Food Microbiology 62 (2017) 32e38

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Food Microbiology
journal homepage: www.elsevier.com/locate/fm

Tolerance of Clostridium perfringens biofilms to disinfectants

commonly used in the food industry
Audrey Charlebois, Mario Jacques, Martine Boulianne, Marie Archambault*
Facult
e de Medecine V
et
erinaire, D
epartement de Pathologie et Microbiologie, Centre de Recherche en Infectiologie Porcine et Aviaire (CRIPA), Universit
e de
Montreal, Saint-Hyacinthe, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and
Received 22 October 2015 various enterotoxemia in animal species. Recently, it was shown to form mono-species biofilms, a
Accepted 14 September 2016 structured community of bacterial cells enclosed in a self-produced extracellular matrix. Biofilms have
Available online 15 September 2016
been associated with tolerance to antibiotics, disinfectants, and physical and environmental stresses.
Very little is known about the tolerance of C. perfringens biofilm toward disinfectants. In the present
Keywords:
study, susceptibilities of C. perfringens biofilms to five types of commonly used disinfectants on farms and
Clostridium perfringens
in food processing environments were analysed. In this paper, we show that C. perfringens mono-species
Biofilms
Disinfectants
biofilms can protect the bacterial cells from the action of potassium monopersulfate, quaternary
Food microbiology ammonium chloride, hydrogen peroxide and glutaraldehyde solutions. However, sodium hypochlorite
solution was shown to be effective on C. perfringens biofilms. Our investigation of dual-species biofilms of
C. perfringens with the addition of Staphylococcus aureus or Escherichia coli demonstrated that overall, the
mono-species biofilm of C. perfringens was more tolerant to all disinfectants than the dual-species bio-
films. For the anaerobic grown biofilms, the mono-species biofilm of C. perfringens was more tolerant to
sodium hypochlorite and quaternary ammonium chloride than the dual-species biofilms of C. perfringens
with S. aureus or E. coli. This study demonstrates that C. perfringens biofilm is an effective protection
mechanism to disinfectants commonly used on farms and in food processing environments.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction disease appears to be on the rise because of removal of antibiotic

Clostridium perfringens is a Gram-positive, aerotolerant anaer-
obic spore-forming bacterium that causes a wide variety of diseases
in humans and animals, primarily as a result of its ability to produce
many different toxins (Markey et al., 2013). In humans,
C. perfringens is responsible for gas gangrene, enteritis necroticans,
food poisoning, and antibiotic-associated diarrheas (Myers et al.,
2006). Currently, C. perfringens type A food poisoning ranks as the
second most commonly reported foodborne illness in Canada
(Thomas et al., 2013). In poultry, avian-specific C. perfringens strains
cause necrotic enteritis, an economically significant poultry disease
that costs the global industry over $2 billion annually in losses and
control measures (Stanley et al., 2014). In some countries, this
* Corresponding author.
E-mail addresses: audrey.charlebois@umontreal.ca (A. Charlebois), mario.
jacques@umontreal.ca (M. Jacques), martine.boulianne@umontreal.ca
(M. Boulianne), marie.archambault@umontreal.ca (M. Archambault).
growth promoters (Stanley et al., 2014). C. perfringens is also a cause
of various enterotoxemia in other animal species. Isolates of animal
origin constitute a risk for transmission to humans through the
food chain.
In order to persist in the environment, many bacteria have
evolved the ability to form biofilms (Davey and O'Toole, 2000;
Jefferson, 2004). In fact, the predominant organizational state of
bacteria in nature is biofilms (Costerton, 1999). Important features
of cells in biofilms include: aggregation in suspension or on solid
surfaces, increased antibiotic tolerance, and resistance to physical
and environmental stresses (Davey and O'Toole, 2000; Davies,
2003; Hall-Stoodley and Stoodley, 2009). It is now generally
accepted that the biofilm growth mode induces bacterial tolerance
to disinfection that can lead to substantial economic and health
concerns (Bridier et al., 2011). Although the precise mechanism of
such tolerance remains unclear, a review has recently discussed the
subject as a multifactorial process involving the spatial organiza-
tion of the biofilm (Bridier et al., 2011). More recently, we, and
others, have described the formation of biofilms in C. perfringens

http://dx.doi.org/10.1016/j.fm.2016.09.009
0740-0020/© 2016 Elsevier Ltd. All rights reserved.
 A. Charlebois et al. / Food Microbiology 62 (2017) 32e38 33

(Charlebois et al., 2014; Varga et al., 2006). We demonstrated that  de Montre

the biofilm formed by C. perfringens could protect the cells from an
exposure to atmospheric oxygen and to high concentrations of
antibiotics and anticoccidial agents (Charlebois et al., 2014). It has
also been observed that the biofilm formed by C. perfringens could
protect the cells from an exposure to 10 mM of hydrogen peroxide
even though this bacterium is catalase-negative (Varga et al., 2006).
The capacity of C. perfringens to be part of dual- or multi-species
biofilm has recently been reviewed (Pantaleon et al., 2014) and
C. perfringens biofilm was detected in many types of multi-species
biofilm including biliary stents (Leung et al., 2000; Pantaleon
et al., 2014). However, susceptibilities of C. perfringens mono- and
dual-species biofilms exposed to most disinfectants are currently
unknown. This study was undertaken to investigate the tolerance
of C. perfringens mono- and dual-species biofilms to disinfectants
used in farms and food processing environments.
2. Materials and methods
2.1. Bacterial strains
C. perfringens strains used in this study are described in Table 1.
Commensal strains of C. perfringens (n ¼ 15) from poultry were
recovered from the normal intestinal microbiota of animals taken
at five processing plants located in the province of Que bec, Canada
and are part of our C. perfringens culture collection. Strains of
clinical origin (n ¼ 15) were from cases of necrotic enteritis from
chickens. These strains were kindly provided by Dr Martine Bou-
lianne and the Clinical Laboratory of Molecular Diagnostic of the
Universite al (St-Hyacinthe, Quebec, Canada). Thawed
strains were grown on Columbia agar with 5% sheep blood (Oxoid,
Nepean, Ontario, Canada) and then incubated in an anaerobic
chamber at 35  C. Escherichia coli EcL 17606 and Staphylococcus
aureus E452b3 were kindly provided by the Clinical Bacteriology
 de Montreal.
Laboratories of the Universite
2.2. Disinfectants
Five types of disinfectants commonly used on farms and in the
food processing industry were tested in this study at concentra-
tions suggested by the manufacturers: a quaternary ammonium
chlorideebased disinfectant (1%) (Aseptol 2000, S.E.C. Repro Inc.,
bec, Canada), a potassium monop-
Ange-Gardien-de-Rouville, Que
toquinol N.-A. Inc., Lavaltrie,
ersulfate solution (1%) (Virkon, Ve
bec, Canada), a sodium hypochlorite solution (0.27%) (Lavo
Que
Pro6, Lavo Inc., Montre al, Que
bec, Canada), a hydrogen peroxide
(H2O2) solution (10%) (Sigma, Oakville, Ontario, Canada), and a
glutaraldehyde-based disinfectant (2%) (Gluterate, Germiphene
corp., Brantford, Ontario, Canada). Solutions were prepared
following the manufacturer's instructions in sterile distilled water.
Hydrogen peroxide solution was prepared fresh daily in sterile
distilled water from a 30% H2O2 stock solution. The glutaraldehyde-
based disinfectant was supplied as a ready-to-use solution.
2.3. Mono- and dual-species biofilms
Mono-species biofilms were cultured as previously described
(Charlebois et al., 2014) to obtain optimal biofilm growth of

Table 1
Bacterial strains used in this study.

Bacteria Origin Strains Type Mean optical density values (OD) Biofilm formationa Source

Clostridium perfringens Commensal c2614_B A 0.026 Weak (Charlebois et al., 2014)

Commensal c2628_A A 0.022 Weak (Charlebois et al., 2014)
Commensal c2643_A A 0.052 Weak (Charlebois et al., 2014)
Commensal c2645_A A 0.036 Weak (Charlebois et al., 2014)
Commensal c2647_A A 0.032 Weak (Charlebois et al., 2014)
Commensal c2649_A A 0.028 Weak (Charlebois et al., 2014)
Commensal c2650_A A 0.090 Weak (Charlebois et al., 2014)
Commensal c2651_A A 0.033 Weak (Charlebois et al., 2014)
Commensal c2652_A A 0.044 Weak (Charlebois et al., 2014)
Commensal c2654_A A 0.038 Weak (Charlebois et al., 2014)
Commensal c2662_A A 0.068 Weak (Charlebois et al., 2014)
Commensal c2725_A A 0.072 Weak (Charlebois et al., 2014)
Commensal C3039_A A 0.075 Weak (Charlebois et al., 2014)
Commensal C3080_A A 0.009 Weak (Charlebois et al., 2014)
Commensal C3143_A A 0 No (Charlebois et al., 2014)
Clinical DUR-109-L469 A 0.231 Moderate This study
Clinical DUR-109-B468 A 0.228 Moderate This study
Clinical DUR-106-E170 A 0.078 Weak This study
Clinical DUR-106-D170 A 0.090 Weak This study
Clinical DUR-106-C170 A 0.078 Weak This study
Clinical DUR-106-B170 A 0.076 Weak This study
Clinical DUR-106-A170 A 0.084 Weak This study
Clinical FBC-3200A-5 A 0.588 High This study
Clinical FBC-3200A-3 A 0.110 Weak This study
Clinical FBC-3200L-410 A 0.084 Weak This study
Clinical FBC-3200K-410 A 0.100 Weak This study
Clinical FBC-3200J-410 A 0.096 Weak This study
Clinical FBC-3200E-176 A 0.093 Weak This study
Clinical FBC-3200D A 0.353 High This study
Clinical FBC-3200B A 0.298 Moderate This study
Clinical ATCC13124 A 0.024 Weak This study
Escherichia coli Clinical EcL 17606 N/A 0.218 Moderate This study
Staphylococcus aureus Commensal E452b3 N/A 0.969 High This study
a
Strains are divided into four categories (no biofilm producer, weak biofilm producer, moderate biofilm producer and strong biofilm producer) based upon the previously
calculated optical density (OD) values measured at 570 nm: OD  ODc ¼ no biofilm producer; ODc < OD  2X ODc ¼ weak biofilm producer; 2X ODc < OD  4X
ODc ¼ moderate biofilm producer; 4X ODc < OD ¼ strong biofilm producer. ODc is defined as three standard deviations (SD) above the mean OD of the negative control.
34 A. Charlebois et al. / Food Microbiology 62 (2017) 32e38
C. perfringens. Briefly, overnight blood agar cultures of C. perfringens
were resuspended to a density of 0.5 MacFarland standard in
Tryptic soy broth (TSB) (BD, Ontario, Canada) supplemented with
10 mM of filter-sterilized glucose (Sigma). Two hundred mL of cul-
tures were added in 96-well polystyrene tissue culture plates
(Costar® #3595, Corning Incorporated, Corning, NY, USA) and
incubated anaerobically at 44  C for 6 d in a sealed container. After
incubation, medium containing planktonic cells was removed.
Biofilms were washed twice with MiliQ water to remove unat-
tached cells and air dried for 5 min at room temperature.
C. perfringens ATCC 13124 was used in the dual-species biofilms
along with Escherichia coli EcL 17606 and Staphylococcus aureus
E452b3 which are both facultative anaerobes (Table 1). Dual-
species biofilms were cultured as described above except that
100 mL of each culture were added per well for the dual-species
biofilms and plates were incubated anaerobically or aerobically at
35  C for 24 h in sealed containers. These conditions were used to
allow the optimal biofilm growth of all three microorganisms.
Mono-species biofilms for all three microorganisms were also
grown under these conditions to allow comparison with dual-
species biofilms. To quantify the biofilm formation for both
mono- and dual-species biofilms, the crystal violet assay was used
as described elsewhere (Varga et al., 2008). Strains were catego-
rized as per their biofilm production as described previously
(Barbosa et al., 2013; Charlebois et al., 2014; Esteban et al., 2010)
with the scale elaborated by Stepanovic et al. (Stepanovic et al.,
2007). With this method, strains are divided into four categories
(no biofilm producer, weak biofilm producer, moderate biofilm
producer and strong biofilm producer) based upon the previously
calculated optical density (OD) values measured at 570 nm:
OD  ODc ¼ no biofilm producer; ODc < OD  2X ODc ¼ weak
biofilm producer; 2X ODc < OD  4X ODc ¼ moderate biofilm
producer; 4X ODc < OD ¼ strong biofilm producer. ODc is defined as
three standard deviations (SD) above the mean OD of the negative
control.
2.4. Mono-species biofilms spore formation
Mono-species biofilms of C. perfringens ATCC 13124 were
cultured as described above and also with a few modifications to
promote spore formation. Briefly, C. perfringens were resuspended
at a density of 0.5 MacFarland in Duncan-Strong sporulation me-
dium (Duncan and Strong, 1968). Two hundred mL of cultures were
added in 96-well polystyrene tissue culture plates then incubated
anaerobically at 44  C for 6 d in a sealed container. After incubation,
medium containing planktonic cells was removed. Mono-species
biofilms were washed twice with MiliQ water to remove unat-
tached cells and air dried for 5 min at room temperature. Mono-
species biofilms were scraped and then transferred on micro-
scope slides which were analysed by dark field microscopy or
stained with malachite green and analysed by bright field micro-
scopy to visualize and count spores.
2.5. Disinfectant treatments of planktonic and biofilm cells
Plates containing the C. perfringens biofilms were treated for
10 min with 100 mL/well of sterile PBS, or with quaternary ammo-
nium chloride, sodium hypochlorite, glutaraldehyde, potassium
monopersulfate or H2O2 solutions. After treatments, disinfectants
were removed and wells were washed two times with PBS. A vol-
ume of 100 mL of sterile PBS was added into each well. Fifty mL of
BacTiterGlo (Promega, Madison, WI, USA) was added in each well
and the ATP levels were measured by luminometry, in accordance
with the manufacturer's instructions, after an incubation of
5 min at room temperature. For the planktonic cultures, 100 mL of a
culture resuspended to a density of 0.5 MacFarland standard was
transferred in a 96-well polystyrene tissue culture plates (Costar®
#3595, Corning Incorporated) and then treated with 100 mL of 2X
solutions of disinfectants for 10 min. Cultures were then centri-
fuged and cells were washed two times with PBS. Cells were
resuspended into 500 mL of sterile PBS. One hundred mL of the
culture was distributed in three wells and the viability was
measured with the BacTiterGlo kit as described above. Two washes
were used to remove residual sanitizer instead of a neutralization
step because the Dey-Engley neutralizing broth interfered with the
ATP assay. Percentages of bacterial survival were calculated by
dividing the ATP level of disinfectant-treated sample by the ATP
level of the non-treated sample. All strains were done in triplicates
and two independent experiments were performed to obtain the
viability mean percentages of the 30 strains for the five
disinfectants.
For the dual-species biofilms part, since E. coli and S. aureus are
likely to contribute to ATP levels better than C. perfringens in aer-
obic condition, bacterial counts were done to assess the degree of
killing of each species after disinfection. Briefly, after treatments,
disinfectants were discarded and 1 mL of Dey Engley neutralizing
media (BD) was added to each well. Biofilms were scraped then
plates were incubated for 10 min at room temperature. The me-
dium was recovered and serial dilutions were done (up to 106).
One hundred mL of each dilution were plated onto MacConkey agar
(Oxoid) for E. coli, Mannitol Salt agar (Oxoid) for S. aureus or
Tryptose Sulphite Cycloserine agar (Oxoid) for C. perfringens. Plates
were incubated anaerobically at 35  C for 24 h. After incubation,
colonies on each type of plates were counted to determine the
population of each microorganism and results were expressed as
log10 CFU/mL.
2.6. Statistical analysis
A Student t-test was used to compare the planktonic and the
biofilm cells viability means after disinfections. The same test was
used to compare viability before and after disinfection for the dual-
species biofilms. A p < 0.05 was considered to be significant.
3. Results
3.1. Mono- and dual-species biofilms
Out of C. perfringens strains tested (n ¼ 30), most were able to
form biofilm (n ¼ 29) in the conditions used in this study. The
optical density values at 570 nm (OD570) ranged from 0.009 to 0.353
(Table 1). C. perfringens strains were divided into four categories: no
biofilm producer (n ¼ 1), weak biofilm producer (n ¼ 24), moderate
biofilm producer (n ¼ 4) and strong biofilm producer (n ¼ 2), based
upon the above calculated OD570 values (Table 1). Moderate and
strong biofilm producers were found in clinical strains of chickens
whereas in commensal strains, no and weak biofilm producers
were observed. C. perfringens ATCC 13124, a weak biofilm producer,
was used in the dual-species biofilms along with Escherichia coli EcL
17606 and Staphylococcus aureus E452b3 which were both classi-
fied as moderate and strong biofilm producers, respectively
(Table 1). All dual-species biofilms contained both microorganisms
after incubation, with an average of 5.4  106 CFU/mL for
C. perfringens, 5.3  107 CFU/mL for E. coli and 2.2  108 CFU/mL for
S. aureus. As for the single-species biofilms, they contained an
average of 1.1  106 CFU/mL (C. perfringens), 2.9  107 CFU/mL
(E. coli) and 3.3  108 CFU/mL (S. aureus).
 A. Charlebois et al. / Food Microbiology 62 (2017) 32e38 35

3.2. Disinfectant treatments of mono-species biofilms and spore
formation
C. perfringens cells in biofilms had significantly higher viability
means (p values between 8.7  109 and 2.6  106) compared to
those observed for planktonic cells when treated with potassium
monopersulfate, quaternary ammonium chloride, hydrogen
peroxide and glutaraldehyde solutions (Fig. 1A, C, D, E and
Tables S1eS5). More specifically, the viability mean percentages of
planktonic cells of C. perfringens dropped to 0.8 ± 0.6%, 2.0 ± 1.6%,
1.8 ± 2.0% and 6.7 ± 5.4%, after treatments with potassium
monopersulfate, quaternary ammonium chloride, hydrogen
peroxide and glutaraldehyde solutions, respectively. Whereas, the
viability mean percentages of C. perfringens cells in biofilms were,
respectively, 19.3 ± 11.6%, 50.1 ± 14.2%, 51.5 ± 12.4% and
46.7 ± 13.0% when exposed to these disinfectants. Interestingly, the
viability mean percentage that we observed with sodium hypo-
chlorite was significantly higher (p ¼ 2.5  104) for planktonic
cells (14.1 ± 10.4%) than for cells in biofilms (3.8 ± 2.7%).
Also, no significant difference (p ¼ 0.17) was observed in
viability mean percentages between biofilms of C. perfringens
strains of clinical and commensal origins after exposition to dis-
infectants (Fig. 2) but variabilities were observed between repli-
cates. No significant difference (p ¼ 0.43) was found in viability
mean percentages following disinfectant treatments between bio-
films of C. perfringens strains forming weak and moderate biofilms.
Low numbers of strains forming high and no biofilms did not allow
statistical comparison. No spore was detected within the biofilms
formed by C. perfringens (data not shown).
3.3. Disinfectant treatments of dual-species biofilms
All disinfectants were further tested with multi-species biofilms
of C. perfringens to assess their effectiveness on mixed biofilm
matrix. Overall, mono- and dual-species biofilms were susceptible
to all disinfectants, with diminutions between 3.41 and 8.23
log10 CFU/mL for aerobic biofilms and between 2.87 and 7.42
log10 CFU/mL for anaerobic biofilms (Table 2 and Table S6).

Fig. 1. Viability of C. perfringens ATCC 13124 planktonic cells compared to cells in biofilm following exposure to disinfectants. Planktonic cultures and 6-days-old biofilms of
C. perfringens ATCC 13124 exposed to [A] potassium monopersulfate (1%), [B] sodium hypochlorite (0.27%), [C] quaternary ammonium chloride (1%), [D] hydrogen peroxide (10%), or
[E] glutaraldehyde (2%) for 10 min. Results are expressed as percentages of the control not exposed to disinfectants. Differences in survival of planktonic cells versus biofilm were
compared using Student's t-test. *, p < 0.05. The error bars represent standard deviations. Results presented are the mean of triplicates from two independent experiments for the
strain C. perfringens ATCC 13124. Data for all other strains are described in Supplemental Tables S1eS5.
36 A. Charlebois et al. / Food Microbiology 62 (2017) 32e38

Fig. 2. Viability of C. perfringens strains of clinical and commensal origins in biofilm following exposure to disinfectants. Viability of 6-days-old biofilms of C. perfringens
strains exposed to [A] potassium monopersulfate (1%), [B] sodium hypochlorite (0.27%), [C] quaternary ammonium chloride (1%), [D] hydrogen peroxide (10%) or [E] glutaraldehyde
(2%) for 10 min. Results are expressed as percentages of the control not exposed to disinfectants. No statistical difference was observed between clinical and commensal strains
using the Student's t-test. Each dot represents the mean of the triplicates for each strain. The bars represent the mean values of each group.

Table 2
Bacterial survival of mono-species biofilms grown in anaerobic conditions compared to dual-species biofilms following exposure to disinfectants.

C. perfringens E. coli S. aureus C. perfringens þ E. coli dual C. perfringens þ S. aureus dual

biofilms biofilms

C. perfringens E. coli C. perfringens S. aureus

Non-treated 6.16 (0.14) 7.42 (0.19) 8.75 (0.10) 6.26 (0.68) 7.05 (0.05) 6.72 (0.15) 8.37 (0.20)
Quaternary ammonium chloride (1%) <1* 2.65 (0.65)* 4.43 (0.12)* <1* 1.35 (0.35)* 1.24 (0.24)* 3.31 (0.55)*
Potassium monopersulfate (1%) <1* 4.15 (0.59)* 4.30 (0.06)* <1* 4.18 (0.86)* <1* 4.12 (0.17)*
Sodium hypochlorite (0.27%) <1* 1.15 (0.15)* 4.17 (0.20)* <1* 2.11 (0.07) <1* 3.32 (0.50)*
Hydrogen peroxide (10%) <1* <1* 2.48 (0.01)* <1* <1* <1* 1.35 (0.35)*
Glutaraldehyde (2%) <1* <1* 1.97 (0.07)* <1* 1.65 (0.05)* <1* 2.13 (1.13)*

Data are shown as mean log10 CFU per mL (standard deviation). All biofilms were done in triplicates. Differences in number of survivors between non-treated and treated cells
were compared using the Student T-test. *p < 0.05.
 A. Charlebois et al. / Food Microbiology 62 (2017) 32e38
4. Discussion
In this study, five types of disinfectants commonly used on
farms and in food processing environments were analysed for their
abilities to reduce the viability of C. perfringens cells engulfed in a
biofilm compared to their planktonic counterparts. C. perfringens
cells in biofilms had significantly higher viability means (p < 0.05)
compared to those observed for planktonic cells when treated with
potassium monopersulfate, quaternary ammonium chloride,
hydrogen peroxide and glutaraldehyde solutions. For other bacte-
ria, biofilm growth mode has been associated with bacterial toler-
ance to disinfection (Bridier et al., 2011; Grobe et al., 2002; Pereira
et al., 2001). Different studies have shown that it is likely due to
multifactorial processes involving the spatial organization of the
biofilm and a review has recently discussed these mechanisms
(Bridier et al., 2011). Indeed, the multiple layers of cells and
extracellular polymeric substance (EPS) may constitute a complex
and thick structure within which biocides penetrate with difficulty
to reach internal layers. Apart from diffusion limitations that may
occur, the penetration of biocide into microbial biofilms is also
controlled by the reaction of the disinfectant with biofilm compo-
nents such as organic matter, inorganic particles and cell debris
(Grobe et al., 2002; Pereira et al., 2001). Furthermore, the EPS is
charged and can therefore bind to biocides before they reach the
target cell, while the metabolism of cells within biofilms is different
from that of planktonic cells (Bridier et al., 2011; Vilain et al., 2004).
This study revealed that sodium hypochlorite was effective
against C. perfringens biofilms. This data is consistent with other
studies where sodium hypochlorite was shown to be active against
bacteria in biofilms (Davison et al., 2010; Saravanan et al., 2006;
Wang et al., 2012). Sodium hypochlorite has been proposed as an
effective mean to depolymerize and remove the EPS that protects
bacteria against disinfection (Lomander et al., 2004). Sodium
hypochlorite-based disinfectants are the most commonly used re-
agents in food processing plants because they are generally effec-
tive against a wide variety of bacteria (Wang et al., 2012). It has
been shown that hydrogen peroxide was able to penetrate and kill a
subset of the cells only in a biofilm formed by catalase-deficient
Pseudomonas aeruginosa (Stewart et al., 2000). It was demon-
strated that P. aeruginosa wild-type biofilm were protected from
hydrogen peroxide penetration by catalase-mediated destruction
of the biocide (Stewart et al., 2000). Recent studies have shown that
in P. aeruginosa, Burkholderia pseudomallei, and Vibrio cholerae,
genes coding for protective enzymes against oxidizing stress were
under the control of quorum sensing (Bridier et al., 2011). It is
tempting to speculate that biofilm tolerance of some commensal
strains of C. perfringens to hydrogen peroxide could be due to the
presence of oxidative stress enzymes in the matrix and under
quorum sensing regulation, but this hypothesis warrants further
investigations using transcriptomic studies. Interestingly, Vidal
et al. (2015) have recently linked C. perfringens biofilm formation
and the Agr-like quorum sensing system (Vidal et al., 2015).
Also, no significant difference (p > 0.05) was found in viability
mean percentages following disinfectant treatments between bio-
films of C. perfringens strains forming weak, moderate or strong
biofilms. One could speculate that strong biofilm producers should
be more tolerant to biocides. However, many studies in other
bacteria have shown that transport limitations are important
mechanisms that contribute to the resistance of biofilms to disin-
fectants that seem related to physicochemical interactions between
the biocide and EPS or bacterial cells rather that steric hindrance
inside the biofilm (Bridier et al., 2011). Also, it was shown that
despite biocide effective penetration into a biofilm, only a low level
of inactivation was achieved (Stewart et al., 2001). For example,
Staphylococcus aureus biofilm tolerance to quaternary ammonium
37
compound has been largely attributed to phenotypic modifications
to cells rather than to the protective presence of the EPS matrix
(Campanac et al., 2002). These phenotypic adaptations are linked
with the expression of particular genes in response to their envi-
ronment (Bridier et al., 2011). During the adhesion step, studies
have shown that genes coding flagellar proteins are repressed
while other genes coding for EPS and curli are induced in E. coli and
Pseudomonas putida (Prigent-Combaret et al., 2000; Sauer and
Camper, 2001). Following the adhesion, bacteria develop into a
biofilm with a three-dimensional structure where cells located at
the periphery have access to nutrients and oxygen, while those in
internal layers have poorer environments containing more meta-
bolic waste elements (Bridier et al., 2011). It has been stated that
this chemical heterogeneity is the source of the physiological het-
erogeneity (Stewart and Franklin, 2008) where cells with distinc-
tive metabolic rates are present throughout the biofilm. This
induces modifications to membrane composition and in defense
mechanism expressions leading to an increase in tolerance of
bacteria to disinfectants (Sabev et al., 2006; Taylor et al., 2000).
Also, it has been reported that a small part of the bacterial popu-
lation within a biofilm may develop into a highly protected state
with drastic resistance and referred to as persisters (Harrison et al.,
2005; Lewis, 2005). They may therefore contribute to disinfectant
protection in the biofilm (Harrison et al., 2005; Lewis, 2005). These
studies illustrate that many mechanisms are involved in biofilm
tolerance to biocides (Bridier et al., 2010). More recently, biofilm
tolerance to sanitization was shown to be strain-dependent in
Shiga toxin-producing Escherichia coli (STEC) regardless of the se-
rotypes and that curli expression appeared to play a critical role in
STEC biofilm formation and tolerance to sanitizers (Wang et al.,
2012).
The capacity of C. perfringens to be part of dual- or multi-species
biofilm has recently been reviewed (Pantaleon et al., 2014) and
C. perfringens biofilm was detected in some types of multi-species
biofilm (Leung et al., 2000; Pantaleon et al., 2014). Surprisingly,
no data is available on tolerance of C. perfringens dual-species
biofilms to disinfectants. This raises the question whether
C. perfringens are able to form multi-species biofilms that protect
the embedded bacteria from toxic molecules coming from the
environment. Conditions used for dual-species biofilm formation
were modified to allow growth of all bacteria. Results showed that
all dual-species biofilms contained both microorganisms after in-
cubation and bacterial counts indicated that no significant
competition occurred in dual-species biofilms between
C. perfringens and E. coli or S. aureus. It is tempting to suspect that no
or little competitive interaction occurred between species in our
dual-species biofilms. Interestingly, C. perfringens was able to grow
a biofilm in aerobic conditions. In these new conditions, all biofilms
were susceptible to disinfectants but S. aureus and E. coli in biofilm
were more resistant to disinfection than C. perfringens cells in
biofilm. In general, multi-species biofilms were shown to be more
tolerant to disinfection than single species biofilms (Luppens et al.,
2008; Simoes et al., 2009). In contrast, Behnke and Camper (2012)
demonstrated that Pseudomonas aeruginosa dual-species biofilm
with Burkholderia cepacia was less tolerant to disinfection than
their mono-species counterparts (Behnke and Camper, 2012). This
could be due to antagonist interactions between species composing
the biofilm, with the production of antimicrobial compounds such
as toxins, bacteriolytic enzymes, bacteriophages, antibiotics or
bacteriocins (Tait and Sutherland, 2002). Interestingly, potassium
monopersulfate was found to be effective against mono and dual-
species biofilms. This has been previously reported for Salmonella
mono-species biofilms (Moretro et al., 2009) but to our knowledge,
this is the first description of such effectiveness against mixed
biofilms.
38 A. Charlebois et al. / Food Microbiology 62 (2017) 32e38
Results from this study showed that the effect of disinfection on
C. perfringens biofilms was different depending on the temperature
used to produce the biofilm. This could be due to the effect of
temperature on the ability of this bacterium to form biofilm.
Indeed, it was shown previously that for C. perfringens the optimal
temperature for biofilm formation was 44  C (Charlebois et al.,
2014). This modulation of temperature on biofilm formation was
also described for other bacteria (Barbosa et al., 2013; Ramli et al.,
2012; Rode et al., 2007).
In conclusion, this study reports for the first time that biofilms of
C. perfringens can protect the embedded bacteria from toxic mole-
cules such as disinfectants. Results also showed that the effect of
disinfection on C. perfringens biofilm depends on the temperature
used to produce the biofilm.
Acknowledgements
This work was funded by a grant from the Natural Sciences and
Engineering Research Council of Canada to M. Archambault
(RGPIN-191461).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.fm.2016.09.009.
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