The Characterization of OXA-232 Carbapenemase-Producing ST437 Klebsiella pneumoniae in China

Carbapenem-resistant Klebsiella pneumoniae (CRKP) was epidemic around the world and become a global threat to public health. The most important carbapenem-resistant mechanism is producing carbapenemases, especially Klebsiella pneumoniae carbapenemase (KPC), which is prevalent in the international clonal complex CC11. The high-risk multidrug-resistant CC11 is widespread worldwide, and KPC-producing and (New Delhi metallo) NDM-producing strains had been reported in this clonal complex before; moreover, cases with the CC11 strain faced more severe forms of drug resistance and treatment challenges than other clonal complexes. In this study, we identified an OXA-232-producing ST437 Klebsiella pneumoniae isolate in China, which belonged to CC11. The isolate was resistant to β-lactams, aminoglycosides, and fluoroquinolones but susceptible to fosfomycin, tigecycline, and colistin. The blaOXA-232 gene was located on a 6141 bp ColKP3-type nonconjugative plasmid, and the plasmid was transformed by chemical transformation successfully. This is the first report of OXA-232-producing ST437 K. pneumoniae in China, a new clone of high-risk multidrug-resistant CC11.


Introduction
With the spread of carbapenemase genes, the emergence and dissemination of carbapenem-resistant Klebsiella pneumoniae (CRKP) is an escalating global threat. e most important carbapenem-resistant mechanism is producing carbapenemases, which includes KPC, NDM, VIM, IMP, and OXA-48like. A nationwide survey conducted in China showed that acquisition of two carbapenemase genes, bla KPC-2 and bla NDM , was responsible for phenotypic resistance in 90% of the CRE strains tested (58% and 32%, respectively), among which several major strain types, such as ST11 of K. pneumoniae, were identified [1]. Currently, local clonal disseminations of OXA-48-type carbapenemases in China have been reported [2][3][4], but we are short of precise data concerning the epidemiology of this carbapenemase.
Clonal complex 11 (CC11) denotes the STs differed from ST11 by one housekeeper gene, including ST11, ST258, ST340, ST437, ST757, ST855, and others. CC11 is an epidemic K. pneumoniae clonal complex [1], and cases with the CC11 strain faced more severe forms of drug resistance and treatment challenges than other clonal complexes [8]. KPCproducing [1] and NDM-producing [9] CC11 have been reported in China, and no one reported OXA-48-typeproducing CC11 clone in China although a clonal dissemination of OXA-48-producing ST147 and ST383 [4] as well as two clonal disseminations of OXA-232-producing ST15 have been reported in China [2,3]. is is the first report of OXA-232-producing ST437 K. pneumoniae in China, a new clone of high-risk multidrug-resistant CC11. e aim of this study was to determine the antimicrobial resistance profile, and identify the carrier plasmid and the flanking region of the bla OXA-232 gene.

Materials and Methods
2.1. Isolation and Identification. K. pneumoniae NB5306 was recovered from a blood culture from a 72-year-old female patient. e patient hospitalized (Day 0) in hematology ward in Ningbo First Hospital, China, with the history of aplastic anemia. In order to prevent nosocomial infection, ceftriaxone/sulbactam (3 g q8 h) was administrated after admission for 2 days, and then administration was changed to biapenem (0.3 g q8 h) for 10 days; the patient was stable and had no invasive procedure. However, the patient had a fever at Day 20, and the highest temperature was up to 40°C, with white blood cell count being 0.60 × 10 9 /L, neutrophil% 20.0%, and c-reactive protein (CRP) 37.20 mg/L. Blood culture was examined immediately on Day 20, and the empiric therapy was administrated with imipenem (0.5 g q6 h), vancomycin (0.5 g q12 h), and voriconazole (0.2 g q12 h). During the treatment, the temperature of patient was sustained, and her condition continued to deteriorate; therefore, the patient gave up further treatment on Day 22 and returned home. Unfortunately, we were informed that the patient died at home two days after discharge by phone follow-up. K. pneumoniae NB5306 was recovered from blood culture and was identified with VITEK ® 2 automated microbial identification system.

Antimicrobial Susceptibility Testing.
Antimicrobial susceptibility testing of MICs was performed by the agar dilution method or broth microdilution method (for tigecycline and colistin), with susceptibility defined according to Clinical and Laboratory Standards Institute (CLSI) (M100-S28) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) (version 8.1, for tigecycline and colistin) guidelines.

Plasmid Conjugation and Chemical Transformation.
Plasmid conjugation of the bla OXA-232 gene was attempted by conjugation experiments at 37°C using rifampicin-resistant E. coli EC600 or sodium azide-resistant E. coli J53 as recipient, and imipenem (0.12 mg/L) associated with rifampicin (700 mg/L) or sodium azide (300 mg/L) was used for selection. e chemical transformation was performed when conjugation failed. Plasmid harboring bla OXA-232 gene was transferred into E. coli DH5α by chemical transformation with imipenem (0.12 mg/L) for selection.

S1 PFGE and Southern Bolt.
Genomic DNA was digested with S1-nuclease (Takara, Otsu, Japan) and electrophoresed on a PFGE system (Bio-Rad, Hercules, CA, USA) for 10 h at 14°C, with run conditions of 6 V/cm and pulse times from 2.16 s to 63.8 s. DNA fragments were transferred to a positively charged nylon membrane (Millipore, Billerica, MD, USA) and then hybridized with a digoxigenin-labelled bla OXA-232 -specific probe. e fragments then were detected with an NBT/BCIP color detection kit (Roche, Mannheim, Germany).

e Primer Walking Sequencing.
e plasmid containing bla OXA-232 was obtained by Axygen plasmid miniprep (Axygen Biosciences, USA), initial round of sequencing was from a known sequence at one end of the template, and initial primers were bla OXA-48-F (5′-ATGCGTGTAT-TAGCCTTATCGGCT-3′) and bla OXA-48-R (5′-CTAGG-GAATAATTTTTTCCTGTTTGAG-3′); then, each subsequent round was initiated from a new primer, which is based on the end of the sequence obtained from the previous reaction. Finally, the full length of the plasmid was sequenced after some rounds of reactions. e replicon of plasmid was identified by plasmid finder 2.1 (https://cge.cbs. dtu.dk/services/PlasmidFinder/).

Results of Plasmid Conjugation and Chemical
Transformation. NB5306 carried a wild-type ompK35 but a novel ompK36 variant, a highly conserved domain within the region of loop 3 (DVLPEFGGDTDTYGFDNFLQSRA NGV).

Discussion
e OXA-232 carbapenemase-producing strains emerging had been frequently reported to have association with travelling to India. For example, Potron et al. reported that two K. pneumoniae and one E. coli harboring OXA-232 were recovered from three patients transferred from India to France in 2011 [7]. Findlay et al. reported that OXA-232 was found in isolates from patients reporting travel to India across the UK between 2007 and 2014 [10]. Jeong et al. reported that clonal and horizontal spread of the bla OXA-232 gene among Enterobacteriaceae in a Korean hospital was attributed to an index patient who was likely colonized during a prior hospitalization in India [11]. But some reported OXA-232 strains were not strongly relevant to the history of recent travel abroad. For example, Mancini et al. reported that 6 K. pneumoniae were recovered in 2017 from 5 different patients, who reported no recent travel abroad [12]. Abdul Momin et al. reported that 5 OXA-232-producing K. pneumoniae isolates were recovered from 5 patients in Brunei; all patients were hospitalized locally and had no history of recent travel [13]. In this study, the patient was hospitalized locally and reported no travel abroad history.
bla OXA-232 K. pneumoniae was found in France [7] and China [2], and this study remained susceptible to colistin and tigecycline, but NB5306 showed different drug resistance profiles to carbapenems compared with strain RAN, which was the first K. pneumoniae strain harboring bla OXA-232 [7], with NB5306 MICs of ertapenem was 8 mg/L, imipenem was 0.25 mg/L, and meropenem was 2 mg/L, while RAN was resistant to ertapenem, imipenem, and meropenem, with MIC >32 mg/L [7]. NB5306-D obtained bla OXA-232 by chemical transformation, and the MIC of ertapenem was 1 mg/L, in which we conjectured that the ompK36 variant might play a role in charge of MIC of ertapenem [26].
Among studies involving OXA-232, only Jeong et al. reported that the bla OXA-232 gene was located on a conjugative ColE-type plasmid 6141 bp in size, and this plasmid was successfully transferred from K. pneumoniae H16 to E. coli J53 by conjugation [11]. However, this investigation and other studies reported that bla OXA-232 gene was located on a nonconjugative ColKP3-type plasmid 6141 bp in size [3,7,22]. Nonconjugative plasmids are incapable of initiating conjugation; however, they can be transferred with the assistance of conjugative plasmids. e same kind of 6 kb plasmids harboring bla OXA-232 were also found in Europe, North America, Asia, and North Africa [20]. It is speculated that 6 kb plasmid is likely disseminated in the whole world.
e study provides the emergence of an OXA-232 K. pneumoniae belonging to CC11. Further surveillance and investigations are needed to have better understanding of potential transmission and evolution of OXA-232 carbapenemase-producing CRKP in the world.

Data Availability
e draft genome sequence of K. pneumoniae NB5306 and the complete nucleotide sequence of the plasmid carrying blaOXA-232 have been deposited in GenBank under accession numbers QYCO00000000 and MK105834, respectively.

Ethical Approval
We obtained the approval letter of Ningbo First Hospital Ethics Committee (Approval no. 2019-R044). Institutional approval was obtained by Clinical Research Management Committee of Ningbo First Hospital to publish the case details.

Consent
Written informed consent was obtained from the patient's son.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Xingbei Weng and Qiucheng Shi contributed equally to this work.