Serratiopeptidase reduces the invasion of osteoblasts by Staphylococcus aureus

Finding new strategies to counteract periprosthetic infection and implant failure is a main target in orthopedics. Staphylococcus aureus, the leading etiologic agent of orthopedic implant infections, is able to enter and kill osteoblasts, to stimulate pro-inflammatory chemokine secretion, to recruit osteoclasts, and to cause inflammatory osteolysis. Moreover, by entering eukaryotic cells, staphylococci hide from the host immune defenses and shelter from the extracellular antibiotics. Thus, infection persists, inflammation thrives, and a highly destructive osteomyelitis occurs around the implant. The ability of serratiopeptidase (SPEP), a metalloprotease by Serratia marcescens, to control S. aureus invasion of osteoblastic MG-63 cells and pro-inflammatory chemokine MCP-1 secretion was evaluated. Human osteoblast cells were infected with staphylococcal strains in the presence and in the absence of SPEP. Cell proliferation and cell viability were also evaluated. The release of pro-inflammatory chemokine MCP-1 was evaluated after the exposure of the osteoblast cells to staphylococcal strains. The significance of the differences in the results of each test and the relative control values was determined with Student’s t-test. SPEP impairs their invasiveness into osteoblasts, without affecting the viability and proliferation of bone cells, and tones down their production of MCP-1. We recognize SPEP as a potential tool against S. aureus bone infection and destruction

Serratiopeptidase: a well-known metalloprotease with a new non-proteolytic activity against S. aureus biofilm

Background The use of indwelling medical devices is associated with a significant risk of infections by Staphylococcus aureus (S. aureus) which possesses a variety of virulence factors including many toxins and the ability to invade eukaryotic cells or to form biofilm on biotic and abiotic surfaces. The virulence factors above described are often related to proteins exposed on the bacterial surface. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases. Previously reports evaluated the anti-infective properties of serratiopeptidase (Spep), an extracellular metalloprotease produced by Serratia marcescens ATCC 21074 (E-15), in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. However, to date its mechanism of action is unknown. Methods Spep gene was PCR amplified and cloned into expression vector pET28b(+). The mutant EspepA was constructed from plasmid pET28b-Spep applying the one-step overlap extension PCR strategy. There sulting plasmids were costransformed in EcBL21(DE3) cells with the plasmid pRuW4inh1 harboring the Erwinia chrysanthemi secretion system. Bacterial pellets and supernatants were collected and analyzed by SDS-PAGE and zymography. The unambiguous identification and a detailed structure characterization of both the wild type and the mutant Spep were obtained by mass spectrometric analyses. The resultant supernatants sterilized by filtration were separately used to condition biofilm formation of S. aureus. Quantification was based on crystal violet method. Results In this work we constructed Spep mutant by substituting the glutamic acid in the catalytic site with a residue of alanine. In this manner we were able to evaluate the anti-biofilm activity of Spep mutant in absence of proteolytic activity. As expected, this mutant did not display protease activity but it retained its anti-biofilm properties, suggesting that this action is independent by enzymatic activity. Conclusions New knowledge obtained from data reported in this paper calls attention to a novel mechanism of action of Spep. This protein could be developed as a potential “antipathogenic agent” capable to impair the ability of S. aureus to form biofilm on prostheses, catheters and medical devices, exploiting a mechanism different from the proteolytic activity

Ultrasound affects minimal inhibitory concentration of ampicillin against methicillin resistant Staphylococcus aureus USA300

Antimicrobial resistance is one of the most serious global public health problems. Therefore, novel strategies are needed to counteract bacterial resistance development. The aim of the present study was to enhance the activity of antibiotics to bacteria by using ultrasound. Ultrasound reduced the dosage of ampicillin required to impair bacterial viability

Virulence of MRSA USA300 is enhanced by sub-inhibitory concentration of two different classes of antibiotics.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 is responsible of many kinds of infections of skin and soft-tissue. Antibiotic resistance, biofilm formation and the ability to adhere and invade are virulence factors that contribute to MRSA pathogenesis. In some cases, decreased bioavailability of antibiotics in systemic circulation could result; in these conditions sub-therapeutic levels of the antibiotics may be established, exposing bacteria to sub-inhibitory concentrations. On the basis of several published scientific data it is fair to assume that all these events could induce an increase of bacterial virulence. In the present study, we investigated this process by measuring the effects of low doses of two different classes of antibiotics on some virulence features of MRSA USA300 isolate, like the ability to adhere and invade eukaryotic cells. Results obtained strongly support the importance of the respect of a correct dosage of antibiotic in therapy to escape the insurgence of more virulent phenotypes