The remarkable metabolic adaptability and physiological plasticity of Pseudomonas aeruginosa underpin its persistence and success as a prominent opportunistic pathogen in clinical settings. This bacterium possesses an array of intrinsic and acquired virulence determinants that substantially complicate the management of healthcare-associated infections, including a functional type III secretion system, alginate capsular polysaccharide, type IV pili, and a potent capacity for biofilm development1. According to estimates by the National Institutes of Health (NIH), microbial biofilms contribute to approximately 65% of all acute microbial infections and up to 80% of chronic infectious diseases1,2. Within biofilm architectures, bacterial cells are encased in a self-produced extracellular polymeric matrix that impedes antimicrobial penetration, protects against host immune defenses, and fosters physiological tolerance2.

P. aeruginosa employs multiple multifaceted mechanisms that confer resistance to clinically vital antimicrobial classes. These mechanisms include the inducible production of chromosomally encoded AmpC β-lactamase, an enzyme that hydrolyzes penicillins and cephalosporins3; transcriptional downregulation, mutational disruption, or total loss of the OprD outer membrane porin, which selectively mediates carbapenem entry into the bacterial cell4,5; and the active extrusion of antimicrobial agents via multidrug resistance-nodulation-division (RND) efflux pumps, predominantly MexAB–OprM and MexCD–OprJ (the latter partially encoded by mexD)4,5. Furthermore, mobile genetic elements frequently mediate the acquisition of metallo-β-lactamases (MBLs), such as New Delhi metallo-β-lactamase (NDM), conferring broad resistance against almost all β-lactams, including carbapenems5.

Molecular typing methods are essential for elucidating the transmission dynamics, genetic diversity, and clonal relatedness of clinical pathogens. Reference approaches in molecular epidemiology include multilocus sequence typing (MLST), whole-genome sequencing (WGS), and pulsed-field gel electrophoresis (PFGE)6. While MLST and WGS represent sequence-based modalities and PFGE is a macrorestriction band-based technique, each provides high discriminatory resolution for tracking epidemic lineages6. However, routine clinical implementation of these advanced tools remains severely constrained in many resource-limited hospital laboratories due to high instrumentation costs, specialized bioinformatic requirements, and prolonged turnaround times7.

Transfer RNA intergenic spacer PCR (tDNA-PCR) has emerged as a rapid, accessible, and reproducible PCR-based fingerprinting method8,11. This technique utilizes consensus primers complementary to conserved flanking tRNA gene sequences to amplify the variable spacer regions located between adjacent tRNA loci. Because bacterial lineages exhibit pronounced structural variations in both the length and nucleotide sequence of these intergenic spacers, electrophoretic resolution of the resulting amplicons generates distinct, strain-specific banding patterns8,11. Multiple studies have demonstrated that tDNA-PCR delivers robust discriminatory performance, making it a viable preliminary surveillance tool when sequencing-based technologies are unavailable8. Accordingly, this study aimed to investigate the antimicrobial resistance patterns, biofilm-forming capacity, distribution of key resistance and virulence genes (mexD, oprD, pelA, and bla_NDM-1), and genetic diversity of clinical P. aeruginosa isolates using tDNA-PCR fingerprinting.

In this hospital-based investigation, P. aeruginosa accounted for 30.7% of 260 clinical specimens, corroborating its standing as a major opportunistic pathogen in healthcare settings. This recovery rate aligns closely with surveillance studies conducted in Duhok, Iraq, which reported a 29.3% isolation frequency12, and in Baghdad, Iraq, where P. aeruginosa constituted 36.0% of clinical isolates13. In contrast, a lower prevalence (19.3%) was reported from specialized hospital settings in Ethiopia14. The clinical ubiquity and persistence of P. aeruginosa stem from its minimal nutritional requirements, tolerance to physical desiccation, intrinsic antimicrobial resistance, and propensity to colonize medical devices and moist hospital reservoirs15.

Analysis by infection site revealed that burn wounds were the primary clinical source (32.5%), consistent with findings from northern Iran reporting a 36.1% prevalence in burn units16. Severe thermal injuries disrupt the cutaneous epithelial barrier, impair local cellular immunity, and necessitate prolonged hospitalization and extensive therapeutic intervention, thereby establishing an optimal niche for P. aeruginosa colonization and invasive disease. Conversely, studies in Addis Ababa, Ethiopia, documented a lower frequency (12.8%) among burn patients17, reflecting regional differences in infection control protocols and patient populations.

The antimicrobial susceptibility profile revealed pronounced resistance to frontline antipseudomonal β-lactams, particularly aztreonam (75.0%), cefepime (75.0%), and ticarcillin–clavulanate (66.3%), alongside elevated resistance to imipenem (50.0%) and ciprofloxacin (50.0%). These findings mirror high resistance rates reported across the Middle East and North Africa; for instance, an Egyptian surveillance study recorded resistance exceeding 80% to multiple β-lactams and aminoglycosides18. In contrast, Said et al.12 observed that 78.9% of isolates remained imipenem-susceptible, while Asamenew et al.14 reported lower resistance to cefepime (51.0%), ceftazidime (50.0%), imipenem (28.4%), and ciprofloxacin (14.9%). The elevated resistance observed in our center reflects intense selective pressures driven by empirical broad-spectrum antibiotic usage.

A notable observation in this study was the higher resistance rate to cefepime (75.0%) compared to ceftazidime (25.0%). In typical AmpC β-lactamase hyperproducers, cross-resistance between both extended-spectrum cephalosporins is customary. However, preferential resistance to cefepime while retaining ceftazidime susceptibility has been documented previously19,20. This phenotypic discordance is frequently governed by active multidrug efflux mechanisms, specifically the upregulation of MexCD–OprJ or MexXY–OprM efflux systems, combined with mutations in regulatory genes (e.g., nfxB) that selectively diminish intracellular cefepime accumulation without conferring concomitant high-level ceftazidime resistance19,20.

Biofilm formation was confirmed in 72.5% of isolates, with 42.5% displaying a strong biofilm phenotype. These rates are congruent with reports by Farhan et al.21 (80.0% biofilm formers) and Abou Elez et al.18 (100% biofilm formers, 73.1% strong). Biofilm development serves as a formidable survival adaptation that thwarts antimicrobial efficacy by creating a physical barrier to antibiotic diffusion, establishing oxygen- and nutrient-depleted microenvironments that promote metabolic dormancy, and shielding bacteria from host complement and phagocytic clearance22.

Molecular analysis identified the mexD efflux gene in 68.7% of isolates. Overexpression of the MexCD–OprJ multidrug efflux pump contributes significantly to elevated minimum inhibitory concentrations (MICs) against fluoroquinolones, zwitterionic cephalosporins (cefepime), and macrolides, often acting synergistically with target mutations or enzymatic degradation23. The porin gene oprD was detected in 42.5% of isolates, while pelA was detected in 37.5%. We identified a statistically significant association between pelA PCR positivity and the strong biofilm phenotype (P = 0.0035), corroborating the structural necessity of the Pel exopolysaccharide in maintaining biofilm matrix integrity and cell–cell cohesion24,25. Nevertheless, the overall prevalence of pelA (37.5%) was lower than the total biofilm-forming rate (72.5%). This discrepancy underscores that biofilm biogenesis in P. aeruginosa is a multifactorial process coordinated by alternative redundant pathways, including the Psl polysaccharide operon, alginate biosynthesis machinery, type IV pili, flagella, and bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling circuits26. Consequently, biofilm development cannot be ascribed solely to a single genetic locus.

The metallo-β-lactamase gene bla_NDM-1 was absent across all 80 isolates. This finding indicates that carbapenem resistance in this cohort was mediated by non-NDM mechanisms. Resistance to carbapenems in P. aeruginosa frequently arises from mutational inactivation of OprD, stable derepression of chromosomal AmpC, or overexpression of MexAB–OprM, as well as the potential presence of other transferable carbapenemase genes such as bla_VIM, bla_IMP, or bla_OXA variants26,27. Interestingly, oprD PCR amplicons were significantly more frequent among imipenem-resistant isolates (P < 0.001). This phenomenon is consistent with published evidence demonstrating that positive PCR amplification of an oprD fragment does not guarantee the synthesis of an intact, functional porin channel28,29. Previous molecular investigations have demonstrated that PCR-positive oprD alleles in carbapenem-resistant isolates frequently harbor disruptive point mutations, frame-shifts, premature stop codons, or insertion elements that truncate or abrogate OprD protein expression28,29. Similarly, the association between mexD detection and cefepime resistance was not statistically significant (P = 0.2101). Standard PCR demonstrates gene presence rather than transcriptional expression or functional pump assembly; hence, PCR detection alone is insufficient to predict phenotypic resistance30,31.

Genotypic fingerprinting by tDNA-PCR revealed 49 distinct patterns among the 80 isolates, including 27 unique genotypes and 22 clustered genotypes encompassing 53 strains (clustering rate = 66.25%). Both Simpson’s Diversity Index and the Hunter–Gaston Discriminatory Index reached 0.986. These results demonstrate extensive genetic polymorphism among clinical strains while simultaneously confirming the existence of clonal dissemination across the institution. Crucially, identical fingerprint profiles were identified in isolates recovered from different anatomical sources (burns, wounds, urine, ear). This observation points to cross-transmission or the circulation of shared environmental clones across distinct hospital departments. Similar molecular diversity patterns have been documented in Brazil11 (eight genotypes among clinical isolates) and Egypt8 (21 genotypes, seven clusters, Simpson's index 0.921). The higher diversity index observed here (0.986) underscores the polyclonal composition of clinical P. aeruginosa in this healthcare center.

The high propensity for biofilm development identified herein emphasizes the urgent clinical necessity for non-conventional therapeutic modalities to overcome chronic recalcitrant infections. Novel adjunctive strategies under active investigation include bacteriophage therapy, synthetic antimicrobial peptides, and quorum-sensing inhibitors32,33. Several recent studies have shown that bacteriophage–antibiotic combinations exert potent synergistic effects, degrading extracellular matrix architecture, reducing biofilm biomass, and clearing established P. aeruginosa infections34,35. Incorporating these biological and molecular strategies into conventional antimicrobial regimens represents a promising avenue for mitigating persistent infections.

This study has several limitations that should be acknowledged. First, biofilm production was assessed using the semi-quantitative tube adherence assay rather than the quantitative microtiter plate assay or confocal laser scanning microscopy. Although the tube method is cost-effective and suited for screening, its visual scoring involves potential subjectivity and inter-observer variability. Second, conventional PCR assays only established the presence or absence of target gene regions, without providing nucleotide sequence integrity, mutational analysis, or quantitative transcriptional expression levels (e.g., via RT-qPCR). Third, although tDNA-PCR demonstrated high discriminatory capacity, it offers lower resolution and inter-laboratory standardization than whole-genome sequencing or multilocus sequence typing. Finally, the study was conducted at a single tertiary hospital, which may limit the generalizability of the findings to broader geographical regions.

This study demonstrates a high prevalence of multidrug resistance and biofilm-forming capability among clinical P. aeruginosa isolates recovered from diverse hospital infections. Resistance to aztreonam and cefepime was particularly prominent. Biofilm formation was documented in 72.5% of isolates and was significantly associated with the presence of the pelA gene. The absence of bla_NDM-1, coupled with the detection of mexD and oprD, indicates that chromosomal mechanisms and non-NDM pathways drive carbapenem and β-lactam resistance in this setting. Molecular fingerprinting by tDNA-PCR revealed extensive genetic diversity (HGDI = 0.986) while identifying shared clonal clusters circulating across different clinical services. tDNA-PCR represents a rapid, economical, and discriminative molecular epidemiological tool for tracking clinical P. aeruginosa in resource-constrained environments.

ATM: Aztreonam; CAZ: Ceftazidime; c-di-GMP: Bis-(3′-5′)-cyclic dimeric guanosine monophosphate; CIP: Ciprofloxacin; CLSI: Clinical and Laboratory Standards Institute; df: Degrees of freedom; DNA: Deoxyribonucleic acid; dNTP: Deoxynucleotide triphosphate; FEP: Cefepime; HGDI: Hunter–Gaston Discriminatory Index; IPM: Imipenem; IRB: Institutional Review Board; LB: Luria–Bertani / Lysogeny broth; MBL: Metallo-β-lactamase; MEM: Meropenem; MIC: Minimum inhibitory concentration; MLST: Multilocus sequence typing; NDM-1: New Delhi metallo-β-lactamase-1; NIH: National Institutes of Health; PBS: Phosphate-buffered saline; PCR: Polymerase chain reaction; PFGE: Pulsed-field gel electrophoresis; RND: Resistance-nodulation-division; SDD: Susceptible-dose dependent; SDI: Simpson’s Diversity Index; SPSS: Statistical Package for the Social Sciences; STROBE: Strengthening the Reporting of Observational Studies in Epidemiology; TAE: Tris-acetate-EDTA; TBE: Tris-borate-EDTA; tDNA-PCR: Transfer RNA intergenic spacer polymerase chain reaction; TIM: Ticarcillin–clavulanate; TSB: Trypticase soy broth; TZP: Piperacillin/tazobactam; UTI: Urinary tract infection; UV: Ultraviolet; WGS: Whole-genome sequencing

The authors express their sincere gratitude and appreciation to the administration, medical staff, and laboratory personnel of Al-Hakeem Teaching Hospital (Najaf, Iraq) for their generous assistance, technical support, and facilitation of clinical specimen collection throughout this study.

All authors contributed significantly to the conception, study design, collection of clinical specimens, laboratory investigations, data analysis, and manuscript preparation. All authors critically reviewed, revised, and approved the final manuscript.

None.

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

The study protocol was approved by the Institutional Review Board and Ethics Committee of Al-Hakeem Teaching Hospital, Najaf Health Directorate, Iraq (IRB Approval No. 3668, approved on January 30, 2025). The requirement for informed consent was waived by the committee because the research involved routine diagnostic clinical specimens and did not disclose any personal patient-identifying information.

Not applicable.

During the preparation of this manuscript, the authors used artificial intelligence assistance solely for language editing, grammatical correction, and scientific style refinement to improve the readability and clarity of the paper. The authors thoroughly reviewed and edited the resulting text and take full responsibility for the integrity and accuracy of the content of the published article.

The authors declare that they have no competing interests.