Hafnia alvei and Hafnia paralvei, ubiquitous commensals in the human and animal gut as well as food environments, can cause opportunistic infections [1, 2]. These species are resistant to first-generation aminopenicillins and cephalosporins due to chromosomal AmpC β-lactamase (ACC type) and susceptible to carbapenems and fourth-generation cephalosporins [1, 2]. The resistance mechanisms in Hafnia species, including carbapenem resistance due to outer-membrane protein loss [3] and the presence of carbapenemases, such as bla_NDM−1, bla_VIM−1 [4], and bla_OXA−48 [5], are emerging. However, class A carbapenemase genes have not been reported in any Hafnia species.

We isolated a KPC-producing H. paralvei strain from the bile of a 71-year-old male patient with hilar cholangiocarcinoma who presented to CHA Bundang Medical Center, Seongnam, Korea, in April 2024. After accidentally removing the percutaneous transhepatic bile drainage tube, the patient developed a fever (38.2°C). Following tube replacement and bile culture, H. alvei and Klebsiella pneumoniae were identified using a Bruker MALDI Biotyper (Bruker Daltonics GmbH, Germany). Given the prior detection of carbapenem-resistant Enterobacterales, empirical treatment with colistin and meropenem was initiated. C-reactive protein decreased from 37 mg/L to 23 mg/L after 3 days of antimicrobial therapy, during which the fever also subsided. Antibiotic-susceptibility testing was performed using an AST N413 card (bioMérieux, Marcy-l’Étoile, France) and interpreted according to the CLSI guidelines [6]. Minimum inhibitory concentration results confirmed carbapenem-resistance in both strains. The presence of KPC-type class A carbapenemase genes was identified using carbapenemase-inhibition testing and the Xpert Carba-R Assay (Cepheid, Sunnyvale, CA, USA). The Institutional Review Board of CHA Bundang Medical Center approved this study (approval number: 2024-08-024) and waived the requirement for informed consent.

For molecular epidemiological analysis, genomic DNA was extracted using the Maxwell RSC Tissue DNA Kit (Promega, Madison, WI, USA) and whole-genome sequencing (WGS) was conducted by Macrogen (Seoul, Korea) using the PacBio Sequel IIe platform (Pacific Biosciences, Menlo Park, CA, USA). Genome assembly and annotation were performed using the Microbial Genome Analysis pipeline (Pacific Biosciences, Menlo Park, CA, USA) and PROKKA software, respectively. Genome-based identification was performed using the TrueBac ID cloud system. (www.truebacid.com). Antimicrobial-resistance genes were analyzed using ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/). Plasmid-incompatibility groups were identified using PlasmidFinder (https://cge.cbs.dtu.dk/services/PlasmidFinder/), and multilocus sequence typing (MLST) of whole-genome data was performed using the MLST-2.0 Server (https://cge.food.dtu.dk/services/MLST/). Genomic data are available under GenBank accession numbers CP194438–CP194440 and CP186712–CP186713 (Table 1).

The strain was initially identified as H. alvei (identification score: 2.33) via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. However, TrueBac analysis showed the highest average nucleotide identity (ANI; 95.9%) with H. paralvei. Although three species other than H. paralvei exceeded the cut-off value (98.7%) for 16S rRNA-based identification, their ANI values (<95%) did not meet the species-level identification criteria. The genomic and plasmid characteristics of bla_KPC-2-producing K. pneumoniae and H. paralvei strains determined by WGS are presented in Table 1 and Fig. 1.

bla_KPC-2 was detected in both H. paralvei CRS2402 and K. pneumoniae CRS2403. KPC genes have not been reported in Hafnia spp., indicating possible acquisition from K. pneumoniae. Genes encoding type-IV secretion system components (PtlH, VirB4, VirB8, VirB9, and VirD4) were identified in the 48-kb plasmid (IncX3), suggesting potential conjugative transfer [7]. IncX3 plasmids, predominantly found in Enterobacterales, show high conjugation ability, stability, lack of fitness cost, and enhanced biofilm formation and can carry bla_NDM, bla_KPC, and bla_OXA-181 [8]. In K. pneumoniae, KPC genes are most commonly associated with large IncFII, IncN, and IncL/M plasmids, which typically carry multiple resistance determinants [9]. However, the IncX3 plasmid identified here is compact, highly transferable, and usually carries bla_NDM rather than bla_KPC [8]. Thus, bla_KPC-2 on the IncX3 plasmid is a rare finding. In K. pneumoniae, the most prevalent sequence types associated with IncX3 plasmids are ST307, ST11, and ST15 [8], suggesting that the 48-kb IncX3 plasmid from K. pneumoniae CRS2403 was transferred to H. paralvei CRS2402 via conjugation.

Although the clinical significance of H. paralvei remains uncertain because of its co-isolation with K. pneumoniae, the identification of a bla_KPC-2-carrying strain suggests the potential dissemination of carbapenemase genes to uncommon Enterobacterales species. This report represents the first demonstration of a KPC-2-producing H. paralvei strain, likely acquired through horizontal gene transfer from a co-isolated K. pneumoniae. Our findings highlight the role of IncX3 plasmids in carbapenemase dissemination in Enterobacterales, as well as the role of WGS in identifying the genetic determinants underlying antimicrobial resistance [10]. Since the transfer of resistance between different bacterial species can present novel therapeutic challenges, continuous surveillance is essential to manage and control antimicrobial resistance within healthcare settings.