Genomic Characterization of VIM and MCR Co-Producers: The First Two Clinical Cases, in Italy

Background: the co-production of carbapenemases and mcr-genes represents a worrisome event in the treatment of Enterobacteriaceae infections. The aim of the study was to characterize the genomic features of two clinical Enterobacter cloacae complex (ECC) isolates, co-producing VIM and MCR enzymes, in Italy. Methods: species identification and antibiotic susceptibility profiling were performed using MALDI-TOF and broth microdilution methods, respectively. Transferability of the blaVIM- and mcr- type genes was verified through conjugation experiment. Extracted DNA was sequenced using long reads sequencing technology on the Sequel I platform (PacBio). Results: the first isolate showed clinical resistance against ertapenem yet was colistin susceptible (EUCAST 2020 breakpoints). The mcr-9.2 gene was harbored on a conjugative IncHI2 plasmid, while the blaVIM-1 determinant was harbored on a conjugative IncN plasmid. The second isolate, resistant to both carbapenems and colistin, harbored: mcr-9 gene and its two component regulatory genes for increased expression on the chromosome, mcr-4.3 on non-conjugative (yet co-transferable) ColE plasmid, and blaVIM-1 on a non-conjugative IncA plasmid. Conclusions: to our knowledge, this is the first report of co-production of VIM and MCR in ECC isolates in Italy.


Introduction
The ECC (Enterobacter cloacae complex) is composed of six species including E. cloacae and subspp, E. kobei, E. nimipressuralis, E. ludwigii, E. asburiae and E. hormaechei [1]. Carbapenem resistant E. cloacae complex (CREC) prevalence has increased significantly during recent years [2]. While colistin is considered as the last resort antibiotic for treating infections due to multi-drug resistant strains, increased reports of plasmid mediated mcr genes coding for colistin resistance in Enterobacterales represent a challenging and alarming situation [3]. Until now, ten variants of the mcr gene, mcr-1-mcr-10, have been identified [4]. The mcr-4.3 was reported for the first time in Singapore in 2014 on a ColE10 plasmid from a clinical E. cloacae isolate [5], and MCR-9 was initially described in 2010 in USA, in a clinical Salmonella enterica isolate [6]. On the other hand, among carbapenemase producers, the first detection of metallo-β-lactamase VIM-1 enzyme, was reported in a Pseudomonas aeruginosa strain isolated in 1997, in Italy [7]. Up until 30 September 2020, 73 bla VIM variants were overall reported in the National Database of Antibiotic Resistant Organisms (https://www.ncbi.nlm.nih.gov/pathogens/antimicrobial-resistance/).
Here we report the first two Italian clinical cases, involving ECC isolates co-producing: VIM-1 and MCR-9 in the first case and VIM-1, MCR-9 and MCR-4.3 in the second one.

Case Presentation, Antimicrobial Susceptibility Test and Molecular Investigations
The first strain, Enterobacter cloacae (ENCL_3849), was isolated from a 91 years old female patient admitted to "Istituto Geriatico Milanese" on the 2nd of March 2017 in Milan, Italy. The patient suffered from chronic health complications such as Type II diabetes mellitus, hypothyroidism, sever bilateral gonarthrosis and a risk of falls. From March until the end of June two courses of ceftriaxone were given. On the 7th of July, the blood culture was positive for a multidrug resistant (MDR) Enterobacter cloacae. The blood culture was repeated on the 13th and was still positive. On the 16th, the patient suffered from hyperpyrexia and hypotension and was treated with piperacillin. The patient was discharged on the 8th of August. The second strain, Enterobacter kobei (ENCB_IB2020), was isolated from a rectal swab (for routine screening purposes) of a 56 years old male patient on the 14th of December 2019 in Modena, Italy.

Conjugation/Transformation Assay
The ability of the plasmids harboring the mcr genes and the bla VIM-1 gene to conjugate was tested through conjugation experiments. The conjugation was performed in Mueller Hilton (MH) broth (OXOID, Hampshire, UK) using E. coli A15 r Azi as the recipient.
Transconjugants for ENCL_3849 were selected on MH agar (OXOID, Hampshire, UK) plates supplemented with sodium azide (150 mg/L) (Sigma-Aldrich, St. Louis, MO, USA) and ampicillin (1000 mg/L) (Sigma-Aldrich, St. Louis, MO, USA). For ENCB_IB2020, transconjugants were selected on MH agar plates supplemented with sodium azide (150 mg/L), meropenem (2 mg/L) (Sigma-Aldrich, St. Louis, MO, USA) and colistin (2 mg/L) (Sigma-Aldrich, St. Louis, MO, USA). The presence of bla VIM-1 , and mcr-like genes in the transconjugants was confirmed through PCR. MICs for transconjugants were performed using the broth-microdilution method. Isolates that failed to transfer the mcr genes of interest through conjugation were subjected to transformation; plasmids were extracted using Qiagen Maxi kit (Qiagen, Hilden, Germany) and the competent E. coli DH5α cells were used as the recipient. Transformants were selected on MH agar (OXOID, Hampshire, UK) with 2 mg/L colistin. Transformants were confirmed to be MCR producers through PCR.

Whole-Genome Sequencing (WGS)
For genomic characterization, genomic DNA was extracted using NucleoSpin Microbial DNA kit (Macherey-Nagel, Duren, Germany) and sheared using the Hydropore-long on Megaruptor 2 (Diagenode). Microbial multiplexing library preparation was performed without size selection according to the manufacturer's instructions. The multiplexed library was sequenced using long reads sequencing technology using the Sequel I platform (Pacific Biosciences, Menlo Park, CA, USA) for a 10 h movie run.

Whole-Genome-Sequencing-Data Analysis
Assembly was performed using the "Microbial Assembly" pipeline offered by the SMRT Link v9.0. with the default settings (minimum seed coverage of 30×). In-silico multilocus sequence typing of the strains (MLST) and of the plasmids when applicable (pMLST) was performed; antibiotic resistant genes, plasmid replicons and integrons were detected upon uploading the assemblies to PubMLST (https://pubmlst.org/organisms/enterobact er-cloacae), Plasmid MLST [21], ResFinder 4.1 and CARD [22,23], PlasmidFinder 2.1 [24] and INTEGRALL [25] respectively. BRIG v.0.95 was used to produce figures of comparison of the circular plasmids' sequences. Genome annotation was done using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Species identification of the isolates were confirmed with the NCBI database upon submitting the sequences to GenBank.

Isolates Susceptibility Profiles
Both strains showed resistance against ampicillin, cefotaxime, ceftazidime, and piperacillin-tazobactam. Moreover, ENCB_IB2020 showed clinical resistance to carbapenems and colistin while ENCL_3849 was susceptible to colistin and resistant to ertapenem. The minimum inhibitory concentrations (MICs) of two strains are shown in Table 1.

Plasmid Transferability
For ENCL_3849, the conjugation experiment was successful and MALDI-TOF species identification and PCR of bla VIM-1 and mcr-9 on the transconjugants confirmed the results. Moreover, the transconjugant showed similar antibiotic susceptibility profile as the donor. The PBRT kit for plasmid typing confirmed the presence of two incompatibility groups in the transconjugant: IncN and IncHI2. However, the conjugation experiment in ENCB_IB2020 was not successful. Transformation of the mcr bearing plasmid was successful at a low frequency, however it was not stable and the plasmid was lost upon re-streaking.

1.
The circular map of p3849_IncN_VIM-1 (pink) against pOW16C2 (turquoise). The outer most curved segments red and purple) correspond to antibiotic resistance genes, the mer region, and the toxin-antitoxin system.
Moreover, a ColE plasmid (pIB2020_ColE_MCR; 12,808 bp) that harbored mcr-4.3 gene coding for colistin resistance was found. The mcr-4.3 gene was bound with an IS26 upstream in opposite orientation followed by a Type II toxin/antitoxin system downstream. Moreover, the plasmid harbored mobA and mobX, two genes responsible for the plasmid mobility/cotransferability with a conjugative plasmid as reported elsewhere [27] (Figure 2).
Moreover, a ColE plasmid (pIB2020_ColE_MCR; 12,808 bp) that harbored mcr-4.3 gene coding for colistin resistance was found. The mcr-4.3 gene was bound with an IS26 upstream in opposite orientation followed by a Type II toxin/antitoxin system downstream. Moreover, the plasmid harbored mobA and mobX, two genes responsi-ble for the plasmid mobility/co-transferability with a conjugative plasmid as reported elsewhere [27] (Figure 2).

Discussion and Conclusion
The reports of VIM and MCR co-production in ECC isolates are increasing, as described elsewhere [30][31][32][33][34]. The presence of mcr-9-like genes bound by two insertion se-

Discussion and Conclusion
The reports of VIM and MCR co-production in ECC isolates are increasing, as described elsewhere [30][31][32][33][34]. The presence of mcr-9-like genes bound by two insertion sequences as in p3849_IncHI2_mcr and as reported in Sadek et al. 2020 andBitar et al. 2020 will not express colistin resistance and such plasmids can circulate silently until detected. On the other hand, IB2020 had mcr-9 on the chromosome in this gene's cassette: mcr-9-wubC-qseC-qseB-exeA. This gene's cassette was detected to express colistin resistance as presented in our IB2020 isolate [32,34]. Our results confirm and highlight some important aspects: the presence of mcr-9 determinants together with the two-component regulatory genes, can increase the gene expression, leading to colistin resistance whether on a plasmid or on the chromosome. The association of metallo-β-lactamases and increased colistin resistance largely reduce the numbers of therapeutic options available against severe Gram-negative infections. In particular, with the new combination strategies approved by the US Food & Drug Administration (FDA), the only available options against severe infections are Aztreonam-Avibactam and Cefiderocol, not degraded by metalloβ-lactamases [35,36]. This is evident from the MICs threat that limited the therapeutic option in our case to few antibiotics ( Table 1). The presence of mcr-4.3 encoding ColE plasmid and another mcrgene (mcr-9), is alarming. The ColE plasmid also harbored the genes necessary for its mobilization/co-transfer, which indicates that this plasmid is able to co-transfer with a conjugative plasmid, leading to its further dissemination. Moreover, the IncA and IncN plasmids represent self-conjugative plasmids, with a high tendency to acquire different antibiotic resistance islands, which may eventually lead to extremely drug resistant phenotypes and to the spread of different resistance genes in heterogeneous plasmid environment [36][37][38].
Finally, to our knowledge, these are the first cases of mcr-4.3, mcr-9, bla VIM-1 and mcr-9.2 and bla VIM-1 genes in ECC strains isolated from clinical cases, in Italy.

Data Availability Statement:
The nucleotide sequences of the chromosome and plasmids of ENCB_IB2020 and ENCL_3849 were deposited in GenBank and the following accession numbers have been assigned respectively: CP059480-CP059486 and CP052870-CP052875.