Comparative genomic characterization of multidrug-resistant Citrobacter spp. strains in Fennec fox imported to China

Background To investigate the antimicrobial profiles and genomic characteristics of MDR-Citrobacter spp. strains isolated from Fennec fox imported from Sudan to China. Methods Four Citrobacter spp. strains were isolated from stool samples. Individual fresh stool samples were collected and subsequently diluted in phosphate buffered saline as described previously. The diluted fecal samples were plated on MacConkey agar supplemented with 1 mg/l cefotaxime and incubated for 20 h at 37 °C. Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (MALDI–TOF–MS) was used for identification. Antimicrobial susceptibility testing was performed using the broth microdilution method. Whole-genome sequencing was performed on an Illumina Novaseq-6000 platform. Acquired antimicrobial resistance genes and plasmid replicons were detected using ResFinder 4.1 and PlasmidFinder 1.3, respectively. Comparative genomic analysis of 277 Citrobacter genomes was also performed. Results Isolate FF141 was identified as Citrobacter cronae while isolate FF371, isolate FF414, and isolate FF423 were identified as Citrobacter braakii. Of these, three C. braakii isolates were further confirmed to be extended-spectrum β-lactamases (ESBL)-producer. All isolates are all multidrug resistance (MDR) with resistance to multiple antimicrobials. Plasmid of pKPC-CAV1321 belong to incompatibility (Inc) group. Comparative genomics analysis of Citrobacter isolates generated a large core-genome. Genetic diversity was observed in our bacterial collection, which clustered into five main clades. Human, environmental and animal Citrobacter isolates were distributed into five clusters. Conclusions To our knowledge, this is the first investigation of MDR-Citrobacter from Fennec Fox. Our phenotypic and genomic data further underscore the threat of increased ESBL prevalence in wildlife and emphasize that increased effort should be committed to monitoring the potentially rapid dissemination of ESBL-producers with one health perspective. Supplementary Information The online version contains supplementary material available at 10.1186/s13099-021-00458-w.

hospital-acquired infections and sometimes even animal infections [3][4][5][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 AmpCproducing 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][14][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 crosstransmissions 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][25][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.

Isolation and identification
In this work, four Citrobacter spp. isolates resistant to cephalosporins were cultivated by selective medium plates. Among these strains, isolate FF141 was identified as Citrobacter cronae while isolate FF371, isolate FF414, and isolate FF423 were identified as Citrobacter braakii (Additional file 1: Figure S1). Of these, three C. braakii isolates were further confirmed to be ESBL-producer (Table 1).

Comparative genomics analysis of Citrobacter isolates
Roary matrix-based gene sequence analysis generated 60,923 total genes and a large core-genome of 1578 gene clusters of 277 whole genomes. The whole-genome phylogeny (Fig. 2) revealed a population structure. Genetic diversity was observed in our bacterial collection, which clustered into five main clades. Interestingly, C. braakii isolates have close relatedness with a clinical isolate (SAMD00112928, C. freundii) from Vietnam and an environmental strain (SAMN11928073, C. freundii), while C. cronae FF141 showed a high similarity with a clinical isolate from Nigeria (SAMN13830004, C. freundii) and a clinical isolate from India (SAMN06660612, C. freundii) (Fig. 2, Additional file 3: Table S2).

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] 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 (bla CMY-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 ESBLproducing 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 bla CTX-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 SAM N120 2490         The year of the isolation is labelled in the outer ring. The source of the strains is presented in the middle ring. The location of the isolates is colored in the inner ring. Isolates identified in this study were colored in red. NA, details regarding the region of the strains is not available. The bar shows 100,000 nucleotide substitution per position 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 ESBLencoding 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.