Amauri Braga Simonetti

Nanobacteria-like particles: a threat to cell cultures

The main goal of this study is to alert researchers who work with cell cultures for the risk of contamination by structures called nanobacteria (NB). NB are tiny structures with size varying from 80 to 500 nm, commonly occurring in clusters and producing a biofilm which contains carbonate or hydroxyl apatite. The most likely source of cell culture contamination by such organisms is serum used as supplement in culture media. The presence of NB leads to a progressive culture deterioration with accumulation of granules (probably phagocytized NB) in cytoplasmic vacuoles, an increasing number of dead cells in the supernatant and degeneration of cells that remained attached to the bottom of the vessel. NB can also be found in culture supernatants where they are found in clusters with variable size and displaying brownian movement. In this study, 19 cell lineages, 8 batches of sera and 1 batch of growth supplement from different sources were analyzed. Samples from sera were cultured in Eagle’s Minimum Essential Medium (E-MEM) or incubated directly at 37oC. Tests carried out to detect the presence of extracellular bacteria, Mycoplasma sp and viruses were all negative. Analysis by scanning electron microscopy (SEM) revealed tiny oval structures less than 500 nm in size, isolated or in small groups, in all material analyzed except in one fetal bovine serum batch.

Isolamento e identificação de amebas de vida livre potencialmente patogênicas em amostras de ambientes de hospital público da cidade de Porto Alegre, RS

A study on the presence of free-living amoebae in a public hospital was developed in the city of Porto Alegre, State of Rio Grande do Sul. Dust and biofilms were collected using sterile swabs that had been prepared for this study, from 15 hospital environments, including the intensive care center, pediatric intensive care unit, kitchen, emergency room, outpatient surgical center, clinical surgical center, water storage tanks, taps and six drinking fountains for general use, every month from July 2004 to March 2005. The FLAs were isolated by culturing, using non-nutrient agar medium with the addition of heat-killed Escherichia coli. The protozoa were identified by morphological observation of cysts and trophozoites, in accordance with Pages morphological criteria (1988). Among the 135 samples collected from the 15 environments, 47 (35%) were positive for FLAs. Of these, thirty-four percent presented morphological characteristics particular to the genus Acanthamoeba.

The biology of malarial parasite in the mosquito: a review

The purpose of this review is to summarize the biology of Plasmodium in the mosquito including recent data to contribute to better understanding of the developmental interaction between mosquito and malarial parasite. The entire sporogonic cycle is discussed taking into consideration different parasite/vector interactions and factors affecting parasite development to the mosquito.

CONJUNCTIVAL ENDOGENOUS MICROBIOTA IN PATIENTS SUBMITTED TO CATARACT SURGERY

Bacterial isolation, identification and antimicrobial susceptibility tests were carried out in ocular material collected with swab and polimethylmethacrylate (PMMA) or silicone intraocular lenses (IOL) from forty six patients submitted to cataract surgery. Seventy six isolates and seven different microorganisms were identified. Coagulase-negative staphylococci (CNS) were the predominant microorganisms isolated from swabs (71.4% of cases), PMMA lenses (81.3%) and silicon lenses (77.8%). Coagulase-negative staphylococci isolates revealed high resistance to penicillin G followed by tetracycline, chloramphenicol and aminoglicosides. However, these isolates displayed great susceptibility to vancomycin, cephalothin and ofloxacin. Except for penicillin G, Staphylococcus aureus was very sensitive to the antimicrobial agents including oxacillin. Among Gram-negatives, Proteus mirabilis was prevalent and presented high resistance to tetracycline and chloramphenicol. Enterococcus isolates were vancomycin sensitive.

Redução de cromo hexavalente por bactérias isoladas de solos contaminados com cromo

The reduction of Cr(VI) to Cr(III) decrease the toxic effect of this metal in the environment, because Cr(III) is insoluble to the biological membranes. The microbial reduction of Cr(VI) it is an alternative to reduce the environmental impacts caused by this metal used in several industrial processes. The objective of this research was to select microorganisms from chromium contaminated soil and to characterize their ability to reduce Cr(VI). The activity of reduction of Cr(VI) for the isolated was quantified with s-diphenylcarbazide. A group of 20 chromium resistant bacteria were isolated; six of these were able to reduce 100mg L -1 Cr(VI) in 24 hours. The isolated bacteria, from contaminated soil can remediate chromate and presented potential for other studies seeking their application in bioremediation processes.

Aderência bacteriana in vitro a lentes intra-oculares de polimetilmetacrilato e de silicone

PURPOSE: To evaluate bacterial adherence to silicone and polymetylmethacrylate (PMMA) intraocular lenses as a risk factor for postsurgery endophthalmitis by using an in vitro model with three potentially pathogenic microorganisms. METHODS: In vitro experiments were carried out with the Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis (clinical isolate) and Pseudomonas aeruginosa ATCC 27853 strains including growth curves, tests to verify capsule production, hydrophobicity and adherence to different materials as well as optical microscopy, scanning electronic microscopy (SEM) and atomic force microscopy (AFM). RESULTS: No relation between capsule production, adherence of the strains tested and amount of microorganisms was observed; no statistically significant differences were detected between S. aureus and S. epidermidis adherence to polymetylmethacrylate and silicone intraocular lenses; P. aeruginosa was the most adherent microorganism to both materials. This adherence pattern was confirmed by SEM, while biofilm production by the three strains was visualized by AFM. CONCLUSIONS: In vitro experiments showed no differences of bacterial adherence between PMMA and silicone lenses, but P. aeruginosa displayed a greater level of adherence in relation to staphylococci. All three strains were shown to produce biofilm. Silicone was shown to be more hydrophobic when compared to polymethylmethacrylate.

Growth of Paecilomyces variotii in B0 (diesel), B100 (biodiesel) and B7 (blend), degradation and molecular detection

The introduction of biodiesel to diesel may allow the fuel to be more susceptible to microorganism growth, especially during incorrect storage. To evaluate the effect of adding biodiesel in pure diesel on the growth of Paecilomyces variotii, microcosms containing pure diesel (B0), blend diesel/biodiesel (B7) and pure biodiesel (B100) were used. In microcosm with minimal mineral medium and B0, B7 or B100, after 60 days, the biomass (dry weight) formed at interface oil-water in B7 and B100 was significantly higher when compared to that of B0. Infrared analysis showed reduction of the carbonile fraction in B7 and B100 suggesting formation of intermediate compounds in B7. To monitor possible contamination of fuel storage tank by P. variotii samples were collected and analysed by specific-PCR assay for detection of P. variotii spores in the aqueous phase. This method was able to detect a minimum of 103 spores ml-1, corresponding to 0.0144 ng µl-1 of DNA. Specificity was tested against Aspergillus fumigatus and Pseudallescheria boydii.

Phylogenetic characterization of bovine parainfluenza 3 from contaminated cell cultures and field isolates from Brazil

Genomic fragments of the HN and L genes from Brazilian bovine parainfluenza 3 virus (bPIV-3) isolated as contaminants from cell cultures and clinical specimens were amplified by reverse transcription-polymerase chain reaction (RT-PCR), sequenced using specific degenerate primers and analyzed by phylogenetic comparison with reference strains of bPI3V. The Brazilian isolates revealed a high degree of genomic when compared to SF4/32 prototype strain, within the recently proposed genotype A of bPIV-3.

Detecção do vírus da cinomose canina por RT-PCR utilizando-se oligonucleotídeos para os genes da fosfoproteína, hemaglutinina e neuraminidase

The reverse transcription-polymerase chain reaction was used to detect canine distemper virus (CDV). Four oligonucleotide pairs were selected (P1, P2, N1, H1), based on the sequences of the phosphoprotein, hemagglutinin and nuraminidase genes for assay standardization, and three CDV vaccine strains were used as positive controls. Three viral isolates from dogs with canine distemper and four samples from animals clinically suspected of distemper were analysed. No amplification was detected in suspected samples. Results obtained by using P1 and N1 oligonucleotides were superior to those with H1 ones. P2 oligonucleotides were considered inadequate for CDV detection. Amplicons resulting from amplification of P1, N1 and H1 oligonucleotides were submitted to cleavage by restriction endonucleases and restriction patterns of viral samples were compared to that of Lederle strain used as reference. A similar restriction pattern was observed in all analysed samples.