Transferable IncX3 plasmid harboring blaNDM-1, bleMBL, and aph(3’)-VI genes from Klebsiella pneumoniae conferring phenotypic carbapenem resistance in E. coli

Background The dissemination of carbapenem resistance via carbapenemases, such as the metallo-β-lactamase NDM, among Enterobacterales poses a public health threat. The aim of this study was to characterize a plasmid carrying the blaNDM-1 gene, which was extracted from a clinical Klebsiella pneumoniae uropathogen from an Egyptian patient suffering from a urinary tract infection. Methods and results The recovered plasmid was transformed into competent E. coli DH5α which acquired phenotypic resistance to cefoxitin, ceftazidime, and ampicillin/sulbactam, and intermediate sensitivity to ceftriaxone and imipenem (a carbapenem). Whole plasmid sequencing was performed on the extracted plasmid using the DNBSEQ™ platform. The obtained forward and reverse reads were assembled into contigs using the PRINSEQ and PLACNETw web tools. The obtained contigs were uploaded to PlasmidFinder and ResFinder for in silico plasmid typing and detection of antimicrobial resistance genes, respectively. The final consensus sequence was obtained using the Staden Package software. The plasmid (pNDMKP37, NCBI accession OK623716.1) was typed as an IncX3 plasmid with a size of 46,160 bp and harbored the antibiotic resistance genes blaNDM-1, bleMBL, and aph(3’)-VI. The plasmid also carried mobile genetic elements involved in the dissemination of antimicrobial resistance including insertion sequences IS30, IS630, and IS26. Conclusions This is Egypt’s first report of a transmissible plasmid co-harboring blaNDM-1 and aph(3’)-VI genes. Moreover, the respective plasmid is of great medical concern as it has caused the horizontal transmission of multidrug-resistant phenotypes to the transformant. Therefore, new guidelines should be implemented for the rational use of broad-spectrum antibiotics, particularly carbapenems. Supplementary Information The online version contains supplementary material available at 10.1007/s11033-023-08401-9.


Introduction
Antimicrobial resistance (AMR) has been on the rise globally over the last decade, in addition to a shortage of functional antimicrobials, and a lack of novel ones. Such resistance has a major impact on treatment outcomes, resulting in higher antimicrobial costs, longer hospital stays, greater hospital expenses, and higher fatality rates [1]. In an effort to overcome the increasing rates of resistance to penicillins, cephalosporins, aminoglycosides, and fluoroquinolones, the use of carbapenems has increased in recent years [2,3]. Regrettably, the extensive usage of carbapenems has led to the development of carbapenem-resistance (CR) particularly, in Gram-negative bacteria (GNB) such as Enterobacterales, Acinetobacter spp., and Pseudomonas spp. This has resulted in a global public health crisis due to the rapid spread of CR and the scarcity of novel antimicrobials [4][5][6].
The World Health Organization (WHO) announced in 2017 a global priority pathogens list for which new antibiotics are urgently needed, among which carbapenem-resistant Enterobacterales (CRE) that are co-resistant to 3rd generation cephalosporins were listed as critical priority pathogens [7]. Infections caused by CRE pathogens have caused substantial morbidity and mortality, and are considered as a rising healthcare threat [8,9]. Carbapenemase-producing K. pneumoniae represents the fastest-growing threat to antibiotic resistance in terms of morbidity and mortality [10][11][12].
Among the different mechanisms of CR, carbapenemases represent the greatest threat in terms of AMR spread, due to their ability to inactivate most β-lactams, and the fact that they are encoded by genes conferred by mobile genetic elements (MGEs) such as transposons, insertion sequences, or plasmids, which are capable of interspecies and intraspecies horizontal transfer [13,14]. Plasmids are extrachromosomal, self-replicating DNA units that encode nonessential but usually useful traits for their host. Several copies of one or more plasmids may be present within a bacterial cell [15]. Many plasmids encode genes for resistance to antimicrobial agents and heavy metals, virulence factors, production of toxins, attachment to intestinal mucosa, and for new pathways of degradation [16]. Acquisition of such plasmids enables the host bacterium to adapt to environmental changes, such as exposure to antibiotics, rapidly and effectively [17]. Plasmid replicon typing was established in order to facilitate their identification and study [18].
The New Delhi metallo-β-lactamase (NDM), a carbapenemase that belongs to Ambler class B β-lactamases, was discovered in 2008 when a Swedish patient who had traveled to New Delhi, India, acquired a urinary tract infection caused by a carbapenem-resistant K. pneumoniae. The strain isolated from his urine was a metallo-β-lactamase (MBL) producer but was negative for previously known MBL genes. This led to the discovery of the bla NDM-1 gene [19]. It was later revealed that NDM β-lactamases confer resistance to almost all β-lactam antimicrobials (except aztreonam), including carbapenems which are often used as last-resort treatment options for multidrug-resistant (MDR) and extended-spectrum β-lactamase (ESBL) producersassociated infections [20,21].
Genes coding for NDM enzymes are highly transmissible as they are often located on plasmids harboring several other antimicrobial resistance determinants, thus, NDMproducing bacteria are often resistant to aminoglycosides and fluoroquinolones, a fact which has posed challenges in the clinical treatment of infections caused by CRE [20,22,23].
In this study, we report the sequence of a transmissible IncX3 plasmid carrying a carbapenemase resistance gene (bla NDM-1 ), bleomycin resistance gene (ble MBL ), and aminoglycoside resistance gene (aph(3')-VI), which was extracted from a carbapenem-resistant K. pneumoniae clinical isolate recovered from the urine of an Egyptian patient suffering from a urinary tract infection.

Collection of the clinical isolate
Carbapenem-resistant K. pneumoniae isolate (code: 37.AK) was obtained from El-Demerdash Tertiary Care Hospital's Microbiology laboratory in Cairo, Egypt. According to the hospital's records, the isolate was recovered from the urine sample of a male patient admitted to the hospital with significant bacteriuria (uropathogens > 10 5 cfu/ml) in May 2020. The identification of the isolate was carried out using phenotypic and cultural characteristics [24]. Identification of the isolate was further confirmed by using the commercially available MIKROLATEST ® ID Kit ENTEROtest 24 N (Erba Lachema, The Czech Republic) [25] following the manufacturer's instructions. The patient had no history of international travel. Written and oral informed consents were attained from the patient. The study was approved by the Ethics Committee of the Faculty of Pharmacy Ain Shams University (EN-REC-ASU-2019-98) and was in accordance with the Declaration of Helsinki.

Detection of carbapenemase production
Blue-Carba test was performed as described by Pires et al. for the detection of carbapenemases directly from bacterial cultures [28]. The modified carbapenem inactivation method (mCIM) was also used to detect the production of carbapenemases as recommended by the CLSI 2020 [27]. The reference strain E. coli ATCC ® 25,922 was used as control.

Amplification of some plasmid-encoded carbapenemase genes
The plasmid DNA was extracted using the GeneJet Plasmid Miniprep Kit (catalog number: K0502 Thermo Fisher Scientific, Lithuania). The bla NDM , bla KPC , bla OXA-48, bla VIM , and bla IMP carbapenemase genes were amplified using the plasmid extract as a template for PCR and the appropriate primers synthesized by Invitrogen ® (Thermo Fisher Scientific, UK), and DreamTaq™ Green PCR Master Mix (Thermo Fisher Scientific, Lithuania). Gel electrophoresis was used to analyze the PCR products as previously reported [29]. Table 1 shows the primers used for PCR amplification of the tested carbapenemase-encoding genes, their annealing temperatures, and the expected product sizes of the tested genes.

Transformation
Chemical transformation was performed according to the protocol described by Sambrook and Russell [29]. E. coli DH5α is a standard susceptible strain free from antimicrobial resistance genes. The preparation of competent E. coli DH5α cells was carried out via the modified Hanahan method [30]. The plasmid extract was used to transform the competent E. coli DH5α. The transformant was cultured on an LB/ampicillin agar plate at a concentration of 100 µg/ml. Untransformed E.coli DH5α was used as a negative control. Blue-Carba test, mCIM test, and antimicrobial susceptibility testing of the transformant were carried out, as well as the determination of the minimum inhibitory concentrations (MICs) of meropenem and imipenem by broth microdilution method following CLSI guidelines [27,31]. The EDTAmodified carbapenem inactivation method (eCIM) is a phenotypic test used to differentiate MBLs (e.g. NDM, VIM, and IMP carbapenemases) from serine carbapenemases (e.g. OXA-48, and KPC carbapenemases) in Enterobacterales isolates showing positive mCIM test results. This test was carried out and interpreted as mentioned in the CLSI guidelines [27] to determine the type of carbapenemase enzyme produced by the transformant. The plasmid DNA was then extracted from the transformant (TS37.AK) to be used as a template for PCR amplification for confirming the presence of carbapenemase genes on the transformed plasmid using the primers listed in Table 1.

Plasmid sequencing and bioinformatic analysis
The transformant was sub-cultured 2 successive times on LB/ampicillin (100 µg/ml), followed by a third time on LB/

Antimicrobial susceptibility, carbaenemaseproduction, and PCR amplification of carbapenemase-encoding genes
The results of antimicrobial susceptibility, Blue-Carba test, mCIM, and PCR amplification of carbapenemase-encoding genes of the carbapenem-resistant K. pneumoniae clinical isolate (37.AK) were previously published [36]. In terms of antimicrobial susceptibility to carbapenems, the isolate was resistant to meropenem, imipenem, and ertapenem, and was susceptible to doripenem.

Transformation
The The plasmid sequence was uploaded to the plasmid database PLSDB (https://ccb-microbe.cs.uni-saarland.de/ plsdb/) (accessed on 3 December 2022) to search the database for plasmids with nucleotide sequences similar to our plasmid (pNDMKP37). The search strategy used was "Mash dist." (for long sequences e.g., contigs or long reads), and the parameters were maximum p-value of 0.05 and maximum distance of 0.05. The creation of the circular image and comparison with other reported similar plasmids were performed using the BLAST Ring Image Generator (BRIG) tool v0.95 (https://sourceforge.net/projects/brig/)(accessed on 3 December 2022) [35].

Nucleotide sequence accession number and data availability
The plasmid pNDMKP37 sequence project has been deposited in the GenBank under BioProject PRJNA878540, sample number SAMN30732688, and with the plasmid sequence accession number OK623716.1. The Sequence Read Archive (SRA) data are available from GenBank under the accession number SRR21492341. ble MBL coding for bleomycin resistance protein BRP MBL , and aph(3')-VI gene coding for APH(3')-VI (aminoglycoside 3′-phosphotransferase). The final obtained consensus sequence length was 46,160 bp with 46.28% G + C content. The plasmid contained 58 open reading frames (ORFs) (37 genes of known functions and 21 genes of hypothetical proteins, Fig. 1). The insertion sequence IS30 family transposase (98.98% similarity to ISAba125) was found upstream of bla NDM-1 , and bleomycin resistance gene, ble MBL , was located downstream. The genes dsb and trpF were found downstream of ble MBL , all of which are common genetic contexts of bla NDM gene variants [37]. Other MGEs were found in the genetic environment of bla NDM-1 , including the insertion sequences IS630 and IS26, which can promote the mobilization of bla NDM-1 between plasmids or chromosomes.

Discussion
The most effective antimicrobials for treating infections caused by MDR bacteria are carbapenems. This class of antimicrobials exhibits a wide spectrum of activity against both Gram-negative and Gram-positive bacteria. The overuse of carbapenems in many nations has accelerated the emergence of carbapenem resistance, resulting in a worldwide public health crisis [38]. Increasing numbers of Enterobacterales (especially Klebsiella spp.) and lactose non-fermenters are acquiring and producing carbapenemases. NDMs, which confer resistance to carbapenems and other β-lactam antibiotics, have been increasingly reported across the world since their first report.
In this study we report an IncX3 transmissible plasmid carrying 3 resistance genes: the carbapenemase resistance gene (bla NDM-1 ) coding for NDM-1 carbapenemase, bleomycin resistance gene (ble MBL ) coding for bleomycin resistance protein BRP MBL , and aminoglycoside resistance gene (aph(3')-VI) coding for aminoglycoside 3′-phosphotransferase APH(3')-VI. The plasmid was extracted from a carbapenem-resistant K. pneumoniae clinical isolate recovered from the urine of an Egyptian patient suffering from a urinary tract infection. In our previous study involving the same transformant (TS37.AK), the PCR amplification results of the transferable plasmid revealed the presence of genes coding for carbapenemases in the plasmid extract. However, after whole plasmid sequencing in the current study, the sequencing data confirmed that this plasmid carried the bla NDM-1 gene coding for NDM-1 carbapenemase, as well as other resistance determinants namely the ble MBL gene coding for bleomycin resistance protein BRP MBL , and aph(3')-VI gene coding for APH(3')-VI (aminoglycoside 3′-phosphotransferase).

The assembled consensus sequences
Clean forward and reverse sequence reads were assembled into 6 contigs. The plasmid belonged to the IncX3 plasmid incompatibility group and was found to carry 3 resistance genes, namely bla NDM-1 coding for NDM-1 carbapenemase, bla  , and bla OXA-232 genes were associated with ISEcp1 elements [39]. In our study, although bla OXA-48 gene was detected by PCR in the plasmid extract of the transformant (TS37.AK), neither the gene nor its usual genetic context were detected on the sequenced plasmid. To confirm that the bla OXA-48 gene not being in the plasmid was not a bioinformatics processing error, whole genome DNA templates of the transformant were prepared as described by Doyle et al. [40], and the supernatant was used as a template for PCR amplification of bla OXA-48 and bla NDM genes. This was done to detect the possibility that the bla OXA-48 could be carried on a MGE that was not incorporated in the sequenced plasmid and was instead chromosomally incorporated. The PCR results confirmed the presence of the bla NDM gene and the absence of the bla OXA-48 gene from the whole genome of the transformant, although the bla OXA-48 gene was previously detected in the initial plasmid extract of the transformant by PCR and subsequent sanger sequencing of the resulting amplicon [36]. It was thus speculated that the MGE carrying the bla OXA-48 gene might have been lost from our plasmid after repeated subculturing of the transformant, which could be proved by whole genome sequencing of the clinical isolate and the transformant.
Generally, the bla NDM gene variants share two common features pertaining to their genetic environment: the bleomycin resistance gene (ble MBL ) is always downstream of bla NDM , and the ISAba125 insertion sequence (either intact or truncated) is always upstream. Further downstream of ble MBL , there is often a set of several genes, including trpF (which encodes a phosphoribosylanthranilate isomerase), and dsb (which encodes a twin-arginine translocation pathway signal sequence domain protein) [37]. This was evident in the sequence of the plasmid in the present study (pND-MKP37), where ble MBL was located downstream of bla NDM-1 , and the insertion sequence IS30 family transposase (98.98% similarity to ISAba125) was found upstream. Additionally, the genes trpF and dsb were found downstream of ble MBL . Other MGEs were found in the genetic context of bla NDM-1 , including insertion sequences IS630 and IS26, which can promote the mobilization of bla NDM-1 between plasmids or chromosomes.
As for the bla OXA-48 gene, it is generally associated with an upstream IS1999 element, and a downstream composite transposon Tn1999 [39]. Other bla OXA-48-like genes have been found on plasmids in association with other insertion sequences, for example the bla OXA-163 gene was located downstream of an ISEcl4 element, and bla OXA-181 , pneumoniae strain RIVM_C018652; AC, CP068835.1), and pNDM5-LDR (K. pneumoniae strain LDR; AC, MK308632.1). The red ring represents the plasmid used as a reference for the alignment (pND-MKP37); the size of the reference is indicated in the center of the panel. The genes coding for APH (3ʹ)-VI, NDM-1, and BRP MBL proteins are marked in orange in the outer ring, and hypothetical proteins are marked in blue. AC, GenBank accession code and amikacin) and quinolones (ciprofloxacin and levofloxacin), however, such resistance was not conferred to the transformant by the transformed plasmid. This is probably because resistance mechanisms to such antimicrobial agents was chromosomally-mediated in the parent clinical isolate.

Conclusions
In this study, a transmissible plasmid co-harboring bla NDM-1 and aph(3')-VI genes was detected in a K. pneumoniae clinical isolate in Egypt. The plasmid (pNDMKP37) was typed as an IncX3 plasmid with a size of 46,160 bp and harbored antibiotic resistance genes against carbapenems (bla NDM-1 ), bleomycin (ble MBL ), and aminoglycosides (aph(3')-VI). Upon transformation of the pNDMKP37 in E. coli DH5α, it conferred phenotypic resistance against ceftazidime, cefoxitin, and ampicillin/sulbactam, and intermediate sensitivity to ceftriaxone and imipenem. Moreover, the respective plasmid poses a great medical concern as it was responsible for the horizontal transmission of multidrug-resistant phenotypes to the transformant, particularly phenotypic resistance to carbapenems. New guidelines should be implemented for the rational use of broad-spectrum antibiotics, particularly carbapenems.
It was noticed that the parent K. pneumoniae clinical isolate (37.AK) was resistant to aminoglycosides (gentamicin Data Availability All the data supporting the findings are included in the manuscript. The plasmid pNDMKP37 sequence project has been deposited in the GenBank under BioProject PRJNA878540, sample number SAMN30732688, and with the plasmid sequence accession number OK623716.1. The Sequence Read Archive (SRA) data are available from GenBank under the accession number SRR21492341. Acknowledgments:

Declarations
Competing interests 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.

Ethics approval
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Faculty of Pharmacy Ain Shams University Ethics Committee (ENREC-ASU-2019-98).

Consent to participate
Informed consent was obtained from the patient after explaining the purpose of the study.
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