Abstract

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 (Dong et al., 2018). 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 colistin-resistance 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 plasmid-mediated resistance-nodulation-division (RND) efflux pump, tmexCD1-toprJ1, conferring resistance to tigecycline, quinolones, cephalosporins, and aminoglycosides was identified in K. pneumoniae (Lv et al., 2020). 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.

Materials and Methods

Results and Discussion

Strain T698-1 isolated from the intestinal sample of the patient was identified as K. pneumoniae by both MALDI-TOF MS and whole genome sequencing. It was resistant to aminoglycoside (gentamycin), fluoroqinolone (ciprofloxacin), polymyxin (colistin), tetracycline, phenicols (florfenicol, chloramphenicol), sulfonamide (trimethoprime-sulfamethoxazole), and some β-lactams (ceftazidime, cefotaxime, cefoperazone/sulbactam, ampicillin, aztreonam), but remained susceptible to carbapenems (meropenem, imipenem) and tigecycline (Table 1). In line with the resistance phenotypes, no carbapenemase gene was detected in strain T698-1, while ESBL genes including bla_DHA–1 and bla_CTX–M–15 were detected. Meanwhile, strain T698-1 was negative for the tigecycline resistance determinant tet(X), but positive for the colistin resistance gene mcr-8.2 and the RND-type efflux pump gene cluster tmexCD1-toprJ1, which is associated with resistance to tetracyclines, quinolones, cephalosporins, and aminoglycosides. Despite carrying tmexCD1-toprJ1, strain T698-1 was susceptible to tigecycline, quite possibly due to the low expression level of this gene.

The genome of strain T698-1 (BioProject number: PRJNA747739) was assembled into four complete circularized contigs, including a 5,265,236 bp chromosome (CP079781) encoding 5207 predicted ORFs with a GC content of 57.6% and three multi-drug resistant (MDR) plasmids (pKPT698-tmexCD, pKPT698-mcr, pKPT698-tetA). Strain T698-1 belonged to ST1326 (gapA-infB-mdh-pgi-phoE-rpoB-tonB allele number 3-3-1-1-1-1-16), which is closely related to the endemic K. pneumoniae clones ST258 (3-3-1-1-1-1-79) and ST11 (3-3-1-1-1-1-4) in clonal group (CG) 258 with only one allele (tonB) variance among the three STs. According to the previous study, CGs were defined as groups for which MLST profiles showed only one allelic mismatch with at least one other member of the group, suggesting strain T698-1 belonged to the notorious CG258 clonal group (Bialek-Davenet et al., 2014). To date, 77 immunologically distinct Klebsiella capsule types (K1-K77) have been defined by serology, and comparative analysis of the full-length capsule synthesis loci (K-loci) extracted from whole genome sequences identified 134 distinct K-loci (KL1-KL134) (Wyres et al., 2016). Strain T698-1 belonged to KL102, which has not been verified with traditional immunoelectrophoresis techniques. Apart from the bla_SHV–11 gene conferring intrinsic ampicillin resistance in K. pneumoniae, the chromosome of strain T698-1 carried acquired antimicrobial resistance genes sul1 and aadA2, which were bordered by mobile elements (IS26-IS6100-orf1-sul1-orf2-aadA2-IS26). Fluoroquinolone resistance-associated mutations including S80I in ParC and S83I in GyrA were detected in the chromosome of strain T698-1 (Wyres et al., 2019).

Plasmid pKPT698-tmexCD (CP079784) was 291,624 bp in length, with an average G + C content of 46.8%. It is an IncHI1B/FIB plasmid comprising 330 predicted ORFs. One plasmid, p18-29-MDR (MK262712), with similar backbone of 99.94% identity at 99% coverage to pKPT698-tmexCD was retrieved in the NCBI nr database by BLASTn analysis (Figure 1A). Plasmids pKPT698-tmexCD and p18-29-MDR, both from K. pneumoniae, shared the same backbone and plasmid replication genes, indicating that they might be originated from a common ancestor and have undergone evolution separately. A total of 18 different resistance genes containing tmexCD1-toprJ1, aadA1, aadA2, cmlA1, sul3, aac(3)-IVa, aph(4)-Ia, aph(3′)-Ic, strA (two copies), strB (two copies), mph(E), msr(E), armA, sul1, bla_DHA–1 and qnrB4, and an truncated IS102-like gene, tet(B), were identified on plasmid pKPT698-tmexCD. Antimicrobial resistance genes were located on a 65,340 bp MDR-encoding region bordered by the TnAs1 transposon. A further BLAST search of this region in the NCBI nr database returned seven plasmid hits (CP076031, MK262712, MN099026, MT647838, AP023338, CP075257, CP075287) with >99.7% identity and ≥95% coverage, indicating this MDR region was most likely acquired by horizontal gene transfer, and it was transmissible among diverse plasmid backbones. The MDR region harbored an array of mobile elements including TnAs1, Tn5393, ISEc28, ISEc29, ISEc59, IS903, ISCR1, IS1, 5 copies of IS26, and an IntI1 integrase, suggesting active genetic recombination could occur in this region (Figure 1B). The RND-type efflux pump gene cluster, tmexCD1-toprJ1, was first reported to be located on the transposon Tn5393 of plasmid pHNAH8I-1 (MK347425), which was considered to be associated with its translocation (Lv et al., 2020). On plasmid pKPT698-tmexCD, the upstream Tn5393 and its adjacent genetic elements were disrupted by an IS26, constituting the genetic context of IS26-tnfxB1-tmexC1-tmexD1-toprJ1-Tn5393:tnpA-Tn5393:tnpR-strA-strB, which was slightly distinct from that reported previously (Lv et al., 2020; Sun et al., 2020). Predicted 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 (Lv et al., 2020; Sun et al., 2020; Wan et al., 2020; Li et al., 2021; Wang C.-Z. et al., 2021; Wang Q. 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 (Lv et al., 2020; Wan et al., 2020; Hirabayashi et al., 2021; Wang Q. 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 (Lv et al., 2020), further proving the inefficacy of TMexCD1-TOprJ1 in T698-1.

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FIGURE 1

(A) Circular plasmid map of pKPT698-tmexCD. Green, blue, and red arrows indicate the antimicrobial resistance genes, mobile elements, and other predicted ORFs, respectively. The names of antimicrobial resistance genes, mobile elements are labeled alongside the corresponding arrows. The multi-drug resistant (MDR) region is indicated with dotted line frame. (B) Linear comparisons of the MDR region in plasmids pKPT698-tmexCD and p18-29-MDR. Cyan, blue, and yellow arrows indicated the antimicrobial resistance genes, mobile elements, and other predicted ORFs, respectively.

The second plasmid, pKPT698-tetA (CP079782), is a 138,834 bp, IncFIB plasmid which encodes 167 predicted ORFs with a G + C content of 52.3%. BLASTn analysis in the NCBI nr database returned no hits with similar backbones (>85% coverage), and the most closely related plasmid (pE196_IMP6, 250,392 bp, AP019405) exhibited 99.49% identity to pKPT698-tetA at 80% coverage. pKPT698-tetA harbored multiple antimicrobial resistance genes including aac(6′)Ib-cr, arr-3, dfrA27, aadA16, sul1, and catA2 located in a 23,779 bp MDR region bordered by the transposable element Tn2. The MDR region is >99.95% identical to its counterparts on plasmid pKp21774-135 (MG878868) from K. pneumoniae and plasmid pM206-NDM1 (AP018830) from Enterobacter xiangfangensis at 100% coverages, suggesting this region was acquired by horizontal gene transfer (Figure 2A).

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FIGURE 2

Circular plasmid maps of pKPT698-tetA (A) and pKPT698-mcr (B). Green, blue, and red arrows indicate the antimicrobial resistance genes, mobile elements, and other predicted ORFs, respectively. The names of antimicrobial resistance genes, mobile elements are labeled alongside the corresponding arrows. (A) The MDR region of pKPT698-tetA including aac(6′)Ib-cr, arr-3, dfrA27, aadA16, sul1, and catA2 was bordered by the transposable element Tn2. (B) pKPT698-mcr carried bla_CTX–M–15 (two copies), bla_TEM–1B, mcr-8.2 with mobile elements such as IS903B and ISEcl1 inserting into this segment.

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 (Wang et al., 2018; Zhang et al., 2021). In line with the previous findings, the mcr-8.2 gene on pKPT698-mcr 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 (Lv et al., 2020), with an extremely high prevalence of 52.4% in animals and 0.08~2.5% in patients (Lv et al., 2020; 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 (Sun et al., 2020). The simultaneous presence of NDM carbapenemases further promoted the emergence of pan-drug resistant bacteria, especially in K. pneumoniae with high genome plasticity (Sun et al., 2020). 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.

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

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References