Dissemination of Carbapenem-resistant Klebsiella pneumoniae clinical isolates with various combinations of Carbapenemases (KPC-2, NDM-1, NDM-4, and OXA-48) and 16S rRNA Methylases (RmtB and RmtC) in Vietnam

Methods Twenty-seven clinical isolates of carbapenem-resistant Klebsiella pneumoniae with MICs ≥4 mg/L for imipenem or meropenem were obtained from inpatients in a hospital in Vietnam. Antimicrobial susceptibility tests and whole genome sequencing were performed. Multilocus sequence typing and the presence of drug resistant genes were determined and a maximum-likelihood phylogenetic tree was constructed by SNP alignment of whole genome sequencing data. Results All the isolates harbored one of genes encoding carbapenemases, including KPC-2, NDM-1, NDM-4 and OXA-48. Of the isolates, 13 were resistant to arbekacin with MICs ≥256 mg/L and to amikacin with MICs ≥512 mg/L. These isolates harbored a gene encoding a 16S rRNA methylase, either RmtB or RmtC. Eighteen and 4 isolates belonged to international clones, ST15 and ST16, respectively. None of the isolates had colistin-resistant factors. Conclusion Carbapenem-resistant K. pneumoniae isolates belonged to international clones spread in a medical setting in Vietnam, and that these isolates harbored genes encoding various combinations of carbapenemases and 16S rRNA methylases. This is the first report of KPC-2, NDM-4 and OXA-48 producers in a medical setting in Vietnam.


Background
Emergence of carbapenemase-producing Klebsiella pneumoniae isolates has become serious problems worldwide [1]. These isolates produce several carbapenemases belonging to class A, B, and D, including KPCs, NDMs and OXA-48, respectively [2]. KPC-1 was initially found in a carbapenem-resistant strain K. pneumoniae 1534, which was collected in a surveillance during 1996 to 1997 in the United States hospitals [3]. NDM-1 was initially identified in K. pneumoniae and Escherichia coli in 2009 in Sweden [4]. Since then, NDM-1-producing Enterobacteriaceae have been reported worldwide [5]. OXA-48 was first identified in K. pneumoniae 11,978, which was isolated in 2001 in Turkey [6].

Bacterial strains and antimicrobial susceptibility
Twenty-seven K. pneumoniae isolates with minimum inhibitory concentrations (MICs) ≥4 mg/L for imipenem or meropenem were obtained from 27 inpatients treated at a hospital, Vietnam, from from February 2014 to April 2015. Of them, 22 isolates were from respiratory tracts, 3 from pus samples, 1 from a bile sample, and 1 from a urine sample. The isolates were phenotypically identified and species identification was confirmed by 16S rRNA sequencing. MICs were determined using the microdilution method, according to the guidelines of the Clinical Laboratory Standards Institute (M100-S25). The colistin MICs were also determined by Etest in colistin-resistant isolates evaluated by broth microdilution method.

MLST and phylogenetic analysis
Multilocus sequence types (MLSTs) were deduced as described in the protocols of the Institut Pasteur MLST (IP-MLST) (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html) databases. Clonal complexes (CC) were determined by eBURST version 3 (http://eburst.mlst.net). Single nucleotide polymorphisms (SNPs) of the genome sequences of all carbapenem-resistant isolates tested were identified by comparisons with the sequence of NDM-1 producing ST15 K. pneumoniae PMK1, (Gen Bank accession no. CP008929), with all the reads of each isolate aligned against the PMK1 sequence using CLC Genomic Workbench version 9.0.1. SNP concatenated sequences were aligned using MAFFT (http:/mafft.cbrc.jp/ alignment/server/). Phylogenetic trees were constructed from the SNP concatemers. Models and parameters used for the phylogenetic analyses were computed using j-Model Test-2.1.4. A maximum-likelihood phylogenetic tree was constructed from SNP alignment with PhyML 3.0.

Pulsed-field gel electrophoresis and southern hybridization
The plasmids in each ST strain were extracted and pulsedfield gel electrophoresis was performed as describing previously [8]. Probes for bla KPC-2 , bla NDMs and bla OXA-48 were amplified by PCR using the primer sets as follows; KPC-F-TCGCTAAACTCGAACAGG and KPC-R-TTAC TGCCCGTTGACGCCCAATCC for bla KPC-2 , NDM-F-T TGGCCTTGCTGTCCTTG and NDM-R-ACACCAGTG ACAATATCACCG for bla NDMs and OXA-48-F-TGTT TTTGGTGGCATCGAT and OXA-48-R-GTAAMRATGC TTGGTTCGC for bla OXA-48 , respectively. Signal detection was carried out using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Applied Science, Indianapolis, IN).

Nucleotide sequence accession numbers
The whole genome sequences of all 27 isolates have been deposited at GenBank as accession numbers DRA005275.

Genetic environments surrounding genes encoding carbapenemases
The genetic structure surrounding bla KPC-2 , bla NDM-1 , bla NDM-4 and bla OXA-48 were shown in Fig. 1. The genomic structure surrounding bla KPC-2 was identical with that of Aeromonas hydrophila strain WCHAH01 plasmid pKPC2 (GenBank accession no. KR014106), which was isolated in China.
All isolates harboring bla NDM-1 tested had the same genetic structure surrounding bla NDM-1 (Fig. 1), which was identical with that of plasmid pRIH26 in NDM-1 producing K. pneumoniae isolated from a patient in 2012 in Rhode Island, the United States [9]. This patient had returned to the United States after a hospitalization in Vietnam [9].
All isolates harboring bla NDM-4 tested had the same genetic structure surrounding bla NDM-4 (Fig. 1), which was identical to that of NDM-1 producing K. pneumoniae strain KP4 plasmid pKP04NDM isolated in China (GenBank accession no. KU314941).
The bla KPC-2 , bla NDM-1 , bla NDM-4 and bla OXA-48 in each ST strain will be all located on plasmids and the sizes of the plasmids were shown in Table 1.

Discussion
To our knowledge, this is the first report of the whole genome based molecular epidemiological analysis of carbapenem-resistant K. pneumoniae in Vietnam. Our study suggests that carbapenem-producing ST15 K. pneumoniae have been spreading in medical settings in Vietnam. A NDM-1 producing K. pneumoniae clinical isolate in Vietnam was firstly obtained from a urinary tract of a 62-year-old man in 2010 [10]. This is the first report of NDM-4 or OXA-48 producing K. pneumoniae in Vietnam. NDM-4 was firstly detected in E. coli I5, which was recovered from a urine sample of a patient hospitalized in 2010 in India [11]. Since then, NDM-4 producers were reported in Enterobacter cloacae in Sri Lanka [12], E. coli in India [13], Italy [14] and Vietnam [15], and K. pneumoniae in Japan [16]. NDM-4 possessed increased hydrolytic activity toward carbapenems and several cephalosporins compared to NDM-1 [11]. NDM-4 with an amino acid substitution at position 130 (Met to Leu) showed increased hydrolytic activity toward carbapenems and several cephalosporins compared to NDM-1 [11].
ST15 will be an emerging high-risk multidrug-resistant clone with carbapenem-encoding genes, including bla KPCs , bla NDMs and bla OXAs . Outbreaks caused by ST15 OXA-48 produces were reported in France and Spain [2]. When Diancourt et al. [17] developed a MLST for K. pneumoniae Fig. 2 a Molecular phylogeny of the 27 K. pneumoniae strains. A maximum-likelihood phylogenetic tree was constructed from the 27 carbapenem-resistant isolates. The isolates belonging to ST15 formed the largest clade among the 27 isolates. b Molecular phylogeny of the 18 K. pneumoniae strains belonging to ST15. A maximum-likelihood phylogenetic tree was constructed from the 18 carbapenem-resistant isolates. Of them, 16 belonging to subclade A harbored bla OXA-48 and 2 belonging to subclade B harbored bla  in 2005, they already detected ST15 isolates from several countries in Europe, such as Austria, France, Portugal and Poland, and the most of the isolates were resistant to ceftazidime and ciprofloxacin. ST15 K. pneumoniae isolates were reported to spread in medical settings in 2005 in Hungary [2]. ST15 K. pneumoniae isolates were detected in other European countries, including Bulgaria, Croatia, Czech Republic, Denmark, Hungary, Italy, Netherlands, and Spain [2]; they were also detected in Asian countries, including China, South Korea, Malaysia, Singapore, Thailand, and Vietnam [2]; in African countries, including Côte d'Ivoire, Madagascar, Morocco, and Senegal [18]. These ST15 isolates frequently produced ESBL, including CTX-M, SHV-28 and TEM variants [19], and moreover, they became to produce various carbapenemases, including KPCs, OXA-48, NDMs and VIM-4 [19]. One of the wellrecognized high-risk clones is CC258 which is frequently associated with KPCs-producing K. pneumoniae known as a high-risk clone [19], and these isolates were reported many countries, such as the United States, Greek, Norway, Sweden, Italy, Poland, Canada, Brazil and Korea [19]. ST11, a related clone ST258, was reported in KPCs-producing isolates mainly in China, but also in NDMs-producers from Czech Republic, Switzerland, Thailand, Australia, the United States, the United Arab Emirates and Greece [19].
Etest seems a more reliable method to measure colistin MICs than broth microdilution method [20,21]. In the present study, 11 isolates were resistant to colistin with MICs 4-32 mg/L by broth microdilution method, although none of the isolates had colistin-resistant factors. Our previous study indicated that Enterobacteriacae isolates showed lower colistin MICs by Etest than by broth microdilution method [20]. It is necessary to find feasible susceptibility testing methods of determining the MICs of polymyxins for clinical laboratories.

Conclusions
This study showed that carbapenem-resistant K. pneumoniae isolates belonged to international clones spread, and that these isolates harbored genes encoding various combinations of carbapenemases and 16S rRNA methylases, in a medical setting in Vietnam.