BACKGROUND: Long-term treatment with trimethoprim-sulfamethoxazole (SXT) can lead to the formation of small-colony variants (SCVs) of Staphylococcus aureus. However, the mechanism behind SCVs formation remains poorly understood. In this study, we explored the phenotype and omics-based characterization of S. aureus SCVs induced by SXT and shed light on the potential causes of SCV formation.
METHODS: Stable SCVs were obtained by continuously treating S. aureus isolates using 12/238 µg/ml of SXT, characterized by growth kinetics, antibiotic susceptibility testing, and auxotrophism test. Subsequently, a pair of representative strains (SCV and its parental strain) were selected for genomic, transcriptomic and metabolomic analysis.
RESULTS: Three stable S. aureus SCVs were successfully screened and proven to be homologous to their corresponding parental strains. Phenotypic tests showed that all SCVs were non-classical mechanisms associated with impaired utilization of menadione, heme and thymine, and exhibited slower growth and higher antibiotic minimum inhibitory concentrations (MICs), compared to their corresponding parental strains. Genomic data revealed 15 missense mutations in 13 genes in the representative SCV, which were involved in adhesion, intramolecular phosphate transfer on ribose, transport pathways, and phage-encoded proteins. The combination analysis of transcriptome and metabolome identified 35 overlapping pathways possible associated with the phenotype switching of S. aureus. These pathways mainly included changes in metabolism, such as purine metabolism, pyruvate metabolism, amino acid metabolism, and ABC transporters, which could play a crucial role in promoting SCVs development by affecting nucleic acid synthesis and energy metabolism in bacteria.
CONCLUSIONS: This study provides profound insights into the causes of S. aureus SCV formation induced by SXT. The findings may offer valuable clues for developing new strategies to combat S. aureus SCV infections.

Small colony variant (SCV) is a bacterial phenotype closely related to persistent and recurrent infections. SCVs are mutations that occur within bacterial populations, resulting in a change in bacterial morphology and the formation of small colonies. This morphological change may enhance bacterial resistance to antibiotics and contribute to persistent and recurrent infections.
We isolated Klebsiella pneumoniae (KPN) and its SCV from a child with recurrent respiratory tract infections. KPN and SCV were treated with subinhibitory concentrations of antibiotics. growth curves, serum resistance experiments, macrophage phagocytosis experiments and whole genome sequencing were used to characterize KPN and SCV.
After treating KPN and SCV with subinhibitory concentrations of antibiotics, we found that ciprofloxacin induced the SCV transition to the mucoid phenotype. We found that the growth of mucoid Klebsiella pneumoniae was significantly slower than maternal strain and SCV though growth curves. Serum resistance experiments showed that mucoid strains had significantly higher serum resistance compared to maternal strain and SCV. Macrophage phagocytosis experiments revealed that SCV had significantly higher intracellular survival rates compared to maternal strain and mucoid strains. Differential gene analysis of three strains revealed that the mucoid strain contained DNA polymerase V subunit UmuC gene on the plasmid, while the SCV strain had an additional IcmK family IV secretion protein on its plasmid.
Our study showed the SCV of KPN changed to a mucoid colony when exposed to subinhibitory concentrations of ciprofloxacin. The higher resistance of serum of mucoid colonies was possibly related to the UmuC gene, while the increased intracellular survival of SCV may be related to the IcmK family type IV secretion proteins.

OBJECTIVE: This study aimed to explore the role of small colony variants (SCVs) of Staphylococcus aureus in intraosseous invasion and colonization in patients with periprosthetic joint infection (PJI).
METHODS: A PJI diagnosis was made according to the MusculoSkeletal Infection Society (MSIS) for PJI. Bone and tissue samples were collected intraoperatively and the intracellular invasion and intraosseous colonization were detected. Transcriptomics of PJI samples were analyzed and verified by polymerase chain reaction (PCR).
RESULTS: SCVs can be isolated from samples collected from chronic PJIs intraoperatively. Transmission electron microscopy (TEM) and immunofluorescence (IF) showed that there was more S. aureus in bone samples collected from chronic PJIs, but much less in bone samples from acute PJIs, providing a potential mechanism of PJI. Immunofluorescence results showed that SCVs of S. aureus were more likely to invade osteoblasts in vitro. Furthermore, TEM and IF also demonstrated that SCVs of S. aureus were more likely to invade and colonize in vivo. Cluster analysis and principal component analysis (PCA) showed that there were substantial differences in gene expression profiles between chronic and acute PJI. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these differentially expressed genes were enriched to chemokine-related signal pathways. PCR also verified these results.
CONCLUSIONS: Our study has shown that the S. aureus SCVs have a greater ability to invade and colonize in bone, resulting in S. aureus remaining in bone tissues long-term, thus explaining the pathogenesis of chronic PJI.Cite this article: Bone Joint Res 2022;11(12):843-853.

Staphylococcus aureus small colony variants (SCVs) can cause persistent infections. However, the genomes and transcriptomes of S. aureus SCVs remain poorly understood. A pair of isogenic wild-type and SCV methicillin-resistant S. aureus (MRSA) strains (IE1 and IE2, respectively) were isolated from a patient with prosthetic valve infectious endocarditis. The SCV strain IE2 grew more slowly than the wild-type strain, and serum killing and mouse lethality assays revealed that the virulence of SCV strain IE2 was decreased. Whole-genome sequencing of the SCV and wild-type strains revealed 15 mutations in nine genes associated with metabolism, virulence and DNA repair, including serine/threonine-protein kinase PrkC (prkC), glycerol-3-phosphate acyltransferase (plsY), 2-deoxyribose-5-phosphate aldolase (deoC), extracellular adherence protein (eap), iron compound ABC uptake transporter substrate-binding protein (sstD), RecU Holliday junction resolvase (recU), excinuclease ABC subunit B (uvrB), type I restriction-modification system, M subunit (hsdM) and smooth muscle caldesmon. Sequencing of RNA transcripts revealed that expression levels of 321 genes were upregulated and 582 genes were downregulated in SCV strain IE2 compared with IE1. Most of the differentially expressed genes were involved in metabolism. Expression levels of several genes involved in the pathways to which plsY, deoC, eap and sstD belonged were changed, associated with the metabolism and virulence of S. aureus. In conclusion, the reduced growth rate and decreased virulence of MRSA SCV strains may be related to mutations in and downregulation of genes associated with metabolism and virulence, especially plsY, deoC, eap and sstD.