Representative Targeted Molecular And Genomic Characterization Of Virulence Genes In Staphylococcus aureus From Diabetic Foot Infections

Summary

This study characterized the mechanisms of antimicrobial resistance, virulence determinants, and genomic features of Staphylococcus aureus isolates recovered from diabetic foot infections using antimicrobial susceptibility testing, PCR-based virulence profiling, and whole-genome sequencing.

Abstract

Diabetic foot infections (DFIs) represent a major public health concern, and methicillin-resistant Staphylococcus aureus (MRSA) is among the most clinically significant pathogens. This study investigated the prevalence of virulence genes (cna and hlg), antimicrobial resistance profiles, and representative genomic features of multidrug-resistant S. aureus isolates recovered from patients with DFIs. A cross-sectional observational study was conducted on 125 patients with diabetic foot infections between January and December 2024. Antimicrobial susceptibility testing was performed using the Kirby–Bauer disk diffusion method and cefoxitin screening according to Clinical and Laboratory Standards Institute (CLSI) guidelines, with S. aureus ATCC 25923 used as the quality-control strain. Vancomycin susceptibility was confirmed by minimum inhibitory concentration (MIC) testing. Polymerase chain reaction (PCR) was used to detect the virulence genes (cna and hlg) and blaOXA-group I genes. Whole-genome sequencing (WGS) was performed on two representative isolates, including one multidrug-resistant (MDR) isolate and one extensively drug-resistant (XDR) isolate, using a de novo sequencing approach to generate draft genome assemblies. Among 125 clinical specimens, bacterial growth was observed in 90 samples (72%), of which 45 isolates (50%) were identified as S. aureus. Among these isolates, 35/45 (77.78%) were classified as MRSA, and 36/45 (80%) were multidrug resistant. The hlg gene was detected in all isolates, whereas the cna gene was identified in 13/45 (28.89%) isolates. No blaOXA-group I genes were detected. Genomic analysis identified multiple resistance-associated genes, including blaZ, tet(38), norA, and vanT, together with CRISPR-Cas elements and plasmid-associated resistance determinants. These findings highlight the high prevalence of multidrug-resistant S. aureus in DFIs and support the importance of continued genomic surveillance of clinically relevant resistant strains.

Introduction

Numerous colonization and virulence characteristics render the gram-positive bacterium Staphylococcus aureus (S. aureus) a prevalent opportunistic pathogen capable of causing a wide range of infections, including impetigo, wound infections, furuncles, skin abscesses, bacteremia, and infective endocarditis¹. Some of these infections may progress to severe disease and require hospitalization, particularly in patients with diabetes mellitus, immunosuppression, malignancy, urinary catheterization, or human immunodeficiency virus infection¹.

Methicillin-resistant S. aureus

Protocol

1. Study Patients

This study was approved by the Ethical Approval Committee of the University of Anbar, Ministry of Higher Education and Scientific Research, Iraq (Approval No. 145), Ramadi City, Anbar Governorate, Iraq, on December 24, 2023. All procedures involving human participants were performed in accordance with the institutional ethical guidelines of the University of Anbar. Written informed consent was obtained from all participants prior to enrollment.

A cross-sectional observational study was conducted from January to December 2024 at Ramadi Teaching Hospitals and affiliated private clin....

Results

Specimen processing and culture positivity

A total of 125 clinical specimens were collected and analyzed in this study. Bacterial growth was observed in 90/125 samples (72%). These culture-positive specimens were subsequently processed for bacterial identification and antimicrobial susceptibility testing. Descriptive statistics, including frequencies and percentages, were used to summarize the distribution of bacterial isolates among all clinical specimens and culture-positive.......

Discussion

Diabetic foot infections (DFIs) caused by multidrug-resistant (MDR) microorganisms are increasing globally, with poor glycemic control and inadequate foot hygiene contributing to increased morbidity and mortality^18,19,20,21. Although DFIs are frequently polymicrobial, Staphylococcus aureus remains one of the predominant pathogens isolated from these infections, with the prevalence of m.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Abricate Software	GitHub (T. Seemann)	Open-source	Virulence gene detection
Agarose	Invitrogen, USA	16500-500	Gel electrophoresis
Agencourt AMPure XP Kit	Beckman Coulter	A63881	DNA purification and size selection
Amikacin (30 µg) Disc	Oxoid, UK	CT0107B	AST
Antibiotic Discs (Ampicillin 20 µg)	Oxoid, UK	CT0003B	Antimicrobial susceptibility testing
Blood Agar Base	Oxoid, UK	CM0055	Isolation of Gram-positive and Gram-negative bacteria
Brain Heart Infusion (BHI) Broth	Oxoid, UK	CM1135	Culturing and storage of isolates
Cefoxitin (30 µg) Disc	Oxoid, UK	CT0119B	MRSA detection
Cefuroxime (30 µg) Disc	Oxoid, UK	CT0026B	AST
Ciprofloxacin (5 µg) Disc	Oxoid, UK	CT0425B	AST
Clindamycin (2 µg) Disc	Oxoid, UK	CT0064B	AST
DNBSEQ Platform	BGI, China	Various	Whole genome sequencing
Erythromycin (15 µg) Disc	Oxoid, UK	CT0020B	AST
FastQC	Simon Andrews at the Babraham Institute (Babraham Bioinformatics)	NA	queueing system
Gel Documentation System	Bio-Rad, USA	Gel Doc™ XR+	Imaging of electrophoresis gels
Gentamicin (10 µg) Disc	Oxoid, UK	CT0024B	AST
Glycerol (Molecular Grade)	Sigma-Aldrich	G5516	Used at 20% for long-term isolate preservation
GoTaq Green Master Mix (2×)	Promega, USA	M7122	PCR amplification mix
Levofloxacin (5 µg) Disc	Oxoid, UK	CT1587B	AST
Linezolid (30 µg) Disc	Oxoid, UK	CT1716B	AST
MacConkey Agar	Oxoid, UK	CM0007	Selective medium for Gram-negative bacteria
Mannitol Salt Agar (MSA)	Oxoid, UK	CM0085	Selective/differential medium for Staphylococcus spp.
Meropenem (10 µg) Disc	Oxoid, UK	CT0774B	AST
MgCl2 Solution	Promega, USA	Various	PCR reaction component
Mueller-Hinton Agar	Oxoid, UK	CM0337	Used for Kirby–Bauer disc diffusion testing
MUSCLE Alignment Tool	Open-source	Various	Protein sequence alignment
Nuclease-Free Water	Promega, USA	P1193	PCR reaction component
Nutrient Agar	Oxoid, UK	CM0003	General-purpose growth medium
Oxacillin (1 µg) Disc	Oxoid, UK	CT0159B	AST
PathogenWatch	Wellcome Sanger Institute	Online Tool	Species ID & AMR gene detection
PATRIC / BV-BRC	Bacterial Bioinformatics Resource Center	Online Platform	Genome assembly & annotation
Piperacillin/Tazobactam (100/10 µg) Disc	Oxoid, UK	CT1628B	AST
Proksee Genome Visualization Tool	University of Toronto	Online Tool	Genome map visualization
Quantus Fluorometer	Promega, USA	E6150	DNA quantification
Qubit Fluorometer	Thermo Fisher Scientific	Q33238	DNA quantification for WGS
RAxML Software	Open-source	Various	Phylogenetic analysis
S. aureus ATCC 25923	ATCC, USA	25923	Quality control strain for AST
Safe Red Stain	iNtRON Biotechnology	Various	DNA gel stain
SaMag Bacterial DNA Extraction Kit	Sacace Biotechnologies, Italy	Various	Genomic DNA extraction
SaMag-12 Automated Extraction System	Sacace Biotechnologies, Italy	SMG-12	Automated nucleic acid extractor
Sterile Cotton Swabs	Various manufacturers	Various	Used for deep swab technique sample collection
Sterile Normal Saline	Various manufacturers	Various	Used for washing ulcer surface before sampling
Sterile Scalpel Blades	Various manufacturers	Various	Used for ulcer debridement prior to sampling
Tigecycline (15 µg) Disc	Oxoid, UK	CT1849B	AST
Trimethoprim-Sulfamethoxazole (25 µg) Disc	Oxoid, UK	CT0052B	AST
UV Transilluminator	Various manufacturers	Various	Visualization of PCR products
Vancomycin (30 µg) Disc	Oxoid, UK	CT0588B	AST
VITEK 2 Compact	BioMérieux, France	Various	Automated identification system
VITEK 2 GN ID Cards	BioMérieux, France	Various	Identification of Gram-negative bacteria