Abstract
BackgroundAlthough generally known as a human commensal, Staphylococcus epidermidis is also an opportunistic pathogen that can cause nosocomial infections related to foreign body materials and immunicompromized patients. Infections are often caused by multidrug resistant (MDR) lineages that are difficult and costly to treat, and can have a major adverse impact on patients’ quality of life. Heterogeneity is a common phenomenon in both carriage and infection, but present methodology for detection of this is laborious or expensive. In this study, we present a culture-independent method, labelled Epidome, based on an amplicon sequencing-approach to deliver information beyond species level on primary samples and to elucidate clonality, population structure and temporal stability or niche selection of S. epidermidis communities. MethodsBased on an assessment of >800 genes from the S. epidermidis core genome, we identified genes with variable regions, which in combination facilitated the differentiation of phylogenetic clusters observed in silico, and allowed classification down to lineage level. A duplex PCR, combined with an amplicon sequencing protocol, and a downstream analysis pipeline were designed to provide subspecies information from primary samples. Additionally, a probe-based qPCR was designed to provide valuable absolute abundance quantification of S. epidermidis. ResultsA combined laboratory and bioinformatic approach was successfully designed and validated on isolates representing skin commensals and on genomic mock communities to validate primer specificity and reproducibility using technical replicates. The method was furthermore applied to a sample set of primary skin and nasal samples, revealing a high degree of heterogeneity in the S. epidermidis population at both sampling sites with resolution down to distinct lineages. Additionally, the qPCR, with a detection limit of <10 copies/µL, showed a high degree of variation in absolute abundance of S. epidermidis.ConclusionsThe Epidome method is designed for use on primary samples to obtain information on S. epidermidis abundance and diversity beyond species-level to answer important questions regarding the emergence and dissemination of nosocomial lineages, investigating clonality of S. epidermidis communities, population dynamics, and niche selection. Our targeted-sequencing method allows rapid differentiation and identification of clinically important nosocomial lineages in primary low-biomass samples such as skin samples.
Active antimicrobial efflux in Staphylococcus epidermidis: building up of resistance to fluoroquinolones and biocides in a major opportunistic pathogen
