Plasmid Mediated mcr-1.1 Colistin-Resistance in Clinical Extraintestinal Escherichia coli Strains Isolated in Poland

Objectives: The growing incidence of multidrug-resistant (MDR) bacteria is an inexorable and fatal challenge in modern medicine. Colistin is a cationic polypeptide considered a “last-resort” antimicrobial for treating infections caused by MDR Gram-negative bacterial pathogens. Plasmid-borne mcr colistin resistance emerged recently, and could potentially lead to essentially untreatable infections, particularly in hospital and veterinary (livestock farming) settings. In this study, we sought to establish the molecular basis of colistin-resistance in six extraintestinal Escherichia coli strains. Methods: Molecular investigation of colistin-resistance was performed in six extraintestinal E. coli strains isolated from patients hospitalized in Medical University Hospital, Bialystok, Poland. Complete structures of bacterial chromosomes and plasmids were recovered with use of both short- and long-read sequencing technologies and Unicycler hybrid assembly. Moreover, an electrotransformation assay was performed in order to confirm IncX4 plasmid influence on colistin-resistance phenotype in clinical E. coli strains. Results: Here we report on the emergence of six mcr-1.1-producing extraintestinal E. coli isolates with a number of virulence factors. Mobile pEtN transferase-encoding gene, mcr-1.1, has been proved to be encoded within a type IV secretion system (T4SS)-containing 33.3 kbp IncX4 plasmid pMUB-MCR, next to the PAP2-like membrane-associated lipid phosphatase gene. Conclusion: IncX4 mcr-containing plasmids are reported as increasingly disseminated among E. coli isolates, making it an “epidemic” plasmid, responsible for (i) dissemination of colistin-resistance determinants between different E. coli clones, and (ii) circulation between environmental, industrial, and clinical settings. Great effort needs to be taken to avoid further dissemination of plasmid-mediated colistin resistance among clinically relevant Gram-negative bacterial pathogens.


INTRODUCTION
The growing incidence of multidrug-resistant (MDR) bacteria is an unavoidable challenge in modern medicine. Constant selection of MDR bacteria significantly contributes to the reduction of available therapeutic options. Colistin, also referred to as polymyxin E, is a cationic polypeptide considered a "last-resort" antimicrobial for treating infections caused by MDR Gram-negative bacterial pathogens, along with carbapenems and tigecycline (Livermore et al., 2011). Colistin was originally introduced in the 1950s for the treatment of infections caused by Gram-negative bacteria; however, polymyxins fell out of favor in the middle of the 1970s due to high rates of nephro-and neurotoxicity coupled with the advent of less toxic antibacterial agents. Nevertheless, by the mid-1990s (El-Sayed Ahmed et al., 2020), polymyxins were reintroduced into clinical practice due to the emergence of extensively drug-resistant (XDR) Gram-negative bacteria, and currently serve a critical role in the antimicrobial armamentarium (Kaye et al., 2016). Moreover, colistin often stands as the last antimicrobial agent retaining activity against carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii (Olaitan et al., 2014). Unfortunately, bacterial resistance to polymyxin E emerged rapidly, and could potentially lead to essentially untreatable infections, particularly in the hospital setting where aforementioned XDR microorganisms frequently cause lifethreatening infections in the most vulnerable patient populations (Kaye et al., 2016).
Colistin is capable of interacting with lipid A moiety of the lipopolysaccharide (LPS), thereby expelling Ca 2+ and Mg 2+ ions from phosphate groups and resulting in disruption of the negatively charged outer membrane (OM) of Gram-negative bacteria. Therefore, the ability of bacteria to resist killing by antimicrobial cationic polypeptides often entails modification of the OM (LPS modification resulting in reduced OM net negative charge). Polymyxin resistance has increased gradually within the last few years, and knowledge on a wide variety of possible chromosomal or acquired resistance mechanisms is still expanding (Olaitan et al., 2014). Most mechanisms conferring polymyxin resistance are directed at modifications of the lipid A moiety of the LPS, which is the primary target of colistin. Most genetic alterations, either chromosomal or acquired, entail a common lipid A modification pathway with 4-amino-4-deoxy-L-arabinose (L-Ara4N) and/or phosphoethanolamine (pEtN) addition. The most important target of L-Ara4N is the 4phosphate group of lipid A, but it can also be added to the 1-phosphate group or 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) (Baron et al., 2016). Substitution of the phosphate groups by L-Ara4N is followed by significant reduction of net negative charge of lipid A to 0, while pEtN modifications are associated with a net charge decrease from −1,5 to −1 (Nikaido, 2003). Therefore, L-Ara4N modification seems to be the most effective, owing to the nature of the OM charge modification (Olaitan et al., 2014). In Enterobacteriaceae, the aforementioned modifications of lipid A can result from mutation in the two-component systems (TCSs) such as PhoPQ, BasSR (PmrAB), small feedbackinhibition peptide MgrB, as well as from plasmid-mediated determinants (i.e., mcr gene encoding pEtN transferase) (Zeng et al., 2016;Litrup et al., 2017;Roer et al., 2017;Torpdahl et al., 2017;Osei Sekyere, 2019). The first plasmid-mediated polymyxin resistance gene, termed as mcr-1 (currently mcr-1.1) was identified in China in November 2015, and was subsequently reported all over the world, in both retrospective and prospective studies (Liu et al., 2016). The earliest, so far described, mcr-producing strains date back to the end of the previous century, in the 1980s (Shen et al., 2016). The earliest mcr-producing bacterial strain of clinical origin was a Shigella sonnei isolated from a pediatric patient in Vietnam, in 2008(Pham Thanh et al., 2016. The various mcr variants (mcr-1 to mcr-10) have been so far, identified in various species of Gram-negative pathogens originating from animals, meat, food products, environmental, and human sources (Partridge et al., 2018;Nang et al., 2019). The emergence of plasmid-mediated pEtN transferase-encoding genes is a matter of serious concern due to the potential for rapid dissemination via horizontal gene transfer. Broad distribution of mcr genes in multidrugresistant hospital strains would be especially dangerous in clinical settings, and could possibly result in wide dissemination of pandrug-resistant bacteria and untreatable infections. Here, we report on the emergence of mcr-1.1-harboring IncX4 plasmid in six extraintestinal Escherichia coli strains of clinical origin isolated in University Hospital of Bialystok, Poland between 2016 and 2018.

Clinical Isolates Used in the Study
Colistin-resistant E. coli isolates were obtained during microbiological screening of infected patients hospitalized in Medical University Hospital in Bialystok, between 2016 and 2018. Extraintestinal solates originated from postoperative wound swab, bedsore swab, perianal abscess swab, pharyngeal swab, bronchial aspirate, and endotracheal tube secretion. Clinical characteristics of the patients colonized by extraintestinal mcr-1.1-producing E. coli strains are presented in Table 1.

Whole Bacterial DNA Extraction and Sequencing
Whole bacterial DNA from six clinical extraintestinal E. coli was isolated from Luria Broth overnight cultures with use of silica column-based Genomic Mini AX Bacteria kit (A&A Biotechnology). Purified bacterial DNA was sequenced using both short-and long-read methodologies (Illumina and Oxford Nanopore Technology).
In the first step of molecular analysis, Nextera XT library preparation kit and Nextera XT Indexes (Illumina) were used for previously quantified bacterial DNA, which was simultaneously fragmented and tagged with sequencing adapters in a singletube enzymatic reaction. Quality and quantity of libraries were assessed by fluorometry (Qubit, Thermo Fisher Scientific) and chip electrophoresis (2100 Bioanalyzer, Agilent). FASTQ reads were generated with the use of MiSeq Reagent Kit v3 (600 cycles) and MiSeq analyzer (Illumina).
In the next step, DNA libraries were prepared with the use of a Ligation Sequencing Kit (SQK-LSK109) with Native Barcoding Expansion (EXP-NBD104). Quality and quantity of libraries were assessed by fluorometry (Qubit, Thermo Fisher Scientific) and chip electrophoresis (2100 Bioanalyzer, Agilent). FASTQ reads were generated with the use of Spot-ON Flow Cell (FLO-MIN106D R9 Version) and MinION Mk1b analyzer (Oxford Nanopore Technology).

Raw Data Quality Assessment and Downstream Bioinformatics
After quality assessment and quality filtering, reads were trimmed (Trimmomatic in case of Illumina reads), and demultiplexed with Porechop in case of long ONT reads (Bolger et al., 2014). Full structures of bacterial chromosomes and plasmids were recovered using Unicycler hybrid assembler which utilizes spades.py, racon, makeblastdb, tblastn, bowtie2, samtools, bcftools, and pilon (Wick et al., 2017). Alignment and mapping of nucleotide sequences were performed using Geneious 10.0.9 software (Biomatters Ltd., Auckland, New Zealand). A RAST (Rapid Annotation using Subsystem Technology)-annotated genomes were subjected to subsequent in silico analyses with use of PlasmidFinder, Resfinder, Virulence Finder (Carattoli et al., 2014;Brettin et al., 2015;Bortolaia et al., 2020).

Strain Phylogenomics
The final assembled genome sequence data were uploaded to the Type (Strain) Genome Server (TYGS), a free bioinformatics platform available under https: //tygs.dsmz.de, for a whole genome-based taxonomic analysis (Meier-Kolthoff and Göker, 2019). For the phylogenomic inference, all pairwise comparisons among the set of genomes were conducted using the Genome BLAST Distance Phylogeny approach (GBDP) and accurate intergenomic distances inferred under the algorithm 'trimming' and distance formula d5 (Meier-Kolthoff et al., 2013). Phylogenomic tree inferred with FastME 2.1.6.1 (Lefort et al., 2015) from GBDP distances calculated from genome sequences.

IncX4 Plasmids Phylogenomics
For the purpose of IncX4 plasmid phylogenomic inference, 100 similar sequences from the BLAST database were aligned and analyzed with the use of Clustal Omega (clustalo 1.2.4). Resulting phylogenetic tree was visualized using iTOL v6 1 (Letunic and Bork, 2021). Structural comparison between colistin-conferring plasmids harbored by studied extraintestinal E. coli isolates and IncX4 plasmid sequences deposited in NCBI was prepared using BLAST Ring Image Generator (BRIG) -default parameters with 90/70 as upper/lower threshold (Alikhan et al., 2011).

Transconjugation Assays
Electrotransformation of the IncX4 plasmid into the recipient E. coli strain was performed in order to confirm its influence on colistin-resistance phenotype in clinical E. coli isolates. To determine whether mcr-1.1 gene was located on pMUB-MCR 33.3 kbp IncX4 plasmid, transconjugation experiments were performed, with plasmid profiles preparation using Plasmid Mini AX kit (A&A Biotechnology), and subsequent electrotransformation into plasmid-free and colistin-sensitive E. coli TOP10 strain. Electrotransformation with subsequent selection of the transformants on the Luria-Bertani medium containing 1 mg/L colistin was conducted for the E. coli TOP10 strain.

RESULTS
Extraintestinal E. coli isolates incorporated into the described study presented a relatively similar antimicrobial resistance pattern (66.66%; 4 of 6).  to amoxicillin/clavulanic acid (MIC > 32 mg/L), and presented the highest colistin MIC = 16 mg/L. Detailed antimicrobial susceptibility testing results of six extraintestinal E. coli strains are presented in Table 2.
Whole-genome sequencing with use of a hybrid assembly approach allowed us to recover full genome and mobilome structures of tested extraintestinal colistin-resistant E. coli strains. Detailed characteristics of the sequenced genomes are presented in Table 3.
Moreover, all six colistin-resistant extraintestinal E. coli strains were equipped with a relatively rich plasmidic panel (Table 4). Interestingly, all of the tested strains harbored a IncX4 33.3 kbp plasmid pMUB-MCR, with the mobile pEtN transferase-encoding gene, mcr-1.1, which has been proved to be encoded within a type IV secretion system (T4SS), next to the PAP2-like membrane-associated lipid phosphatase gene (Figures 1, 2). The biological consequences of pMUB-MCR IncX4 plasmid possession were evaluated with the use of a transconjugation assay. The E. coli TOP10 electrotransformants carrying the 33.3 kbp IncX4 plasmid showed a MIC of colistin of 2 mg/L, which corresponded to a 16-fold increase as compared to the recipient E. coli TOP10 strain. These data confirmed that 33.3 kbp IncX4 pMUB-MCR conjugative plasmid is responsible for colistin-resistance in six extraintestinal clinical E. coli strains isolated from patients hospitalized in Medical University Hospital in Bialystok, Poland. Moreover, phylogenomics of IncX4 plasmids bearing mobile pEtN transferase-encoding genes is presented in Figure 3.
The MLST approach was utilized in order to evaluate the molecular relatedness of tested extraintestinal colistin-resistant E. coli strains. Among 6 tested clinical strains, two were proven to be clonally related, namely E. coli MIN11 and MIN12, which belonged to ST-10. Those two strains were isolated within an interval of 36 days, in the Department of

Beta-Lactam Resistance
In the present study, all tested strains presented isolated resistance to amoxicillin-clavulanate.
FIGURE 2 | BRIG alignment of IncX4 plasmids recovered from different Enterobacterales. Structural comparison between colistin-conferring plasmids harbored by studied extraintestinal E. coli isolates and IncX4 plasmid sequences deposited in NCBI was prepared using BLAST Ring Image Generator (BRIG). The alignment includes the mcr-1.1. sequence (red fragment), and the one IncX4 mcr-1-bearing plasmid harbored by E. coli MIN6 described in our study (green inner circle).
Extraintestinal E. coli strains also possess a number of virulence factors, such as long polar fimbriae, heat-stable toxin EAST-1 or enterobactin siderophore. All genes encoding virulence factors are listed in Table 5.

DISCUSSION
In this paper we sought to investigate the mechanism of colistin-resistance in six extraintestinal E. coli strains isolated from patients hospitalized in Medical University Hospital, Bialystok, Poland. Full structures of bacterial chromosomes and plasmids were recovered with use of both short-and long-read sequencing technologies and Unicycler hybrid assembly. Results of antimicrobial resistance testing were in accordance with genomic and mobilome screening for antimicrobial resistance determinants. All tested extraintestinal E. coli strains harbored an IncX4 33.3 kbp plasmid pMUB-MCR, with the mobile pEtN transferase-encoding gene, mcr-1.1. Moreover, its influence on colistin-resistance phenotype was confirmed by transconjugation assays.
In the present study, we report the first detailed description of mcr-containing IncX4 plasmid harbored by clinical E. coli strains in Poland. In six extraintestinal E. coli strains, mcr-1.1 was found within a type IV secretion system (T4SS) contained within a 33.3 kbp IncX4 plasmid that is known to be involved in the disseminating of multiple mcr variants. It is widely accepted that some type IV secretion systems (T4SSs) in pathogenic Gram-negative bacteria are utilized in order to translocate virulence factors into the host cell, mediate downregulation of the hosts innate immune response genes and an increase bacterial uptake and survival within macrophages and epithelial cells (Gokulan et al., 2013). Moreover, T4SSs could be also responsible for horizontal gene transfer (Juhas et al., 2008), thus contributing to genome plasticity and the evolution of pathogens through dissemination of antibiotic resistance and virulence genes (Juhas et al., 2008). Conjugative T4SSs are often encoded on self-transmissible plasmids coupled with genes that provide selective advantages for the cell such as antibiotic resistance, virulence factors or other metabolic functions that enhance survival (Wallden et al., 2010). The 33 kbp IncX4 plasmid was proven to be highly transmissible, showing 10 2 -10 5fold higher transfer frequencies relative to epidemic IncFII plasmid (Lo et al., 2014;Xavier et al., 2016). Moreover, Lo and colleagues proved that 33 kbp IncX4 plasmid carriage is associated with relatively low fitness cost, which makes it a highly effective vehicle for drug resistance determinants (Wu et al., 2018). Interestingly, it has been also recently reported that IncX4 plasmid can be relatively easily and stably maintained in host bacteria (Beyrouthy et al., 2017). In fact, IncX4 plasmids have been recently shown to harbor multiple mcr variants, CTX-M extended spectrum β-lactamase, as well as the 33.3 kbp IncX4 vehicles without any drug-resistance determinants (Lo et al., 2014;Chen et al., 2019). Similar IncX4 mcr-containing plasmids are reported as increasingly disseminated mainly among E. coli isolates ( Table 6), suggesting that it is becoming an "epidemic" plasmid, responsible for (i) disseminating colistin-resistance determinants between different E. coli clones, and (ii) circulating between environmental, industrial, and clinical settings. The phylogenomics of IncX4 plasmids bearing mobile pEtN transferase-encoding genes is presented in Figure 3. FIGURE 4 | Whole-genome sequence-based GBDP tree of mcr-producing E. coli complete genomes available at NCBI. Tree inferred with FastME 2.1.6.1 [6 (Lefort et al., 2015)] from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula d5.
The first European environmental mcr-producing E. coli strain was obtained from Italian diarrhoeic veal calves in 2005 (Haenni et al., 2016), whereas the first European strain of clinical origin harboring plasmid-mediated colistin-resistance gene, was described in Denmark in a Salmonella Typhimurium ST34 strain with an mcr-3 variant .
Here, we describe mobile pEtN transferase, mcr-1.1, encoded within a T4SS-containing 33.3 kbp IncX4 plasmid, pMUB-MCR. Similar IncX4 plasmids harboring different mcr variants were recently reported all over the world, with particular reference to animal breeding farms and environmental settings. Global dissemination of similar mcr-harboring IncX4 plasmids in Enterobacterales is presented in Table 6. So far, IncX4 mcrharboring strains have been reported mainly in animal breeding farms, meat industry, and natural environments. Incidence of IncX4 mcr-harboring strains originating from clinical sources has been recently reported, in Switzerland (E.  (Table 6; Zajac et al., 2019). Furthermore, the first clinical occurrence of a mcr-producing pathogen in Poland was reported by Izdebski et al. (2016). Clinical E. coli ST-617 strain, a member of ST-10 complex, possesses ∼250 kbp plasmid carrying mcr-1.1 and bla CMY−2 -containing IncA/C2 plasmids (∼160 kbp).
In conclusion, the increasing prevalence of plasmids responsible for colistin-resistance, often carrying other determinants of drug resistance, may possibly lead to formation of pandrug-resistant bacterial lineages. Great effort needs to be taken to avoid further dissemination of plasmid-mediated colistin resistance among clinically relevant Gram-negative pathogens.

ETHICS STATEMENT
This molecular investigation uses strains obtained from collection of strains deposited in Department of Microbiological Diagnostics and Infectious Immunology, Medical University of Bialystok, Poland. The Bioethics Commission of the Medical University in Bialystok did not require the study to be reviewed or approved by an ethics committee because apart from the strains from the Department's collection, no data enabling patient identification was used in the study.

AUTHOR CONTRIBUTIONS
PiM, DS, JN, and ET substantially contributed to the conception of the submitted research manuscript, designing and validation of the experiments, and data acquisition and interpretation (antimicrobial susceptibility testing, short-read sequencing, longread sequencing, and preparation of the figures and tables). PaM, AG, AS, and DS wrote the main manuscript. PiM was responsible for library preparation, WGS, and bioinformatics. PaM, DG, DI, PS, AZ, PR, PW, THa, IS, KM, RC, and JD contributed to the validation of the designed experiments and data acquisition and interpretation. THr, BK, JK, JG, SC, and PR were responsible for the medical care of the patient. All authors reviewed the manuscript.