Background
The worldwide increase and spread of infections caused by multidrug-resistant (MDR) Gram-negative bacteria of human and animal origin is a significant global public health burden in recent decades [
1,
2]. Enterobacteriaceae are common bacteria and usually associated with different types of community- and hospital-acquired infections and sometimes even animal infections [
3‐
6]. Thus, antimicrobial resistance (AMR) in these bacteria has significantly potential impacts on the control of AMR from the perspective of the One Health concept [
7,
8]. Among these organisms, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae are recognized as the most prevalent group of pathogens due to their mobility [
9,
10]. Treatment of infections caused by ESBL- and AmpC-producing Enterobacteriaceae strains is challenging, due to the emergence and spread of carbapenem resistance in ESBL-producing Enterobacteriaceae isolates, which is of particular clinical relevance [
11,
12].
The ESBLs are usually carried by mobile genetic elements, such as a variety of self-transferring plasmids, which can be transferred to other bacteria [
13‐
15]. Thus far, several ESBL types have been identified in Enterobacteriaceae isolates of which the CTX-M, TEM, and SHV β-lactamases are the most prevalent groups [
16,
17]. It is noteworthy that ESBL- and carbapenemase-producing Enterobacteriaceae occurring in animals has become a public-health issue in recent years [
4,
17]. Zoonotic pathogens contributed to the cross-transmissions of ESBL-producing Enterobacteriaceae [
18]. Previously, we also reported the detection of the transmission of AMR across human, animals and environmental compartments in China [
8,
19,
20].
Citrobacter spp. are common Gram-negative bacilli and widely found in water, food, soil, and intestines of animals and humans [
21]. AMR encoding genes are frequently reported in
Citrobacter freundii, and it became a reservoir of antibiotic resistance genes in recent years [
22,
23]. In addition, MDR
C. freundii has been reported in numerous hosts including but are not limited to humans [
24‐
26]. In this work, we identified four MDR
Citrobacter isolates in Fennec fox imported from Sudan to China. Antimicrobial susceptibility tests, conjugation experiments, whole-genome sequencing, and comparative genomic analysis were performed to study the molecular characteristics of these MDR strains.
Discussion
The wide dissemination of MDR Enterobacteriaceae is a global concern with one health perspective [
27]. In clinical settings,
Citrobacter spp. isolates represent up to 6% of all isolated Enterobacteriaceae from clinical specimens [
28]. Members of the genus
Citrobacter are reported to associate with nosocomial infections for a high mortality rate [
29]. The
Citrobacter genus is most closely related to
Escherichia coli and
Salmonella, and is divided into 11 different genomospecies [
30]. There is some information indicating a high prevalence of MDR Enterobacteriaceae in wild animals [
31,
32]. Although previous studies suggest that fur animals are potential reservoirs of AMR [
5,
33], little is known about the antimicrobial patterns, and genomic characteristics of MDR
Citrobacter isolates from wildlife. In the present study, we first described the isolation of MDR-
Citrobacter strains from Fennec fox. We subsequently obtained the antimicrobial resistance profiles and genomic information by AST and WGS. We also identified antimicrobial resistance and virulence-associated genes.
Citrobacter spp. isolates can have chromosomal AmpC β-lactamases, as well as plasmid encoded carbapenemases, which results in ineffective of many antimicrobial agents for treatment [
20,
22,
34,
35].
C. cronae was identified as a new
Citrobacter species from human stool samples very recently [
28], the antimicrobial profiles of
C. cronae are largely unknow. In this study, we first identified an AmpC β-lactamase encoding gene (
blaCMY-98) in
C. cronae, which provides a glimpse of antimicrobial insight into this species. Occurrence of ESBL-producing
C. braakii isolated from animals and food product are occasionally reported [
36]. Our detection of three ESBL-producing
C. braakii isolates from Fennec fox further suggests the risk of zoonotic potential MDR
C. braakii from animals and animal products deservedly garners considerable attention.
Previous investigations found that CTX-M-55 became one of the prevalent ESBL type detected among clinical, animals, and environment in some countries [
13,
14,
37,
38]. Very recently, the occurrences of CTX-M-55-producing
Escherichia coli were also increasingly reported in environment and diverse animal species in Europe [
39,
40]. Our previous work confirmed that CTX-M-55-producing
Escherichia coli was the most prevalent ESBL-producer from Fennec fox [
5]. These findings further strengthened that wildlife may act as potential reservoirs and vectors of CTX-M-55, although some of these isolates carried
blaCTX-M-55 genes on the chromosome.
Interestingly, the diverging clonality of the human, environmental and animal
Citrobacter isolates was confirmed by the fact that strains originating in these three sources distributed into five clusters. It is still not sure whether the ecological and animal strains are highly related to the human strains in terms of genetic phylogeny. The previous investigation highlighted the challenges associated with species designation of
Citrobacter by core genome analysis, particularly in regards to
Citrobacter freundii, which did not constitute a discrete phylogenetic group [
41]. As we noted in our data,
C. cronae and
C. braakii strains were clustered into the same clade, which suggests further accurate taxonomic inquiry is needed to clarify the lineage of
Citrobacter members.
Conclusion
In summary, this investigation involved the first survey of MDR Citrobacter isolates in Fennec fox. We characterized the phenotypic characteristics and genomic basis of MDR Citrobacter strains. Fennec fox may serve as a common vector for the rapid dissemination of ESBL-encoding genes via animal contact and thereby threaten public health. Our findings further underscore the threat of increased ESBL prevalence in Enterobacteriaceae, and improved multisectoral surveillance for ESBL-producing Citrobacter is warranted.
Methods and materials
Bacterial identification and isolation of Citrobacter strains
We collected 168 stool samples of wild Fennec fox imported from North Africa to China [
5]. Stool samples were cultured by MacConkey agar supplemented with 1 mg/l cefotaxime as described previously [
5]. Bacterial identification was conducted by MALDI–TOF MS (Bruker, Bremen, Germany) as described [
15]. Confirmation of ESBL-producing isolations was further performed by a standard double-disk diffusion method as defined by the Clinical and Laboratory Standards Institute (CLSI) (
https://clsi.org/).
Antimicrobial susceptibility testing (AST)
Susceptibility to 16 antibiotics (amikacin, aztreonam, cefpirome, cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, florfenicol, fosfomycin, gentamicin, imipenem, meropenem, piperacillin–tazobactam, polymyxin E, tigecycline, tobramycin, and trimethoprim–sulfamethoxazole) for four MDR Citrobacter isolates were evaluated. The MICs were determined via an agar dilution method for all antibiotics except for colistin and tigecycline, for which a broth microdilution method was used according to the CLSI standards. E. coli ATCC 25922 was used as control.
Whole-genome sequencing (WGS) and bioinformatics analysis
Genomic DNA was extracted from four MDR
Citrobacter isolates using the Qiagen Blood/Tissue kit (Qiagen, Hilden, Germany) [
6]. The sequencing library was prepared by using Illumina Nextera XT kit (Illumina, San Diego, CA, USA) and sequenced using the Illumina NovaSeq 6000-PE150 platform (Illumina). Paired reads were then assembled into scaffolds using Velvet version 1.2.10 [
42]. Acquired antimicrobial resistance genes and plasmid replicons were performed using the CGE server (
http://www.genomicepidemiology.org). Antibiotics Resistance Genes (ARGs) were identified using the ResFinder 4.1 database [
43]. Genotyping was performed to query the seven domesticated genes (
aspC, clpX, fadD, mdh, arcA, dnaG, and lysP) via the MLST database (
https://pubmlst.org/organisms/citrobacter-spp). We further created a core genome-based phylogenetic tree using 4
Citrobacter genomes sequenced in this study and 272 randomly selected publicly available
Citrobacter genomes (Additional file
1: Table S1). The isolate collection includes strains from clinical (n = 159) and the environment (n = 133) sources that were widely distributed over time and geographical locations.
Citrobacter genomes were annotated using Prokka [
44] and RAST tool [
45]. The core genes were identified using Prokka [
44] and maximum likelihood-based phylogenetic reconstruction was performed with Roary [
46]. Phylogenetic tree visualizations were generated by using iTOL (
https://itol.embl.de/).
Plasmid characterization
The transferability of plasmids carrying MDR encoding genes was determined by filter mating as described previously [
19]. Animal isolates and
Escherichia coli J53 were used as donors and acceptors, respectively. The animal isolates and J53 strains were mixed in (LB) broth at a ratio of 1:3 and incubated at 37 °C for 18 h. Transduction and binding were selected on MacConkey agar plates containing cefotaxime (2 μg/ml) and sodium azide (150 μg/ml) for 12 h. Susceptibility test was performed to determine the horizontal transferability of drug resistance, and the corresponding transduction conjugate was confirmed by PCR amplification and pulsed field gel electrophoresis (PFGE) (Additional file
2: Table S2).
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