Increasing antibiotic resistance of methicillin-resistant (MRSA), as found in the well-characterized USA 300 LAC strain, renders MRSA strains difficult to treat and may result in high mortality rates [2, 4]. necrotizing fasciitis, and pneumonia [1C3]. Increasing antibiotic resistance of methicillin-resistant (MRSA), as found in the well-characterized USA 300 LAC strain, renders MRSA strains difficult to treat and may result in high mortality rates [2, 4]. Vancomycin is used for empiric treatment [5] but displays various problems, such as slow bactericidal activity, poor tissue penetration, and no activity against bacterial virulence factors [6C8]. Moreover, vancomycin-resistant strains have developed [9C10]. Thus, additional treatment strategies are necessary to improve clinical response and to reduce further antibiotic resistance development. Ideally, these additive therapeutic strategies should target virulence directly thereby disarming the bacteria and rendering them more susceptible to host innate immune defenses. Efficient colonization and avoidance of host defense after invasion are facilitated by numerous staphylococcal virulence factors, including proteases, lipases, hyaluronidase, collagenase, as well as deoxyribonuclease (nuclease) [11C13]. Two active thermostable nucleases (Nuc1 and Nuc2), encoded by different open reading frames, are present in the genome [14]. The transcript levels are at their maximum during the postexponential growth phase, whereas those of are high during the early exponential phase and decline afterward [15]. Nuclease activity measurements showed a strong correlation with the transcription levels suggesting Nuc1 to be the major nuclease [15]. Furthermore, a recent study showed that Nuc1 transcription and production is regulated in vitro and in Metiamide vivo by the 2-component system SaeRS [16]. Production of Nuc1 is conserved across methicillin-susceptible and Metiamide MRSA strains and is therefore ST6GAL1 used as a unique marker to distinguish from other staphylococcal species [17C18]. It has been shown that the expression and secretion of nucleases by various bacterial species allows bacteria to evade killing by the innate immune system through degradation of neutrophil extracellular traps (NETs) [19C21]. NETs consist of extracellular DNA fibers, histones, antimicrobial peptides and cell-specific proteases designed to entrap and kill various microbes [22]. In addition to NETs evasion streptococcal nucleases were shown to allow bacteria to circumvent Toll-like receptor 9Cmediated recognition via degradation of unmethylated CpG-rich bacterial DNA [23]. Various nuclease activity detection methods have been developed, such as gel electrophoresis, enzyme-linked immunosorbent assays, or filter binding. However, they all have some disadvantages, such as being time consuming or discontinuous or requiring handling of radioactive material. To overcome such obstacles, fluorescence-based assays have been developed using DNA or RNA target molecules labeled with a fluorophore and a quencher. In the uncleaved state, Metiamide the quencher is in spatial proximity to the fluorophore. On cleavage of the target molecule, the quencher is no longer in the vicinity of the fluorophore, and an increase in fluorescence can be measured [24C29]. Owing to its excellent bioavailability, the protein synthesis inhibitor clindamycin is commonly used in clinics, in particular for skin Metiamide and soft-tissue infections. However, owing to its bacteriostatic action, clindamycin is not the treatment of choice for bacteremia. Clindamycin at low concentrations was shown to inhibit toxic-shock syndrome exotoxin production [30]. Transcription of most exoproteins is consistently inhibited. Levels of messenger RNA (mRNA) are affected by protein inhibitors, most likely via inhibition of global regulators [31]. For example, -hemolysin expression is impaired Metiamide by clindamycin [32], paralleled by decreased mRNA expression [33]. In addition to antibiotics, immunoglobulin may be used in severe staphylococcal infections as an adjunct treatment. Immunoglobulin contains antibodies capable of neutralizing and/or binding exoproteins of [34C35]. We hypothesized that clindamycin as well as immunoglobulin could be used to treat infections, aiming to attenuate staphylococcal virulence factors and thus rendering the bacteria more susceptible to host clearance. We present a simple-to-perform assay to quantify nuclease activity and show that both clindamycin and immunoglobulin efficiently blocked staphylococcal Nuc1. This.