Production of plasmid-encoding NDM-1 in clinical Raoultella ornithinolytica and Leclercia adecarboxylata from China

Raoultella ornithinolytica YNKP001 and Leclercia adecarboxylata P10164, which harbor conjugative plasmids pYNKP001-NDM and pP10164-NDM, respectively, were isolated from two different Chinese patients, and their complete nucleotide sequences were determined. Production of NDM-1 enzyme by these plasmids accounts for the carbapenem resistance of these two strains. This is the first report of blaNDM in L. adecarboxylata and third report of this gene in R. ornithinolytica. pYNKP001-NDM is very similar to the IncN2 NDM-1-encoding plasmids pTR3, pNDM-ECS01, and p271A, whereas pP10164-NDM is similar to the IncFIIY blaNDM-1-carrying plasmid pKOX_NDM1. The blaNDM-1 genes of pYNKP001-NDM and pP10164-NDM are embedded in Tn125-like elements, which represent two distinct truncated versions of the NDM-1-encoding Tn125 prototype observed in pNDM-BJ01. Flanking of these two Tn125-like elements by miniature inverted repeat element (MITE) or its remnant indicates that MITE facilitates transposition and mobilization of blaNDM-1 gene contexts.


Introduction
Raoultella ornithinolytica is widely found in aquatic environments, insects and fishes. R. ornithinolytica is able to convert histidine to histamine (scombroid toxin) and is thus known to cause fish poisoning. Symptoms primarily manifest with facial flushing, dizziness, vomiting, diarrhea, dyspnea, headache, urticarial, and generalized pruritus and commonly subside in a few hours (Kanki et al., 2002). Infections by R. ornithinolytica are exceedingly rare in humans and have been reported as bloodstream, urinary tract and soft tissue infections in adults and as fatal neonatal infections. Most adult cases are linked with underlying diseases, especially malignancies (Morais et al., 2009;Mau and Ross, 2010;Solak et al., 2011;Hadano et al., 2012;Haruki et al., 2014;Chun et al., 2015).
NDM is an Ambler class B metallo-β-lactamase that confers resistance to nearly all β-lactam antibiotics, including carbapenems, and bla NDM genes have been identified in a large array of Enterobacteriaceae species Johnson and Woodford, 2013;Dortet et al., 2014). Both R. ornithinolytica and L. adecarboxylata are members of Enterobacteriaceae. In this study, we analyzed complete nucleotide sequences of two different NDM-1-encoding plasmids, pYNKP001-NDM, and pP10164-NDM, recovered from R. ornithinolytica YNKP001 and L. adecarboxylata P10164, respectively, of clinical origin in China.

Bacterial Strains and Identification
The use of human specimens and all related experimental protocols were approved by the Committee on Human Research of indicated institutions and carried out in accordance with the approved guidelines. Informed consent was obtained from the indicated patients. All bacterial strains were subjected to species identification by BioMérieux VITEK 2, Bruker MALDI Biotyper, and 16S rRNA gene sequencing. For 16S rRNA gene sequence determination, nearly the complete coding region of 16S rRNA gene was amplified by PCR with the universal primers 27f (AGAGTTTGATCCTGGCTCAG) and 1492r (TACCTTGTTACGACTT) (Frank et al., 2008). The major carbapenemase and ESBL genes (Table S1) were subjected to PCR detection. All PCR amplicons were sequenced on an ABI 3730 Sequencer with the same primers for PCR.

Plasmid Conjugal Transfer
Plasmid conjugal transfer experiments were carried out with Escherichia coli EC600 (rifampin-resistant) or TB1 (streptomycin-resistant) used as recipient and the bla NDMpositive strain YNKP001 or P10164 as donor. Then, 3 ml of overnight culture of each of donor and recipient bacteria were mixed together, harvested and resuspended in 80 µl of brain heart infusion (BHI) broth (BD Biosciences). The mixture was spotted on a 1-cm 2 filter membrane placed on BHI agar (BD Biosciences) plates. The plates were incubated for mating at 37 • C for 12-18 h. Bacteria were washed from filter membrane and spotted on Muller-Hinton (MH) agar (BD Biosciences) plates containing 1000 µg/ml rifampin or 250 µg/ml streptomycin and 200 µg/ml ampicillin for selection of bla NDM -positive E. coli transconjugants.

Determination of Minimum Inhibitory Concentration (MIC)
The MIC values of the indicated bacterial strains were tested by VITEK 2 according to the manufacturer's instructions, and antimicrobial susceptibility was assessed by the Clinical and Laboratory Standards Institute (CLSI) standards.

Determination of Plasmid DNA Sequences
The chromosome DNA-free plasmid DNA was isolated from the cell cultures of indicated E. coli transconjugant using a Qiagen large construct kit and then sequenced using the wholegenome shotgun strategy in combination with Illumina HiSeq 2500 sequencing technology. The contigs were assembled with Velvet, and the gaps were filled through combinatorial PCR and Sanger sequencing with an ABI 3730 Sequencer. The genes were predicted with GeneMarkS and further annotated by BLASTP against UniPort and NR databases.

Nucleotide Sequence Accession Numbers
The complete sequences of plasmids pP10164-NDM and pYNKP001-NDM were submitted to GenBank under accession numbers KP900016 and KP900017, respectively.

Results
Characterization of R. ornithinolytica YNKP001 and L. adecarboxylata P10164 Raoultella ornithinolytica YNKP001 was recovered in November 2010 from the blood specimens of a 4-year-old child with acute encephalitis, bronchitis and tympanitis from a hospital in Kunming City in China. L. adecarboxylata P10164 was isolated in August 2012 from the sputum specimens of a 43year-old male with pneumonia admitted to a teaching hospital in Chongqing City in China. Both patients received empirical intravenous administration with ceftazidime for at least 1 week, but their symptoms did not improve. Subsequent antimicrobial susceptibility tests indicated that both strains were resistant to multiple β-lactams, including imipenem, and meropenem, but remained susceptible to fluoroquinolones. The patients then received intravenous administration with moxifloxacin and were cured and discharged approximately 10 days after initiating antimicrobial treatment.

Comparative Genomics of pYNKP001-NDM
As revealed by high-throughput sequencing with a mean coverage fold of 143, plasmid pYNKP001-NDM was 41,190 bp in size with a mean GC content of 50.8% and contained 54 open reading frames (ORFs) (Figure 1A). The replication module present on pYNKP001-NDM belonged to the IncN2 incompatibility group . This plasmid was mostly similar to another NDM-encoding IncN2 plasmid, pTR3 (Chen et al., 2012), with genetic differences in only three single nucleotide polymorphisms (SNPs).
Further linear genomic comparison (Figure 2A) was performed with all four NDM-encoding IncN2 plasmids, namely, pYNKP001-NDM, pTR3 (Chen et al., 2012), pNDM-ECS01 (Netikul et al., 2014), and p271A . The former three plasmids essentially had the same genomic organization. The genome of each plasmid could be divided into the backbone and accessory module. The backbone was composed of genes responsible for plasmid replication (repA), stability (stbABC, ssb, korB, klcA, etc.) and conjugal transfer (tra). p271A lacked a 5.2 Kb region found in pYNKP001-NDM, pTR3, and pNDM-ECS01. This region corresponded to the CUP (conserved upstream repeat)-controlled regulon commonly found in the IncN1 and IncN2 plasmids, and the loss of this region might be due to recombination between CUPs .
The four plasmids shared a single conserved accessory module, which was sequentially organized as an intact miniature inverted repeat element (MITE), a bla NDM−1 -containing Tn125like element, a MITE remnant, an intact ISSen4 element with 26 bp inverted repeats (IRs) at both sides, a truncated aphA6 (aminoglycoside resistance) gene, and an intact Tn5403 element with 39 bp IRs at both ends ( Figure 3A). The Tn125 prototype was sequentially organized as ISAba125, bla NDM−1 , ble MBL (bleomycin resistance), trpF, dsbC, cutA, groES, groEL, ISCR27, and ISAba125; Tn125 was a typical composite transposon (Tn) containing two flanking copies of ISAba125 (each with 26 bp IRs at both ends); Tn125 could be inserted into a site downstream of aphA6, leaving 3 bp direct repeats (DRs) at both ends, as observed in pNDM-BJ01 (Hu et al., 2012;Poirel et al., 2012) (Figure 3A). Compared with the Tn125 prototype, the Tn125-like element in pYNKP001-NDM lacked the entire fragment of dsbC, cutA, groES, groEL, ISCR27, and ISAba125. In addition, the upstream copy of ISAba125 was a truncated version lacking the left IR and was interrupted by ISEc33 (with 17 bp IRs at both ends) ( Figure 3A).
The flanking of a genetic context (e.g., class 1 integron) by two identical MITEs has been recently characterized as a mechanism for mobilizing antimicrobial resistance determinants via MITE-mediated transposition or homologous recombination (Poirel et al., 2009;Domingues et al., 2013;Zong, 2014). Notably, the Tn125-like element in pYNKP001-NDM was embedded between an intact 257 bp MITE and a 55 bp MITE remnant ( Figure 3A). The intact MITE contained 39 bp IRs and could potentially form a long stem-loop RNA structure (Delihas, 2011), whereas the downstream MITE remnant corresponded to the 3 ′ -terminal 55 bp fragment (including the right IR) of the intact MITE.

Comparative Genomics of pP10164-NDM
Plasmid pP10164-NDM was fully sequenced, with a mean coverage fold of 248. Moreover, it was 99,276 bp in size, with a mean GC content of 55%, and contained 101 ORFs (Figure 1B). pP10164-NDM was assigned to the IncFII Y incompatibility group encoding two different replication proteins IncFII Y (repA) and IncFIB (repB) (Villa et al., 2010). Comparative genomics analysis was performed with the only two characterized NDMencoding IncFII Y plasmids: pP10164-NDM and pKOX_NDM1 (Huang et al., 2013) (Figure 2B). pP10164-NDM differed from pKOX_NDM1 in 214 SNPs, 11 single-nucleotide indels, and three large deletions (11285 bp, 180 bp, and 45 bp, respectively). pP10164-NDM and pKOX_NDM1 had very similar backbones composed of genes responsible for plasmid replication (repA and repB), stability (pmaAB, psiAB, klcA, yub, etc.) and conjugal transfer (tra); the 45 bp deletion (nucleotide position 102082 to 102126 in pKOX_NDM1; located within the traD gene) was the only observed structural difference between the backbones of pP10164-NDM and pKOX_NDM1 ( Figure 2B). pP10164-NDM contained a single accessory module that was 38,098 bp in size, in which the 11285 bp and 180 bp deletions (nucleotide positions 10620-21904 and 37157-37336 in pKOX_NDM, respectively) compared with the counterpart of pKOX_NDM1 were located ( Figure 3B). The accessory module of pKOX_NDM1 harbored three copies of 256 bp MITEs highly similar to the above-mentioned 257 bp MITE, constituting a linear structure organized as the 11285 bp region (MITE plus 11029 bp region), MITE, Tn125-like element, and MITE ( Figure 3B). Homologous recombination mediated by the first two copies of MITE appeared to lead to the insertion of the 11285 bp region into pKOX_NDM1 relative to pP10164-NDM ( Figure 3B). In addition to the first copy of MITE, the 11285 bp region still contained rmtC (16S rRNA methylase for aminoglycoside resistance), ISCR14 and an uncharacterized Tn (tniB, tniA, and tniA) ( Figure 3B).
The bla NDM−1 -harboring Tn125-like element was flanked by the second and third copies of MITE, indicating a similar mechanism of MITE-mediated insertion to the Tn125-like element ( Figure 3B). Compared with the Tn125 prototype in pNDM-BJ01, the Tn125-like element in pP10164-NDM ( Figure 3B) exhibited absence of a downstream copy of ISAba125, as well as truncation of ISCR27 and an upstream copy of ISAba125.
In addition, three potential composite Tns, with the characteristic of being flanked by two separate copies of IS5 or IS1, were identified within the accessory module of pP10164-NDM or pKOX_NDM1, but none of the flanking DRs as target sites could be found.

Discussion
Horizontal transfer of plasmid-borne bla NDM genes enabled NDM enzymes to be rapidly spread in Enterobacteriaceae, less frequently in Acinetobacter, and rarely in Pseudomonas Johnson and Woodford, 2013;Dortet et al., 2014). Klebsiella pneumoniae and E. coli are the most commonly described bla NDM -carrying Enterobacteriaceae species, and bla NDM genes have been described in many other enterobacterial species, such as K. oxytoca, K. ozaenae,  Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Proteus mirabilis, Morganella morganii, Providencia spp., Serratia marcescens, and Salmonella enterica (Ou et al., 2014;Biedenbach et al., 2015). These NDM-producing bacteria have been shown to cause hospital-and community-acquired infections (Ou et al., 2014;Biedenbach et al., 2015). bla NDM−1positive bacteria are frequently found in environmental settings, indicating environmental origins of bla NDM genes in human pathogens, whereas use of antimicrobial drugs in an indiscriminate manner in some countries makes bla NDM−1positive bacteria spread easily and pose a serious public health threat Isozumi et al., 2012). This is the first report of bla NDM in L. adecarboxylata. The clinical L. adecarboxylata isolate characterized herein harbors a conjugative IncFII Y plasmid pP10164-NDM that encodes the NDM-1 enzyme. pP10164-NDM is highly similar to pKOX_NDM1, which is recovered from a nosocomial K. oxytoca strain from a patient following medical transfer from a hospital in Jiangxi to Taiwan, China (Huang et al., 2013). There are only two reports of bla NDM in R. ornithinolytica, one from India (Khajuria et al., 2013) and the other from China (Zhou et al., 2014), both of which were confined to surveillance of bla NDM genes. This work presents extended evidence that the production of NDM-1 by a conjugative IncN2 plasmid pYNKP001-NDM accounts for the carbapenem resistance of a clinical R. ornithinolytica isolate from China. pYNKP001-NDM is very similar to pTR3 (identified in K. pneumoniae from a Chinese patient) (Chen et al., 2012), pNDM-ECS01 (in E. coli from a Thai patient) (Netikul et al., 2014) and p271A (in E. coli from a patient following medical transfer from a hospital in Bangladesh to Australia) .
The bla NDM−1 genes of pYNKP001-NDM and pP10164-NDM are embedded in Tn125-like elements, which are characterized as two distinct truncated versions of the prototype NDM-encoding Tn125, as observed in pNDM-BJ01 (Hu et al., 2012).
The Tn125-like elements with a large array of derivatives represent the major genetic platforms for bla NDM genes across host bacteria Mcgann et al., 2012;Zhang et al., 2013;Zong and Zhang, 2013;Fiett et al., 2014;Jones et al., 2014;Mataseje et al., 2014). Notably, flanking of Tn125-like elements by MITE or its remnant as observed herein indicates that MITE facilitates transposition and mobilization of bla NDM−1 gene contexts. Figure S1 | Detection of carbapenemase activity. In the presence of any carbapenemase, relevant carbapenems are hydrolyzed and transformed into its carboxylic form, leading to a pH decrease, which is detected by a color change of phenol red solution (red to yellow-orange). Ambler class A carbapenemases are at least partially inhibited by tazobactam, whereas class B carbapenemases (metallo-ß-lactamases) are inhibited by divalent cation chelators, such as EDTA. There is no available chemical inhibitor for class D carbapenemases. In this study, the bla NDM−1 -positive strains R. ornithinolytica YNKP001 and L. adecarboxylata P10164, E. coli YNKP001-NDM-TB1 and P10164-NDM-EC600, and K. pneumoniae ATCC BAA-2146 (positive control) (Rasheed et al., 2013) exhibited class B carbapenemase activity. As expected, E. coli EC600 and DH10B had no carbapenemase activity.