Farnesol antimicrobial role as biofilm cell detachment inducer in S. epidermidis biofilms

Objetives: Farnesol is a naturally-occurring sesquiterpene that was originally isolated from essential oils found in many plants has been described to have antimicrobial potential against several bacteria, including S. epidermidis. However, farnesol mechanism of action is not yet fully understood and some contradictory findings have been reported. We recently described that while farnesol was not efficient at killing biofilm bacteria, a strong reduction on biofilm biomass was detected, and we hypothesize that farnesol could be inducing biofilm detachment. Here, we address this hypothesis. Methods: To test our hypothesis we used 36 representative clinical strains of S. epidermidis from different parts of the world and characterized them in terms of genetic variability, biofilm formation and on the effect of farnesol on biofilm physiology and gene expression. Results: Farnesol had no bactericidal effect on stationary phase populations equal or above 108 CFU/mL. In exponential phase planktonic bacteria, farnesol showed a bacteriostatic effect after cell density reached 108 CFU/mL. In any of the growth phases studied, farnesol was effective in killing above 90% of bacteria in 4 h when cell density was 107 CFU/mL or below. Confocal microscopy and flow citometry analysis confirmed that in biofilms bacteria were not killed by farnesol but nevertheless cell wall integrity was affected. Gene expression studies revealed differential responses to farnesol, depending on the bacterial strain tested. Farnesol cell detachement from biofilms was also straindependent. Conclusions: We found that while farnesol cannot kill high density bacterial communities, such as biofilms, it was nevertheless able to induce biofilm detachment in 50% of the strains that formed biofilm

Development of a rapid method for Proteus spp. detection in urine by peptide nucleic acid fluorescence in situ hybridisation (PNA)

The need to avoid empirical treatment of patients, and the fact that urine samples are among the most numerous of specimen types sent for microbiology studies, have prompted many researchers to explore methods to limit the time and expense of urine culture processing. The aim of this work was to develop a new peptide nucleic acid fluorescence in situ hybridization (PNA FISH) method for the rapid detection of Proteus spp., a genus related to the emergence of complicated urinary tract infectios, especially for immuno-compromised patients. Methods: The PNA probe was designed, optimized, tested on representative strains of the genus and other related strains, and, finally, a PNA FISH method was developed for application in urine samples. Results: The PNA FISH method was optimized, and laboratory testing on representative strains from the Proteus genus and several related bacterial species, showed experimental specificity and sensitivity both of 100% (sensitivity, 95% CI, 81.5 – 100 and specificity, 95% CI, 91.4 - 100). Then, the PNA FISH method was adapted to the detection of Proteus in urine. Artificial urine samples were contaminated with decreasing pathogen concentrations and the PNA FISH method was able to detect, in approximately 2 hours, as low as 1x10^4 CFU/mL, a concentration considered indicative of infection for catheter associated urinary tract infections (CAUTI’s). Conclusions: PNA FISH is a very sensitive, specific and rapid method for Proteus detection in urine and it could be a reliable alternative to the currently used culture-based techniques as it may avoid the need for empirical antibiotic treatment

Mapping the packaging cleavage determinants in the phage-dependent Staphylococcus aureus pathogenicity island SaPI1

Members of the SaPI family of Staphylococcus aureus pathogenicity islands are highly mobile, superantigen-encoding genetic elements that depend upon specific helper bacteriophages for their horizontal transfer. Following helper phage infection, these elements excise, replicate, and package their genomes in virions provided by the helper phage. One of the novel features of this process is the use of a SaPI-encoded small subunit of terminase, which replaces the phage small terminase subunit and redirects terminase packaging specificity to SaPI DNA. The SaPI genome, like that of the helper phage, is packaged from concatemers by a headful packaging mechanism, resulting in encapsidated DNA molecules that exhibit terminal redundancy and partial circular permutation. In this study we have localized the site of initial cleavage in SaPI1, a prototype member of this family, and have defined a small region of the SaPI1 genome sufficient for specific packaging. The cleavage site was initially localized to an intergenic region upstream of SaPI1 operon 1 by identification of submolar fragments in restricted SaPI1 virion DNA. Ligation of linkers to the ends of SaPI virion DNA, followed by amplification and sequencing of the linker/SaPI1 junction, further defined the sites of initial cleavage. A fragment encompassing this region was cloned into a plasmid vector co-expressing SaPI1 small terminase and shown to confer high frequency plasmid transduction by helper phage 80α. The critical determinants for SaPI1-specific packaging were further defined by deletion analysis of the cloned fragment. These results demonstrate that the sequence required for SaPI1-specific packaging maps to a small region upstream of the promoter for SaPI1 operon 1. This is strikingly different from the pac site used by the helper phage terminase, which, like other phage pac sites, maps to within the small terminase gene itself

Comparison of RNA extraction methods from biofilm samples of Staphylococcus epidermidis and other bacterial pathogens

Due to its ability to form compact biofilms, Staphylococcus epidermidis, a normal inhabitant of the human skin and mucosa, has emerged as a leading cause of nosocomial infections, particularly, in patients with indwelling medical devices. The quantification of specific messenger RNA (mRNA) from these biofilms is crucial to understand the switching to the pathogenic mode of life and the interaction with the host immune system. There are a vast range of commercial available Kits to extract RNA from bacterial cells. Distinct extraction methods can yield different RNA quantity and purity and the success of RNA-based analysis depends to a great extent on that. Herein, five different commercial available Kits, namely: FastRNA Pro®Blue from MPBio, RNAspin Mini from GEHealthcare, PureZol RNA isolation reagent from Bio-Rad, PureLinkTM RNA Mini Kit from Invitrogen and GenJETTM from Fermentas were tested using S. epidermidis and other gram positive (L. monocytogenes) and gram negative (E. coli and S. Enteritidis) biofilm-forming bacteria. Regarding the quantity and purity of mRNA isolated from S. epidermidis biofilms, the best method is, clearly, the MPBio isolating almost 25-70 times more RNA than the other tested methods. Concerning the RNA purity only the kits from Fermentas and Bio-Rad showed values below the accepted range for pure RNA. In conclusion, depending on the nature of our sample, these RNA extraction kits could not present the best performance. This way tests should be done in order to get high RNA yield and purity for downstream applications

Dopamine up-regulates Th17 phenotype from individuals with generalized anxiety disorder

AbstractOur objective was to evaluate the effect of stress-related dose of dopamine (DA) on the in vitro proliferation and cytokine production in polyclonally-activated T cells from healthy individuals or individuals with generalized anxiety disorder (GAD). Our results demonstrated that cell cultures from GAD group proliferated less following T cell activation, as compared with control group. The addition of DA reduced the proliferative response in cell cultures from healthy but not from GAD individuals. The cytokine profile in GAD individuals revealed Th1 and Th2 deficiencies associated with a dominant Th17 phenotype, which was enhanced by DA. A similar DA-induced immunomodulation was also observed in PPD-activated cell cultures from GAD individuals. Unlike the control, DA-enhanced Th17 cytokine production in GAD individuals was not affected by glucocorticoid. In conclusion, our results show that the T cell functional dysregulation in GAD individuals is significantly amplified by DA. These immune abnormalities can have impact in increasing the susceptibility of individuals with anxiety disorders to infectious diseases and inflammatory/autoimmune disorders

Sequence determinants for DNA packaging specificity in the S. aureus pathogenicity island SaPI1

The SaPIs and their relatives are a family of genomic islands that exploit helper phages for high frequency horizontal transfer. One of the mechanisms used by SaPIs to accomplish this molecular piracy is the redirection of the helper phage DNA packaging machinery. SaPIs encode a small terminase subunit that can be substituted for that of the phage. In this study we have determined the initial packaging cleavage sites for helper phage 80α, which uses the phage-encoded small terminase subunit, and for SaPI1, which uses the SaPI-encoded small terminase subunit. We have identified a 19 nt SaPI1 sequence that is necessary and sufficient to allow high frequency 80α transduction of a plasmid by a terminase carrying the SaPI1-encoded small subunit. We also show that the hybrid enzyme with the SaPI1 small terminase subunit is capable of generalized transduction.This work was performed in part under FDA's Medical Countermeasures Initiative, Contract #HHSF2232010000521 and NIH 1R56 AI081837 (to GEC). JCB was supported by the Portuguese Institute Fundacao para a Ciencia e Tecnologia (FCT), PhD Fellowship SFRH/BD/66250/2009. E.K.R. was supported by the National Academies National Research Council; his contribution to this study was carried out at NICHHD, NIH, Bethesda, MD, in the laboratory of the late Robert A. Weisberg. Plasmid pCN51 (in strain NRS613) and strain RN4282 (NRS145) were obtained through the Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA) program supported under NIAID/NIFI contract #HHSN272200700055C

Variability of RNA quality extracted from biofilms of foodborne pathogens using different kits impacts mRNA quantification by qPCR

The biofilm formation by foodborne pathogens is known to increase the problem related with surface disinfection procedure in the food processing environment and consequent transmission of these pathogens into the population. Messenger RNA has been increasingly used to understand the action and the consequences of disinfectants in the virulence on such biofilms. RNA quality is an important requirement for any RNA-based analysis since the quality can impair the mRNA quantification. Therefore, we evaluated five different RNA extraction kits using biofilms of the foodborne pathogens Listeria monocytogenes, Escherichia coli, and Salmonella enterica. The five kits yielded RNA with different quantities and qualities. While for E. coli the variability of RNA quality did not affect the quantification of mRNA, the same was not true for L. monocytogenes or S. enterica. Therefore, our results indicate that not all kits are suitable for RNA extraction from bacterial biofilms, and thus, the selection of RNA extraction kit is crucial to obtain accurate and meaningful mRNA quantification.AF and JCB acknowledge the financial support of individual grants SFRH/BD/62359/2009 and SFRH/BD/66250/2009, respectively. The authors acknowledge the gift of bacterial strains to Joana Azeredo and Maria Olivia Pereira.

45S rDNA external transcribed spacer organization reveals new phylogenetic relationships in Avena genus

Research ArticleThe genus Avena comprises four distinct genomes organized in diploid (AA or CC), tetraploid (AABB or AACC) and hexaploid species (AACCDD), constituting an interesting model for phylogenetic analysis. The aim of this work was to characterize 45S rDNA intergenic spacer (IGS) variability in distinct species representative of Avena genome diversity±A. strigosa (AA), A. ventricosa (CvCv), A. eriantha (CpCp), A. barbata (AABB), A. murphyi (AACC), A. sativa (AACCDD) and A. sterilis (AACCDD) through the assessment of the 5' external transcribed spacer (5'-ETS), a promising IGS region for phylogenetic studies poorly studied in Avena genus. In this work, IGS length polymorphisms were detected mainly due to distinct 5'-ETS sequence types resulting from major differences in the number and organization of repeated motifs. Although species with A genome revealed a 5'-ETS organization (A-organization) similar to the one previously described in A. sativa, a distinct organization was unraveled in C genome diploid species (C-organization). Interestingly, such new organization presents a higher similarity with other Poaceae species than A-genome sequences, supporting the hypothesis of C-genome being the ancestral Avena genome. Additionally, polyploid species with both genomes mainly retain the A-genome 5'-ETS organization, confirming the preferential elimination of C-genome sequences in Avena polyploid species. Moreover, 5'-ETS sequences phylogenetic analysis consistently clustered the species studied according to ploidy and genomic constitution supporting the use of ribosomal genes to highlight Avena species evolutive pathways.info:eu-repo/semantics/publishedVersio

Prevention of methamphetamine-induced microglial cell death by TNF-α and IL-6 through activation of the JAK-STAT pathway

<p><b>Abstract</b></p> <p><b>Background</b></p> <p>It is well known that methamphetamine (METH) is neurotoxic and recent studies have suggested the involvement of neuroinflammatory processes in brain dysfunction induced by misuse of this drug. Indeed, glial cells seem to be activated in response to METH, but its effects on microglial cells are not fully understood. Moreover, it has been shown that cytokines, which are normally released by activated microglia, may have a dual role in response to brain injury. This led us to study the toxic effect of METH on microglial cells by looking to cell death and alterations of tumor necrosis factor-alpha (TNF-α) and interleukine-6 (IL-6) systems, as well as the role played by these cytokines.</p> <p><b>Methods</b></p> <p>We used the N9 microglial cell line, and cell death and proliferation were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and incorporation of bromodeoxyuridine, respectively. The TNF-α and IL-6 content was quantified by enzyme-linked immunosorbent assay, and changes in TNF receptor 1, IL-6 receptor-alpha, Bax and Bcl-2 protein levels by western blotting. Immunocytochemistry analysis was also performed to evaluate alterations in microglial morphology and in the protein expression of phospho-signal transducer and activator of transcription 3 (pSTAT3).</p> <p><b>Results</b></p> <p>METH induced microglial cell death in a concentration-dependent manner (EC₅₀ = 1 mM), and also led to significant morphological changes and decreased cell proliferation. Additionally, this drug increased TNF-α extracellular and intracellular levels, as well as its receptor protein levels at 1 h, whereas IL-6 and its receptor levels were increased at 24 h post-exposure. However, the endogenous proinflammatory cytokines did not contribute to METH-induced microglial cell death. On the other hand, exogenous low concentrations of TNF-α or IL-6 had a protective effect. Interestingly, we also verified that the anti-apoptotic role of TNF-α was mediated by activation of IL-6 signaling, specifically the janus kinase (JAK)-STAT3 pathway, which in turn induced down-regulation of the Bax/Bcl-2 ratio.</p> <p><b>Conclusions</b></p> <p>These findings show that TNF-α and IL-6 have a protective role against METH-induced microglial cell death via the IL-6 receptor, specifically through activation of the JAK-STAT3 pathway, with consequent changes in pro- and anti-apoptotic proteins.</p