Pathogenicity Factors of Staphylococcus Pettenkoferi in Foot Wounds and Osteitis in Diabetic Patients

NCT06688084 | Active Not Recruiting

• 1 other identifier: NIMAO/2023-2/CM-01
• Study Type: observational
• Target: 230
• Locations: 1 country
• Sites: 1

Brief Summary

Gram-positive cocci, particularly Staphylococcus aureus and coagulase-negative staphylococci (SCoN), are the bacteria most frequently isolated from diabetic foot ulcers. Although studies have been carried out on the role of S. aureus in the unfavorable evolution of these wounds, no studies have focused on the role of SCoN. Of the fifty or so SCoN species, not all have the same virulence potential. The role of Staphylococcus pettenkoferi is unknown, yet this bacterium is the 7th most frequently identified in diabetic foot ulcers, suggesting that it may also be involved in the pathophysiology of these infections. At Nîmes University Hospital, this bacterium is mainly identified in samples from diabetic foot ulcers or osteitis in our laboratory and 80% of the bacteria present are in biofilms.It is essential to understand the mechanisms governing these bacterial interactions and establish the true pathogenic potential of these bacteria. Recently, the Nîmes team showed that a strain of S. pettenkoferi (SP165) isolated from foot osteitis in a diabetic patient had real virulence potential. SP165 could not only produce biofilm, but could also survive in human blood, human keratinocytes and murine and human macrophages. It also caused significant embryonic mortality in a zebrafish model. A second study of 29 isolates from Nîmes University Hospital subsequently demonstrated that there were two predominant clones with different virulences. Three biofilm production profiles (rapidly and highly biofilm-producing, slowly biofilm-producing and non-biofilm-producing) and two zebrafish profiles (highly and moderately lethal) were reported by phenotypic and genomic analyses on this panel of strains. Genes for resistance, virulence and biofilm production were also found on their genomes.

Conditions

Diabetic Foot Ulcer

Keywords

Diabetes; Foot; Infection; Staphylococcus pettenkoferi; Osteitis

Outcome Measures

Primary Outcomes (1)

• Genetic diversity of Staphyloccocus pettenkoferi strains isolated from osteitis and non-diabetic wounds, blood cultures and nasal carriage.

  Number of clades estimated via typing by complete sequencing of the bacterial genomes of all S. pettenkoferi strains and analysis of phylogenetic distances (whole genome and core genome single nucleotide polymorphisms).

  Day 0 to 3 months

Secondary Outcomes (45)

• Genetic diversity of Staphyloccocus pettenkoferi strains isolated from osteitis and non-diabetic wounds, blood cultures and nasal carriage.

  3 - 6 months

• Resistome in the strain population according to sample origin:osteitis

  3 - 6 months

• Resistome in the strain population according to sample origin: non-diabetic wounds

  3 - 6 months

• Resistome in the strain population according to sample origin: diabetic wounds

  3 - 6 months

• Resistome in the strain population according to sample origin: blood cultures

  3 - 6 months

Interventions

Molecular epidemiology of the collection OTHER

Next-generation genomic sequencing (MICRO\&BIO platform, MiSeq, Illumina) and bioinformatics analyses. After validating data quality, genomes will be re-assembled using Spades software (version 3.15.4) and CLC® (Qiagen), then compared with the National Center for Biotechnology Information GenBank database. They will also be analysed on the online Type-Strain Genomes Server platform to accurately identify the bacteria. Genome annotation will be done using the DDBJ Fast Annotation and Submission Tool. Single nucleotide polymorphisms (SNP) analysis based on the alignment of whole genomes and core-genomes will be carried out using version 7 of MAFFT (multiple alignment using fast Fourier transform) software, then quantified using snp-dists (version 0.6.3). This approach will enable the molecular epidemiology of the collection of strains in France and other European countries to be clarified, and the main circulating clades to be assessed, particularly depending on the origin of samples.

Study of the resistome of the S. pettenkoferi population OTHER

The resistome will be assessed by identifying known and described resistance genes in staphylococci using bioinformatics analyses of sequenced complete bacterial genomes. A search for plasmids will also be made using various specific software packages (ResFinder, CARD, PointFinder, Plasmidfinder, in-house pipeline).

Study of the virulome of the S. pettenkoferi population OTHER

The virulome of the strains will be studied based on the genomic sequencing data obtained and with reference to the literature on Staphylococci. Various specific software packages (Pathogenfinder, Virulencefinder, in-house pipeline) will be used for this purpose.

Study of the resistance profiles of all S. pettenkoferi isolates OTHER

Antibiotic resistance will be assessed using a Sensititer® plate (Thermo ScientificTM, Illkirch) to evaluate the minimum inhibitory concentration of isolates against a series of antibiotics commonly used for S. pettenkoferi infections, including new molecules currently on the market or in development, such as Ceftaroline, Ceftobiprole, Dalbavancin, Delafloxacin, Tedizolid and Oritavancin. Minimum inhibitory concentration values will be interpreted in accordance with the recommendations of the Antibiogram Committee of the Société Française de Microbiologie (https://www.sfm-microbiologie.org/2021/04/23/casfm-avril-2021-v1-0/).

Selection of strains for in-depth phenotypic study OTHER

Different strains of S. pettenkoferi isolated from foot osteitis in diabetic patients and non-diabetic osteitis will be selected according to the clades identified and phenotypically characterised by further analysis: * Observation of culture characteristics (culture requirements, growth time, colony size and haemolytic capacity), (n=20) * Determination of growth curves by measuring absorbance at 600 nm using the Infinite M Mano automatic absorbance reader (TECAN, Lyon), (n=20) * Study of the ability to form biofilm in the presence (antibiofilmogram) (n=85) or absence (BioFilm Ring® test, Biofilm Control, St Beauzire) (n=85) of various antibiotics commonly used for the treatment of S. pettenkoferi infection during osteitis.

Selection of strains (n=3) for the virulence study OTHER

Three strains of S. pettenkoferi isolated from foot osteitis in diabetic patients will be selected on the basis of the clades identified and the study of the virulome in order to be analysed on : * In vitro cell culture models of macrophages (RAW 264.7) and osteoblasts (MC3T3-E1) to assess the invasion potential of these isolates by measuring internalisation rates (TECAN, Lyon) and LDH release (CyQUANT LDH Cytotoxicity kit), a sign of cellular toxicity of the isolate. * An in vivo wound model on diabetic zebrafish (Dario rerio) with evaluation of their survival at 48h in the presence of the isolates by immersion. These models were developed at the MICRO\&BIO Platform and the INSERM 1047 VBIC unit.

Eligibility Criteria

• Sex: all
• Healthy Volunteers: No
• Age Groups: Child (0-17), Adult (18-64), Older Adult (65+)
• Sampling Method: Non-Probability Sample

Study Population

The collection of strains at the Nîmes University Hospital site from 9 European laboratories collected between 2015 and 2023 for a total of 108 months was carried out between November 2021 and August 2023.

You may qualify if:

• Not applicable for this research on a ready-constituted collection of strains of Staphylococcus pettenkoferi

You may not qualify if:

• Not applicable for this research on a ready-constituted collection of strains of Staphylococcus pettenkoferi

Sponsors & Collaborators

• Centre Hospitalier Universitaire de Nīmes: lead
• Staphylococcal National Reference Center, Lyon, France: collaborator
• Rennes University Hospital: collaborator
• Nantes University Hospital: collaborator
• Universität Tübingen: collaborator
• Örebro University, Sweden: collaborator
• Charles University, Czech Republic: collaborator
• University Hospital Muenster: collaborator

Study Sites (1)

Nîmes University Hospital

Nîmes, Gard, 30029, France

Location

Biospecimen

Retention: SAMPLES WITHOUT DNA

This is an international multi-center translational research project, based on a collection of strains (strain library) consisting of 230 S. pettenkoferi isolates collected between 2015 and 2023 from 9 European centres, (Reference 2021U1047-002).

MeSH Terms

Conditions

Diabetic Foot; Diabetes Mellitus; Infections; Osteitis

Condition Hierarchy (Ancestors)

Diabetic Angiopathies; Vascular Diseases; Cardiovascular Diseases; Foot Ulcer; Leg Ulcer; Skin Ulcer; Skin Diseases; Skin and Connective Tissue Diseases; Diabetes Complications; Endocrine System Diseases; Diabetic Neuropathies; Glucose Metabolism Disorders; Metabolic Diseases; Nutritional and Metabolic Diseases; Bone Diseases; Musculoskeletal Diseases

Study Officials

• Chloé MAGNAN, Dr.

  Nîmes University Hospital, FRANCE

  PRINCIPAL INVESTIGATOR

Study Design

• Study Type: observational
• Observational Model: COHORT
• Time Perspective: OTHER
• Target Duration: 2 Years
• Sponsor Type: OTHER
• Responsible Party: SPONSOR

Study Record Dates

• First Submitted: November 9, 2024
• First Posted: November 14, 2024
• Study Start: January 1, 2024
• Primary Completion: December 31, 2025
• Study Completion: May 1, 2026
• Last Updated: November 17, 2025

Record last verified: 2025-11

Locations

FR (1)