Interconnected Reservoirs: Virulence & Biofilm Traits of ESBL-Klebsiella pneumoniae in Municipal Wastewater & Agricultural Systems

Extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-KP) is an important antimicrobial-resistant pathogen, and wastewater may serve as a reservoir for its persistence and dissemination. This study investigated the virulence-associated genes, biofilm-forming capacity and genomic relatedness of ESBL-KP isolates recovered from wastewater and livestock farm environments in southeastern North Carolina. A cross-sectional study was conducted between May and September 2025 at two wastewater treatment plants (WWTPs) and two livestock farms. ESBL-KP isolates recovered from wastewater, animal feces, and water samples were characterized using PCR, whole-genome sequencing and crystal violet biofilm assays. Genomic relatedness was assessed using phylogenomic analysis. Data were analyzed using descriptive statistics and Fisher’s exact test. ESBL-KP was detected in 15.4% (n = 69/449) of samples, with the highest prevalence observed in WWTPs (75.4%, n = 52) followed by poultry farms (21.7%, n = 15). The most frequent virulence genes were mrkD (30/69), entB (26/69), K2 (21/69), and rmpA (21/69). Significant variation in gene distribution by sample type was observed for mrkD (p = 0.0013) and entB (p = 0.0011). Biofilm formation varied by sample type, with strong biofilm predominating in influent (n = 20) and sludge (n = 8), although no significant differences were detected across sample types (p = 0.357). Phylogenetic analysis revealed that one poultry farm isolate was clonally related to wastewater isolates, differing by 1–3 single nucleotide polymorphisms (SNPs) and sharing the virulence genes mrkA, iutA, and fimH. Overall, environmental ESBL-KP isolates exhibited widespread virulence potential and robust biofilm-forming capacity, while phylogenetic evidence demonstrated clonal relatedness between poultry farm and wastewater isolates and sharing mrkA, iutA, and fimH virulence genes. These findings highlight wastewater and agricultural systems as genetically related reservoirs for clinically relevant ESBL-KP strains and underscore the need for strengthened One Health-based surveillance to monitor and mitigate their environmental dissemination.