Comparison of microbial numbers in soils by using various culture media and temperatures

The influence of different media and incubation temperatures on the quantification of microbial populations in sorghum, eucalyptus and forest soils was evaluated. Microbial growth was compared by using complex (tryptone soybean agar, TSA, casein-starch, CS, and Martin) and saline (Thorton, M3, Czapeck) media and incubation temperatures of 25 and 30° C. Higher numbers of total bacterial. and fungal colony-forming units (CFU) were observed in sorghum soils, and of spore-forming and Gram-negative bacteria in forest soils than other soils. Actinomycetes counts were highest in forest soil when using CS medium at 30° C and in sorghum soil at 25° C in M3 medium. Microorganism counts were dependent on the media and incubation temperatures. The counts at temperatures of 30° C were significantly higher than at 25° C. Microbial quantification was best when using TSA medium for total. and spore-forming bacteria, Thorton for Gram-negative bacteria, M3 for actinomycetes, and Martin for fungi. © 2005 Elsevier GmbH. All rights reserved.

Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

Background: Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli ( Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli. Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells.Results: Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-beta-D-thiogalactopyranoside ( IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo, showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture.Conclusion: The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.

Oral microbial colonization in patients with systemic lupus erythematous: correlation with treatment and disease activity

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

MICROBIAL COUNTS of DARK RED LATOSOL SAMPLES STORED AT DIFFERENT TEMPERATURES

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

In vitro antibacterial efficacy of endodontic irrigants against Enterococcus faecalis

Objective. The objective of this study was to compare the in vitro antimicrobial activity of 2% chlorhexidine gel against Enterococcus faecalis with sodium hypochlorite in 2 different concentrations (1.5% and 5.25%).Study design. Eighty human lower premolars with single root canals were prepared, autoclaved, and infected for 7 days with E. faecalis monocultures. The roots were then separated into 5 experimental groups according to the irrigant solution used during the standardized preparation. To assess the antimicrobial action of the irrigant solutions, 3 microbial samples were taken: S1-initial (before the biomechanical preparation), S2-posttreatment (immediately after the biomechanical preparation), and S3-final (7 days after the biomechanical preparation). The microbiological samples were plated to count the colony-forming units (CFU).Results. The 2% chlorhexidine gel and 5.25% sodium hypochlorite significantly reduced the E. faecalis CFU in the posttreatment and final microbiological samples. The 1.5% sodium hypochlorite also reduced the E. faecalis CFU immediately after the root canal instrumentation, but the E. faecalis CFU increased in the final sample showing no statistical difference from the control group.Conclusion. The 2% chlorhexidine gluconate gel and 5.25% sodium hypochlorite were effective in eliminating E. faecalis even 7 days after the instrumentation; moreover, the higher the concentration of sodium hypochlorite the better its antimicrobial action.

Comparative study of the clinical and anti-microbial efficacy of tongue cleaners

Candida species have frequently been isolated from the oral cavities of a variety of patients, such as elderly people, dentures users, immunocompromised and health patients. Yeasts may be associated with immune response and local factors such as poor oral hygiene. It was evaluated effectiveness of tongue cleaner showing which types would be preferred by patients, changes in tongue coating and in saliva yeasts counting. Thirty patients were selected and randomly distributed into three groups. This crossover blind study evaluated the effect of tongue cleaning using: a plastic and a steel tongue scraper and a nylon soft-bristle toothbrush. All patients were instructed to use the cleaners twice a day for one week (fifteen-day wash-out period). Saliva and tongue coating samples were collected from each patient from each test period, the yeasts were counted by colony forming units per mL (CFU/ mL) and the species were identified. The patients were questioned about cleaner preference. An increase in the percentage of patients with no tongue coating after scraping was observed. A reduction in the mean number of Candida species in tongue coating was observed only after nylon soft-bristle toothbrush cleaner. Candida albicans was the prevalent species. Volunteers preferred to the steel tongue scraper (60%). Tongue cleaners reduced the tongue coating and the mean number of saliva's yeasts. Degree of tongue coating favors the Candida species colonization.

Late-onset sepsis: Epidemiology, evaluation, and outcome

Late-onset neonatal sepsis is a common serious problem in preterm infants in neonatal intensive care units. Diagnosis can be difficult because clinical manifestations are not specific and none of the available laboratory tests can be considered an ideal marker. For this reason, a combination of markers has been proposed. Complete blood count and acute-phase reactants evaluated together help in diagnosis. C-reactive protein is a specific but late marker, and procalcitonin has proven accurate, although it is little studied in newborns. Blood, cerebrospinal fluid, and urine cultures always should be obtained when late-onset sepsis is suspected. Blood culture, the gold standard in diagnosis, is highly sensitive but needs up to 48 hours to detect microbial growth. Various cytokines have been investigated as early markers of infection, but results are not uniform. Other diagnostic tests that offer promise include: neutrophil surface markers, granulocyte colony-stimulating factor, toll-like receptors, and nuclear factor kappa B. The greatest hope for quick and accurate diagnosis lies in molecular biology, using real time polymerase chain reaction combined withDNAmicroarray. Sepsis and meningitis may affect both the short- and long-term prognosis for newborns. Mortality in neonatal meningitis has been reduced in recent years, but short-term complications and later neurocognitive sequelae remain. Late-onset sepsis significantly increases preterm infant mortality and the risk of cerebral lesions and neurosensory sequelae, including developmental difficulties and cerebral palsy. Early diagnosis of late-onset sepsis contributes to improved neonatal prognosis, but the outcome remains far from satisfactory. © 2010 by the American Academy of Pediatrics.

Mineral water: A microbiological approach

The microbiological quality of bottled mineral water of various domestic brands sold in Brazil was investigated, with particular focus on the heterotrophic plate count (HPC). Neither total coliforms nor Escherichia coli were found in any 1.5 L bottle samples. Total coliforms were found in 2.9% of the small bottles, while in 20 L bottles the presence of total coliforms and E. coli was demonstrated in 15.5 and 2.4% of samples, respectively. Pseudomonas aeruginosa was detected in 4.3, 4.5 and 9.5% of small, 1.5 and 20 L bottles, respectively. In 36.4% of the samples of 1.5 L bottles, the HPC was above 500 cfu/mL. This percentage of samples with an HPC above 500 cfu/mL increased to 52.0 and 61.9% in small and 20 L bottles, respectively. Higher contamination by total coliforms, E. coli, P. aeruginosa and HPCs occurred in 20 L bottles. In conclusion, several samples in this study were outside the international quality standard for mineral water and the large number of samples with high HPCs shows that more work must be done on the use of HPC in mineral water and the damaging effects that these microorganisms may cause to humans. The bottled mineral water was confirmed as a particularly important public health problem, due to the poor microbiological quality of the products that are marketed. © IWA Publishing 2012.

Photodynamic inactivation of Streptococcus mutans and Streptococcus sanguinis biofilms in vitro

The purpose of this study was to evaluate specific effects of photodynamic inactivation (PDI) using erythrosine (ER) and Rose Bengal (RB) photosensitizers and a blue light-emitting diode (LED) on the viability of Streptococcus mutans and Streptococcus sanguinis biofilms. Biofilms were grown in acrylic disks immersed in broth to production of biofilms, inoculated with microbial suspension (106 cells/mL) and incubated for 48 h. After the formation of biofilms, the effects of the photosensitizers ER and RB at a concentration of 5 μM for 5 min and blue LED (455 ± 20 nm) for 180 s, photosensitizers alone and conjugated were evaluated. Next, the disks were placed in tubes with sterile physiological solution (0.9 % sodium chloride) and sonicated for to disperse the biofilms. Tenfold serial dilutions were carried and aliquots seeded in brain heart infusion agar which were then incubated for 48 h. Then the numbers colony-forming units per milliliter (CFU/mL; log 10) were counted and analyzed statistically (ANOVA, Tukey test, P ≤ 0.05). Significant decreases in the viability of all microorganisms were observed for biofilms exposed to PDI mediated by both photosensitizers. The reductions with RB and ER were, 0.62 and 0.52 log10 CFU mL -1 for S. mutans biofilms (p = 0.001), and 0.95 and 0.88 log 10 CFU mL-1 for S. sanguinis biofilms (p = 0.001), respectively. The results showed that biofilms formed in vitro by S. mutans and S. sanguinis, were sensitive to PDI using a blue LED associated with photosensitizers ER or RB, indicating its use in the control of caries and periodontal diseases. © 2012 Springer-Verlag London Ltd.

Quantitative variations in heterotrophic plate count and in the presence of indicator microorganisms in bottled mineral water

Quantitative variations in heterotrophic plate count (HPC) and in the presence of indicator microorganisms in 0.5, 1.5 and 20-L bottles of different brands of Brazilian mineral water were analyzed during their shelf life. No variations were identified in the presence of indicator microorganisms, but quantitative variations in HPC were observed in some brands, which suggests that changes may be occurring in the water quality during storage. The aim of this study was also to evaluate the quality of the bottled mineral waters and the presence of enterococci and Pseudomonas aeruginosa were verified in six and two bottles, respectively, which is in disagreement with the microbiological quality criteria established in the current legislation. Although no limit is set for HPC in mineral water, this study relies on the limit of 500 colony-forming units per mL of sample (CFU/mL). Seventy-two bottles presented levels above 500 CFU/mL and up to 560,000 CFU/mL. This study showed that the control of HPC (<500 CFU/mL) for non-returnable packaging seems to be adequate to ensure the quality of mineral water during storage. The high values of HPC and its variations detected during storage seem to fully justify the need for a reevaluation of the use of HPC in bottled mineral water quality management. More detailed studies on the potential health risk of HPC and its variations in mineral water are also needed. © 2012 Elsevier Ltd.

Experimental infection of commercial layers with wild or attenuated Salmonella Gallinarum mutant strains: Anatomic pathology, total blood cell count and serum protein levels

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

Prevenção da contaminação cruzada em odontologia por meio de terapia fotodinâmica (TFD) utilizando LED azul e curcumina

Pós-graduação em Odontologia - FOAR

Avaliação do efeito bactericida do ozônio associado ao propilenoglicol em canais radiculares contaminados com enterococcus faecalis em diferentes períodos de tempo de armazenagem

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Identificação e caracterização da microbiota lática isolada de queijo mussarela de búfala

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

Produção e aplicação de enzimas na redução de biofilme de Candida albicans

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)