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Genetic characterization of blaNDM-harboring plasmids in carbapenem-resistant Escherichia coli from Myanmar

  • Yo Sugawara ,

    Roles Data curation, Investigation, Writing – original draft

    akeda@biken.osaka-u.ac.jp (YA); y1yo2yo3@biken.osaka-u.ac.jp (YS)

    Affiliation Thailand–Japan Research Collaboration Center on Emerging and Re-emerging Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

  • Yukihiro Akeda ,

    Roles Conceptualization, Investigation, Writing – original draft

    akeda@biken.osaka-u.ac.jp (YA); y1yo2yo3@biken.osaka-u.ac.jp (YS)

    Affiliations Thailand–Japan Research Collaboration Center on Emerging and Re-emerging Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Japan, Division of Infection Control and Prevention, Osaka University Hospital, Suita, Japan

  • Noriko Sakamoto,

    Roles Data curation, Investigation

    Affiliation Thailand–Japan Research Collaboration Center on Emerging and Re-emerging Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

  • Dan Takeuchi,

    Roles Data curation, Investigation

    Affiliation Thailand–Japan Research Collaboration Center on Emerging and Re-emerging Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

  • Daisuke Motooka,

    Roles Data curation

    Affiliation Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

  • Shota Nakamura,

    Roles Data curation

    Affiliation Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

  • Hideharu Hagiya,

    Roles Investigation

    Affiliation Division of Infection Control and Prevention, Osaka University Hospital, Suita, Japan

  • Norihisa Yamamoto,

    Roles Investigation

    Affiliation Division of Infection Control and Prevention, Osaka University Hospital, Suita, Japan

  • Isao Nishi,

    Roles Investigation

    Affiliation Laboratory for Clinical Investigation, Osaka University Hospital, Suita, Japan

  • Hisao Yoshida,

    Roles Investigation

    Affiliation Division of Infection Control and Prevention, Osaka University Hospital, Suita, Japan

  • Kazuhisa Okada,

    Roles Conceptualization

    Affiliation Thailand–Japan Research Collaboration Center on Emerging and Re-emerging Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

  • Khwar Nyo Zin,

    Roles Investigation

    Affiliation Clinical Laboratory Department, Yangon General Hospital, Yangon, Myanmar

  • Mya Mya Aye,

    Roles Investigation

    Affiliation Bacteriology Research Division, Department of Medical Research, Yangon, Myanmar

  • Kazunori Tonomo,

    Roles Supervision

    Affiliation Division of Infection Control and Prevention, Osaka University Hospital, Suita, Japan

  • Shigeyuki Hamada

    Roles Conceptualization, Supervision, Writing – review & editing

    Affiliation Thailand–Japan Research Collaboration Center on Emerging and Re-emerging Infections, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

Correction

6 Dec 2017: The PLOS ONE Staff (2017) Correction: Genetic characterization of blaNDM-harboring plasmids in carbapenem-resistant Escherichia coli from Myanmar. PLOS ONE 12(12): e0189435. https://doi.org/10.1371/journal.pone.0189435 View correction

Abstract

The bacterial enzyme New Delhi metallo-β-lactamase hydrolyzes almost all β-lactam antibiotics, including carbapenems, which are drugs of last resort for severe bacterial infections. The spread of carbapenem-resistant Enterobacteriaceae that carry the New Delhi metallo-β-lactamase gene, blaNDM, poses a serious threat to public health. In this study, we genetically characterized eight carbapenem-resistant Escherichia coli isolates from a tertiary care hospital in Yangon, Myanmar. The eight isolates belonged to five multilocus-sequence types and harbored multiple antimicrobial-resistance genes, resulting in resistance against nearly all of the antimicrobial agents tested, except colistin and fosfomycin. Nine plasmids harboring blaNDM genes were identified from these isolates. Multiple blaNDM genes were found in the distinct Inc-replicon types of the following plasmids: an IncA/C2 plasmid harboring blaNDM-1 (n = 1), IncX3 plasmids harboring blaNDM-4 (n = 2) or blaNDM-7 (n = 1), IncFII plasmids harboring blaNDM-4 (n = 1) or blaNDM-5 (n = 3), and a multireplicon F plasmid harboring blaNDM-5 (n = 1). Comparative analysis highlighted the diversity of the blaNDM-harboring plasmids and their distinct characteristics, which depended on plasmid replicon types. The results indicate circulation of phylogenetically distinct strains of carbapenem-resistant E. coli with various plasmids harboring blaNDM genes in the hospital.

Introduction

Carbapenems are broad-spectrum antibiotics used as the last line of defense against multidrug-resistant bacteria; however, infections with carbapenem-resistant Enterobacteriaceae (CRE) have been increasingly reported since the early 2000s [1], [2]. CRE are resistant to most commonly prescribed antibiotics; therefore, CRE infections are associated with poor prognosis and pose a serious threat in clinical settings [3]. Carbapenem resistance in Enterobacteriaceae is primarily due to carbapenemases; plasmid-borne β-lactamases that hydrolyze carbapenems [4]. Several types of carbapenemases, such as KPC, OXA-48, VIM, IMP, and New Delhi metallo-β-lactamase (NDM), have been identified worldwide [5].

Routine worldwide surveillance is essential to understand and prevent CRE transmission; however, in many countries, including Myanmar, surveillance is limited or non-existent. Recently, Myat et al. reported the isolation of three Escherichia coli and three Klebsiella pneumoniae strains harboring blaNDM genes in a screening of 592 blood cultures in three hospitals in Yangon, Myanmar [6]. No other carbapenemase-encoding genes were identified in their study, suggesting that NDM rather than other carbapenemases is prevalent in Myanmar. The blaNDM genes are usually located adjacent to or in between mobile genetic elements, including transposons and insertion sequences, which facilitate transposition between replicons [7], [8]. Accordingly, blaNDM genes are currently found in an array of plasmid replicon types, such as IncF, IncX3, IncL/M, and IncH, as well as in plasmids with a broad-host-range, including IncA/C2. Consequently, the blaNDM gene has spread from the putative original reservoir, Acinetobacter, to enteric bacteria [9]. Additionally, accumulation of nucleotide substitutions in blaNDM has produced several variants from the originally identified enzyme NDM-1 [10]. Organisms harboring blaNDM genes have been detected not only in hospitals, but also in the environment in some Asian countries [11], [12]. Therefore, it is necessary to characterize the bacterial host, the blaNDM-harboring plasmid, and the genetic environment associated with blaNDM in order to understand the gene acquisition mechanism, track its spread, and investigate possible preventive measures [9].

In this study, we genetically characterized eight carbapenem-resistant E. coli isolates from a tertiary care hospital in Yangon, Myanmar, using whole-genome sequencing (WGS). Comparative analysis highlighted the diversity of nine plasmids carrying blaNDM, providing insight into an evolutionary relationship with the already identified blaNDM plasmids.

Materials and methods

Bacterial isolates

E. coli isolates were obtained from clinical blood or urine specimens collected from patients at Yangon General Hospital, Yangon, Myanmar, from April to August 2015. Ethical approval for the collection of patient specimens was obtained from the Ethics Committee of Osaka University Graduate School of Medicine and the Department of Medical Research, Myanmar, with a waiver of informed consent. All samples were anonymized before analysis. Specimens were cultured on blood agar plates at 37°C overnight, and each single colony formed was subjected to species identification and antimicrobial-susceptibility testing using a VITEK2 automated system (Sysmex bioMérieux, Marcy l’Etoile, France). Enterobacteriaceae strains with minimum inhibitory concentration (MIC) > 2 μg/mL for meropenem were collected and stored at −80°C in phosphate-buffered saline supplemented with 25% glycerol. Isolates were also grown on M-ECC [13], CHROMagar ECC (CHROMagar Microbiology, Paris, France) supplemented with 0.25 μg/mL meropenem and 70 μg/mL ZnSO4, and analyzed using an API 20E system (Sysmex bioMérieux) and an EIKEN dry plate (Eiken Chemical, Tokyo, Japan) to reconfirm species identity and antimicrobial susceptibility.

WGS analysis

Isolates were subjected to WGS using PacBio RSII (Pacific Biosciences, Menlo Park, CA, USA) and MiSeq systems (Illumina, San Diego, CA, USA). To prepare genomic DNA, bacterial isolates were cultured overnight in brain-heart-infusion broth (BD Bacto, Franklin Lakes, NJ, USA) supplemented with 0.25 μg/mL meropenem, and DNA was isolated using a PowerSoil DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA). To prepare libraries for PacBio RSII, 10 μg DNA was sheared using g-TUBE (Covaris, Woburn, MA, USA) to obtain 10,000–20,000-bp fragments, which were ligated to SMRTbell adapters (Pacific Biosciences). The fragments were processed with a DNA template prep kit 1.0 and a DNA/polymerase binding kit (P6v2; Pacific Biosciences) according to manufacturer’s instructions. Then, the fragments were sequenced with a DNA sequencing kit C4 (Pacific Biosciences), and the sequence reads were assembled de novo using HGAP version 2.1.0 with default parameters [14]. The sequences were submitted to the DDBJ/Genbank/EMBL database under accession numbers DRX064631 (M105), DRX064632 (M107), DRX064633 (M109), DRX064634 (M110), DRX064635 (M213), DRX064636 (M214), DRX064638 (M216), and DRX064639 (M217). To prepare libraries for MiSeq analysis, 200 ng of genomic DNA was sheared using Covaris S220 (Covaris) and processed with KAPA library preparation kits (KAPA Biosystems, Boston, MA, USA). Paired-end sequencing with 250-bp reads was performed using a MiSeq version 2 500 cycle kit (Illumina). Reads were mapped to the sequence generated from PacBio RSII using mapping software in CLC Genomics Workbench version 7.5 (CLC Bio, Aarhus, Denmark), and the consensus sequence was extracted for further analysis. The plasmid sequences were submitted to the DDBJ/Genbank/EMBL database under accession numbers AP018136 (pM105_FII), AP018137 (pM105_mF), AP018138 (pM107_FII), AP018139 (pM109_FII), AP018140 (pM110_FII), AP018141 (pM110_X3), AP018142 (pM213_X3), AP018143 (pM214_AC2), AP018144 (pM214_FII), AP018145 (pM216_AC2), AP018146 (pM216_X3), and AP018147 (pM217_FII).

A phylogenetic tree of the E. coli isolates based on single-nucleotide polymorphisms (SNPs) was constructed using CSI Phylogeny 1.3 [15]. Multilocus-sequence typing, plasmid-replicon typing, and identification of resistance genes were carried out using MLST 1.8 [16], PlasmidFinder 1.3 [17], and ResFinder 2.1 [18], respectively. Genomic sequences were annotated with RASTtk [19] and MiGAP [20], and genetic structure was compared in EasyFig [21]. A maximum-likelihood tree for IncFII plasmids was constructed using MEGA7 [22].

Bacterial conjugation and transformation

All blaNDM-harboring plasmids identified in this study were transferred to laboratory strains by conjugation or transformation and the presence of blaNDM and the plasmid-replicon type was confirmed by PCR [2325].

Bacterial conjugation was carried out as described previously [26], with some modifications. Luria-Bertani (LB) broth cultures of E. coli isolates in the early exponential phase were mixed with the recipient strain, E. coli ML4909 [27], at a 1:10 ratio. The bacterial mixture was pelleted by centrifugation, transferred onto nitrocellulose membranes on an LB agar plate, and incubated at 37°C for 6 h. Transconjugants were selected on the LB plate supplemented with 2 μg/mL meropenem and 100 μg/mL rifampicin.

For transformation, plasmids were extracted from overnight cultures of the isolates using the Plasmid Midi kit (Qiagen, Hilden, Germany). HST08 (Takara Bio, Shiga, Japan), a derivative of E. coli K12, was electroporated with the extracted plasmids using a Gene Pulser Xcell System (Bio-Rad, Hercules, CA, USA), and transformants were selected on brain-heart-infusion agar (BD Bacto) supplemented with 0.25 μg/mL meropenem.

Results and discussion

Characterization of carbapenem-resistant E. coli

Carbapenem-resistant E. coli strains (n = 8) were isolated at Yangon General Hospital, Yangon, Myanmar, from April to August 2015. Patients that tested positive for carbapenem-resistant E. coli were admitted to the following wards: hematology (n = 5), surgery (n = 2), and physical medicine (n = 1). Six isolates were obtained from blood specimens derived from patients in the hematology (M105, M109, M110, M214, and M217) and physical medicine (M107) wards, and two were isolated from urine derived from patients in the surgery ward (M213 and M216). All eight isolates were non-susceptible to not only β-lactams, including carbapenems, but also most other antibiotics tested, including aminoglycoside, quinolone, and chloramphenicol (Table 1). Interestingly, isolates from urine were highly resistant to chloramphenicol (MIC > 128), unlike isolates from blood (MIC: 8–32). Colistin appeared to be the only drug effective against all of the isolates examined, with fosfomycin representing a viable alternative for all strains, except for M110 (MIC: 16). By database searches using ResFinder and PlasmidFinder, all isolates were found to carry different types of plasmids encoding several antimicrobial-resistance determinants, including β-lactamases, supporting their multidrug-resistance phenotype (Fig 1B and S1 Table). Phylogenetic analysis based on genome-wide SNPs showed that the isolates belong to different previously defined phylogroups [28], with some belonging to pathogenic phylogroups D and F and others belonging to the commensal and less virulent phylogroups A and B1 (Fig 1A). The isolates were further classified into five multilocus-sequence types; no predominant sequence types were found (Fig 1B). Notably, the five isolates from the hematology ward are not closely related genetically, except for M105 and M109, indicating that different resistant strains disseminated in the ward. M105 and M109 differ in the types of NDM and blaNDM-harboring plasmid (Fig 1B), suggesting that they had acquired the plasmids independently. This is in contrast to clonal expansion of other drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus, that cause nosocomial infections. A database search with WGS and Sanger-sequencing sequence data indicated that the isolates harbor different blaNDM variants (blaNDM-1, blaNDM-4, blaNDM-5, and blaNDM-7) (Fig 1B) carried by nine distinct plasmids, of which eight belong to the known replicon types IncA/C2 (n = 1), IncX3 (n = 3), and IncFII (n = 4). The remaining plasmid is comprised of replication genes of three replicon types (IncFIA, FIB, and Q1). Additionally, isolate M214 harbors two different types of blaNDM-carrying plasmids (IncFII and A/C2).

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Table 1. MICs of antimicrobial agents for E. coli clinical isolates from Myanmar.

https://doi.org/10.1371/journal.pone.0184720.t001

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Fig 1. Phylogeny of carbapenem-resistant E. coli isolated in Myanmar and features of their blaNDM-harboring plasmids.

(A) Phylogenetic tree of carbapenem-resistant E. coli isolates based on WGS. Reference genomes and several other available genomes were included for comparison. Phylogroups of the reference strains are indicated in boxes. (B) Multilocus-sequence types, NDM types, plasmid-replicon types, and antimicrobial-resistance profiles determined by database search. In strain M214, NDM types are indicated by numbers on black-shaded boxes.

https://doi.org/10.1371/journal.pone.0184720.g001

IncA/C2 plasmids

IncA/C2 plasmids are broad-host-range plasmids that are predominantly found among NDM-1 producers [29,30]. Indeed, a plasmid we designated as pM214_A/C2 represents an IncA/C2 plasmid harboring blaNDM-1 (Fig 2A). A BLAST search indicated that pM214_A/C2 shares the highest degree of similarity with pNDM-1_Dok01 (GenBank accession no. AP012208.1), a blaNDM-1 plasmid isolated from a clinical E. coli isolate in Japan, but of Indian origin, with 99% identity and 93% query coverage [31]. Both plasmids encode blaNDM-1 in the ARI-A region, a resistance island designated for IncA/C2 [32]; however, the gene clusters containing blaNDM-1 are inverted between the two plasmids. In pM214_A/C2, the resistance island consists of two parts: an ISAba125-mediated composite transposon (Tn125) and a Tn1548-like transposon. The former region, which harbors blaNDM-1, completely matches that in pNDM-BJ01 (GenBank accession no. JQ001791.1) (Fig 2B), which was isolated from a clinical isolate of Acinetobacter lwofii [33]. Notably, Tn125 is often detected in Acinetobacter; however, the intact form of this transposon has never been found in Enterobacteriaceae [34]. This gene cluster is bracketed by IS26-insertion sequences in pM214_ A/C2, suggesting that the plasmid acquired blaNDM-1 from pNDM-BJ01 or a closely related plasmid via a single IS26-mediated gene transfer. Additionally, the Tn1548-like transposon is located adjacent to Tn125 (Fig 2B). This 15,525-bp fragment contains a class 1 integron comprising several antimicrobial-resistance genes and is identical to an unrelated plasmid (pCTX-M-3, GenBank accession no. AF550415.2) [35], except for one nucleotide. This cluster is partially conserved in other IncA/C2 plasmids, such as pNDM-1_Dok01 and pM216_A/C2, isolated from strain M216. Therefore, a Tn1548-like transposon might have contributed to the ARI-A island in the putative ancestor of these plasmids, with prototypic features being well conserved in pM214_A/C2. However, pM216_A/C2 encodes neither blaNDM-1 nor blaCMY-4, both of which are present in pM214_A/C2, but not in the putative precursor plasmid pR148 (GenBank accession no. JX141473.1) [36]. Moreover, the gene array in the ARI-A resistance island significantly differs from that in pM214_A/C2 (Fig 2A), suggesting that these two plasmids were derived from different precursors.

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Fig 2. IncA/C2 plasmids harboring blaNDM-1.

(A) Comparison of IncA/C2 plasmids isolated in this study with similar and/or putative backbone plasmids pNDM-1_Dok01 and pR148. Homologous regions are shaded dark gray. Genes are indicated in colors as follows: blaNDM-1, red; other β-lactamases, purple; other antimicrobial-resistance genes, orange; IS26, blue; other mobile genetic elements, cyan; other accessory genes, light gray. The origins of the indicated plasmids are shown in parentheses. pM214_ A/C2 and pNDM-1_Dok01 harbor the class C β-lactamase gene, blaCMY-4. (B) Schematic representation of the genetic context of blaNDM-1 in pM214_ A/C2 and in other plasmids.

https://doi.org/10.1371/journal.pone.0184720.g002

IncX3 plasmids

We detected three IncX3 plasmids encoding NDM variants: the blaNDM-4-harboring plasmids pM213_X3 and pM216_X3 and the blaNDM-7-harboring plasmid pM110_X3. The three plasmids are highly similar to previously reported IncX3 plasmids: the blaNDM-4-harboring plasmid pJEG027 (GenBank accession no. KM400601.1) [37], the blaNDM-5-harboring plasmid pNDM_MGR194 (KF220657.1) [38], and the blaNDM-7-harboring plasmids pKpN01-NDM7 (CP012990.1) [39] and pOM26-1 (KP776609.1), suggesting they share a common ancestor (Fig 3A and 3B). Notably, pJEG027 was isolated from a K. pneumoniae isolate from an Australian patient hospitalized in Myanmar before returning to Australia [37]. In these IncX3 plasmids, the blaNDM genes are encoded in three distinct structures with various lengths of upstream insertion sequences as shown in Fig 3A. Additionally, all of the plasmids found in the survey lack a 92-bp fragment upstream of taxD as compared with pJEG027 and pNDM_MGR194 (Fig 3A, arrow) and as previously observed in the blaNDM-7 plasmids pKpN01-NDM7 and pOM26-1 [37]. The IncX3 plasmids do not carry antimicrobial-resistance genes other than blaNDM and apparently represent early steps in the evolution and spread of the blaNDM gene [38]. Nonetheless, these plasmids encode distinct NDM variants. Therefore, it is likely that blaNDM variants have evolved via nucleotide substitutions within the IncX3 plasmid [37], [39]. This process might be driven by selection for stronger resistance, given that NDM-4, -5, and -7 exhibit higher β-lactamase activity than does NDM-1 [4042].

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Fig 3. IncX3 plasmids harboring blaNDM.

(A) Schematic representation of the blaNDM-4 plasmid pM216_X3 and comparison of the genetic context of blaNDM in IncX3 plasmids. Genes are indicated in colors as defined in Fig 2, except that genes involved in conjugal transfer are depicted in green. A 92-bp deletion that is found in pM110_X3, pM213_X3, pM216_X3, pKpN01-NDM7, and pOM26-1 is indicated by the arrow. (B) Evolutionary relationship of fully sequenced IncX3 plasmids harboring blaNDM as inferred from genetic variances. Encoded NDM variants are shown in parentheses along with geographical origin. The plasmids identified in this study are boxed. pJEG027, pNDM-MGR194, and pKpN01-NDM7 were isolated from K. pneumoniae isolates and are shaded gray. pOM26-1 was isolated from an E. coli isolate. SNS, single nucleotide substitution.

https://doi.org/10.1371/journal.pone.0184720.g003

IncFII plasmids

IncFII was the most predominant type of blaNDM-harboring plasmid in the isolates investigated. Here, we isolated the blaNDM-4-harboring plasmid pM109_FII and the blaNDM-5-harboring plasmids pM107_FII, pM214_FII, and pM217_FII (Fig 4A). In contrast to IncX3 plasmids, these plasmids carry other β-lactamases, as well as resistance genes against several antibiotics, including aminoglycosides, macrolides, sulphonamides, and trimethoprims. The overall genetic structure of these plasmids resembles that of known blaNDM IncFII plasmids, such as pGUE-NDM (GenBank accession no. JQ364967.1) [43], pMC-NDM (HG003695.1) [44], and pCC1409-1 (KT725789.1)/pCC1410-1 (KT725788.1) [45], which were found in clinical isolates originating from India, Congo, and United Arab Emirates, respectively. Nevertheless, the genetic structure of the IncFII plasmids is relatively diverse as compared to those of IncX3 plasmids, as we found several insertions/deletions and clear traces of genetic mobilization via IS26. pM109_FII, p214_FII, and p217_FII contain a complete transfer operon and were successfully transferred to E. coli ML4909 by conjugation. However, a fragment was missing from pM107_FII (Fig 4A, arrowhead), rendering the transfer operon incompetent.

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Fig 4. IncFII plasmids harboring blaNDM.

(A) Comparison of IncFII plasmids harboring blaNDM in Myanmar isolates. Homologous regions are shaded gray, with the percent identity shaded according to the color bar. The arrowhead indicates a deletion in pM107_FII that encompasses several genes involved in conjugal transfer. The arrow indicates a deletion of a gene cluster carrying blaNDM-5 in pM105_FII. Genes are indicated in colors as defined in Fig 2, except that genes involved in conjugal transfer and toxin-antitoxin modules are illustrated in green and black, respectively. (B) Schematic representation of the genetic context of blaNDM. Identical regions are shaded gray, whereas arrows denote gene-cluster insertions. (C) Maximum-likelihood tree of IncFII plasmids harboring blaNDM. Encoded NDM variants are shown in parentheses along with geographical origin. IncFII plasmids lacking blaNDM and found in Myanmar isolates were also included and are indicated in italics. Bootstrap support values based on 1000 replications are indicated at the branching points. All the plasmids, except for pCC1409-1 and pCC1410-1, which were identified in K. pneumoniae isolates, were isolated from E. coli.

https://doi.org/10.1371/journal.pone.0184720.g004

Concerning the genetic context of blaNDM, pM109_FII is distinct from other IncFII Myanmar plasmids, but similar to the blaNDM-1 plasmid pGUE-NDM (Fig 4B). Both plasmids share a 12-kbp resistance-gene region surrounding blaNDM. In pM109_FII, an additional gene cassette bracketed by two IS26 sequences containing the class A β-lactamase gene blaTEM-1 and the aminoglycoside resistance-coding gene rmtB is located downstream of this region. Interestingly, this gene cassette was also found in the blaNDM region of other plasmids, including pMC-NDM (Fig 4B). pM109_FII differs from other IncFII Myanmar plasmids in that it lacks the macrolide-resistance gene ermB and the toxin-antitoxin module pemI-pemK (Fig 4A and 4B).

pM107_FII and pM214_FII clustered with pCC1409-1 and pCC1410-1 in the phylogenetic tree, whereas pM217_FII was assigned to a different branch (Fig 4C). The transfer operon in the former four plasmids differs slightly from that in other IncFII plasmids, with pM214_FII and pM217_FII sharing 81% nucleotide identity in this region. Interestingly, the transfer operon in pM214_FII is completely identical to that in known plasmids, such as pC15-1a [46]. Therefore, the transfer region in pM214_FII and related plasmids might be unrelated to other plasmids, possibly as a result of recombination.

Additionally, we found two IncFII plasmids lacking blaNDM (pM110_FII and pM105_FII). pM110_FII encodes resistance genes against quinolone and tetracycline and is phylogenetically distinct from other IncFII plasmids harboring β-lactamases and macrolide-resistance genes (Figs 1B and 4C). pM105_FII, a plasmid identified in strain M105, clustered with the blaNDM-5-harboring plasmids pM107_FII and pM214_FII. Indeed, pM105_FII is very similar to pM214_FII, except for the region surrounding blaNDM-5 (Fig 4A, arrow), with 99.3% nucleotide identity outside of this region. Therefore, it is likely that pM105_FII had been encoded previously, but recently lost blaNDM-5 along with other neighboring genes. This region is bracketed by two IS26 sequences, implying that IS26 was involved in the loss of this fragment [47]. Additionally, the M105 isolate encodes blaNDM-5 on a multireplicon IncF plasmid designated as pM105_mF, and, notably, the genetic context of blaNDM-5 in this plasmid is identical to that in pM214_FII and related plasmids (Fig 5). Furthermore, the resistance island in pM107_FII and pM214_FII bracketed by two IS26 sequences is conserved in pM105_mF (Fig 5, yellow). This implies that pM105_mF likely acquired blaNDM-5 from the pM105_FII precursor or related IncFII plasmids via IS26.

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Fig 5. Circular map of plasmid pM105_mF.

The outermost circle indicates size in bp. The next circle indicates composite transposons (dark gray) and regions homologous to the blaNDM-5 region in pM214_FII (yellow) and pV228-a (dark green). The third and fourth circles show coding sequences transcribed clockwise and anti-clockwise, respectively. Genes are indicated in colors as defined in Fig 2, except that genes involved in plasmid replication and maintenance are illustrated in green. R-M, R-S, and R-R denote restriction-modification genes (M, methylase; S, specificity; and R, restriction). The innermost circle shows GC content.

https://doi.org/10.1371/journal.pone.0184720.g005

Other plasmids

Multiple replicons might broaden the plasmid host range and enable the maintenance of incompatible replicons [24]. To the best of our knowledge, the pM105_mF plasmid backbone comprising IncFIA-, FIB-, and Q1-replication genes (Fig 1B, designated FIA-FIB-Q1) has not been previously reported. A BLAST search revealed a partial match to pV228-a (Fig 5, dark green), with 99% identity, but query coverage of only 54%. pV228-a, containing IncFIA-, FIB-, and FII-replication genes, has been identified in an E. coli strain isolated from a sewage-treatment plant in India [48], and similar multireplicon F plasmids have been found in three other Myanmar isolates (Fig 1B), with one containing an IncFII-replication gene (FIA-FIB-FII-Q1). These plasmids commonly encode several antimicrobial-resistance determinants, such as blaTEM-1, and resistance genes against sulphonamide, trimethoprim, and tetracycline. Collectively, these data indicate that multireplicon plasmids with several antimicrobial-resistance genes might be widespread in Myanmar.

Although it is unclear whether the blaNDM-5 gene cluster was transferred directly from a precursor of pM105_FII onto pM105_mF, our findings demonstrated that mobilization of blaNDM between plasmids resulted in a novel resistance plasmid. Additionally, the number of antimicrobial-resistance genes in pM105_mF was higher than that in IncFII plasmids harboring blaNDM-5 (Fig 1B and S1 Table), suggesting that recombination increased the number of resistance genes encoded on the same plasmid as blaNDM. As shown in Fig 1B, several multidrug-resistant plasmids, such as multireplicon F plasmids, co-exist with blaNDM plasmids in all isolates. Therefore, clinical settings in Myanmar appear to provide a breeding ground for novel resistance plasmids, and thus require more vigilance from national and local public health authorities.

Conclusions

We genetically characterized clinical carbapenem-resistant E. coli isolates from Myanmar and discovered multiple-resistance plasmids and several NDM variants in the diverse genetic backgrounds of the bacteria, even within the eight isolates examined. These findings suggest dissemination of NDM via multiple introductions and/or prolonged presence in the hospital allowing for recombination and dissemination via horizontal gene transfer into various genetic backgrounds. The situation seems to be similar to that reported in India, where NDM is already endemic and is found not only in healthcare settings, but also in the community [11], [49]. Therefore, further surveillance, including both the hospital and the community, is warranted to understand the dissemination of CRE in Myanmar.

Supporting information

S1 Table. Antimicrobial resistance determinants carried by the plasmids identified in E. coli clinical isolates in Myanmar.

https://doi.org/10.1371/journal.pone.0184720.s001

(DOCX)

Acknowledgments

We are grateful to Yoshikazu Ishii, Masato Suzuki and Mizue Anda for helpful discussion and comments. We also thank Yumi Sasaki and Kazuhiro Maeda for technical assistance on antimicrobial susceptibility testing.

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