Emergence of a novel mobile colistin resistance gene, mcr-8, in NDM-producing Klebsiella pneumoniae

The rapid increase in carbapenem resistance among gram-negative bacteria has renewed focus on the importance of polymyxin antibiotics (colistin or polymyxin E). However, the recent emergence of plasmid-mediated colistin resistance determinants (mcr-1, -2, -3, -4, -5, -6, and -7), especially mcr-1, in carbapenem-resistant Enterobacteriaceae is a serious threat to global health. Here, we characterized a novel mobile colistin resistance gene, mcr-8, located on a transferrable 95,983-bp IncFII-type plasmid in Klebsiella pneumoniae. The deduced amino-acid sequence of MCR-8 showed 31.08%, 30.26%, 39.96%, 37.85%, 33.51%, 30.43%, and 37.46% identity to MCR-1, MCR-2, MCR-3, MCR-4, MCR-5, MCR-6, and MCR-7, respectively. Functional cloning indicated that the acquisition of the single mcr-8 gene significantly increased resistance to colistin in both Escherichia coli and K. pneumoniae. Notably, the coexistence of mcr-8 and the carbapenemase-encoding gene blaNDM was confirmed in K. pneumoniae isolates of livestock origin. Moreover, BLASTn analysis of mcr-8 revealed that this gene was present in a colistin- and carbapenem-resistant K. pneumoniae strain isolated from the sputum of a patient with pneumonia syndrome in the respiratory intensive care unit of a Chinese hospital in 2016. These findings indicated that mcr-8 has existed for some time and has disseminated among K. pneumoniae of both animal and human origin, further increasing the public health burden of antimicrobial resistance.


Introduction
Klebsiella pneumoniae, belonging to the family Enterobacteriaceae, is a bacterial pathogen commonly associated with nosocomial infections, including pneumonia, bloodstream infection, urinary tract infection, and hepatic abscess, especially among immunocompromised patients 1 . Carbapenem was used as the drug of choice for the treatment of infections caused by multidrug-resistant K. pneumoniae. However, the increased prevalence of carbapenem-resistant K. pneumoniae (CRKP) greatly compromised the efficacy of carbapenem antibiotics 2 .
Colistin has been considered a last resort antibiotic, used either alone or in combination with other drugs, for the treatment of serious infections caused by CRKP 3 . In recent years, multiple studies have indicated that the prevalence of colistin resistance has increased rapidly among Enterobacteriaceae 4 . In 2015, Liu et al. 5 identified the first mobile colistin resistance gene, mcr-1, in Enterobacteriaceae, mainly Escherichia coli and K. pneumoniae. In a short time, mcr-1 and its slightly altered gene variants (mcr-1.1 to mcr-1.12) were ubiquitously identified in various Enterobacteriaceae of different origins in over 40 countries across five different continents [6][7][8][9][10] . Several other MCR homologs (MCR-2, MCR-3, MCR-4, and MCR-5) were subsequently identified in Enterobacteriaceae. Very recently, two MCR homologs (MCR-6 and MCR-7) were annotated and deposited into GenBank, and the mcr-7.1 gene was detected in K. pneumoniae of chicken origin in China 11 . To date, mcr-2 has only been detected in E. coli and Salmonella isolates from European countries 12,13 , whereas mcr-3 has been widely identified in Enterobacteriaceae (mainly E. coli) and Aeromonas spp. from Asia, Europe, and North America 14 . mcr-4 and mcr-5 were first characterized in Salmonella and E. coli from European countries 15,16 and further detected in Enterobacter cloacae and E. coli isolates from Asia (Singapore and Japan) 17,18 . Thus far, only mcr-1 and mcr-3 have been reported in K. pneumoniae, and the prevalence of these two genes is relatively low 5,6 . In this study, we characterized a novel mobile colistin resistance gene, mcr-8, in a K. pneumoniae isolate and then identified the coexistence of mcr-8 and bla NDM in K. pneumoniae isolates from both animals and humans.
Conjugation assays showed that the colistin resistance determinant in K. pneumoniae KP91 can be transferred into the recipient E. coli strain J53. The transconjugant J53-pKP91 had a pulsed-field gel electrophoresis (PFGE) pattern identical to that of E. coli J53 but distinct from that of KP91. S1-PFGE indicated that J53-pKP91 differed from E. coli J53 by the presence of a single~90-kb plasmid, designated pKP91 (Fig. 1). These results confirmed the presence of an uncharacterized colistin resistance determinant located on a conjugative plasmid in K. pneumoniae KP91.
Identification and functional confirmation of mcr-8 in K. pneumoniae and E. coli To characterize the colistin resistance determinants, we used Single Molecule Real-Time (SMRT) sequencing to generate the complete sequence of the plasmid isolated from transconjugant J53-pKP91. SMRT sequencing revealed that plasmid pKP91 was 95,983 bp in size, with a GC content of 50.16%, which differed from the GC content of the genome (56.33%) (Fig. 2). pKP91 was identified as a typical IncFII-type plasmid and contained genes involved in plasmid stability, formation of a type-IV pilus, and conjugative transfer (Fig. 2). BLASTn analysis identified a 1698-bp open-reading frame (ORF) encoding a
The presence of mcr-8 in K. pneumoniae of human origin BLASTp analysis of MCR-8 against the NCBI database revealed a protein sequence with 99% amino-acid identity and 99% query coverage to MCR-8 in a clinical CRKP strain WCHKP1845, originally isolated from the sputum of a patient with pneumonia in the respiratory intensive care unit at West China Hospital, Chengdu, China, in May 2016. In the corresponding paper, the authors claimed that colistin resistance was conferred by an unknown mechanism in strain WCHKP1845 26 . These analyses suggest that mcr-8 may be responsible for the colistin resistance of this strain, and the clinical isolate WCHKP1845 with ST1 was not related to the pig-origin KP91 with ST42.

Structure prediction and genetic environment analysis of MCR-8
Similar to previously characterized MCR proteins, MCR-8 was predicted using RaptorX (Xu Group, Chicago, IL, USA) to contain two domains (Fig. 4a). Domain 1 (residues 1-234) contained five transmembrane α-helices (Fig. 4b), whereas domain 2 (residues 235-565) contained the putative catalytic center. i-Tasser homology modeling revealed that the best-fit structure in the Protein Data Bank for MCR-8 was 5FGN, which was the first characterized full-length lipid A phosphoethanolamine transferase from Neisseria meningitidis 27 .
Genetic environment analysis revealed that two complete insertion sequences of IS903B, originating from E. coli, were located up-and downstream of mcr-8 in pKP91. In addition, the 50-bp IRR and 50-bp IRL (Fig. 5) were identical to those surrounding IS903B described in the ISfinder database (https://www-is.biotoul.fr/index.php). IS903B was absent from the 13.47-kb NCBI contig from clinical WCHKP1845, whereas a complete insertion sequence, ISEcl1, originating from Enterobacter cloacae, was present downstream of mcr-8 in WCHKP1845 (Fig. 5). These findings revealed that several IS elements (IS903B, ISEcl1) may play a pivotal role in the dissemination of mcr-8 among Enterobacteriaceae.
Coexistence of mcr-8 and carbapenemase genes in K. pneumoniae isolates In total, we examined 53 colistin-and carbapenemresistant K. pneumoniae isolates derived from pigs (n = 28) and chickens (n = 25). Of these, four (pig = 3, chicken = 1) isolates were positive for mcr-8, whereas five (pig = 3, chicken = 2) were positive for mcr-1. In addition, we detected the presence of bla NDM but not the bla VIM , bla IMP , bla OXA-48 , or bla KPC genes in 47 of the carbapenemresistant K. pneumoniae isolates. mcr-8 and bla NDM were shown to coexist in three isolates. These findings indicated that the novel mcr-8 gene has disseminated among K. pneumoniae and that the prevalence and coexistence of mcr-8 and bla NDM in K. pneumoniae may further threaten public health through either the food chain or environmental routes.

Phylogenetic typing analysis
To determine whether the mcr-1and mcr-8-positive K. pneumoniae isolates were genetically related, five mcr-1and five mcr-8-positive isolates, including KP91, along with 44 mcr-negative carbapenem-resistant K. pneumoniae isolates, were selected for PFGE analysis with XbaI digestion. Using a cutoff of 80% pattern similarity, the 54K. pneumoniae isolates were grouped into 13 clusters (PFGE patterns represented by multiple strains) and 17 unique PFGE patterns (PFGE patterns represented by a single strain) (Fig. 6). Overall, the K. pneumoniae isolates from different regions were genetically diverse, suggesting that the mcr-8-positive K. pneumoniae isolates were also genetically diverse and that mcr-8 could disseminate among different K. pneumoniae isolates, mainly by horizontal transmission.

Discussion
The rapid emergence of mobile colistin resistance genes represents a paradigm shift in colistin-resistant mechanisms, which were long dominated by chromosomal mutations and vertical transmission. To date, mcr-1 and mcr-3 have frequently been detected in Enterobacteriaceae from different regions 28 , whereas mcr-2, mcr-4, and mcr-5 have been rarely reported. Although all of these mcr genes have been identified in various Enterobacteriaceae and non-Enterobacteriaceae species, E. coli remains the predominant host. A small number of reports have indicated the presence of mcr-1 and mcr-3 in K. pneumoniae at relatively low detection rates 6,18,29 . In this study, we identified a novel mobile colistin gene, mcr-8, carried on an IncFII-type conjugative plasmid in K. pneumoniae. The presence of mcr-8 (7.54%, 4/53) among the colistin-resistant CRKP isolates indicates that this novel mobile colistin resistance gene may already be widely disseminated among K. pneumoniae isolates of livestock origin.
In conclusion, we identified a novel gene, mcr-8, conferring resistance to colistin, which is considered the last resort drug for the treatment of carbapenem-resistant Enterobacteriaceae, especially CRKP. The coexistence of mcr-8 and bla NDM was noted in K. pneumoniae from both food-producing animals and human clinical samples and poses a serious public health concern. Therefore, further surveillance may help us understand the prevalence and dissemination of this novel antibiotic resistance gene.

Materials and methods
Bacterial isolation and susceptibility testing All K. pneumoniae isolates were recovered from pig fecal matter and chicken cloacae samples collected from Shandong Province, China, according to a previously described protocol 30 . Isolates were identified as described Fig. 3 WebLogo of amino-acid sequences of MCR-1-8. A logo represents each column of the alignment in a stack of letters, with the height of each letter proportional to the observed frequency of the corresponding amino acid, and the overall height of each stack proportional to the sequence conservation, measured in bits, at that position. Red asterisks indicate the six conserved cysteine residues that form three disulfide bonds. Black asterisks indicate the conserved active sites among MCR-1-8. The logo was built using WebLogo software (http://weblogo.berkeley.edu/logo.cgi) previously 27 . The MICs of the K. pneumoniae isolates to colistin, tetracycline, meropenem, ceftazidime, amoxicillin-clavulanate, trimethoprim-sulfamethoxazole, amikacin, gentamicin, streptomycin, florfenicol, and ciprofloxacin were determined using the broth microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute (document VET01-A4), and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www. eucast.org).

Conjugation assay
Conjugation assays were performed according to a previously described method 31 . In brief, the azideresistant E. coli strain J53 was used as the recipient, and donor and recipient strains were mixed at a ratio of 1:3 on a microporous membrane for 12 h. The mixtures were collected and then plated on Luria-Bertani agar containing azide (100 μg/ml) and colistin (2 μg/ml).

Genome sequencing and assembly
Genomic DNA was extracted from K. pneumoniae isolate KP91 using a Wizard Genomic DNA Purification Kit (Promega, Beijing, China) and used as template for 150-bp paired-end whole-genome sequencing using the Illumina HiSeq 2500 System (Annoroad, Beijing, China). The draft genome was assembled using the CLC Genomics Workbench 9.0 (CLC Bio-Qiagen, Aarhus, Denmark). The conjugative plasmid was extracted from E. coli transconjugant strain J53-pKP91 using a Plasmid Midi Kit (OMEGA, Norcross, GA, USA) and then sequenced using both the Illumina HiSeq platform and the PacBio RSII System (Sinobiocore, Beijing, China). Plasmid assembly was performed using the Hierarchical Genome Assembly Process (HGAP) and Quiver as part of the SMRT analysis program (version 2.3) using the HGAP3 protocol and then corrected using Pilon.

Detection of genes conferring resistance to colistin and carbapenem
The presence of the five colistin resistance genes, mcr-1 to -5, in the K. pneumoniae isolates was determined by PCR and Sanger sequencing. We also detected the presence of the carbapenem resistance genes bla NDM , bla VIM , bla IMP , bla OXA-48 , and bla KPC in the K. pneumoniae isolates by PCR analysis. The primers and conditions used for the PCR assays are listed in Table S1.
PFGE, S1 nuclease-PFGE, and Southern blotting PFGE analysis of K. pneumoniae isolates was performed using XbaI as the restriction endonuclease, as previously described 34,35 . Salmonella enterica serovar Braenderup H9812 digested with XbaI was used as the reference marker. The PFGE results were analyzed using InfoQuest software version 4.5 (Bio-Rad Laboratories, Hercules, CA, USA). S1 nuclease-PFGE was performed to determine the plasmid profiles. The genomic location of mcr-8 was indicated by Southern hybridization using a digoxigeninlabeled mcr-8 probe according to the manufacturer's instructions for the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics, Basel, Switzerland).

Accession number
The complete nucleotide sequence of plasmid pKP91 has been deposited in GenBank under accession no. MG736312.