Biblioteca Digital

Objective: To determine the E. faecalis biofilm formation on the surface of five adhesive systems (AS) and its relationship with roughness. Study Design: The formation of E. faecalis biofilms was tested on the surface of four dual-cure AS: AdheSE DC, Clearfil DC Bond, Futurabond DC and Excite DSC and one light-cure antimicrobial AS, Clearfil Protect Bond, after 24 hours of incubation, using the MBEC high-throughput device. Results: E. faecalis biofilms grew on all the adhesives. The least growth of biofilm was on Excite DSC, Clearfil Protect Bond, and the control. Futurabond DC resulted in the greatest roughness and biofilm amount. There was a close relationship between the quantity of biofilm and roughness, except for Clearfil Protect Bond, which showed little biofilm but high roughness. Conclusion: None of the tested AS prevented E. faecalis biofilm formation, although the least quantity was found on the surface of Clearfil Protect Bond.

As infecções causadas por leveduras representam um grave problema em Saúde Pública. O objetivo desta pesquisa é o estudo aderência de Candida spp. (C. albicans ATCC18804, C. albicans SC5314, C. albicans cepa de campo, C. glabrata ATCC2001) sobre cateter urinário de Látex siliconizado (LS) e Silicone total (ST); avaliação da sensibilidade ao M-EDTA® dos biofilmes formados e quantificação dos genes ALS1 e ALS3 expressos por células sésseis de C. albicans. Os biofilmes de Candida spp. foram produzidos sobre cateteres urinários (LS e ST) segmentados em 1,0 cm de comprimento e separadamente introduzidos em poços de placas de poliestireno, sendo incubados a 35±1°C em contato com as suspensões de leveduras por 6, 24 e 48 e 72 horas. Os segmentos de cateter foram examinados por MEV, por cultura microbiológica, para avaliação da viabilidade celular e redução do sal de tetrazólio (XTT), para medir a atividade metabólica das células em biofilme. A sensibilidade das células sésseis ao M-EDTA® foi determinada após a exposição dos biofilmes ao produto; a expressão dos genes ALS1 e ALS3 de C. albicans SC5314 foi analisada por reação em cadeia de polimerase quantitativo (qPCR) e o resultado da recuperação de células viáveis de biofilme de Candida spp. foi expresso em log média ± desvio-padrão. Do biofilme de C. albicans ATCC18804 formado sobre LS...

Em consultórios odontológicos são utilizadas canetas rotatórias que durante seu uso entram em contato com a microbiota oral, podendo trazer conseqüências para o próprio paciente ou para outros que utilizarem o mesmo equipamento, se não houver um tratamento apropriado para sua reutilização. Para avaliar a eficiência de diferentes tratamentos utilizados rotineiramente na limpeza e desinfecção de equipamentos odontológicos, este trabalho descreve a produção de biofime in vitro em superfície de canetas odontológicas e a eficiência dos biocidas glutaraldeído, ácido peracético e álcool 70%, do detergente enzimático e da lavagem ultra-sônica para remoção do biofilme induzido, utilizando amostras de Pseudomonas aeruginosa e Staphylococcus aureus. Para a padronização dos métodos, além de curvas de crescimento de ambos os micro-organismos, foram realizados testes de adesão em cupons obtidos pelo corte das canetas. Foram testados diferentes tempos de incubação para a produção do biofilme, cujo valor máximo foi obtido em 14 dias. A avaliação da formação de biofilme foi realizada pelo método de contagem de bactérias viáveis (CBV), por microscopia eletrônica de varredura (MEV) e pelo método Cristal Violeta. A eficiência dos tratamentos na remoção do biofilme foi determinada pela diferença entre o número de células aderidas aos cupons submetidos ao tratamento e os cupons não submetidos. Maior remoção foi observada nos cupons tratados com ácido peracético...

O advento do uso de cateteres venosos centrais na prática médica trouxe muitos benefícios aos pacientes, porém está relacionado a um aumento na incidência de infecções por microrganismos multirresistentes. Além disso, freqüentemente ocorre colonização por bactérias produtoras de biofilme. Estes microrganismos se aderem ao material abiótico desses dispositivos intravenosos, ficando protegidos sob a matriz exopolissacarídica do biofilme. Isso faz com que sistema imunológico e antimicrobianos sejam incapazes de ter sua ação plena e, muitas vezes, não atingem os microrganismos mais internos. O motivo deste insucesso é porque muitos desses agentes biológicos e farmacológicos apresentam propriedades físico-químicas incompatíveis com a penetração nesta matriz. Com o objetivo de determinar quais antimicrobianos são mais adequados para uso quando o microrganismo é produtor de biofilme e quais as propriedades físico-químicas que estão diretamente relacionadas à penetração do antimicrobiano na matriz polissacarídica, utilizou-se método colorimétrico com cristal violeta em microplacas modificado para obtenção de concentração inibitória mínima em biofilme (MBIC) e método já padronizado para concentração inibitória mínima (MIC). Para isso foram testados 10 antimicrobianos em Staphylococcus spp.: rifampicina...

Introduction: The aim of this study was to compare Enterococcus faecalis biofilm formation on different substrates. Methods: Cell culture plates containing growth medium and E. faecalis (ATCC 29212) were used to grow biofilm on bovine dentin, gutta-percha, hydroxyapatite, or bovine bone. Substrates were incubated at 37°C for 14 or 21 days, and the medium was changed every 48 hours. After the growth induction periods, specimens (n = 5 per group and per induction period) were stained by using Live/Dead, and the images were analyzed under a confocal microscope. The total biovolume (μm3), live bacteria biovolume (μm3), and substrate coverage (%) were quantified by using the BioImage-L software. Results obtained were analyzed by nonparametric tests (P =.05). Results: Biofilm formation was observed in all groups. Gutta-percha had the lowest total biovolume at 14 days (P <.05) and hydroxyapatite the highest at 21 days (P <.05). No significant difference was observed in green biovolume at 14 days. At 21 days, however, hydroxyapatite had the highest volume (P <.05). The percentages of coverage were similar among all substrates at 21 days (P >.05), but at 14 days, bovine bone presented the highest coverage (P <.05). Conclusions: E. faecalis was capable of forming biofilm on all substrates during both growth periods; hydroxyapatite presented the highest rates of biofilm formation. The type of substrate influenced the biofilm characteristics...

This work proposes and discusses a simple flow cell reactor that provides a means to monitoring biofilm growth by periodical removing biofilm-attached slides for off-line, nondestructive and destructive biofilm analysis without the stoppage of the flow. With this flow cell, biofilm growth and respiratory activity can be easily followed, either in well defined laboratory conditions or in an industrial environment. The reproducible and typical biofilm development curves obtained validated this flow cell and confirmed its potential for different biofilm-related studies, which can include biocidal treatment.

Candida dubliniensis is an opportunistic yeast that can cause serious systemic diseases. This recently found yeast has been mismatched with Candida albicans once it presented similar genotypic and phenotypic characteristics. In the normal body flora, these yeasts exist predominantly in the form of biofilm. The main goal of this work was the evaluation of the ability for initial adhesion and biofilm formation by both Candida dubliniensis (two strains) and Candida albicans (one strain). To mimic the oral conditions the biofilms were formed on acrylic coupons in artificial saliva and using Sabouraud Dextrose Broth (SDB) as control medium. The biofilms were formed during 72 hours and compared in terms of total number of cells (using crystal violet) and activity (measured with XTT). In terms of initial adhesion, the three strains showed no statistical differences. However, the biofilm formed by Candida dubliniensis 7988 behaved similarly to the biofilm of Candida albicans, while the biofilm formed by Candida dubliniensis 7987 had a distinct evolution, either in SDB or saliva. The results show that Candida dubliniensis biofilm formation differs among strains and is not directly correlated with initial adhesion.

The study of biofilm ecology and interactions might help to improve our understanding of their resistance mechanisms to control strategies. Concerns that the diversity of the biofilm communities can affect disinfection efficacy have led us to examine the effect of two antimicrobial agents on two important spoilage bacteria. Studies were conducted on single and dual species biofilms of Bacillus cereus and Pseudomonas fluorescens. Biofilms were formed on a stainless steel rotating device, in a bioreactor, at a constant Reynolds number of agitation (ReA). Biofilm phenotypic characterization showed significant differences, mainly in the metabolic activity and both extracellular proteins and polysaccharides content. Cetyl trimethyl ammonium bromide (CTAB) and glutaraldehyde (GLUT) solutions in conjunction with increasing ReA were used to treat biofilms in order to assess their ability to kill and remove biofilms. B. cereus and P. fluorescens biofilms were stratified in a layered structure with each layer having differential tolerance to chemical and mechanical stresses. Dual species biofilms and P. fluorescens single biofilms had both the highest resistance to removal when pre-treated with CTAB and GLUT, respectively. B. cereus biofilms were the most affected by hydrodynamic disturbance and the most susceptible to antimicrobials. Dual biofilms were more resistant to antimicrobials than each single species biofilm...

Staphylococcus epidermidis is now well established as a major nosocomial pathogen, associated with indwelling medical devices. Its major virulence factor is related with the ability to adhere to indwelling medical devices and form biofilms. In this study, the biofilm matrix of four S. epidermidis clinical isolates was extracted and the polysaccharides and proteins content was quantified. The results were correlated with the total biofilm biomass (determined by crystal violet assay) and cellular metabolic activity (evaluated with XTT reduction assay). According to the results, the exopolymers studied play an important role not only on structure and biofilm biomass but also on cellular activity. Thus, the strain forming biofilms with the highest level of polysaccharides (S. epidermidis 1457) also formed thicker biofilms but with the lowest metabolic activity. The protein concentration also varied among strains, with the biofilm matrix of S. epidermidis 9142 presenting a higher concentration of proteins comparing to the remaining strains. This fact indicates the different levels of importance that matrix proteins can hold on biofilm composition among strains albeit overall, it is suggested that extracellular protein production it is not a determinative factor for biofilm total biomass.; Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/19265/2004...

This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC ¼ 0.55 + 0.20 mM for planktonic cells; MBC ¼ 1.7 + 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA...

Staphylococcus epidermidis has recently become known as a common cause of nosocomial infections, predominantly in patients with indwelling medical devices. Although, S. epidermidis infections only rarely develop into life-threatening diseases, they are very frequent and difficult to treat due to the ability of this bacterium to adhere to the surfaces of indwelling medical devices and form biofilms. A biofilm is a three‐dimensional microbial structure consisting of a multicellular community composed of cells embedded in a matrix composed, at least partially, of material synthesized by the sessile cells in the community. When S. epidermidis cells are in a biofilm they are more resistant to antibiotics and to the immune system. The importance of biofilms in the pathogenesis of the S. epidermidis infections is becoming more understandable, consequently several studies are needed, in order to develop effective methods for biofilm control. The use of (bacterio)phages to eradicate biofilms has been considered a potentially valuable approach. Phages are virus that infect bacteria and are the most abundant organisms on Earth. They are generally very efficient antibacterial agents and possess many advantages over antibiotics. Our aim is to search for virulent phages with broad host range for S. epidermidis biofilm therapy. Using wastewater treatment plants raw effluents we were able to isolate 5 phages. Their activity against 40 clinical S. epidermidis isolates with different genetic profiles was screened and was found to be different ranging from 46% to 95% of positive results. Further morphologic and genetic characterization of these isolated phages is now being performed. Preliminary results show that...

In the present study, the protein profiles of the cell wall (CW) extracts of eight Staphylococcus epidermidis strains were analysed. The protein pattern of the extracellular matrix (EM) of four S. epidermidis good biofilm producer strains was also obtained. The main goal was to relate these protein patterns with their bacterial adhesion and biofilm forming ability. For this purpose, 2 h adhesion and 8 d-old biofilm assays were carried out, with the adhered cells and biomass quantified by microscopic observation and crystal violet quantification, respectively. CW and EM proteins were visualized by SDS-PAGE. According to the results, a significant percentage of the CW proteins detected was common to all the strains studied. However, the most adhering strains expressed a high number of proteins associated with the initial adhesion process, and the strongest biofilm producers expressed proteins that were absent in the protein profile of the strains that produced lower amounts of biofilm. Therefore, the present results demonstrate the importance of such proteins in adhesion and biofilm formation processes. Some of the CW proteins were also present in the EM protein pattern, though to a smaller amount, as well as several exoenzymes important for the host immune system invasiveness.

In the past half century, bacterial vaginosis (BV) has been a controversial topic in medical microbiology, and despite the wealth of information on this topic, the etiological agent has not yet been definitively identified [1]. The first advances on BV pointed Gardnerella vaginalis as the infectious causative agent of BV [2] but soon after it was found that G. vaginalis was also present in healthy women [3]. Additionally, G. vaginalis was not able to cause BV consistently. Furthermore, other microorganisms started to be associated with BV, and this resulted in a shift in the paradigm to that of a multispecies infection. However, epidemiological data revealed inconsistencies with this latter theory [4]. A couple of years ago the first descriptions of multispecies biofilm communities were described in BV [5]. Interestingly, G. vaginalis was present in most cases and accounted for the majority of the biofilm biomass. Further studies demonstrated that biofilm-forming G. vaginalis presented higher tolerance to external stresses [6]. Taking these data into consideration, we hypothesized that strains of G. vaginalis that were able to form biofilms could be the causative agent of BV. To test our hypothesis, we isolated more than 30 bacterial species from BV patients and also several strains of G. vaginalis from healthy women...

Isothiocyanates (ITCs) are plant secondary metabolites with a range of biological effects including antimicrobial activity. This study reports the activity of two ITCs [allylisothiocyanate (AITC) and 2-phenylethylisothiocyanate (PEITC)] on bacterial motility and prevention of biofilm formation by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes. AITC caused total inhibition of swimming (P. aeruginosa) and swarming (E. coli, P. aeruginosa) motilities. PEITC caused total inhibition of swimming (E. coli, P. aeruginosa and L. monocytogenes) and swarming (E. coli and P. aeruginosa) motilities. Colony spreading of S. aureus was completely inhibited with PEITC. Total biofilm prevention was observed for E. coli with AITC. AITC and PEITC had no preventive effects in biofilm formation by S. aureus and L. monocytogenes, respectively. Significant preventive action with AITC on biofilm formation by P. aeruginosa (90%) and by L. monocytogenes (61%), and with PEITC on biofilm formation by S. aureus (75%) was verified. In terms of viability, AITC and PEITC promoted reductions higher than 87% for all the biofilms tested. In conclusion, these molecules demonstrated potential to inhibit bacterial motility and to prevent biofilm formation of pathogenic bacteria.

Nitrate removal from water has been accomplished by heterotrophic biofilms using organic carbon as a source of reducing power. To overcome the natural limitation in organic carbon in water, a poly-epsilon-caprolactone based biofilm carrier that serves simultaneously as a biofilm carrier and as a source of organic carbon was developed and tested in the present work. The feasibility of the new biofilm carrier for nitrate removal from water was evaluated in a packed bed reactor. The combination of size and structure provided a carrier element having high surface area and void volume, 1,170 m(2)/m(3) and 67 %, respectively. A maximum denitrification rate of 4.4 mg N-NO3-/(L.h) (9.2 mg N-NO3-/(m(2).h)) was achieved in the packed bed reactor at 20 degrees C and pH 7.0. Main advantages of the biofilm carrier developed in the present work are its mechanical stability in water even after biofilm formation and controlled release of organic carbon by enzymatic reactions. The proposed biotechnology to remove nitrate from groundwater is robust and easy to operate.

An experimental model was proposed to study biofilm formation by Listeria monocytogenes ATCC 19117 on AISI 304 (#4) stainless steel surface and biotransfer potential during this process. In this model, biofilm formation was conducted on the surface of stainless steel coupons, set on a stainless steel base with 4 divisions, each one supporting 21 coupons. Trypic Soy Broth was used as bacterial growth substrate, with incubation at 37 ºC and stirring of 50 rpm. The number of adhered cells was determined after 3, 48, 96, 144, 192 and 240 hours of biofilm formation and biotransfer potential from 96 hours. Stainless steel coupons were submitted to Scanning Electron Microscopy (SEM) after 3, 144 and 240 hours. Based on the number of adhered cells and SEM, it was observed that L. monocytogenes adhered rapidly to the stainless steel surface, with mature biofilm being formed after 240 hours. The biotransfer potential of bacterium to substrate occurred at all the stages analyzed. The rapid capacity of adhesion to surface, combined with biotransfer potential throughout the biofilm formation stages, make L. monocytogenes a potential risk to the food industry. Both the experimental model developed and the methodology used were efficient in the study of biofilm formation by L. monocytogenes on stainless steel surface and biotransfer potential.

Biofouling of membranes demands costly periodic cleaning and membrane replacement. A sustainable and environmentally friendly solution for maintenance is not available and would be of great interest for many purposes including economical. As complex biofilm formation by environmental strains is the major cause of biofouling and biofilm formation in most cases are controlled by N-Acylhomoserine lactone (AHL)mediated Quorum Sensing (QS). An effort was made to understand the appropriateness of 2(5H)-furanone, to use against biofouling of membranes. QS inhibition activity by 2(5H)-furanone was studied using bioindicator strains and known AHLs of different acyl chain lengths. The biofilm inhibition was studied by growth analysis on polystyrene plate of Aeromonas hyrdrophila, an environmental biofilm strain isolated from a bio-fouled reverse osmosis (RO) membrane. Results showed a QS inhibition activity against a wide range of AHLs and also biofilm formation by 2(5H)-furanone, which is believed to act as a potential quorum inhibition agent in a bacterial biofilm community.

Streptococcus mutans (S. mutans), harboring biofilm formation, considered as a main aetiological factor of dental caries. Gtf genes play an important role in S. mutans biofilm formation. The purpose of this study was to investigate the effect of Lactobacillus acidophilus-derived biosurfactant on S. mutans biofilm formation and gtfB/C expression level (S. mutans standard strain ATCC35668 and isolated S. mutans strain (22) from dental plaque). The Lactobacillus acidophilus (L. acidophilus) DSM 20079 was selected as a probiotic strain to produce biosurfactant. The FTIR analysis of its biosurfactant showed that it appears to have a protein-like component. Due to the release of such biosurfactants, L. acidophilus was able to interfere in the adhesion and biofilm formation of the S. mutans to glass slide. It also could make streptococcal chains shorter. Using realtime RT-PCR quantitation method made it clear that gtfB and gtfC gene expression were decreased in the presence of L. acidophilus-derived biosurfactant fraction. Several properties of S. mutans cells (the surface properties, biofilm formation, adhesion ability and gene expression) were changed after L. acidophilus-derived biosurfactant treatment. It is also concluded that biosurfacant treatment can provide an optional way to control biofilm development. On the basis of our findings...

Staphylococcus epidermidis which exists in healthy human skin as a commensal inhabitant is also an important pathogen forming biofilms on many surfaces and recently, increased resistance traits were suggested to be acquired in biofilm environments. In this study; clinical Prevalences, antibiotic resistances and biofilm formations of S. epidermidis strains were determined and comparison of all these findings with each other was carried out in order to take precautions against them and figure out if high biofilm forming S. epidermidis strains display multi drug resistance. According to our results; samples of wound and blood were the most S. epidermidis isolated clinical materials (40%; 35%) and cardiothoracic surgery was the most S. epidermidis observed service unit. All of these strains were sensitive to vancomycin, however 65% of them showed resistance to all β-lactam antibiotics (Penicillin, Oxacillin, Amoxicilin / Clavulonic acid), used in this study and 60% of all S. epidermidis strains were found as multi drug resistant. When the results of strong biofilm forming S. epidermidis strains are examined; they were isolated from sample of blood and service unit of cardiovascular surgery in highest frequency and 80% of them were β-lactam resistant whereas 100% of them were multi drug resistant. One of these multi drug resistant strains which was resistant to maximum amount of different antimicrobial classes...

Proteus mirabilis strains ability to form biofilm is a current topic of a number of research worldwide. In this study the biofilm formation of P. mirabilis strains derived from urine of the catheterized and non-catheterized patients has been investigated. A total number of 39 P. mirabilis strains isolated from the urine samples of the patients of dr Antoni Jurasz University Hospital No. 1 in Bydgoszcz clinics between 2011 and 2012 was used. Biofilm formation was evaluated using two independent quantitative and qualitative methods with TTC (2,3,5-triphenyl-tetrazolium chloride) and CV (crystal violet) application. The obtained results confirmed biofilm formation by all the examined strains, except quantitative method with TTC, in which 7.7% of the strains did not have this ability. It was shown that P. mirabilis rods have the ability to form biofilm on the surfaces of both biomaterials applied, polystyrene and polyvinyl chloride (Nelaton catheters). The differences in ability to form biofilm observed between P. mirabilis strains derived from the urine of the catheterized and non-catheterized patients were not statistically significant.