The Effect of Tannin-Rich Witch Hazel on Growth of Probiotic Lactobacillus plantarum.

Probiotic bacteria help maintain microbiome homeostasis and promote gut health. Maintaining the competitive advantage of the probiotics over pathogenic bacteria is a challenge, as they are part of the gut microbiome that is continuously exposed to digestive and nutritional changes and various stressors. Witch hazel that is rich in hamamelitannin (WH, whISOBAXTM) is an inhibitor of growth and virulence of pathogenic bacteria. To test for its effect on probiotic bacteria, WH was tested on the growth and biofilm formation of a commercially available probiotic Lactobacillus plantarum PS128. As these bacteria are aerotolerant, the experiments were carried out aerobically and in nutritionally inadequate/poor (nutrient broth) or adequate/rich (MRS broth) conditions. Interestingly, despite its negative effect on the growth and biofilm formation of pathogenic bacteria such as Staphylococcus epidermidis, WH promotes the growth of the probiotic bacteria in a nutritionally inadequate environment while maintaining their growth under a nutritionally rich environment. In the absence of WH, no significant biofilm is formed on the surfaces tested (polystyrene and alginate), but in the presence of WH, biofilm formation was significantly enhanced. These results indicate that WH may thus be used to enhance the growth and survival of probiotics.

Vaccine development for the prevention of staphylococcal mastitis in dairy cows.

Staphylococci are the most common and costly mammary disease of dairy cattle worldwide. Target of RNAIII Activating Protein (TRAP), a membrane associated 167AA protein, is highly conserved among staphylococci and was shown in Staphylococcus aureus to be involved in bacterial stress response. The aims of this study were to test the safety and efficacy of recombinant TRAP (rTRAP) vaccine in dairy animals. The vaccine was safe as 2-3 subcutaneous injections of rTRAP (54-100 µg) with adjuvant ISA 206 to cows and goats did not lead to any abnormal symptoms of sensitivity to the vaccine. The rTRAP vaccine was immunogenic and caused the induction of a humoral immune response that remained high for at least 160 d post second immunization. The rTRAP vaccine was efficacious; at parturition only 13.5% (5/37) heifers in the immunized group were infected with Staphylococcus chromogenes as compared to 42.9% (18/42) in the non-immunized group. Additionally, when cows were immunized in mid-lactation, the difference between somatic cell count (SCC) in immunized and control animals was profound (45 ± 7 vs. 470 ± 194, respectively). At the same time, the difference in milk yield was also evident (48.3 ± 1.4 L d(-1)vs. 44.3 ± 0.9 L d(-1), respectively). Put together, these studies indicate the value of the rTRAP vaccine in preventing new udder infections by staphylococci, which significantly lead to lowered SCC and some increase in milk yield. TRAP is conserved among all strains and species and is constitutively expressed in any strain of S. aureus or CNS tested so far, including those isolated from cows. TRAP may thus serve as a universal anti-staphylococcus vaccine.

Isolation of agr quorum sensing autoinducers.

Autoregulation of genes is often associated with quorum sensing systems where bacteria produce and secrete molecules that allow the cells to communicate with one another, leading to the activation of certain genes at certain population densities. Here we describe the identification of the agr as a quorum sensing system in Staphylococcus aureus and the isolation of agr autoinducers and inhibitors by northern blotting, real-time RT-PCR, and ß-lactamase reporter cells assays.

Molecular mechanisms of RIP, an effective inhibitor of chronic infections.

Non-healing bacterial infections are often associated with the formation of a biofilm, where bacteria are more resistant to conventional treatment modalities and to host immune responses. We show here that RNAIII inhibiting peptide (RIP), a linear heptapeptide, is very effective in treating severe polymicrobial infections, including drug-resistant staphylococci like MRSA. By functional genomics studies (microarray analysis) on Staphylococcus aureus, we show here that RIP downregulates the expression of genes involved in biofilm formation and toxin production, and upregulates genes involved in stress response. This pattern of gene regulation may explain why RIP has been so effective in treating severe infections and hopefully through the addition of RIP to existing protocols, a new way of tackling chronic persistent infections will be established.

YhgC protects Bacillus anthracis from oxidative stress.

Bacillus anthracis can cause lethal inhalational anthrax and can be used as a bioweapon due to its ability to form spores and to survive under various environmental stress conditions. YhgC in bacilli are structural homologues of TRAP, a protein involved in stress response in staphylococci. To test the role of YhgC in B. anthracis, yhgC gene was deleted in B. anthracis strain Sterne and parent and mutant strains tested. Immunolocalization studies indicated that YhgC is clustered both on the cell surface and within the cytoplasm. Phenotypic analyses indicated that YhgC is an important factor for oxidative stress tolerance and for macrophage infection in vitro. Accordingly, transcriptomics studies indicated that yhgC has a profound effect on genes encoding for stress response regulatory proteins where it negatively regulates the expression of genes encoding for Class I and Class III stress response proteins belonging to the regulons hrcA (hrcA, grpE, dnaK, dnaJ, groEL and groES) and ctsR (ctsR, mcsA, mcsB, clpC/mecB, clpP1). Proteomics studies also indicated that YhgC positively regulates the expression of ClpP-2 and camelysin, which are proteins involved in adaptive responses and pathogenesis in various Gram-positive bacteria. Put together, these results suggest that YhgC is important for the survival of B. anthracis under oxidative stress conditions and thus inhibition of YhgC may compromise the ability of the bacteria to survive within the host.

YhgC protects Bacillus anthracis from oxidative stress.

Bacillus anthracis can cause lethal inhalational anthrax and can be used as a bioweapon due to its ability to form spores and to survive under various environmental stress conditions. YhgC in bacilli are structural homologues of TRAP, a protein involved in stress response in staphylococci. To test the role of YhgC in B. anthracis, yhgC gene was deleted in B. anthracis strain Sterne and parent and mutant strains tested. Immunolocalization studies indicated that YhgC is clustered both on the cell surface and within the cytoplasm. Phenotypic analyses indicated that YhgC is an important factor for oxidative stress tolerance and for macrophage infection in vitro. Accordingly, transcriptomics studies indicated that yhgC has a profound effect on genes encoding for stress response regulatory proteins where it negatively regulates the expression of genes encoding for Class I and Class III stress response proteins belonging to the regulons hrcA (hrcA, grpE, dnaK, dnaJ, groEL and groES) and ctsR (ctsR, mcsA, mcsB, clpC/mecB, clpP1). Proteomics studies also indicated that YhgC positively regulates the expression of ClpP-2 and camelysin, which are proteins involved in adaptive responses and pathogenesis in various Gram-positive bacteria. Put together, these results suggest that YhgC is important for the survival of B. anthracis under oxidative stress conditions and thus inhibition of YhgC may compromise the ability of the bacteria to survive within the host.

Prospecting gene therapy of implant infections.

Infection still represents one of the most serious and ravaging complications associated with prosthetic devices. Staphylococci and enterococci, the bacteria most frequently responsible for orthopedic postsurgical and implant-related infections, express clinically relevant antibiotic resistance. The emergence of antibiotic-resistant bacteria and the slow progress in identifying new classes of antimicrobial agents have encouraged research into novel therapeutic strategies. The adoption of antisense or "antigene" molecules able to silence or knock-out bacterial genes responsible for their virulence is one possible innovative approach. Peptide nucleic acids (PNAs) are potential drug candidates for gene therapy in infections, by silencing a basic gene of bacterial growth or by tackling the antibiotic resistance or virulence factors of a pathogen. An efficacious contrast to bacterial genes should be set up in the first stages of infection in order to prevent colonization of periprosthesis tissues. Genes encoding bacterial factors for adhesion and colonization (biofilm and/or adhesins) would be the best candidates for gene therapy. But after initial enthusiasm for direct antisense knock-out or silencing of essential or virulence bacterial genes, difficulties have emerged; consequently, new approaches are now being attempted. One of these, interference with the regulating system of virulence factors, such as agr, appears particularly promising.

OpuC--an ABC transporter that is associated with Staphylococcus aureus pathogenesis.

RIP is a novel antibiotic against staphylococci. It acts at least in part by competing with RNAIII activating protein (RAP) by downregulating TRAP histidine phosphorylation, and by downregulating the expression of the acessory gene regulator (agr). While much is known about the function of the agr as a quorum sensing system that regulates virulence, not much is known about TRAP. TRAP is a 167-kDa protein that is highly conserved among staphylococci and is involved in DNA protection from stress. TRAP is membrane-associated but does not have a transmembrane domain, and thus it may be bound to the membrane through other proteins. To search for these proteins, protein-protein interaction studies were carried out using a bacterial two-hybrid system, and OpuCA was discovered as a TRAP-binding protein. OpuCA is an ATP binding-cytoplasmic (ABC) domain of an OpuC ABC transporter. S. aureus OpuC- mutant strain was constructed and shown to be less tolerant to salt stress, and was defective in choline uptake. OpuC- cells were less pathogenic and showed reduced TRAP phosphorylation and agr activity, did not respond to RAP, and were defective in biofilm formation in vitro and in vivo. These results suggest that OpuC acts as a transporter and also plays a role in S. aureus pathogenesis.

TRAP plays a role in stress response in Staphylococcus aureus.

Staphylococci are common pathogens of implant-related infections. RIP is a heptapeptide (YSPWTNF-NH2 ) that was shown to be very effective in preventing and treating antibiotic-resistant staphylococcal infections, in healing polymicrobial wounds, and in enhancing the effect of commonly used antibiotics. How the peptide negatively affects the survival of the bacteria in the host is not yet known. In staphylococci, RIP was shown to suppress toxin production by inhibiting the expression of agr and production of RNAIII. RIP was also shown to suppress the phosphorylation of TRAP (target of RNAIII-activating peptide), whose function was not clear. Here we show that mutant S. aureus TRAP- cells were more sensitive to oxidative stress and had higher rates of spontaneous and adaptive (agr) mutations. Furthermore, recombinant TRAP protected DNA from oxidative damage caused by hydroxyl radicals. Put together, these results suggest that TRAP is involved in DNA protection from stress. RIP may thus suppress pathogenesis through multiple independent molecular mechanisms involving both suppression of virulence and suppression of stress response.

RNAIII-inhibiting peptide enhances healing of wounds infected with methicillin-resistant Staphylococcus aureus.

Quorum sensing is a mechanism through which a bacterial population receives input from neighboring cells and elicits an appropriate response to enable survival within the host. Inhibiting quorum sensing by RNAIII-inhibiting peptide (RIP) has been demonstrated as a very effective mode of prevention and therapy for device-associated staphylococcal infections and was tested here for healing of wounds that are otherwise resistant to conventional antibiotics. Wounds, established through the panniculus carnosus of BALB/c mice, were inoculated with 5 x 10(7) CFU of methicillin-resistant Staphylococcus aureus. Mice were treated with Allevyn, RIP-soaked Allevyn (containing 20 microg RIP), daily intraperitoneal teicoplanin (7 mg/kg of body weight), Allevyn and teicoplanin, and RIP-soaked Allevyn and daily intraperitoneal teicoplanin. The main outcome measures were quantitative bacterial culture and histological examination with assessment of microvessel density and of vascular endothelial growth factor (VEGF) expression in tissue sections. Treatment with RIP-soaked Allevyn together with teicoplanin injection greatly reduced the bacterial load to 13 CFU/g (control untreated animals had 10(8) CFU/g bacteria). All other treatments were also significantly effective but only reduced the bacterial load to about 10(3) CFU/ml. Histological examination indicated that only treatment with RIP-soaked Allevyn with teicoplanin injection restored epithelial, granulation, and collagen scores, as well as microvessel density and VEGF expression, to the levels found with uninfected mice. In conclusion, we observed that RIP may be useful for the management of infected wounds and that it could represent an exciting and future alternative to the conventional antibiotics, at present considered the gold-standard treatments for methicillin-resistant S. aureus infections.

Discovery of a quorum-sensing inhibitor of drug-resistant staphylococcal infections by structure-based virtual screening.

Staphylococci are a major health threat because of increasing resistance to antibiotics. An alternative to antibiotic treatment is preventing virulence by inhibition of bacterial cell-to-cell communication using the quorum-sensing inhibitor RNAIII-inhibiting peptide (RIP). In this work, we identified 2',5-di-O-galloyl-d-hamamelose (hamamelitannin) as a nonpeptide analog of RIP by virtual screening of a RIP-based pharmacophore against a database of commercially available small-molecule compounds. Hamamelitannin is a natural product found in the bark of Hamamelis virginiana (witch hazel), and it has no effect on staphylococcal growth in vitro; but like RIP, it does inhibit the quorum-sensing regulator RNAIII. In a rat graft model, hamamelitannin prevented device-associated infections in vivo, including infections caused by methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains. These findings suggest that hamamelitannin may be used as a suppressor to staphylococcal infections.

RNAIII-inhibiting peptide affects biofilm formation in a rat model of staphylococcal ureteral stent infection.

Ureteral stents coated with the quorum-sensing inhibitor RNAIII-inhibiting peptide (RIP) were implanted in rat bladders and shown to suppress Staphylococcus aureus formation on the stent and in urine and was especially effective when combined with teicoplanin. Coating ureteral stents with RIP thus increases the efficacy of teicoplanin in preventing ureteral stent-associated staphylococcal infections.

Treatment of Staphylococcus aureus biofilm infection by the quorum-sensing inhibitor RIP.

The quorum-sensing inhibitor RIP inhibits staphylococcal TRAP/agr systems and both TRAP- and agr-negative strains are deficient in biofilm formation in vivo, indicating the importance of quorum sensing to biofilms in the host. RIP injected systemically into rats has been found to have strong activity in preventing methicillin-resistant Staphylococcus aureus graft infections, suggesting that RIP can be used as a therapeutic agent.

RNAIII-inhibiting-peptide-loaded polymethylmethacrylate prevents in vivo Staphylococcus aureus biofilm formation.

Staphylococci, common orthopedic pathogens, form antibiotic-resistant biofilms. Polymethylmethacrylate (PMMA) beads loaded with the quorum-sensing inhibitor RNAIII-inhibiting peptide (RIP) were implanted in rats and shown to prevent methicillin-resistant Staphylococcus aureus infection. RIP release was bimodal, typical of previously-tested antibiotics. These results suggest that RIP-PMMA warrants further evaluation for management of orthopedic infections caused by staphylococci.

RNAIII-inhibiting peptide in combination with the cathelicidin BMAP-28 reduces lethality in mouse models of staphylococcal sepsis.

A mouse model of staphylococcal sepsis was used to evaluate the efficacy of RNAIII-inhibiting peptide (RIP) combined with the cathelicidin BMAP-28. Preliminary in vitro studies showed that both peptides, alone or combined, were able to inhibit the lipoteichoic acid-induced production of tumor necrosis factor alpha and nitric oxide by RAW 264.7 cells. For in vivo experiments, the main outcome measures were lethality, quantitative blood cultures, and detection of tumor necrosis factor alpha and interleukin 6 plasma levels. BALB/c mice were injected i.v. with 2.0 x 10(6) colony-forming units of live Staphylococcus aureus ATCC 25923 or with 5.0 x 10(8) heat-killed cells of the same strain. All animals were randomized to receive i.v. isotonic sodium chloride solution, 10-mg/kg RIP, alone or in combination with 2-mg/kg BMAP-28, 7-mg/kg imipenem, or 7-mg/kg vancomycin, immediately and at 6 hours after bacterial challenge. In in vivo experiments performed with live bacteria, all compounds reduced lethality rates and bacteremia when compared with controls. In general, combined-treated groups had significantly lower bacteremia when compared with single-treated groups. Lowest lethality rates and bacteremia were obtained when RIP was administered in combination with BMAP-28 or vancomycin. In the experiments performed using heat-killed organisms, only BMAP-28 demonstrated significant efficacy on lethality rates and cytokines plasma levels when compared with controls. RIP combined with BMAP-28 exhibited the highest efficacy on all main outcome measurements. These data were observed on both immediate and delayed treatments. These results highlight the capacity of RIP and BMAP-28 to reduce the septic effects of bacterial cell components and exotoxins, and suggest their potential use in the treatment of severe staphylococcus-associated sepsis.

RNAIII-inhibiting peptide significantly reduces bacterial load and enhances the effect of antibiotics in the treatment of central venous catheter-associated Staphylococcus aureus infections.

BACKGROUND: Medical devices used in clinical practice are often associated with biofilm-associated staphylococcal infections. METHODS: An in vitro antibiotic susceptibility assay of Staphylococcus aureus biofilms using 96-well polystyrene tissue-culture plates was performed to test the effects of RNAIII-inhibiting peptide (RIP), ciprofloxacin, imipenem, and vancomycin. Efficacy studies were performed using a rat model of central venous catheter (CVC)-associated infection. Twenty-four hours after implantation, the catheters were filled with RIP (1 mg/mL). Thirty minutes later, rats were challenged, via the CVC, with 1.0 x 10(6) cfu of S. aureus strain Smith diffuse. The antibiotic-lock technique was begun 24 h later. RESULTS: Minimum inhibitory concentrations of antibiotics in biofilms were at least 4-fold higher than those against the freely growing planktonic cells. When they were first treated with RIP, the cells in biofilms became as susceptible to antibiotics as did planktonic cells. These data were confirmed by the in vivo studies. In particular, when CVCs were treated with both RIP and antibiotics, the biofilm bacterial load was further reduced to 1 x 10(1) cfu/mL, and bacteremia was not detected, suggesting that there was 100% elimination of bacteremia and a 6 log10 reduction in biofilm bacterial load. CONCLUSION: RIP significantly reduces bacterial load and enhances the effect of antibiotics in the treatment of CVC-associated S. aureus infections.

Transcriptional profiling of target of RNAIII-activating protein, a master regulator of staphylococcal virulence.

Staphylococcus aureus is a gram-positive bacterium that is part of the normal healthy flora but that can become virulent and cause infections by producing biofilms and toxins. The production of virulence factors is regulated by cell-cell communication (quorum sensing) through the histidine phosphorylation of target of RNAIII-activating protein (TRAP), which is a 21-kDa protein that is highly conserved among staphylococci. Using microarray analysis, we show here that the expression and phosphorylation of TRAP upregulate the expression of most, if not all, toxins known to date, as well as their global regulator agr. In addition, we show here that the expression and phosphorylation of TRAP are also necessary for the expression of genes known to be necessary for the survival of the bacteria in a biofilm, like arc, pyr, and ure. TRAP is thus demonstrated to be a master regulator of staphylococcal pathogenesis.

Prevention of staphylococcal biofilm-associated infections by the quorum sensing inhibitor RIP.

Staphylococcus aureus and Staphylococcus epidermidis associated with implantable medical devices, are often difficult to treat with conventional antimicrobials. Formation of a biofilm and subsequent production of toxins are two distinct mechanisms considered important in foreign body infections. Staphylococcal virulence is caused by a complex regulatory process, which involves cell-to-cell communication through the release and response to chemical signals in a process known as quorum sensing. We explored the possibility of preventing infections by interfering with biofilm formation and toxin production using the quorum sensing inhibitor ribonucleic-acid-III-inhibiting peptide. In our studies ribonucleic-acid-III-inhibiting peptide prevented graft-associated infections caused by all species of staphylococci tested so far, including methicillin resistant S. aureus and S. epidermidis. Ribonucleic-acid-III-inhibiting peptide also enhances the effects of antibiotics and cationic peptides in the clearance of normally recalcitrant biofilm infections. Ribonucleic-acid-III-inhibiting peptide is nontoxic, highly stable, and no resistant strains have been found so far, suggesting that ribonucleic-acid-III-inhibiting peptide may be used to coat medical devices or used systemically to prevent infections. When the target of ribonucleic-acid-III activating protein activity is disrupted, biofilm formation is reduced under flow and static conditions and genes important for toxin production or biofilm formation are down-regulated. These in vitro data help explain why ribonucleic-acid-III-inhibiting peptide seems to be effective in preventing staphylococcal infections.

Engineering approaches for the detection and control of orthopaedic biofilm infections.

Artificial joints are subject to chronic infections associated with bacterial biofilms, which only can be eradicated by the traumatic removal of the implant followed by sustained intravenous antibiotic therapy. We have adopted an engineering approach to develop electrical-current-based approaches to bacterial eradication and microelectromechanical systems that could be embedded within the implanted joint to detect the presence of bacteria and to provide in situ treatment of the infection before a biofilm can form. In the former case we will examine the combined bactericidal effects of direct and indirect electrical fields in combination with antibiotic therapy. In the latter case, bacterial detection will occur by developing a microelectromechanical-systems-based biosensor that can "eavesdrop" on bacterial quorum-sensing-based communication systems. Treatment will be effected by the release of a cocktail of pharmaceutical reagents contained within integral reservoirs associated with the implant, including a molecular jamming signal that competitively binds to the bacteria's quorum sensing receptors (which will "blind" the bacteria, preventing the production of toxins) and multiple high dose antibiotics to eradicate the planktonic bacteria. This approach is designed to take advantage of the relatively high susceptibility to antibiotics that planktonic bacteria display compared with biofilm envirovars. Here we report the development of a generic microelectromechanical systems biosensor that measures changes in internal viscosity in a base fluid triggered by a change in the external environment.

RNAIII-inhibiting peptide improves efficacy of clinically used antibiotics in a murine model of staphylococcal sepsis.

RNAIII-inhibiting peptide (RIP, YSPWTNF-NH2) is a quorum-sensing peptide inhibitor that prevents Staphylococcus aureus toxin production and biofilm formation. A mouse sepsis model was used to test the efficacy of RIP alone or in combination with conventional antibiotics in suppressing S. aureus-induced sepsis. Mice were injected intravenously with 3.0x10(6)CFU of S. aureus ATCC 25923 or with 3.0x10(6)CFU of S. aureus strain Smith diffuse. All animals were randomized to receive intravenously isotonic sodium chloride solution as a control, or 20 mg/kg RIP alone or combined with 20 mg/kg cefazolin, 10 mg/kg imipenem, or 10 mg/kg vancomycin immediately or 6 h after bacterial challenge. Main outcome measures were bacteremia and lethality. All compounds reduced lethality when compared to controls. Although, in general combined-treated groups had significant lower bacterial counts when associated to singly-treated groups only the combination between RIP and vancomycin with respect to cefazolin gave a statistically significant decrease in the lethality rate. Lowest lethality rates (10%) and bacteremia (<10(2)CFU/ml) were obtained when RIP was administered in combination with vancomycin. Because RIP can be synergistic with current antibiotic therapies and help to reduce S. aureus exotoxins production, it can be considered a promising agent to associate with antibiotics for further clinical research into treatment of sepsis.