Genomic Characterization of A Multidrug-resistant Citrobacter freundii Strain ZT01-0079 Co-Producing blaNDM-1 and blaSHV-12 using MiSeq and MinION

Background: The emergence of multi-drug resistant Citrobacter freundii poses daunting challenges to the treatment of clinical infections. The purpose of this study was to characterize the genome of a C. freundii strain with an IncX3 plasmid encoding both the bla NDM-1 and bla SHV-12 genes. Methods: Strain ZT01-0079 was isolated from a clinical urine sample. The Vitek2 system was used for identication and antimicrobial susceptibility testing. The presence of bla NDM-1 was detected by PCR and sequencing. Conjugation experiments and Southern blotting were performed to determine the transferability of the bla NDM-1 - carrying plasmid. Nanopore and Illumina sequencing were performed to better understand the genomic characteristics of the strain. Results: Strain ZT01-0079 was identied as C. freundii, and the coexistence of bla NDM-1 and multiple drug resistance genes was conrmed. Electrophoresis and Southern blotting showed that bla NDM-1 was located on a ~53kb IncX3 plasmid. The NDM-1-encoding plasmid was successfully transferred at a frequency of 1.68×10 −3 . Both bla NDM-1 and bla SHV-12 were located on the self-transferable IncX3 plasmid. Conclusion: The rapid spread of the IncX3 plasmid highlights the importance of continuous monitoring of the prevalence of NDM-1-encoding Enterobacteriaceae. Mutations of existing carbapenem resistance genes will bring formidable challenges to clinical treatment.

The coexistence of NDM and other resistance genes on a single plasmid is being reported with increasing frequency and confers high-level carbapenem resistance, which poses daunting challenges to clinical management [7,11,[14][15][16][17].
Citrobacter freundii, a member of the Enterobacteriaceae family,is a constituent of the commensal intestinal microbiota of animal and humans. However, it can cause diarrhea, sepsis, meningitis, respiratory and urinary tract infections, and can serve as a reservoir of antibiotic resistance genes. In recent years, due to the abuse of antibiotics, C. freundii has acquired increasing resistance to common antibiotics. In addition, bla NDM-1 -positive C. freundii has been reported in numerous countries that include but are not limited to China [2][3][4][5], India, Pakistan, France, Sweden, Australia, UK, USA [7], and South Africa [9]. We report a carbapenem-resistant strain of C. freundii with coexistent bla NDM-1 and bla SHV-12 genes on a transferrable IncX3 plasmid. Antimicrobial susceptibility tests, conjugation experiments, and wholegenome sequencing were performed to study the molecular characteristics of the multi-drug resistant strain.

Bacterial identi cation and isolation
Strain ZT01-0079 was isolated from a clinical urine sample in 2018, in Guangzhou, China. Species identi cation was conducted by using the VITEK2 compact system (Bio Merieux, France) and con rmed by 16S rDNA sequencing. The entire bla NDM and bla SHV genes were ampli ed with primers as described previously [18] and sequenced. All experiments were conducted in accordance with relevant regulations and approved by the Chinese PLA Center for Disease Control and Prevention.

Southern blotting and Conjugation experiment
A DNA fragment of strain ZT01-0079 was prepared by electrophoresis and S1 endonuclease. Subsequent Strain ZT01-0079 and Escherichia coli strain J53 were used as donors and acceptors, respectively. Conjugation experiments were performed by lter mating and broth, and the mixture was then incubated at 37 ℃ for 18h. The transconjugant was selected on a MacConkey agar plate containing meropenem (4 μg/ml) and sodium azide (100 μg/ ml) at 37 ℃ for 12h. PCR ampli cation and sequencing were performed to determine the presence of bla NDM-1 and bla SHV-12 genes in the transconjugant.
Whole genome sequencing and comparative genome analysis.
Genomic DNA was extracted from strain ZT01-0079 using the QIAamp DNA Mini Kit (Qiagen, Inc, USA).
Genomic and plasmid sequences of strain ZT01-0079 have been deposited into GenBank under accessions CP055247 and CP022050.

Discussion
The prevalence of NDM-1-producing bacteria is receiving increasing attention as a threat to global health. Most bla NDM-1 -positive isolates of diverse species of Enterobacteriaceae show high-level resistance to βlactam antibiotics. Concurrently, the bla NDM-1 gene has also spread in many environmental and animal reservoirs, such as sewage, rivers, soil, and many mammals and poultry in Asia and the Middle East [2,9,12,20]. However, reports on C. freundii are still rare. Therefore, we investigated the genomic characteristics of multidrug-resistant C. freundii (strain ZT01-0079) isolated from a clinical urine specimen. This strain has the IncX3 plasmid pZY-NDM1 that co-harbors bla NDM-1 and bla SHV-12 .
Compared with other IncX3 plasmids [21], pZY-NDM1 has a higher transfer rate, which may be an important contributor to the rapid dissemination of the bla NDM-1 gene.
Coexistent bla NDM-1 and other β-lactamase genes in IncX3 plasmids, which have a broad host range, mediate resistance to broad-spectrum antibiotics such as carbapenems and cephalosporins, and can be transferred between Enterobacteriaceae [21]. Notably, there are few reports of such plasmids in C. freundii, which is emerging as an important opportunistic pathogen causing increasingly di cult to treat infections.

Conclusions
In summary, we characterized the genomic basis of multi-drug resistance in C. freundii strain ZT01-0079. MLST revealed that strain ZT01-0079 belongs to ST19. ZT01-0079 carried multiple bla genes, which increased the level of carbapenem resistance. pZY-NDM1carries both the bla NDM-1 and bla SHV-12 genes; is transferable; and may thereby serve as a common vector for the rapid dissemination of carbapenemaseencoding genes. Our ndings further underscore the threat of increased NDM-1 prevalence in Enterobacteriaceae, and emphasize that increased resources and effort should be dedicated to monitor the potentially rapid spread of NDM-1-encoding plasmids.  Table   Table 1 Antibiotic susceptibilities of ZT01-0079 and transconjugants