Staphylococcus aureus biofilms are extremely difficult to treat. They provide a protected niche for the bacteria, rendering them highly recalcitrant toward host defenses as well as antibiotic treatment. Bacteria within a biofilm are shielded from the immune system by the formation of an extracellular polymeric matrix, composed of polysaccharides, extracellular DNA (eDNA), and proteins. Many antibiotics do not readily penetrate biofilms, resulting in the presence of subinhibitory concentrations of antibiotics. Here, we show that subinhibitory concentrations of clindamycin triggered a transcriptional stress response in S. aureus via the alternative sigma factor B ( B ) and upregulated the expression of the major biofilm-associated genes atlA, lrgA, agrA, the psm genes, fnbA, and fnbB. Our data suggest that subinhibitory concentrations of clindamycin alter the ability of S. aureus to form biofilms and shift the composition of the biofilm matrix toward higher eDNA content. An understanding of the molecular mechanisms underlying biofilm assembly and dispersal in response to subinhibitory concentrations of clinically relevant antibiotics such as clindamycin is critical to further optimize antibiotic treatment strategies of biofilm-associated S. aureus infections. Staphylococcus aureus is a major cause of both health care-related and community-associated (CA) infections. The Grampositive human-pathogenic bacterium produces and secretes a range of toxins and enzymes leading to acute infections such as bacteremia and skin abscesses (1, 2). In addition, most S. aureus strains are capable of biofilm formation and can persist in host tissues such as the bone, leading to chronic osteomyelitis, or on implanted medical devices such as vascular catheters, vascular grafts, heart valves, and prosthetic joints (3-5). Biofilm-associated infections are extremely difficult to treat, and these chronic or relapsing infections typically require prolonged antibiotic treatment or removal of the device (6-8). Antibiotic resistance of bacteria within a biofilm may result from slow growth, phenotypic heterogeneity, persister cell formation, and inactivation or reduced penetration of the antibiotic (9, 10). Diffusion of the antibiotic through biofilm cell clusters is dependent on the thickness and the composition of the extracellular polymeric matrix (9, 11). The slow transport within biofilms suggests that the bacteria may encounter subinhibitory concentrations of antibiotics. Previous studies have shown that low doses of different antibiotics trigger biofilm formation (12, 13) and lead to dramatic alterations in bacterial gene expression in S. aureus (14).Biofilm formation proceeds in at least three phases: initial attachment, biofilm maturation, and dispersal (15, 16). Initial surface attachment is dependent on bacterial surface molecules such as the S. aureus murein hydrolase AtlA, teichoic acids, and fibronectin-binding proteins (FnBPs) (17)(18)(19)(20). After attachment to the surface, the bacteria multiply and produce the extracellular...
Treatment failure in biofilm-associated bacterial infections is an important healthcare issue. In vitro studies and mouse models suggest that bacteria enter a slow-growing/non-growing state that results in transient tolerance to antibiotics in the absence of a specific resistance mechanism. However, little clinical confirmation of antibiotic tolerant bacteria in patients exists. In this study we investigate a Staphylococcus epidermidis pacemaker-associated endocarditis, in a patient who developed a break-through bacteremia despite taking antibiotics to which the S. epidermidis isolate is fully susceptible in vitro. Characterization of the clinical S. epidermidis isolates reveals in-host evolution over the 16-week infection period, resulting in increased antibiotic tolerance of the entire population due to a prolonged lag time until growth resumption and a reduced growth rate. Furthermore, we observe adaptation towards an increased biofilm formation capacity and genetic diversification of the S. epidermidis isolates within the patient.
Staphylococcus aureus causes chronic and relapsing infections, which may be difficult to treat. So-called small colony variants (SCVs) have been associated with chronic infections and their occurrence has been shown to increase under antibiotic pressure, low pH and intracellular localization. In clinics, S. aureus isolated from invasive infections often show a dysfunction in the accessory gene regulator (agr), a major virulence regulatory system in S. aureus. To assess whether intracellular environment and agr function influence SCV formation, an infection model was established using lung epithelial cells and skin fibroblasts. This allowed analyzing intracellular survival and localization of a panel of S. aureus wild type strains and their isogenic agr knock out mutants as well as a natural dysfunctional agr strain by confocal laser scanning microscopy (CLSM). Furthermore, bacterial colonies were quantified after 1, 3, and 5 days of intracellular survival by time-lapse analysis to determine kinetics of colony appearance and SCV formation. Here, we show that S. aureus strains with an agr knock out predominantly resided in a neutral environment, whereas wild type strains and an agr complemented strain resided in an acidic environment. S. aureus agr mutants derived from an intracellular environment showed a higher percentage of SCVs as compared to their corresponding wild type strains. Neutralizing acidic phagolysosomes with chloroquine resulted in a significant reduction of SCVs in S. aureus wild type strain 6850, but not in its agr mutant indicating a pH dependent formation of SCVs in the wild type strain. The in-depth understanding of the interplay between intracellular persistence, agr function and pH should help to identify new therapeutic options facilitating the treatment of chronic S. aureus infections in the future.
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