A new anti-infective strategy to reduce the spreading of antibiotic resistance by the action on adhesion-mediated virulence factors in Staphylococcus aureus.

Staphylococcus aureus is a flexible microbial pathogen frequently isolated from community-acquired and nosocomial infections. S. aureus expresses a wide array of secreted and cell surface-associated virulence factors, including proteins that promote adhesion to damaged tissue and to the surface of host cells, and that bind proteins in blood to help evade immune responses. Furthermore, surface proteins have a fundamental role in virulence related properties of S. aureus, including biofilm formation. The present study evaluates the anti-infective capabilities of a secreted protein of Serratia marcescens (serratiopeptidase, SPEP), in impairing some staphylococcal virulence-related properties, such as attachment to inert surfaces and adhesion/invasion on eukaryotic cells. SPEP seems to exert its action by modulating specific proteins. It is not assessed if this action is due to the proteolytic activity of SPEP or to a specific mechanism which triggers an out/inside signal. 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, SdrD, Sbi, EF-Tu and EF-G. EF-Tu and EF-G are known to perform a variety of function, 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 impairs the ability of this pathogen to adhere to prostheses, catheters and medical device

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

Colonic epithelial ion transport is not affected in patients with diverticulosis

<p>Abstract</p> <p>Background</p> <p>Colonic diverticular disease is a bothersome condition with an unresolved pathogenesis. It is unknown whether a neuroepithelial dysfunction is present. The aim of the study was two-fold; (1) to investigate colonic epithelial ion transport in patients with diverticulosis and (2) to adapt a miniaturized Modified Ussing Air-Suction (MUAS) chamber for colonic endoscopic biopsies.</p> <p>Methods</p> <p>Biopsies were obtained from the sigmoid part of the colon. 86 patients were included. All patients were referred for colonoscopy on suspicion of neoplasia and they were without pathological findings at colonoscopy (controls) except for diverticulosis in 22 (D-patients). Biopsies were mounted in MUAS chambers with an exposed area of 5 mm². Electrical responses to various stimulators and inhibitors of ion transport were investigated together with histological examination. The MUAS chamber was easy to use and reproducible data were obtained.</p> <p>Results</p> <p>Median basal short circuit current (SCC) was 43.8 μA·cm^-2(0.8 – 199) for controls and 59.3 μA·cm^-2(3.0 – 177.2) for D-patients. Slope conductance was 77.0 mS·cm^-2(18.6 – 204.0) equal to 13 Ω·cm²for controls and 96.6 mS·cm^-2(8.4 – 191.4) equal to 10.3 Ω·cm²for D-patients. Stimulation with serotonin, theophylline, forskolin and carbachol induced increases in SCC in a range of 4.9 – 18.6 μA·cm^-2, while inhibition with indomethacin, bumetanide, ouabain and amiloride decreased SCC in a range of 6.5 – 27.4 μA·cm^-2, and all with no significant differences between controls and D-patients. Histological examinations showed intact epithelium and lamina propria before and after mounting for both types of patients.</p> <p>Conclusion</p> <p>We conclude that epithelial ion transport is not significantly altered in patients with diverticulosis and that the MUAS chamber can be adapted for studies of human colonic endoscopic biopsies.</p

Anti-biofilm activities from marine cold adapted bacteria against staphylococci and Pseudomonas aeruginosa

Microbial biofilms have great negative impacts on the world’s economy and pose serious problems to industry, public health and medicine. The interest in the development of new approaches for the prevention and treatment of bacterial adhesion and biofilm formation has increased. Since, bacterial pathogens living in biofilm induce persistent chronic infections due to the resistance to antibiotics and host immune system. A viable approach should target adhesive properties without affecting bacterial vitality in order to avoid the appearance of resistant mutants. Many bacteria secrete antibiofilm molecules that function in regulating biofilm architecture or mediating the release of cells from it during the dispersal stage of biofilm life cycle. Cold-adapted marine bacteria represent an untapped reservoir of biodiversity able to synthesize a broad range of bioactive compounds, including anti-biofilm molecules. The anti-biofilm activity of cell-free supernatants derived from sessile and planktonic cultures of coldadapted bacteria belonging to Pseudoalteromonas, Psychrobacter, and Psychromonas species were tested against Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa strains. Reported results demonstrate that we have selected supernatants, from cold-adapted marine bacteria, containing non-biocidal agents able to destabilize biofilm matrix of all tested pathogens without killing cells. A preliminary physico-chemical characterization of supernatants was also performed, and these analyses highlighted the presence of molecules of different nature that act by inhibiting biofilm formation. Some of them are also able to impair the initial attachment of the bacterial cells to the surface, thus likely containing molecules acting as anti-biofilm surfactant molecules. The described ability of cold-adapted bacteria to produce effective anti-biofilm molecules paves the way to further characterization of the most promising molecules and to test their use in combination with conventional antibiotics

Sesquiterpene Induction by the Balsam Woolly Adelgid (Adelges piceae) in Putatively Resistant Fraser Fir (Abies fraseri)

Fraser fir, Abies fraseri (Pursh) Poir., is a tree endemic to the Southern Appalachians and is found only in a few isolated populations at high elevations. Fraser firs are also cultivated on a commercial scale as Christmas trees. The species is imperiled by an introduced insect, the balsam woolly adelgid, Adelges piceae Ratzeburg (BWA). The insect severely damages Christmas tree crops and has caused substantial Fraser fir mortality in natural stands. Foliar terpenoids are one mechanism of host plant defense against invading insects and may be one focus of future Christmas tree breeding efforts. This study examines the correlation of foliar terpenoids with Fraser fir performance when infested with BWA. GC-MS and GC-FID analysis of artificially infested Fraser fir foliage reveals that increased concentrations of four terpenoid compounds are associated with BWA infestations. Foliar concentrations of two sesquiterpenes, camphene and humulene, are significantly higher in putatively resistant Fraser fir clones than in more susceptible clones after sustained adelgid feeding for a period of 20 weeks. Although it is unclear if the induction of these sesquiterpenes in the host fir is directly contributing to adelgid resistance, these compounds could serve as effective indicators while screening for BWA resistance in future Christmas tree breeding programs