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Álvarez-Pérez et al. BMC Veterinary Research (2023) 19:238
https://doi.org/10.1186/s12917-023-03801-2 BMC Veterinary Research
*Correspondence:
José L. Blanco
jlblanco@ucm.es
1Department of Animal Health, Faculty of Veterinary Medicine,
Complutense University of Madrid, Madrid, Spain
2Laboratorio de Biología Molecular y Microbiología, Instituto Tecnológico
Agrario de Castilla y León, Valladolid, Spain
Abstract
Background Previous studies have demonstrated that fidaxomicin, a macrocyclic lactone antibiotic used to treat
recurrent Clostridioides difficile-associated diarrhea, also displays potent in vitro bactericidal activity against Clostridium
perfringens strains isolated from humans. However, to date, there is no data on the susceptibility to fidaxomicin of C.
perfringens strains of animal origin. On the other hand, although combination therapy has become popular in human
and veterinary medicine, limited data are available on the effects of antibiotic combinations on C. perfringens. We
studied the in vitro response of 21 C. perfringens strains obtained from dogs and cats to fidaxomicin and combinations
of fidaxomicin with six other antibiotics.
Results When tested by an agar dilution method, fidaxomicin minimum inhibitory concentrations (MICs)
ranged between 0.004 and 0.032 µg/ml. Moreover, the results of Etest-based combination assays revealed that
the incorporation of fidaxomicin into the test medium at a concentration equivalent to half the MIC significantly
increased the susceptibility of isolates to metronidazole and erythromycin in 71.4% and 61.9% of the strains,
respectively, and the susceptibility to clindamycin, imipenem, levofloxacin, and vancomycin in 42.9–52.4% of the
strains. In contrast, ¼ × MIC concentrations of fidaxomicin did not have any effect on levofloxacin and vancomycin
MICs and only enhanced the effects of clindamycin, erythromycin, imipenem, and metronidazole in ≤ 23.8% of the
tested strains.
Conclusions The results of this study demonstrate that fidaxomicin is highly effective against C. perfringens strains of
canine and feline origin. Although fidaxomicin is currently considered a critically important antimicrobial that has not
yet been licensed for veterinary use, we consider that the results reported in this paper provide useful baseline data to
track the possible emergence of fidaxomicin resistant strains of C. perfringens in the veterinary setting.
Keywords Antibiotic combination, Cat, Clostridium perfringens, Dog, Fidaxomicin
In vitro activity of daxomicin
and combinations of daxomicin with other
antibiotics against Clostridium perfringens
strains isolated from dogs and cats
Sergio Álvarez-Pérez1, Blanca Anega1, José L. Blanco1*, Marta Hernández2 and Marta E. García1
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 2 of 6
Álvarez-Pérez et al. BMC Veterinary Research (2023) 19:238
Background
e Gram-positive, spore-forming, toxin-producing
anaerobe Clostridium perfringens is a common entero-
pathogen of humans and diverse animals, including dogs
and cats [1–3]. Previous studies have demonstrated that
C. perfringens strains from different sources often show
resistance or decreased susceptibility to diverse antibiot-
ics, including first-line anti-anaerobic drugs such as met-
ronidazole [4–10].
Fidaxomicin is a macrocyclic lactone antibiotic that
targets RNA polymerase and has bactericidal activity
against Clostridioides dicile (formerly Clostridium dif-
cile) and other clostridia, including C. perfringens [11–
15]. In general, most C. perfringens strains analyzed to
date have shown low minimum inhibitory concentrations
(MICs) to fidaxomicin (typically ≤ 0.004–0.06µg/ml) [11,
12, 14]. Furthermore, fidaxomicin has multiple benefits
compared to other antibiotics used to treat clostridial
gastrointestinal infections, such as its good safety and
tolerability profile, its low fecal binding and minimal sys-
temic absorption, and the fact that it has minimal effect
on the normal gut microbiota [11, 13, 15]. Nevertheless,
the elevated acquisition cost is a major drawback of fidax-
omicin (e.g., USD 3845.44 vs. USD 23.28 for a 10-day
course of fidaxomicin and vancomycin, respectively [16]),
even when it has been claimed that, in some contexts,
fidaxomicin use might reduce total healthcare costs with
respect to vancomycin or metronidazole [17–19].
Economical aspects have also precluded the use of
fidaxomicin in veterinary medicine, as well as the inclu-
sion of this antibiotic in the World Health Organiza-
tion’s list of critically important antimicrobials (CIAs)
for human medicine [20] and the European Union’s list
of antimicrobials reserved for treatment of certain infec-
tions in humans [21]. However, given the zoonotic poten-
tial often attributed to C. perfringens [22], it is important
to provide baseline data on the susceptibility of animal
isolates of this pathogen to fidaxomicin, either alone or
in combination with other antimicrobial drugs. Accord-
ingly, in this study we analyzed the in vitro response of
C. perfringens strains from dogs and cats to fidaxomi-
cin and several combinations of fidaxomicin with other
antibiotics.
Results
e MICs to fidaxomicin obtained by the Clinical and
Laboratory Standards Institute (CLSI) agar dilution
method [23] for the C. perfringens strains analyzed in this
study (n = 21) ranged from 0.004 to 0.032µg/ml (median
value: 0.008 µg/mL; Table S1). Furthermore, the fidax-
omicin MIC distributions obtained for strains of dif-
ferent toxinotype and animal origin were overlapping:
toxinotype A, 0.004 to 0.032µg/ml (n = 19); toxinotype F,
0.008 to 0.016µg/ml (n = 2); dog origin, 0.008 to 0.032µg/
ml (n = 19); and cat origin, 0.004 to 0.016µg/ml (n = 4).
On the other hand, the MICs to the other antibiotics
used in the combination assays (see below) were as fol-
lows: clindamycin, < 0.016 to 1µg/ml; erythromycin, 0.5
to 4µg/ml; imipenem, 0.032 to ≥ 32 µg/ml; levofloxacin,
0.125 to 2µg/ml; metronidazole, 4 to 64µg/ml; and van-
comycin, 0.5 to 2µg/ml (Table S1).
Table 1 shows an overview of the results obtained
in the combination assays of fidaxomicin with the
aforementioned six antibiotics (see detailed results
in Table S1). When tested at concentrations equiva-
lent to half the MICs determined by the agar dilution
agar (BBA + 1/2F medium, see Methods), fidaxomicin
significantly enhanced the effect of the other antibiot-
ics tested for ≥ 42.9% of the C. perfringens strains, with
the highest frequency of significant MIC reduction
being detected between fidaxomicin and metronidazole
(71.4% of strains; Table1). Moreover, the combinations
of fidaxomicin with clindamycin, imipenem, erythro-
mycin, and metronidazole, yielded variable outcomes
(i.e., those instances in which different categorical
results –significant increase or decrease of the MIC val-
ues, or non-significant MIC variation– were observed
in two replicates of the combination assay) for 9.5 to
19% of strains (Table1). In contrast, significant activity
Table 1 Overview of the results of the assays testing the
interaction of fidaxomicin with other antibiotics against
Clostridium perfringens isolates from dogs and cats (n = 21)
Test
mediumaCombined
antibioticbOutcome of the interactionc
Signicant
activity
enhancement
Non-
signicant
MIC
variation
Variable
result
BBA + 1/2F Clindamycin 10 (47.6%) 9 (42.9%) 2 (9.5%)
Erythromycin 13 (61.9%) 5 (23.8%) 3 (14.3%)
Imipenem 9 (42.9%) 8 (38.1%) 4 (19%)
Levofloxacin 10 (47.6%) 11 (52.4%) 0 (0%)
Metronidazole 15 (71.4%) 3 (14.3%) 3 (14.3%)
Vancomycin 11 (52.4%) 10 (47.6%) 0 (0%)
BBA + 1/4F Clindamycin 5 (23.8%) 12 (57.1%) 4 (19%)
Erythromycin 1 (4.8%) 19 (90.5%) 1 (4.8%)
Imipenem 1 (4.8%) 20 (95.2%) 0 (0%)
Levofloxacin 0 (0%) 21 (100%) 0 (0%)
Metronidazole 4 (19%) 16 (76.2%) 1 (4.8%)
Vancomycin 0 (0%) 21 (100%) 0 (0%)
a BBA + 1/2F and BBA + 1/4F refer to B rucella blood agar wit h hemin and vitamin
K (BBA) supplem ented with daxomici n at ½ × and ¼ × the minimum inhibitory
concentrati on (MIC) determined by t he CLSI agar dilution met hod, respective ly
b Etest strip s placed on BBA + 1/2F and BBA + 1/4F in the combinati on assay
c Frequency (and percentage) of each outcome. Signicant activity
enhancement: ≥3 two-fold reduction in the MIC when compared to control
plates containing no daxomicin (BBA + 0F); non-signicant MIC variation: ≤2
two-fold MIC change compared to BBA + 0 F; variable result: cases in which
dierent categorical results (signicant activity enhancement, signicant
activity reduction, or non-signicant MIC variation) were observed in two
replicates o f the combination assay
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Álvarez-Pérez et al. BMC Veterinary Research (2023) 19:238
enhancement was remarkably less frequent for all anti-
biotic combinations tested when fidaxomicin was pres-
ent in BBA at a quarter of the MIC of the tested strains
(BBA + 1/4F medium, see Methods; Table1). Significant
activity reduction (i.e., MIC increase) could not be con-
firmed for any antibiotic combination tested; neverthe-
less, two strains that yielded a variable result (namely,
G/05P1 and M/14P3) showed a > 2-fold dilution increase
in the MIC to clindamycin in one of the test replicates
in BBA + 1/4F. Finally, it was found that for all antibi-
otic pairs except fidaxomicin-clindamycin the frequency
of each outcome of the combination assay significantly
depended on the concentration of fidaxomicin included
in the test medium (P = 0.331 for fidaxomicin-clindamy-
cin; P < 0.001 for all other antibiotic combinations).
Discussion
Although fidaxomicin is not currently used in veterinary
medicine, given the widespread occurrence of antibiotic-
resistant C. perfringens in animals and the environment
(see, e.g. [4–6, 9]) and the ‘One Health’ approach pro-
posed for the study of other clostridia of similar ecology
(e.g., C. dicile [24, 25] and Clostridium botulinum [26]),
animal strains of C. perfringens should also be tested for
in vitro susceptibility to fidaxomicin. However, to our
knowledge, no other previous studies have addressed this
issue. To fill in this research gap, we analyzed the in vitro
effect of fidaxomicin against 21C. perfringens strains of
canine and feline origin. Our results showed than, when
tested by the CLSI agar dilution method [23], fidaxomi-
cin MICs were low (0.004–0.032 µg/ml), which agrees
with the results of previous studies testing C. perfringens
strains of human origin and the potent bactericidal activ-
ity that this antibiotic has against C. dicile and other
clostridia sensu lato, e.g., Clostridium butyricum, Clos-
tridium paraputricum, Paraclostridium bifermentans
(formerly Clostridium bifermentans), and Terrisporobac-
ter glycolicus (formerly Clostridium glycolicum) [11, 12,
14]. Similarly, unpublished data from our research group
indicates that strains isolated from intensively-raised pigs
and Iberian pigs also display low fidaxomicin MICs (typi-
cally ≤ 0.032µg/ml; García M.E. et al., unpublished data).
In contrast, other species such as Clostridium innocuum,
Enterocloster bolteae (formerly Clostridium bolteae),
Hungatella hathewayi (formerly Clostridium hathewayi),
and omasclavelia ramosa (formerly Clostridium ramo-
sum) seem to be intrinsically resistant to fidaxomicin,
and a human isolate of C. perfringens with a MIC value of
64µg/ml has been found in Japan [12, 14].
On the other hand, antibiotic combination therapy has
become popular in human and veterinary medicine as a
strategy to enhance the efficacy of antibiotic treatments
against diverse bacterial pathogens while reducing the
undesirable side effects of such treatments and slowing
down the development of resistance [27–29]. To our
knowledge, the susceptibility of C. perfringens to antibi-
otic combinations has never been assessed, even when,
for example, some combinations of metronidazole with
vancomycin, macrolides, quinolones, beta-lactams, and/
or rifaximin are often used or have been tested in clinical
trials to treat a variety of digestive disorders in humans
and pets [30–34]. In the present study, we analyzed the
in vitro response of C. perfringens to combinations of
fidaxomicin with other six antibiotics and found that the
outcome of the interaction assays depended on the com-
bined antibiotics, the concentration of fidaxomicin in the
test medium, and the strain. In particular, the incorpora-
tion of fidaxomicin into the test medium at half the MIC
determined by the agar dilution method significantly
enhanced the activity (i.e., decreased the MIC values) of
clindamycin, erythromycin, levofloxacin, imipenem, met-
ronidazole, and vancomycin in > 40% of the tested strains.
In contrast, concentrations of fidaxomicin equivalent to a
quarter of the MIC resulted in non-significant variation
oflevofloxacin or vancomycin MICs and reduced the fre-
quency of significant activity enhancement of clindamy-
cin, erythromycin, imipenem, and metronidazole. Similar
strain-, compound-, and/or concentration-dependent
antibiotic combination effects have been reported for
other bacteria (e.g., carbapenemase-producing entero-
bacteria [35], methicillin-resistant Staphylococcus aureus
[36], and vancomycin-resistant enterococci [37]), which
makes it difficult to generalize about the effects of a par-
ticular antibiotic combinations against a given pathogen
and highlights the need for baseline data such as those
reported here.
A limitation of this study is that the results of combina-
tion assays are generally interpreted using the fractional
inhibitory concentration index (FICI; which is calcu-
lated using the following formula: FICI = MICAB/MICA
+ MICBA/MICB, where MICA and MICB are the MICs of
drugs A and B when acting alone and MICAB and MICBA
are the MICs of drugs A and B when acting in combina-
tion, respectively) and by interpreting the possible results
in terms of ‘synergy’ (FICI ≤ 0.5), ‘antagonism’ (FICI > 4),
and ‘indifference’ or ‘no interaction’ (FICI > 0.5–4) [38,
39]. Alternative definitions of these concepts have been
proposed by other authors for those cases where one of
the antibiotics is included in an agar medium at a fixed
concentration (i.e., fidaxomicin in the present study) but
there is a concentration gradient of the other antibiotic
used in the combination assay (e.g., created by using Etest
strips) [40, 41]. However, in absence of a clear consensus
for these alternative definitions of synergy and antago-
nism, we have interpreted our results in terms of signifi-
cant activity enhancement, significant activity reduction,
or non-significant MIC variation. Furthermore, the lim-
ited number of C. perfringens strains of toxinotype F and
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Álvarez-Pérez et al. BMC Veterinary Research (2023) 19:238
cat origin tested in this study (two and four, respectively),
preclude a reliable analysis of toxinotype- and host-based
differences in fidaxomicin susceptibility and combina-
tion effects. Additionally, although the strains included
in this study were genetically diverse (see Methods), we
acknowledge that our limited selection of strains might
not represent the whole intra-species genetic diversity of
C. perfringens and, therefore, future studies should con-
firm if our conclusions are applicable to strains of other
genetic backgrounds. Despite these limitations of our
study and the fact that fidaxomicin is currently consid-
ered a CIA that should be reserved for the treatment of
certain infections in humans and, accordingly, that it has
not yet been licensed for veterinary use, we consider that
the results here reported provide useful baseline data to
track the possible emergence of fidaxomicin resistant
strains of C. perfringens in the veterinary setting. In any
case, as already done for other CIAs, the eventual use of
fidaxomicin in the treatment of animal infections should
follow the guidelines and recommendations of inter-
national, national, and/or regional agencies for medici-
nal products and animal health (see, for example, Refs.
[42–44]).
Conclusion
Although the use of fidaxomicin is currently restricted to
human medicine, the results of this study demonstrate
that this antibiotic is also highly effective against C. per-
fringens toxinotype A and F strains of canine and feline
origin. Moreover, our results reveal that the occurrence
of in vitro combination effects between fidaxomicin and
other antibiotics against C. perfringens is compound-,
concentration-, and strain-dependent. Future research
should clarify if these conclusions can also be applied to
other toxinotypes of C. perfringens and/or strains from
other sources and genetic backgrounds.
Methods
Strains
A total of 21C. perfringens strains obtained from fecal
samples of dogs (n = 17 strains) and cats (n = 4) attended
between 24 and 2015 and 1 December 2015 at different
primary care veterinary clinics located in the Madrid
region, Spain (see details in Álvarez-Pérez et al. [5, 45])
were included in the present study. e selection of
strains was mainly done based on their MIC values to
fidaxomicin and other antibiotics (see Results), so as
to have a representation of strains with different anti-
biotic susceptibility profiles in the combination assays
described below. All strains had been primarily recov-
ered in Columbia blood agar (bioMérieux, Marcy l’Etoile,
France) and stored at − 70°C as cell suspensions in brain
heart infusion broth (BHI; Pronadisa, Madrid, Spain)
supplemented with 25% glycerol (Panreac, Barcelona,
Spain). PCR toxinotyping revealed that 19 of the stud-
ied strains, including 15 strains from dogs and the four
strains from cats, belonged to toxinotype A (they only
had the cpa gene encoding for C. perfringens alpha toxin),
whereas the other two strains of canine origin should be
classified as toxinotype F (besides cpa, these strains also
had the cpe gene enconding C. perfringens enterotoxin)
[5, 45, 46]. Furthermore, all studied strains belonged to
different amplified fragment length polymorphism geno-
types [5, 45].
Fidaxomicin susceptibility testing
In vitro susceptibility to fidaxomicin (0.001 to 0.125µg/
ml; Sigma-Aldrich, Madrid, Spain) was determined by
the agar dilution method, which was performed by fol-
lowing the CLSI guidelines for anaerobes [23], using
dimethyl sulfoxide (DMSO; Sigma-Aldrich) as the anti-
biotic solvent and Brucella blood agar with hemin (5µg/
ml; Sigma-Aldrich), vitamin K (1µg/ml; Sigma-Aldrich),
and 5% v/v of defibrinated sheep blood (Oxoid Ltd.,
Basingstoke, UK) (BBA) as culture medium,. All strains
were tested at least twice on different days, and drug-free
plates were included as growth controls.
Combination assays
Analysis of the response of C. perfringens strains to
combinations of fidaxomicin with other six antibiotics,
namely clindamycin, erythromycin, imipenem, levofloxa-
cin, metronidazole, and vancomycin was performed by
an Etest-based method. Briefly, based on the MIC results
determined by the agar dilution method (see above), two
series of 90-mm-diameter plates containing 20 ml of
BBA supplemented with fidaxomicin at ½ × MIC and ¼ ×
MIC concentrations (hereafter referred to as BBA + 1/2F,
BBA + 1/4F, respectively) were prepared. Additionally,
plates containing 20 ml of BBA plus 1% v/v of DMSO but
no fidaxomicin (BBA + 0F) were used as controls. Assay
plates were immediately used or stored at 4°C and used
within 24h.
Cotton-tipped sterile swabs (Aptaca Spa., Canelli,
Italy) were dipped in cell suspensions (McFarland stan-
dard of 1, prepared in BHI from 2 to 3-day old cultures),
which were spread onto the surface of the BBA + 1/2F,
BBA + 1/4F, and BBA + 0 F plates. Clindamycin, eryth-
romycin, imipenem, levofloxacin, metronidazole, and
vancomycin Etest strips (bioMérieux) were laid on the
surface of inoculated plates. All plates were incubated
under strictly anaerobic conditions (< 0.1% of oxygen
after 2.5 h; GENbox anaer, bioMérieux) at 37 °C and
read after 48 h. e effect of each antibiotic combina-
tion on each C. perfringens strain was tested twice on dif-
ferent days. e drug concentration shown on the Etest
strip at the outer border of the elliptical inhibition halo
was recorded as the MIC. High off-scale values were
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Page 5 of 6
Álvarez-Pérez et al. BMC Veterinary Research (2023) 19:238
converted to the next highest concentration, whereas low
off-scale MICs were left unchanged. A MIC reduction or
increase of at least three two-fold dilutions in the pres-
ence of fidaxomicin in both test replicates was regarded
as proof of significant activity enhancement or signifi-
cant activity reduction, respectively, and ≤ 2 two-fold
MIC changes were interpreted as non-significant MIC
variation.
Data analysis
Statistical analysis of results was performed using R
v.4.2.2. e Fisher’s exact test for count data with simu-
lated P-value (two-sided, based on 104 replicates) was
used for the analysis of categorical data where appro-
priate. P-values < 0.05 were considered statistically
significant.
Abbreviations
BBA Brucella blood agar with hemin and vitamin K
BBA + 0 F BBA with no fidaxomicin
BBA + 1/2F BBA supplemented with fidaxomicin at half the MIC
BBA + 1/4F BBA supplemented with fidaxomicin at a quarter the MIC
BHI brain heart infusion broth
CIA critically impor tant antimicrobial
CLSI Clinical Laboratory and Standards Institute
DMSO dimethyl sulfoxide
MIC minimum inhibitory concentration
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12917-023-03801-2.
Supplementary Material 1
Authors’ contributions
Conceptualization and resources: all authors. Investigation, formal analysis,
and data curation: BA, SA-P. Writing — original draft preparation: SA-P, JLB, MH,
MEG. Writing — review and editing: all authors. Supervision: MEG, SA-P, MH.
Funding: MEG, SA-P.
Funding
This work was funded by project grant [PID2019-108071RR-C22] and a ‘Ramón
y Cajal’ contract [RYC2018-023847‐I, awarded to Sergio Álvarez Pérez], both of
which were funded by the Spanish Ministry of Science and Innovation. The
funder body had no role in the design, analysis, and reporting of the study.
Data availability
The relevant datasets supporting the conclusions of this article are included
within the article or in the supplementary materials.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 18 April 2023 / Accepted: 30 October 2023
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