Diabetes mellitus poses significant healthcare challenges worldwide, with diabetic foot ulcers (DFUs) being among the most serious complications due to their high risk of infection, poor healing, and potential for amputation [1,2]. Pseudomonas aeruginosa-a ubiquitous, opportunistic, Gram negative rod-thrives in environments ranging from soil and water to hospital settings, especially affecting immunocompromised individuals [3].

Morphologically, P. aeruginosa measures 1.5–3 µm by 0.5–0.8 µm, is motile via a single polar flagellum, and exhibits twitching/swarming motility through type IV pili [1]. Although primarily aerobic, it can grow anaerobically under nitrate-reducing conditions [4]. Its virulence is multifaceted-exotoxins, enzymes, biofilm-forming capacity, and quorum sensing all contribute to its ability to evade immune defences and resist antibiotics, particularly within chronic wounds [2,5].

Clinically, P. aeruginosa is frequently isolated from DFUs, with a global prevalence of ~16.6% and higher rates in Asia (~18.5%) and Africa (~16.3%) versus Western regions (~11.1%) [6,7]. In Pakistan, its prevalence in DFUs ranges from 22.3% to 46.6%, depending on region and study [8-10].

Various intrinsic and acquired resistance mechanisms-like low outer-membrane permeability, efflux pumps (e.g., MexAB-OprM), AmpC β-lactamase hyperproduction, and horizontal gene transfer of ESBL genes (bla_TEM, bla_SHV, bla_OXA) and carbapenemases-have contributed to its multidrug resistance [11-13].

Table 1: Presents Global Prevalence Data.

Worldwide prevalence of Pseudomonas spp. in hospital settings

A novel ESBL gene, bla CTXM GP1, has recently garnered attention. Though classical ESBLs are well studied in P. aeruginosa, GP1 has not been widely investigated in DFU isolates from South Asia. Its presence and expression may contribute to multidrug resistance phenotypes, especially under selective pressure from antibiotic exposure [14-15].

Objectives

This study aims to:

Isolate P. aeruginosa from pus samples of diabetic patients.

Assess antibiotic susceptibility profiles.

Determine the presence of bla CTXM GP1 via PCR.

Sample Collection

From September 2024 to May 2025, 90 pus samples were obtained from diabetic foot ulcer patients at hospitals across Punjab province. Standard aseptic techniques were used, and samples were transported to the lab under recommended conditions.

Microbiological Identification

Samples were cultured on nutrient agar, MacConkey agar, and cetrimide agar. Colony morphology and pigment production were examined, followed by Gram staining and biochemical assays (oxidase, motility, pigment tests) to confirm P. aeruginosa identity.

Antibiotic Susceptibility Testing

The Kirby–Bauer disk diffusion method, per CLSI guidelines, was used to assess susceptibility against cefotaxime (CTX, 30µg), cefoxitin (FOX, 30µg), ciprofloxacin (CIP, 5µg), and imipenem (IMP, 10µg). P. aeruginosa ATCC 27853 served as the quality control strain.

Molecular Detection of Bla CTXM GP1

DNA was extracted using a commercial kit. PCR was performed with primers specific to bla CTXM GP1, under optimized cycling conditions. Products were electrophoresed on a 1.5% agarose gel; the absence of expected bands was determined as negative.

Data Analysis

Data were tabulated and analyzed for prevalence and resistance rates. The PCR results were compared against AST findings to explore correlations.

Isolation Rate

Out of 90 pus samples, P. aeruginosa was confirmed in 45 cases (50%). Identification was validated by colony characteristics, Gram reaction, and biochemical tests.

Antibiotic Resistance Profile

AST revealed that all isolates (100%) were resistant to cefotaxime and cefoxitin. Ciprofloxacin sensitivity was 85%, while imipenem showed 100% efficacy.

Molecular Results

PCR assays for bla CTXM GP1 returned negative for all 45 isolates, indicating absence of the gene.

Summary

aeruginosa isolation: 50% (45/90)

Resistance: CTX and FOX – 100%

Sensitivity: CIP – 85%, IMP – 100%

bla CTXM GP1: not detected in any isolate

This study confirmed a 50% isolation rate of P. aeruginosa from DFUs-higher than some reported studies (˜17–46%) but aligned with regional data [9,10]. The variation reflects differences in sampling, antibiotic exposure, and healthcare settings.

The universal resistance to cefotaxime and cefoxitin aligns with global reports of ESBL- and AmpC-mediated β-lactam resistance, exacerbated by efflux pumps and reduced permeability [12,16]. High ciprofloxacin sensitivity matches regional trends, although fluoroquinolone resistance remains an emerging concern [17]. The sustained imipenem effectiveness suggests carbapenems remain viable first-line agents for severe P. aeruginosa DFIs in Punjab [18-21].

Interestingly, bla CTXM GP1 was not detected in any isolates. Despite ESBL-like resistance phenotypes, the absence of this gene implies that other resistance mechanisms-AmpC hyperexpression, porin modifications, efflux pump overexpression-are the main drivers [22-25]. This highlights the need to broaden molecular investigations beyond classically targeted ESBL genes.

Clinically, these findings suggest imipenem and ciprofloxacin are appropriate empirical therapies in our region. However, the high cephalosporin resistance underscores the need for culture-based susceptibility testing before prescribing [26-30].

Limitations

No screening for other ESBL genes (e.g., bla_TEM, bla_SHV).

Absence of minimum inhibitory concentration (MIC) data for more refined resistance patterns.

Lack of sequencing to confirm genetic variants or regulatory mutations.

Future Directions

Broader surveillance incorporating multiplex PCR for multiple β-lactamase genes, efflux-pump expression analysis, and molecular sequencing would clarify the genetic basis of resistance. Regular AST monitoring and infection control are essential; stewardship programs should be reinforced to curb cephalosporin resistance and prevent carbapenem misuse [31-35].

This study confirms the prominence of P. aeruginosa in DFUs in Punjab, Pakistan (50% prevalence), with universal resistance to cefotaxime and cefoxitin. The lack of bla CTXM GP1 suggests alternative resistance pathways at work. Imipenem and ciprofloxacin remain effective treatments. Implementation of molecular monitoring, targeted AST, and robust antimicrobial stewardship is recommended to manage resistance trends and prevent treatment failures.

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