Differentiation between Staphylococcus aureus and coagulase-negative Staphylococcus species by real-time PCR including detection of methicillin resistants in comparison to conventional microbiology testing.

BACKGROUND Staphylococcus aureus has long been recognized as a major pathogen. Methicillin-resistant strains of S. aureus (MRSA) and methicillin-resistant strains of S. epidermidis (MRSE) are among the most prevalent multiresistant pathogens worldwide, frequently causing nosocomial and community-acquired infections. METHODS In the present pilot study, we tested a polymerase chain reaction (PCR) method to quickly differentiate Staphylococci and identify the mecA gene in a clinical setting. RESULTS Compared to the conventional microbiology testing the real-time PCR assay had a higher detection rate for both S. aureus and coagulase-negative Staphylococci (CoNS; 55 vs. 32 for S. aureus and 63 vs. 24 for CoNS). Hands-on time preparing DNA, carrying out the PCR, and evaluating results was less than 5 h. CONCLUSIONS The assay is largely automated, easy to adapt, and has been shown to be rapid and reliable. Fast detection and differentiation of S. aureus, CoNS, and the mecA gene by means of this real-time PCR protocol may help expedite therapeutic decision-making and enable earlier adequate antibiotic treatment.

BIOCHEMICAL CHARACTERIZATION AND CLINICAL SIGNIFICANCE OF COAGULASE-NEGATIVE STAPHYLOCOCCI

Although coagulase-negative staphylococci (C-NS) have been implicated in certain human infections, they are generally regarded as contaminants and their clinical significance is questioned. To assess their role as pathogens, 205 isolates of C-NS from wounds, and body fluids (blood, urine, pleural and peritoneal fluids, etc.) were studied. Patient's charts were reviewed and using strict criteria a determination was made regarding the clinical significance of these isolates. The organisms were then identified using the scheme of Kloos and Schleifer to determine if certain species of C-NS were associated with specific infections. S. epidermidis sensu stricto accounted for 81% of the C-NS isolated; the frequency of other species was S. haemolyticus (6%), S. hominis (5%), S. capitis (4%), S. warneri (3%), and others (1%). Only two isolates were novobiocin resistant; neither was identified as S. saprophyticus. Using these criteria, 22% of C-NS were considered to be clinically significant and the majority of these (93%) were due to S. epidermidis. The most common source of the clinically relevant C-NS isolates was from wounds. These data suggest that identifying C-NS species other than S. epidermidis may be of limited value in predicting clinical significance.^ In addition, selected pathogenic and non-pathogenic strains of C-NS were compared for their ability to adhere to human cells in vitro. Although the results were not conclusive, it appeared that pathogenic C-NS adhered more avidly than non-pathogenic C-NS to buccal cells. Experiments with HeLa cells showed no difference between pathogenic and non-pathogenic C-NS in adherence abilities. ^

Resonadores piezoeléctricos de cizalladura: estudio de la formación de biofilms y caracterización de fluidos magneto‐reológicos

La presente tesis revisa y analiza algunos aspectos fundamentales relativos al comportamiento de los sensores basados en resonadores piezoeléctricos TSM (Thickness Shear Mode), as�� como la aplicación de los mismos al estudio y caracterización de dos medios viscoelásticos de gran interés: los fluidos magnetoreológicos y los biofilms microbianos. El funcionamiento de estos sensores está basado en la medida de sus propiedades resonantes, las cuales varían al entrar en contacto con el material que se quiere analizar. Se ha realizado un análisis multifrecuencial, trabajando en varios modos de resonancia del transductor, en algunas aplicaciones incluso de forma simultánea (excitación pulsada). Se han revisado fenómenos como la presencia de microcontactos en la superficie del sensor y la resonancia de capas viscoelásticas de espesor finito, que pueden afectar a los sensores de cuarzo de manera contraria a lo que predice la teoría convencional (Sauerbrey y Kanazawa), pudiéndonos llevar a incrementos positivos de la frecuencia de resonancia. Además, se ha estudiado el efecto de una deposición no uniforme sobre el resonador piezoeléctrico. Para ello se han medido deposiciones de poliuretano, modelándose la respuesta del resonador con estas deposiciones mediante FEM. El modelo numérico permite estudiar el comportamiento del resonador al modificar distintas variables geométricas (espesor, superficie, no uniformidad y zona de deposición) de la capa depositada. Se ha demostrado que para espesores de entre un cuarto y media longitud de onda aproximadamente, una capa viscoelástica no uniforme sobre la superficie del sensor, amplifica el incremento positivo del desplazamiento de la frecuencia de resonancia en relación con una capa uniforme. Se ha analizado también el patrón geométrico de la sensibilidad del sensor, siendo también no uniforme sobre su superficie. Se han aplicado sensores TSM para estudiar los cambios viscoelásticos que se producen en varios fluidos magneto-reológicos (FMR) al aplicarles distintos esfuerzos de cizalla controlados por un reómetro. Se ha podido ver que existe una relación directa entre diversos parámetros reológicos obtenidos con el reómetro (fuerza normal, G’, G’’, velocidad de deformación, esfuerzo de cizalla…) y los parámetros acústicos, caracterizándose los FMR tanto en ausencia de campo magnético, como con campo magnético aplicado a distintas intensidades. Se han estudiado las ventajas que aporta esta técnica de medida sobre la técnica basada en un reómetro comercial, destacando que se consigue caracterizar con mayor detalle algunos aspectos relevantes del fluido como son la deposición de partículas (estabilidad del fluido), el proceso de ruptura de las estructuras formadas en los FMR tanto en presencia como en ausencia de campo magnético y la rigidez de los microcontactos que aparecen entre partículas y superficies. También se han utilizado sensores de cuarzo para monitorear en tiempo real la formación de biofilms de Staphylococcus epidermidis y Eschericia coli sobre los propios resonadores de cristal de cuarzo sin ningún tipo de recubrimiento, realizándose ensayos con cepas que presentan distinta capacidad de producir biofilm. Se mostró que, una vez que se ha producido una primera adhesión homogénea de las bacterias al sustrato, podemos considerar el biofilm como una capa semi-infinita, de la cual el sensor de cuarzo refleja las propiedades viscoelásticas de la región inmediatamente contigua al resonador, no siendo sensible a lo que sucede en estratos superiores del biofilm. Los experimentos han permitido caracterizar el módulo de rigidez complejo de los biofilms a varias frecuencias, mostrándose que el parámetro característico que indica la adhesión de un biofilm tanto en el caso de S. epidermidis como de E. coli, es el incremento de G’ (relacionado con la elasticidad o rigidez de la capa), el cual viene ligado a un incremento de la frecuencia de resonancia del sensor. ABSTRACT This thesis reviews and analyzes some key aspects of the behavior of sensors based on piezoelectric resonators TSM (Thickness Shear Mode) and their applications to the study and characterization in two viscoelastic media of great interest: magnetorheological fluids and microbial biofilms. The operation of these sensors is based on the analysis of their resonant properties that vary in contact with the material to be analyzed. We have made a multi-frequency analysis, working in several modes of resonance of the transducer, in some applications even simultaneously (by impulse excitation). We reviewed some phenomena as the presence of micro-contacts on the sensor surface and the resonance of viscoelastic layers of finite thickness, which can affect quartz sensors contrary to the conventional theory predictions (Sauerbrey and Kanazawa), leading to positive resonant frequency shifts. In addition, we studied the effect of non-uniform deposition on the piezoelectric resonator. Polyurethane stools have been measured, being the resonator response to these depositions modeled by FEM. The numerical model allows studying the behavior of the resonator when different geometric variables (thickness, surface non-uniformity and deposition zone) of the deposited layer are modified. It has been shown that for thicknesses between a quarter and a half of a wavelength approximately, non-uniform deposits on the sensor surface amplify the positive increase of the resonance frequency displacement compared to a uniform layer. The geometric pattern of the sensor sensitivity was also analyzed, being also non-uniform over its surface. TSM sensors have been applied to study the viscoelastic changes occurring in various magneto-rheological fluids (FMR) when subjected to different controlled shear stresses driven by a rheometer. It has been seen that there is a direct relationship between various rheological parameters obtained with the rheometer (normal force, G', G'', stress, shear rate ...) and the acoustic parameters, being the FMR characterized both in the absence of magnetic field, and when the magnetic field was applied at different intensities. We have studied the advantages of this technique over the characterization methods based on commercial rheometers, noting that TSM sensors are more sensitive to some relevant aspects of the fluid as the deposition of particles (fluid stability), the breaking process of the structures formed in the FMR both in the presence and absence of magnetic field, and the rigidity of the micro-contacts appearing between particles and surfaces. TSM sensors have also been used to monitor in real time the formation of biofilms of Staphylococcus epidermidis and Escherichia coli on the quartz crystal resonators themselves without any coating, performing tests with strains having different ability to produce biofilm. It was shown that, once a first homogeneous adhesion of bacteria was produced on the substrate, the biofilm can be considered as a semi-infinite layer and the quartz sensor reflects only the viscoelastic properties of the region immediately adjacent to the resonator, not being sensitive to what is happening in upper layers of the biofilm. The experiments allow the evaluation of the biofilm complex stiffness module at various frequencies, showing that the characteristic parameter that indicates the adhesion of a biofilm for the case of both S. epidermidis and E. coli, is an increased G' (related to the elasticity or stiffness of the layer), which is linked to an increase in the resonance frequency of the sensor.

Designing surfaces that kill bacteria on contact

Poly(4-vinyl-N-alkylpyridinium bromide) was covalently attached to glass slides to create a surface that kills airborne bacteria on contact. The antibacterial properties were assessed by spraying aqueous suspensions of bacterial cells on the surface, followed by air drying and counting the number of cells remaining viable (i.e., capable of growing colonies). Amino glass slides were acylated with acryloyl chloride, copolymerized with 4-vinylpyridine, and N-alkylated with different alkyl bromides (from propyl to hexadecyl). The resultant surfaces, depending on the alkyl group, were able to kill up to 94 ± 4% of Staphylococcus aureus cells sprayed on them. A surface alternatively created by attaching poly(4-vinylpyridine) to a glass slide and alkylating it with hexyl bromide killed 94 ± 3% of the deposited S. aureus cells. On surfaces modified with N-hexylated poly(4-vinylpyridine), the numbers of viable cells of another Gram-positive bacterium, Staphylococcus epidermidis, as well as of the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli, dropped more than 100-fold compared with the original amino glass. In contrast, the number of viable bacterial cells did not decline significantly after spraying on such common materials as ceramics, plastics, metals, and wood.

Estudo comparativo da microflora bacteriana de doentes portadores de próteses totais acrílicas mucosuportadas e implantosuportadas

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Infection risk associated with a closed luer access device

The potential for microbial contamination associated with a recently developed needleless closed luer access device (CLAD) (Q-Syte™ Becton Dickinson, Sandy, UT, USA) was evaluated in vitro. Compression seals of 50 multiply activated Q-Syte devices were inoculated with Staphylococcus epidermidis NCTC 9865 in 25% (v/v) human blood and then disinfected with 70% (v/v) isopropyl alcohol followed by flushing with 0.9% (w/v) sterile saline. Forty-eight of 50 (96%) saline flushes passed through devices that had been activated up to a maximum of 70 times remained sterile. A further 25 Q-Syte CLADs that had undergone multiple activations were challenged with prefilled 0.9% (w/v) sterile saline syringes, the external luer tips of which had been inoculated with S. epidermidis NCTC 9865 prior to accessing the devices. None of the devices that had been accessed up to 70 times allowed passage of micro-organisms, despite challenge micro-organisms being detected on both the syringe tip after activation and the compression seals before decontamination. These findings suggest that the Q-Syte CLAD may be activated up to 70 times with no increased risk of microbial contamination within the fluid pathway. The device may also offer protection from the external surface of syringe tips contaminated with micro-organisms. © 2005 Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Evaluation of a 2% chlorhexidine gluconate in 70% isopropyl alcohol skin disinfectant

The efficacy of a new skin disinfectant, 2% (w/v) chlorhexidine gluconate (CHG) in 70% (v/v) isopropyl alcohol (IPA) (ChloraPrep®), was compared with five commonly used skin disinfectants against Staphylococcus epidermidis RP62A in the presence or absence of protein, utilizing quantitative time-kill suspension and carrier tests. All six disinfectants [70% (v/v) IPA, 0.5% (w/v) aqueous CHG, 2% (w/v) aqueous CHG, 0.5% (w/v) CHG in 70% (v/v) IPA and 10% (w/v) aqueous povidone iodine (PI)] achieved a log10 reduction factor of 5, in colony-forming units/mL, in a suspension test (exposure time 30 s) in the presence and absence of 10% human serum. Subsequent challenges of S. epidermidis RP62A in a biofilm (with and without human serum) demonstrated reduced bactericidal activity. Overall, the most effective skin disinfectants tested against S. epidermidis RP62A were 2% (w/v) CHG in 70% IPA and 10% (w/v) PI. These results suggest that enhanced skin antisepsis may be achieved with 2% (w/v) CHG in 70% (v/v) IPA compared with the three commonly used CHG preparations [0.5% (w/v) aqueous CHG, 2% (w/v) aqueous CHG and 0.5% (w/v) CHG in 70% (v/v) IPA]. © 2005 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Mediators of gram-positive septic shock

Septic shock can occur as a result of Gram-negative or Gram-positive infection and involves a complex interaction between bacterial factors and the host immune system producing a systemic inflammatory state that may progress to multiple organ failure and death. Gram-positive bacteria are increasingly becoming more prevalent especially Staphylococcus epidermidis in association with indwelling devices. Lipopolysaccaride (LPS) is the key Gram-negative component involved in this process, but it is not clear which components of Gram-positive bacteria are responsible for progression of this often fatal disease. The aim of this thesis was to investigate the effect of bacterial components on the immune systems. Lipid S, a short chain form of lipoteichoic acid (LTA) found to be excreted from bacteria during growth in culture medium was examined along with other Gram-positive cell wall components: LTA, peptidoglycan (PG) and wall teichoic acids (WTA) and LPS from Gram-negative bacteria. Lipid S, LTA, PG and LPS but not WTA all stimulated murine macrophages and cell lines to produce significant amounts of NO, TNF-a, IL-6 and IL-1 and would induce fever and tissue damage seen in inflammatory diseases. Lipid S proved to be the most potent out of the Gram-positive samples tested. IgG antibodies in patients serum were found to bind to and cross react with lipid S and LTA. Anti-inflammatory antibiotics, platelet activating factor (PAF), PAF receptor antagonists and monoclonal antibodies (mAbs) directed to LTA, CD14 and toll-like receptors were utilised to modulate cytokine and NO production. In cell culture the anti-LTA and the anti-CD14 mAbs failed to markedly attenuate the production of NO, TNF-a, IL-6 or IL-1, the anti-TLR4 antibody did greatly inhibit the ability of LPS to stimulate cytokine production but not lipid S. The tetracyclines proved to be the most effective compounds, many were active at low concentrations and showed efficacy to inhibit both lipid S and LPS stimulated macrophages to produce NO.

Characterisation of coagulase-negative staphylococci associated with endocarditis

Coagulase-negative staphylococci are major aetiological agents of prosthetic valve endocarditis and an occasional cause of native valve disease. It is currently unclear how this group of usually avirulent microorganisms produces an infection associated with high rates of morbidity and mortality. The aim of this thesis was to investigate whether there are specific genotypes and/or phenotypes of coagulase-negative staphylococci with a propensity to cause infective endocarditis and to investigate any identified virulence factors as markers of infection. In this study, strains of endocarditis-related coagulase-negative staphylococci were genotyped by determining their macrorestriction genomic profile using pulsed-field gel electrophoresis. The strains were also investigated for phenotypic characteristics that predisposed the microorganisms to infect heart valves. By comparing coagulase-negative staphylococcal strains recovered from endocarditis patients with isolates from other significant infections (prosthetic device-related osteomyelitis and catheter-associated sepsis), no specific genotype or phenotype with a predilection to cause endocarditis was identified. However, the majority of the endocarditis-associated and other infection strains expressed the potential virulence factors lipase and esterase. Another approach to the investigation of virulence determinants used patient's serum to screen a Staphylococcus epidermidis NCTC 11047 genomic DNA library for cellular and secreted staphylococcal products that were expressed in vivo. The characterisation of two clones, which reacted with serum collected from a S. epidermidis-related endocarditis patient identified a staphylococcal pyruvate dehydrogenase complex E2 subunit and a novel secreted protein with homology to a Staphylococcus aureus staphyloxanthin biosynthesis protein and a secreted protein of unknown function described in Staphylococcus carnosus. Investigation of the secreted protein previously undetected in S. epidermidis, termed staphylococcal secretory antigen (SsaA), identified a potential marker of S. epidermidis-related endocarditis.

Studies on biofilm growth of Pseudomonas aeruginosa PAO1

The development of in vitro techniques to model the surface-associated mode of growth is a prerequisite to understanding more fully the physiological changes involved in such a growth strategy. Key factors believed to influence bacterial persistence in chronic infections are those of the biofilm mode of growth and slow growth rate. Methods for controlling Pseudomonas aeruginosa biofilm population growth rates were investigated in this project. This microorganism was incompatible with the in vitro 47mm diameter membrane filter-based biofilm technique developed for the study of Escherichia coli and Staphylococcus epidermidis by Gilbert et al (Appl. Environ. Microbiol. 1989, 55, 1308-1311). Two alternative methods were designed. The first comprised a 25mm diameter cellulose acetate membrane filter supported in an integral holder. This was found to be limited to the study of low microbial population densities with low flow rates. The second, based on a cylindrical cellulose fibre depth filter, permitted rapid flow rates to be studied and allowed growth rate control of biofilm and eluted cells. Model biofilms released cells to the perfusing medium as they grew and divided. The viability of released cells was reduced during, and shortly after, inclusion of ciprofloxacin in the perfusate. Outer membrane profiles of biofilm populations exhibited at least two bands not apparent in planktonic cells grown in batch and chemostat culture, and LPS profiles of biofilm populations showed variation with growth rate. Cell surface hydrophobicity of resuspended biofilm cells varied little with growth rate, whilst it decreased markedly for cells released from the biofilms as growth rate increased. Cells released from the biofilm were more hydrophilic than their sessile counterparts. Differing growth rates, LPS profiles and hydrophobicity are proposed to have a bearing on the release of cells from the adherent population.

Studies of skin antisepsis and enhanced penetration of chlorhexidine

Current evidence-based guidelines recommend that 2% (w/v) chlorhexidine digluconate (CHG), preferentially in 70% (v/v) isopropyl alcohol (IIPA), is used for skin antisepsis prior to incision of the skin. In this current study, the antimicrobial efficacy of CHG, six essential oils [tea tree oil (TTO), thymol, eucalyptus oil (EO), juniper oil, lavender oil and citronella] and novel benzylidenecarboxamidrazone and thiosemicarbazone compounds were determined against a panel of microorganisms commonly associated with skin infection (Staphylococcus epidermidis, S. aureus, meticillin-resistant S. aureus, Propionibacterium acnes, Acinetobacter spp., Pseudomonas aeruginosa and Candida albicans) The results demonstrated synergistic activity of CHG in combination with EO against biofilm cultures of S. epidermidis, with significantly reduced concentrations of CHG and EO required to inhibit biofilm growth compared to CHG or EO alone. Skin permeation of CHG was subsequently investigated using an in vitro human skin model (Franz cell) and the penetration profile was determined by serial sectioning of the full thickness human skin. Two percent (w/v) CHG in aqueous solution and in 70% (v/v) IPA demonstrated poor skin permeation; however, the skin permeation was significantly enhanced in combination with 5% - 50% (v/v) EO. Detectable levels of CHG did not permeate through full thickness skin in 24 h. Skin permeation of 2% (w/v) CHG in 70% (v/v) IPA in the presence of 10% (v/v) EO was subsequently studied. The results demonstrated a significantly enhanced skin penetration of CHG after a 2 min application, with CHG detected at significant levels to a depth of 600 m with CHG in combination with EO and IPA compared to 100 m with IPA alone. Combination antisepsis comprising CHG and EO may be beneficial for skin antisepsis prior to invasive procedures to reduce the number of microorganisms on and within the skin due to enhanced skin penetration of CHG and improved efficacy against S. epidermidis in a biofilm mode of growth.

Antibacterial activity of low amperage and low voltage electric current (DC)

Infection is a major clinical problem associated with the use of intravenous catheters.The efficacy of a direct electric current (10µA, 9V) via electrode-conducting carbon impregnated catheters to prevent colonisation of catheters by micro-organisms was investigated. The range of organisms susceptible to 10µA was determined by a zone of inhibition test. The catheters acting as the anode and the cathode were inserted into a nutrient agar plate inoculated with a lawn of bacteria. There was no zone of inhibition observed around the anode. Organisms susceptible to 10µA at the cathode were Staphylococcus aureus (2 strains), Staphylococcus epidermidis (5 strains), Escherichia coli and Klebsiella pneumoniae (2 strains each), and one strain of the following micro-organisms: Staphylococcus hominis, Proteus mirabilis, Pseudomonas aeruginosa and Candida albicans. The zones ranged from 6 to 16 mm in diameter according to the organisms under test. The zone size was proportional to the amperage (10 - 100 µA) and the number of organisms on the plate. Ten µA did not prevent adhesion of staphylococci to the cathode nor did it affect their growth in nutrient broth. However, it was bactericidal to adherent bacteria on the cathodal catheter and significantly reduced the number of bacteria on the catheter after 4 to 24 h application of electricity. The antimicrobial activity of low amperage electric current under anaerobic conditions and in the absence of chloride ions against bacteria attached to the surface of a current carrying electrode was also investigated.The mechanisms of the bactericidal activity associated with the cathode were investigated with S. epidermidis and S. aureus. The inhibition zone was greatly reduced in the presence of catalase. There was no zone around the cathode when the test was carried out under anaerobic conditions. Hydrogen peroxide was produced at the cathode surface under aerobic conditions, but not in the absence of oxygen. A salt-bridge apparatus was used to demonstrate further that hydrogen peroxide was produced at the cathode, and chlorine at the anode. The antimicrobial activity of low amperage electric current under anaerobic conditions and in the absence of chloride ions against bacteria attached to the surface of a current carrying electrode was also investigated. Antibacterial activity was reduced under anaerobic conditions, which is compatible with the role of hydrogen peroxide as a primary bactericidal agent of electricity associated with the cathode. A reduction in chloride ions did not significantly reduce the antibacterial activity suggesting chlorine plays only a minor role in the bactericidal activity against organisms attached to anodal electrode surfaces. The bactericidal activity of electric current associated with the cathode and H202 was greatly reduced in the presence of 50 μM to 0.5 mM magnesium ions in the test menstrum. Ten μA applied via the catheters did not prevent the initial biofilm growth by the adherent bacteria but reduced the number of bacteria in the biofilm by 2 log order aiter 24 h. The results suggested that 10 μA may prevent the colonisation of catheters by both the extra~ and intra-luminal routes. The localised production of hydrogen peroxide and chlorine and the intrinsic activity due to electric current may offer a useful method for the eradication of bacteria from catheter surfaces.

Delivery of the antibiotic gentamicin sulphate from precipitation cast matrices of polycaprolactone

Microporous, poly(ε-caprolactone) (PCL) matrices were loaded with the aminoglycoside antibiotic, gentamicin sulphate (GS) using the precipitation casting technique by suspension of powder in the PCL solution prior to casting. Improvements in drug loading from 1.8% to 6.7% w/w and distribution in the matrices were obtained by pre-cooling the suspension to 4°C. Gradual release of approximately 80% of the GS content occurred over 11 weeks in PBS at 37°C and low amounts of antibiotic were measured up to 20 weeks. The kinetics of release could be described effectively by the Higuchi model with the diffusion rate constant (D) increasing from of 1.7 to 5.1 μg/mg matrix/day0.5 as the drug loading increased from 1.4% to 8.3% w/w. GS-loaded PCL matrices retained anti-bacterial activity after immersion in PBS at 37°C over 14 days as demonstrated by inhibition of growth of S. epidermidis in culture. These findings recommend further investigation of precipitation-cast PCL matrices for delivery of hydrophilic molecules such as anti-bacterial agents from implanted, inserted or topical devices. © 2005 Elsevier B.V. All rights reserved.

Efeito imunomodulador e atividade antimicrobiana de heterofucanas de Sargassum filipendula

Fucans, sulphated polysaccharides that contain L-fucose in its constitution, obtained from species of Phaeophyceae of the Sargassum kind, display several biological activities. Heterofucans from Sargassum filipendula are bioactive molecules that contain strong antiproliferative and antioxidant activity. However, their immunomodulatory and antimicrobial activities have not yet been examined. In this context, the aim of this research was to evaluate the heterofucans as for their immunomodulatory capacity and antimicrobial action against Leishmania infantum, Trichomonas vaginalis, Staphylococcus epidermidis and Klebsiella pneumonia (KPC). The five heterofucans obtained from S. filipendula show activities that are distant as stimulants of the immune system and microbial agent. The SF0.5V, SF0.7V amd SF1.0V heterofucans were capable of acting in the activation of murine and human macrophages. In addition to that, SF0.5V has shown antibiofilm activity of S. epidermides and SF0.7V and 1.0V almost completely inhibited the survival of the protozoan T. vaginalis. Results such as this one, reflect the broad range of action of the sulphated polysaccharides obtained from seaweeds, especially from the species S.filipendula

Extração, caracterização e prospecção de atividades farmacológicas de polissacarídeos sulfatados da macroalga verde Caulerpa prolifera

This study aimed to extract, characterize and conduct a prospective analysis of pharmacological activities of sulfated polysaccharides from green seaweed Caulerpa prolifera. Seven fractions (CP-0.3/CP-0.5/CP-0.7/CP-0.9/CP-1.1/CP-1.5/CP-2.0) were obtained from C. prolifera by alkaline proteolysis followed by sequential precipitation in acetone. The physicochemical analyzes indicated that C. prolifera synthesizes a homogalactan (CP-0.9) and different populations of sulfated heteropolysaccharides. In the analysis of anticoagulant activity, all fractions except CP-0.3, influenced the intrinsic coagulation pathway. All fractions showed antioxidant activity in six different assays being more pronounced in hydrogen peroxide scavenging assay, especially CP-0.3, CP-0.7 and CP-0.9 (which obtained 61% of hydrogen peroxide scavenging), in ferric chelation assay (especially CP-0.9 with 56% chelation) and cupric chelation assay (especially CP-2.0 with 78% chelation). With respect to immunomodulatory activity, the presence of CP-0.3, CP-0.7 and CP-0.9 showed an immunogenic potential, increasing the production of nitric oxide (NO) by 48, 142 and 163 times, respectively. Conversely, the NO synthesis fell 73% after the activation of macrophages by LPS, incubated concurrently with CP-2.0. The anti-adipogenic activity of the fractions was also evaluated and CP-1.5 was able to reduce the differentiation of pre-adipocytes (3T3-L1) into adipocytes by 60%, without affecting the cell viability. The fractions CP-0.3, CP-0.5 and CP-0.9 reduced the viability of the HeLa cells (human cervical adenocarcinoma) by 55% and CP-1.5 reduced the viability of the 786-0 cells (human renal adenocarcinoma) by 75%. Leishmanicidal activity and microbicide effect against Carbapenem-resistant Klebsiella pneumoniae (KPC) have not been identified. However, the viability of Staphylococcus epidermidis was reduced by 23.8% in the presence of CP -1.5. All fractions were able to change the formation of calcium oxalate crystals. CP-0.3, CP-0.5 and CP-1.1 only promoted the formation of COD type crystals with a very small size (1 μm). Confocal microscopy and zeta potential data of crystals formed in the presence of the samples showed that the polysaccharides present in the fractions must interact with calcium ions present throughout the crystal lattice, affecting the growth and morphology of crystals The results described herein indicate that the fractions rich in polysaccharides obtained from the green seaweed C. prolifera present a multi therapeutic potential, and subsequent purification steps, as well as research on the mechanisms of action by which these polymers act should be investigated.