Production of TNF-a by primary cultures of human keratinocytes challenged with Loxosceles gaucho venom

Primary cultures of human keratinocytes were challenged with increasing doses from 10 ng/mL to 2 mg/mL of Loxosceles gaucho venom, responsible for dermonecrotic lesion in humans. TNF-a was investigated by bioassay and ELISA in the supernatant of the cultures challenged with 100 ng/mL, 500 ng/mL, 1 and 2 mg/mL of venom. TNF-a was detected by bioassay in the supernatant of cultures challenged with 100 ng/mL, after 6 h. The cytokine was detected by ELISA in the supernatant of the cells challenged with doses of l mg/mL, after 6 and 12 h. The results point out the capacity of this venom to activate the keratinocytes in primary cultures to produce TNF-a. The production of cytokines could contribute to the local inflammatory process in patients bitten by Loxosceles sp.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 µg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 µg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.

The use of the antifungal agent miconazole as an inhibitor of Blastocystis hominis growth in Entamoeba histolytica/E. dispar cultures

In regions with high prevalence, Blastocystis hominis is frequently found in association with Entamoeba histolytica/E. dispar in xenic cultures. Its exacerbated growth is often superimposed on the growth of amebas, thus impeding the continuation of the amebas in the culture, within a few generations. The present study reports on the excellent efficacy (100%) of the antifungal agent miconazole in eliminating B. hominis from cultures of E. histolytica/E. dispar, thereby maintaining the integrity of the trophozoites of the amebas. Nystatin presented low efficacy (33.3%).

Histopathology, serology and cultures in the diagnosis of cryptococcosis

Cryptococcosis is one of the most common opportunistic fungal infections in patients with acquired immunodeficiency syndrome (AIDS). We report 13 cases of cryptococcal infection based on histopathology, serology and cultures. Epidemiological analysis, histochemical techniques of hematoxilin and eosin (HE) and Grocot's silver (GMS), as well special histochemical techniques such as Mayer's mucicarmine (MM) and Fontana-Masson (FM), cryptococcal antigen test (CrAg) and isolation on fungal media: Sabouraud's (SAB), brain-heart infusion agar (BHI) and canavanine-glycine-bromothymol blue (CGB) agar were analyzed. Unsatisfactory staining results by MM stain associated to negative titers by CrAg test, which FM stain confirmed that capsule-deficient Cryptococcus infections were observed in four cases. Eight isolated cases were identified as follows: six cases were infection with Cryptococcus neoformans and two cases were Cryptococcus gattii.

Apoptotic-like activity of staurosporine in axenic cultures of Trypanosoma evansi

Trypanosoma evansi is a blood protozoan parasite of the genus Trypanosoma which is responsible for surra (Trypanosomosis) in domestic and wild animals. This study addressed apoptotic-like features in Trypanosoma evansi in vitro. The mechanism of parasite death was investigated using staurosporine as an inducing agent. We evaluated its effects through several cytoplasmic features of apoptosis, including cell shrinkage, phosphatidylserine exposure, maintenance of plasma membrane integrity, and mitochondrial trans-membrane potential. For access to these features we have used the flow cytometry and fluorescence microscopy with cultures in the stationary phase and adjusted to a density of 10(6) cells/mL. The apoptotic effect of staurosporine in T. evansi was evaluated at 20 nM final concentration. There was an increase of phosphatidylserine exposure, whereas mitochondrial potential was decreased. Moreover, no evidence of cell permeability increasing with staurosporine was observed in this study, suggesting the absence of a necrotic process. Additional studies are needed to elucidate the possible pathways associated with this form of cell death in this hemoparasite.

SUSCEPTIBILITY TO ANTIBIOTICS IN URINARY TRACT INFECTIONS IN A SECONDARY CARE SETTING FROM 2005-2006 AND 2010-2011, IN SÃO PAULO, BRAZIL: DATA FROM 11,943 URINE CULTURES

Introduction: Urinary tract infection (UTI) has a high incidence and recurrence, therefore, treatment is empirical in the majority of cases. Objectives: The aim of this study was to analyze the urine cultures performed at a secondary hospital, during two periods, 2005-2006 and 2010-2011, and to estimate the microbial resistance. Patients and methods: We analyzed 11,943 aerobic urine cultures according to basic demographic data and susceptibility to antibiotics in accordance with the Clinical and Laboratory Standards Institute (CLSI) for Vitek 1 and 2. Results: Most of our cohort consisted of young adult females that were seen at the Emergency Department. E. coli was the most frequent (70.2%) among the 75 species isolated. Resistance of all isolates was ≥ 20% for trimethoprim/sulfamethoxazole (TMP/SMX), norfloxacin, nitrofurantoin, cefazolin and nalidixic acid. Although E. coli was more susceptible (resistance ≥ 20% for TMP/SMX and nalidixic acid) among all of the isolates, when classified by the number and percentage of antibiotic resistance. Global resistance to fluoroquinolones was approximately 12%. Risk factors for E. coli were female gender and an age less than 65 years. Men and patients older than 65 years of age, presented more resistant isolates. Extended spectrum beta-lactamases (ESBL) were identified in 173 out of 5,722 Gram-negative isolates (3.0%) between 2010 and 2011. Conclusion: E. coli was the most frequent microbe isolated in the urine cultures analyzed in this study. There was a significant evolution of bacterial resistance between the two periods studied. In particular, the rise of bacterial resistance to fluoroquinolones was concerning.

SUSCEPTIBILITY OF Candida spp. ISOLATED FROM BLOOD CULTURES AS EVALUATED USING THE M27-A3 AND NEW M27-S4 APPROVED BREAKPOINTS

The high mortality rates associated with candidemia episodes and the emergence of resistance to antifungal agents necessitate the monitoring of the susceptibility of fungal isolates to antifungal treatments. The new, recently approved, species-specific clinical breakpoints (SS-CBPs)(M27-S4) for evaluating susceptibility require careful interpretation and comparison with the former proposals made using the M27-A3 breakpoints, both from CLSI. This study evaluated the susceptibility of the different species of Candida that were isolated from candidemias based on these two clinical breakpoints. Four hundred and twenty-two isolates were identified and, among them, C. parapsilosis comprised 46.68%, followed by C. albicans (35.78%), C. tropicalis (9.71%), C. glabrata (3.55%), C. lusitaniae (1.65%), C. guilliermondii (1.65%) and C. krusei (0.94%). In accordance with the M27-A3 criteria, 33 (7.81%) non-susceptible isolates were identified, of which 16 (3.79%) were resistant to antifungal agents. According to SS-CBPs, 80 (18.95%) isolates were non-susceptible, and 10 (2.36%) of these were drug resistant. When the total number of non-susceptible isolates was considered, the new SS-CBPs detected 2.4 times the number of isolates that were detected using the M27-A3 interpretative criteria. In conclusion, the detection of an elevated number of non-susceptible species has highlighted the relevance of evaluating susceptibility tests using new, species-specific clinical breakpoints (SS-CBPs), which could impact the profile of non-susceptible Candida spp. to antifungal agents that require continuous susceptibility monitoring.

The un-infectivity of the PF cultivated strain of trypanosoma cruzi to mice. An evaluation through a one year period by blood cultures and histopathology

Trypanosoma cruzi of the cultivated PF strain when injected in mice per subcutaneous route, in adequate doses, is able to induce an efficient sterile immunization in the animais (for at least one year) as determined by whole blood cultures and histopathology.

Detection of viable and viable nonculturable Vibrio cholerae O1 through cultures and immunofluorescence in the Tucumán rivers, Argentina

Vibrio cholerae has been sporadically isolated from rivers in Tucumán, Argentina, since the outbreak in 1991. The aim of this study was to determine the environmental reservoir of the bacterium in these rivers, assessing the presence of Vibrio cholerae non-O1 and O1 (the latter both in its viable culturable and non culturable state) and its relationship to environmental physicochemical variables. 18 water samplings were collected in the Salí River (in Canal Norte and Banda) and the Lules River between 2003 and 2005. Physical-chemical measurements (pH, water temperature, electrical conductivity and dissolved oxygen) were examined. Vibrio cholerae was investigated with conventional culture methods and with Direct Immunofluorescence (DFA-VNC) in order to detect viable non culturable organisms. All isolated microorganisms corresponded to Vibrio cholerae non-O1 and non-O139 (Lules 26%, Canal Norte 33% and Banda 41%). The majority was found during spring and summer and correlated with temperature and pH. Non culturable Vibrio cholerae O1 was detected year round in 38 of the 54 water samples analyzed. Application of the Pearson correlation coefficient revealed that there was no relationship between positive immunofluorescence results and environmental physicochemical parameters. Genes coding for somatic antigen O1 were confirmed in all DFA-VNC-positive samples, whereas the virulence-associated ctxA and tcpA genes were confirmed in 24 samples.

Characterization of a new coagulase-negative Staphylococcus species (Staphylococcus pettenkoferi) isolated from blood cultures from a hospitalized patient in Porto Alegre, Brazil

INTRODUCTION: Staphylococcus pettenkoferi was originally isolated and described by Trülzsch et al (2002). In this study, we characterized two isolates of this newly described species. METHODS: Blood cultures were initially processed using the BacT/ALERT® device, and the isolates were initially characterized using the Vitek2 identification system. RESULTS: The initial characterization revealed slow-growing Gram-positive cocci that formed opaque colonies on sheep blood agar. Other phenotypic/genotypic tests were performed. CONCLUSIONS: We would like to emphasize that this new staphylococcus species is phenotypically similar to other CoNS, especially S. auricularis. This could potentially lead to misidentification of these uncommon species.

The induction of root and shoot morphogenesis in Phaseolus vulgaris tissue cultures

Exogenous concentrations of bean seed extract prepared from seeds pretreated in aerated water, homogenized in Veliky and Martin's 67-V salt solution, filtered, and added to the culture medium at proper concentrations promote callus proliferation, root morphogenesis, and shoot morphogenesis in leaf explains of Phaseolus vulgaris var. Bico de Ouro. The activity of the bean seed factor is dependent upon the period of pretreatment in aerated water.

Atividade antibacteriana do Aspergillus niger van Tieghem, 1867; pesquisas em 14 amostras da National Collection of Type Cultures (M. R. C.)

1. Pesquisamos a atividade antibacteriana em 14 amostras de Aspergillus niger da National Collection of Type Cultures. 2. Em meio de Raulin e Mosseray, sete amostras apresentaram atividade total, nunca superior a 1:10, contra Staphylococcus aureus nº 553, sendo que as amostras 1.161 e 2.390 permaneceram ativas por mais de 40 dias. 3. A utilização do meio de Czapek-Dox com 5% de "corn-steep" não melhorou os resultados obtidos com o meio de Raulin e Mosseray. 4. No meio de levedo peptonado, todas as amostras apresentaram-se inativas.

Rapid detection by transmission electron microscopy of mycoplasma contamination in sera and cell cultures

Transmission electon microscopy has been employed for the rapid detection of mycoplasma in sera and cell cultures. High speed centrifugation of sera or low speed centrifugation of cell debris, followed by negative staining of the resuspended pellet, detected mycoplasma contamination more frequently than a culture method followed by direct fluorescence (DAPI), which was used as a control procedure. The appearance of the mycoplasma cell border and content gives some information about particle viability.