Emergence of an ST1326 (CG258) Multi-Drug Resistant Klebsiella pneumoniae Co-harboring mcr-8.2, ESBL Genes, and the Resistance-Nodulation-Division Efflux Pump Gene Cluster tmexCD1-toprJ1 in China

CG258 is the dominant carbapenemase-producing Klebsiella pneumoniae clone worldwide and treatment of infections caused by this clone relies largely on the last-line antibiotics, colistin, and tigecycline. However, the emergence and global dissemination of mcr and tmexCD1-toprJ1 genes have significantly compromised their clinical applications. CG258 K. pneumoniae carrying both mcr and tmexCD1-toprJ1 have not been reported. A colistin-resistant strain T698-1 belonging to ST1326, a member of CG258, was isolated from the intestinal sample of a patient and characterized by the antimicrobial susceptibility testing, conjugation assay, WGS and bioinformatics analysis. It was resistant to colistin, tetracycline, aminoglycoside, fluoroqinolone, phenicols, sulfonamide, and some β-lactams, and positive for mcr-8.2, tmexCD1-toprJ1, and ESBL genes (blaDHA–1 and blaCTX–M–15). The tmexCD1-toprJ1 gene cluster was located in an multi-drug resistant (MDR) region flanked by TnAs1 elements on an IncHI1B/FIB plasmid. The genetic context of tmexCD1-toprJ1 was slightly distinct from previously reported Tn5393-like structures, with an IS26 element disrupting the upstream Tn5393 and its adjacent genetic elements. The mcr-8.2 gene was inserted into the backbone of an IncFII/FIA plasmid with the genetic context of ISEcl1-mcr-8.2-orf-ISKpn26. To our knowledge, this is the first report of co-occurrence of mcr-8.2 and tmexCD1-toprJ1 in a CG258 K. pneumoniae strain. Though this strain is tigecycline sensitive, the acquisition of colistin and tigecycline resistance determinants by the endemic CG258 K. pneumoniae clone still poses a serious public health concern. CG258, which became resistant to multiple last resort antibiotics, would be the next emerging superbug.


INTRODUCTION
Klebsiella pneumoniae is an opportunistic pathogen causing both community-and hospital acquired severe infections such as bacteremia, respiratory tract infections, and liver abscess (Gu et al., 2018;David et al., 2019). The extensive use of antibiotics has led to the emergence and rapid dissemination of multidrug resistant K. pneumoniae, particularly those resistant to last-line antibiotics including carbapenems, colistin, and tigecycline (Navon-Venezia et al., 2017). The majority of carbapenem-resistant K. pneumoniae producing KPC worldwide belong to the notorious CG258 clonal group, with ST258 and ST11 being the dominant sequence types . Treatment regimens for carbapenem-resistant K. pneumoniae are mainly reliant on colistin and tigecycline, which are classified as critically important antimicrobials by the WHO (World Health Organization) (He et al., 2019). However, the clinical potential of both antibiotics has been significantly compromised by the global dissemination of plasmid-mediated colistinresistance genes (mcr) and the mobile tigecycline-resistance genes (variants of the tet(X), tet(A), tet(K), and tet(M) genes), respectively (Linkevicius et al., 2016;Liu et al., 2016;He et al., 2019;Xu et al., 2021). Recently, a novel plasmidmediated resistance-nodulation-division (RND) efflux pump, tmexCD1-toprJ1, conferring resistance to tigecycline, quinolones, cephalosporins, and aminoglycosides was identified in K. pneumoniae . These plasmid-mediated resistance determinants are highly transmissible, presenting a severe challenge for clinical management. In this study, we reported an ST1326 multidrug resistant K. pneumoniae isolate, belonging to CG258, harboring resistance determinants including ESBL genes, mcr-8.2, and tmexCD1-toprJ1 from a patient with critical illness. The emergence of colistin and tigecycline resistance determinants in the endemic K. pneumoniae clone constituted a true public threat.

Strain Origin and Antimicrobial Susceptibility Testing
An 84-year-old female patient with a previous history of pneumonia was admitted to Department of Respiratory Medicine in Dali Bai Autonomous Prefecture People's Hospital on May 12, 2020 (Day 1) due to chronic cough. The patient experienced sudden loss of consciousness during hospitalization and received invasive mechanical ventilation via tracheal intubation. Anti-infective treatment with cefoperazone-sulbactam and caspofungin was also applied. She suffered from recurrent fungal and bacterial infections, fever, and gastro-intestinal bleeding. The drugs were switched to imipenem-cilastatin and ceftazidime 2 months later according to the patient's condition. However, the patient responded poorly to the treatment and discharged on Day 96 with critical illness. On Day 104, a multidrug-resistant K. pneumoniae isolate (T698-1) was recovered from her intestinal samples for hospital infection surveillance. The species identity of T698-1 was confirmed by a matrix-assisted laser desorption ionization-time of flight mass spectrometer (MALDI-TOF MS) (Bruker, Germany). Antimicrobial susceptibility testing was conducted using the broth microdilution method and interpreted according to the CLSI guideline (CLSI, 2020).

Plasmid Elimination Assay
The plasmid elimination experiment was performed using sodium dodecyl sulfate (SDS, 20%) as previously described with some modifications (Liu et al., 2017). Briefly, a single colony of strain T698-1 was picked and grown in 5 ml LB broth at 37 • C overnight. Then 100 µl bacterial solution was transferred to fresh LB broth containing 20% SDS and incubated at 37 • C. The overnight culture was spread on LB plates, the colonies on which were streaked onto drug-free LB plates and LB plates containing 2 µg ml −1 colistin or 2 µg ml −1 tigecycline simultaneously. The colonies losing plasmids were further confirmed by PCR.

Identification of Mutations in Colistin-Resistance Genes
Identification of mutations in mgrB gene was conducted using Kleborate version 2.0.4 (Lam et al., 2021). Mutations in amino acid sequences of PmrA/PmrB and PhoP/PhoQ were identified by aligning with a colistin-susceptible reference genome of the strain NJST258_1 (Genbank accession number: CP006923) reported previously (Deleo et al., 2014).

Whole Genome Sequencing and Bioinformatics Analysis
To decipher the genomic characterization, the genome of T698-1 was extracted from overnight cultures by using the PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, United States) and sequenced using both short-read sequencing (2 × 150 bp) by Illumina Hiseq 2500 platform and long-read sequencing by Oxford Nanopore Technologies MinION platform . Hybrid assembly of both sequencing reads was constructed using Unicycler v 0.4.4 (Wick et al., 2017). Complete genome sequence was annotated by the RAST tool and modified manually (Overbeek et al., 2013). Genotyping including species identification, multi-locus sequence typing, serotyping, identification of antimicrobial resistance genes, and virulence genes was conducted using Kleborate v2.0.4 (Lam et al., 2021). Plasmid replicons were analyzed using PlasmidFinder v2.1 (Carattoli et al., 2014). Insertion sequences (ISs) were identified using ISfinder (Siguier et al., 2006). The genetic contexts of mcr and tmexCD1-toprJ1 genes were analyzed by comparing with similar items available in the NCBI nr (non-redundant) database. Plasmid comparisons and genetic context comparisons were visualized using BRIG and Easyfig (Alikhan et al., 2011;Sullivan et al., 2011).
to have originated from the chromosome of Pseudomonas sp., the RND efflux pump gene cluster tmexCD1-toprJ1, as well as its variants tmexCD2-toprJ2 and tmexCD3-toprJ3, has been reported from a wide diversity of species including Pseudomonas sp., K. pneumoniae, Klebsiella quasipneumoniae, Raoultella ornithinolytica, and Proteus mirabilis Sun et al., 2020;Wan et al., 2020;Li et al., 2021;Wang C.-Z. et al., 2021;. Diverse mobile genetic elements including plasmids, insertion sequences, transposons, integrative, and conjugative elements (ICEs) were reported to have contributed to its wide dissemination Wan et al., 2020;Hirabayashi et al., 2021;. Plasmid elimination was performed to demonstrate the influence of TMexCD1-TOprJ1 and MCR-9 to tigecycline and colistin resistance, respectively. Despite trying three times, we failed to get a single plasmid-eliminated derivative strain and only the strain T698-1-PC of which both pKPT698-tmexCD and pKPT698-mcr were cured of was obtained. The T698-1-PC showed the same or close MIC value to tigecycline (≤0.5 mg/L), tetracycline (32 mg/L), quinolones (>4 mg/L), gentamycin (>16 mg/L), and cephalosporins (>16 mg/L) as the original strain T698-1 and those antibacterial agents were the substrate spectrums of TMexCD1-TOprJ1 for K. pneumoniae , further proving the inefficacy of TMexCD1-TOprJ1 in T698-1.
The third plasmid, pKPT698-mcr (CP079783), is a 90,695 bp, IncFII/FIA plasmid which encodes predicted 112 ORFs with a G + C content of 50.1%. It was 99.97% identical to plasmid pZZW20-88K (accession: CP058962) at 98% coverage. Antimicrobial resistance genes carried by pKPT698-mcr include bla CTX−M−15 (two copies), bla TEM−1B , and mcr-8.2 ( Figure 2B). Several mobile elements such as IS903B and ISEcl1 were reported to have played pivotal roles in the dissemination of mcr-8 among Enterobacteriaceae Zhang et al., 2021). In line with the previous findings, the mcr-8.2 gene on pKPT698mcr was associated with the genetic context of ISEcl1-mcr-8.2-orf -ISKpn26 (Zhang et al., 2021). Both pKPT698-tmexCD and pKPT698-mcr were shown to be non-conjugative under laboratory conditions. The elimination of pKPT698-mcr in strain T698-1-PC caused a marked decrease of MIC values to colistin from 64 mg/L in T698-1-PC to 1 mg/L in T698-1, indicating mcr-8.2 genes mediated colistin resistance in T698-1. Besides, we further screened common chromosomal mutations that caused colistin resistance. The result from Kleborate showed that no mutations associated with resistance to colistin were detected in mgrB gene. Compared with the colistin-susceptible genome of the strain NJST258_1, T698-1 did not possess any amino acid mutations of PmrA/PmrB, and PhoP/PhoQ. Therefore, these data suggested that the mcr-8.2-bearing plasmid in this strain is responsible for colistin resistance.
The plasmid-borne tigecycline-resistant RND efflux pump gene cluster tmexCD1-toprJ1 was first reported in K. pneumoniae in China in 2020 , with an extremely high prevalence of 52.4% in animals and 0.08∼2.5% in patients Sun et al., 2020). This indicated that we should be alert to the further dissemination of TmexCD1-ToprJ1-positive K. pneumoniae in hospital despite that only one K. pneumoniae carrying TmexCD1-ToprJ1 was detected. Worse still, the K. pneumoniae strains even plasmids co-harboring tmexCD1-toprJ1 and mcr have been prevailing in China, and some strains belonged to the ST11 clone, the predominant and high risk clone of CRKP strains in China . The simultaneous presence of NDM carbapenemases further promoted the emergence of pan-drug resistant bacteria, especially in K. pneumoniae with high genome plasticity . The MDR strain T698-1 was susceptible to tigecycline and carbapenems but conferred resistance to colistin on account of mcr-8.2, a variant of the mcr-8 colistin resistance gene identified in 2020 (Ma et al., 2020), suggesting that future nosocomial infections surveillance pay more attention to K. pneumoniae.

CONCLUSION
In conclusion, we reported the emergence of a ST1326 MDR K. pneumoniae strain in a hospital in China, which belonged to the endemic CG258 clone -and carried mcr-8.2, ESBL genes, and the RND efflux pump gene cluster tmexCD1-toprJ1. K. pneumoniae of CG258 was commonly shown to be associated with carbapenem resistance. The acquisition of colistin and tigecycline resistance determinants by CG258 K. pneumoniae may pose a serious public health concern.

DATA AVAILABILITY STATEMENT
The complete genome sequence of strain T698-1 was deposited in the GenBank database under the BioProject number: PRJNA747739, with accession numbers CP079781-CP079784.

ETHICS STATEMENT
The study was approved by the Ethics Committee of Second Affiliated Hospital, Zhejiang University School of Medicine (2020-319). Written informed consent was obtained from the patients for the publication of any potentially identifiable data.

AUTHOR CONTRIBUTIONS
CL and YW performed the experiments and wrote the original draft. YF and ZS contributed to data analysis and manuscript writing. LH, ND, YZ, and JL helped with the experiments. RZ helped editing the manuscript and contributed to study design. GC put forward the conception, designed the study, and edited the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This study was funded by the National Natural Science Foundation of China (Nos. 81861138052, 82072341, and 81871705).