New Delhi Metallo-β-Lactamase 1-Producing Klebsiella pneumoniae ST719 Isolated from a Neonate in China

Bacterial isolation and identification

The NDM-1-producing K. pneumoniae strain KP14003 was isolated from the blood sample of a 16-day neonate in Beijing, China, in 2014. Species identification was performed with VITEK II automated system (bioMérieux, France). The common genes encoding carbapenemases and extended-spectrum β-lactamases (ESBLs) were screened by PCR. ¹² The entire bla_NDM gene was amplified with previously described primers and confirmed by sequencing. ¹³ Written informed consent was obtained from the parents of the neonate. All experimental protocols were approved by the institutional ethics committees of Academy of Military Medical Sciences.

Antimicrobial susceptibility testing

Antibiotic susceptibility testing was performed using the VITEK 2 Compact system (bioMérieux). The minimum inhibitory concentration (MIC) results of ampicillin, amoxicillin/clavulanic acid, piperacillin, cefazolin, ceftazidime, ceftriaxone, cefepime, amikacin, gentamicin, levofloxacin, ciprofloxacin, aztreonam, imipenem, meropenem, trimethoprim-sulfamethoxazole, tetracycline, and nitrofurantoin were interpreted following the Clinical and Laboratory Standards Institute guidelines. ¹⁴ E. coli ATCC25922 was used as quality control.

Southern blotting and conjugation experiment

Bacterial genomic DNA was prepared in agarose plugs and digested with S1 nuclease (Takara, Dalian, China). Linearized plasmids and partially digested genomic DNA were separated by pulsed-field gel electrophoresis (PFGE) through the CHEF-DR III system (Bio-Rad, Hercules). The plasmid DNA was transferred onto a positively charged nylon membrane (Roche) as described previously. ¹⁵ The membrane fixed by UV exposure was hybridized with PCR-generated probes (digoxigenin labeled) specific to bla_NDM-1.

Conjugation experiment was performed by broth and filter mating using the strain KP14003 as the donor and E. coli J53 AzR (resistant to sodium azide) as the recipient. ¹⁶ Donor and recipient organisms were incubated in 4 mL Luria-Bertani for 18 hours. The transconjugants were selected on trypticase soy agar plates containing 2 μg/mL imipenem and 300 μg/mL sodium azide after 18 hours of incubation. The acquired drug resistances were assessed by antimicrobial susceptibility testing. Transferability of plasmids was evaluated by the S1-PFGE experiment with the transconjugants as described above. The bla_NDM-1 gene and common ESBL genes in the tranconjugants were screened by PCR amplification.

Whole-genome sequencing and plasmid assembly

Genomic DNA of strain KP14003 was extracted from cultured bacterium using the QIAamp DNA minikit (Qiagen, Inc., Valencia, CA). Whole-genome sequencing was performed using both the Illumina Hiseq 2500 platform at Novogene Company (Beijing, China) and the MinION sequencer (Nanopore, Oxford, United Kingdom). The de novo hybrid assembly of short Illumina reads and long MinION reads was performed using Unicycler (v0.4.8), including a polishing step with Pilon. ¹⁷ The genomic sequence was annotated using RAST. ¹⁸ The sequence type (ST) of strain KP14003 was assigned by uploading the assembled genome onto the MLST web server. ¹⁹ Plasmid replicon types were identified using PlasmidFinder. ²⁰ Capsular serotype was identified using Kaptive. ²¹ Virulence factors were identified by aligning the assembled genome against the sequences for 80 virulence genes provided by the Pasteur Institute.

Nucleotide sequence accession numbers

The complete sequences of the chromosome and plasmids of strain KP14003 have been deposited in GenBank under the accession no. CP041934 (chromosome), CP041935 (plasmid unnamed1), CP041936 (plasmid unnamed2), CP041937 (plasmid unnamed3), and CP041938 (pNDM-KP14003).

Microbiological and genetic features of strain KP14003

Strain KP14003 was recovered from the blood sample of a 16-day neonate through routine surveillance in Beijing, China, in 2014. The bacterial isolate was identified as K. pneumoniae using the VITEK 2 Compact system (BioMérieux). Antimicrobial susceptibility testing showed that KP14003 was resistant to ampicillin, amoxicillin-clavulanic acid, trimethoprim-sulfamethoxazole, piperacillin, aztreonam, imipenem, and cephalosporins, but still susceptible to meropenem, amikacin, gentamicin, ciprofloxacin, levofloxacin, tetracycline, and nitrofurantoin (Table 1). Presence of bla_NDM-1 was identified by PCR amplification and confirmed by sequencing. S1 PFGE revealed that KP14003 contained four different plasmids (∼60, ∼105, ∼175, and ∼230 kb) (Fig. 1). Southern blotting indicated that bla_NDM-1 was carried by the ∼60 kb plasmid, which was transferable to E. coli strain J53 at a frequency of 2.14 × 10⁻³ per donor cell. The transconjugants acquired majority of the antimicrobial resistances of KP14003, but were susceptible to trimethoprim-sulfamethoxazole. Further analysis revealed the nontransferability of the plasmid harboring trimethoprim-sulfamethoxazole resistance genes sul1 and dfrA1. The presence of the bla_NDM-1-carrying plasmid alone in the transconjugants was identified by S1-PFGE. Interestingly, PCR amplification and sequencing revealed that a bla_SHV-12 gene was transferred to E. coli J53 simultaneously, indicating the coexistence of bla_NDM-1 and bla_SHV-12 on the same plasmid.

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FIG. 1.

(A) The S1-PFGE pattern of strain KP14003 and southern blotting for the bla_NDM-1 gene. (B) S1-PFGE pattern of Escherichia coli J53 transconjugants. Lanes: Marker, Salmonella serotype Braenderup strain H9812; 1, PFGE pattern for S1-digested plasmid DNA of strain KP14003; 2, southern blot analysis with the probe specific to bla_NDM-1; 3, PFGE pattern for S1-digested plasmid DNA of E. coli J53 transconjugants. Black arrows indicate the plasmids of strain KP14003. PFGE, pulsed-field gel electrophoresis. Color images are available online.

K. pneumoniae KP14003 belonged to ST ST719 and capsular serotype K9. The whole-genome analysis for virulence factors revealed the presence of the mrk gene cluster encoding type 3 fimbriae and the iutA gene encoding the aerobactin receptor in strain KP14003. A hybrid de novo assembly of short Illumina reads and long MinION reads indicated the bla_SHV-27 gene on the chromosome and revealed the coexistence of bla_TEM-1, aac(3)-IId, aadA5, dfrA1, and sul1 genes on an IncH plasmid of 287,790 bp. The bla_TEM-1 gene was flanked by an upstream IS110 and a downstream ISCfr1.

Characterization of plasmid pNDM-KP14003

The plasmid carrying bla_NDM-1 had 60,125 bp in length and was designated as pNDM-KP14003. It has 58 open reading frames with an average GC content of 49.1%. pNDM-KP14003 belonged to the incompatibility group IncX3. BLAST search indicated that the sequence of pNDM-KP14003 was identical with that of plasmid pNDM-BJ03 in Enterobacter cloacae from China. ²² It also presented high similarity with plasmids pNDM-HN380 (96% coverage and 99% identity) ²³ and pEC27-1 (95% coverage and 99% identity) (CP020090). The above plasmids shared a typical IncX3 plasmid backbone of 30.2 kb, which contained a set of core genes for partition (parA), replication (bis and repB), maintenance (topB and hns), and conjugation/T4SS (pilX genes) (Fig. 2).

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FIG. 2.

Linear comparison of plasmids pNDM-HN380, pNDM-BJ03, pNDM-KP14003, pEC27-1, and pNDM-BJ01 (Tn125). The open reading frames are shown with arrows. The orange, pink, gold, and cyan arrows indicate genes related to conjugation/T4SS, maintenance, partition, and replication, respectively. The bla_NDM-1 gene is presented in red. The insert sequences IS3000, ISAba125, and IS26 are presented in green, purple, and brown, respectively. The accessory modules are indicated in blue. Other genes of the backbone are indicated in dark gray. Homology regions among different plasmids are denoted in light gray. Color images are available online.

The 29.9-kb genetic load region in pNDM-KP14003 was inserted between the res gene and the hns gene. It consisted of an IS4, a truncated tnpA, an IS3000, a 7.9-kb bla_NDM-1-carrying Tn125 remnant, and an IS26-bla_SHV-12 composite transposon. Compared with the prototype Tn125, the Tn125 remnant in pNDM-KP14003 had undergone a truncation of the upstream ISAba125 by IS3000 and a deletion of the downstream copy, suggesting possible transposition events. No flanking direct repeats of Tn125 could be detected. The genetic structure of bla_NDM-1 is different from that in pNDM-HN380, of which the upstream ISAba125 was interrupted by an IS5. The IS26-bla_SHV-12 composite transposon included bla_SHV-12, ygbI, and ygbJ, which were flanked by two oppositely oriented copies of IS26. The inversion of the transposon had been observed in pNDM-HN380, while the insertion of an additional copy of IS26 in pEC27-1. Interestingly, there existed a 6.9-kb conjugative fragment integrated into the mpr gene, which was interrupted into two fragments in pNDM-KP14003 compared with plasmid pNDM-HN380. This conjugative fragment contained stbA (plasmid transfer), frmRAB operon (degeneration of formaldehyde), and tnpA, which was homologous to that of plasmid pKPN1482 (CP020846) and was likely introduced through the horizontal transfer.