Takuhiro Matsumura ORCID iD: 0000-0002-3747-829X

Yukako Fujinaga ORCID iD: 0000-0001-8623-1376

Accepted Article
Effects of antibiotics on the viability of and toxin
production by Clostridium botulinum

Masahiro Yutani, Takuhiro Matsumura, Yukako Fujinaga

Department of Bacteriology, Graduate School of Medical Sciences, Kanazawa

University, 13-1, Takara-machi, Kanazawa, Ishikawa 920-8640, Japan

Correspondence

Yukako Fujinaga

Email: yukafuji@med.kanazawa-u.ac.jp

Tel: +81-76-265-2200

Running title

Effects of antibiotics on Clostridium botulinum

Subject Sections and Specified Fields

Bacteriology – Antibacterial agents and chemotherapy

This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process,
which may lead to differences between this version and the Version of Record.
Please cite this article as doi: 10.1111/1348-0421.12928.

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 Funding statement

This work was supported by JSPS KAKENHI Grant Numbers 18H02654,

Accepted Article
18K07107 and AMED Grant Number 20wm0325025h0001.

Acknowledgements

We thank S. Akagi and Y. Koino for their technical support. This work was

supported by JSPS KAKENHI Grant Numbers 18H02654, 18K07107 and AMED

Grant Number 20wm0325025h0001.

Conflict of interest disclosure

The authors declare that there is no conflict of interests.

Ethics approval statement

not relevant

Patient consent for publication statement

not relevant

Co-auther information

Takuhiro Matsumura

Affiliation: Department of Bacteriology, Graduate School of Medical Sciences,

Kanazawa University

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 E-mail: t-matsu@med.kanazawa-u.ac.jp

Yukako Fujinaga
Accepted Article
Affiliation: Department of Bacteriology, Graduate School of Medical Sciences,

Kanazawa University

E-mail: yukafuji@med.kanazawa-u.ac.jp

ORCID ID

Yukako Fujinaga: http:orcid.org/0000-0001-8623-1376

Abstract

Clostridium botulinum causes infant and adult intestinal botulism by

colonizing in the intestine and producing botulinum neurotoxin (BoNT).

Antimicrobial agents are not currently used for treatment due to the potential

facilitation of BoNT production and bacterial cell lysis, which releases toxins into

the intestinal lumen.

In this study, we analyzed effects of four antibiotics on the viability of and

BoNT production by four C. botulinum group I strains. Our results indicate that

metronidazole rapidly reduced their viability without enhancing BoNT production.

Antibiotics with these properties may promote elimination of C. botulinum from the

intestines while maintaining low levels of BoNT.

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 Key words

antibiotics, botulinum neurotoxin, botulism, Clostridium botulinum, toxin

Accepted Article
production

Clostridium botulinum is a Gram-positive, obligately anaerobic, and

spore-forming bacterium. C. botulinum and related bacteria produce a proteinous

neurotoxin (botulinum neurotoxin, BoNT) that causes the paralytic disease botulism.

In infant botulism and adult intestinal botulism, spore(s) of C. botulinum

germinate(s) in the intestinal tract of a compromised host and vegetative cells then

multiply and produce BoNT1. BoNT (in forms of complexes with non-toxin

associated proteins) is absorbed from intestinal epithelia and causes flaccid

paralysis. Severe cases may be fatal due to breathing difficulties caused by paralysis

of the respiratory muscles.Antitoxin therapy with equine anti-BoNT antiserum is the

major option for botulism. However, equine anti-BoNT antiserum is associated with

a risk of severe allergic diseases, such as anaphylaxis2. This serotherapy cannot be

used for infant patients. In the United States, human derived anti-BoNT

immunoglobulin preparation, Botulism Immune Globulin Intravenous, BabyBIG, is

administered to infant patients3. Except for the United States, BabyBIG and other

anti-BoNT human antibody preparations are not approved. Therefore, safe and

effective human antibody preparation is required to be developed.

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 In infant and adult intestinal botulism, C. botulinum transiently colonizes

the intestines of patients and continues to produce toxins during this period. Even
Accepted Article
after the resolution of paralysis, C. botulinum and BoNT may exist in the feces4, 5. In

duration that the living C. botulinum exists in the intestine, the bacterium can

multiply again and cause relapse6. To address this problem, approaches to eliminate

the bacterium safely could be effective in combination with anti-BoNT serum

treatment. However, at present, to avoid the risk of worsening symptoms,

antimicrobial agents are not used for this infectious disease because they kill

bacterial cells, which may promote the release of BoNT4. Furthermore, bacteria

exposed to antibiotics may alter their physiology by, for example, up-regulating the

expression of virulence factors7.

An antibiotic that decreases the viability of C. botulinum without inducing

the production of BoNT is a potent agent for the safe and rapid elimination of this

bacterium from the intestines of patients. In the present study, we assessed four

different classes of antibiotics for their ability to decrease the viability of C.

botulinum and to restrain BoNT production.

C. botulinum strains are divided into four groups (I to IV) based on their

physiological properties. Group I is composed of proteolytic strains, which include

serotype-A BoNT-producing strains (type A), a part of serotype-B BoNT-producing

strains (type B), and a part of serotype-F BoNT-producing strains. The majority of

reported cases of infant botulism have been caused by type A and B strains

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 belonging to group I4. Therefore, we used C. botulinum type A strains 62A and

7I03-H and type B strains Okra and Osaka05 in the present study. Strain 62A is one
Accepted Article
of the most well-studied strains of this bacterium, however, the source of the isolate

is unclear8, 9. Strain Okra was derived from a case of food-borne botulism10 and

strains 7I03-H and Osaka05 from cases of infant botulism11, 12. All four strains

belong to group I.

We selected the following four antibiotics: ampicillin (Amp), vancomycin

(Van), metronidazole (MNZ), and fidaxomicin (FDX), and examined their effects on

the viability of and BoNT production by C. botulinum strains. Amp is a beta-lactam

antibiotic that inhibits cell wall synthesis by binding to penicillin-binding proteins

that crosslink peptidoglycan. Amp is effective against Gram-positive and some

Gram-negative bacteria. Van belongs to the glycopeptide family and inhibits cell

wall synthesis by binding to the D-alanyl-D-alanine end of the murein monomer, a

component of peptidoglycan. Van is effective against Gram-positive bacteria. MNZ

is used to against anaerobic bacteria and is reduced to form nitroso radicals, which

induce damage to and inhibit the synthesis of DNA. FDX inhibits bacterial RNA

polymerase. FDX exhibits strong antibacterial activity against some species of the

genus Clostridium and Clostridioides difficile13. These four antibiotics exhibit

bactericidal activity. Van, MNZ, and FDX are used to treat C. difficile infections14.

We measured the minimal growth inhibitory concentrations (MIC) of

these antibiotics against C. botulinum. The MIC assay was performed according to

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 the serial dilution method using 96-well microplates. The cultivation of the

bacterium was initiated from spores. Spore suspensions of C. botulinum were

Accepted Article
prepared using the following procedure. Each strain was anaerobically cultured in

TPGY broth overnight. The overnight culture was spread onto TPGY agar plates and

incubated for one week. The spores that formed on agar plates were flushed out by

ice-cold sterile water and washed using ice-cold sterile water ten times. The spore

suspension prepared was stored at -80ºC until used. In the MIC assay, the spore

suspension was inoculated into brain heart infusion (BHI) broth and incubated

overnight. The overnight culture was diluted to an OD600 of 0.1 in fresh BHI broth

containing the antibiotic at a concentration of the two-fold dilution series. After a

48-h incubation, MIC was measured based on the visual turbidity of the well. In the

present study, all cultivations were performed anaerobically at 37ºC.

The results obtained are shown in Table 1. In the present study, the MIC

values of FDX against C. botulinum strains ranged between 7.8 and 62.5 ng/mL.

FDX has a narrow spectrum and exhibits strong antimicrobial activity, particularly

against Clostridium phylogenetic cluster I (including C. botulinum and C.

perfringens) and cluster XI (previously contained C. difficile, formerly classified

into the genus Clostridium)15. Yanagihara et al. showed that FDX had a MIC90 value

of 8 ng/mL against clinical isolates of C. perfringens (n = 30) and 120 ng/mL against

those of C. difficile (n = 50)15. The present results indicated that strains of C.

botulinum group I exhibited hypersensitivity to FDX. Strain 7I03-H showed strong

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 resistance against Amp, with a MIC value >32.0 µg/mL. We confirmed that strain

7I03-H produced beta-lactamase using the Cefinase paper disc (Becton Dickinson,
Accepted Article
Franklin Lakes, NJ) test (data not shown). Barash et al. showed a beta-lactamase

positive single strain of C. botulinum type A isolated from a patient with infant

botulism in US16. The MIC values of Van and MNZ against the four strains tested

were 8 µg/mL. Against clinical isolates of C. difficile (n = 716), which were isolated

in the US and Europe, the MIC90 values of Van and MNZ were 2 and 1 µg/mL,

respectively17.

Next, we examined the effects of these four antibiotics on the viability of

and toxin production by C. botulinum. In infant (adult intestinal) botulism, the

development of symptoms indicates that the causative organism C. botulinum has

been multiplying and producing BoNT in the intestines of patients. In an in vitro

culture, C. botulinum actively produces BoNT in the stationary phase18. Therefore,

in the present study, we added antibiotics to the in vitro culture of C. botulinum in the

late logarithmic to stationary phases and investigated their effects on cell viability

and BoNT production. Although strains 62A and Okra were not derived from cases

of infant botulism, they were previously shown to colonize and produce BoNT in the

gut of suckling mice within a specific time period and in the gut of adult mice whose

intestinal-microflora-disrupted adult mice19 – 21. Therefore, we used C. botulinum

strain 62A in this experiment. FDX, Amp, Van, and MNZ were used at

concentrations that were approximately 5-fold higher than MIC. In bacterial

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 viability and toxin production assays, the spore suspension was inoculated into TPM

(toxin-producing medium containing 2% peptone, 5% yeast extract, 0.5%

Accepted Article
D-glucose, and 0.025% sodium thioglycolate, pH 7.0) and incubated until the culture

reached an OD600≈2.0. This preculture was diluted to an OD600 of 0.1 in fresh TPM.

After an incubation for 6 h, the antibiotics were individually added and the

incubation was continued. The number of viable cells was 1.03 × 109 CFU/mL after

the 6-h incubation, and the culture entered the stationary phase in the absence of

antibiotics (Fig. 1A). In the presence of FDX, Van, or MNZ, the viability of the

bacterium decreased. FDX decreased viability from 1.03 × 109 to 1.22 × 106

CFU/mL 4 h after its addition. Van decreased viability from 1.03 × 109 to 5.62 × 106

CFU/mL 6 h after its addition. MNZ rapidly decreased the viability of C. botulinum

from 1.03 × 109 to 2.81 × 106 CFU/mL within 15 minutes of its addition. Amp

slightly decreased viability from 1.03 × 109 to 2.44 × 108 CFU/mL 6 h after its

addition. Although Amp is a bactericidal antibiotic, clear bactericidal effects were

not observed in this assay. This may have been due to the timing of the addition of

the drug being between the late logarithmic growth and stationary phases, and the

growth of the bacterium, which requires cell wall synthesis, the target of Amp, being

slower22. Van is a cell wall synthesis inhibitor; however, it reduced viability more

strongly than Amp. This difference may be due to Van increasing cell membrane

permeability23, 24 and inhibiting RNA synthesis25 in addition to its inhibition of cell

wall synthesis.

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 These cultures were collected chronologically and analyzed by Western

blotting for the relative quantification of BoNT. C. botulinum produces BoNT after
Accepted Article
the late log-phase18. BoNT molecules are synthesized as a single peptide chain of

150 kDa, which is nicked by protease and divided into a light chain (LC) of 50 kDa

and a heavy chain (HC) of 100 kDa. The two chains are crosslinked by disulfide

bonds. The results obtained are shown in Fig. 1B and C. After a 6-h incubation,

immediately before the addition of antibiotics, small amounts of BoNT, as a single

peptide without nicking, were detected in the whole culture of strain 62A. The

amount of BoNT increased over time, indicating that the toxin was produced in the

stationary phase. After a 9-h incubation, some BoNT molecules were separated into

LC and HC by nicking.

The amount of BoNT was significantly higher in the presence of FDX than in that of

the control at all time points after the addition of the antibiotic despite decreases in

viable cells. These results indicated that FDX enhanced the production of BoNT in

surviving cells and/or during cell death. Amp also increased BoNT production over

that by the control; however, smaller increases were observed than in the presence of

FDX. In the presence of Amp, the viability slightly decreased to 2.44 × 108 CFU/mL

6 h after its addition. Therefore, the enhancement of toxin production by a single live

cell of the bacterium was considered to be small. Van suppressed toxin production

compared to the control from 3 to 6 h after its addition. In the presence of Van, the

viability of 62A was 6.87 × 107 CFU/mL 2 h after its addition, 1.29 × 107 CFU/mL 4

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 h after its addition, and 5.62 × 106 CFU/mL 6 h after its addition. Therefore, BoNT

continued to be produced at a low level in the presence of Van due to a decrease in

Accepted Article
the number of viable cells capable of producing the toxin. The amount of the toxin

slightly increased or remained unchanged after the addition of MNZ, suggesting that

BoNT proteins were not synthesized de novo. MNZ rapidly decreased viability

within 15 min of its addition. Based on these results, MNZ possesses the most

advantageous properties among these four antibiotics.

We then examined the dose dependency of the effects of MNZ on the

viability of and toxin production by strain 62A. Culture conditions and methods to

assay viability and toxin production were the same as those described above. MNZ

was added to a culture of strain 62A in TPM at 10, 50, or 100 µg/mL after a 6-h

incubation. The results are shown in Fig. 2. MNZ exerted dose-dependent effects

against strain 62A. MNZ decreased the viability of strain 62A from 1.33 × 109 to

1.17 × 108 CFU/mL at 10 µg/mL, to 2.20 × 106 CFU/mL at 50 µg/mL, and to 1.27 ×

103 CFU/mL at 100 µg/mL (Fig. 2 A). At any concentration of MNZ, decreases in

viability were equally induced within 15 minutes of its addition. MNZ reduced the

production of BoNT at 50 µg/mL or more (Fig. 2B and C). At sublethal

concentrations or sub MIC values, antibiotics induced physiological responses to

bacteria, e.g., the up- or down-regulation of amino acid biosynthesis, purine

nucleotide biosynthesis, and the expression of virulence factors26. In our

experiments, MNZ did not stimulate the production of BoNT by C. botulinum strain

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 62A at 10 µg/mL, a concentration that was similar to MIC (Fig. 2B and C). Further

experiments to confirm whether MNZ does not stimulate the BoNT production at
Accepted Article
any conditions, including in vivo, will define the advantage of MNZ.

We also investigated the effects of MNZ on the viability of and BoNT

production by the 7I03-H, Okra, and Osaka05 strains using the same methods as

those described above. MNZ rapidly decreased the viability of strains 7I03-H and

Osaka05 at 50 µg/mL from 7.67 × 108 to 3.00 × 105 CFU/mL (7I03-H) or 6.13 × 108

to 2.02 × 105 CFU/mL (Osaka05), respectively, within 15 minutes of its addition

(Fig. 3A and G). The effects of MNZ on the viability of strain Okra were weaker than

those against the other strains, decreasing the viable bacterial cell count from 3.78 ×

109 to 5.42 × 108 CFU/mL at 50 µg/mL and from 3.78 × 109 to 1.50 × 106 CFU/mL at

100 µg/mL (Fig. 3D). In the MIC assay, MNZ was added to the culture in the early

logarithmic phase, and strain Okra was as sensitive to MNZ as the other three strains,

with a MIC value of 8 µg/mL against all four strains. MNZ suppressed toxin

production by these three strains (Fig. 3B, C, E, F, H, and I). These results revealed

that MNZ rapidly decreased the viability of the four strains in C. botulinum group I

used in this study and maintained toxin production at low levels.

Several studies have been made on MICs of antibiotics against C.

botulinum16, 27, 28. However, no study to date has examined the effect of antibiotics on

the time-course change of viability and on the BoNT production. In the present

study, we investigated the effects of antibiotics on the viability of and toxin

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 production by C. botulinum group I strains in the stationary phase. FDX decreased

the viability of strain 62A and concurrently stimulated toxin production. On the other
Accepted Article
hand, MNZ rapidly decreased the viability of strain 62A and the three other strains in

group I without enhancing toxin production. The death of C. botulinum cells within a

short time period may be very important for the suppression of toxin production by

MNZ by reducing the time required for the synthesis of toxin proteins. Antimicrobial

agents possessing these properties may promote the elimination of C. botulinum

from the intestinal tracts of patients while maintaining low production levels of

BoNT. However, we do not intend to recommend the clinical use of MNZ forthwith

for following reasons; i) according to a report, the concentration of MNZ is

approximately 1 µg/g in feces from human adults administrated 200–400 mg of

MNZ orally29. If the value is considered to be approximately 1 µg/mL in large

intestine, this concentration is lower than MIC determined in this study. ii) in vivo

study is required to verify the efficiency and safety.

In the present study, a limited number of strains and antibiotics were

examined. Accumulation of data that using more strains and antibiotics will

contribute to the development of safe and feasible antibiotic method for intestinal

infection of C. botulinum. By using suitable antibiotic(s) in combination with

antibody treatment, early clearance of C. botulinum and prevention of recurrence

will be achieved in clinically safe manner.

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 Acknowledgements

We thank S. Akagi and Y. Koino for their technical support. This work was
Accepted Article
supported by JSPS KAKENHI Grant Numbers 18H02654, 18K07107 and AMED

Grant Number 20wm0325025h0001.

Disclosure

The authors declare that there is no conflict of interests.

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Figures

Figure 1. Effect of the antibiotics on C. botulinum 62A viability and toxin

production. (A) Colony-forming units were assayed after incubations for 0, 6, 8, 10,

and 12 h (none, Amp, and Van) or 0, 6, 6.25 (6 h 15 min), 6.5 (6 h 30 min), 7, 8, 10,

and 12 h (FDX and MNZ). (B) BoNT in a whole culture collected at the indicated

time point was detected by Western blotting and (C) quantified by densitometry

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 analysis on the Western blot band in panel (B). The values are presented as relative

density against the sample after 6 h of incubation. Three independent experiments

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were performed and representative data of the bacterial viability (A) and western

blotting (B) were shown. Data of densitometry analysis is shown as mean with SEM

(n = 3) (C).

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 Figure 2. Dose-dependent effects of MNZ on C. botulinum strain 62A viability

and toxin production. (A) Colony-forming units were assayed after incubations for
Accepted Article
0, 6, 6.25 (6 h 15 min), and 12 h. (B) BoNT in a whole culture collected at the

indicated time point was detected by Western blotting and (C) quantified by

densitometry analysis on the Western blot band in panel (B). The values were

presented as relative density against the sample after 6 h of incubation. Three

independent experiments were performed and representative data of the bacterial

viability (A) and western blotting (B) were shown. Data of densitometry analysis

was shown as mean with SEM (n = 3) (C).

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 Figure 3. MNZ decreases the viability of and suppresses toxin production by C.
botulinum group I strains other than strain 62A. The viability of and toxin
production by C. botulinum strain 7I03-H (A, B, C), Okra (D, E, F), and Osaka05 (G,
Accepted Article
H, I) in the presence of MNZ were assayed. (A, D, G) Colony-forming units were
assayed after incubations for 0, 6, 6.25 (6 h 15 min), and 12 h. (B, E, H) BoNT
contained in a whole culture collected at the indicated time point was detected by
Western blotting and (C, F, I) quantified by densitometry analysis on the Western
blot band. The values were presented as relative density against the sample after 6 h
of incubation. Three independent experiments about each strain were performed and
representative data of the bacterial viability (A, D, G) and western blotting (B, E, H)
were shown. Data of densitometry analysis were shown as mean with SEM (n = 3)
(C, F, I).

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 Table 1. MIC of antibiotics against strains in C. botulinum group I.
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strain of C. botulinum
Antibiotics

(abbreviation)
62A 7I03-H Okra Osaka05

fidaxomicin (FDX) 0.0078 0.0078 0.0625 0.0156

ampicillin (Amp) 0.0156 > 3200.0 < 0.0031 0.00625

vancomycin (Van) 8.0 8.0 8.0 8.0

metronidazole (MNZ) 8.0 8.0 8.0 8.0

MIC values are showed in µg/mL.

List of abbreviation

Amp – ampicillin

BoNT – botulinum neurotoxin

CFU – colony forming units

FDX – fidaxomicin

HC – heavy chain

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 LC – light chain

MIC – minimum growth inhibitory concentration

Accepted Article
MNZ – metronidazole

Van – vancomycin

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