Abstract
Purpose
Klebsiella pneumoniae, a gram-negative bacterium, poses a severe hazard to public health, with many bacterial hosts having developed resistance to most antibiotics in clinical use. The goal of this study was to look into the development of resistance to both ceftazidime–avibactam and carbapenems, including imipenem and meropenem, in a K. pneumonia strain expressing a novel K. pneumoniae carbapenemase-2 (KPC-2) variant, referred to as KPC-49.
Methods
After 1 day of incubation of K1 on agar containing ceftazidime–avibactam (MIC = 16/4 mg/L), a second KPC-producing K. pneumoniae strain (K2) was recovered. Antimicrobial susceptibility assays, cloning assays, and whole genome sequencing were performed to analyse and evaluate antibiotic resistance phenotypes and genotypes.
Results
K. pneumoniae strain (K1), that produced KPC-2, was susceptible to ceftazidime–avibactam but resistant to carbapenems. The K2 isolate harboured a novel blaKPC-49 variant, which differs from blaKPC-2 by a single nucleotide (C487A), and results in an arginine-serine substitution at amino acid position 163 (R163S). The mutant K2 strain was resistant to both ceftazidime–avibactam and carbapenems. We demonstrated the ability of KPC-49 to hydrolyse carbapenems, which may be attributed to high KPC-49 expression or presence of an efflux pump and/or absence of membrane pore proteins in K2. Furthermore, blaKPC-like was carried on an IncFII (pHN7A8)/IncR-type plasmid within a TnAs1-orf-orf-orf-orf-orf-orf-ISKpn6-blaKPC-ISKpn27 structure. The blaKPC-like gene was flanked by various insertion sequences and transposon elements, including the Tn3 family transposon, such as TnAs1, TnAs3, IS26, and IS481-ISKpn27.
Conclusion
New KPC variants are emerging owing to sustained exposure to antimicrobials and modifications in their amino acid sequences. We demonstrated the drug resistance mechanisms of the new mutant strains through experimental whole genome sequencing combined with bioinformatics analysis. Enhanced understanding of laboratory and clinical features of infections due to K. pneumoniae of the new KPC subtype is key to early and accurate anti-infective therapy.
Introduction
The worldwide dissemination of carbapenem-resistant Enterobacteriaceae (CRE), particularly carbapenem-resistant K. pneumoniae (CRKP), poses a significant risk to public health. CRKP can cause various infections, such as urinary tract infections, bloodstream infections, and pneumonia, leading to high morbidity and mortality.Citation1 Prevention and control of K. pneumoniae infection are becoming more challenging due to the development of antibiotic resistance. In addition to their resistance against most β-lactam antibiotics, including carbapenems, additional non-β-lactam antibiotic resistance genes, such as those for aminoglycosides and quinolones, are also present in the CRKP.Citation2 The synthesis of carbapenemases encoded by the blaKPC gene is the leading cause of CRKP.Citation3 In particular, K. pneumoniae carbapenemase 2 (KPC-2) and K. pneumoniae carbapenemase 3 (KPC-3) have been frequently associated with the development of CRKP. Therefore, the development of newer, more effective antibiotics that can overcome this resistance is of high clinical importance in managing CRKP infections.
Inhibiting class A, class C, and some class D β-lactamases is the function of the novel enzyme inhibitor avibactam. Following the demonstration that ceftazidime–avibactam therapy significantly increased antibiotic activity against CRE, the Food and Drug Administration (FDA) and European Medicines Agency (EMA) authorized it for clinical use in 2015.Citation4,Citation5 The combined use of avibactam and ceftazidime (ceftazidime–avibactam) has provided an effective treatment for K. pneumoniae infections. However, new variants resistant to ceftazidime–avibactam are being found in clinical settings, with their emergence being attributed to mutations in the blaKPC gene in CRE.Citation6–9 Mutations in KPC, such as mutations in Asp179Tyr, Val240Gly, Ala240Val, Ala177Glu, Thr243Met, Pro169Leu, Asn179Asp, Tyr179Asp, Gln169Leu, and Gly130Ser, are the most important mechanism leading to ceftazidime–avibactam resistance, a widely reported phenomenon.Citation7,Citation10–13 In addition, combination of several resistance mechanisms, such as membrane pore protein mutations, increases KPC expression. Moreover, an increased efflux pump activity can also produce ceftazidime–avibactam resistance.Citation14 These findings motivated us to conduct selection experiments using ceftazidime–avibactam to discover further mutations that may be accountable for the increasing rates of ceftazidime–avibactam-resistant variants.
Materials and Methods
Bacterial Isolates and Single-Step Mutant Selection
The parental strain of K. pneumoniae (K1) harboured blaKPC-2. The bacterial strains were identified by matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The selection procedure was performed as previously described.Citation15 A 109 CFU/mL bacterial sample from an overnight broth culture was distributed on Mueller–Hinton agar (MHA) containing ceftazidime–avibactam (fixed at 4 mg/L). The concentrations of ceftazidime–avibactam were 2 to 16 times greater than the minimum inhibitory concentration (MIC) as demonstrated previously by the CLSI broth microdilution method. After continuous observation for several days, the strains growing on all plates were recorded, saved, and passed for 10 consecutive times. Three consecutive ceftazidime–avibactam concentrations were measured on the obtained strains. Mutants were found to have MIC values four times greater than those of the original strain.
Antimicrobial Susceptibility Tests and Identification of Carbapenem Resistance Genes
The MICs of meropenem, imipenem, ceftazidime–avibactam, ceftazidime, cefpodoxime, amikacin, polymyxin B, tigecycline, sulfamethoxazole, ciprofloxacin, and aztreonam were determined using the broth microdilution method. Antimicrobial breakpoints were determined according to the standards recommended by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) for polymyxin B,Citation16 the FDA for tigecycline,Citation17 and the American Clinical and Laboratory Standards Institute (CLSI) for other antimicrobial agents.Citation18 Carbapenemase was initially identified via phenotyping, immunochromatography, and the carbapenemase inhibition enhancement assay using phenylboronic acid (PBA) and EDTA (PBA-EDTA assay).Citation19 PCR and whole genome sequencing (WGS) were performed to confirm the isolation of carbapenemase. Escherichia coli strain ATCC 25922 and K. pneumoniae strain ATCC 700603 served as controls for MIC testing.
Cloning Experiments
The blakpc gene was amplified using the Q5® High-Fidelity 2X Master Mix. KPC-F-EcoRI (5′-CCATGATTACGAATTGTGCGCGGAACCCCTATTTG-3′) and KPC-R-BamH (5′-CGACTCTAGAGGATCCAATAGATGATTTTCAGCCTTAC-3′) were used as primer pairs. The cycling parameters were: 98 °C for 30s, 98 °C for 30s, 63 °C for 30s, and 72 °C for 60s for 30 cycles. The plasmid pHSG398 cloning site was exposed using restriction endonuclease and recombinantly reacted with purified PCR products using 2X Hieff Clone Enzyme Premix; then, the recombinant plasmid was transferred into the recipient E. coli DH5α strain via chemical transformation. The potential transformants were selected using MHA plates containing 50 mg/L of chloramphenicol (for pHSG398). PCR and sanger sequencing were then used to confirm the results.
Whole Genome Sequencing and Data Analysis
The Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) was used to extract genomic DNA. The whole genome sequence was submitted to next-generation sequencing (NGS) with the Illumina NovaSeq 6000 system (Oxford Genomics Centre, Oxford, UK) with 2×150 base pair (bp) paired-end reads and long-read high-throughput sequencing (LRS) on a PacBio Sequel II system.Citation20 The long-reads generated by the PacBio Sequel II system were assembled using the Canu genome assemblerCitation21 and combined with the short-reads from the Illumina NovaSeq 6000 sequencing using PilonCitation22 to obtain the whole genome and complete plasmid sequences. The coding sequence (CDS) of the assembled sequences was predicted using the microbial-gene finding system Glimmer (http://ccb.jhu.edu/software/glimmer/index.shtml).Citation23 Multi-locus sequence typing (MLST) was conducted using the MLST 2.0 software (https://cge.food.dtu.dk/services/MLST/).Citation24 Plasmid replicon types were determined directly using the PlasmidFinder tool.Citation25 Antimicrobial resistance genes were identified using ResFinder 4.0 (https://cge.cbs.dtu.dk/services/ResFinder-4.0/)Citation26 and the Comprehensive Antibiotic Resistance Database 1.1.3 (https://card.mcmaster.ca/).Citation27 Comparative genomic circle maps were constructed using the Basic Local Alignment Search Tool BLAST Ring Image Generator (BRIG) (http://sourceforge.net/projects/brig).Citation28 The insertion sequences (IS) and genomic islands (GI) on the plasmids were predicted using ISfinder (www-is.biotoul.fr)Citation29 and IslandViewer 4 (https://www.pathogenomics.sfu.ca/islandviewer/upload/),Citation30 respectively. Covariance analysis of the plasmids was performed using Easyfig (http://easyfig.sourceforge.net/).Citation31 The autonomous transmissibility of plasmids was analysed using oriTfinder (https://tool-mml.sjtu.edu.cn/oriTfinder/instruction.html).Citation32
Results
Antimicrobial Susceptibility in Parental and Mutant Strains
The parental strain (K1) was of the sequence type 859 (ST859) and carried the blaKPC-2 gene. It exhibited resistance to aminoglycosides, quinolones, cephalosporins, and carbapenems. The MIC values of imipenem and meropenem were 128 mg/L and above 128 mg/L, respectively. K1 was still susceptible to tigecycline, polymyxin B, and ceftazidime–avibactam. The ceftazidime–avibactam-resistant mutant (K2) was isolated after incubating K1 on MHA plates containing 16/4 mg/L ceftazidime–avibactam overnight. Although K2 exhibited an eight-fold reduction in the MIC of IMP and above a two-fold reduction in the MIC of MEM compared with that of K1, it was still resistant to both antibiotics. Importantly, K2 was also resistant to ceftazidime–avibactam with a MIC value of 64 mg/L ().
Table 1 Antimicrobial Susceptibility Results by Micro-Broth Dilution for KPC-Producing K. pneumoniae Isolates, KPC-Producing E. coli Transformants (pKPC-2-TM, pKPC-49-TM) and the E. coli DH5α-pHSG398
Genes Associated with Antibiotic Resistance and Plasmid Traits in Parental and Mutant Strains
In both K1 and K2, blaKPC-like was carried on the IncFII(pHN7A8)/IncR plasmid (plasmid B), which had a gene size of 109,251 bp with a guanine-cytosine (GC) content of 54.49%. K1 and K2 contained the same antibiotic resistance genes conferring resistance to β-lactams, including blaKPC-like, blaTEM-1B, blaSHV-12, as well as the rmtB gene conferring resistance to aminoglycosides. Antibiotic resistance-associated genes, including efflux pump-associated genes acrAB-tolC, kpnEF, and kpnGH, as well as marA and ramA, which positively regulate the AcrAB-TolC efflux pump, were found in both K1 and K2. Genes related to membrane permeability, including ompK35 and ompK36, were not detected. Using K1 as the reference genome, investigation of single nucleotide polymorphisms (SNPs) with NGS revealed one mutation of blaKPC-like on IncFII(pHN7A8)/IncR in K2. The unique blaKPC-49 mutation differed from the parental strain expressing blaKPC-2 by a single nucleotide alteration (Nucleotide 487, C-A), resulting in the substitution of arginine for serine at amino acid position 163 (R163S).
The Nucleotide BLAST (BLASTn) search found that plasmid B had the highest alignment score in the whole sequence with the previously reported plasmid pB (CP069172.1)Citation33 with query coverage and identity of 96% and 99.98%, respectively, and with pZHKPC1 (OM928502.1)Citation34 with query coverage and identity of 95% and 100% respectively.
As shown in , the findings of the BLASTn search suggested that plasmid B, pKP20194e-p2 (CP054728.1) and pKP58-2 (CP041375.1) had the highest alignment score at 44,910–54,954 bp, where blaTEM-1B, rmtB, and surrounding mobile genetic elements (MGEs) were expressed. In contrast, plasmid B, p12085-KPC (MN842292.1) and pKPC-L388 (CP029225.1) showed the highest alignment scores in the segment of 74531–10054 bp, where blaKPC-like, blaSHV-12 and surrounding MGEs were encoded. The plasmid PKP048 (FJ628167.2) was obtained from Nucleotide database screening for the blaKPC-2-harbouring K. pneumoniae in 2010.Citation35 The comparison revealed that plasmid B had high similarity to PKP048 in the region spanning between the blaSHV-12 and holE genes, and blaKPC-2 was in this region. According to the alignment results on a BLAST search, all plasmids under comparison were from K. pneumoniae, excluding p16055-KPC, which was derived from S. marcescens.
Figure 1 Circular map between plasmid B and the 10 comparative plasmids investigated in this study. The outermost circle represents plasmid B. Protein-coding sequences are denoted by arrows.
As shown in , plasmid B contained two genomic islands (GIs) between 84,004 and 99,319 bp and the critical carbapenem-resistance gene, blaKPC-2, was carried on the first of the two GIs (GI 1). Upstream of blaKPC-2 were TnAs1, ISKpn6, a gene encoding a replication protein, klcA, and three genes encoding hypothetical proteins, whilst the ISKpn27 insertion sequence was located downstream. The structure was “TnAs1-orf-orf-orf-orf-orf-ISKpn6-blaKPC-ISKpn27” from 84,004 to 90,918 bp. Furthermore, GI 2 was 5749 bp in length, spanning from 9,3570 to 99,319 bp, with TnAs3 and IS5075 flanking the two ends. It also included seven gene clusters encoding mercury resistance in the sequence of merE-merD-merA-merC-merP-merT-merR. The GI 1 region was very similar to the IncFII/IncR plasmids of K. pneumoniae, including pZHKPC1 (OM928502.1), pHS2953-KPC (MT875328.1), p12085-KPC (MN842292.1) and pKP0294e-p2 (CP054728.1). It was also present in the same plasmid type, p16055-KPC (MN823985.1), from S. marcescens. Similar structures were also found in the IncN plasmids, pL901 (CP045256.1) and pT211 (CP017083.1), isolated from Proteus mirabilis.
Figure 2 Detailed structure and comparison of the blaKPC-2 gene clusters within the strains. Green arrow indicates transposase genes and red arrows show resistance genes. Arrows indicate the direction of translation of the coding genes. The larger picture depicts a genome-wide comparison of plasmid B with the near-source plasmids, and the smaller picture shows two conserved genomic islands (Islands 1 and 2).
The oriTfinder tool was used to determine whether horizontal transfer elements, including the origin of transfer site (oriT), relaxase gene, type IV coupling protein (T4CP), and type IV secretion system (T4ss) were present in plasmid B. Importantly, plasmid B expressed part of the T4ss but not oriT, relaxase, or T4CP. Given that plasmid B did not express all four MGEs, it was concluded that plasmid B is a non-mobile plasmid.
Results of Cloning Experiments
We successfully obtained pKPC-2 and pKPC-49 recombinant plasmids through cloning experiments and transferred them into E. coli DH5α strains to produce the transformants, pKPC-2-TM and pKPC-49-TM. The comparative results indicated that the MIC values of pKPC-49-TM against carbapenems (imipenem and meropenem) were reduced, whereas the MIC value of ceftazidime–avibactam was significantly increased. The variations in the susceptibility profiles of the donors and transformants are listed in .
Discussion
In the present study, we have identified a novel KPC-2 variant, sequence matched as KPC-49 by in-vitro single-step mutant selection. This variant was formerly identified as the KPC-3 variant.Citation36 The newly discovered variant in ST859 conferred resistance to both ceftazidime–avibactam and several carbapenems, distinguishing it from the KPC-49 variant in E. coli mentioned above. The KPC-49 variant in E. coli regained susceptibility to imipenem and meropenem. However, strain K2 was still resistant to imipenem and meropenem, and the pKPC-49-TM was susceptible to IMP and MEM in our study. According to the cloning experiment results, the MICs of imipenem and meropenem in the K1 and K2 strains were higher than those of the transformants (pKPC-2-TM and pKPC-49-TM). The reason for this phenomenon might be the existence of other carbapenem resistance mechanisms, including high expression of KPC, over-production of efflux pumps, and loss of outer membrane porins (OMPs), which can render bacteria more resistant to carbapenems. The enzyme inhibition enhancement assay and bioinformatics analysis supported these possibilities. The enzyme inhibition enhancement test of strain K2 suggested that the diameter of the imipenem inhibition circle was expanded by ≥5 mm after adding phenylboronic acid to strain K2 compared with that of the single drug. The KPC-49 variant likely allows bacteria to retain the hydrolytic ability of carbapenemase, and the high expression of KPC-49 in strain K2 is responsible for its resistance to carbapenems.
We also found that the K1 and K2 strains did not encode OmpK35 and OmpK36, two major OMPs of K. pneumoniae. The absence or deficiency of OMPs, along with the production of extended-spectrum beta-lactamases and/or AmpC, contribute significantly to carbapenem resistance in K. pneumoniae. Notably, this is also the mechanism of carbapenem resistance in K. pneumoniae that do not produce carbapenemases.Citation37
In addition, bioinformatic investigation of antimicrobial resistance genes indicated that K1 and K2 strains expressed efflux pumps, including AcrAB-TolC from the resistance-nodulation-division family, KpnGH from the major facilitator superfamily, and KpnEF from the small multidrug resistance family. The genes marA and ramA positively control the efflux pump of AcrAB-TolC.Citation38,Citation39 Eliminating the AcrAB-TolC pump of K. pneumoniae could impact resistance, fitness, and virulence.Citation40 High resistance to quinolones and decreased susceptibility to carbapenems have been linked to the efflux pump AcrAB-TolC in K. pneumoniae.Citation41 Srinivasan et al reported that the insertion of an inactivated kpnGH gene fragment increased the susceptibility of K. pneumoniae to antibiotics such as ceftazidime, ciprofloxacin, and ertapenem.Citation42 They used specific efflux genes kpnE and kpnF to encode the efflux pump KpnEF, which recognises a variety of substrates such as ceftriaxone, erythromycin, and tetracycline in K. pneumoniae strain NTUH-K2044. When the kpnEF gene was knocked out, the sensitivity of the KpnEF pump to the previously mentioned antibiotics was reduced.Citation43 marA and ramA both promote expression of AcrAB, thereby enhancing the ability of K. pneumoniae to eliminate antibiotics via the AcrAB-TolC efflux pump.Citation38,Citation39
Our analysis revealed that the KPC-49 variant was carried on an IncFII/IncR type blaKPC-bearing plasmid. IncFII/IncR type plasmids are frequently isolated from K. pneumoniae, including the pZHKPC1,Citation34 pHS2953-KPC, and p12085-KPC plasmids, as well as from other gram-negative bacteria, including the p16055-KPC plasmid from S. marcescens.
Plasmid B, on which blaKPC was expressed, was analysed using oriTfinder to determine its capacity for autonomous propagation. According to relevant research, plasmids can be classified into three categoriesCitation32: 1) Conjugative plasmids containing relaxase, T4CP, and T4SS; 2) mobilisable plasmids containing only relaxase; and 3) non-mobilisable plasmids which lack relaxase. An analysis of the autonomous transmissibility of plasmid B using oriTfinder found that plasmid B did not have oriT, relaxase, and T4CP. Plasmid B can be categorised as a non-mobile plasmid since it lacks the necessary components for a mobile plasmid. A plasmid lacking a splice structure can transfer transposons, integrins, and its related resistance genes to another recipient bacterium with the help of another plasmid or by phage transduction or transformation.
In addition to plasmids, other MGEs, including insertion sequences, transposons, integrins, GIs, and integrated splice elements, play a critical role in the acquisition and dissemination of drug-resistant genes.Citation44 GI 1 of plasmid B contained the carbapenem resistance gene, blaKPC-2, in the TnAs1-orf-orf-orf-orf-orf-ISKpn6-blaKPC-2-ISKpn27 unit, which showed high similarity to the IncFII/IncR-type replicon of K. pneumoniae and was also present in the IncFII/IncR-type replicons of S. marcescens and IncN-type replicons of P. mirabilis. This suggests that GI 1 may not only be transferred horizontally between different bacterial species via plasmids but also in a transposable manner, binding to other types of replicons to achieve transfer of the drug resistance gene blaKPC. In addition to blaKPC, the plasmid encoded additional resistance genes, including blaTEM-1B, rmtB, and blaSHV-12. These resistance genes were surrounded by a range of insertion sequences and transposon elements, including the Tn3-family transposon, including TnAs1, TnAs3, IS26, IS5-IS903B, S91-IS1294, IS1182-ISKpn6, IS481-ISKpn27, IS110-IS5075, and IS1-ISKpn14, and they may also be transmitted through these MGEs.
In the present study, we analysed the development of resistance to ceftazidime–avibactam and carbapenems, including imipenem and meropenem, in K. pneumoniae expressing a novel KPC-2 variant (KPC-49) through bioinformatics analysis and cloning assays. Of concern is that strain K2 remains resistant to imipenem and meropenem. It was found that factors associated with this situation may involve high expression of KPC49, efflux pumps, and absence of associated OMPs, with one or more contributing to the outcome. We can further clarify this by relevant experiments in future studies.
Conclusion
The over-prescription and inappropriate use of antibiotics in clinical care has led to the emergence of multiple drug-resistant strains that are difficult to eradicate and slow progress in the treatment of related diseases. Resistance genes can be transmitted through multiple mobile components. These have contributed to the global antibiotic resistance crisis. Technological innovations, such as WGS and NGS combined with bioinformatics analysis, provide valuable analytical tools for studying antimicrobial resistance. By combining traditional detection techniques with modern technologies, we have gained a more comprehensive and specific understanding of the drug-resistant phenotype, resistance mechanism, and transmission risk of the new KPC subtype K. pneumoniae. This will play a key role in early and precise clinical anti-infective treatment. It will also help to further combat microbial drug resistance.
Ethics Approval
The Qingdao Municipal Hospital’s Ethics Committee approved the whole plan for this study’s research. The authors of this paper have not performed any studies on humans or animals. The need for informed consent was waived because this study was about bacteria and did not involve patients.
Author Contributions
All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.
Disclosure
In this work, the authors state that they have no conflicts of interest.
Acknowledgments
The authors would like to thank the Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China, for its technical help, including provision of instruments, equipment and relevant experimental material.
Additional information
Funding
References
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