Staphylococcus aureus is one of the most common pathogens that infects the airways of patients with cystic fibrosis (CF) and contributes to respiratory failure. Recently, livestock-associated methicillin-resistant S. aureus (LA-MRSA), usually cultured in farm animals, were detected in CF airways. Although some of these strains are able to establish severe infections in humans, there is limited knowledge about the role of LA-MRSA virulence in CF lung disease. To address this issue, we analyzed LA-MRSA, hospital-associated (HA-) MRSA and methicillin-susceptible S. aureus (MSSA) clinical isolates recovered early in the course of airway infection and several years after persistence in this hostile environment from pulmonary specimens of nine CF patients regarding important virulence traits such as their hemolytic activity, biofilm formation, invasion in airway epithelial cells, cytotoxicity, and antibiotic susceptibility. We detected that CF LA-MRSA isolates were resistant to tetracycline, more hemolytic and cytotoxic than HA-MRSA, and more invasive than MSSA. Despite the residence in the animal host, LA-MRSA still represent a serious threat to humans, as such clones possess a virulence potential similar or even higher than that of HA-MRSA. Furthermore, we confirmed that S. aureus individually adapts to the airways of CF patients, which eventually impedes the success of antistaphylococcal therapy of airway infections in CF.

Staphylococcus aureus small-colony variants (SCVs) are associated with chronic, persistent, and relapsing courses of infection and are characterized by slow growth combined with other phenotypic and molecular traits. Although certain mechanisms have been described, the genetic basis of clinical SCVs remains often unknown. Hence, we adapted an episomal tool for rapid identification and investigation of putative SCV phenotype-associated genes via antisense gene silencing based on previously described Tnl0-encoded tet-regulatory elements. Targeting the SCV phenotype-inducing enoyl-acyl-carrier-protein reductase gene (fabI), plasmid pSN1-AS‘fabI’ was generated leading to antisense silencing, which was proven by pronounced growth retardation in liquid cultures, phenotype switch on solid medium, and 200-fold increase of antisense ‘fabI’ expression. A crucial role of TetR repression in effective regulation of the system was demonstrated. Based on the use of anhydrotetracycline as effector, an easy-to-handle one-plasmid setup was set that may be applicable to different S. aureus backgrounds and cell culture studies. However, selection of the appropriate antisense fragment of the target gene remains a critical factor for effectiveness of silencing. This inducible gene expression system may help to identify SCV phenotype-inducing genes, which is prerequisite for the development of new antistaphylococcal agents and future alternative strategies to improve treatment of therapy-refractory SCV-related infections by iatrogenically induced phenotypic switch. Moreover, it can be used as controllable phenotype switcher to examine important aspects of SCV biology in cell culture as well as in vivo.