Coexistence of blaOXA-48 and Truncated blaNDM-1 on Different Plasmids in a Klebsiella pneumoniae Isolate in China

Objectives: To describe the genetic environment, transferability, and antibiotic susceptibility of one clinical Klebsiella pneumoniae isolate harboring both blaOXA-48 and blaNDM-1 on different plasmids from a Chinese hospital. Methods: The isolate was subjected to antimicrobial susceptibility testing and multilocus sequence typing using Etest and PCR. The plasmids harboring blaOXA-48 and blaNDM-1 were analyzed through conjugation experiments, S1-nuclease pulsed-field gel electrophoresis, and hybridization with specific probes. Plasmid DNA was sequenced using Pacbio RS II and annotated using RAST. Results: K. pneumoniae RJ119, carrying both blaOXA-48 and blaNDM-1, was resistant to almost all carbapenems, cephalosporins, fluoroquinolone, and aminoglycosides and belonged to ST307. blaOXA-48 was located on a 61,748-bp IncL/M conjugative plasmid, which displayed overall nucleotide identity (99%) to pKPN-E1-Nr.7. blaNDM-1 was located on a 335,317-bp conjugative plasmid, which was a fusion of a blaNDM-1-harboring InA/C plasmid pNDM-US (140,825 bp, 99% identity) and an IncFIB plasmid pKPN-c22 (178,563 bp, 99% identity). The transconjugant RJ119-1 harboring blaNDM-1 was susceptible to carbapenem, and there was an insertion of IS10 into the blaNDM-1 gene. Conclusion: This is the first report of the coexistence of blaOXA-48 and blaNDM-1 in one K. pneumoniae clinical isolate in China. OXA-48 in RJ119 contributed to the majority to its high resistance to carbapenems, whereas NDM-1 remained unexpressed, most likely due to the insertion of IS10. Our results provide new insight for the relationship between genetic diagnosis and clinical treatment. They also indicate that increased surveillance of blaOXA-48 is urgently needed in China.


INTRODUCTION
The emergence of carbapenem-resistant Enterobacteriaceae (CRE) has become a challenge to clinical therapy because of the rapid worldwide dissemination of multidrug resistance. The most important carbapenemase genes in Enterobacteriaceae are those encoding for KPC, VIM, IMP, NDM, and OXA-48 (Queenan and Bush, 2007). NDM-1 was first reported in 2008 as being produced by a Klebsiella pneumoniae isolate from a Swedish patient who had returned from India (Yong et al., 2009). Alarmingly, it was often found on large conjugative plasmids along with additional antibiotic resistance determinants (Hudson et al., 2014). NDM-1-producing K. pneumoniae isolates are considered as endemic in many countries including India, Pakistan, Bangladesh (Nordmann and Poirel, 2014) and show sporadic spread in the USA (Rasheed et al., 2013), Canada (Lowe et al., 2013), Colombia (Ocampo et al., 2016), Greece (Spyropoulou et al., 2016), Singapore (Ling et al., 2015), and China (Qu et al., 2015). OXA-48 was first identified in a K. pneumoniae isolate from Turkey in 2001 (Poirel et al., 2003). Since then, endemic spread of OXA-48-producing K. pneumoniae isolates has been reported in countries such as Turkey, Morocco, Libya, Egypt, Tunisia, and India (Nordmann and Poirel, 2014). The coexistence of genes for at least two classes of carbapenemases in K. pneumoniae also has been reported worldwide, including KPC-3 and VIM-2 in Italy, KPC-2 and VIM-24 in Colombia, NDM-1 and KPC-2 in Brazil, and NDM-1 and OXA-181 in Singapore (Lee et al., 2016).
We studied the genetic environment, transferability, and antibiotic susceptibility of a clinical isolate of K. pneumoniae harboring the bla OXA−48 and bla NDM−1 genes on different plasmids, which was isolated from a Chinese hospital.

Bacterial Isolates and Antimicrobial Susceptibility Testing
Klebsiella pneumoniae RJ119, carrying both the bla OXA−48 and bla NDM−1 genes, was isolated from the wound of a female burn patient at Ruijin Hospital, Shanghai, in September 2015. The previous travel history of the patient was not recorded. The genes were detected by the Cepheid Xpert Carba-R assay (Cepheid, Sunnyvale, CA, USA), a qualitative diagnostic test that was designed for rapid detection and differentiation of the bla KPC , bla NDM , bla VIM , bla OXA−48 , and bla IMP−1 genes. The minimum inhibitory concentrations (MICs) of ceftazidime, cefotaxime, ceftriaxone, cefepime, aztreonam, piperacillin/tazobactam, ertapenem, meropenem, imipenem, trimethoprim/sulfamethoxazole, ciprofloxacin, and amikacin in the different strains isolated from the patient were determined by using the Etest (bioMérieux, France), and the results were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute [CLSI] (2014). The CLSI does not have interpretative criteria for tigecycline; therefore, European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used 1 . Escherichia coli ATCC 25922 was used as a quality control reference strain.
This study was approved by Ruijin Hospital Ethics Committee (Shanghai Jiao Tong University School of Medicine), and the Review Board exempted the requirement for informed consent because this retrospective study only focused on bacteria and did not affect the patients.

DNA Preparation and Sequencing
Plasmid DNA was extracted from the RJ119 strain using the QIAGEN Midi Kit (Qiagen, Hilden, Germany). DNA was sequenced using Pacbio RS II (Pacific Biosciences, Menlo Park, CA, USA). The reads were de novo assembled using HGAP 3.0 of SMRT TM Pipe (Supplementary Images 1). Protein-coding genes were initially identified and annotated using RAST (Aziz et al., 2008). Insertion elements (IS) and antimicrobial resistance genes were identified using IS Finder 3 and ResFinder 4 , respectively. PlasmidFinder 5 was used to detect and type the plasmid. BLAST 6 searches were used to identify related plasmids carrying bla OXA−48 and bla NDM−1 to guide PCR-based gap closure and Sanger sequencing to assemble contigs into complete plasmids.

Nucleotide Sequence Accession Numbers
The complete sequence of plasmids pRJ119-NDM1 and pRJ119-OXA48 in K. pneumoniae RJ119 has been deposited in GenBank under the accession numbers KX636095 and KX636096, respectively (Supplementary Datasheet File 1).

DISCUSSION
Carbapenemase genes often spread worldwide through clonal expansion in several pathogenic strains (Lee et al., 2016). Over 50% of NDM-producing K. pneumoniae isolates from India belong to either ST11 or ST147 (Lascols et al., 2013), and ST11 has also been associated with OXA-48-like enzymes from isolates found in Argentina, Turkey, and Spain (Oteo et al., 2013). The K. pneumoniae RJ119 isolate in our study belonged to ST307, which is not the part of the major clonal complex (Supplementary Images 2) and most prevalent in Pakistan and Korea (Habeeb et al., 2013;Park et al., 2015).
not present in pOXA-48; (ii) Tn1999.2 in pKPN-E1-Nr.7 was only the inversion of the sequence in pRJ119-OXA48, and the korC gene was not present in pRJ119-OXA48; and (iii) the sequence of Tn1999.2 was homologous to that in pOXA48-PM. Therefore, we speculated that pRJ119-OXA48 was an existence form in the evolution of bla OXA−48 . Recent studies have reported the emergence of OXA-48-producing K. pneumoniae isolates and nosocomial outbreak of infections in Taiwan and Beijing, respectively (Ma et al., 2015;Guo et al., 2016). bla OXA−48 found in Taiwan was located on a 160-kb IncA/C plasmid, which was identical to the pKP OXA -48N1 plasmid found in France (Berger et al., 2013). bla OXA−48 found in Beijing was located on the IncL/M plasmid. These data from epidemiological investigations, coupled with those of our current report, show that the bla OXA−48 genes found in mainland China and Taiwan are from two different genetic backgrounds, and emphasize the need for increased surveillance of bla OXA−48 in China.
In China, bla NDM−1 was first identified in Acinetobacter baumannii. Subsequently, more bla NDM−1 genes were discovered in Enterobacteriaceae, especially in K. pneumoniae, and the gene was found carried on plasmids of various sizes (Qin et al., 2014;Qu et al., 2015;Zhang et al., 2015). In our study, bla NDM−1 was located on a 335-kb large plasmid. Based on the BLAST results, we suspected that it might be the fusion of a bla NDM−1 -harboring InA/C plasmid, pNDM-US, and an IncFIB plasmid, pKPN-c22 (Figure 3). Previous studies have suggested that IncX3-type plasmids contributed to the dissemination of bla NDM−1 in China (Ho et al., 2012;Qu et al., 2015;Yang et al., 2015). However, in our study, the first half of pRJ119-NDM1 (contain bla NDM−1 ) was found belonging to the IncA/C group and identical to pNDM-US, the second part of pRJ119-NDM1 showed 99% identity to pKPN-c22, a plasmid detected in the USA. We speculated that the pRJ119-NDM1 plasmid might have been introduced from America after the fusion. Moreover, IS1-IS2-IS1, the repeat sequence joint, might play an important role in the fusion of the two plasmids. The fusion of these two plasmids, each harboring an extraordinary number of resistance genes, might enhance antibiotic resistance. However, notably, the transconjugant RJ119-1, harboring truncated bla NDM−1 , was susceptible to carbapenem, which might have been due to the insertion of IS10 into the gene. Molecular-based diagnostic methods such as PCR, microarrays, and sequence-based diagnostics are now used in clinical applications due to their celerity and accuracy and are beginning to permeate clinical diagnostic laboratories in many countries (Okeke et al., 2011). However, in the case of our present study (unexpressed bla NDM−1 due to the insertion of IS10), because the PCR results did not match with the actual clinical situation, they may mislead clinical treatment.

CONCLUSION
This is the first report on the coexistence of bla OXA−48 and bla NDM−1 in one K. pneumoniae clinical isolate in China. The production of OXA-48 in RJ119 contributed to the majority to its high resistance to carbapenems, whereas NDM-1 remained unexpressed, most likely due to  the insertion of IS10. This study provides insight on the relationship between genetic diagnosis and clinical treatment in cases similar to the one in this study. Our findings also indicate the possibility of further spread of bla OXA−48 in China and emphasize the need for intensive surveillance and precautions.

AUTHOR CONTRIBUTIONS
JS and YD designed experiments; LX and KZ carried out experiments; JS, LH, and XG analyzed experimental results; YC and YD analyzed sequencing data; LX and JS wrote the manuscript.