Coexistence of blaNDM–5 and tet(X4) in international high-risk Escherichia coli clone ST648 of human origin in China

The emergence of pathogens is conferring resistance to last-resort therapies such as tigecycline, colistin, and carbapenems, limiting the therapeutic options, and raising concerns about the emergence of new “superbugs.” This study reports the first incident of a bla_NDM–5 and tet(X4) co-harboring Escherichia coli with resistance to carbapenem and tigecycline recovered as the causative agent of a urinary tract infection in a 94-year-old patient. The E. coli strain ECCL209 carries multiple resistance genes [i.e., bla_TEM–1B, bla_NDM–5, bla_CMY–2, aadA22, florR, erm(B), mph(A), erm(42), lnuG, qnrS1, and sul2] and exhibits resistance to almost all clinically used antibiotics. MLST analysis found that the strain belongs to ST648, considered a worldwide high-risk pandemic clone. Moreover, multiple plasmid incompatibility types were detected, i.e., IncHI1A, IncHI1B, IncFII, IncFIA, IncFIB, IncQ1, Col, and IncX4. Genetic analysis revealed that bla_NDM–5 and tet(X4) genes were localized on two hybrid plasmids with multiple replicons. Continuous monitoring studies are suggested to quantify the antimicrobial resistance and assess the dissemination of such superbugs into a human healthcare setting.

Introduction

Antimicrobial resistance (AMR) has been an emerging and increasing threat to global health (World Health Organization [WHO], 2014; Su et al., 2017). A report from 2016 predicted that global fatalities from infectious diseases caused by AMR will rise from 0.7 to 10 million by 2050, with a vast estimated inaction cost of US$100 trillion between 2016 and 2050 (O’Neill, 2016). The antibiotic-resistant bacteria (ARB) of particular interest are multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) (Basak et al., 2016). These ARBs are called superbugs, and they can cause severe bacterial infections due to their acquired and intrinsic resistance mechanisms and render the efficacy of many existing antibiotics (Potter et al., 2016; Acolatse et al., 2022).

Of particular concern is AMR among Gram-negative bacterial species, especially the carbapenem-resistant Escherichia coli (CRE), which is the leading cause of urinary tract infections (UTIs) and is challenging to treat with last-resort carbapenem antibiotics. Carbapenems were developed to tackle bacteria producing extended-spectrum β-lactamases (ESBLs). However, Gram-negative bacteria have become resistant to this group of drugs by developing and/or acquiring bla genes encoding carbapenem hydrolyzing enzymes, named carbapenemases (Codjoe and Donkor, 2017; Nordmann and Poirel, 2019). Among the newly emerging carbapenemases, New Delhi Metallo-β-lactamase (NDM) is very important due to its widespread dissemination and allelic variations (Suay-García and Pérez-Gracia, 2021). The pathogens harboring these genes resist almost all β-lactam antibiotics (Wu et al., 2019). Tigecycline and colistin were relatively effective and used as the last-resort treatments to treat such infections caused by MDR and XDR bacteria (He et al., 2019). However, the recent discoveries of plasmid-mediated colistin resistance genes (mcr-1 to mcr-10) and/or the tigecycline resistance genes tet(X1) to tet(X15) among Enterobacteriaceae, especially in CRE, predict a return to the pre-antibiotic era and pose a severe threat to public health (He et al., 2019; Hussein et al., 2021). Furthermore, the co-occurrence of tet(X4) and mcr-1 as well as the combination of tet(X4) and bla_NDM–5 genes in tigecycline- colistin- and carbapenem-resistant E. coli strains recovered from animals in China, posing a significant threat to public health, which requires urgent monitoring in terms of its prevalence (He et al., 2020; Sun et al., 2021; Lu et al., 2022).

To the best of our knowledge, herein, we identified the first case of XDR E. coli isolate co-harboring plasmid-mediated bla_NDM–5 and tet(X4) genes from a clinical sample from a human patient.

Materials and methods

Sample collection and identification

During a routine surveillance project on AMR, an E. coli isolate ECCL209 was recovered from a 94-year-old man, admitted for >6 months in the respiratory and critical care department at Shantou Hospital, Guangdong Province, China. The patient was diagnosed with UTI. The E. coli strain ECCL209 was identified by automated mass spectrometry systems (VitekMS, bioMerieux, Marcy I’Etoile, France) and further confirmed by PCR utilizing the primers specific to the uidA gene as reported previously (Shafiq et al., 2019).

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was accomplished by Vitek 2 COMPACT (bioMerieux, Marcy I’Etoile, France) with AST-N334 cards for the following antimicrobial agents: amikacin (AMK), cefoperazone/sulbactam (SCF), cefepime (FEP), cefoxitin (FOX), cefotaxime (CTX), ertapenem (ETP), imipenem (IMP), amoxicillin/clavulanic acid (AMC), cefuroxime (CXM), ceftriaxone (CRO), ceftazidime (CAZ), piperacillin/tazobactam (TZP), ticarcillin/clavulanic (TCC), ceftazidime-avibactam (CZA), ciprofloxacin (CIP), doxycycline (DOX), tigecycline (TIG), aztreonam (ATM), minocycline (MIC), tobramycin (TOB), trimethoprim/sulfamethoxazole (SXT), and colistin (COL). Antibiotic susceptibility for levofloxacin (LEV) was determined using Levofloxacin Susceptibility Test Paper (Thermo Scientific™ Oxoid™, Leicestershire, United Kingdom). Results for all antibiotics were interpreted following the standard of the Clinical and Laboratory Standard Institute (CLSI M100; 31st edition) guidelines, except imipenem, ertapenem, amoxicillin-clavulanic acid, and ceftazidime/avibactam for which the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were considered.¹

Detection of antibiotic resistance genes

Detection of common ESBL genes (i.e., bla_TEM, bla_CTX–M, and bla_SHV), carbapenemases (bla_NDM, bla_KPC, bla_IMP, bla_VIM, and bla_OXA), and tigecycline-resistant genes tet (X3 and X4) was performed using PCR to identify resistance genes. All the primers used in this study are summarized in Supplementary Table 1.

Mating assay

Conjugation experiments were performed according to a previously described method (Shafiq et al., 2019). The donor strain [bla_NDM and tet(X4)-positive E. coli] was diluted to the 0.5 McFarland standard and mixed with rifampicin-resistant recipient strain (E. coli C600) at a ratio of 1:1, respectively, on the microporous membrane. After cultures were incubated at 37°C for 12–14 h, the mixtures were collected and streaked on freshly made Luria-Bertani (LB) agar plates containing tigecycline (2 mg/L), meropenem (2 mg/L), and rifampicin (300 mg/L). The presence of bla_NDM and tet(X4) in transconjugants was confirmed by PCR and corresponding resistance phenotyping. The number of positive transconjugants per recipient calculated the transfer frequency of conjugation.

Whole-genome sequencing with Illumina and Nanopore

To determine the genomic background, the ECCL209 E. coli strain was subjected to whole-genome sequencing (WGS) on the Illumina Miseq and Oxford Nanopore MinION platforms. The total DNA of E. coli strain ECCL209 was collected from fresh overnight cultures using a DNA kit (QIAamp^® DNA Mini Kit, Germany) according to the manufacturer’s guidelines. The quality and quantity of extracted genomic DNA were measured and confirmed using a Nanodrop OD-1000 spectrophotometer (Thermo-Scientific^®). DNA libraries were constructed using NEBNext^® UltraTM DNA Library Prep Kit for Illumina (NEB, USA) and sequenced using an Illumina MiSeq sequencer (Illumina, San Diego, CA, USA). For the Nanopore platform, a Rapid Barcoding Sequencing Kit was used to construct the libraries and sequenced with a mini device (MinION), as previously reported (Maestri et al., 2019). Guppy base-calling software version 2.2 was used to generate fast5 files harboring the 1D DNA sequence from fast5 files. The quality of raw data from paired-end sequencing was checked using FastQC (version 0.11.6). Fastp (version 0.23.2) (Chen et al., 2018) was performed for the quality filtering to remove the low-quality reads, adapters, and poly-G tails. De novo assembly was accomplished using SPAdes (version 3.15.3) and Flye (version 2.8.3) with default parameters.

Assembly annotation and genetic analysis of Escherichia coli ECCL209

The assembled genomes were subjected to determine the resistome, virulome, MLST, serotype, mobile genetic elements (MGEs), and plasmidome using online search tools such as ResFinder 4.0; VirulenceFinder 2.0, MLST 2.0, SerotypeFinder 2.0, MobileElementFinder, and PlasmidFinder 2.0, at the Center for Genomic Epidemiology (CGE).² Genome annotation and visualization were performed using Prokka (version 1.14.6) and Proksee.³ Plasmid replicons were identified using Abricate (version 1.0.1)⁴ from the assemblies. EasyFig (version 2.2.2) was used to compare and visualize the region of interest between similar sequences. The sequence similarity search was performed using BLAST against the NCBI nucleotide database. The significant hits were investigated, and related information, including the source organisms and hosts, was visualized along the BLAST result tree using ggtree version 3.4.

Results and discussion

The E. coli strain exhibited resistance against 19 antimicrobial agents, an XDR phenotype (Magiorakos et al., 2012), including SCF, FEP, FOX, CTX, ETP, IMP, AMC, LEV, TZP, CXM, CRO, CAZ, TCC, CZA, CIP, DOX, TIG, ATM, and MIC, while susceptible to AMK, TOB, SXT, and COL. The susceptibility data are shown in Supplementary Table 2. The E. coli isolate ECCL209 was resistant to carbapenems and tigecycline and harbored bla_TEM, bla_NDM, and tet(X4) genes, amplified by PCR and subsequently confirmed by Sanger sequencing.

To determine the transmissibility of bla_NDM and tet(X4) genes, we performed conjugation experiments with a recipient E. coli strain C600. The outcomes of conjugation proved that the bla_NDM and tet(X4) genes in donor E. coli isolate ECCL209, with their corresponding resistance against imipenem and tigecycline, were successfully moved to the recipient strain C600, suggesting that bla_NDM and tet(X4) genes were located on conjugative plasmids. The cotransfer of bla_NDM and tet(X4) was at a frequency of (1.67 ± 0.2) × 10^–1 to (3.12 ± 0.1) × 10^–3 cells per recipient.

The main comprehensive results from the WGS analysis of Illumina and Nanopore are summarized in Table 1. The ECCL209 isolate was assigned as serotype O83:H42 using SerotypeFinder 2.0,⁵ which is an extraintestinal pathogenic E. coli (ExPEC) primarily found in samples from animals, indicating their possible transmission from animal to humans (Abreu-Salinas et al., 2020; Shafiq et al., 2021a,b, 2022). MLST analysis revealed that E. coli isolate ECCL209 in this study belonged to sequence type (ST648), which had been previously reported to carry bla_CTX–M–, bla_CMY–2–, bla_NDM–, bla_OXA–48–, and mcr-1 encoding genes and caused a significant proportion of infections in humans (Hornsey et al., 2011; Poirel et al., 2018; Chowdhury et al., 2022). This clonal lineage has emerged as a pandemic high-risk clone, being globally reported in humans, animals, and the environment (Hornsey et al., 2011; Fernandes et al., 2018; Furlan et al., 2020; Chowdhury et al., 2022; Landolsi et al., 2022). To the best of our knowledge, this is the first report of ST648, with bla_NDM and tet(X4).

TABLE 1

Table 1. Genomic characteristics of Escherichia coli ST648 strain isolated from human origin.

Our resistome results confirmed aminoglycosides (aadA22), amphenicols (floR), β-lactams (bla_TEM–1B, bla_NDM–5, and bla_CMY–2), sulfonamides (sul2), macrolides [ermB, mphA, erm(42), and lnuG], quinolones (qnrS1), and tetracycline-resistant genes [tet(X4) and tetM]. Moreover, we found chromosomal mutations in parE (p. S458A), parC (p. S801), and gyrA (p. S83L, p. D87N), which encodes high-level resistance to fluoroquinolones (Mohsin et al., 2019). Multiple plasmids were detected in the E. coli ECCL209 strain, including, IncHI1A, IncHI1B, IncFII, IncFIA, IncFIB, IncQ1, Col, and IncX4. Detection of multiple plasmid types reflects the strains’ severity because all these replicons identified have the ability of horizontal transfer and play a vital role in spreading AMR genes (Rodríguez-Beltrán et al., 2021).

Regarding virulence genes, the presence of iutA (ferric aerobactin receptor), terC (tellurium ion resistance protein), IpfA (long polar fimbriae), traT (outer membrane protein complements resistance), air (enteroaggregative immunoglobulin repeat protein), sitA (iron transport protein), hra (heat resistance agglutinin), yfcV (fimbrial protein), iucC (aerobactin synthetase), eilA (Salmonella HilA homolog), and chuA (outer membrane hemin receptor) were identified in E. coli strain ECCL209. These virulence genes could enhance bacterial pathogenicity, and a recent study also described their direct interaction with ARGs in terms of bacterial survivability, which need to be disclosed in future studies (Zhang et al., 2019).

To further understand the genetic contexts of bla_NDM–5 and tet(X4), we carried out long-read sequencing of E. coli ECCL209 isolate with the Oxford Nanopore MinION platform to obtain complete genome sequences. This assembled genome had four contigs, with a total length of 5,411,927 bp and an average G + C content of 50.31%. Bioinformatic analysis revealed that isolate ECCL209 harbored a chromosome and three circular plasmids comprising pECCL209-tetX4-190-kb, pECCL209-blaNDM5-157-kb, and pECCL209-blaCMY2-36-kb.

pECCL209-tetX4 was a 190,682-bp plasmid co-fused with IncHI1A, IncHI1B, and IncFIA, forming multiple replicon plasmids. Similarly, a fusion plasmid has been previously reported from China recently, where a tet(X4) gene was located in Enterobacter cloacae on a hybrid plasmid (∼190 kb) with IncFIA, IncHI1A, and IncHI1B replicons (Wu et al., 2022). This high homology of plasmids from animal and human origin suggests that tet(X4)-carrying plasmids could be conjugated from E. cloacae to E. coli. The BLASTn search was performed against the NCBI database to examine the sequence similarity of pECCL209-tetX4-190-kb and pECCL209-blaNDM5-157-kb. Phylogenetic analysis revealed that the pECCL209-tetX4 plasmid was similar to other bacterial strains with ≥90% query coverage. Most of the plasmid sequences matched with pECCL209-tetX4 were from animal origins, while this is the first human-origin E. coli plasmid harboring tet(X4) resistant gene (Figure 1A). The result showed that the pECCL209-tetX4-like plasmid might have been widely spread in different species of Enterobacteriaceae. pECCL209-tetX4-190-kb displayed a mosaic structure harboring five AMR genes, including flor (phenicol resistance), qnrS1 (quinolone resistance), bla_TEM–1B (β-lactam resistance), aadA22 (aminoglycosides resistance), and lnu(G) (lincosamide resistance), and the MGEs found in the MDR region were IS26, ISVsa3, IS6, and IS91 (Figure 1B). The backbone of plasmid pECCL209-tetX4-25-kb from this study harboring a tet(X4) gene showed >99% nucleotide homology and 100% query coverage to several other tet(X4) carrying plasmids in Klebsiella pneumoniae and E. coli reported from animal origins, including plasmid p3Z-5L-2-X4 (GeneBank accession no. CP072517.1) in Klebsiella quasipneumoniae and pTKPN_3-186k-tetX4 (GeneBank accession no. MZ773211.1) in K. pneumoniae, while plasmid pYUGZP1-tetX (GeneBank accession no. MW439255.1) in E. coli of unknown animal origin (Figure 1C). This high similarity and dissemination of this type of plasmid suggest that plasmids harboring tet(X4) had been widely propagated in animals (He et al., 2020; Sun et al., 2021; Lu et al., 2022). Moreover, the tet(X4) genetic context in plasmid pECCL209-tetX4 of this study showed a resemblance with the above three plasmids from the NCBI database, showing that the tet(X4) gene tends to be adjacent to the upstream rdmC gene and flanked by a complete IS1R element.

FIGURE 1

Figure 1. Structure of the tet(X4)-carrying plasmid and comparison of the genetic context of tet(X4). (A) BLAST tree comparison of plasmid pECCL209-tetX4 with other homologous plasmids available in the NCBI database. (B) Structure of the tet(X4)-carrying plasmid pECCL209. (C) Sequence comparison of the genetic context of a plasmid carrying tet(X4) gene from different sources. The arrows showed the direction of the transcription. Regions of >99% of homology are displayed by gray shading.

Similarly, pECCL209-blaNDM5 was a 157,741-bp hybrid plasmid with three replicon types, i.e., IncFII, IncFIA, and IncFIB. This plasmid showed ≥90% sequence identity with other bla_NDM–5-carrying plasmids in K. pneumoniae plasmid pEH13_2 (GeneBank accession no. CP089099.1) and E. coli plasmid pYSP8-1-CTX-M-14 (GeneBank accession no. CP037912.1) of human and animal origin, respectively, suggesting that bla_NDM–5-carrying plasmids had widely disseminated in China (Figure 2A). The bla_NDM–5 gene resided in a complex region of the plasmid pECCL209-blaNDM5-157,741-bp. The plasmid carried other resistance genes, including aadA22 (aminoglycosides resistance), erm(B), erm(42), mph(A) (macrolides resistance), tet(M) (tetracycline resistance), and sul2 (sulfonamide resistance), and MGEs found in the MDR region, including IS26, ISVsa3, IS5, ISEc9, ISKox3, and IS91 (Figure 2B). The bla_NDM–5 gene was located within a 10.8-kb region, which was highly similar (99% identity) to E. coli plasmid pGZ3_NDM5 (GeneBank accession no. CP017981.1) obtained from patient urine with intra-abdominal infections and Salmonella enterica plasmid unnamed2 (GeneBank accession no. CP019444.1) collected from a patient stool in China. In the plasmid backbone of pECCL209-blaNDM5-10kb, IS5 was inserted with ISAba125 upstream of bla_NDM–5, and the ble_MBL trpF, dsbD, and IS26 were located downstream from bla_NDM–5 as shown Figure 2C. Interestingly, on sequence alignment of our plasmid pECCL209-blaNDM5 with other identical plasmids ≥60% BLAST query coverage found that the bla_NDM–5 region in other strains was mostly missing as shown in Figure 3, suggesting that bla_NDM–5 in pECCL209-blaNDM5 was captured from other mobile elements.

FIGURE 2

Figure 2. Structure of the bla_NDM–5-carrying plasmid and comparison of the genetic context of bla_NDM–5. (A) BLAST tree comparison of plasmid pECCL209-blaNDM5 with other homologous plasmids available in the NCBI database. (B) Structure of the bla_NDM–5-carrying plasmid pECCL209. (C) Sequence comparison of the genetic context of a plasmid carrying bla_NDM–5 gene from different sources. The arrows showed the direction of the transcription. Regions of >99% of homology are displayed by gray shading.

FIGURE 3

Figure 3. Linear alignment of the selected bla_NDM gene comparison with other homologous plasmids available in the NCBI database.

Conclusion

As far as we know, this is the first report that emphasized the emergence of high-risk E. coli clone ST648 of a human origin, which carries the mobile carbapenem and tigecycline resistance determinants bla_NDM–5 and tet(X4), respectively. Regardless of their low prevalence rate in humans and animal-associated sources, the mobile plasmid-mediated resistance genes in such superbugs can pose a significant threat to public health. Therefore, continuous monitoring of such MDR and XDR bacteria in humans, animals, and the environment should be considered under the aegis of the One Health approach and to guide the deployment of public health interventions before clinical cases increase.

Data availability statement

The sequence data mentioned in this present study were deposited to the GenBank NCBI database under the BioProject PRJNA850111 with accession number: SRR19844396.

Ethics statement

Ethical approval was provided by the Human Research Ethics Committee of Shantou Central Hospital and Shantou University Medical College (Ref 047 and SUMC-2021-51, respectively). Consent forms from the patients were waived by the Ethical Committee as all the clinical samples were obtained from the hospital laboratory.

Author contributions

MS and XJ designed the experiments. MS, MZ, and XL performed the experiments. MS wrote the original manuscript. BP and YY helped in the analysis. JH, HB, FY, and AA reviewed and edited the manuscript. All authors read and approved the final manuscript.

Funding

This study was graciously supported by the National Natural Science Foundation of China for International Young Scientists (No. 42150410383) and the 2020 Li Ka Shing Foundation Cross-Disciplinary Research Grant (project number: 2020LKSFG03E).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2022.1031688/full#supplementary-material

Supplementary Table 1 | List of primers used in this study.

Supplementary Table 2 | Antibiotic susceptibility profile of Escherichia coli strain ECCL209.

Footnotes

1. ^ http://www.eucast.org/clinical_breakpoints/, accessed on 30 July 2022.
2. ^ http://www.genomicepidemiology.org/services/, accessed on 30 June 2022.
3. ^ https://proksee.ca/
4. ^ https://github.com/tseemann/abricate
5. ^ http://www.genomicepidemiology.org/services/, accessed on 30 July 2022.

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