Molecular studies of Staphylococcus epidermidis

Abstract

Staphylococcus epidermidis is a conspicuous member of the human microbiome, widely present on human skin. The shift in modern medicine towards invasive procedures has favoured its emergence as a significant nosocomial pathogen, especially in the setting of prosthetic devices. Despite being the most genetically diverse of the staphylococcal species, with 989 S. epidermidis multilocus sequence types characterised thus far, a single lineage, ST2, accounts for most clinical disease. Unfortunately, existing typing schemes lack the sensitivity to offer any epidemiological insights beyond this. Furthermore, the absence of dedicated molecular tools coupled with the genetically intractable nature of S. epidermidis has hindered the study of its molecular genetics, pathogenesis and treatment.

This thesis utilised new genome sequencing technologies to provide a modern perspective on the global molecular epidemiology of S. epidermidis with a focus on antimicrobial resistance. The project began with the functional analysis of the first complete genome of a multidrug resistant ST2 S. epidermidis. Then extended globally, to uncover the previously unrecognised international spread of three multidrug-resistant, hospital-adapted lineages of S. epidermidis (two ST2 and one ST23) that emerged in recent decades. Acquisition of a dual D471E plus I527M RpoB substitution was common to all three lineages and demonstrated to have become fixed in the populations. Analysis of isolates from 96 institutions in 24 countries identified the RpoB D471E plus I527M combination as the most common cause of rifampicin resistance in S. epidermidis, accounting for 86.6% of mutations. Furthermore, these substitutions were found to occur almost exclusively in isolates from the ST2 and ST23 lineages. By breaching lineage-specific DNA methylation restriction modification (RM) barriers, then performing site-specific mutagenesis, these rpoB mutations were shown to confer both rifampicin resistance and reduced susceptibility to the glycopeptide antibiotics vancomycin and teicoplanin. Importantly, these findings suggest that current clinical practice has contributed to the generation of rifampicin and vancomycin resistance and spread of the three multidrug-resistant S. epidermidis lineages, warranting the review of staphylococcal treatment guidelines.

The final work in this thesis is the first comprehensive analyses of the barriers to the uptake of foreign DNA in S. epidermidis. Contrary to current assumptions, analyses revealed that the type I RM systems in S. epidermidis are not lineage specific like Staphylococcus aureus, instead demonstrating considerable diversity even within a single genetic lineage. This diversity can be attributed to marked differences in the gene arrangement, chromosomal location and movement of type I RM systems in S. epidermidis compared to S. aureus. Findings indicated that genetic manipulation of S. epidermidis must be tailored to each strain of interest. Using Escherichia coli plasmid artificial modification to express S. epidermidis hsdMS, the restriction barriers in S. epidermidis were readily overcome, with electroporation efficiencies equivalent to modification-deficient mutants. Through functional experiments, the use of genomic data to predict both the activity of type I RM systems and the potential for a strain to be electroporation proficient were demonstrated, and an efficient approach to the genetic manipulation of any S. epidermidis strain of interest, including those that have hitherto been intractable is presented.

Collectively, the works in this thesis offer new insights into the molecular epidemiology, resistance mechanisms, treatment recommendations and genetic manipulation of S. epidermidis. The method outlined for the genetic manipulation of any given S. epidermidis isolate should prove valuable in assisting future molecular studies and advancing knowledge about this significant nosocomial pathogen.