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

Authors Mouftah SF, Pál T, Darwish D, Ghazawi A, Villa L, Carattoli A, Sonnevend Á

Received 29 March 2019

Accepted for publication 27 May 2019

Published 21 June 2019 Volume 2019:12 Pages 1729—1742


Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Dr Joachim Wink

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Shaimaa F Mouftah,1,* Tibor Pál,1,* Dania Darwish,1 Akela Ghazawi,1 Laura Villa,2 Alessandra Carattoli,3 Ágnes Sonnevend1,4

1Department of Medical Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates; 2Department of Infectious Diseases, Istituto Superiore di Sanitá, Rome, Italy; 3Department of Molecular Microbiology, University of Rome La Sapienza, Rome, Italy; 4Department of Medical Microbiology and Immunology, Medical School, University of Pécs, Pécs, Hungary

*These authors contributed equally to this work

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



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.25 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.714 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.1220

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.1,2,710 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).2224 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 in1,2224 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.2527 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).3134

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 (, and submitted to GenBank (Accession numbers are shown in Table 2).

Table 1 Distribution of IncX3 plasmid carrying isolates among strains expressing different carbapenem resistance mechanisms

Table 2 Characteristics of carbapenemase producing Enterobacteriaceae harboring IncX3 type plasmids with carbapenemase genes

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).


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.

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 (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 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).

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 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.


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.


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.


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.


The authors report no conflicts of interest in this work.


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Supplementary materials

Table S1 Antibiotic susceptibility of clinical isolates carrying carbapenemases on IncX3 plasmids and their single IncX3 plasmid containing derivatives

Table S2 IncX3 plasmids (retrieved from GenBank in January 2019) from different geographical regions with unique backbone sequences and beta-lactamase genes carried

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