Dr. Martin Gencik, FA für Medizinische Genetik
Brünnlbadgasse 15, 1090 Wien
Tel: 01/95 80 164, Fax: 01/89 07 943
e-mail: mikrobiom@medgene.at, Web: mikrobiom.medgene.at
RESULT WOUND MICROBIOME
Indication
ICD-10-Code L98. 499 for non-pressure-related chronic ulcers of the skin

Diagnosis
Woundinfection with Fusobacterium necrophorum (67% of the total microbiome). Wohlfahrtiimonas chitiniclastica (9%) infects wounds by transmission with fly larvae.

Results
Basic parameter of the microbioms
Measured values of the properties of the bacterial population.

Wound bed microbiome basic parameter
Microbiome resilience (Shannon diversity): good

Resilience = 4.2

The species diversity is in the range of 75 % of the reference samples.
Shannon-diversity compared to the reference population (white line = 50th percentile). The black bar/arrow shows the value for this sample. 75 % of the healthy samples fall within the green range. A minority of the reference samples have values in the yellow/red range. Shannon alpha diversity is a statistical, quantitative value that combines the number of bacterial sequences detected (richness) and the evenness of their distribution. A higher value indicates more, well-represented bacteria, which is generally associated with a healthy microbiome. All measurements for a sample are compared to the total population.
Microbiome species richness: good
Species richness = 23.0
The species richness was in the range of 75 % of the reference samples.
Microbiome species evenness good
Species evenness = 0.5
The species similarity was in the range of 75 % of the reference samples.
Number of species compared to the reference population (white line = 50th percentile). The black bar/arrow shows the value for this sample. 75 % of the healthy samples fall into the green range. A minority of the reference samples have values in the yellow/red range. Healthier commensal species are associated with a more robust microbial population. The measurements of the individual samples are compared with the general population.
Equality of species compared to the population (white line = 50th percentile). The black bar/arrow shows the value for this sample. 75 % of the population falls into the green range. A minority of the samples have values in the yellow/red range. A more even representation of commensal species is associated with healthier biodiversity. The measured values of the individual samples are compared with the general population.
Dr. Martin Gencik, FA für Medizinische Genetik
Brünnlbadgasse 15, 1090 Wien
Tel: 01/95 80 164, Fax: 01/89 07 943
e-mail: mikrobiom@medgene.at, Web: mikrobiom.medgene.at
Bacterial strains with potentially pathogenic species
There are only a few groups of bacteria that are associated with healthy skin, including Firmicutes and Bacteriodetes. Under the right circumstances, almost any bacterium can begin to proliferate and lead to infection. Pathogenic germs are particularly common in the Fusobacteriota and Pseudomonadota strains.
Percentage of Fusobacteria in the total microbiome: bad
Fusobacteria = 67.827%
The proportion of Fusobacteria is higher than in 95% of the reference samples.

Percentage of Fusobacteria compared to the reference population. The black bar/arrow shows the value for this sample. Healthier samples generally have lower Fusobacteria levels. Values in the yellow/red area are higher than 75% or more of the population. The phylum Fusobacteria contains several known human pathogens. Certain Fusobacteria can promote inflammation and contribute to infections and diseases. The individual areas are based on the reference population.
Types of Fusobacteria in the swab sample
The Fusobacteria strain contains several known human pathogens. Certain Fusobacteria can promote inflammation and contribute to infections and diseases. The individual areas are based on the reference population.
Dr. Martin Gencik, FA für Medizinische Genetik
Brünnlbadgasse 15, 1090 Wien
Tel: 01/95 80 164, Fax: 01/89 07 943
e-mail: mikrobiom@medgene.at, Web: mikrobiom.medgene.at
Percentage of Proteobacteria in the total microbiome: good
Proteobacteria = 13.662%
Proteobactera were found in the order of 75 % of the normal population.
Percentage of Proteobacteria compared to the reference poulation (white line = 50. percentile). The black bar/arrow shows the value for this sample. 75 % of the population falls into the green range. A minority of the reference samples have values in the yellow/red range. The phylum Proteobacteria contains several known human pathogens. High levels of Proteobacteria can promote inflammation and contribute to infection and disease.
Types of Proteobacteria in the swab sample
The Proteobacteria strain contains several known human pathogens. High concentrations of proteobacteria can promote inflammation and contribute to infections and diseases.
Dr. Martin Gencik, FA für Medizinische Genetik
Brünnlbadgasse 15, 1090 Wien
Tel: 01/95 80 164, Fax: 01/89 07 943
e-mail: mikrobiom@medgene.at, Web: mikrobiom.medgene.at
Types of bar chart
Relative distribution of bacterial species.
Types Bar chart wound bed swab
Dr. Martin Gencik, FA für Medizinische Genetik
Brünnlbadgasse 15, 1090 Wien
Tel: 01/95 80 164, Fax: 01/89 07 943
e-mail: mikrobiom@medgene.at, Web: mikrobiom.medgene.at
The most frequently detected types of bacteria

Fusobacterium necrophorum: 66.67%
Potentially pathogenic
Fusobacterium necrophorum has been found in pharyngeal abscesses near the tonsils, which can lead to sepsis and/or infection of the jugular vein if left untreated. Further complications may include intestinal and urogenital tract infections. The pathogen can be found in small quantities in healthy people.
Wohlfahrtiimonas chitiniclastica: 9.16%
Potentially pathogenic
A rare zoonotic pathogen associated with bacteraemia and sepsis has been linked to infected wounds and cellulitis following infection with fly larvae.
Bacteroides fragilis: 7.73%
Potentially pathogenic
A rare zoonotic pathogen associated with bacteraemia and sepsis has been linked to infected wounds and cellulitis following infection with fly larvae.
Streptococcus dysgalactiae: 2.31%
Rare bacterium
The significance of Streptococcus dysgalactiae is not precisely defined. It is not considered a common pathogen in wounds.
Peptostreptococcus anaerobius: 1.7%
Probably commensal
Peptostreptococcus anaerobius is an anaerobic bacterium that is part of the normal flora of the human gastrointestinal tract, oral cavity and urogenital tract. It can be involved in anaerobic infections, including skin and soft tissue infections.
Parvimonas micra: 1.67%
Potentially pathogenic
Parvimonas micra is an anaerobic gram-positive bacterium, hich is commonly found in the human oral cavity and gastrointestinal tract. It may be associated with infections, including skin and soft tissue infections, particularly in the context of mixed anaerobic infections.
Dr. Martin Gencik, FA für Medizinische Genetik
Brünnlbadgasse 15, 1090 Wien
Tel: 01/95 80 164, Fax: 01/89 07 943
e-mail: mikrobiom@medgene.at, Web: mikrobiom.medgene.at
Species Sunburst Diagram - Total Microbiome
The colour discs represent the relative representation of the bacterial taxa. The centre ring is the lowest resolution (domain/kingdom), followed by phylum, class, order, family, genus, species, the last outer ring is the individual amplicon sequence variant (ASV) of the species DNA sequences. Note that strains usually have multiple ASVs. The last number in the ASV ring is the percent identity (PID) to the named species. Novel strains usually have a lower PID than known strains. Closely related strains have a PID of more than 98%.
Species Sunburst Diagram- Wound bed
Dr. Martin Gencik, FA für Medizinische Genetik
Brünnlbadgasse 15, 1090 Wien
Tel: 01/95 80 164, Fax: 01/89 07 943
e-mail: mikrobiom@medgene.at, Web: mikrobiom.medgene.at
Species Principal components Spatial diagram - Total microbiome
Each dot represents a different microbiome summarised in several dimensions calculated from its bacterial taxa, which is referred to as principal component analysis. Healthy and unhealthy samples are shown, with more diverse samples tending to be in the centre of the diagram. The colours of the dots refer to the Shannon Diversity Microbiome Resilience. Lighter colours have higher values for microbiome resilience, darker colours have less diversity and uniformity and therefore lower resilience.
PCA dysbiosis Space diagram- wound bed swab sample
Dr. Martin Gencik, FA für Medizinische Genetik
Brünnlbadgasse 15, 1090 Wien
Tel: 01/95 80 164, Fax: 01/89 07 943
e-mail: mikrobiom@medgene.at, Web: mikrobiom.medgene.at
Species table - total microbiome
List of all detected species with relative frequency information
Species table - wound bed swab
Dr. Martin Gencik, FA für Medizinische Genetik
Brünnlbadgasse 15, 1090 Wien
Tel: 01/95 80 164, Fax: 01/89 07 943
e-mail: mikrobiom@medgene.at, Web: mikrobiom.medgene.at
General Information

Interpretation
The analysis of the wound microbiome reveals a marked dominance of Fusobacterium necrophorum (66.67%), accompanied by other relevant bacteria such as Wohlfahrtiimonas chitiniclastica (9.16%), Bacteroides fragilis (7.73%), Streptococcus dysgalactiae (2.31%), Peptostreptococcus anaerobius (1.7%), and Parvimonas micra (1.67%). The results suggest a potentially severe mixed infection involving both anaerobic and facultative anaerobic pathogens.

1. Fusobacterium necrophorum This anaerobic, gram-negative bacterium is known for its association with necrotizing infections and sepsis. It is frequently found in deep soft tissue infections and can cause severe tissue damage due to its virulence factors such as leukotoxins and hemolysins (Bolstad et al., 2022). The high prevalence of F. necrophorum in this wound microbiome suggests a central pathogenic contribution.

2. Wohlfahrtiimonas chitiniclastica This gram-negative species is typically associated with wound infections, particularly in cases mediated by fly larvae. It is often an opportunistic pathogen thriving in necrotic tissue and is linked to polymicrobial infections (Tóth et al., 2021). Its presence may indicate environmental contamination or secondary colonization.

3. Bacteroides fragilis As a member of the Bacteroides group, B. fragilis plays a significant role in mixed infections. This gram-negative anaerobe is known for producing toxins that promote inflammatory responses and its ability to develop antibiotic resistance (Wexler, 2021). Its presence may contribute to the chronic persistence of the infection.

4. Streptococcus dysgalactiae This beta-hemolytic streptococcus belongs to the group of pyogenic bacteria. It is a common cause of soft tissue infections and plays a role in exacerbating tissue inflammation and impairing wound healing. Additionally, it can cause systemic complications through its production of superantigens (Barnham et al., 2020).

5. Peptostreptococcus anaerobius Peptostreptococcus anaerobius is a strictly anaerobic, gram-positive coccus frequently found in chronic wound infections. Its presence indicates a hypoxic environment that provides ideal growth conditions for anaerobic bacteria (Brook, 2022).

6. Parvimonas micra This anaerobic, gram-positive coccus is known for its involvement in mixed infections, particularly in chronic wounds and dental infections. It can delay wound healing through biofilm production and the induction of inflammation (Murphy & Frick, 2023).

Overall Assessment and Clinical Significance The composition of the microbiome suggests a polymicrobial infection predominantly involving anaerobic pathogens. The high prevalence of Fusobacterium necrophorum and Wohlfahrtiimonas chitiniclastica indicates the potential for both tissue damage and systemic complications. The presence of Bacteroides fragilis and other anaerobic cocci points to an established infection that may be difficult to treat due to biofilm formation and resistance mechanisms.

Personalized Supportive Therapies
Diagnostics: Complementary culture and resistance tests are essential to plan specific therapeutic approaches. These tests enable targeted treatment adjustments based on the identified pathogens and their antibiotic resistances.
Therapy: The use of broad-spectrum antibiotics covering anaerobes should be considered, followed by targeted therapy based on test results. Thorough wound cleansing and debridement are necessary to reduce bacterial load and promote healing. Long-term management: Monitoring for signs of systemic complications such as sepsis or osteomyelitis. In addition to conventional treatment methods like antibiotics and surgical debridement, alternative strategies such as probiotic and prebiotic approaches are increasingly being explored. These therapies aim to positively influence the microbiome, displace pathogenic bacteria, and promote healing.

1. Probiotic Therapies: Probiotics contain live microorganisms that can provide health benefits when administered in adequate amounts. Specific bacteria are being studied for wound therapy that exhibit antimicrobial properties or support wound healing.

Lactobacillus species: Various Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus reuteri, inhibit pathogenic germs like Fusobacterium necrophorum through the production of lactic acid and bacteriocins. Additionally, they promote epithelial regeneration and exhibit anti-inflammatory effects (Peral et al., 2020).

Bacillus subtilis: This probiotic organism produces enzymes such as amylases and proteases that can break down biofilms, a common cause of chronic wound infections (Wang et al., 2022).

Topical Probiotics: Probiotic creams or gels containing live bacteria can be applied directly to the wound. Studies show that these applications can reduce infections and accelerate healing (Sharifi-Rad et al., 2021).

2. Prebiotic Therapies: Prebiotic therapies utilize nutrients that stimulate the growth of health-promoting microorganisms. They can be applied locally to wounds or administered systemically.

Inulin and fructooligosaccharides (FOS) promote the growth of beneficial bacteria such as Lactobacillus and Bifidobacterium, which have anti-inflammatory effects and can displace pathogenic germs (Gibson et al., 2021).

Medical honey, such as Manuka honey, has antibacterial properties and supports the colonization of beneficial bacteria. The sugars and enzymes it contains promote a healthy microbiome in the wound (Maddocks et al., 2020).

3. Synbiotic Approaches: Synbiotics combine probiotics and prebiotics to achieve synergistic effects. These can be applied, for example, in the form of topical products containing both live microorganisms and their specific nutrients.

4. Phage-Assisted Probiotic Therapy: The combination of probiotics and bacterium-specific phages that selectively kill pathogenic bacteria is an innovative approach. This strategy can maintain a healthy microbial community while combating infections (Kortright et al., 2020).

5. Additional Experimental Approaches: Microbiome transplantation: Preliminary studies are investigating whether the application of healthy skin microbiome material can help displace pathogenic microorganisms in wounds. Antimicrobial peptides: These can be expressed in probiotic bacteria to specifically eliminate pathogenic microorganisms.

Conclusion: Probiotic and prebiotic approaches offer promising potential for the treatment of wound infections, particularly in chronic or antibiotic-resistant cases. They can restore balance in the wound microbiome, promote healing, and reduce the need for antibiotics. Further clinical studies are required to validate dosages, application methods, and long-term effects.

Literature Personalized Supportive Therapies

Peral, M. C., et al. (2020). Probiotic use in wound healing: A review. Wound Repair and Regeneration.
Wang, Y., et al. (2022). Bacillus subtilis as a biofilm disruptor in wound infections. Frontiers in Microbiology.
Gibson, G. R., et al. (2021). The role of prebiotics in microbiome modulation and wound healing. Advances in Nutrition.
Maddocks, S. E., et al. (2020). Manuka honey and its prebiotic effects in wound healing. Journal of Wound Care.
Kortright, K. E., et al. (2020). Phage therapy in the context of microbiome-based wound treatments. Nature Reviews Microbiology.
Materials
Sterile swab, Essener Kreisel
Localization: Calf
Description: open abrasion wound
Wound infection: redness, warmth, odor
Therapies: mechanical wound treatments, no antibiotics

Methods
Skin swab sample (collected with flocked sampling swabs, BioCat), stabilized with DNA/RNA Shield (ZymoResearch). Transport fluid stabilized for laboratory transport. DNA isolation was performed using a ZymoBIOMICS DNA Miniprep Kit (ZymoResearch). DNA barcoding and library preparation were carried out with the Shoreline Wave StrainID Kit (Intus Biosciences) and the LSK-114 Amplicon Ligation Kit (Oxford Nanopore). The 2.5 kb base pair PCR products were sequenced using a MinION Mk1C and analyzed with the Titan-1 Bioinformatics Pipeline (Intus Biosciences).

Literature
Literature Wound Microbiome

References used.

1. Bolstad, A. I., Jensen, H. B., & Bakken, V. (2022). Pathogenic mechanisms of Fusobacterium necrophorum. Clinical Infectious Diseases.
2. Tóth, E. M., & Márialigeti, K. (2021). The role of Wohlfahrtiimonas chitiniclastica in human infections. BMC Microbiology.
3. Wexler, H. M. (2021). Bacteroides: Biology and Pathogenesis. ASM Press.
4. Barnham, M. R., et al. (2020). Streptococcus dysgalactiae: Emerging pathogen in soft tissue infections. Journal of Clinical Microbiology.
5. Brook, I. (2022). The role of anaerobic bacteria in infections. Anaerobe.
6. Murphy, E. C., & Frick, I. M. (2023). Biofilm and pathogenicity of Parvimonas micra. Current Opinion in Microbiology.

Notes: We would like to point out that the results of molecular genetic tests should always be considered and interpreted in a clinical context.

Dr. med. Martin Gencik Dr. phil. Alfred Schöller

Specialist in Medical Genetics Biologist