Confocal laser scanning microscopy analysis of S. epidermidis biofilms exposed to farnesol, vancomycin and rifampicin

Staphylococcus epidermidis is the major bacterial species found in biofilm-related infections on indwelling medical devices. Microbial biofilms are communities of bacteria adhered to a surface and surrounded by an extracellular polymeric matrix. Biofilms have been associated with increased antibiotic tolerance to the immune system. This increased resistance to conventional antibiotic therapy has lead to the search for new antimicrobial therapeutical agents. Farnesol, a quorum-sensing molecule in Candida albicans, has been described as impairing growth of several different microorganisms and we have previously shown its potential as an adjuvant in antimicrobial therapy against S. epidermidis. However, its mechanism of action in S. epidermidis is not fully known. In this work we better elucidate the role of farnesol against S: epidermidis biofilms using confocal laser scanning microscopy (CLSM). Findings 24 h biofilms were exposed to farnesol, vancomycin or rifampicin and were analysed by CLSM, after stained with a Live/Dead stain, a known indicator of cell viability, related with cell membrane integrity. Biofilms were also disrupted by sonication and viable and cultivable cells were quantified by colony forming units (CFU) plating. Farnesol showed a similar effect as vancomycin, both causing little reduction of cell viability but at the same time inducing significant changes in the biofilm structure. On the other hand, rifampicin showed a distinct action in S. epidermidis biofilms, by killing a significant proportion of biofilm bacteria. Conclusions While farnesol is not very efficient at killing biofilm bacteria, it damages cell membrane, as determined by the live/dead staining, in a similar way as vancomycin.. Furthermore, farnesol might induce biofilm detachment, as determined by the reduced biofilm biomass, which can partially explain the previous findings regarding its role as a possible chemotherapy adjuvant.(undefined

S. epidermidis response to human blood and its cellular and soluble components

Staphylococcus epidermidis, a normal inhabitant of healthy human skin and mucosae, can cause persistent and relapsing infections due to its ability to adhere to medical devices and form biofilms. Hence, S epidermidis is considered one of the most important medical device-associated nosocomial agents, being particularly associated with vascular catheters. Although the biofilms formed on these catheters are in constant contact with human blood, their mutual interaction is poorly understood. Here, we evaluated the expression of genes associated with biofilm formation (icaA, aap, bhp), immune evasion (icaA, mprF, sepA) and programmed cell death (lrgB), as well as biofilm structure and viability, upon bacterial interaction with human blood and its components. We observed that contact with human blood increased the transcription of icaA and bhp but decreased aap, sepA and lrgB gene expression, when compared with plasma. In contrast, no significant transcriptional alterations were detected upon contact with purified mononuclear cells, whereas purified polymorphonuclear cells lead to increased bhp and mprF gene expression. Furthermore, human blood reduced by 50% the number of viable cells within the biofilm and induced significant alterations in its structure, with the creation of a fibre-like matrix. In conclusion, our study reveals that S. epidermis biofilms adapt to particular environmental stress by changing the expression of specific genes and by altering their structure. Despite these overall observations, significant variability was found between different blood donors, suggesting that particularities of the host immune system may strongly affect the outcome of S. epidermidis infections

Lactobacillus crispatus represses vaginolysin expression by BV associated Gardnerella vaginalis and reduces cell cytotoxicity

Using a chemically-defined medium simulating genital tract secretions, we have shown that pre-adhering Lactobacillus crispatus to Hela epithelial cells reduced cytotoxicity caused by Gardnerella vaginalis. This effect was associated to the expression of vaginolysin and was specific to L. crispatus interference, as other vaginal facultative anaerobes had no protective effect.This work was supported by Portuguese National Funds (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684). JC, and MER acknowledge the financial support of individual Grants SFRH/BD/93963/2013, and SFRH/BPD/95401/2013 respectively. NC is an Investigador FCT.info:eu-repo/semantics/publishedVersio

Farnesol induces cell detachment from established S. epidermidis biofilms

Antibiotic resistance is a serious problem in Staphylococcus epidermidis infections as many clinical isolates of this organism are resistant to up to eight different antibiotics. The increased resistance to conventional antibiotic therapy has lead to the search for new antimicrobial therapeutic agents. Farnesol, an essential oil found in many plants, has been shown to be active against S. epidermidis. Using a type control strain we recently described that although farnesol was not efficient at killing biofilm bacteria, a strong reduction on biofilm biomass was detected, and we hypothesize that farnesol could, somehow, induce biofilm detachment. In this report, to test our hypothesis we used 36 representative clinical strains of S. epidermidis from different geographic locations and characterized them in terms of genetic variability by multilocus sequence typing and staphylococcal chromosome cassette mec. Strains were tested for biofilm formation, and the presence of ica, bhp and aap genes was determined. Stronger biofilms had always the presence of ica operon but often co-harbored bhp and aap genes. Farnesol was then used in biofilm-forming strains, and biofilm detachment was detected in half of the strains tested. Furthermore, we also showed that farnesol inability to kill biofilm bacteria was not the result of the biofilm structure but was related to high cell density. Our results demonstrate, for the first time, that the biomass reduction previously found by us, and many other groups, is the result not of cell killing but instead is the result of biofilm detachment.We thank Herminia de Lencastre for reviewing the manuscript. Support for this work was provided by project P-99911 from Fundacao Calouste Gulbenkian and CONCORD-HEALTH-F3-2008/Project Number 222718/European Commission. This work was also supported by Fundacao para a Ciencia e a Tecnologia through grant #PEst-OE/EQB/LA0004/2011 awarded to ITQB

Dormancy within Staphylococcus epidermidis biofilms: an immunoproteomic characterization

Dormant bacteria within biofilms contribute to biofilm heterogeneity. Consequently, physiological heterogeneity of biofilms may influence host immune response and tolerance to antibiotics. Recently, we described an in vitro model to modulate dormancy in S. epidermidis biofilms. Here, we present a study based on immunoproteomics, where we compared the reactive profile of S. epidermidis biofilm proteins with prevented and induced dormancy, to human sera. A total of 19 immunoreactive proteins were identified by MALDI-TOF/TOF. Most of these proteins present molecular functions, such as catalytic activity and ion binding. CodY and GpmA proteins were more reactive to sera when biofilm dormancy was induced, while FtnA and ClpP were more reactive when dormancy was prevented. This is the first work identifying protein immunoreactivity differences between bacterial biofilms with induced or prevented dormancy. Considering the importance of dormancy within biofilms, further studies on these proteins may provide insights into the mechanisms related to dormancy and help improving current understanding on how dormancy affects the host immune response

Effect of farnesol on structure and composition of staphylococcus epidermidis biofilm matrix

Staphylococcus epidermidis is the most frequent cause of nosocomial sepsis and catheter-related infections in which biofilm formation is considered to be one of the main virulence mechanisms. Moreover, their increased resistance to conventional antibiotic therapy enhances the need to develop new therapeutical agents. Farnesol, a natural sesquiterpenoid present in many essential oils, has been described as impairing bacterial growth. The aim of this study was to evaluate the effect of farnesol on the structure and composition of biofilm matrix of S. epidermidis. Biofilms formed in the presence of farnesol (300 μM) contained less biomass, and displayed notable changes in the composition of the biofilm matrix. Changes in the spacial structure were also verified by confocal scanning laser microscopy (CSLM). The results obtained by the quantification of extracellular polymers and by wheat germ agglutinin (WGA) fluorescent detection of glycoproteins containing β(1→4)-N-acetyl-d-glucosamine support the hypothesis that farnesol causes disruption of the cytoplasmic membrane and consequently release of cellular content.Fernanda Gomes and Pilar Teixeira fully acknowledge the financial support of Fundacao para a Ciencia e Tecnologia (FCT) through the grants SFRH/BD/32126/2006 and SFRH/BPD/26803/2006, respectively

Staphylococcus aureus immunodominant surface antigen B is a cell-surface associated nucleic acid binding protein

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>immunodominant surface antigen B (IsaB) elicits an immune response during septicemia and is generally classified as a virulence factor, but its biological function remains completely undefined. In an attempt to identify staphylococcal RNA-binding proteins, we designed an RNA Affinity Chromatography assay and subsequently isolated IsaB.</p> <p>Results</p> <p>Western analysis indicated that IsaB was both secreted and cell-surface associated. Gel Shift analysis confirmed the RNA binding activity but revealed that IsaB bound to any nucleic acid without sequence specificity. IsaB exhibited the highest affinity for double-stranded DNA followed by single-stranded DNA and RNA. Because extracellular DNA has been shown to play a role in biofilm formation, we investigated the biofilm-forming capacity of an isogenic <it>isaB </it>deletion mutant but we found that IsaB did not contribute to biofilm formation under any conditions tested.</p> <p>Conclusion</p> <p>IsaB is an extracellular nucleic acid binding protein, with little to no sequence specificity, but its role in virulence remains unclear.</p

Optimizing a qPCR Gene Expression Quantification Assay for S. epidermidis Biofilms: A Comparison between Commercial Kits and a Customized Protocol

Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psmβ1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used

Holocene paleo-earthquakes recorded at the transfer zone of two majorfaults: the Pastores and Venta de Bravo fault (Trans-Mexican Volcanic Belt).

We present evidence of fi ve late Holocene earthquake ruptures observed at two paleoseismological trenches in the Laguna Bañí sag pond (Trans-Mexican Volcanic Belt, central Mexico). The trenches exposed two fault branches of the western termination of the Pastores fault, one of the major fault systems within the central Trans-Mexican Volcanic Belt. The site was studied by combining geomorphological and structural approaches, volcanic mapping, ground-penetrating radar, and paleoseismological analysis. The study revealed that coseismic surface rupture was noncharacteristic, and that the exposed fault branches had not always moved simultaneously. The fault tip has ruptured at least 5 times within the past 4 k.y., and the rupture events followed and preceded the deposition of an ignimbrite. The close temporal relationship of the seismic rupture with the volcanic activity of the area could be the result of volcanism triggered by faulting and its associated seismicity. The relatively high recurrence of seismic events (1.1 2.6 k.y.) and the noncharacteristic fault behavior observed at this tip of the Pastores fault suggest that the fault might have been active as a primary fault rupturing along segments of variable length or depth, and/or that the fault ruptured eventually as a secondary fault. The secondary ruptures would likely be related to earthquakes produced at major neighboring faults such as the Acambay fault, which moved during the 1912 Acambay earthquake, or the Venta de Bravo fault. A relatively large slip rate estimated for this fault branch (0.23 0.37 mm/yr) leads us to contemplate the possible connection at depth between the Pastores and the Venta de Bravo faults, increasing the maximum expected magnitude for central Mexico