Andrea Cellini

Protease treatment affects both invasion ability and biofilm formation in Listeria monocytogenes

Listeria monocytogenes is a notably invasive bacterium associated with life-threatening food-borne disease in humans. Several surface proteins have been shown to be essential in the adhesion of L. monocytogenes, and in the subsequent invasion of phagocytes. Because the control of the invasion of host cells by Listeria could potentially hinder its spread in the infected host, we have examined the effects of a protease treatment on the ability of L. monocytogenes to form biofilms and to invade tissues. We have chosen serratiopeptidase (SPEP), an extracellular metalloprotease produced by Serratia marcescens that is already widely used as an anti-inflammatory agent, and has been shown to modulate adhesin expression and to induce antibiotic sensitivity in other bacteria. Treatment of L. monocytogenes with sublethal concentrations of SPEP reduced their ability to form biofilms and to invade host cells. Zymograms of the treated cells revealed that Ami4b autolysin, internalinB, and ActA were sharply reduced. These cell-surface proteins are known to function as ligands in the interaction between these bacteria and their host cells, and our data suggest that treatment with this natural enzyme may provide a useful tool in the prevention of the initial adhesion of L. monocytogenes to the human gut.

Bacterial biofilm formation inhibitory activity revealed for plant derived natural compounds

Use of herbal plant remedies to treat infectious diseases is a common practice in many countries in traditional and alternative medicine. However to date there are only few antimicrobial agents derived from botanics. Based on microbiological screening tests of crude plant extracts we identified four compounds derived from Krameria, Aesculus hippocastanum and Chelidonium majus that showed a potentially interesting antimicrobial activity. In this work we present an in depth characterization of the inhibition activity of these pure compounds on the formation of biofilm of Staphylococcus aureus as well as of Staphylococcus epidermidis strains. We show that two of these compounds possess interesting potential to become active principles of new drugs.

A new anti-infective strategy to reduce adhesion-mediated virulence in Staphylococcus aureus affecting surface proteins.

Staphylococcus aureus is a flexible microbial pathogen frequently isolated from community-acquired and nosocomial infections. The use of indwelling medical devices is associated with a significant risk of infection by this bacterium 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 present study evaluates the anti-infective properties of serratiopeptidase, a secreted protein of Serratia marcescens, in impairing virulence-related staphylococcal properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. SPEP seems to exert its action by modulating specific proteins. Proteomic studies performed on surface proteins extracted from SPEP-treated S. aureus cultures revealed that a number of proteins are affected by the treatment. Among these we found the adhesin/autolysin Atl, FnBP-A, SecA1, Sbi, EF-Tu, EF-G, and alpha-enolase. EF-Tu, EF-G and alpha-enolase are known to perform a variety of functions, depending on their cytoplasmic or surface localization. All these factors can facilitate bacterial colonization, persistence and invasion of host tissues. Our results suggest that SPEP could be developed as a potential “anti-infective agent” capable to hinder the entry of S. aureus into human tissues, and also impair the ability of this pathogen to form biofilm on prostheses, catheters and medical devices.

Anti-biofilm activity of pseudoalteromonas haloplanktis tac125 against staphylococcus epidermidis biofilm: Evidence of a signal molecule involvement?

Staphylococcus epidermidis is recognized as cause of biofilm-associated infections and interest in the development of new approaches for S. epidermidis biofilm treatment has increased. In a previous paper we reported that the supernatant of Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 presents an anti-biofilm activity against S. epidermidis and preliminary physico-chemical characterization of the supernatant suggested that this activity is due to a polysaccharide. In this work we further investigated the chemical nature of the anti-biofilm P. haloplanktis TAC125 molecule. The production of the molecule was evaluated in different conditions, and reported data demonstrated that it is produced in all P. haloplanktis TAC125 biofilm growth stages, also in minimal medium and at different temperatures. By using a surface coating assay, the surfactant nature of the anti-biofilm compound was excluded. Moreover, a purification procedure was set up and the analysis of an enriched fraction demonstrated that the anti-biofilm activity is not due to a polysaccharide molecule but that it is due to small hydrophobic molecules that likely work as signal. The enriched fraction was also used to evaluate the effect on S. epidermidis biofilm formation in dynamic condition by BioFlux system.

Holo and apo-transferrins interfere with adherence to abiotic surfaces and with adhesion/invasion to HeLa cells in Staphylococcus spp.

Staphylococcus aureus and Staphylococcus epidermidis are the major cause of infections associated with implanted medical devices. Colonization on abiotic and biotic surfaces is often sustained by biofilm forming strains. Human natural defenses can interfere with this virulence factor. We investigated the effect of human apo-transferrin (apo-Tf, the iron-free form of transferrin, Tf) and holo-transferrin (holo-Tf, the iron-saturated form) on biofilm formation by CA-MRSA S. aureus USA300 type (ST8-IV) and S. epidermidis (a clinical isolate and ATCC 35984 strain). Furthermore S. aureus adhesion and invasion assays were performed in a eukaryotic cell line. A strong reduction in biofilm formation with both Tfs was obtained albeit at very different concentrations. In particular, the reduction in biofilm formation was higher with apo-Tf rather than obtained with holo-Tf. Furthermore, while S. aureus adhesion to eukaryotic cells was not appreciably affected, their invasion was highly inhibited in the presence of holo-Tf, and partially inhibited by the apo form. Our results suggest that Tfs could be used as antibacterial adjuvant therapy in infection sustained by staphylococci to strongly reduce their virulence related to adhesion and cellular invasion.

Evaluation of contact lens multipurpose solutions on bacterial biofilm development.

Objectives: No sooner are contact lenses (CLs) inserted into the eyes than lipids, proteins, and glycoproteins rapidly accumulate on their surface, thus favoring the adhesion of commensal bacteria and biofilm formation. Infections may be caused by the proliferation of indigenous flora or other opportunistic pathogens. Our purpose was to evaluate the activity and the capacity of different CL solutions to interfere with the mechanisms of biofilm formation and stability and use of a system to study dynamically biofilm development. Methods: We evaluated the antibiofilm activity of three different multipurpose solutions (MPSs): Regard, Biotrue, and OPTI-FREE PureMoist on four bacterial species (Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus). Static biofilm assay was first performed to analyze the effect of MPSs. Dynamic assays were performed with the BioFlux system to analyze the effect of the OxyChlorite solution Regard on the biofilm formation. Results: Our studies show that MPSs are able to completely inhibit biofilm formation of Staphylococcus species and of S. marcescens after only 4 hr of incubation. Moreover, a reduction of biofilm formation by Pseudomonas was noted. Best results on P. aeruginosa were obtained with Regard. Regard was also used for dynamic assay, revealing its ability to disaggregate the mature biofilm. Regard completely inhibited biofilm formation by S. epidermidis and slowed down biofilm development by P. aeruginosa. Conclusions: Our findings indicate that the CL solutions tested were all able to reduce biofilm formation. Furthermore, the BioFlux system was proven to be useful for the evaluation of the effectiveness of CL solutions against microbial biofilm formation.

Comparison of anti-bacterial prophylactic properties of two different vascular grafts: action of anti-bacterial graft coating and systemic antibiotic treatment.

The main problem associated with artificial vascular devices is the risk of bacterial infections, mostly sustained by coagulase negative staphylococci or Staphylococcus aureus. Many efforts have been made to identify materials refractory to bacterial adhesion. The aim of our study is to verify the antimicrobial properties of two kinds of vascular prosthesis to prevent early onset infections and the efficacy of the concomitant action of a systemic antibiotic treatment. Adult male Wistar rats were used. We subcutaneously implanted in four groups a silver-coated prosthesis fragment, and a rifampicin-soaked prosthesis fragment in the remaining four groups. We inoculated in the site of implant a high bacterial burden of S. aureus in four groups and a low burden in the remaining groups. Systemic levofloxacin was administered for seven days in four groups representing the two kinds of prosthesis; after 21 days the rats were sacrificed, prosthesis fragments were sonicated and the corresponding supernatants were plated for bacterial counts. The rifampicin-soaked prostheses explanted from rats treated with levofloxacin were sterile, regardless of the bacterial inoculum. In other groups some prostheses were colonized. In the experimental rat model used, the action of local and systemic antibiotic treatment was able to reduce colonisation of artificial prostheses.

Effect of betamethasone in combination with antibiotics on gram positive and gram negative bacteria.

Betamethasone is an anti-inflammatory steroid drug used in cases of anaphylactic and allergic reactions, of Alzheimer and Addison diseases and in soft tissue injuries. It modulates gene expression for anti-inflammatory activity suppressing the immune system response. This latter effect might decrease the effectiveness of immune system response against microbial infections. Corticosteroids, in fact, mask some symptoms of infection and during their use superimposed infections may occur. Thus, the use of glucocorticoids in patients with sepsis remains extremely controversial. In this study we analyzed the in vitro effect of a commercial formulation of betamethasone (Bentelan) on several Gram positive and Gram negative bacteria of clinical relevance. It was found to be an inhibitor of the growth of most of the strains examined. Also the effect of betamethasone in combination with some classes of antibiotics was evaluated. Antibiotic-steroid combination therapy is, in such cases, superior to antibiotic-alone treatment to impair bacterial growths. Such effect was essentially not at all observable on Staphylococcus aureus or Coagulase Negative Staphylococci (CoNS).

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.

Adhesive behaviour and virulence of coagulase negative staphylococci isolated from Italian cheeses.

Coagulase-negative staphylococci (CoNS) belong to saprophytic microbiota on the skin and mucous membranes of warm-blooded animals and humans, but are also isolated from foodstuffs such as meat, cheese, and milk. In other circumstances, some CoNS can act as pathogens. Thus the presence of CoNS may not be an immediate danger to public health, but can become a risk factor. In particular antibiotic-resistant genes could be transferred to other potentially pathogenic microorganisms. Furthermore, CoNS are known to be strong biofilm producers and this is also a risk factor for public health. The aim of the present work was to determine the genotypic and phenotypic profiles of 106 CoNS belonging to four different species isolated from five different Italian cheeses for the presence of some adhesion and virulence features. In order to verify a possible correlation between the formation of biofilm and staphylococcal virulence factors, we checked the presence of adhesin genes by PCR and we investigated the ability of these strains to make biofilm at different temperatures. Furthermore, in some conditions, we analyzed surface proteins and autolytic pattern of selected strains. In conclusion, we checked the presence of norA and mecA genes responsible for fluoroquinolones and methicillin resistance, respectively. We found resistant genes in a proportion of the food isolates in amounts of 9.4% (mecA) and 5.7% (norA). These data support the importance to continuously examine the microbiota not only for the creation of a database but also to safeguard public health.