Genomic Insights into Plasmid Mediated Dissemination of bla_KPC-2 and bla_CTX-M-14 in a Fecal ST595 Serratia Marcescens Isolate

Introduction

Serratia marcescens is a Gram-negative bacillus belonging to the Enterobacteriaceae family, recognized for causing nosocomial infections, particularly in immunocompromised patients. Known for causing a variety of infections, including pneumonia, urinary tract infections, and notably, bloodstream infections, S. marcescens has become an emerging threat due to its ability to acquire and disseminate resistance genes.¹ The rise of carbapenem-resistant Enterobacteriaceae (CRE), particularly those producing Klebsiella pneumoniae carbapenemase (KPC), poses a severe threat to public health because of limited treatment options.² The dissemination of the bla_KPC-2 gene is primarily facilitated through plasmids, which can transfer between different bacterial species, exacerbating the spread of resistance. CTX-M-14 is an extended-spectrum β-lactamase (ESBL) enzyme, frequently detected in Enterobacteriaceae and contributing to resistance against third generation cephalosporins.³ While S. marcescens carrying bla_KPC-2 has been described in clinical infections in China (eg, ICU isolates), stool carriage in this species remains rare in the Chinese literature to date. These gaps emphasize the importance of stool-based genomic surveillance and motivate the present analysis of a fecal isolate.”

This study focuses on the genomic characterization of S. marcescens L4843, a bla_KPC-2- and bla_CTX-M-14-co-carrying isolate recovered from a fecal sample in China. Understanding the genetic context of bla_KPC-2 and other resistance determinants within this isolate is crucial for developing effective strategies to combat CRE.

Materials and Methods

The S. marcescens L4843 strain was isolated from a fecal sample of a patient at a tertiary hospital in Hangzhou, China in 2023. The isolate was cultured on MacConkey agar, and species identification was confirmed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS).

The minimum inhibitory concentrations (MICs) for various antibiotics were determined using the broth microdilution method, following the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The antibiotics tested included carbapenems (imipenem, meropenem), β-lactams (ceftazidime, cefepime), aminoglycosides (gentamicin, amikacin), and fluoroquinolones (ciprofloxacin, levofloxacin).

Genomic DNA of S. marcescens L4843 was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The short-read library was prepared using Nextera DNA Flex Library Prep Kit (Illumina Inc., San Diego, CA, USA) for WGS according to the manufacturer’s instructions and sequenced on a NovaSeq instrument (Illumina Inc., San Diego, CA, USA). The long-read library was prepared using a Ligation Sequencing Kit 1D (Oxford Nanopore Technologies, Oxford, UK) and sequenced with the MinION sequencer.⁴ De novo genome assembly was carried by Unicycler v.0.4.8⁵ and the genome was subsequently annotated using Prokka 1.14.5.⁶ Species identification was corroborated using SpeciesFinder v.2.0 (https://cge.cbs.dtu.dk/services/Species-Finder/). PlasmidFinder and ResFinder were used to identify plasmid replicons and resistance genes, respectively.⁷ Integrons were detected using the Integron Finder tool (https://github.com/gem-pasteur/Integron_Finder). Specifically, the SQK-LSK109 kit and R9.4 flow cell (FLO-MIN106) were used for sequencing. MLST analysis was performed using the PubMLST database (https://pubmlst.org/bigsdb?db=pubmlst_serratia_seqdef). Default settings were used for all bioinformatics tools unless otherwise specified. Putative transconjugants were screened by PCR for both bla_KPC-2 and bla_CTX-M-14.

Results and Discussion

The isolate S. marcescens L4843 demonstrated high-level resistance to carbapenems, with MIC values for imipenem and meropenem exceeding 16 μg/mL. Resistance to other β-lactams, aminoglycosides, and fluoroquinolones was also observed, confirming the multidrug-resistant (MDR) phenotype of this isolate.

The genome of S. marcescens L4843 comprises one chromosome and three plasmids. It comprised 5,377,884 bp with a GC content of 59.8%. The genome contained 5,083 coding sequences, 95 tRNA genes, and 6 rRNA operons. MLST analysis identified the isolate as sequence type ST595. The genome of L4843 harbors multiple antimicrobial resistance genes, including bla_KPC-2, aac(3)-IId and aac(6’)-Ic conferring resistance to carbapenems and aminoglycosides, respectively. Other resistance genes identified included bla_SRT-1, bla_CTX-M-14, and bla_LAP-2 (Table S1), which provide resistance to beta-lactams. The presence of these genes correlates with the observed multidrug-resistant (MDR) phenotype, including resistance to imipenem and meropenem. All public repository accessions and direct hyperlinks for the S. marcescens L4843 genome and associated datasets (BioProject, BioSample, and WGS assembly) are compiled in (Table S2).

Plasmid analysis of bla_KPC-2-carrying repB(R1701) plasmid pL4843-KPC2 provided comprehensive insights into the genetic environment surrounding the bla_KPC-2 gene (Figure 1A). The bla_KPC-2 gene is embedded within a complex genetic environment on the plasmid, which includes multiple insertion sequences (IS) and transposons. The bla_KPC-2 gene is flanked by IS26 and ISKpn27 elements, facilitating its mobility and potential horizontal transfer. The presence of multiple IS elements (IS26, ISKpn27, ISKpn19) and transposons (TnAs1) indicates a high potential for genetic rearrangements and the acquisition of additional resistance genes. Under the selection used, only bla_KPC-2 was detected in transconjugants, indicating that bla_CTX-M-14 did not co-transfer.

Figure 1 (A) Circular BLAST Ring Image Generator map of pL4843-KPC2 (innermost lavender ring) aligned against three reference plasmids: CP047692 (light blue ring), CP047686 (cornflower blue ring) and MT269826 (medium blue ring). Open reading frames (ORFs) are depicted as red arrows on each ring, with the blaKPC-2 gene labeled in black. Arrows around the circle correspond sequentially to the various genetic elements. (B) Linear comparative alignment generated with Easyfig, showing the pL4843-KPC2 backbone (top track) aligned beneath against CP047692, CP047686 and MT269826. Homologous regions are indicated by shaded arcs: regions with ≥ 99% identity in medium blue. ORFs are shown as colored arrows to reflect its predicted functional category. Mint green: mobile element–associated genes (eg, transposases and insertion sequences). Red: antibiotic resistance genes (eg, blaKPC‑2). Medium blue: conjugation, replication, and stability genes. Orange: hypothetical proteins.

Comparative genomics showed that pL4843-KPC2 shares a highly conserved backbone to three previously reported bla_KPC-2-bearing plasmids: pC110-KPC (CP047692.1) and p2838-KPC (CP047686.1), both from clinical S. marcescens isolates, and pBSI030-KPC2 (MT269826), from a clinical Klebsiella pneumoniae strain. Pairwise alignments demonstrated 100% nucleotide identity across 79% of the pC110-KPC and p2838-KPC backbones, and 99.9% identity over 80% of pBSI030-KPC2 (Figure 1B), yet exhibits sequence divergence outside this conserved core, warranting pL4843-KPC2 as a novel structural variant. These findings underscore both the remarkable stability of the repB(R1701) scaffold and its ability to mediate interspecies dissemination of carbapenemase genes.

Comparative genomics demonstrated that pL4843-KPC2 shares a conserved IncR–IncFII-like backbone with three previously reported bla_KPC-2-bearing plasmids: pC110-KPC (CP047692.1) and p2838-KPC (CP047686.1), both originating from clinical S. marcescens isolates, and pBSI030-KPC2 (MT269826), derived from a K. pneumoniae clinical strain. Pairwise alignments showed 100% nucleotide identity over 79% of the pC110-KPC sequence and p2838-KPC sequence, whereas homology to pBSI030-KPC2 is 99.9% identity across 80% coverage (Figure 1B). This confirming its novel structural variant. These findings underscore both the stability of the repB(R1701) backbone and its capacity for interspecies dissemination of carbapenemase genes.

Beyond stool carriage, S. marcescens carrying bla_KPC-2 and bla_CTX-M-14 has been reported from non-fecal clinical specimens in China and elsewhere, underscoring clinical relevance. However, as of September 2025, we found no published report of human fecal S. marcescens carrying bla_KPC-2 in China, and existing Chinese CRE carriage studies do not specifically document such stool isolates. Within this context, L4843 adds evidence for a potential intestinal reservoir, which-together with the close backbone homology between pL4843-KPC2 and plasmids reported from Klebsiella pneumoniae-supports a risk of interspecies transfer and highlights the value of targeted stool-based surveillance.

Conclusion

In summary, this study provides a comprehensive genomic characterization of a KPC-2- and CTX-M-14-producing S. marcescens isolate from a fecal sample. We highlight the complexity and mobility of resistance mechanisms within this pathogen. The presence of bla_KPC-2 on a transferrable plasmid underscores the role of mobile genetic elements in the dissemination of carbapenem resistance.⁸ In conjunction with the apparent scarcity of published fecal S. marcescens carrying bla_KPC-2 in China, our findings highlight the intestinal reservoir as a plausible waypoint for plasmid-mediated and potentially interspecies dissemination of β-lactam resistance, reinforcing the need for integrated stool-based surveillance and infection-control strategies.