Characterization of Plasmid Co-Harboring NDM-1 and SHV-12 from a Multidrug-Resistant Citrobacter freundii Strain ZT01-0079 in China.

BACKGROUND: The emergence of multidrug-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 identification 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 identified as C. freundii, and the coexistence of bla NDM-1 and multiple drug resistance genes was confirmed. 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 the bla NDM-1 and bla SHV-12 genes 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.

Background

New Delhi metallo-β-lactamase-1 (NDM-1) is an enzyme capable of hydrolyzing most β-lactam antibiotics. Since its first detection in 2009 in Klebsiella pneumoniae,1 21 NDM variants have been reported from various Enterobacteriaceae species.2–10 The increasing prevalence of NDM-producing bacteria is due primarily to intra- and inter-species exchanges of a variety of self-transferring plasmids. 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

Citrobacter freundii, a member of the Enterobacteriaceae family, is a constituent of the commensal intestinal microbiota of animal and humans.12 However, it can cause diarrhea, sepsis,13 meningitis,14 respiratory15 and urinary tract,16 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.17 In addition, blaNDM-1-positive C. freundii has been reported in numerous countries that including but are not limited to China,2–5 India,6,7 Japan,8 Denmark9 and South Africa.10 We report a carbapenem-resistant strain of C. freundii with coexistent blaNDM-1 and blaSHV-12 genes on a transferrable IncX3 plasmid. Antimicrobial susceptibility tests, conjugation experiments, and whole-genome sequencing were performed to study the molecular characteristics of the multidrug-resistant strain.

Methods

Bacterial Identification and Isolation

Strain ZT01-0079 was isolated from a urine sample of a patient with dysuria in 2018, in Guangzhou, China. Species identification was conducted by using the VITEK2 compact system (BioMerieux, France) and confirmed by 16S rDNA sequencing. The blaNDM gene was detected by PCR amplification as previously described.18 The isolate was obtained by conventional collection, and verbal consent was obtained as no personal information is included. All experiments were conducted in accordance with relevant regulations and approved by the Chinese PLA Center for Disease Control and Prevention.

Susceptibility Testing

The VITEK2 system (BioMérieux, France) with AST-GN09 card (bioMérieux) was used for minimum inhibitory concentrations (MICs) determinations of strain ZT01-0079 and transconjugants. The E-test method was used to determine the MIC value of meropenem. Results were interpreted in accordance with the Clinical and Laboratory Standards Institute (CLSI)19 guidelines. The following agents were tested: amikacin, aztreonam, nitrofurantoin, ciprofloxacin, piperacillin, gentamicin, cefepime, ceftriaxone, ceftazidime, tobramycin, imipenem, levofloxacin and sulfamethoxazole/trimethoprim.

S1-PFGE, Southern Blotting and Conjugation Experiment

The genomic DNA of strain ZT01-0079 was prepared in an agarose plug with S1 endonuclease (Takara, Dalian, China). PFGE separates DNA fragments by the CHEF-DR III system (Bio-Rad, Hercules, USA). PFGE runs for 15 h under the conditions of a gradient of 6.0 V/cm, a pulse time of 0.20–26.0 s and an angle of 120°. The plasmid DNA was transferred to a positively charged nylon membrane (Solabio, China) and hybridized with a digoxigenin-labeled blaNDM-1 specific probe.

Strain ZT01-0079 and Escherichia coli strain J53 were used as donors and recipients, respectively. The ZT01-0079 and J53 strains were mixed in (LB) broth at a ratio of 1:3 and incubated at 37°C for 18 h. Transconjugants were selected on MacConkey agar plates containing meropenem (4 μg/mL) and sodium azide (150 μg/mL) for 12 h. A susceptibility test was performed to determine the horizontal transferability of drug resistance, and the corresponding transconjugants were confirmed by PCR and sequencing of the NDM gene and 16s rRNA gene, S1-PFGE and southern blotting. The frequency of conjugation was calculated as the ratio of transconjugants to recipient cells.

Whole Genome Sequencing and Comparative Genome Analysis

Genomic DNA was extracted from strain ZT01-0079 using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Whole-genome sequencing was performed using the Illumina HiSeq 2500 sequencer in Novogene (Beijing, China), with a coverage of 109X. The nanopore sequencing library was prepared using the rapid sequencing kit SQK-RAD004 (Oxford Nanotechnology, UK). The mixed assembly of Illumina and Nanopore (Oxford Nanotechnology, UK) reads was performed using Unicycler20 (v0.4.7). PlasmidFinder21 () was used to determine the plasmid replicon type. Genotyping was performed to query the seven domesticated genes (aspC, clpX, fadD, mdh, arcA, dnaG and lysP) via the multi-locus sequence typing MLST web service22 ().

Nucleotide Sequence Accession Numbers23

The complete sequence of the ZT01-0079 strain and plasmids pZY-1, pZY-2 and pZY-NDM1 have been deposited in GenBank with accession numbers CP055247, CP055248, CP055249 and CP022050.

Results

Microbiological Features of Strain ZT01-0079

Strain ZT01-0079 was identified as C. freundii. Susceptibility testing showed that C. freundii ZT01-0079 exhibited resistance to aztreonam, nitrofurantoin, ciprofloxacin, piperacillin, cefepime, ceftriaxone, ceftazidime, tobramycin, levofloxacin, and imipenem (Table 1). S1-PFGE revealed that C. freundii ZT01-0079 contained three plasmids (~143kb, ~89kb and ~53kb) (Figure 1). Conjugation and southern blotting showed that the blaNDM-1 gene was located on the ~53kb plasmid and only the NDM-1-carrying plasmid was successfully transferred to J53 at a frequency of 1.69×10−3 events per donor cell. Transconjugants acquired resistance to aztreonam, piperacillin, cefepime, ceftriaxone, ceftazidime, and imipenem (Table 1). The E-test showed that strain ZT01-0079 (MIC = 8) and transconjugants (MIC = 8) are both resistant to meropenem. In silico, MLST found that C. freundii ZT01-0079 belongs to sequence type 19 (ST19). In addition to blaNDM-1, C. freundii ZT01-0079 carried multiple resistance genes including blaSHV-12, blaCTX-M-55, blaCMY-152, aph (3ʹ’)-Ib, aph (6)-Id, blaTEM-1B, sul2, floR, tet(A) and aac(3)-IId. Both the 89kb plasmid (named pZY-1) and the 143kb plasmid (named pZY-2) carried resistance genes including aph(3ʹ’)-Ib, aph(6)-Id (aminoglycoside resistance) and sul2 (sulfonamide resistance). pZY-1 also carried the blaCTX-M-55 (fluoroquinolone resistance), blaTEM-1B (cephalosporin resistance), floR (florfenicol resistance), tet(A) (tigecycline resistance) genes. pZY-2 also carried the aac(3)-IId (aminoglycoside resistance) gene, while the 53kb plasmid (named pZY-NDM1) carried the blaNDM-1 and blaSHV-12 genes (carbapenem resistance).

Table 1

Antibiotic Susceptibilities of C. freundii ZT01-0079, Transconjugant and E. coli J53 (Recipient)

Antimicrobial	MIC (μg/mL)
	ZT01-0079	Transconjudant	Recipients (E.coli J53)
Amikacin	8	≤2	≤2
Aztreonam	≥64	16	≤1
Nitrofurantoin	128	≤16	≤16
Ciprofloxacin	≥4	≤0.25	≤0.25
Piperacillin	≥128	64	≤4
Gentamicin	≥16	≤1	≤1
Cefepime	≥64	8	≤4
Ceftriaxone	≥64	≥64	≤4
Ceftazidime	≥64	≥64	≤1
Tobramycin	≥16	≤1	≤1
Imipenem	≥16	≥16	≤1
Levofloxacin	≥8	≤0.25	≤0.25
Sulfamethoxazole-trimethoprim	≤20	≤20	≤20

Figure 1

S1-PFGE pattern for strain ZT01-0079 and Southern blot analysis of blaNDM-1 genes. Lane marker, Salmonella serotype Braenderup strain H9812 as a reference size standard; Lane 1, PFGE result of S1-digested plasmid DNA of strain ZT01-0079; Lane 2, PFGE patterns for S1-digested plasmid DNA of the transconjugants J53.; Lane 3, Southern blotting of strain ZT01-0079 with the probes specific to the blaNDM-1 gene. The black arrows indicate the plasmid pZY-NDM1.

Genetic Analysis of pZY-NDM1

Plasmid pZY-NDM1 is a 53573 bp circular plasmid with an average GC content of 49.24% and has 76 open reading frames. pZY-NDM1 belongs to type IncX3. BLAST comparison disclosed that the genome sequence of pZY-NDM1 shared >99% similarity with plasmids pNDM-HK3694 (Genbank accession JX104760.1) and p309074-NDM (Genbank accession MH909346.1). These three plasmids share a typical IncX plasmid backbone composed of replication (repB), plasmid stability (parA, parB), plasmid maintenance (hns, top) and type IV secretion system (taxA, taxB, taxC, VirD1, VirD2, VirD3-4, VirD5 and VirD6, VirD8, VirD9, VirD10, VirD11) encoding regions (Figure 2A). The 19.66 kb genetic load region of pZY-NDM1 located between the hns and resolvease genes, contained Tn3, IS3000, the transposon Tn125 variant encoding blaNDM-1 and a mobile element containing blaSHV-12 and ISL3. The transposon Tn125 variant served as a mobile element for the transfer of blaNDM-1 and comprised IS5, ΔISAba125, blaNDM-1, the bleomycin resistance gene ble, trpF, tat, cutA, groES and groEL as previously described.23 Compared with p309074-NDM, the pZY-NDM1 plasmid has a deletion of an 891bp ΔISAba125 fragment, which is located between IS5 and IS3000. Compared with traditional Tn125, the upstream of the pZY-NDM1 plasmid lacks ISAba125, and the downstream ISAba125 is replaced to ΔISAba125 and IS5 (Figure 2B).

Figure 2

Schematic diagram of (A) plasmid comparison: plasmids p309074-NDM; pZY-NDM1 and pNDM-HK3694 and (B) Resistance area analysis: plasmids p309074-NDM; pZY-NDM1 and pNDM-HK3694. The open reading frames are indicated by arrows. The black, green, purple, and yellow arrows represent genes associated with replication, plasmid stability, plasmid maintenance and the type IV secretion system. The blaSHV-12 gene is shown in pink, while the blaNDM-1 gene is shown in red. The accessory modules are shown in blue. Other genes of the backbone are shown in dark gray. Homology regions among different plasmids are denoted by light gray.

The blaSHV-12-carrying transposon was located in two opposite IS26 elements and composed of ΔygbJ, ygbI and blaSHV-12. Compared with pNDM-HK3694, the blaSHV-12-carrying transposon of pZY-NDM1 was reversed. In the transposon Tn125 variant, the two ends of the blaSHV-12-carrying transposon are divided into two incomplete ΔumuD1 and ΔumuD2, which may serve as insertion sites of regions containing the blaSHV-12-carrying transposons, Tn125 variant and tnpA (Figure 2B).

Discussion

The prevalence of NDM-1-producing bacteria is receiving increasing attention as a threat to global health. Most blaNDM-1-positive isolates of diverse species of Enterobacteriaceae show high-level resistance to β-lactam antibiotics. Concurrently, the blaNDM-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.2,9,12,24 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 blaNDM-1 and blaSHV-12. Plasmids belonging to the IncX3 group usually have a wide host range and self-conjugation.12,25,26 Host-range plasmids usually appear with variable frequencies ranging from 10−3 to 10−6.27 pZY-NDM1 showed a relatively high transfer frequency, proving its great potential for cross-species transfer.

The coexistence of blaNDM-1 and other β-lactamase genes in IncX3 plasmids, which have a broad host range, mediates resistance to broad-spectrum antibiotics, such as carbapenems and cephalosporins, and can be transferred between Enterobacteriaceae.28 Notably, there are few reports of such plasmids in C. freundii, and there is a potential risk of spreading this plasmid and monitoring should be strengthened.

Conclusions

In summary, we characterized the genomic basis of multidrug resistance in C. freundii ZT01-0079 strain. MLST revealed that strain ZT01-0079 belongs to ST19. ZT01-0079 carried multiple bla genes, which increased the level of carbapenem resistance. The transferable plasmid pZY-NDM1 carries blaNDM-1 and blaSHV-12 genes, and may thereby serve as a common vector for the rapid dissemination of carbapenemase-encoding genes. Our findings further underscore the threat of increased NDM-1 prevalence in Enterobacteriaceae and emphasize that increased resources and effort should be dedicated to monitoring the potentially rapid spread of NDM-1-encoding plasmids.