Plasmid analysis of NDM metallo-β-lactamase-producing Enterobacterales isolated in Vietnam

Carbapenem-resistant Enterobacterales (CRE) represent a serious threat to public health due to the lack of treatment and high mortality. The rate of antimicrobial resistance of Enterobacterales isolates to major antimicrobials, including carbapenems, is much higher in Vietnam than in Western countries, but the reasons remain unknown due to the lack of genomic epidemiology research. A previous study suggested that carbapenem resistance genes, such as the carbapenemase gene blaNDM, spread via plasmids among Enterobacterales in Vietnam. In this study, we characterized blaNDM-carrying plasmids in Enterobacterales isolated in Vietnam, and identified several possible cases of horizontal transfer of plasmids both within and among species of bacteria. Twenty-five carbapenem-nonsusceptible isolates from a medical institution in Hanoi were sequenced on Illumina short-read sequencers, and 13 blaNDM-positive isolates, including isolates of Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Morganella morganii, and Proteus mirabilis, were further sequenced on an Oxford Nanopore Technologies long-read sequencer to obtain complete plasmid sequences. Almost identical 73 kb IncFII(pSE11)::IncN hybrid plasmids carrying blaNDM-1 were found in a P. mirabilis isolate and an M. morganii isolate. A 112 kb IncFII(pRSB107)::IncN hybrid plasmid carrying blaNDM-1 in an E. coli isolate had partially identical sequences with a 39 kb IncR plasmid carrying blaNDM-1 and an 88 kb IncFII(pHN7A8)::IncN hybrid plasmid in a C. freundii isolate. 148–149 kb IncFIA(Hl1)::IncA/C2 plasmids and 75–76 kb IncFII(Yp) plasmids, both carrying blaNDM-1 were shared among three sequence type 11 (ST11) isolates and three ST395 isolates of K. pneumoniae, respectively. Most of the plasmids co-carried genes conferring resistance to clinically relevant antimicrobials, including third-generation cephalosporins, aminoglycosides, and fluoroquinolones, in addition to blaNDM-1. These results provide insight into the genetic basis of CRE in Vietnam, and could help control nosocomial infections.


Introduction
Carbapenems have a broad spectrum of antimicrobial activity and are reserved for the treatment of infections caused by multidrug-resistant Gram-negative bacteria including Enterobacterales. The increase of cases infected by carbapenem-resistant Enterobacterales (CRE) is a serious threat to public health due to limited management of severe infections and risk of increased mortality [1,2]. Carbapenem-hydrolyzing β-lactamase (carbapenemase) genes, such as bla NDM , bla KPC , and bla  , that confer resistance to a broad range of β-lactams including third-generation cephalosporins and carbapenems are predominantly encoded on conjugative plasmids and have been transferred among Enterobacterales around the world [3]. There was a report that out of 27 carbapenem-resistant Klebsiella pneumoniae isolated from patients in a medical institution in Hanoi, Vietnam from 2014 to 2015, two isolates belonging to sequence type 395 (ST395) harbored bla NDM-1 , and five isolates belonging to ST15, ST16, and ST2353 harbored bla NDM-4 [4]. There was another report that six K. pneumoniae isolates with bla NDM-1 were isolated from water samples in Hanoi, Vietnam in 2011 and belonging to ST283 [5]. Furthermore, we previously detected bla NDM in 68.1% (47/69) of CRE isolates from 45 patients in a medical institution in Hanoi, Vietnam from 2010 to 2012, and some of isolates could be shown to transfer bla NDM to Escherichia coli [6], suggesting that bla NDM had spread via plasmids among Enterobacterales in medical institutions and communities in Vietnam.
In this study, we report the detailed structures of bla NDM-1 -carrying plasmids, and co-existing AMR genes and MGEs on the plasmids in Enterobacterales isolated in Vietnam. This knowledge provides insight into genetic characteristics and potential transmissions of the plasmids among Enterobacterales in Vietnam for the first time.

Putative transfer of bla NDM-1 -carrying plasmids between Enterobacterales isolated in Vietnam
To investigate the similarities of the bla NDM-1 -carrying plasmids in more detail, long-read sequencing was performed for 12 bla NDM-1 -positive Enterobacterales isolates, including seven K. pneumoniae belonging to ST11 (three isolates), ST395 (three isolates), and ST656 (one isolate), two C. freundii, one E. coli belonging to ST405, one M. morganii, and one P. mirabilis isolates, and one bla NDM-4 -positive K. pneumoniae isolate belonging to ST15 were further sequenced with the long-read sequencer. Hybrid analysis using short-read and long-read sequencing data resulted in complete sequences of 13 bla NDM -carrying plasmids that ranged from 39.2 kb to 345.7 kb sizes (covered by at least x125 long-read sequencing data; S2 Table). Interestingly, many bla NDM -carrying plasmids co-carried other clinically relevant AMR genes, such as 16S rRNA methyltransferases genes (9/13, 69.2%) and quinolone resistance genes (6/ 13, 46.2%) on the same plasmid (S2 Table).

Discussion
The Viet Nam Resistance (VINARES), a nationwide surveillance network consisting of 16 central and provincial-level medical institutions was established in Vietnam in 2013 [18]. According to VINARES data, the rates of resistance of K. pneumoniae to third-generation cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones were 66.4%, 17.1%, 29.5%, and 53.0%, respectively, in Vietnam [19], whereas the rates were 13.6%, 0.5%, 6.5%, and 8.7%, respectively, in the United Kingdom in 2013 [20], and 17.2%, 4.3%, 4.5%, and 16.8%, respectively, in the United States in 2010 [21]. Though the rates of resistance of Enterobacterales to major antimicrobials are much higher in Vietnam than in Western countries, the genetic causes remain unknown.
In this study, we performed comprehensive genetic analysis on plasmids carrying bla NDM carbapenemase genes in Enterobacterales isolated from patients in Vietnam. We focused on carbapenem-nonsusceptible Enterobacterales isolates harboring bla NDM , one of the most prevalent carbapenemase genes in the world, and completely sequenced 12 plasmids carrying bla NDM-1 from K. pneumoniae (ST11, ST395, and ST656), E. coli (ST405), C. freundii, M. morganii, and P. mirabilis isolates and one plasmid carrying bla NDM-4 from K. pneumoniae isolate (ST15). Nearly identical plasmids carrying bla NDM-1 were detected in different bacterial species of this study, suggesting that they represent common and important AMR plasmids disseminated in Vietnam (Figs 2, 4 and 5). Moreover, the co-existence of clinically relevant AMR genes, such as ESBL genes (e.g., bla CTX-M , bla OXA , and bla TEM ), aminoglycoside resistance genes (e.g., armA and rmt), and fluoroquinolone resistance genes (e.g., qep and qnr), with bla NDM-1 was observed in several plasmids, such as P. mirabilis pMH13-009N_1 (Fig 2) and K. pneumoniae pMH15-289M_1 (Fig 4). Hence, the spread of the plasmids both within and among species of bacteria would have caused an increase in multidrug-resistant bacteria and the infected hosts in Vietnam, and will pose a threat to public health.
Almost identical IncFII(pSE11)::IncN hybrid plasmids, pMH13-009N_1 and pMH16-367M_1, were found in P. mirabilis and M. morganii isolates, respectively (Fig 2A and 2B). These plasmids had identical sequences including a set of conjugation-associated T4SS genes with the IncN plasmid from S. enterica isolated in the United States (Fig 3A). IncN plasmids are prevalent in the microbiota of animals, and disseminate AMR genes such as bla CTX-M-1 and qnr [22], suggesting that IncN plasmids with the same origin of P. mirabilis pMH13-009N_1 and M. morganii pMH16-367M_1 could be disseminated in communities, including humans, animals, and the environment, in Vietnam and other countries.
The IncFII(pRSB107)::IncN hybrid plasmid pMH13-051M_1 in E. coli, had partial sequence identity with the IncR plasmid pMH17-012N_1 and the IncFII(pHN7A8)::IncN hybrid plasmid pMH17-012N_2 both in C. freundii (Fig 2C and 2D). Rearrangement of conjugation genes plays an important role in the dissemination of AMR genes between bacteria [23]. Because known IncR plasmids are non-transferable [24], an IncR plasmid carrying AMR genes (e.g. C. freundii pMH17-012N_1) and another plasmid carrying conjugation genes (e.g. C. freundii pMH17-012N_2) could be assumed to have been fused into a single plasmid with both AMR and conjugation genes (e.g. E. coli pMH13-051M_1) in a bacterium. Subsequently the plasmid was propagated by conjugation to another bacterium. There is an increasing number of reports of IncR plasmids carrying various AMR genes and the pool of AMR genes on IncR plasmids is thought to spread to transmissible plasmids via recombination, contributing to the high evolutionary plasticity of bacterial genomes [25]. Further analysis on the possible recombination events are needed to understand the ways of transmission of these plasmids between bacterial isolates and species.
bla NDM-1 was found on several hybrid plasmids, such as IncFII(pSE11)::IncN, IncFII (pRSB107)::IncN hybrid plasmids (Fig 2A and 2C), and IncFIA(Hl1)::IncA/C2 hybrid plasmids from three K. pneumoniae ST11 isolates (Fig 4). Hybrids of multiple replicons belonging to different incompatibility groups represent a plasmid strategy for expansion of host range and dissemination of acquired AMR genes among bacteria [27]. The IncFIA(Hl1)::IncA/C2 hybrid plasmids or IncFII(Yp) plasmids were reported from other regions, including the United States, Europe, and Asia (Figs 4 and 5). IncFIA(Hl1)::IncA/C2 plasmids and IncFII (Yp) plasmids were detected in K. pneumoniae in this study, suggesting that these plasmids and/or bacterial isolates would have also been spreading in medical institutions and community in Vietnam.
MLST analysis is helpful to estimate whether bacterial isolates belonging to the same STs were originated from the same clones and spread within the medical institution, and wholegenome SNV-based analysis enables us to estimate more accurate genetic relationship. According to a report [29], SNVs accumulated in K. pneumoniae ST258 isolates at a rate of 3.9 SNVs per year. Our analysis of SNVs of K. pneumoniae ST11 isolates (Fig 4) revealed 214 SNVs in MH16-398D relative to MH15-289M. This suggests that different ST11 clones are present in the medical institutions and communities. In support of this theory, a report [30] showed that K. pneumoniae isolates from medical institutions, sewage, canals, and agricultural waste water were intermixed in the phylogenetic classification. Because ST11 is one of major international epidemic clones of K. pneumoniae [31], an ST11 clone carrying AMR plasmid could have disseminated in a medical institution or local community in Hanoi, Vietnam. Another three ST395 isolates of K. pneumoniae (Fig 5) are very clonal based on the rate of SNV accumulation [29] and could be disseminated from the same clone within the medical institution because only a few SNVs were present in MH15-191M and MH13-055M relative to MH15-208H.

Conclusion
Plasmid analysis in this study shows the complex structures and diversity of plasmids carrying bla NDM-1 in Vietnam. Hybrid analysis with both Illumina short-read and ONT long-read sequencing is a promising method for detecting important AMR plasmids in CRE isolates and the real-time analysis is useful for controlling nosocomial infections.

Bacterial isolates
A total of 122 ESBL-producing Enterobacterales isolates were collected from selected specimens, including blood, sputum, urine, and pus from both in-patients and out-patients with infections in daily diagnosis in a reference medical institution in Hanoi, Vietnam between 2013 and 2017. All bacterial isolates were excluded from duplicates from the same patient. The medical institution has 700 beds, and includes 13 surgical departments and 10 internal medicine departments. The medical services are provided for both military and civil patients. Bacterial species identification, prediction of ESBL activity, and antimicrobial susceptibility testing were performed by Vitek 2 (BioMèrieux) and E-test (BioMèrieux). ESBL gene(s)-harboring isolates could also harbor other important AMR genes, including carbapenemase gene(s). According to the Clinical and Laboratory Standards Institute (CLSI) 2020 guidelines, breakpoints of meropenem and imipenem are �1 μg/mL (susceptible), 2 μg/mL (intermediate), �4 μg/mL (resistant), respectively. Carbapenemase activity was examined by CarbaNP test according to the CLSI 2020 guidelines. Major carbapenemase genes (bla NDM , bla KPC , bla OXA-48 , bla IMP , and bla VIM ) were detected by a multiplex PCR method [32]. Twenty-five carbapenem-nonsusceptible and carbapenemase-positive isolates (19 K. pneumoniae, two E. coli, two C. freundii, one M. morganii, and one P. mirabilis) were subjected to the whole-genome sequencing analysis described below.

Preparation of genomic DNA
For short-read sequencing, genomic DNAs of bacterial isolates were extracted with the phenol-chloroform method, and purified with QIAquick PCR Purification Kit (QIAGEN) according to the manufacturer's instructions. For long-read sequencing, high-molecular-weight genomic DNAs of bacterial isolates were extracted with MagAttract HMW DNA Kit (QIA-GEN) according to the manufacturer's instructions. The extracted genomic DNAs were quantified with a Qubit 2.0 fluorometer (Thermo Fisher Scientific).

Whole-genome sequencing and bioinformatics analysis
Whole-genome sequencing was performed to examine the prevalence of important AMR genes on plasmids. Long-read sequencing is useful for constructing complete sequences of whole plasmids and tracking horizontal transfer of AMR plasmids during nosocomial infections [33]. Recently, long-read nanopore sequencing using MinION nanopore sequencer from Oxford Nanopore Technologies (ONT) has been applied to such investigations [34,35].
Whole genome sequencing using MiniSeq or HiSeq systems (Illumina) was performed for phylogenetic and MLST analysis, and screening of acquired AMR genes and plasmid replicon types. Library for Illumina sequencing (insert size of 500-900 bp) was prepared using Nextera XT DNA Library Prep Kit (Illumina). Paired-end sequencing was performed using MiniSeq (2 x 150 bp) or HiSeq 4000 systems (2 x 150 bp). Trimming and de novo assembly of paired-end reads was performed using CLC Genomics Workbench v12.0 (QIAGEN) with default parameters. A maximum-likelihood phylogenetic tree was generated by PhyML from a concatenated core-gene alignment consisting of 13,305 SNVs constructed using the Roary pipeline (https:// sanger-pathogens.github.io/Roary/). For PhyML, we used the following parameters that indicate the GTR +G4 model of DNA substitution with estimation of the shape parameter of the gamma distribution by maximizing the likelihood: -m GTR -c 4 -a e.
Twelve Enterobacterales isolates with bla NDM-1 (seven K. pneumoniae, one E. coli, two C. freundii, one M. morganii, and one P. mirabilis) and one K. pneumoniae isolate with bla NDM-4 were further sequenced on MinION (ONT) using the SQK-RAD002 or SQK-RBK001 kits and R9.4 flowcells according to the manufacturer's instructions to obtain complete sequences of plasmids carrying carbapenem resistance genes. De novo assembly was performed using Canu v1.5 [36] and Miniasm [37] with default parameters. The overlap region in the assembled contig was detected by genome-scale sequence comparison using LAST (http://last.cbrc.jp) and was trimmed manually. Illumina paired-end reads were mapped onto the resulted circular sequences, and error correction was performed by extracting consensus of mapped reads using CLC Genomics Workbench v12.0 with default parameters.
Coding sequences (CDS) annotation using RASTtk on the PATRIC v3.6.3 server (https:// www.patricbrc.org) with default parameters. Sequence variant detection was performed using CLC Genomics Workbench v12.0 with default parameters of Fixed Ploidy Variant Detection (Ploidy = 1). Linear comparison of sequences was performed using BLAST with the default settings (the nucleotide collection database and the megablast program), and visualized by Easyfig (http://mjsull.github.io/Easyfig/). bla NDM-1 , other important AMR genes (ARG), type IV secretion system (T4SS)-associated genes involved in conjugation that were detected by SecReT4 program [38], and mobile gene elements (MGEs) were identified manually from CDS annotations and basically analyzed by comparing the sequences analyzed in previous studies.
Draft genome and complete plasmid sequences of carbapenem-nonsusceptible Enterobacterales isolated in Vietnam have been deposited at GenBank/EMBL/DDBJ under BioProject number PRJDB6655.
Supporting information S1 Table. Bacterial species and isolates, specimen types for the bacterial isolation, years in which the bacteria were isolated, and minimum inhibitory concentrations (MICs) of meropenem (MEM) and imipenem (IPM) of isolates are shown. Also, sequence types of multilocus sequence typing (MLST) analysis determined from genomes, carbapenemase genes detected by ResFinder in genomes, sizes and contigs of genomes, coverages in short-read sequencing, Illumina sequencing platforms, and accession numbers of genomes are shown. According to the CLSI 2020 guidelines, Breakpoints of meropenem and imipenem are as follows: �1 μg/mL, susceptible; 2 μg/mL, intermediate; �4 μg/mL, resistant (R). (TIFF) S2 Table. Bacterial species and isolates, and bla NDM -carrying plasmids are shown. Also, replicon types detected by PlasmidFinder and antimicrobial resistance genes (ARGs) detected by ResFinder in plasmids, sizes of plasmids, and coverages in long-read sequencing, and accession numbers of plasmids are shown. (TIFF)