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Updated: Jun 29, 2025
Development and Assessment of Intracellular Infection Models for Staphylococcus aureus
Published on: January 17, 2025
Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine
Dalida Bivona1, Carmelo Bonomo1, Lorenzo Colombini2
1Medical Molecular Microbiology and Antibiotic Resistance Laboratory (MMARLab), Department of Biomedical and Biotechnological Sciences (BIOMETEC), University of Catania, 95123 Catania, Italy.
Abstract:
Intracellular survival and immune evasion are typical features of staphylococcal infections. USA300 is a major clone of methicillin-resistant S. aureus (MRSA), a community- and hospital-acquired pathogen capable of disseminating throughout the body and evading the immune system. Carnosine is an endogenous dipeptide characterized by antioxidant and anti-inflammatory properties acting on the peripheral (macrophages) and tissue-resident (microglia) immune system. In this work, RAW 264.7 murine macrophages were infected with the USA300 ATCC BAA-1556 S. aureus strain and treated with 20 mM carnosine and/or 32 mg/L erythromycin. Stable small colony variant (SCV) formation on blood agar medium was obtained after 48 h of combined treatment. Whole genome sequencing of the BAA-1556 strain and its stable derivative SCVs when combining Illumina and nanopore technologies revealed three single nucleotide differences, including a nonsense mutation in the shikimate kinase gene aroK. Gene expression analysis showed a significant up-regulation of the uhpt and sdrE genes in the stable SCVs compared with the wild-type, likely involved in adaptation to the intracellular milieu.
Insights
Carnosine and erythromycin treatment induced stable small colony variants (SCVs) in methicillin-resistant Staphylococcus aureus (MRSA) USA300. These SCVs exhibited genetic mutations, including in the aroK gene, and altered gene expression, aiding intracellular survival.
Area of Science:
- Microbiology
- Immunology
- Genetics
Background:
- Staphylococcal infections, particularly from methicillin-resistant Staphylococcus aureus (MRSA) USA300, are characterized by intracellular survival and immune evasion.
- Carnosine, an endogenous dipeptide, possesses antioxidant and anti-inflammatory properties, modulating immune responses in macrophages and microglia.
Purpose of the Study:
- To investigate the effect of carnosine and erythromycin on the formation of stable small colony variants (SCVs) in MRSA USA300.
- To identify genetic and gene expression changes associated with SCV formation under combined treatment.
Main Methods:
- Infection of RAW 264.7 murine macrophages with MRSA USA300.
- Treatment with carnosine and/or erythromycin.
- Whole genome sequencing (Illumina and nanopore) of wild-type and SCV strains.
- Gene expression analysis.
Main Results:
- Combined carnosine and erythromycin treatment induced stable SCV formation in MRSA USA300 after 48 hours.
- Whole genome sequencing identified three single nucleotide differences between wild-type and SCVs, including a nonsense mutation in the aroK gene.
- Gene expression analysis revealed significant up-regulation of uhpt and sdrE genes in SCVs.
Conclusions:
- Carnosine and erythromycin combination therapy can induce stable SCV formation in MRSA USA300.
- The aroK gene mutation and altered expression of uhpt and sdrE may contribute to MRSA adaptation and survival within the intracellular environment.
