRF =

3.37 ± 0.46), and the NAP1 isolate LIBA-5758

(LRF = 3.58 ± 0.40). With an LRF that differed

by almost two orders of magnitude (1.77 ±

0.47), LIBA-5761 exhibited the highest level of

in vitro resistance to DCC overall (Fig. 1). The

NAP

CR1

isolates were invariably more resistant

to DCC than strain CD630 (ANOVA, P < 0.05).

Bioinformatic analyses: Compared to

CD630, all three NAP

CR1

isolates exhibited

a large deletion in the aminoacid sequence of

BclA1. Besides, LIBA-5761 (Cluster II) and

LIBA-6276 (Cluster III), showed a deletion of

three aminoacid residues in BclA2 (Fig. 2, Fig.

3) No differences were observed in the other

analyzed genes.

DISCUSSION

This is the first investigation that addresses

the response to disinfectants of endospores

from the C. difficile NAP

CR1

strain. In agree-

ment with our working hypothesis, and pos-

sibly in line with its epidemic potential, the

NAP

CR1

strain was found to be more resistant

to DCC than strain CD630. This conclusion

was reached by an in vitro experiment; hence

it could be strengthened by performing similar

tests on surfaces contaminated with different

Fig. 1. Activity of DCC against C. difficile endospores. Endospore preparations from three subtypes of the NAP

CR1

strain

(LIBA-5761, LIBA-2945, LIBA-6276), a NAP1 strain (LIBA-5758), and a reference laboratory strain (CD630), were

tested by triplicate using a dilution-neutralization method. Results were expressed as average log

reduction factors (LRF),

whereby an LRF = 5 indicates a 99.999 % reduction in the original number of endospores. A disinfectant should reach this

threshold value to be considered effective. Error bars represent standard deviations. Different letters above the bars indicate

a statistically significant difference at P < 0.05 (one-way ANOVA).

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Fig. 2. Alignment of the predicted BclA1 protein sequences of strain 630 (reference, top) and three subtypes of the NAP

CR1

strain. Agreements and deletions to the reference sequence appear masked or as hyphens, respectively. The numbering of

the sequence alignment is based on the sequence of strain 630.

Fig. 3. Alignment of the predicted BclA2 protein sequences of strain 630 (reference, top) and three subtypes of the NAP

CR1

strain. Agreements and deletions to the reference sequence appear masked or as hyphens, respectively. The numbering of

the sequence alignment is based on the sequence of strain 630.

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loads and types of organic matter. In most

cases, DCC did not reach the LRF ≥ 5 thresh-

old required for a disinfectant agent to be

considered effective under our experimental

conditions (5 min exposure time, 1 000 ppm).

This apparent caveat can be solved by the

application of DCC in higher concentrations

and for longer contact times (Barbut, 2015). In

any case, it is advisable to stimulate the devel-

opment of alternative compounds on account

of the toxicity and corrosiveness of DCC and

other chlorine-release agents (Ungurs et al.,

2011; Balsells et al., 2016).

This study shows that no disinfection

protocol works equally well for all C. difficile

strains and even for isolates of the same strain.

This finding emphasizes the importance of

evaluating multiple field-isolates together with

reference strains. Moreover, it justifies continu-

ous typing and testing of the circulating isolates

at a given time and place, as they may have been

exposed to undetermined selective pressures.

The susceptibility of one NAP

CR1

subtype

to DCC was unexpectedly low (LIBA-5761,

Cluster III). However, this trait could not be

linked to a distinct profile of sequence variants

in spore- and sporulation-related genes. Of all

genes studied, only those for the collagen-like

exosporium proteins BclA1 and BclA2 showed

sequence polymorphisms. C. difficile has three

bclA genes (bclA1, bclA2, and bclA3), and their

collagen-like regions are somewhat similar to

those found in other sporulated bacteria such

as Bacillus cereus and B. anthracis (Pizarro-

Guajardo et al., 2014). BclA1 has been charac-

terized to some extent, but even less is known

about BclA2 and BclA3.

BclA1 forms stable high molecular mass

complexes with other exosporium proteins

(Pizarro-Guajardo et al., 2014) and increas-

es the hydrophobicity of the exosporium in

B. anthracis (Phetcharaburanin et al., 2014).

Based on this knowledge, we predict that the

observed deletions in bclA1 and bclA2 lead

to the formation of a more robust exosporium

layer through increased cross-linking and water

repellency, hampering the accessibility of DCC

to its internal target in endospores (Cortezzo

et al., 2004). This notion could be confirmed

through a comprehensive comparison of the

composition of the exosporium of strains

CD630 and NAP

CR1

electron microscopy

and proteomics. On the other hand, since iso-

lates with identical gene deletion profiles dis-

played different levels of DCC susceptibility, it

is plausible that the phenotype of LIBA-5761

is multifactorial and influenced by physiologi-

cal factors and the activity of efflux pumps or

ABC transporters.

Our results highlight the importance

of continuously evaluating the efficacy of

deployed disinfection agents against circulating

strains and hint to a potential role of exospo-

rium proteins in resistance to disinfectants in

C. difficile. The relevance of these findings is

twofold. On the one hand, it may guide deci-

sion-makers and contribute to resource optimi-

zation in hospitals. On the other, it deepens our

understanding of the functional diversity of C.

difficile and its role in disease control.

Ethical statement: The authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that they

followed all pertinent ethical and legal proce-

dures and requirements. All financial sources

are fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

This work was supported by the Ministry

of Science, Technology, and Telecommunica-

tions of the Republic of Costa Rica (FI-094-

13, Cd-MAN). We acknowledge Pablo Vargas

and Robin Cárdenas for their skillful technical

assistance. María del Mar Gamboa and Carlos

Quesada contributed with scientific advice dur-

ing the execution of the project. The authors

also thank Adriana Badilla for commenting

early versions of the manuscript. No potential

conflicts of interest are declared.

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RESUMEN

Resistencia de esporas de Clostridioides difficile

de la cepa NAP

CR1

(Clostridiales: Peptostreptococcaceae)

al dicloroisocianurato de sodio

Introducción: Clostridioides difficile es una causa

importante de diarrea a nivel hospitalario y comunitario.

Esta bacteria se transmite por medio de la ingestión de

endosporas, las cuales son difíciles de erradicar por su

resistencia intrínseca a diferentes agentes químicos de

desinfección. La cepa de referencia CD630 está bien

caracterizada, es poco virulenta, no ha causado brotes,

y es altamente susceptible a los desinfectantes. Además,

pertenece al mismo clado MLST y es filogenéticamente

muy cercana a la cepa NAP

CR1

. Sin embargo, solo la últi-

ma ha causado brotes en Costa Rica y se ha convertido en

una cepa endémica en varios hospitales locales. La cepa

NAP

CR1

causa enfermedad por mecanismos poco usuales

y es genotípicamente diferente a la cepa CD630. Por lo

tanto, su potencial epidémico podría estar influenciado por

cambios fenotípicos en sus esporas, como una resistencia

incrementada a los desinfectantes.

Objetivo: Determinar si la cepa NAP

CR1

presenta

mayor resistencia que CD630 a un desinfectante de alta

eficacia utilizado a nivel hospitalario y dilucidar posibles

mecanismos a nivel genómico.

Métodos: Se utilizó el método de dilución-neutra-

lización para evaluar la actividad esporicida in vitro del

dicloroisocianurato de sodio (DCC) contra esporas de 3

subtipos de la cepa NAP

CR1

(LIBA-2945, LIBA-5761, y

LIBA-6276), CD630 y un aislamiento representativo de

la cepa epidémica e hipervirulenta NAP1 (LIBA-5758).

Esta caracterización fenotípica fue complementada con

una búsqueda genómica de polimorfismos en 15 genes

relacionados con la estructura de la endospora o el proceso

de esporulación.

Resultados: El DCC a una concentración final de

0.1 % (p/v) erradicó las endosporas de la cepa CD630 con

gran eficacia (factor de reducción logarítmica; FRL ≥ 5) y

eliminó parcialmente las de las cepas NAP

CR1

(FRL prome-

dio = 1.77-3.64) y NAP1 (FRL promedio = 3.58). El perfil

de susceptibilidad del aislamiento NAP

CR1

LIBA-5761 fue

único, ya que mostró un mayor nivel de resistencia hacia el

DCC que los otros aislamientos NAP

CR1

y la cepa NAP1

examinada (ANOVA, P < 0.05). Los tres aislamientos

NAP

CR1

mostraron deleciones en bclA1 y los aislamientos

LIBA-5761 y LIBA-6276 tenían deleciones adicionales

en bclA2.

Conclusiones: Nuestros experimentos in vitro con-

firman la resistencia incrementada a los desinfectantes de

la cepa NAP

CR1

y una susceptibilidad diferencial en sus

tres subtipos. Adicionalmente, señalan la importancia de

evaluar continuamente la eficacia de los desinfectantes

contra cepas circulantes y asignan un posible papel en la

resistencia a los desinfectantes gracias a las proteínas del

exosporio de C. difficile.

Palabras clave: endosporas bacterianas; desinfec-

ción; agentes esporicidas; proteínas de exosporio.

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