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MS2-Affinity Purification Coupled with RNA Sequencing in Gram-Positive Bacteria
Published on: February 23, 2021
Comparative RNA-seq-Based Transcriptome Analysis of the Virulence Characteristics of Methicillin-Resistant and
Natacha Couto1, Adriana Belas1, Manuela Oliveira1
1CIISA, Faculdade de Medicina Veterinária, Universidade de Lisboa (FMV-UL), Lisbon, Portugal.
Abstract:
Staphylococcus pseudintermedius is often associated with pyoderma, which can turn into a life-threatening disease. The dissemination of highly resistant isolates has occurred in the last 10 years and has challenged antimicrobial treatment of these infections considerably. We have compared the carriage of virulence genes and biofilm formation between methicillin-resistant and methicillin-susceptible S. pseudintermedius (MRSP and MSSP, respectively) isolates and their in vitro gene expression profiles by transcriptome sequencing (RNA-seq). Isolates were relatively unevenly distributed among the four agr groups, and agr type III predominated in MRSP. Five virulence genes were detected in all isolates. Only the spsO gene was significantly associated with MSSP isolates (P = 0.04). All isolates produced biofilm in brain heart infusion broth (BHIB)-4% NaCl. MSSP isolates produced more biofilm on BHIB and BHIB-1% glucose media than MRSP isolates (P = 0.03 and P = 0.02, respectively). Virulence genes encoding surface proteins and toxins (spsA, spsB, spsD, spsK, spsL, spsN, nucC, coa, and luk-I) and also prophage genes (encoding phage capsid protein, phage infection protein, two phage portal proteins, and a phage-like protein) were highly expressed in the MRSP isolate (compared with the MSSP isolate), suggesting they may play a role in the rapid and widespread dissemination of MRSP. This study indicates that MRSP may upregulate surface proteins, which may increase the adherence of MRSP isolates (especially sequence type 71 [ST71]) to corneocytes. MSSP isolates may have an increased ability to form biofilm under acidic circumstances, through upregulation of the entire arc operon. Complete understanding of S. pseudintermedius pathogenesis and host-pathogen signal interaction during infections is critical for the treatment and prevention of S. pseudintermedius infections.
Insights
Methicillin-resistant Staphylococcus pseudintermedius (MRSP) shows increased expression of virulence genes, aiding its spread. Methicillin-susceptible S. pseudintermedius (MSSP) forms more biofilm, especially under acidic conditions.
Area of Science:
- Veterinary Microbiology
- Infectious Diseases
- Antimicrobial Resistance
Background:
- Staphylococcus pseudintermedius causes pyoderma, potentially leading to severe infections.
- The rise of methicillin-resistant S. pseudintermedius (MRSP) poses significant challenges to antimicrobial treatment.
- Understanding virulence factors and biofilm formation is crucial for managing S. pseudintermedius infections.
Purpose of the Study:
- To compare virulence gene carriage and biofilm formation between MRSP and methicillin-susceptible S. pseudintermedius (MSSP) isolates.
- To analyze the in vitro gene expression profiles of MRSP and MSSP using transcriptome sequencing (RNA-seq).
- To elucidate the mechanisms behind the dissemination and pathogenesis of MRSP.
Main Methods:
- Comparative analysis of virulence gene profiles in MRSP and MSSP isolates.
- Assessment of biofilm formation capacity in various media conditions.
- Transcriptome sequencing (RNA-seq) to compare gene expression between MRSP and MSSP.
- Statistical analysis to identify significant associations (e.g., P-values).
Main Results:
- MRSP isolates showed higher expression of virulence genes (surface proteins, toxins, prophage genes) compared to MSSP.
- MSSP isolates exhibited enhanced biofilm formation in specific media (BHIB, BHIB-1% glucose) and under acidic conditions (arc operon upregulation).
- The spsO gene was significantly associated with MSSP isolates.
Conclusions:
- MRSP may disseminate rapidly due to upregulated surface proteins enhancing adherence to host cells.
- MSSP may possess a greater capacity for biofilm formation under specific environmental conditions.
- Further research into S. pseudintermedius pathogenesis and host-pathogen interactions is vital for effective treatment and prevention strategies.
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