Genetic relationships of Corynebacterium diphtheriae strains isolated from a diphtheria case and carriers by restriction fragment length polymorphism of rRNA genes

In the present study we report the results of an analysis, based on ribotyping of Corynebacterium diphtheriae intermedius strains isolated from a 9 years old child with clinical diphtheria and his 5 contacts. Quantitative analysis of RFLPs of rRNA was used to determine relatedness of these 7 C.diphtheriae strains providing support data in the diphtheria epidemiology. We have also tested those strains for toxigenicity in vitro by using the Elek's gel diffusion method and in vivo by using cell culture method on cultured monkey kidney cell (VERO cells). The hybridization results revealed that the 5 C.diphtheriae strains isolated from contacts and one isolated from the clinical case (nose case strain) had identical RFLP patterns with all 4 restriction endonucleases used, ribotype B. The genetic distance from this ribotype and ribotype A (throat case strain), that we initially assumed to be responsible for the illness of the patient, was of 0.450 showing poor genetic correlation among these two ribotypes. We found no significant differences concerned to the toxin production by using the cell culture method. In conclusion, the use of RFLPs of rRNA gene was successful in detecting minor differences in closely related toxigenic C.diphtheriae intermedius strains and providing information about genetic relationships among them.

Adipocyte Secreted Factors Enhance Aggressiveness of Prostate Carcinoma Cells

Obesity has been associated with increased incidence and risk of mortality of prostate cancer. One of the proposed mechanisms underlying this risk association is the change in adipokines expression that could promote the development and progression of the prostate tumor cells. The main goal of this study was to evaluate the effect of preadipocyte and adipocyte secretome in the proliferation, migration and invasion of androgen independent prostate carcinoma cells (RM1) and to assess cell proliferation in the presence of the adiposity signals leptin and insulin. RM1 cells were co-cultured in with preadipocytes, adipocytes or cultured in their respective conditioned medium. Cell proliferation was assessed by flow cytometry and XTT viability test. Cell migration was evaluated using a wound healing injury assay of RM1 cells cultured with conditioned media. Cellular invasion of RM1 cells co-cultured with adipocytes and preadipocytes was assessed using matrigel membranes. Preadipocyte conditioned medium was associated with a small increase in RM1 proliferation, while adipocytes conditioned media significantly increased RM1 cell proliferation (p<0.01). Adipocytes also significantly increased the RM1 cells proliferation in co-culture (p <0.01). Cell migration was higher in RM1 cells cultured with preadipocyte and adipocyte conditioned medium. RM1 cell invasion was significantly increased after co-culture with preadipocytes and adipocytes (p <0.05). Insulin also increased significantly the cell proliferation in contrast to leptin, which showed no effect. In conclusion, prostate carcinoma cells seem to be influenced by factors secreted by adipocytes that are able to increase their ability to proliferate, migrate and invade.

Picocyanobacteria From a Clade Of Marine Cyanobium Revealed Bioactive Potential Against Microalgae, Bacteria, And Marine

The production of bioactive compounds either toxic or with pharmacological applications by cyanobacteria is well established. However, picoplanktonic forms within this group of organisms have rarely been studied in this context. In this study, the toxicological potential of picocyanobacteria from a clade of marine Cyanobium strains isolated from the Portuguese coast was examined using different biological models. First, strains were identified by applying morphological and molecular approaches and cultured under lab conditions. A crude extract and three fractions reflecting a preliminary segregation of lipophilic metabolites were tested for toxicity with the marine microalga Nannochloropsis sp., the bacteria Pseudomonas sp., the brine shrimp Artemia salina, and fertilized eggs of the sea urchin Paracentrotus lividus. No significant apparent adverse effects were noted against Artemia salina. However, significant adverse effects were found in all other assays, with an inhibition of Nannochloropsis sp. and Pseudomonas sp. growth and marked reduction in Paracentrotus lividus larvae length. The results obtained indicated that Cyanobium genus may serve as a potential source of interesting bioactive compounds and emphasize the importance of also studying smaller picoplanktonic fractions of marine cyanobacteria.

OBTENTION OF RABIES ANTIGEN THROUGH BHK21 CELLS ADHERED TO MICROCARRIERS

Four rabies antigen batches were produced from virus suspensions resulting from BHK21 cells adhered to microcarriers (Cytodex 1), inoculated and cultured in a bioreactor. In parallel the methodology of production of rabies virus through cultures of BHK21 cells in monolayers in bottles was used. The results obtained showed that infecting titles were 106.69 DL50/mL and 107.28 DL50/mL for suspensions cultured in bottles and in the bioreactor, respectively. The viral suspension volumes collected were on average 11,900 per batch from the bioreactor and 800mL per bottle. Ten horses were immunized with the antigen produced in the bioreactor. The means of antirabies antibody titers found were 240 and 212 IU/mL after the initial and the first booster doses, respectively. Rabies antigen with satisfactory infecting titers can be obtained on a large scale by culturing in a bioreactor inoculated BHK21 cells adhered to microcarriers.

Genotoxic effects in occupational exposure to formaldehyde: A study in anatomy and pathology laboratories and formaldehyde-resins production

ABSTRACT – Background: According to the Report on Carcinogens, formaldehyde ranks 25th in the overall U.S. chemical production, with more than 5 million tons produced each year. Given its economic importance and widespread use, many people are exposed to formaldehyde environmentally and/or occupationally. Presently, the International Agency for Research on Cancer classifies formaldehyde as carcinogenic to humans (Group 1), based on sufficient evidence in humans and in experimental animals. Manyfold in vitro studies clearly indicated that formaldehyde can induce genotoxic effects in proliferating cultured mammalian cells. Furthermore, some in vivo studies have found changes in epithelial cells and in peripheral blood lymphocytes related to formaldehyde exposure. Methods: A study was carried out in Portugal, using 80 workers occupationally exposed to formaldehyde vapours: 30 workers from formaldehyde and formaldehyde-based resins production factory and 50 from 10 pathology and anatomy laboratories. A control group of 85 non-exposed subjects was considered. Exposure assessment was performed by applying simultaneously two techniques of air monitoring: NIOSH Method 2541 and Photo Ionization Detection equipment with simultaneously video recording. Evaluation of genotoxic effects was performed by application of micronucleus test in exfoliated epithelial cells from buccal mucosa and peripheral blood lymphocytes. Results: Time-weighted average concentrations not exceeded the reference value (0.75 ppm) in the two occupational settings studied. Ceiling concentrations, on the other hand, were higher than reference value (0.3 ppm) in both. The frequency of micronucleus in peripheral blood lymphocytes and in epithelial cells was significantly higher in both exposed groups than in the control group (p < 0.001). Moreover, the frequency of micronucleus in peripheral blood lymphocytes was significantly higher in the laboratories group than in the factory workers (p < 0.05). A moderate positive correlation was found between duration of occupational exposure to formaldehyde (years of exposure) and micronucleus frequency in peripheral blood lymphocytes (r = 0.401; p < 0.001) and in epithelial cells (r = 0.209; p < 0.01). Conclusions: The population studied is exposed to high peak concentrations of formaldehyde with a long-term exposure. These two aspects, cumulatively, can be the cause of the observed genotoxic endpoint effects. The association of these cytogenetic effects with formaldehyde exposure gives important information to risk assessment process and may also be used to assess health risks for exposed worker

FATAL GROUP A STREPTOCOCCAL TOXIC SHOCK-LIKE SYNDROME IN A CHILD WITH VARICELLA: REPORT OF THE FIRST WELL DOCUMENTED CASE WITH DETECTION OF THE GENETIC SEQUENCES THAT CODE FOR EXOTOXINS SPE A AND B, IN SÃO PAULO, BRAZIL

A previously healthy seven-year-old boy was admitted to the intensive care unit because of toxaemia associated with varicella. He rapidly developed shock and multisystem organ failure associated with the appearance of a deep-seated soft tissue infection and, despite aggressive treatment, died on hospital day 4. An M-non-typable, spe A and spe B positive Group A Streptococcus was cultured from a deep soft tissue aspirate. The criteria for defining Streptococcal toxic shock-like syndrome were fulfilled. The authors discuss the clinical and pathophysiological aspects of this disease as well as some unusual clinical findings related to this case.

New sensing materials of molecularly-imprinted polymers for the selective recognition of Chlortetracycline

As a result of the stressful conditions in aquaculture facilities there is a high risk of bacterial infections among cultured fish. Chlortetracycline (CTC) is one of the antimicrobials used to solve this problem. It is a broad spectrum antibacterial active against a wide range of Gram-positive and Gram-negative bacteria. Numerous analytical methods for screening, identifying, and quantifying CTC in animal products have been developed over the years. An alternative and advantageous method should rely on expeditious and efficient procedures providing highly specific and sensitive measurements in food samples. Ion-selective electrodes (ISEs) could meet these criteria. The only ISE reported in literature for this purpose used traditional electro-active materials. A selectivity enhancement could however be achieved after improving the analyte recognition by molecularly imprinted polymers (MIPs). Several MIP particles were synthesized and used as electro-active materials. ISEs based in methacrylic acid monomers showed the best analytical performance according to slope (62.5 and 68.6 mV/decade) and detection limit (4.1 × 10−5 and 5.5 × 10−5 mol L−1). The electrodes displayed good selectivity. The ISEs are not affected by pH changes ranging from 2.5 to 13. The sensors were successfully applied to the analysis of serum, urine and fish samples.

Effects of molybdate and tungstate on expression levels and biochemical characteristics of formate dehydrogenases produced by desulfovibrio alaskensis NCIMB 13491

Journal of Bacteriology. 2011 Jun; Vol. 193 issue 12 pages 2917-2923

Clinical guideline for diagnosis and management of melioidosis

Melioidosis is an emerging infection in Brazil and neighbouring South American countries. The wide range of clinical presentations include severe community-acquired pneumonia, septicaemia, central nervous system infection and less severe soft tissue infection. Diagnosis depends heavily on the clinical microbiology laboratory for culture. Burkholderia pseudomallei, the bacterial cause of melioidosis, is easily cultured from blood, sputum and other clinical samples. However, B. pseudomallei can be difficult to identify reliably, and can be confused with closely related bacteria, some of which may be dismissed as insignificant culture contaminants. Serological tests can help to support a diagnosis of melioidosis, but by themselves do not provide a definitive diagnosis. The use of a laboratory discovery pathway can help reduce the risk of missing atypical B. pseudomallei isolates. Recommended antibiotic treatment for severe infection is either intravenous Ceftazidime or Meropenem for several weeks, followed by up to 20 weeks oral treatment with a combination of trimethoprim-sulphamethoxazole and doxycycline. Consistent use of diagnostic microbiology to confirm the diagnosis, and rigorous treatment of severe infection with the correct antibiotics in two stages; acute and eradication, will contribute to a reduction in mortality from melioidosis.

Pathogenicity of Entamoeba dispar under xenic and monoxenic cultivation compared to a virulent E. histolytica

Two xenic isolates and cloned cultures of Entamoeba dispar were submitted to monoxenization using Crithidia fasciculata as the associated organism. Growth in monoxenic cultivation and ability of xenic and monoxenic trophozoites to destroy VERO cells and produce lesions in hamster livers were compared to those of a virulent E. histolytica. Parental and cloned E. dispar under monoxenic cultivation showed a remarkable lower growth than the monoxenic E. histolytica and were avirulent in both in vivo and in vitro tests. When xenically cultured, trophozoites of E. dispar showed a moderate lytic activity against VERO cells (1.5 to 41.8% of destruction) but caused severe hepatic lesions in hamsters as those caused by the virulent E. histolytica (29 to 100% in prevalence and 0.86 to 4.00 in lesion degree). Although E. dispar has not been associated with invasive disease in men, the ability of xenic trophozoites to produce prominent tissue damage in experimental conditions has indicated that some strains have a considerable pathogenic potential when in presence of bacteria.

Kinetics of growth of Leishmania (Leishmania) chagasi cycle in McCoy cell culture

The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF) and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like) form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.

Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia

Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 µM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.

Expression of circulating leucocytes before, during and after myiasis by Dermatobia hominis in experimentally infected rats

Expression of circulating white blood cells was investigated in rats (Rattus norvegicus) experimentally infected with larvae of Dermatobia hominis, the human bot fly. Leucocytes were counted prior to infection (control group) as well as at 6, 10, 15, 20 and 28 days post-infection (dpi) and at 7, 15, 30 and 60 days post-larval emergence (dple). Total leucocyte numbers did not differ markedly among the groups. Significant differences were registered when values from control and animals harboring each larval stage of D. hominis were compared; with crescent rank: L1-, L2-, control and L3-infected groups. Leucocyte numbers were significantly higher in the control, 15, 20 or 28 dpi groups than in the 6 dpi animals. Higher counts were observed in control, L2- or L3-infected rats than L1-infected animals. Neutrophils, eosinophils and both large and small lymphocytes were also counted and analyzed. Basophils and monocytes were insufficient in number to permit statistical studies. These results stimulate the continuity of the studies about the host-parasite relationship in the dermatobiosis.

Clinical and Bacteriological Survey of Diabetic Foot Infections in Lisbon

AIMS: An epidemiological survey of diabetic foot infections (DFIs) in Lisbon, stratifying the bacterial profile based on patient demographical data, diabetic foot characteristics (PEDIS classification), ulcer duration and antibiotic therapy. METHODS: A transversal observational multicenter study, with clinical data collection using a structured questionnaire and microbiological products (aspirates, biopsies or swabs collected using the Levine method) of clinically infected foot ulcers of patients with diabetes mellitus (DM). RESULTS: Forty-nine hospitalized and ambulatory patients were enrolled in this study, and 147 microbial isolates were cultured. Staphylococcus was the main genus identified, and methicillin-resistant Staphylococcus aureus (MRSA) was present in 24.5% of total cases. In the clinical samples collected from patients undergoing antibiotic therapy, 93% of the antibiotic regimens were considered inadequate based on the antibiotic susceptibility test results. The average duration of an ulcer with any isolated multi-drug resistant (MDR) organism was 29 days, and previous treatment with fluoroquinolones was statistically associated with multi-drug resistance. CONCLUSIONS: Staphylococcus aureus was the most common cause of DFIs in our area. Prevalence and precocity of MDR organisms, namely MRSA, were high and were probably related to previous indiscriminate antibiotic use. Clinicians should avoid fluoroquinolones and more frequently consider the use of empirical anti-MRSA therapy.

Process engineering of liver cells for drug testing applications

Dissertação para obtenção do Grau de Doutor em Bioengenharia