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

Effect of single versus antibiotic combinations on Staphylococcus epidermidis biofilm viability and on genetic expression of some virulence genes

In this study five clinical isolates strains were used, and nine antibiotics at breakpoint concentrations: vancomycin, tetracycline, rifampicin, gentamicin, cefazolin, cephalothin, levofloxacine, daptomycin and clindamycin were tested. 48 hours biofilms were grown on Calgary Biofilm Device (CBD) and challenged overnight with antibiotics alone and in combination. Biofilm cells viability was determined by colony forming units (cfu). Afterwards, the effect of the most active antibiotics combinations against S. epidermidis biofilm on genetic expression of some genes of interest such as: icaA, icaR, sarA and rsbU was determined by real-time PCR. Although biofilms were generally insensitive to individual antibiotics, they were more susceptible to combinations. Levofloxacine was a constituent of almost all the combinations active against S. epidermidis biofilm pointing to be part of any antibiotic therapy directed against biofilms of these organisms

Virulence gene expression by staphylococcus epidermidis biofilm cells exposed to antibiotics

Staphylococcus epidermidis have become important causes of nosocomial infections, as its pathogenesis is correlated with the ability to form biofilms on polymeric surfaces. Production of poly-N-acetylglucosamine (PNAG) is crucial for S. epidermidis biofilm formation and is synthesized by the gene products of the icaADBC gene cluster. Production of PNAG/polysaccharide intercellular adhesin and biofilm formation are regulated by the alternative sigma factor, σB, and is influenced by a variety of environmental conditions including disinfectants and other antimicrobial substances. The susceptibility of five S. epidermidis strains to antibiotics alone and in double combination was previously tested. Our results demonstrated that some combinations are active and present a general broad spectrum against S. epidermidis biofilms, namely rifampicin–clindamycin and rifampicin–gentamicin. In the present study, it was investigated whether the combination of rifampicin with clindamycin and gentamicin and these antibiotics alone influence the expression of specific genes (icaA and rsbU) of S. epidermidis within biofilms using real-time polymerase chain reaction. The data showed that in most cases the expression of both genes tested significantly increased after exposure to antimicrobial agents alone and in combination. Besides having a similar antimicrobial effect, rifampicin combined with clindamycin and gentamicin induced a lower expression of biofilm-related genes relatively to rifampicin alone. Associated with the advantage of combinatorial therapy in avoiding the emergence of antibiotic resistance, this study demonstrated that it can also cause a lower genetic expression of icaA and rsbU genes, which are responsible for PNAG/polysaccharide intercellular adhesin production, and consequently reduce biofilm formation recidivism, relatively to rifampicin alone.F. Gomes and P. Teixeira fully acknowledge the financial support of Fundacao para a Ciencia e Tecnologia through the grants SFRH/BD/32126/2006 and SFRH/BPD/26803/2006, respectively

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

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

Sondas de ácido péptido nucleico, estojo e método para detectar espécies do género Lactobacillus spp. e/ou Gardnerella spp. e respectivas aplicações

O presente invento refere-se à concepção de duas sondas de ácido péptido nucleíco (PNA) para detectar as bactérias Lactobacillus e/ou Gardnerella spp.. Estas sondas são aplicadas a um processo baseado em técnicas de biologia molecular, nomeadamente de hibridação fluorescente in situ (FISH), aplicáveis no diagnóstico de vaginose bacteriana ou a detecção e quantificação destes géneros bacterianos em diversos tipos de amostras, incluindo sangue, alimentos, biopsias, fezes, água e outras amostras clínicas, ambientais ou provenientes da indústria agrícola ou alimentar

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

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

Comparison of vaginal microbiota fingerprints from healthy and bacterial vaginosis-positive portuguese women

Bacterial Vaginosis (BV) is a common disease in women of reproductive age and is characterized by the substitution of Lactobacillus species,which are predominant in the normal vaginal microbiota,by rapidly proliferating anaerobic bacteria, particularly Gardnerellavaginalis. The aim of this study was to study microbial communities’ structure in the vaginal microbiota of healthy and BV-positive Portuguese women. To this end, DNA obtained from vaginal samples of 22 BV-negative and 19 BV-positive women was analyzed using a PCR-DGGE approach.Total bacterial communities were amplified using general 16S rRNA gene primers. Group-specific primers were also used targeting Lactobacillus and Bifidobacterium genera and G.vaginalis. DGGE profiles were compared using the BioNumericsTM software package (Applied Maths, Belgium). Similarity between DGGE profiles was determined by calculating similarity indices of the densitometric curves of the compared profiles, using the Dice product-moment correlation. Different DGGE profiles could be obtained for BV-positive and BV-negative samples and this was verified for all primers sets utilized, suggesting that alteration of microbial community structure of BV-positive and -negative samples could be detected by PCR-DGGE. DGGE profiles obtained from samples of BV-positive women were more diverse that the ones from healthy women (as determined by a higher number of DGGE bands). The analysis of the standard electrophoretic bands for bacteria reveals an intrinsic diversity even within the two groups studied: similarities in bacterial DGGE profiles vary between 14- 78% and 47-100% in BV-positive and BV-negative samples, respectively. Among the 19 BV-positive women studied 18 were colonized with G. vaginalis. G.vaginalis was not detected in any of the healthy women samples. The analysis of Lactobacillus communities revealed a higher diversity in BV-negative women than in BV-positive ones, which confirms the association of Lactobacillus in healthy vaginal microbial communities. A more thoroughly comparison between BV-negative and BV-positive, including the retrieval of sequencing data from these samples, is necessary for getting more insight on BV influence on vaginal microbiota