European Journal of Cardiovascular Medicine

Diabetic foot ulcers (DFUs) are one of the most debilitating complications of diabetes mellitus and contribute significantly to patient morbidity and mortality [1]. The hyperglycemic state commonly associated with diabetes compromises vascular, immune, and neurological functions, predisposing individuals to chronic wounds and infections [5]. One of the most concerning developments in the management of DFUs is the increasing prevalence of multidrug-resistant organisms (MDROs), particularly extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, which complicate treatment regimens and worsen patient outcomes [2][6].

These infections are often polymicrobial, with Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, and Proteus species frequently implicated [3]. Resistance among these bacteria has been largely attributed to the production of ESBLs, enzymes that hydrolyze beta-lactam antibiotics, rendering many commonly used antibiotics ineffective [4]. Several risk factors, including prolonged antibiotic use, repeated hospitalizations, and poor glycemic control, contribute to the emergence of ESBL-producing pathogens in DFUs [7]. The presence of biofilm-forming organisms further exacerbates resistance by hindering antibiotic penetration and promoting bacterial survival [8].

Despite the widespread use of phenotypic detection methods such as the Potentiated Disc Diffusion Test, challenges remain due to the possibility of false negatives, especially when multiple resistance mechanisms coexist [1]. Molecular techniques like PCR are more specific but may not always be accessible [2]. The rising prevalence of beta-lactamase-producing bacteria necessitates routine screening and effective infection control practices [3]. This study, therefore, focuses on phenotypic detection of ESBL-producing Proteus species and analysis of their antibiotic resistance profiles, to contribute to better-targeted interventions for DFUs [2].

Objectives

1. To detect Extended Spectrum Beta-lactamase (ESBL)-producing Proteus species isolated from diabetic foot ulcer wounds using phenotypic methods.

Study Design and Setting

A cross-sectional study was conducted over one year (March 1, 2024 – March 31, 2025) at the Department of Microbiology, KAHER J.N. Medical College, Belagavi.

Sample Collection and Size

A total of 27 Enterobacteriaceae isolates were obtained, comprising Proteus mirabilis and Proteus vulgaris.

Inclusion and Exclusion Criteria

·         Inclusion: All wound swabs from diabetic foot ulcers.

·         Exclusion: None.

Laboratory Procedures

The swabs were cultured using standard microbiological techniques. The isolates underwent purity checks and were identified using:

·         Catalase test

·         Hanging drop preparation

·         Sugar fermentation

·         Methyl Red (MR) and Voges-Proskauer (VP) tests

·         Nitrate reduction test

ESBL DETECTION

Phenotypic detection of ESBL was performed using the Potentiated Disc Diffusion Method with Ceftazidime (CAZ) and Ceftazidime/Clavulanic acid (CAC). An increase in the zone of inhibition of ≥5 mm around CAC compared to CAZ indicated ESBL production [10].

Antimicrobial Susceptibility Testing (AST)

AST was conducted using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar, following CLSI (M100, 30th Edition) guidelines [10]. A range of antibiotics was tested, including Ampicillin (AMP), Gentamicin (GEN), Piperacillin-Tazobactam (PTZ), Cefotaxime (CTX), and carbapenems (IMP, MRP), among others.

Proteus isolates from diabetic foot ulcers were taken for ESBL detection. The zone of inhibition for the specific drugs was noted. They are tabulated and all the data is analyzed for detection of ESBL producers, and the effective treatment against them.

Table No 1:  Organisms isolated from Diabetic foot ulcer patients (n=92)

Organism Distribution

Out of the 27 Proteus isolates:

P. mirabilis – 18 (66.66%)

P. vulgaris – 9 (33.33%)

Table No 2: Detection of ESBL production.

ESBL Detection

·         ESBL Producers: 17 (63%)

·         Non-ESBL Producers: 10 (37%)

Table No 3: Antimicrobial susceptibility pattern of various bacterial isolates from Diabetic foot ulcer.

Antibiotic Susceptibility Patterns

·         High Resistance: Ampicillin (AMP), Cefotaxime (CTX), Ciprofloxacin (CIP)

·         Moderate Resistance: Gentamicin (GEN), Cotrimoxazole (COT), Cefepime (CPM)

·         High Sensitivity: Amikacin (AK), Meropenem (MRP), Imipenem (IMP), Piperacillin-Tazobactam (PTZ)

The study findings reveal a high prevalence of ESBL-producing Proteus species in diabetic foot infections [4]. P. mirabilis was the predominant organism isolated, consistent with its known pathogenicity in chronic wounds [9]. The 63% ESBL positivity rate among isolates indicates a serious concern regarding antimicrobial resistance. ESBL production significantly limits the effectiveness of beta-lactam antibiotics, particularly third-generation cephalosporins [2].

Resistance to first-line agents such as Ampicillin and Ciprofloxacin observed in this study mirrors global trends in ESBL epidemiology [6][9]. The observed sensitivity to carbapenems and aminoglycosides is promising but warrants cautious use due to the risk of developing carbapenem-resistant organisms [5]. These findings

emphasize the necessity of targeted therapy based on susceptibility results rather than empirical treatment, especially in patients with chronic or non-healing ulcers [4].

The limitations of phenotypic detection, as noted in prior research, point to the need for molecular diagnostics to confirm and characterize resistance genes such as bla_CTX-M, bla_SHV, and bla_TEM [6]. The data reinforce the need for regular surveillance and the implementation of antimicrobial stewardship programs to prevent the spread of multidrug-resistant organisms [7][8].

This study underscores the growing threat of ESBL-producing Proteus species in diabetic foot ulcers. With a high prevalence of resistance to commonly used antibiotics, including third-generation cephalosporins, early identification of ESBL-producing organisms is critical [2]. The effective agents identified—Amikacin, Meropenem, Imipenem, and Piperacillin-Tazobactam—should be used judiciously [10]. These findings call for the integration of routine ESBL screening in clinical practice and support the implementation of antimicrobial stewardship programs to prevent further resistance development [8][9].

Summary

This study highlights the emergence of ESBL-producing Proteus spp. as a major concern in diabetic foot infections. The results advocate for evidence-based antimicrobial therapy, routine resistance surveillance, and improved infection control practices [18][19]. Addressing this growing challenge requires timely diagnostic interventions and the strategic use of effective antibiotics to prevent severe complications in diabetic patients.

Aknowledgement

I gratefully acknowledge the Department of Microbiology, J.N. Medical College and its faculty for providing the resources and intellectual environment crucial to the completion of this research. Their insightful feedback and encouragement were instrumental in the development of this paper. I would also like to thank Dr. Sheetal U. Harakuni, Prof. & Head Dept. of Microbiology for her continuous guidance and support.

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