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Pathogenicity Factors of Staphylococcus Pettenkoferi in Foot Wounds and Osteitis in Diabetic Patients
Sponsor: Centre Hospitalier Universitaire de Nīmes
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.
Official title: Evaluation of Pathogenicity Factors of Staphylococcus Pettenkoferi in Foot Wounds and Osteitis in Diabetic Patients
Key Details
Gender
All
Age Range
Any - Any
Study Type
OBSERVATIONAL
Enrollment
230
Start Date
2024-01-01
Completion Date
2026-05-01
Last Updated
2025-11-17
Healthy Volunteers
No
Conditions
Interventions
Molecular epidemiology of the collection
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
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
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
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
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
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.
Locations (1)
Nîmes University Hospital
Nîmes, Gard, France