First report of plasmid-mediated colistin resistance mcr-8.1 gene from a clinical Klebsiella pneumoniae isolate from Lebanon

Colistin is considered as a last resort treatment for infections caused by multidrug-resistant Enterobacteriaceae. Plasmid-mediated mobile colistin resistance (mcr) genes contributed to the global spread of colistin resistance. This is the first report of plasmid-mediated colistin resistance mcr-8 gene from a clinical Klebsiella pneumoniae K9 isolate recovered from Lebanon. The isolate was characterized phenotypically and genotypically through both short and long read whole-genome sequencing, plasmid typing and conjugation assays. k9 belonged to sequence type 15 and harbored 31 antimicrobial resistance genes. The mcr-8.1 variant was carried on a novel ~ 300 kb multireplicon plasmid having IncFIA, IncR and IncHI1B. The plasmid was conjugative and carried a plethora of antimicrobial resistance determinants. The introduction of novel mcr variants in Lebanon poses an alarming health concern. Surveillance and screening for colistin resistant Enterobacteriaceae and mcr in livestock, animal farms, imported meat and poultry is highly recommended along with monitoring antibiotic use.


Introduction
Colistin is the last-line treatment option for infections caused by carbapenem resistant Enterobacteriaceae such as carbapenem-resistant Klebsiella pneumoniae (CRKP) [1]. The first plasmid mediated colistin resistance gene, mcr-1, was identified in 2015 in Enterobacteriaceae, mainly Escherichia coli and K. pneumoniae, collected from animals and humans in China [2]. To date, nine different plasmid-mediated mcr variants have been described, designated as mcr-1 -9, isolated from humans, animals and the environment [3]. mcr-8 located on an IncFII-type conjugative plasmid was first described in 2018 in China in K. pneumoniae collected from both animals and humans [4]. Shortly after, it was detected in Laos [5] and Algeria [6].
The first report of a colistin resistant K. pneumoniae in Lebanon was in 2017 [7]. mcr-1-positive E. coli from a human clinical isolate in Lebanon has been recently reported [8]. On the other hand, mcr-1 was widely detected in Gram-negative bacilli isolated from poultry [9], swine farms [9] and water sources in Syrian refugee camps [10]. This is accordingly, the first report of the complete sequence of a novel multireplicon conjugative plasmid carrying colistin resistance mcr-8.1 gene variant in an ST-15 K. pneumoniae isolate recovered from Lebanon.

Bacterial isolate
The isolate was recovered from the urine sample of a 50-years old female patient on the 28th of August 2018 at the Clinical Microbiology Laboratory of the American University of Beirut Medical Centre (AUBMC), Lebanon. The identification of the isolate was performed using Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) system (Bruker Daltonik, GmbH, Bremen, Germany). The isolate was identified as Klebsiella pneumoniae and designated as k9.

Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed using the disk diffusion assay on Mueller-Hinton agar and included a panel of 24 antibiotics belonging to 16 different classes (Table S1). The obtained data was interpreted according to the guidelines of the clinical & laboratory standards institute (CLSI) [11]. The minimal inhibitory concentrations (MICs) of ertapenem, imipenem and meropenem were determined using the E-test methodology (AB BIODISK, Solna, Sweden) according to the manufacturer's guidelines. MIC of colistin in k9 and in the colistin resistant E. coli transconjugant was further determined by using the broth microdilution method according to the recommendations from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [12].

DNA extraction
DNA extraction was performed from fresh bacterial colonies using the Nucleospin® Tissue kit (Macherey-Nagel, Germany) according to the manufacturer's instructions.

Conjugation assay
Conjugation was performed with an azide-resistant E. coli J53 strain. Selection of transconjuguants was done on Uriselect agar (Bio-Rad, France) supplemented with 100 mg/L sodium azide and 4 μg/ml colistin. E. coli transconjugants were typed by PBRT to detect plasmid replicons.

Whole-genome sequencing
Genomic libraries were constructed using the Nextera XT DNA library preparation kit with dual indexing (Illumina). The libraries were sequenced on an Illumina MiSeq with 250 bp × 2 read length. Genome assembly was performed de novo using Spades Genome Assembler Version 3.6.0 [13]. Quality control check was performed using FastQC version 0.11.5 [14].
The mcr8-carrying plasmid was sequenced using Pac-Bio long-read sequencing technology on the Sequel platform (Pacific Biosciences, CA, USA). Library preparation was performed according to the manufacturer's instructions for microbial multiplexing. G-tubes (Covaris, USA) were used for DNA shearing, and no size selection was performed.

Genome analysis
The assembled genomes were annotated using the RAST online server (http://rast.nmpdr.org) [15]. MLST 2.0 [16], ResFinder 3.0 [17], PointFinder 3.1.0 [18] and Plasmid Finder 2.1 [19] available on the Centre for Genomic Epidemiology website (www.genomicepidemiology.org) were used to determine the ST, presence of resistance genes and plasmid content, respectively. The sequences encoding for plasmids were then extracted and aligned to references obtained from NCBI as previously described [20]. The ISfinder database (https://www-is.biotoul.fr) [21] was used to identify insertion sequences. In silico capsular typing of the wzi gene was performed using Kaptive (www.kaptive.holtlab.net) [22]. The presence of putative virulence factors was screened using the VF scheme available on http://bigsdb.pasteur.fr. Plas-midsSpades was used to assemble the plasmid sequences [23]. Plasmid sequences were extracted and aligned with corresponding reference strains using BioNumerics software version 7.6.1 (Applied Maths, St-Martens-Latem, Belgium). Blast Ring Image Generator (BRIG) version 0.95 was used to visualize the plasmid comparisons [24].
The presence of known mutations conferring colistin resistance on the pmrA, pmrB, pmrC, pmrD, phoP, phoQ, mgrB, crrA and crrB genes were investigated in silico using both nucleic and amino acid alignments compared to the wild-type sequence of K. pneumoniae MGH 78578 (GenBank accession no. CP000647). SNAP [25] (http://www.hiv.lanl.gov) was used to calculate synonymous and non-synonymous substitution rates based on the codon-aligned nucleotide sequences of pmrA and pmrB against the K. pneumoniae MGH 78578 references.

Antimicrobial susceptibility testing
k9 had a colistin MIC of 10 μg/mL (Table S1). It was resistant to 20 of the tested antibiotics, showed intermediate resistance to cefepime, and remained susceptible to meropenem, imipenem, ertapenem, and fosfomycin and was accordingly classified as being extensively drug resistant (XDR) [27].
The presence of known mutations conferring colistin resistance were also investigated. pmrD, phoP, phoQ and mgrB showed 100% identity when compared to the wildtype reference strain K. pneumoniae MGH 78578, while crrA and crrB were not detected. pmrA and pmrB however, showed 40.1 and 49.1% similarity, respectively, to the wild-type reference strain. The rates of synonymous and non-synonymous substitutions were further investigated. SNAP (http://www.hiv.lanl.gov) revealed a dN/dS ratio of 1.131 for pmrA and 1.181 for pmrB indicating a positive selection for non-synonymous mutations ( Figure  S1).

mcr-8 genetic environment
Blast analysis revealed that the mcr variant was mcr-8. 1 showing 100% identity to mcr-8.1 carried by K. pneumoniae plasmid pKP91 (Accession no. MG736312), a~90kb IncFII-type plasmid obtained from swine fecal material in China [4] and in Raoutella ornithinolytica QDRO2 (Accession no. MK097469.1) [26]  The genetic environment of mcr-8.1 recovered from k9 was further compared with other mcr-8 variants. The amino acid sequences and the genetic environment of mcr-8.1 was most similar to mcr-8.3 and mcr-8.4 then to mcr-8.2, the latter being recovered from K. quasipneumoniae in China [28]. mcr-8.2 also harboured more mutations compared to mcr-8.1 than the other variants (Fig. 1).

Discussion
The plasmid-mediated spread of mcr gene variants represents a major health concern worldwide. In this study, we identified a multireplicon plasmid carrying the mcr-8.1 gene variant in a K. pneumoniae clinical isolate recovered from a urine sample in Lebanon. This represents the first report of mcr-8 from Lebanon. mcr-8.1 showed 100% gene sequence similarity to mcr-8.1 encoded by pKP91 plasmid originating from a K. pneumoniae KP91 collected in 2018 from swine fecal material in China [4]. The detection of colistin resistance determinants in both humans and animals directly supports the One Health perspectives that focus on the spread of drug resistance between the environment, humans and animals [31,32]. Since 2016, nine different plasmidmediated mcr variants have been described (mcr-1 to mcr-9) from human, animal and environmental sources [2][3][4], showing the prevalence of the mcr gene in the wildlife and particularly in surface water samples [33].
Colistin resistant bacteria and the mcr gene family could be transmitted via the food chain, and so its prudent use in both human and veterinary medicine is of paramount importance [35]. Polymyxins were classified as the "Highest Priority Critically Important Antimicrobials" by the World Health Organization (WHO) because of the increasing usage of colistin to treat serious infections in humans in many parts of the world [35]. Further surveillance and screening for mcr-8 in livestock and animal farms in Lebanon as well as in imported meat and poultry is recommended to track the environmental influences contributing to the development and dissemination of resistance determinants propagating on large mobile genetic vehicles.
Additional file 2: Figure S1. SNAP plots with potential synonymous and non-synonymous substitutions in pmrA (A) and pmrB (B). Alignment was performed against the query sequence of K. pneumoniae MGH 78578 wild-type chromosomal genes.