A Novel Real-Time SYBR Green PCR Assay for Integron Profiling in Pseudomonas aeruginosa: Unveiling the Resistance Nexus Between Food and Clinical Isolates

Background: The dissemination of extensively drug-resistant Pseudomonas aeruginosa strains across the human–food interface represents a critical threat to global public health, complicating empirical therapeutic choices and challenging traditional nosocomial restriction paradigms. Objective: This study investigated the prevalence, phenotypic resistome landscapes, and underlying integron-mediated mobilization mechanics of P. aeruginosa across the One Health continuum using an optimized real-time quantitative PCR (qPCR) platform. Methods: A total of 116 samples comprising human clinical specimens (n = 61) and retail food matrices (n = 55) were screened. Phenotypic resistance was profiled against 16 antipseudomonal agents, with polymyxin and colistin resistance validated via reference broth microdilution. A novel real-time SYBR Green qPCR assay backed by derivative melting curve analysis was developed to track Class 1 (intI1) and Class 2 (intI2) integrons. Results: Pseudomonas aeruginosa was recovered from 36.2% (n = 42/116) of total samples, exhibiting a homogeneous distribution across clinical (32.8%) and retail food matrices (40.0%, p = 0.445). Antibiogram characterization revealed a critical resistance burden: 100% of isolates fell into extreme epidemiological tiers, with 71.4% (n = 30) exhibiting an extensively drug-resistant (XDR) phenotype and 28.6% (n = 12) reaching absolute pandrug-resistant (PDR) status. Absolute phenotypic resistance (100%) was recorded across both cohorts for meropenem, ceftazidime, aztreonam, tobramycin, polymyxins, and novel combination therapeutics (ceftazidime–avibactam and ceftolozane–tazobactam). Pairwise tracking demonstrated an exceptionally strong positive correlation in resistance distribution patterns between human and food isolates (Spearman’s rho = 0.948, p < 0.001). Analytical validation of the qPCR assay demonstrated tight, reproducible specific derivative melting peaks (Tm) at 83.2 °C for intI1 and 83.8 °C for intI2. Genotypic screening revealed universal conservation of plasmid-borne intI1 (100%), whereas plasmidic intI2 was variably distributed (61.9%). Crucially, intI2 carriage served as a definitive marker for the transition to complete pandrug resistance, being detected in 100% (n = 12/12) of PDR isolates compared to 46.7% (n = 14/30) of XDR strains (p = 0.001), supported by a strong positive correlation with the PDR phenotype (rho = 0.496, p = 0.0008). Conclusions: These findings reveal an overlapping, extreme resistome across the food-clinical interface and identify intI2 as a genetic tipping point for absolute pandrug resistance. The validated real-time assay provides a highly sensitive, proactive molecular surveillance framework capable of mapping high-risk mobile genetic platforms before they manifest as catastrophic empirical treatment failures.