Emergence of a Multidrug-Resistant Enterobacter hormaechei Clinical Isolate from Egypt Co-Harboring mcr-9 and blaVIM-4.

Abstract: This study describes the first full genomic sequence of an mcr-9 and blaVIM-4-carrying multidrug-resistant Enterobacter hormaechei clinical isolate from Egypt. The strain was isolated in April 2015 from the sputum of a patient in Cairo, Egypt. The mcr-9 and blaVIM-4 genes were identified by PCR screening and DNA sequencing; the isolate was subjected to antimicrobial susceptibility testing, conjugation experiments, and whole genomic sequencing. mcr-9 and blaVIM-4 were carried by an IncHI2 plasmid, pAMS-38a (281,121 bp in size); the plasmid also carried genes conferring resistance against sulfonamides (sul1), quinolones (qnrA1), trimethoprim (dfrA1), β-lactams (blaTEM-1B), aminoglycosides (aac (6')-II, aadA23, aadA2b, and ant(2'')-Ia). The strain was susceptible to colistin (MIC, <0.25 μg/mL); this could be due to the absence of the qseC/qseB regulatory system located downstream of mcr-9 in Enterobacterales, which is involved in the induction of colistin-resistance. The genetic context of mcr-9 and blaVIM-4 was identified as IS1-mcr-9-IS903-pcoS-∆pcoE-rcnA and intI1-blaVIM-4-aac (6')-II-dfrA1-∆aadA23-smr-ISPa21-qacE∆1, respectively. This is the first report of an mcr-9 and blaVIM-4 /IncHI2-carrying multidrug-resistant E. hormaechei clinical isolate from Africa and the Middle East. Plasmids of the IncHI2 group and the two insertion sequences (IS1, and IS903) might be the main vehicles for dissemination of mcr-9. Further screening for mcr-9 is essential for identifying its incidence and to prevent its dissemination.


Introduction
Problems associated with the development and spread of antimicrobial resistance (AMR) in clinical practice are increasing and are currently being viewed as a major threat to public health globally [1,2]. Carbapenem resistance in Enterobacterales is mediated through the expression of a large set of carbapenemase-encoding genes: particularly, Ambler class A β-lactamases (KPC, FRI, GES, SME), Ambler class B metallo-β-lactamases (VIM, NDM, IMP, SIM, GIM, SPM), and Ambler class D enzymes (OXA-type) [3,4]. Verona integron-mediated metallo-β-lactamase (VIM) is located as a gene cassette in a class 1 integron with genes for resistance against trimethoprim or aminoglycosides [5]. The bla VIM-4 gene encodes resistance against all β-lactams except aztreonam and was previously reported in Pseudomonas aeruginosa, Enterobacter cloacae, or other Enterobacterales from Egypt, Kuwait, and United Arab Emirates [5][6][7][8]. Recently, we reported bla VIM-2 and bla VIM-24 from clinical P. aeruginosa strains from Egypt [5].
Colistin is one of the last-resort antibiotics for treating infections caused by carbapenem resistant Gram-negative bacteria. The activity and efficacy of colistin has been challenged by the global spread of the self-transmissible mobilized colistin-resistance genes (mcr). In 2016, the first mcr-1 gene was reported from Escherichia coli and Klebsiella pneumoniae isolated from patients, food, and animals in China [9]. MCR-1 acts by modifying the lipid A part of the lipopolysaccharide in Gram-negative bacteria by adding phosphoethanolamine, which reduces the binding affinity to colistin [9]. As of 7 January 2020, 10 variants of the mcr genes are available in the Bacterial Antimicrobial Resistance Reference Gene Database (https://www.ncbi.nlm.nih.gov/pathogens/isolates#/refgene/); however, only mcr-1 has been previously reported from Egypt [10], and little is yet known about the prevalence of other mcr variants in Egypt.
Here, for the first time, we describe the complete genomic sequence of an E. hormaechei clinical isolate carrying the mcr-9 and bla VIM-4 genes on an IncHI2 plasmid from Africa and the Middle East generated using the Illumina MiniSeq and Oxford Nanopore sequencing platforms.

Antimicrobial Susceptibility Testing (AST)
A standardized broth microdilution (BMD) technique was used to determine the minimum inhibitory concentration (MIC) of various antimicrobial agents according to the standards and interpretive criteria described by the Clinical and Laboratory Standards Institute (CLSI, document M100-S24) and European Committee on Antimicrobial Susceptibility Testing (EU-CAST) (for colistin and tigecycline breakpoints) (http://www.eucast.org). For all experiments, the purified powder of each antibiotic was diluted following CLSI recommendations. E. coli ATCC 25922 was used as a control.

Filter-Mating Conjugation
A Conjugation experiment was performed using the AMS-38 strain and the azide resistant E. coli J53 strain as the donor and recipient, respectively. Due to the thermo-susceptibility of IncHI2 plasmids (i.e., conjugation is impaired at 37 • C), we performed this experiment at 25-30 • C [12]. The transconjugants were selected on LB agar plates containing 100 µg/mL ampicillin and 100 µg/mL sodium azide. Colony-direct PCR was performed using VIM-F and VIM-R [5] and mcr-9-forward and mcr-9-reverse [11] primers to confirm the transfer of the plasmid carrying mcr-9 and bla VIM-4 .

Whole Genome Sequencing (WGS) and Analysis
An overnight bacterial culture of the AMS-38 strain was used to extract the total genomic DNA (gDNA) using a standard proteinase K method. The quality of the isolated gDNA was assessed using a Microdrop (Thermo Scientific™, Multiskan™ Sky Microplate Spectrophotometer, Waltham, MA, USA), Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and a dsDNA fluorescent dye method using DeNovix (Wilmington, DE, USA). The sequencing library was constructed using a Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA.) for Illumina MiniSeq. The DNA library for Oxford Nanopore sequencing was prepared using 400 ng of gDNA according to the Rapid Barcoding Sequencing kit (SQK-RBK004) (Oxford Nanopore Technologies, Oxford, UK). The sequencing libraries were purified using a Promega Size-Selective Purification System (Promega, Madison, WI, USA), and 11 µL of the sequencing library was loaded onto a FLO-MIN106 flow cell and sequenced with the GridION device (Oxford Nanopore Technologies) for 48 h. Hybrid assembly of both Illumina and Nanopore reads was performed using Flye v2.6 (https://github.com/fenderglass/Flye). Additionally, Pilon-v1.23 [13] was used to improve the genome assembly. The assembly and annotation of pAMS-38a were performed using DFAST [14]. The identification of AMS-38 was done with JSpeciesWS (http://jspecies.ribohost.com/jspeciesws/#analyse) using Tetra Correlation Search (TCS) against reference database Genome DB and the pairwise comparison using Average Nucleotide Identity calculation based on BLAST+ (ANIb). Antimicrobial resistance genes were identified by ResFinder-3.2 available at Center for Genomic Epidemiology (https://cge.cbs.dtu.dk/services/ResFinder/). The plasmid Inc groups, pMLST and multilocus sequence typing (MLST) were identified by PlasmidFinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/), (https://cge.cbs.dtu.dk/services/pMLST/) and MLST 2.0 software (https://cge.cbs.dtu.dk/services/MLST/), respectively. The plasmids that were highly similar to pAMS-38a were detected by a Mash (v2.1.1) [15] distance search against the PLSDB v. 2019_10_07 [16]. The BRIG tool was used to perform a circular comparison between the complete sequence of pAMS-38 and the highly similar plasmids detected by a Mash distance search [17]. EasyFig tool was used to visualize and compare the genetic environment of mcr-9 in its harboring plasmids (http://mjsull.github.io/Easyfig/).

Characterization of the Genome of E. hormaechei AMS-38
By combining the short reads obtained from Illumina MiniSeq and the long reads obtained from Oxford Nanopore sequencing, we obtained assemblies with high-quality and sufficient for closing the genome and the plasmids contained in the strain. The chromosome of the AMS-38 strain was 4,914,941 bp in size with an average G + C content of 54.9%. Analyzing the average nucleotide identity (ANI) of the AMS-38 genome showed that the strain belonged to E. hormaechei subsp. steigerwaltii group (99.69% identity (ID) with 97.71% query coverage(QC) to E. hormaechei subsp. steigerwaltii (GCA 001011725) GN02001 followed by Enterobacter sp. MGH86 (99.62% ID with 95.21% QC), followed by E. hormaechei subsp. steigerwaltii 44524 (98.36% ID with 86.91% QC), followed by E. hormaechei subsp. steigerwaltii 44517 (98.36% ID with 86.92 QC)). Analysis of MLST of the strain AMS-38 showed that it belonged to ST133 (allelic profile 11-4-4-13-39-4-9).
Based on the available literature, the frequency of detecting mcr-9-carrying Enterobacterales with susceptibility to polymyxins is high [18,[20][21][22]. Additionally, we speculate a silent dissemination of mcr-9 since its possession confers a decreased susceptibility, not resistance, to colistin as detected in several Enterobacterales [18,[20][21][22]. On the other hand, only two studies detected mcr-9 conferring clinical resistance to colistin [11,19]. Moreover, the expression of mcr-9 was shown to be inducible due to the presence of the downstream qseC/qseB regulatory system [11]. Furthermore, the lack of these two genes downstream of the mcr-9-like gene in its original progenitor, B. gaviniae, suggests that their acquisition likely has been through an independent mobilization event with respect to the acquisition of mcr-9 [11]. Similarly, that event might happen in mcr-9-carrying and colistin susceptible strains leading to the induction and colistin resistance. Until now, the clinical impact of mcr-9, in the colistin-susceptible strains lacking qseC/qseB system, is unknown.

Conclusions
In conclusion, we reported the first complete genomic sequence of an mcr-9and bla VIM-4 -coharboring E. hormaechei strain from Africa and the Middle East. Comparative genomics was used to assess the genetic environment of the mcr-9 gene. Plasmids of the IncHI2 group and the two insertion sequences (IS1, and IS903) might be the main vehicles for dissemination of mcr-9. Screening for mcr-9 and other mcr genes is essential in Egypt to determine its prevalence and to prevent its spread.