RESEARCH ARTICLE

The first isolation of Clostridium difficile RT078/

ST11 from pigs in China
Li-Juan Zhang1,2, Ling Yang1,2, Xi-Xi Gu1,2, Pin-Xian Chen1,2, Jia-Li Fu1,2, Hong-
Xia Jiang ID1,2*
1 National Risk Assessment laboratory for antimicrobial resistance of animal original bacteria, College of
Veterinary Medicine, South China Agricultural University (SCAU), Guangzhou, China, 2 Guangdong
Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, College of
Veterinary Medicine, South China Agricultural University (SCAU), Guangzhou, China

* [email protected]
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a1111111111 Abstract
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We investigated the molecular characteristics and antimicrobial susceptibility of Clostridium
difficile isolated from animals in China. We obtained 538 rectal swabs from pigs, chickens
and ducks in 5 provinces during 2015 and 2016. C. difficile isolates were characterized by
OPEN ACCESS
detection of toxin genes, multilocus sequence typing and ribotyping. And antimicrobial sus-
ceptibility testing was performed using the agar dilution method. Out of 538 samples, 44
Citation: Zhang L-J, Yang L, Gu X-X, Chen P-X, Fu
J-L, Jiang H-X (2019) The first isolation of (8.2%) were C. difficile positive with high prevalence in pigs (n = 31). Among these, 39
Clostridium difficile RT078/ST11 from pigs in (88.6%) were toxigenic including 14 (31.8%) that were A+B+CDT+ and 13 (29.5%) A+B+.
China. PLoS ONE 14(2): e0212965. https://doi.org/ The remaining 12 (27.3%) were A-B+. We identified 7 ST types and 6 PCR ribotypes. The
10.1371/journal.pone.0212965
most predominant type was ST11/RT078 with toxin profile A+B+CDT+ and all were isolated
Editor: Pradeep Dudeja, University of Illinois at from piglets with diarrhea. ST109 isolates possessed two different toxigenic profiles
Chicago, UNITED STATES
(A-B-CDT- and A-B+CDT-) and although it was not the most prevalent sequence type, but it
Received: November 2, 2018 was widely distributed between chickens, ducks and pigs in the 5 provinces. All C. difficile
Accepted: February 12, 2019 isolates were fully susceptible to vancomycin, metronidazole, fidaxomicin, amoxicillin/clavu-
Published: February 26, 2019 lanate and meropenem but retained resistance to 4 or 5 of the remaining antibiotics, espe-
cially cefotaxime, tetracycline, ciprofloxacin, cefoxitin. The RT078/ST11 isolates were
Copyright: © 2019 Zhang et al. This is an open
access article distributed under the terms of the simultaneously resistant to cefotaxime, tetracycline, cefoxitin, ciprofloxacin and imipenem.
Creative Commons Attribution License, which This is the first report of the molecular epidemiology of C. difficile isolated from food animals
permits unrestricted use, distribution, and in China. We identified the epidemic strain RT078/ST11 as the predominate isolate among
reproduction in any medium, provided the original
the animals we screened in our study.
author and source are credited.

Data Availability Statement: All relevant data are

within the paper.

Funding: This work was supported by the National

Natural Science Foundation of China (31272602) Introduction
(H-XJ) and Graduate Student Oversea Study
Program of South China Agriculture University
Clostridium difficile is a strictly anaerobic, spore-forming Gram-positive bacterium that colo-
(2017LHPY029) (LY). The funders had no role in nizes the gastrointestinal tract of humans and animals and cause disease [1]. C. difficile patho-
study design, data collection and analysis, decision genesis is associated with the production of two enterotoxins (A and B) encoded by tcdA and
to publish, or preparation of the manuscript. tcdB on its pathogenicity locus. Some strains also produce a third toxin called binary toxin
Competing interests: The authors have declared (cytolethal distending toxin, CDT) that is associated with increased disease severity and
that no competing interests exist. 30-day mortality [2].

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 Clostridium difficile RT078 in pigs in China

C. difficile has emerged as the most common infectious cause of antibiotic-associated diar-
rhea and healthcare infections in developed countries. This is due to the emergence of hyper-
virulent strain, restriction endonuclease analysis type BI, North American pulsed field type 1
and PCR ribotype 027 (BI/NAP1/027) [1]. This strain produces toxins A, B and binary toxin
CDT (A+B+CDT+) and possesses increased fluoroquinolone resistance and has been responsi-
ble for C. difficile infections (CDI) and outbreaks in North America, Canada and Europe since
2000 [3–6]. Currently, RT027 remains prevalent in North America and Europe,however,
another PCR ribotype 078 is emerging as a significant human pathogen [7, 8].
The RT078 strain also produces toxins A, B and CDT (A+B+CDT+). Its importance as a
human pathogen was first reported in the Netherlands and the incidence of infections caused
by this strain increased from 3% to 13% between 2005 and 2008 [8]. At the same time, a similar
increase with occasional outbreaks were recorded throughout Europe [4]. Recently C. difficile
RT078 has increased to 4.4% of all C. difficile clinical isolates in North America [9]. Compared
with RT027, this strain places lower-age populations at higher risk and is more frequently
community associated than other strains [8]. Strain RT078 has been frequently isolated from
livestock in Europe, United States, Canada, Australia, Japan and Taiwan suggesting that ani-
mals, particularly livestock, might be the reservoir for human CDI [10–12]. In addition, based
on clonal relatedness of isolates derived from piglets in Europe, Taiwan and Japan, possible
route of C. difficile RT078 transmission through piglets trading was suggested [13].
The exact evolutionary and epidemiological relationships between the C. difficile RT078
strains of humans and animals are still unknown due to the lack of discriminatory power of
the current strain typing methods. Standard genotyping methods already highlight the genetic
similarity between human and animal C. difficile RT078 strains and project an increase in zoo-
notic transmission [14–16]. More recently, whole-genome sequencing and core genome sin-
gle-nucleotide polymorphism typing further confirmed this genetic overlap. This study
indicated that asymptomatic farmers and their pigs can be colonized with clonal C. difficile
RT078 [17].
Antibiotic resistance plays an important role in the spread of C. difficile strains and this is
associated with the appearance of novel PCR ribotypes [5, 18]. The spread of the epidemic C.
difficile RT027 and RT078 is mainly due to their fluoroquinolone resistance [5, 18, 19]. Previ-
ous studies have reported that the most common mechanism of fluoroquinolone resistance
among C. difficile isolates are specific mutations in gyrA and gyrB [20, 21]. Recently, an
increasing number of CDI studies in China have identified human-derived RT027 and RT078
strains [22–25]. Since no animal-related C. difficile strains have been reported, it is unclear
whether these clones exist in animals in China, and the antimicrobial susceptibility of animal-
derived strains. Therefore, we isolated C. difficile from fecal samples collected from different
food animals in China to study the molecular epidemiology and antimicrobial resistance phe-
notypes of C. difficile, and further investigated the fluoroquinolone resistant determinants.

Materials and methods

Ethics statement
This study protocol was approved by the South China Agriculture University Animal ethics
committee. The strains were isolated from cloacal swabs of pigs or chickens and ducks and the
owners of the animals gave permission for their animals to be used in this study.

Sample collection and C. difficile culture

During 2015 and 2016, we collected 538 stool samples from food animals from 5 Chinese prov-
inces,which including 398 pig samples, 121 chicken samples and 19 duck rectal swabs. The

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 Clostridium difficile RT078 in pigs in China

samples were taken from five to ten animals per litter, and partial samples from all three reser-
voirs were sampled from the overlapping geographical locations.
Pig samples were taken from 4 different larger farms (total animal number >3000) and 2
small farms (total animal number<1000). Two of the 4 large farms were located in two different
regions (Weifang and Qingdao) in Shandong, and the remaining 2 in Hubei and Guangdong.
The 2 small farms were located in Henan and Jiangsu respectively. Those samples including 164
from nursery pigs (28–40 days old) in Jiangsu (n = 20) and Hubei (n = 144), 105 from piglets
(14–20 days old) with diarrhea in Guangdong, 17 from sows (about 200 days old) in Henan and
112 from pregnant sows (>40 weeks old) from Weifang (n = 62) and Qingdao (n = 50).
We also collected 46, 47 and 28 adult chicken (about 28–40 days old) samples from 3 large-
scale farms in Hubei, Shandong and Jiangsu, respectively. They were all healthy and asymp-
tomatic. In addition, 19 rectal swabs from healthy 45 days old ducks were randomly collected
from one farm in Shandong. All swabs were cryopreserved after collection and delivered rap-
idly to the laboratory.
Stool samples were incubated in C. difficile moxalactam-norfloxacin (CDMN, Oxoid, Cam-
bridge, UK) broth supplemented with 0.1% sodium taurocholate at 37˚C for 7 days. The culture
samples were treated with alcohol to a final concentration of 75% at room temperature for 60 min
before the anaerobic isolation of C. difficile. Then samples were centrifuged and the supernatant
was discarded and the sediment was incubated in C. difficile moxalactam-norfloxacin selective
medium (CDMN, Oxoid, Thermo-Fisher, Pittsburg, PA USA) at 37˚C for 48 h. Presumptive C.
difficile colonies were identified by matrix-assisted laser desorption/ionization–time of flight
(MALDI-TOF) mass spectrometry (Shimadzu-Biotech) using instructions provided by the manu-
facturer. Strains were stored in brain heart infusion broth containing 20% glycerol at -80˚C.

Toxin gene profiling and molecular typing

Genomic DNA was isolated from purified colonies of C. difficile grown on the selective
medium CDMN containing cysteine hydrochloride, norfloxacin and moxalactam for 48h
37˚C under anaerobic conditions. Colonies were suspended in 300 μL water and DNA was
extracted using a TIANamp Bacterial DNA Kit (Tiangen Biotech, Beijing, China) according to
the manufacturer’s instructions. DNA samples were stored at -20˚C.
PCR amplification of tcdA, B, cdtA/B and fragments of gyrA and gyrB gene were carried out
as previously described [21, 26–28]. PCR products were analyzed by electrophoresis through
1.5% agarose. Fragments of gyrA and gyrB gene were directly sequenced, and the results were
compared with the genome of C. difficile strain 630 (Accession number: NC_009089.1).
Multilocus sequence typing (MLST) was performed on all isolates as described previously
using the following gene targets: adk, atpA, dxr, glyA, recA, soda and tpi [29]. All amplified
products were commercially sequenced (Invitrogen, Shanghai China) and DNA sequences
were submitted to the MLST database (http://pubmlst.org/clostridium difficile) to obtain the
sequence type (ST).
PCR ribotyping (PR) was performed based on a previously published protocol [30]. The
PCR products were separated by electrophoresis in 3% agarose gels for 2 h at 100 V and the PR
profiles were analyzed using a CHEF-MAPPER System (Bio-Rad Laboratories, Hercules, CA,
USA) to construct a dendrogram. C. difficile RT078 strain was donated by Professor Haihui
Huang of Fudan University.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing of all isolates was performed using the agar dilution
method according to Clinical and Laboratory Standards Institute guidelines (CLSI) [31].

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 Clostridium difficile RT078 in pigs in China

Bacteroides fragilis ATCC25285 and C. difficile ATCC700057 were used as quality control sam-
ples. The following 17 antibiotics were tested: ceftiofur (CTF), ciprofloxacin (CIP), cefoxitin
(CXT), fidaxomicin (FDX), metronidazole (MTZ), vancomycin (VAN), clindamycin (CLI),
tetracycline (TET), imipenem (IPM), meropenem (MEM), cefotaxime (CTX), erythromycin
(ERY), ampicillin (AMP), chloramphenicol (CHL), amoxicillin-clavulanic acid (AMC) and
moxifloxacin (MXF), fosfomycin (FOS). Interpretation of antimicrobial susceptibility was
based on CLSI guidelines [31] and the European Committee on Antimicrobial Susceptibility
Testing (EUCAST) [32]. For antimicrobial agents where no standard breakpoints were avail-
able from CLSI or EUCAST, resistance was considered as follows: ciprofloxacin, �8 mg/L;
erythromycin, �128mg/L [33].

Statistical analysis
MIC50/90 were carried out using SPSS software (Version 18.0).

Results
Strain isolation and toxin gene identification
We isolated 31 C. difficile strains from 398 pig samples at a rate of 7.8% and these strains pos-
sessed four different toxin type combinations. These included 19 from diarrhea piglets
(Guangdong) and included 14 A+B+CDT+ and 5 nontoxigenic strains. The 9 isolates from
pregnant sows (Shandong) were all (A+B+). The 2 strains from Hubei nursery pigs and the sin-
gle Henan sow isolate were A-B+.
The chicken isolates included 9 (7.4%) positive for C. difficile including 6 from Hubei, 2
from Shandong and 1 from Jiangsu. The 4 isolates from Hubei and the single from Jiangsu
were all A-B+ and the other 2 strains from Hubei along with 2 isolates from Shandong were
A+B+. We also isolated 4 strains of C. difficile from 19 duck samples and all were A-B+
(Table 1).

Multilocus sequence typing (MLST) and PCR ribotypes

The 44 total C. difficile isolates possessed 7 MLST genotypes with sequence type (ST) 11 (14/
44) as the most frequent followed by ST109 and ST35 with 9 members each. There were no
new ST types represented in this group. However, ST11, ST35 and ST238 were only present in
pig samples, ST3 were all derived from chicken samples and ST240 was present only in ducks.
ST48 was present both in pigs and in chickens and interestingly, ST109 was present in all three
animal groups (Table 1).
We also identified 6 different PCR ribotypes. However, due to a lack of strains for standard-
ization, these were given the sequential names GZ1, GZ2, GZ3, GZ4, GZ5 and GZ6. All GZ2
strains were confirmed to be RT078 and all were isolated from piglets with diarrhea in Guang-
dong (Fig 1).

Antimicrobial susceptibility and gyrA, and gyrB mutations

All our C. difficile isolates were fully susceptible to vancomycin, metronidazole, fidaxomicin,
amoxicillin/clavulanate and meropenem, but showed different degrees of resistance to other
antibiotics tested in this study (Table 2). Most of the strains were resistant to three or four anti-
biotics and the most frequent multidrug profile was CIP/CXT/TET/CTX. The strain of the
predominant type RT078 (GZ2) we found in this study was resistant to IMP and possessed the
most prevalent multi-resistance profile (Table 1).

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 Clostridium difficile RT078 in pigs in China

Table 1. Genotypes and antibiotic resistance of C. difficile strains isolated from animal feces.
Toxin Total No. of Multidrug-Resistant Patterns Amino acid ST6¼ RT § Animal
profile Strains substitutions source
Quadruple Drug Quintuple Drug Sextuple Drug Resistance GyrA GyrB
Resistance Resistance
A-, B-, 5 CIP/CXT/TET/ CIP/CXT/TET/CTX/ CIP/CXT/TET/CTX/ERY/ Thr87-Ile Ser366-Ala 109 GZ1 Pig
CTX ERY MXF
1 CIP/CXT/TET/CTX/ERY/ 238 GZ1 pig
MXF
A-, B+, CXT/CLI/CTX/ CIP/CXT/TET/CTX/ERY/ 48 GZ6 Pig
ERY CLI Chicken
7 CIP/CXT/TET/ 240 GZ5 Duck
CTX
4 CIP/CXT/TET/CTX/ Ser366-Ala 109 GZ1 Chicken
MXF Pig Duck
A+, B+, 4 CIP/CXT/TET/ Thr82-Ile 3 GZ4 Chicken
CTX
9 CIP/CXT/TET/ CIP/CXT/TET/CTX/ CIP/CXT/TET/CTX/IPM/ Thr82-Ile 35 GZ3 Pig
CTX IPM ERY
A+,B+, 14 CIP/CXT/TET/ CIP/CXT/TET/CTX/ Ser366-Val 11 GZ2 Pig
CDT+ CTX IPM Ser416-Ala (RT078)

6¼Sequence Type.
§ Ribotype.
CIP, ciprofloxacin; CXT, cefoxitin; TET, tetracycline; CTX, cefotaxime; ERY, erythromycin; CLI, clindamycin; MXF, moxifloxacin; IPM, imipenem.

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We also observed high rates of fluoroquinolone resistance. The resistance rates to CIP were
93% (41/44) and 29% of these were also resistant to MXF. We also identified gyrA and gyrB
amplicons for all isolates and 73% (32/44) of these carried mutations in either GyrA or GyrB.
The 5 A-B- pig isolates were resistant to both MXF (MIC range 16 to 32 mg/L) and CIP (MIC
range 32 to 64 mg/L) and both possessed GyrA (T87I) and GyrB (S366A) mutations.
The 8 A+B+ strains from different sources were resistant to MXF (MIC range 16 to 32 mg/
L) and CIP (MIC range 16 to 64 mg/L) and possessed T82I in the GyrA. The 14 A+B+CDT+
strains isolated from piglets were resistant only to ciprofloxacin (MIC = 16 mg/L) and pos-
sessed two GyrB mutations (S366V, S416A) and none in GyrA. The 5 A-B+ strains from differ-
ent origins were only resistant to CIP (MIC range 16 to 64 mg/L) and possessed a single GyrB
(S366A) mutation (Table 1).

Discussion
In the present study, we isolated 31 C. difficile strains from pigs at a rate of 7.8%, which was sig-
nificantly lower than previously reported rates of 36% ~ 50% [34, 35]. This difference can be
accounted for by the animal ages since the prevalence of C. difficile in pigs decreases with age
[36]. Our study samples were derived from nursery pigs and sows with only a few samples
from piglets. Interestingly, strain RT078 (GZ2/ST11) predominated and these samples were all
obtained from piglets. In addition, we found 9 C. difficile in chickens and 4 C. difficile strains
from ducks with isolate rates at 7.4% (9/121) and 21% (4/19) respectively. Since China is the
largest producer of chickens and ducks for food, the isolation of C. difficile from these animals
indicates a potential public health threat.
Among the 44 C. difficile strains, 39 were toxigenic and the most common toxin genes pro-
file was A+B+CDT+. These accounted for 36% (14/44) of the total and all were identified in iso-
lates of ST11/RT078. It is remarkable that the predominant strain we found was the epidemic

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 Clostridium difficile RT078 in pigs in China

Fig 1. Cluster analyses based on PCR ribotyping of 44 C. difficile isolates. a Weifang, b Qingdao.
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strain RT078, which were all isolated from Guangdong. Strain RT027 and RT078 strains have
acquired unique mechanisms to metabolize low concentrations of the disaccharide trehalose,
and this ability correlates with disease severity [37]. The addition of trehalose to animal feeds
in China is not a common practice and was not used on the farms we examined in this study.

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 Clostridium difficile RT078 in pigs in China

Table 2. Minimum inhibitory concentrations (MICs) for 17 antimicrobial agents against 44 C. difficile animal isolates.
Antimicrobial Resistance breakpoint MIC50 MIC90 Range Resistance
agent (mg/L) (mg/L) (mg/L) (%)
Vancomycin �4b 0.5 0.5 0.03–1 0
Fosfomycin - 8 16 2–128 -
Metronidazole �2b 0.06 0.25 0.03–0.25 0
Fidaxomicin - 0.125 0.125 0.03–0.25 -
Clindamycin �8a 0.015 0.25 0.015->256 13.6
Amoxicillin-clavulanic acid �16a 4 8 0.125–128 2.3
Chloramphenicol �32a 4 8 0.06–32 4.5
Moxifloxacin �4b 2 32 0.125–32 29.5
Cefoxitin �64a 64 128 16–128 97.7
Imipenem �16a 8 16 0.06–64 36.3
Erythromycin �128c 64 128 1->512 45.5
Ciprofloxacin �8c 16 64 <0.015–64 93.2
Meropenem �16a 2 2 0.03–2 0
Ampicillin �2a 1 2 0.5–4 16
Tetracycline �16a 16 64 0.015–128 77.3
Cefotaxime 64a 64 64 0.03->512 95.5
Ceftiofur - 64 128 0.015->512 -

MIC50/90, minimum inhibitory concentration for 50% and 90% of the isolates, respectively.
a
MIC breakpoints for C. difficile recommended by the Clinical and Laboratory Standards Institute [31].
b
MIC breakpoint was based on the recommendation by the European Committee on Antimicrobial Susceptibility Testing [32].
c
MIC breakpoints were calculated as previously reported [33].

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Therefore, it is unlikely that the predominance of the RT078 strain in Guangdong is related to
trehalose.
In humans, RT078 is one of the ten most frequently identified ribotypes and accounts for
4% to 8% of C. difficile clinical isolates in North America and Europe [4, 9]. In China, two
reports have identified RT078 strains isolated from environmental surfaces and clinical
patients in Zhejiang and Beijing [23, 25], our study is the first to report of RT078 isolation
from piglets. C. difficile RT078 exists in a clonal population that often moves between livestock
and human hosts independent of geographic barriers [38]. Although RT078 isolates have not
been reported from humans in Guangdong, the identification of RT078 from piglets suggests a
potential for zoonotic CDI risks in China in the near future.
All RT078 strains obtained from this study were fully susceptible to VAN, MTZ, FDX, CLI,
AMC, CHL, MEN, ERY, AMP and CTF, but co-resistant to CXT, TET, CIP, CTX and IPM.
Swine RT078 isolates reported as resistant to MXF showed the same GyrA mutation, T82I [10,
39]. However, all RT078 isolates in our study were susceptible to MXF and possessed two GyrB
mutations (S366V, S416A). Interestingly, we found a relatively high resistance rate of 93% (13/
14) to imipenem among the RT078 strains, while the overall IMP resistance rate of C. difficile
isolated from Chinese medical clinics is very low [40]. A comparison of human and piglet C.
difficile RT078 strains show that imipenem was the only difference in the antimicrobial resis-
tance spectrum between human and pig isolates, with resistant rates at 29 and 50%, respec-
tively [16]. This was unexpected since imipenem is not used in the swine husbandry.
Another important genotype was the A-B+ strain. We found that 31% (12/39) of the A-B+
strains were obtained from three animal types and distributed over five provinces. The A-B+
strain is the most prevalent strain obtained from clinics in China and it may also be the most

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 Clostridium difficile RT078 in pigs in China

prevalent strain from food animals in China [41, 42]. The strains we identified contained 3 dif-
ferent ST types (ST48, ST240 and ST109). It is worth noting that ST109 was not the dominant
ST type, but possessed the widest geographic and animal host distribution. This suggests clonal
transmission between different animals among different regions of China. We also identified 5
nontoxigenic strains belonging to ST109. Compared with the toxigenic ST109 isolates, these
nontoxigenic ST109 strain were resistant to MXF and CIP and possessed both GyrA (T87I)
and GyrB (S366A) mutations. In contrast, the toxigenic strains that were resistant to moxiflox-
acin and ciprofloxacin possessed single GyrA (T82I) mutations.
The remaining toxigenic strains were A+B+ strains that accounted for 33% (13/39) and pos-
sessed two STs; ST3 (4/13) and ST35 (9/13). The ST3 strains were isolated from chicken stools
while ST35 strains were from pigs. All were CIP resistant and possessed the GyrA mutation
T82I. It is worth noting that ST3 and ST35 are also common in human C. difficile isolates in
China and ST3 was the first dominant strain [22]. This indicates that animals may be potential
reservoirs of C. difficile.
In addition, we tested all C. difficile isolates for their minimal inhibitory concentration
against 17 antimicrobial agents and the results showed that they were serious resistance to
CIP, CTX, CXT and TET, with resistance rate at 93.2%, 95.5%, 97.9% and 77.3% respectively.
Data from 30 studies published from 2012 to 2015 indicate that C. difficile clinical isolates are
very commonly resistant to CLI, CXT, CIP, with resistance rate at 55%, 79%, and 99% [18].
The rate of antibiotic resistance varies widely among studies and may depend on geographic
regions and local or national antibiotic policies, as well as the source of the sample (animal and
human).
The high resistance rate of CIP (a second-generation FQ) in C. difficile was commonly
observed in both this and other studies [18, 22].The fluoroquinolone resistance in Gram-nega-
tive bacteria is primarily the result of mutations in the chromosomal gene encoding the quino-
lone targets gyrA, gyrB, parC and parE. Single mutations in the gyrA gene confers low level
quinolone resistance while high-level fluoroquinolone resistance from gyrA mutations often
requires an additional single or double mutation in parB or parC [43, 44]. However, decreased
susceptibility to fluoroquinolones in C. difficile is associated with the occurrence of a gyrA or
gyrB mutation [10, 21]. Interestingly, human clinical isolates of C. difficile often exhibit Asp to
Asn or Val mutations in 426 of GyrB [40]. Our research found that the primary mutations in
GyrB is Ser to Ala. The C. difficile mutation in GyrB from Ser to Ala has also been reported in
human clinical isolates from different countries [18, 20, 40]. Previous research indicated that
the substitutions found in GyrB characteristic of certain C. difficile types and countries [20]. In
addition, it is noteworthy that we found that A-B- strains and A+B+ strains that showed the
same quinolone resistance phenotypes (both resistant to CIP and MXF at similar levels), but
they possessed different gyrA and gyrB mutations. Moreover, the A-B+ and A+B+CDT+ strains
also carried different mutations in gyrA or gyrB, even though they possessed similar CIP resis-
tance phenotypes (MIC range 16 to 64 mg/L). This suggests that the gyrA and gyrB mutation
sites in C. difficile are not only related to fluoroquinolone resistance but also to toxin produc-
tion ability.

Conclusions
The present study is the first report of hypervirulent strain RT078/ST11 strain from piglets in
China. This strain was multiply resistant to CXT/TET/CIP/CTX/IPM. Our results indicated
that ST109 was the most widely distributed type of C. difficile from food animals in China.
These ST isolates were obtained from different animals and provinces and possessed different
resistance phenotypes. This study provides an important baseline for ongoing long-term

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 Clostridium difficile RT078 in pigs in China

surveillance of antimicrobial resistance and prospective tracking of prominent and emerging

strain types in China.

Acknowledgments
We are grateful to Professor Haihui Huang of Fudan University for providing the C. difficile
RT078 strain.

Author Contributions
Data curation: Hong-Xia Jiang.
Formal analysis: Li-Juan Zhang, Hong-Xia Jiang.
Investigation: Xi-Xi Gu.
Methodology: Li-Juan Zhang, Xi-Xi Gu, Pin-Xian Chen, Jia-Li Fu.
Writing – original draft: Li-Juan Zhang.
Writing – review & editing: Ling Yang, Hong-Xia Jiang.

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