Induction and secretion of elastinolytic and proteolytic activity in cultures of Paracoccidioides brasiliensis

P. brasiliensis parasitizes various human tissues and proteinases exported by this fungus may allow it to metabolize and invade host tissues. The influence of the culture medium on the production of proteinases by P. brasiliensis isolates was studied and the export of these enzymes was followed as a function of culture time. The fungus was grown in neopeptone, BSA, elastin or collagen medium. The culture medium was assayed for azocollytic, elastinolytic and caseinolytic activity. Proteolytic activity was also analysed by electrophoresis of the culture medium on gelatin and casein substrate gels. P. brasiliensis expressed relatively high levels of azocoll, elastin and casein degrading activity in all types of medium, except in neopeptone medium. Generally, expression of azocollytic activity peaked during the third week of culture and caseinolytic activity during the fourth week of culture. Azocollytic activity was highest at pH 4.0 and caseinolytic activity at pH 8.0. Elastinolytic activity was also highest at pH 8.0. This activity, as well as the others, may provide the fungus with a source of carbon and nitrogen and may also be responsible for the invasion of host tissues, such as pulmonary elastic fiber, by P. brasiliensis.

Susceptibility of planktonic cultures of Streptococcus mutans to photodynamic therapy with a light-emitting diode

The objective of this study was to evaluate the effect of photodynamic therapy with erythrosine and rose bengal using a light-emitting diode (LED) on planktonic cultures of S. mutans. Ten S. mutans strains, including nine clinical strains and one reference strain (ATCC 35688), were used. Suspensions containing 10 6 cells/mL were prepared for each strain and were tested under different experimental conditions: a) LED irradiation in the presence of rose bengal as a photosensitizer (RB+L+); b) LED irradiation in the presence of erythrosine as a photosensitizer (E+L+); c) LED irradiation only (P-L+); d) treatment with rose bengal only (RB+L-); e) treatment with erythrosine only (E+L-); and f) no LED irradiation or photosensitizer treatment, which served as a control group (P-L-). After treatment, the strains were seeded onto BHI agar for determination of the number of colony-forming units (CFU/mL). The results were submitted to analysis of variance and the Tukey test (p ≤ 0.05). The number of CFU/mL was significantly lower in the groups submitted to photodynamic therapy (RB+L+ and E+L+) compared to control (P-L-), with a reduction of 6.86 log 10 in the RB+L+ group and of 5.16 log 10 in the E+L+ group. Photodynamic therapy with rose bengal and erythrosine exerted an antimicrobial effect on all S. mutans strains studied.

Women, cultures and development: views from Latin America

Includes bibliography

Photodynamic inactivation of planktonic cultures and biofilms of Candida albicans mediated by aluminum-chloride-phthalocyanine entrapped in nanoemulsions

New drug delivery systems, such as nanoemulsions (NE), have been developed to allow the use of hydrophobic drugs on the antimicrobial photodynamic therapy. This study evaluated the photodynamic potential of aluminum-chloride- phthalocyanine (ClAlPc) entrapped in cationic and anionic NE to inactivate Candida albicans planktonic cultures and biofilm compared with free ClAlPc. Fungal suspensions were treated with different delivery systems containing ClAlPc and light emitting diode. For planktonic suspensions, colonies were counted and cell metabolism was evaluated by XTT assay. Flow cytometry evaluated cell membrane damage. For biofilms, the metabolic activity was evaluated by XTT and ClAlPc distribution through biofilms was analyzed by confocal laser scanning microscopy (CLSM). Fungal viability was dependent on the delivery system, superficial charge and light dose. Free ClAlPc caused photokilling of the yeast when combined with 100 J cm-2. Cationic NE-ClAlPc reduced significantly both colony counts and cell metabolism (P < 0.05). In addition, cationic NE-ClAlPc and free ClAlPc caused significant damage to the cell membrane (P < 0.05). For the biofilms, cationic NE-ClAlPc reduced cell metabolism by 70%. Anionic NE-ClAlPc did not present antifungal activity. CLSM showed different accumulation on biofilms between the delivery systems. Although NE system showed a lower activity for planktonic culture, cationic NE-ClAlPc showed better results for Candida biofilms. Candida albicans biofilm overview after 30 min of contact with free ClAlPc. This study presents the photodynamic potential of aluminum-chloride-phthalocyanine (ClAlPc) entrapped in cationic and anionic nanoemulsions (NE) to inactivate C. albicans planktonic cultures and biofilm comparing with free ClAlPc. The photodynamic effect was dependent on the delivery system, superficial charge and light dose. Cationic NE-ClAlPc and free ClAlPc caused significant reduction in colony counts, cell metabolism and damage to the cell membrane (P < 0.05). However, only the free ClAlPc was able to cause photokilling of the yeast. The anionic NE-ClAlPc did not present antifungal activity. Although NE system showed a lower activity for planktonic culture, cationic NE-ClAlPc showed better results for Candida biofilms. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

Effect of different pre-irradiation times on curcumin-mediated photodynamic therapy against planktonic cultures and biofilms of Candida spp

Objectives: The aim of this study was to evaluate the effects of pre-irradiation time (PIT) on curcumin (Cur)-mediated photodynamic therapy (PDT) against planktonic and biofilm cultures of reference strains of Candida albicans, Candida glabrata and Candida dubliniensis. Materials and methods: Suspensions and biofilms of Candida species were maintained in contact with different concentrations of Cur for time intervals of 1, 5, 10 and 20 min before irradiation and LED (light emitting diode) activation. Additional samples were treated only with Cur, without illumination, or only with light, without Cur. Control samples received neither light nor Cur. After PDT, suspensions were plated on Sabouraud Dextrose Agar, while biofilm results were obtained using the XTT-salt reduction method. Confocal Laser Scanning Microscopy (CLSM) observations were performed to supply a better understanding of Cur penetration through the biofilms after 5 and 20 min of contact with the cultures. Results: Different PITs showed no statistical differences in Cur-mediated PDT of Candida spp. cell suspensions. There was complete inactivation of the three Candida species with the association of 20.0 μM Cur after 5, 10 and 20 min of PIT. Biofilm cultures showed significant reduction in cell viability after PDT. In general, the three Candida species evaluated in this study suffered higher reductions in cell viability with the association of 40.0 μM Cur and 20 min of PIT. Additionally, CLSM observations showed different intensities of fluorescence emissions after 5 and 20 min of incubation. Conclusion: Photoinactivation of planktonic cultures was not PIT-dependent. PIT-dependence of the biofilm cultures differed among the species evaluated. Also, CLSM observations confirmed the need of higher time intervals for the Cur to penetrate biofilm structures. © 2012 Elsevier Ltd. All rights reserved.

Drifter technique: a new method to obtain metaphases in Hep-2 cell line cultures

A linhagem celular Hep-2 é formada por células de carcinoma da laringe e é muito utilizada em modelos de carcinogênese e mutagenêse. Para avaliar o potencial proliferativo desta linhagem, desenvolvemos uma metodologia citogenética (técnica do sobrenadante) para obtenção de metáfases a partir de células que, ao entrarem em mitose, perdem adesão celular, ficando em suspensão no meio de cultura. Através deste procedimento, foram contadas 2000 células, correspondendo a um índice mitótico (IM) de 22.2% . Apesar de o IM obtido por esta técnica não ter sido estatisticamente diferente do IM obtido por outras metodologias citogenéticas clássicas, a técnica do sobrenadante é vantajosa porque elimina o uso de alguns reagentes utilizados na obtenção de metáfases e também diminui o consumo de reagentes de manutenção desta linhagem.

Coagulase-negative staphylococci strains resistant to oxacillin isolated from neonatal blood cultures

Coagulase-negative staphylococci (CoNS) are the microorganisms most frequently isolated from clinical samples and are commonly found in neonatal blood cultures. Oxacillin is an alternative treatment of choice for CoNS infections; however, resistance to oxacillin can have a substantial impact on healthcare by adversely affecting morbidity and mortality. The objective of this study was to detect and characterise oxacillin-resistant CoNS strains in blood cultures of newborns hospitalised at the neonatal ward of the University Hospital of the Faculty of Medicine of Botucatu. One hundred CoNS strains were isolated and the mecA gene was detected in 69 of the CoNS strains, including 73.2% of Staphylococcus epidermidis strains, 85.7% of Staphylococcus haemolyticus strains, 28.6% of Staphylococcus hominis strains and 50% of Staphylococcus lugdunensis strains. Among these oxacillin-resistant CoNS strains, staphylococcal cassette chromosome mec (SCCmec) type I was identified in 24.6%, type II in 4.3%, type III in 56.5% and type IV in 14.5% of the strains. The data revealed an increase in the percentage of CoNS strains isolated from blood cultures from 1991-2009. Furthermore, a predominant SCCmec profile of the oxacillin-resistant CoNS strains isolated from neonatal intensive care units was identified with a prevalence of SCCmec types found in hospital-acquired strains.

Influence of the combination of probiotic cultures during fermentation and storage of fermented milk

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cell cultures of Maytenus ilicifolia Mart. are richer sources of quinone-methide triterpenoids than plant roots in natura

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic determinants of methicillin resistance and virulence among Staphylococcus aureus isolates recovered from clinical and surveillance cultures in a Brazilian teaching hospital

Aims. To quantify the presence of SCCmec types and virulence genes among Staphylococcus aureus colonizing and infecting patients from a teaching hospital. Methods. We analyzed 225 and 84 S. aureus isolates recovered from surveillance and clinical cultures, respectively. Strains were studied for the presence and type of SCCmec, as well as for several virulence genes. Univariate and multivariable analysis were performed in order to identify predictors of invasiveness (defined as isolation from clinical cultures). Results. The presence of SCCmec types III (OR, 2.19, 95% CI, 1.08-4.45) and IV (OR, 5.28 95% CI, 1.35-20.63) and of genes coding for exfoliative toxin B (etb, OR, 6.38, 95% CI, 1.48-27.46) and Panton-Valentine leukocidin (pvl, OR, 2.38, 95% CI, 1.16-4.86) was independently associated with invasiveness. Conclusions. SCCmec types III and IV and virulence genes are associated with greater invasiveness of S. aureus. Patients colonized with methicillin-resistant S. aureus, as well as with strains harboring etb or pvl, may be prone to develop invasive disease. Infection-preventing strategies should be more intensively applied to this group.

ANXA1Ac₂₋₂₆ peptide reduces ID1 expression in cervical carcinoma cultures

Cervical cancer is the second most frequent cancer in women worldwide and is associated with genetic alterations, infection with human papilloma virus (HPV), angiogenesis and inflammatory processes. The idea that inflammation is involved in tumorigenesis is supported by the frequent appearance of cancer in areas of chronic inflammation. On the other hand, the inflammatory response is controlled by the action of anti-inflammatory mediators, among these mediators, annexin A1 (ANXA1), a 37 kDa protein was detected as a modulator of inflammatory processes and is expressed by tumor cells. The study was carried out on the epithelial cancer cell line (SiHa) treated with the peptide of annexin A1 (ANXA1Ac2-26). We combined subtraction hybridization approach, Ingenuity Systems software and quantitative PCR, in order to evaluate gene expression influenced by ANXA1. We observed that ANXA1Ac2-26 inhibited proliferation in SiHa cells after 72h. In these cells, 55 genes exhibited changes in expression levels in response to peptide treatment. Six genes were selected and the expression results of 5 up-regulated genes (TPT1, LDHA, NCOA3, HIF1A, RAB13) and one down-regulated gene (ID1) were research by real time quantitative PCR. Four more genes (BMP4, BMPR1B, SMAD1 and SMAD4) of the ID1 pathway were investigated and only one (BMPR1B) shows the same down regulation. The data indicate the involvement of ANXA1Ac2-26 in the altered expression of genes involved in tumorigenic processes, which could potentially be applied as a therapeutic indicator of cervical cancer.

Chromosome aberrations in lymphocyte cultures from paracoccidioidomycosis patients

Blood cell lymphocyte chromosomes from untreated (UT) and clinically-cured (CC) patients with paracoccidioidomycosis and from healthy (control) people (CO) were studied. The frequency of aneuploid cells in the UT patients was higher than in the CC and CO individuals. The frequency of metaphase cells with premature centromere division was significantly higher in the UT than in the CC and CO group. No structural aberration and no statistically significant difference in the frequency of polyploidy was observed in the three groups studied. Our findings are indicative of an aneugenic (aneuploidy-inducing) action of infection by Paracoccidioides brasiliensis.