Closely Related NDM-1-Encoding Plasmids from Escherichia coli and Klebsiella pneumoniae in Taiwan

Objective Two plasmids carrying bla NDM-1 isolated from carbapenem-resistant Klebsiella pneumoniae (CR-KP) and carbapenem-resistant Escherichia coli (CR-EC) were sequenced. CR-KP and CR-EC were isolated from two Taiwanese patients without travel histories. Methods Complete sequencing of the plasmids (pLK75 and pLK78) was conducted using a shotgun approach. Annotation of the contigs was performed using the RAST Server, followed by manual inspection and correction. Results These similar plasmids were obtained from two patients with overlapping stays at the same hospital. The pLK75 and pLK78 plasmids were 56,489-bp and 56,072-bp in length, respectively. Plasmid annotation revealed a common backbone similar to the IncN plasmid pR46. The regions flanking the bla NDM-1 genes in these plasmids were very similar to plasmid pNDM-HU01 in Japan, which contains a complex class 1 integron located next to an ISCR1 element. The ISCR1 element has been suggested to provide a powerful mechanism for mobilising antibiotic resistance genes. Conclusion Two indigenous NDM-1-producing Enterobacteriaceae cases were identified for the first time in Taiwan, highlighting the alarming introduction of NDM-1-producing Enterobacteriaceae in this region.


Introduction
New Delhi metallo-b-lactamase 1 (NDM-1) is a novel metallo-blactamase (MBL) that was first identified in 2009 [1]. Following the first case, NDM-1 positive bacteria have been detected in the UK, India, Pakistan and Bangladesh during 2008-2009 [2]. Since August 2010, NDM-1 isolates have been reported worldwide, including the United States and Canada, Europe, China, Japan, South Asia, Africa and Australia [3,4]. Because it has rapidly spread worldwide, NDM-1 has become a global public health concern in recent years.
Plasmids encoding bla NDM-1 were initially observed in Klebsiella pneumoniae and Escherichia coli. However, these plasmids can be conjugatively transferred into other species, which facilitates the rapid dissemination of bla NDM-1 into various bacteria. The bla NDM-1 gene has been identified on different types of plasmids (IncA/C, IncF, IncL/M, or untypable), and plasmids carrying bla NDM-1 appear to have a broad host range [5,6]. Many partial and complete plasmid sequences containing the bla NDM-1 gene have been published. Dissemination predominantly involves transfer of the bla NDM-1 gene among promiscuous plasmids and clonal outbreaks. Comparison of these plasmids shows that the regions neighbouring bla NDM-1 are quite variable, most likely reflecting multiple genetic modification events [6]. _EN-REF_4Thus, an understanding of the genetic feature of various resistance determinants carried by these bla NDM-1 plasmids is important for understanding the evolution of bla NDM-1 -carrying plasmids.
In Taiwan, the first case of NDM-1-producing Enterobacteriaceae colonisation was reported in 2010. The patient had been hospitalised for a gunshot injury in India and did not have symptoms or signs of infection during hospitalisation in Taipei [7]. Complete sequencing of the bla NDM-1 -carrying plasmid, pKPX-1, revealed a hybrid replicon of IncR and IncF genes [5]. The first episode of infection caused by NDM-1-producing K. pneumoniae appeared soon thereafter in 2010. The patient received a kidney transplant in China and was admitted to a Taiwan hospital due to abdominal pain and dysuria, and an NDM-1-carrying Klebsiella oxytoca strain was isolated from a pelvic abscess [8]. The bla NDM-1carrying plasmid, pKOX_NDM-1, was determined to carry a backbone with conjugation and replication genes similar to IncFIB and IncFII [6]. Here, we report the sequence annotation of pLK75 and pLK78, two similar bla NDM-1 -encoding plasmids, from two Taiwanese patients without travel histories to foreign countries. Notably, these two NDM-1-producing isolates represent the first indigenous cases in Taiwan [9].

Patient Characteristics
The study protocol was reviewed and approved by the Institutional Review Board of Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan. (TSGHIRB-100-05-205) Written informed consent to participants was provided in this study.
Patient 1 was an 82-year-old male with a history of hypertension, coronary artery disease, Parkinsonism, and pulmonary tuberculosis (under treatment for 3 months prior to the admission) and was a hepatitis B carrier. He was admitted for right lower lung pneumonia. CR-KP was isolated from his sputum specimen after two weeks of hospitalisation with antimicrobial therapy. The pneumonia was resolved after 3 days of ciprofloxacin followed by 6 days of ertapenem treatment.
Patient 2 was a 64-year-old female with a history of hypertension, diabetes mellitus, stroke, and end-stage renal disease with regular haemodialysis. She was admitted for respiratory failure and septic shock due to pneumonia. She received many classes of antimicrobial agents during the long hospitalisation period. CR-EC was isolated from the sputum for months after admission. However, she recovered after 14 days of cefepime treatment.
Because their hospital stays overlapped at the same hospital, a further genetic study and an outbreak investigation were performed.

Conjugation of plasmid and antimicrobial susceptibility of bla NDM-1 donors and their transconjugants
Plasmid transfer was carried out by conjugation. A rifampinresistant strain of E. coli (strain JP-995) was used as the recipient [10]. The recipient and donor strains were separately inoculated into brain heart infusion broth (Oxoid Ltd., Basingstoke, England) and incubated at 37uC for 4 h. The cells were then mixed at a ratio of 1:10 (by volume) and incubated overnight at 37uC. A 0.1ml sample of the overnight broth mixture was then spread onto a MacConkey agar plate containing rifampin (100 mg/ml) and imipenem (1 mg/ml). We did the PCR for transconjugants to confirm the transfer of bla NDM-1 . Only one plasmid was observed in each transconjugants.
Complete sequencing and annotation of plasmids pLK75 from CR-EC and pLK78 from CR-KP Complete sequencing of the plasmids was conducted using a shotgun approach; sequencing of 8-kb pair-end and shotgun libraries prepared from purified plasmid DNA was carried out using a 454 GS Junior (Roche, US). Gap filling between the contigs was accomplished by adding Sanger reads with the aid of Consed [11]. Annotation of the contigs was performed using the RAST Server [12], followed by manual inspection and correction. The complete nucleotide sequences and annotations of plasmids pLK75 and pLK78 were submitted to GenBank and assigned the sequence accession numbers KJ440076 and KJ440075, respectively (sequence S1 and S2). Multilocus sequence typing (MLST) performed for K. pneumoniae isolate was according to Diancourt et al. [13]. Allele sequences and sequence types were verified at http://www.pasteur. fr/recherche/genopole/PF8/mlst/Kpneumoniae.html.

Results and Discussion
The first indigenous NDM-1-producing bacteria in Taiwan To our knowledge, only four NDM-1-positive Enterobacteriaceae isolates have been reported in Taiwan to date. These four isolates were recovered from two Taiwanese patients with travel histories to foreign countries [15]. Because the patients in the present study had no such travel history, these 2 NDM-1producing bacteria are thought to be indigenous.
The patients' characteristics of two NDM-1-producing strains Tables 1 and 2 showed the patient clinical features and antimicrobial susceptibility testing results of the bla NDM-1 -carrying isolates. Both of the patients were elderly and immunocompromised. Both patients stayed in the same hospital for pneumonia, and NDM-1-producing bacteria were cultured from their sputum specimens after usage of several broad-spectrum antibiotics. These two isolates were hospital-acquired and suggested to be colonisation isolates because clinical improvement was achieved without effective antimicrobial therapy and no CR-EC or CR-KP was isolated from other sources of the patients. With strict infection and contact control practices, no further NDM-1-producing Enterobacteriaceae isolates were identified for one year. The isolation dates of these two patients' NDM-1-producing Enterobacteriaceae were within in one week of each other. Moreover, their patterns of antimicrobial susceptibility were quite similar. The transconjugants with a plasmid harbouring NDM-1 showed resistance to all b-lactams. The transconjugants converted from ciprofloxacin-resistant to ciprofloxacin-susceptible, indicating nonplasmid-mediated resistance in the donor strains. Both NDM-1carrying E. coli and K. pneumoniae strains were susceptible to amikacin, colistin, and tigecycline (Table 2).

Genetic features
Complete sequencing was performed for the two circular bla NDM-1 plasmids: pLK75 from E. coli and pLK78 from K. pneumoniae. The pLK75 and pLK78 plasmids were 56,489-bp and 56,072-bp in length, respectively. Annotation of the plasmids revealed a common backbone similar to the IncN plasmid pR46 [16] (Figure 1a, 1b). The majority of plasmid pR46 sequences (,72% of 50,969 bp) were preserved in both pLK75 and pLK78, including the repA gene for plasmid replication, tra genes for conjugal transfer, and genes responsible for plasmid stability. Although the tetA/tetR genes responsible for tetracycline resistance were also identified in these plasmids, a gene cluster responsible for arsenite resistance in pR46 was not found in pLK75 and pLK78. Plasmids pLK75 and pLK78 were similar to each other but contained a 26-kb inversion region located next to the sul1 gene of a class 1 integron downstream of the repA replication origin (Figure 1a, 1b). At the other end of the inversion region were tetA/tetR tetracycline resistance genes flanked by two 244-bp repeat sequences (Figure 1c). This 244-bp repeat region was flanked by inverted repeats (IRs) similar to the IRs of the Tn3 family (Figure 1c). Compared to pLK78, an additional copy of the 244-bp repeat was identified at the other end of the inversion region in pLK75 (Figure 1a). It is likely that the 244-bp Tn3-like repeat sequences may have facilitated the inversion of the 26-kb region. Both of the plasmids carried additional antimicrobial resistance genes to pR46, including bla NDM-1 (metallo-b-lactamase   NDM-1), sul1 (sulphonamide resistance gene), aadA1 (aminoglycoside resistance gene), aadA16 (streptomycin-spectinomycin resistance gene), and ble (bleomycin resistance gene). The IncN plasmid has been shown to encode clinically important resistance determinants, such as bla CTX-M , bla IMP , bla NDM , and bla KPC . This type of plasmid is known to be involved in the transmission of VIM-1, KPC-2, CTX-M-1, and NDM-1 among K. pneumoniae isolates and has also been prevalent in E. coli [17]. Highly efficient transmission of these plasmids may explain the diversity and worldwide spread of bla NDM-1 -carrying Enterobacteriaceae [18].
Genetic environment of bla NDM-1 in pLK75 and pLK78 The flanking region of the bla NDM-1 gene in these plasmids was very similar (,100% identity) to plasmid pNDM-HU01, of which the partially sequenced region, including qacEdelta1, aadA, bla NDM-1 , ble, trpF, ISCR1, and sul1, was reported from an E. coli isolate in Japan (GenBank AB769140). Additionally, the immediate region containing bla NDM-1 , including the upstream intergenic region of a truncated ISAba125, bla NDM-1 , the ble bleomycin resistance gene, and the trpF phosphoribosylanthranilate isomerase gene, represents a 'common neighbourhood' to bla NDM-1 , which is identical in almost all of the reported bla NDM-1containing sequences thus far [5,19]. This region was located next to an ISCR1 element in both plasmids. The genetic environment of the bla NDM-1 region represents a typical complex integron in pLK78 ( Figure 2), with the bla NDM-1 'common neighbourhood' region and the adjacent ISCR1 element located between two sul1 genes associated with a nearby class 1 integron (Figure 2). In pLK78, the gene annotation revealed a complex class 1 integron near the bla NDM-1 . While the two plasmids are almost identical, the complex class1 integron structure in pLK75 is truncated due to a 26-kb inversion. Therefore it is likely that pLK75 and pLK78 were derived from the same molecular ancestor, but pLK75 was altered subsequently by the inversion, which resulted in disruption of the integron structure.
ISCR1 elements may transpose adjacent DNA sequences by a mechanism termed rolling-circle transposition and are responsible for the mobilisation of antibiotic resistance genes [20,21]. It is likely that the ISCR1 element mediated the acquisition of the bla NDM-1 common region to the class 1 integron, presumably through recombination at the sul1 locus, to form the complex class 1 integron structure in pLK78 ( Figure 2). Previous analysis reveal that there is an advantage of bla NDM-1 embedded within integron ISCR1 complex since this element is known to be widely associated with the spreading of antibiotic resistance genes among bacteria [22]. Acquisition of bla NDM-1 to the IncN replicon, presumable with the aid of ISCR1 element, may have bolstered the spreading of the resistance among K. pneumoniae and E. coli.
In conclusion, we obtained the first indigenous NDM-1producing E. coli and K. pneumoniae isolates from patients in Taiwan without travel histories. Comparative analyses of the bla NDM-1 -encoding plasmids from E. coli and K. pneumoniae revealed the importance of tra genes and ISCR1. Considering the clinical association and genetic information, the tight connection of these two carbapenem-resistant Enterobacteriaceae is not surprising. Clinicians should be aware of the possible emergence of an epidemic of carbapenem-resistant Enterobacteriaceae related to the level of plasmid transmission.

Supporting Information
Sequence S1 Complete nucleotide sequences and annotations of plasmids pLK75.