The story begins with a clinical isolate recovered on January 20, 2022, from bronchoalveolar lavage fluid collected from a 58-year-old male patient admitted to the intensive care unit. The isolate, designated KP22-83, belonged to the ST11-KL64 lineage of K. pneumoniae, a sequence type that has become notorious worldwide for its capacity to acquire and disseminate carbapenem resistance. Antimicrobial susceptibility testing revealed a deeply troubling multidrug-resistant phenotype: the isolate showed high minimum inhibitory concentrations (MICs) to meropenem, ceftazidime, aztreonam, doxycycline, tobramycin, amikacin, and ciprofloxacin, while remaining susceptible only to polymyxin B, one of the last-resort antibiotics in clinical use. Most alarmingly, the isolate’s MIC to ceftazidime-avibactam reached 640/4 mg/L, an extraordinarily high value that signaled the presence of an unusual resistance mechanism.

Whole-genome sequencing, performed on the Illumina HiSeq 2,500 platform and assembled with SPAdes, identified the culprit: the blaKPC-287 gene, together with 11 additional antimicrobial resistance-associated genes and 34 virulence-associated genes. The resistance genes included aadA2, blaLAP-2, blaSHV-12, blaTEM-1, catA2, dfrA14, fosA6, qnrS1, rmtB1, sul2, and tet(A), a constellation of determinants spanning aminoglycosides, cephalosporins, phenicols, folate pathway inhibitors, fosfomycin, fluoroquinolones, and tetracyclines. The virulence genes were predominantly involved in siderophore-mediated iron acquisition and bacterial adherence, including the enterobactin biosynthesis and transport genes entA, entB, entE, entS, fepA, fepB, fepC, fepD, and fepG; the yersiniabactin-associated genes fyuA, irp1, irp2, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, and ybtX; the aerobactin-associated genes iucA, iucB, iucC, iucD, and iutA; and the adherence-associated genes fimA, fimE, ompA, ecpA, ecpB, ecpC, ecpD, ecpE, and ecpR. Collectively, these findings characterized KP22-83 as a clinically important multidrug-resistant ST11-KL64 isolate and warranted deeper investigation into the contribution of KPC-287 to its resistance phenotype.

To place KPC-287 in evolutionary context, the team retrieved all KPC protein sequences available in the NCBI database as of May 8, 2026, and aligned them using Clustal Omega. The KPC family now comprises more than 270 assigned variants, and resistance-associated changes are concentrated primarily within three structural regions: the 164–179 omega loop, the 237–243 loop, and the 267–275 loop. Phylogenetic analysis showed that KPC-287 clustered close to KPC-155, indicating a close sequence relationship between the two variants. Nucleotide and amino-acid sequence alignment revealed that KPC-287 differed from KPC-155 by two major changes: a proline-to-glutamine substitution at residue 168 (P168Q) and deletion of alanine at residue 242 (A242del), corresponding to one single-nucleotide substitution and one three-nucleotide deletion, respectively. Both changes fall squarely within the two regions most frequently implicated in the evolution of ceftazidime-avibactam resistance.

Structural analysis using AlphaFold-predicted models added a crucial layer of molecular detail. When the predicted structures of KPC-155 and KPC-287 were superimposed using PyMOL, both variants retained the typical KPC β-lactamase architecture and showed a highly similar global fold, with a root-mean-square deviation (RMSD) of only 0.093. The main structural differences were localized near residue 168 and the Ala242 deletion site. The P168Q substitution may alter local side-chain properties and conformational flexibility, whereas the A242 deletion may affect the arrangement of the adjacent β-strand and loop region. Previous structural studies have established that changes within the omega loop can weaken stabilizing interactions and increase conformational flexibility, potentially improving accommodation of the bulky ceftazidime side chain while altering susceptibility to avibactam, the diazabicyclooctane inhibitor designed to neutralize exactly this class of enzymes. The authors caution, however, that these predicted structural differences do not by themselves establish the individual contribution of each alteration to the resistance phenotype.

That contribution was established through cloning experiments. The DNA fragment containing the native upstream promoter and open reading frame of blaKPC-287 was amplified from KP22-83 genomic DNA and ligated into the pUC19 vector to generate pUC19-KPC-287. This construct was then introduced into two recipient backgrounds: Escherichia coli DH5α and K. pneumoniae ATCC 13883. The results were unambiguous. Introduction of pUC19-KPC-287 increased the ceftazidime-avibactam MIC from less than 0.25/4 mg/L to 64/4 mg/L in both E. coli DH5α and K. pneumoniae ATCC 13883, converting both recipient strains from fully susceptible to highly resistant. In contrast, meropenem MICs remained unchanged after acquisition of pUC19-KPC-287 in both backgrounds. The empty vector had no detectable effect on the susceptibility of either strain. Together, these results establish KPC-287 as a sufficient determinant of high-level ceftazidime-avibactam resistance in both E. coli and K. pneumoniae, while its expression had no measurable effect on meropenem susceptibility, a phenotype that echoes the pattern first described for KPC-31, a D179Y derivative of KPC-3 that was the first clinically described KPC variant associated with ceftazidime-avibactam resistance together with restoration of meropenem susceptibility.

The fitness consequences of KPC-287 carriage were then evaluated through a battery of phenotypic assays. Growth curves were broadly similar among the tested strains, but the pUC19-KPC-287 transformant exhibited slightly lower OD600 values in both DH5α and ATCC 13883 backgrounds, indicating that KPC-287 expression imposed a measurable but limited impairment of bacterial growth. Motility assays on semisolid plates containing 0.3% agar showed that pUC19-KPC-287 carriage significantly reduced migration in both bacterial backgrounds, as indicated by smaller migration diameters after 36 hours of incubation. In vitro competition assays, in which KPC-287-carrying and empty-vector strains were mixed at a 1:1 ratio and serially passaged for 72 hours, showed that relative fitness values remained below 1 and decreased over time in both backgrounds, confirming a competitive disadvantage for the KPC-287-carrying strains. Conversely, the pUC19-KPC-287 transformants produced significantly greater crystal violet-stained attached biomass than the corresponding empty-vector controls in both bacterial backgrounds. The authors note that because no construct carrying an ancestral or alternative KPC allele was included in the same vector and host, the biofilm-associated difference should not be interpreted as a KPC-287 variant-specific effect; it could reflect recombinant expression level, cloning context, host background, or indirect physiological effects.

Perhaps the most clinically consequential finding concerns the mobility of the blaKPC-287 gene itself. Sequence analysis showed that blaKPC-287 was located on a hybrid IncFIIpHN7A8:IncR plasmid, embedded within an insertion sequence-rich region adjacent to ISKpn27, ISSwi1, and IS26. The presence of multiple IS26 copies throughout the plasmid indicated a mosaic multidrug-resistance plasmid architecture with potential for horizontal dissemination and structural rearrangement. Conjugation assays, in which KP22-83 served as the donor and a laboratory-preserved hygromycin-resistant strain YZ6 as the recipient, identified transconjugants carrying blaKPC-287 together with the IncFII and IncR replicon markers, supporting the transferability of the gene in an IncFII/IncR plasmid background under the tested mating conditions. Stability testing added another layer of concern: after 30 consecutive passages, blaKPC-287 remained detectable under both antibiotic-free conditions and subinhibitory ceftazidime-avibactam exposure, with 96 randomly selected colonies per condition analyzed by colony PCR. The gene, in other words, is not only transferable but also stubbornly retained, whether or not the antibiotic combination is present to select for it.

Virulence was evaluated using a Galleria mellonella infection model, in which larvae were infected with K. pneumoniae ATCC 13883, ATCC 13883-pUC19, ATCC 13883-pUC19-KPC-287, KP22-83, or the hypervirulent control strain KP19-2065. KP19-2065 caused the greatest larval mortality, with 40% survival at 72 hours post-infection. Although KP22-83 was less virulent than KP19-2065, it also caused substantial larval mortality, with 50% survival at 72 hours. In contrast, survival was comparable among larvae infected with ATCC 13883, ATCC 13883-pUC19, and ATCC 13883-pUC19-KPC-287, indicating that expression of KPC-287 alone did not measurably enhance larval killing in the ATCC 13883 background. The pronounced virulence observed in KP22-83 is more likely related to its additional virulence-associated determinants, including aerobactin, yersiniabactin, enterobactin, fimbrial, and adherence-associated genes, rather than to KPC-287 itself. The authors acknowledge that the G. mellonella model is useful for preliminary virulence assessment but cannot substitute for mammalian infection models.

Several limitations frame the interpretation of these findings. Cloning in the high-copy-number pUC19 vector may not reflect the native blaKPC-287 expression level, and the observed resistance phenotype may be influenced by gene dosage rather than completely mirroring its effect in the native IncFII/IncR plasmid context. Functional validation was limited to two laboratory hosts, and only one clinical isolate was examined, leaving the prevalence, evolutionary origin, and geographic distribution of KPC-287 unknown. Biofilm was evaluated only by crystal violet staining, and no ancestral or alternative KPC allele was tested in the same vector-host background, so a KPC-287-specific effect on biofilm formation cannot be inferred. Isogenic allele comparisons and mechanistic validation are required to determine whether KPC-287 specifically contributes to biofilm development. Nevertheless, the study identifies KPC-287 as a potentially clinically relevant ceftazidime-avibactam resistance determinant and highlights the urgent need for continuous genomic surveillance and functional characterization of emerging KPC variants. The 2024 World Health Organization Bacterial Priority Pathogens List ranked carbapenem-resistant K. pneumoniae as the highest-priority bacterial threat, and the emergence of variants like KPC-287 that can defeat the therapeutic options designed to counter it only sharpens that concern. As the KPC family continues to expand under therapeutic pressure, the message from this study is clear: the evolution of resistance is not slowing down, and the surveillance of emerging variants must keep pace.

Subject of Research: Characterization of KPC-287 conferring high-level ceftazidime–avibactam resistance in an ST11-KL64 Klebsiella pneumoniae isolate

Article Title: Characterization of KPC-287 conferring high-level ceftazidime–avibactam resistance in an ST11-KL64 Klebsiella pneumoniae isolate

Article References: Gong, Y., Shen, Y., Lu, J., Li, Y., Qin, S., Shi, X., & Tian, X. (2026). Characterization of KPC-287 conferring high-level ceftazidime–avibactam resistance in an ST11-KL64 Klebsiella pneumoniae isolate. Frontiers in Microbiology, 17, Article 1938185. https://doi.org/10.3389/fmicb.2026.1938185

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1938185

Keywords: Characterization, KPC-287, conferring, high-level, ceftazidime, avibactam, resistance, ST11-KL64, Klebsiella, pneumoniae, isolate, scientific research