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. 2019 Jun 21;12:1729–1742. doi: 10.2147/IDR.S210554

Epidemic IncX3 plasmids spreading carbapenemase genes in the United Arab Emirates and worldwide

Shaimaa F Mouftah 1,*, Tibor Pál 1,*, Dania Darwish 1, Akela Ghazawi 1, Laura Villa 2, Alessandra Carattoli 3, Ágnes Sonnevend 1,4,✉
PMCID: PMC6593741  PMID: 31417290

Abstract

Purpose

Plasmids of the incompatibility group X type 3 (IncX3) were described carrying various carbapenemase genes in carbapenemase-producing Enterobacteriaceae (CPE) worldwide and in the United Arab Emirates (UAE), as well. To understand the driving force behind the emergence of such plasmids in the UAE, the relationship between IncX3 plasmids encountered locally and globally was investigated.

Methods

CPE strains isolated in the UAE during 2009–2014 were screened by X3 PCR-based replicon typing. The clonal relationship of CPE carrying IncX3 plasmids was determined by multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Complete sequence of selected IncX3 plasmids was determined. Phylogenetic relationship between the carbapenemase carrying IncX3 plasmids from the UAE and of those reported worldwide was established by comparing the plasmid backbones.

Results

10.2% of the 295 CPE tested were identified to carry IncX3 plasmids: 13 Escherichia coli, 13 Klebsiella pneumoniae, two Enterobacter cloacae, one Citrobacter freundii and one Morganella morganii isolate, respectively. Most of them were non-clonal; with small clusters of triplets and pairs of E. coli and K. pneumoniae, and a cluster of five K. pneumoniae ST11 exhibiting >90% similar PFGE patterns, respectively. The 30 isolates harbored either blaNDM-1, blaNDM-4, blaNDM-5, blaNDM-7, blaOXA-181 or blaKPC-2 carbapenemase genes on IncX3 plasmids. Phylogenetic analysis of the backbone region of IncX3 plasmids carrying various beta-lactamase genes from the UAE (n=23) and that of North-America, Europe, Asia and Australia (n=35) revealed three clusters based on the carbapenemase genes carried: plasmids harboring blaOXA-181 and blaNDM-5 formed two distinct groups, whereas backbones of plasmids with blaNDM-1, blaNDM-4 and blaNDM-7 clustered together. Each cluster contained plasmids of diverse geographical origin.

Conclusion

The findings suggest that different carbapenemase gene carrying IncX3 plasmids encountered in the UAE do not evolve locally, rather are subtypes of this epidemic plasmid emerging in this country due to international transfer.

Keywords: Enterobacterales, carbapenemase genes, IncX3 plasmid, Middle-East

Introduction

Due to the limited therapeutic options remaining to treat these infections, carbapenemase-producing Enterobacteriaceae (CPE) are increasingly important human pathogens associated with high mortality.1,2 Their spread is driven by two major forces: clonal dissemination of a few successful CPE lineages, and horizontal transfer of carbapenemase genes often located on epidemic plasmids spreading in different bacterial species, sources and countries.2–5 Plasmids of the incompatibility group (Inc) X defined as X3 type (IncX3)6 have been reported worldwide in Enterobacterales, associated withblaSHV-12 extended-spectrum beta-lactamase (ESBL), blaKPC-2, −3, blaNDM-1, −4, −5, −7 and blaOXA-181 carbapenemase genes.7–14 IncX3 plasmids were reported to disseminate a variety of blaNDM genes in humans, in animals and in the environment particularly in South East Asia; including China, Hong Kong, South Korea, Myanmar, Vietnam and the Indian Subcontinent.12–20

The Middle-East is considered an endemic region for CPE, with the dominance of class D OXA-48-like, and class B NDM carbapenemases, with sporadic occurrence of class A KPC-2, and class B VIM-4 enzymes.1,2,7–10 In the Arabian Peninsula autochthonous, clonal transmission has been implicated as the main driving force in the emergence of CPE,1 but plasmid-mediated dissemination of blaVIM-4 has also been documented in the region.21 Furthermore, sporadic isolates carrying blaNDM-1, blaNDM-7 and blaKPC-2 on IncX3 plasmids were identified in the United Arab Emirates (UAE).22–24 However, the role of this type of plasmid in the dissemination of CPE, and its possible local evolution have not been systematically studied. Here, we present the comparisons of the complete sequences of IncX3 plasmids carrying various carbapenemase genes encountered in the UAE, and evaluate their relatedness to similar episomes identified worldwide.

Materials and methods

Bacterial strains

Altogether 334 non-repeat carbapenem-resistant Enterobacterales (CRE) strains were tested. They were isolated between April 2009 and December 2014 in 12 hospitals of the UAE and submitted to the Department of Medical Microbiology and Immunology, UAE University, without any patient identifiers, to identify the carbapenemases produced. Strains were stored at −80°C in Tryptic Soy Broth (MAST, Merseyside, UK) containing 20% glycerol. This collection included 90 isolates described earlier in1,22–24 and further 246 CRE isolated between May 2013 and December 2014 in six governmental hospitals of Abu Dhabi Emirate.

Detection of carbapenemase genes and screening for the IncX3 replicon

The presence of the blaNDM, blaOXA-48-like, blaVIM, blaIMP, blaKPC carbapenemase genes, and that of blaSHV were detected as described.25–27 The specific alleles of beta-lactamase genes were determined by direct sequencing of the respective amplicons with the Big Dye Cycle Terminator V.3.1 (Applied Biosystems) using the 3130X Genetic Analyzer (Applied Biosystems). A replicase-specific PCR was used to screen strains for the presence of IncX3 plasmids.6

Antibiotic susceptibility assays and phenotypic detection of carbapenemase production

The antibiotic susceptibility of carbapenemase-producing IncX3 plasmid carrying clinical isolates and their derivatives to cefotaxime, ceftazidime, aztreonam, ertapenem, meropenem, imipenem, ciprofloxacin, gentamicin, amikacin, trimethoprim/sulfamethoxazole, tetracycline and colistin was tested by broth microdilution, while fosfomycin and tigecycline susceptibilities were assessed by agar dilution.28 CLSI clinical breakpoints were used for interpretation for the majority of antibiotics.28 Results for colistin, tigecycline and fosfomycin were interpreted by the EUCAST criteria.29 Carbapenemase production was assessed phenotypically by the CIM test.30

Molecular typing

Carbapenemase-producing IncX3 positive K. pneumoniae, E. coli and E. cloacae isolates were typed using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST).31–34

Characterization of the carbapenemase gene-bearing IncX3 plasmids

Plasmids were isolated by the alkaline lysis method, and detected as described in23 using E. coli 39R861 as plasmids' molecular size standards. Southern blotting of the plasmid electrophoresis gel, and hybridization with IncX3 and the respective carbapenemase gene probes was used to prove the localization of carbapenemase genes on IncX3 plasmid.23

The sequence of each IncX3 plasmids carried by different ST and/or PFGE profiles were further investigated. In case of multiple strains exhibiting the same ST and PFGE profile, plasmids were chosen from strains representing each unique plasmid profiles and/or coding for each distinct carbapenemases.

In mating out assays, a sodium-azide resistant derivative of rifampicin-resistant E. coli J53 (J53RAZ) was used as recipient. Transconjugants were selected on Tryptic Soy Agar containing 8 mg/L−1 ceftazidime and 100 mg/L−1 sodium-azide, or in case of OXA-181 producing clinical isolates on 0.5 mg/L−1 ertapenem and 100 mg/L−1 sodium-azide.35 When transconjugants were not obtained, the IncX3 plasmids were transformed into competent E. coli DH5α or E. coli GM2163.35 For complete plasmid sequencing, plasmid DNA was purified from single plasmid containing E. coli transconjugant or transformant using the Plasmid Maxi Prep kit (Qiagen, Hilden, Germany). The complete sequence of the plasmids was established by next-generation sequencing either by using the 454-Genome Sequencer FLX procedure (Roche Diagnostic, Monza, Milan, Italy) or, commercially, on the Illumina MiSeq platform (performed at the CCIB DNA Core Facility in Massachusetts General Hospital, Cambridge, MA, USA). The gaps between contigs assembled were closed by PCR and direct sequencing of the amplicons. The complete plasmid sequences were assembled with Clone Manager v9.0 (Sci-Ed Software, Cary, NC, US), annotated using Geneious R11.0.4 (Biomatters Ltd., Auckland, New Zealand) and Sequin (http://www.ncbi.nlm.nih.gov/Sequin), and submitted to GenBank (Accession numbers are shown in Table 2).

Table 2.

Characteristics of carbapenemase producing Enterobacteriaceae harboring IncX3 type plasmids with carbapenemase genes

Isolate Plasmid Ref.
Name* Date of isolation Hospital Specimen Species Carbapenemase produced MLST C/Cm/nonC Name Size (bp) Resistance gene(s) GenBank Acc. No
ABC133 12/14/2012 TH Sputum E. coli NDM-7 ST4108 nonC pABC133-NDM 37070 blaNDM-7 KX214671 24
ABC239 8/15/2013 RH Urine E. coli OXA-181 ST410 nonC pABC239-OXA-181 51479 blaOXA-181+ qnrS1 MK412916 This study
ABC264 6/9/2014 TH Unknown E. coli OXA-181 ST410 nonC pABC264-OXA-181 51479 blaOXA-181+ qnrS1 MK412917 This study
ABC356 8/8/2014 MH Urine E. coli OXA-181 ST410 Cm pABC356-OXA-181 51479 blaOXA-181+ qnrS1 MK412918 This study
ABC381 11/4/2014 AAH Rectal swab E. coli OXA-181 ST167 nonC pABC381-OXA-181 51479 blaOXA-181+ qnrS1 MK412919 This study
ABC218 12/25/2012 RH Wound E. coli NDM-7 ST167 C pABC218-NDM 34403 blaNDM-7 KX214670 24
ABC233 7/21/2013 RH Urine E. coli NDM-5 ST167 Cm pABC233-NDM-5 46161 blaNDM-5 MK372390 This study
ABC384 11/5/2014 AAH Urine E. coli NDM-5 ST1284 C pABC384-NDM-5 46161 blaNDM-5 MK372389 This study
ABC54 1/2/2011 TH Urine E. coli NDM-1 ST2206 C pABC54-NDM-1 53023 blaNDM-1+ blaSHV-12 MK372382 This study
BC-13-836 9/24/2013 TH Blood E. coli NDM-1 ST446 C pBC836-NDM-1 52565 blaNDM-1+ blaSHV-11 MK372387 This study
ABC280 7/15/2014 TH Urine E. coli NDM-5 ST448 C pABC280-NDM5 35502 blaNDM-5 MK372392 This study
ABC286 8/15/2014 TH Blood E. coli NDM-5 ST448 NT NT NT NT NT This study
ABC268 6/11/2014 AAH Urine E. coli NDM-5 ST2083 Cm pABC268-NDM-5 45232 blaNDM-5 MK372391 This study
ABC40 10/27/2009 TH Wound E. cloacae NDM-1 ST417 Cm pABC40-NDM-1 54035 blaNDM-1+ blaSHV-12 MK372380 This study
ABC302 2/26/2014 MH Urine E. cloacae NDM-4 ST200 C ABC302-NDM-4 49402 blaNDM-4 MK372388 This study
BC-13-947 7/11/2013 TH Blood K. pneumoniae OXA-181 ST2095 nonC pBC947-OXA-181 51479 blaOXA-181+ qnrS1 MK412920 This study
ABC260 3/31/2014 TH Rectal swab K. pneumoniae OXA-181 ST3545 nonC pABC260-OXA-181 51480 blaOXA-181+ qnrS1 MK412915 This study
ABC369 9/23/2014 TH Abdominal fluid K. pneumoniae NDM-5+ OXA-162 ST307 Cm pABC369-NDM-5 45252 blaNDM-5 MK372393 This study
ABC137 1/14/2013 MH Wound K. pneumoniae NDM-1+ OXA-48 ST1318 Cm pABC137-NDM-1 53022 blaNDM-1+ blaSHV-12 MK372384 This study
ABC141 4/20/2013 MH Unknown K. pneumoniae NDM-1+ OXA-48 ST1318 NT NT NT NT NT This study
ABC155 6/5/2013 SKMC Blood K. pneumoniae NDM-1+ OXA-48 ST1318 NT NT NT NT NT This study
ABC220 10/5/2012 RH Wound K. pneumoniae KPC-2 ST14 C# pABC220-KPC-2 46900 blaKPC-2 MK412914 This study
ABC224 3/17/2013 RH Sputum K. pneumoniae KPC-2 ST14 C# NT NT NT NT This study
ABC52 9/19/2010 TH Sputum K. pneumoniae NDM-1 ST11 C pABC52-NDM-1 52565 blaNDM-1+ blaSHV-12 MK372381 This study
ABC53 9/19/2010 TH Sputum K. pneumoniae NDM-1 ST11 NT NT NT NT NT This study
BC680 7/18/2012 TH Blood K. pneumoniae NDM-1 ST11 NT NT NT NT NT This study
BC700 7/24/2012 TH Blood K. pneumoniae NDM-1 ST11 C pBC700-NDM-1 52565 blaNDM-1+ blaSHV-11 MK372386 This study
BC-13-817 9/17/2013 TH Blood K. pneumoniae NDM-1 ST11 NT NT NT NT NT This study
ABC80 5/8/2011 TH Urine Citrobacter freundii NDM-1 NT Cm pABC80-NDM-1 53023 blaNDM-1+ blaSHV-12 MK372383 This study
ABC140 3/25/2013 MH Perianal swab Morganella morganii NDM-1 NA nonC pABC140-NDM-1 52591 blaNDM-1 MK372385 This study
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Notes: *Strain names with bold indicate those from which the IncX3 plasmid was fully sequenced in this study.

Abbreviations: MLST, Multi-Locus Sequence Typing; ST, sequence type; NA, not applicable; NT, not tested; C, conjugative; Cm, conjugative with transconjugants having multiple plasmids; C#, conjugative, but cannot stably maintained in E. coli J53RAZ; nonC, not self-transmissible.

For comparison, all complete sequences of IncX3 plasmids carrying carbapenemase genes available in GenBank up to January 2019 were downloaded. If identical plasmid backbone sequences, carrying the same carbapenemase gene were identified in multiple isolates from the same country, only one was selected randomly for the phylogenetic analysis.

Plasmid backbones of the UAE IncX3 plasmid sequences and those retrieved from GenBank were aligned by ClustalW, and the evolutionary history was inferred by the Jukes-Cantor genetic distance model with 500x bootstrapping using Geneious R11.0.4 (Biomatters Ltd., Auckland, New Zealand).

Results

Characteristics of strains carrying IncX3 plasmids

Of the 334 isolates screened, 295 were positive for at least one carbapenemase gene by PCR. The remaining 39 were negative by PCR for the five common carbapenemase genes tested, and they were carbapenemase non-producers by the CIM test. The distribution of the 32 IncX3 plasmid carrying isolates among strains with various carbapenem resistance genes is shown in Table 1. The IncX3 positive CPE isolates were variably resistant to 3rd generation cephalosporins, aztreonam, aminoglycosides, ciprofloxacin, co-trimoxasole, tetracycline, tigecycline, colistin and fosfomycin (shown in Table S1). The characteristics of the 30 CPE isolates, in which at least one carbapenemase gene was located on an IncX3 plasmid, are shown in Table 2. Altogether five species of Enterobacterales were identified. One Citrobacter freundii, one Morganella morganii and one Enterobacter cloacae carried blaNDM-1 on IncX3 plasmid, and a further Enterobacter cloacae harbored IncX3-borne blaNDM-4.

Table 1.

Distribution of IncX3 plasmid carrying isolates among strains expressing different carbapenem resistance mechanisms

n All NDM OXA-48-like NDM and OXA-48-like VIM KPC All carbapenemase producer Carbapenemase non-producer
334 89 126 75 3 2 295 39
IncX3 PCR positive 32
(9.6%)		 18
(20%)		 6
(4.8%)		 4
(5.3%)		 0 2
(100%)		 30
(10.2%)		 2
(5.1%)
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Table S1.

Antibiotic susceptibility of clinical isolates carrying carbapenemases on IncX3 plasmids and their single IncX3 plasmid containing derivatives

Strain Carbapenemase produced Wild type/transconjugant/transformant Ertapenem Imimpenem Meropenem Ceftazidime Cefotaxime Aztreonam Ciprofloxacin Gentamicin Amikacin Co-trimoxazole Tetracycline Colistin Tigecycline Fosfomycin
ABC220 KPC-2 WT 256 64 128 >128 >128 >128 64 256 16 >256 4 32 0.5 256
GM3163(pABC220-KPC-2) KPC-2 TF 4 4 2 16 4 >128 ≤0.125 1 1 ≤0.5 ≤0.5 ≤0.5 ≤0.125 16
ABC224 KPC-2 WT 256 64 128 >128 >128 >128 32 256 32 >256 4 32 0.5 128
ABC140 NDM-1 WT 2 32 8 128 32 4 8 2 4 ≤0.5 16 >256 4 >512
DH5α(pABC140-NDM-1) NDM-1 TF 0.5 2 ≤0.25 >128 64 32 ≤0.125 ≤0.5 2 ≤0.6 ≤0.5 ≤0.5 ≤0.125 ≤0.25
ABC40 NDM-1 WT 32 16 16 >128 >128 >128 >64 128 4 ≤0.7 256 2 1 8
J53RAZ(pABC40-NDM-1) NDM-1 TC 0.25 4 4 >128 64 32 ≤0.125 ≤0.5 ≤0.5 ≤0.8 ≤0.5 ≤0.5 ≤0.125 0.5
ABC52 NDM-1 WT 64 32 64 >128 >128 >128 >64 >256 >256 >256 4 ≤0.5 1 4
J53RAZ(pABC52-NDM-1) NDM-1 TC 1 4 8 >128 64 64 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 1
ABC53 NDM-1 WT 64 64 64 >128 >128 >128 >64 >256 >256 >256 4 ≤0.5 2 16
ABC54 NDM-1 WT 8 16 16 >128 128 >128 0.25 1 4 >256 128 ≤0.5 0.25 0.5
DH5α(pABC54-NDM-1) NDM-1 TF 0.25 4 4 >128 64 32 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 0.5
ABC80 NDM-1 WT 8 8 4 >128 128 >128 4 32 1 128 ≤0.5 ≤0.5 0.25 0.5
J53RAZ(pABC80-NDM-1) NDM-1 TC 0.5 8 4 >128 64 32 ≤0.125 1 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 0.5
BC680 NDM-1 WT 16 32 64 >128 >128 >128 >64 4 16 >256 2 ≤0.5 2 4
BC700 NDM-1 WT 16 64 32 >128 >128 >128 >64 2 16 >256 2 ≤0.5 2 4
J53RAZ(pBC700-NDM-1) NDM-1 TC 2 4 4 >128 64 ≤0.25 ≤0.125 1 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 0.5
BC-13-817 NDM-1 WT 16 32 32 >128 >128 >128 >64 4 16 >256 2 ≤0.5 2 4
BC-13-836 NDM-1 WT 4 16 16 >128 >128 ≤0.25 ≤0.125 2 4 ≤0.5 128 ≤0.5 ≤0.125 0.5
DH5α(pBC836-NDM-1) NDM-1 TF 0.25 2 ≤0.25 >128 64 ≤0.25 ≤0.125 ≤0.5 1 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
ABC141 NDM-1+ OXA-48 WT 16 64 32 >128 >128 >128 >64 128 8 >256 >256 4 16 8
ABC155 NDM-1+ OXA-48 WT >256 >128 >128 >128 >128 >128 4 128 4 >256 >256 ≤0.5 2 128
ABC137 NDM-1+ OXA-48 WT 16 64 32 >128 >128 >128 4 128 4 >256 >256 ≤0.5 2 8
J53RAZ(pABC137-NDM-1) NDM-1 TC 0.25 4 4 >128 64 32 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 0.5
ABC302 NDM-4 WT 128 64 128 >128 >128 >128 64 >256 >256 >256 8 ≤0.5 2 16
DH5α(pABC302-NDM-4) NDM-4 TF 0.25 2 ≤0.25 >128 64 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
ABC233 NDM-5 WT 32 16 32 >128 >128 >128 >64 >256 >256 128 1 ≤0.5 ≤0.125 0.5
DH5α(pABC233-NDM-5) NDM-5 TF 0.5 2 ≤0.25 >128 64 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
ABC268 NDM-5 WT 32 16 32 >128 >128 32 >64 64 4 256 1 ≤0.5 0.25 1
DH5α(pABC268-NDM-5) NDM-5 TF 0.25 2 ≤0.25 >128 64 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
ABC280 NDM-5 WT 32 16 16 >128 >128 >128 >64 64 8 >256 1 ≤0.5 0.25 1
J53RAZ(pABC280-NDM-5) NDM-5 TC 0.5 4 8 >128 128 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 1
ABC286 NDM-5 WT 64 8 32 >128 >128 >128 >64 32 4 256 1 ≤0.5 0.25 1
ABC384 NDM-5 WT 64 128 32 >128 >128 >128 >64 64 8 256 >256 ≤0.5 ≤0.125 0.5
J53RAZ(pABC384-NDM-5) NDM-5 TC 0.25 4 4 >128 64 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 1
ABC369 NDM-5+ OXA-162 WT 4 8 8 >128 >128 >128 >64 256 >256 >256 4 ≤0.5 2 4
DH5α(pABC369-NDM-5) NDM-5 TF 0.25 2 ≤0.25 >128 64 ≤0.25 ≤0.125 ≤0.5 1 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
ABC133ETP NDM-7 WT 128 128 128 >128 >128 64 >64 64 8 ≤0.5 >256 ≤0.5 0.25 0.5
DH5α(pABC133-NDM) NDM-7 TF 1 4 2 >128 64 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
ABC218 NDM-7 WT 64 16 32 >128 >128 >128 >64 256 8 256 1 ≤0.5 ≤0.125 0.5
J53RAZ(pABC218-NDM) NDM-7 TF 2 8 8 >128 128 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 1
ABC239 OXA-181 WT 2 0.5 ≤0.25 >128 >128 >128 >64 128 8 >256 >256 ≤0.5 ≤0.125 0.5
DH5α(pABC239-OXA-181) OXA-181 TF 0.5 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
ABC260 OXA-181 WT 128 64 32 2 2 0.5 16 1 1 >256 4 16 2 >512
DH5α(pABC260-OXA-181) OXA-181 TF ≤0.125 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
ABC264 OXA-181 WT 2 1 ≤0.25 >128 >128 >128 >64 4 4 256 >256 ≤0.5 0.25 1
DH5α(pABC264-OXA-181) OXA-181 TF ≤0.125 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
ABC356 OXA-181 WT 2 1 0.5 >128 >128 >128 >64 128 8 >256 >256 ≤0.5 0.25 0.5
DH5α(pABC356-OXA-181) OXA-181 TF 0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
ABC381 OXA-181 WT 16 8 8 >128 128 8 64 2 2 256 256 ≤0.5 0.25 ≤0.25
DH5α(pABC381-OXA-181) OXA-181 TF ≤0.125 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
BC-13-936 OXA-181 WT 0.25 2 0.5 ≤0.25 ≤0.25 ≤0.25 1 2 2 >256 1 ≤0.5 0.25 16
BC-13-947 OXA-181 WT ≤0.125 2 0.5 16 2 64 1 2 2 >256 1 8 0.25 16
DH5α(pBC947-OXA-181) OXA-181 TF ≤0.125 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.125 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
BC-13-970 OXA-181 WT 0.25 2 0.5 0.5 ≤0.25 ≤0.25 1 2 2 >256 1 ≤0.5 0.25 16
DH5α None R ≤0.125 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.125 1 1 ≤0.5 ≤0.5 ≤0.5 ≤0.125 ≤0.25
GM2163 None R ≤0.125 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.125 2 4 ≤0.5 ≤0.5 ≤0.5 ≤0.125 16
J53Raz None R ≤0.125 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.125 1 ≤0.5 ≤0.5 ≤0.5 ≤0.5 ≤0.125 0.5
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The 13 E. coli carried either blaNDM-1, blaNDM-5, blaNDM-7 or blaOXA-181 on IncX3 plasmids. They exhibited limited clonality; a triplet and two pairs of isolates formed PFGE clusters with ≥90% pattern similarity, respectively (Figure S1A). The 13 E. coli belonged to 8 different sequence types (Table 2). The 13 K. pneumoniae were less heterogeneous: five K. pneumoniae ST11 carrying IncX3-borne blaNDM-1 exhibited ≥90% similar PFGE patterns, three NDM-1 and OXA-48 co-producing K. pneumoniae ST1318, with blaNDM-1 being located on IncX3 plasmid, also clustered by PFGE, and the two KPC-2 producer K. pneumoniae ST14 were indistinguishable by PFGE (Figure S1B). The further three K. pneumoniae were of different sequence types; two of them carried blaOXA-181, and one had blaNDM-5 on an IncX3 plasmid. This latter isolate, a K. pneumoniae ST307, co-produced NDM-5 and OXA-162, but blaOXA-162 was not located on the IncX3 plasmid (Table 2).

Figure S1.

Figure S1

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(A) Comparison of pulsed-field gel electrophoresis patterns of Escherichia coli isolates. (B) Comparison of pulsed-field gel electrophoresis patterns of Klebsiella pneumoniae isolates.

Characteristics of IncX3 plasmids carrying carbapenemase genes

Altogether 21 IncX3 plasmids were selected for further analysis. Single plasmid-bearing derivatives obtained by conjugation or by transformation (Table 2) showed varying degrees of non-susceptibility to carbapenems and to 3rd generation cephalosporins and were susceptible to non-beta lactam antibiotics (Table S1).

Complete DNA sequences of the 21 plasmids were obtained and compared to two IncX3 plasmids carrying blaNDM-7 (pABC133-NDM and pABC218-NDM), previously described from the UAE24 (Table 2 and Figure 1).

Figure 1.

Figure 1

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Comparison of IncX3 plasmids from the United Arab Emirates carrying various carbapenemases.

Notes: Grey shades represent regions with ≥99% similarity.

In pABC220-KPC-2, the blaKPC-2 gene was located on a Tn4401b transposon, and no further resistance gene was carried by this plasmid.

The six blaOXA-181 carrying plasmids were >99% similar to each other, and all of them harbored the blaOXA-181 and a qnrS1 quinolone resistance gene in a composite transposon bracketed by IS26.

The genetic load region of the eight blaNDM-1 carrying plasmids was flanked by IS26 and Tn3. The immediate genetic surrounding of the blaNDM-1 between an ISCR27 and a truncated ISAba125 was identical in all eight plasmids. The IS26 bracketed composite transposon upstream of ISCR27 either carried blaSHV-11 (n=2), or blaSHV-12 (n=5), or contained a truncated Tn3 transposase (pABC140-NDM-1). The genetic surroundings of blaNDM-4, blaNDM-5 and blaNDM-7 between IS26 and IS5 were identical.

Although the genetic load regions were different in plasmids having various classes of carbapenemases, the plasmid backbones were highly similar with the notable absence of hns, and variable presence of complete or truncated topB and ATPase genes in pABC280-NDM-5, pABC218-NDM and pABC133-NDM (Figure 1).

Phylogenesis of the carbapenemase gene-bearing IncX3 plasmids

As pABC218-NDM, despite a large deletion in the conserved region, demonstrated to be self-conjugative and sufficiently stable, a 24905 bp long region coding for its replication, partitioning and transfer (from position 1286 to 26190 in GenBank Acc. No. KX214670) was used in the phylogenetic analysis. This backbone region was extracted from all complete IncX3 plasmid sequences from the UAE, and from the complete sequence of 35 IncX3 plasmids from different geographical regions downloaded from GenBank (listed in Table S2).

Table S2.

IncX3 plasmids (retrieved from GenBank in January 2019) from different geographical regions with unique backbone sequences and beta-lactamase genes carried

Resistance genes Country Name GenBank Accession No
blaKPC-2 Hong Kong pKPC-NY79 JX104759
blaNDM-17 China pAD-19R KX833071
blaNDM-4 Myanmar pM216_X3 AP018146
blaNDM-4 Australia pJEG027 KM400601
blaNDM-4 Czech Republic pEncl-922cz MG252892
blaNDM-5 Czech Republic pEsco-5256cz MG252891
blaNDM-5 India pNDM-MGR194 KF220657
blaNDM-5 Hong Kong pNDM-HK2998 MH234508
blaNDM-5 Hong Kong pNDM-HK2967 MH234509
blaNDM-5 South Korea pCREC-591_4 CP024825
blaNDM-7 South Korea pCREC-532_3 CP024833
blaNDM-7 Oman pOM26-NDM KP776609
blaNDM-7 Kuwait pKW53T-NDM KX214669
blaNDM-7 Canada pKpN01-NDM-7 CP012990
blaNDM-7 Myanmar pM110-X3 AP018141
blaNDM-7 China pEC50-NDM-7 KX470735
blaOXA-181 Italy pKP_BO_OXA-181 MG228426
blaOXA-181, qnrS1 China pOXA-181 KP400525
blaOXA-181, qnrS1 Switzerland pKS22 KT005457
blaOXA-181, qnrS1 Germany pOXA-181-IHIT35346 KX894452
blaOXA-181, qnrS1 South Korea pD6-OXA_1_1 MG702491
blaOXA-181, qnrS1 Myanmar pM206-OXA181 AP018831
blaOXA-181, qnrS1 Czech Republic pOXA181_29144 KX523903
blaOXA-181, qnrS1 Lebanon pSTIB_IncX3_OXA_181 MG570092
blaOXA-181, qnrS1 Denmark pAMA1167-OXA-181 CP024806
blaSHV-11 Italy pIncX-SHV JN247852
blaSHV-11, blaKPC-3 Italy p45-IncX3 KT362706
blaSHV-12 Netherlands pEC-393 KX618697
blaSHV-12 Netherlands pEC-125 KX618703
blaSHV-12, aac(6ʹ)-Ib USA pKPN-819 CP008799
blaSHV-12, blaKPC-2 France pKpS90 JX461340
blaNDM-1+ blaSHV-12 China pNDM-HN380 JX104760
blaNDM-1+ blaSHV-12 Hong Kong pNDM-HK3694 MH234505
blaSHV-12, blaTEM-1, qnrS1 Netherlands pEC-NRS18 KX618696
None USA pUCLAOXA232-2 CP012563
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The Neighbor-Joining tree of the 58 IncX3 plasmid backbone sequences (Figure 2) showed three distinct clades. The first contained blaNDM-1, blaNDM-4 and blaNDM-7 carrying plasmids from the UAE and plasmids carrying similar carbapenemase genes of other geographical regions, and a blaSHV-12 carrying plasmid from The Netherlands. The second one included blaNDM-5 carrying plasmids, and the third clade clustered blaOXA-181 carrying IncX3 plasmids originating from various parts of the world with a single outlier of blaOXA-181 carrying IncX3 plasmid (MG228426) from Italy only. Conversely, plasmids harboring blaKPC were distinct from each other.

Figure 2.

Figure 2

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Phylogenetic tree of backbone sequences of IncX3 plasmids from various geographical area.

Notes: The sequences were aligned using ClustalW, and the Neighbor-Joining tree was constructed using the Jukes-Cantor genetic distance model with 500 bootstrap replicates. All positions containing gaps and missing data were eliminated. There was a total of 24,868 positions in the final dataset. Plasmid names printed in bold represent IncX3 plasmids from the UAE, for plasmids retrieved from GenBank the accession number, the beta-lactamase gene carried, and the country of isolation is shown.

Abbreviations: CA, Canada; CH, Switzerland; CN, China; CZ, Czech Republic; DN, Denmark; FR, France; GR, Germany; HK, Hong Kong; IN, India; IT, Italy; KR, South Korea; KW, Kuwait; LB, Lebanon; MY, Myanmar; NL, the Netherlands; OM, Oman; US, United States of America.

Discussion

Our data showed that in CRE isolated in 12 hospitals of the UAE, the overall prevalence of IncX3 plasmids was 9.6%, and in NDM-producer as high as 20%. Importantly, in the 30 CPE, the carbapenemase gene (or one of them in the double carbapenemase producers) was located on an IncX3 type plasmid. This is a prevalence substantially higher than the one reported in human fluoroquinolone or cefotaxime resistant E. coli isolates,7 but considerably lower compared to a report on CRE from Hong Kong (30.3%).12

The CRE isolates carrying IncX3 with a carbapenemase gene were quite diverse. They belonged to five different species of Enterobacterales (K. pneumoniae, E. coli, E. cloacae, C. freundii and M. morgannii). Similar, or even higher diversity of hosts of carbapenemase bearing IncX3 plasmids has been noted in South-East Asian countries.12,16 The majority of CRE isolates carrying carbapenemase-encoding IncX3 plasmids were unrelated. However, PFGE clustering of five K. pneumoniae ST11 harboring blaNDM-1 on IncX3 plasmids, all isolated in the same hospital, suggested clonal dissemination. Interestingly, the two plasmids sequenced from these five isolates carried different blaSHV alleles: blaSHV-12 and blaSHV-11 differing in three nucleotides, otherwise being 100% identical to each other. The combination of carbapenemase carrying IncX3 plasmid and the K. pneumoniae ST11 clone, both considered to have epidemic potential,5 is especially worrisome.

Interestingly, two K. pneumoniae ST14, which were described earlier in,22 carried blaKPC-2, although this clone was found to be the most common NDM- and OXA-48-like producer K. pneumoniae clone in Dubai in a later period, when no KPC-producing isolates were encountered.36

A member of another high-risk K. pneumoniae clone, ST307, was also encountered possessing blaOXA-162 and an IncX3 plasmid-borne blaNDM-5. To the best of our knowledge, blaOXA-162 has not previously been associated with this clone. It is noteworthy that the same ST had been reported earlier from the UAE to carry blaNDM-1 on an IncHI1B plasmid and blaOXA-162 on IncL/M plasmid.35 While that isolate did not harbor an IncX3 plasmid, it was recovered in the same hospital as the current one with the IncX3 blaNDM-5 plasmid, and was also co-harboring a blaOXA-162. Therefore, the possibility of local acquisition of blaNDM-5 carrying IncX3 plasmid cannot be excluded.

A cluster of three OXA-181 producing E. coli ST410 harboring the carbapenemase on IncX3 plasmids was also encountered. Recently, it was established that this sequence type of E. coli is also an emerging high-risk clone.37 The three E. coli ST167 isolates carried three different carbapenemases: blaNDM-5, blaNDM-7 and blaOXA-181, all located on IncX3 plasmids (Table 2). This clone is considered to be an epidemic NDM-5-producing E. coli clone in China38 and was shown to carry IncX3 plasmid-borne blaNDM-5 in the Czech Republic, too.39 It was also reported to harbor blaNDM-7 on IncX3 plasmid from France40 and India.41 However, E. coli ST167 with blaOXA-181 carrying IncX3 plasmid has not been encountered yet, although a single locus variant of ST167 was reported to carry this carbapenemase gene from São Tomé and Príncipe.42

It has been suggested that the wide dissemination of IncX3 plasmids is due to its highly efficient conjugal transfer, contributing to its spread within clinical settings, as well as in the environment.12,16 Based on our studies we cannot comment on these observations, since several of our plasmids co-transferred with other episomes, and some were non-conjugative, despite genes for conjugal transfer were apparently present and intact in all but one plasmid of our collection (pABC133-NDM described in24). Similarly, we cannot comment on the role of the environmental dissemination suggested earlier,12,24,43 as the current study included human isolates only.

Since many, but not all, carbapenemase carrying IncX3 plasmids resided in international high-risk clones of Enterobacteriaceae, we compared the conserved regions of plasmids from the UAE to the ones reported earlier from various countries (Table S2) to evaluate whether these plasmids occur in the UAE as a result of local evolution, or rather as a consequence of international transfer. The analysis identified clades exhibiting good correlation with the carbapenemase genes carried (Figure 2), ie close phylogenetic relationship of IncX3 plasmids harboring blaNDM-1, blaNDM-4 and blaNDM-7 from the UAE and from different countries of the Middle-East, Asia, Europe and North-America was observed. On the other hand, blaNDM-5 carrying plasmids from the UAE, Czech Republic, China, Hong Kong, India and South Korea formed a distinct clade. Previously, based on the high degree of synteny among the complete NDM-IncX3 plasmid sequences, the evolution of blaNDM alleles within the IncX3 plasmid was suggested.12 Our findings partially support this hypothesis with the notion that certain blaNDM alleles, notably that of NDM-5, are located on plasmids with a more distantly related backbone, suggestive of multiple uptakes of blaNDM genes by these plasmids.

blaOXA-181 carrying IncX3 plasmids encountered in the UAE, as well as in Lebanon, Germany, Denmark, Czech Republic, Switzerland, China, South Korea and Myanmar formed another distinct clade with a single outlier (MG228426) from Italy, only. The KPC-IncX3 plasmids were phylogenetically heterogeneous: while two blaKPC-2 harboring plasmids from Hong Kong and from France mapped relatively close (JX104759 and JX461340), the backbone of the plasmid coding for the same allele from of the UAE (pABC220-KPC-2) and that of an Italian plasmid carrying blaKPC-3 (KT362706) were distant.

Conclusion

Phylogenetic analysis, clustering backbones of IncX3 plasmids of diverse geographical origin based on the carbapenemase gene carried, suggests that these plasmids disseminate across the continents. Consequently, the emergence of different carbapenemase carrying IncX3 plasmids in the UAE is likely not the result of local evolution, but due to the international transfer of such plasmids. Moreover, finding of high-risk K. pneumoniae and E. coli clones in the UAE, harboring these plasmids, warrants further studies to better understand the role of the epidemic plasmids and clones in the emergence and spread of CPE in the country highly exposed to international travel and trade.

Acknowledgments

This work was supported by grants from the College of Medicine and Health Sciences, United Arab Emirates University 31M251 and United Arab Emirates University UPAR-31M235 awarded to ÁS.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary materials

References

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