Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil

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

Inhibitory effect of essential oils against Lactobacillus rhamnosus and starter culture in fermented milk during its shelf-life period

The use of essential oils in foods has attracted great interest, due to their antagonistic action against pathogenic microorganisms. However, this action is undesirable for probiotic foods, as products containing Lactobacillus rhamnosus. The aim of the present study was to measure the sensitivity profile of L. rhamnosus and a yogurt starter culture in fermented milk, upon addition of increasing concentrations of cinnamon, clove and mint essential oils. Essential oils were prepared by steam distillation, and chemically characterised by gas chromatography-mass spectrometry (GC-MS) and determination of density. Survival curves were obtained from counts of L. rhamnosus and the starter culture (alone and in combination), upon addition of 0.04% essential oils. In parallel, titratable acidity was monitored over 28 experimental days. Minimum inhibitory concentration values, obtained using the microdilution method in Brain Heart Infusion medium, were 0.025, 0.2 and 0.4% for cinnamon, clove and mint essential oils, respectively. Cinnamon essential oil had the highest antimicrobial activity, especially against the starter culture, interfering with lactic acid production. Although viable cell counts of L. rhamnosus were lower following treatment with all 3 essential oils, relative to controls, these results were not statistically significant; in addition, cell counts remained greater than the minimum count of 10(8)CFU/mL required for a product to be considered a probiotic. Thus, although use of cinnamon essential oil in yogurt makes starter culture fermentation unfeasible, it does not prevent the application of L. rhamnosus to probiotic fermented milk. Furthermore, clove and mint essential oil caused sublethal stress to L. rhamnosus.

The copper resistance operon copAB from Xanthomonas axonopodis pathovar citri: gene inactivation results in copper sensitivity

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker and the completion of the Xac genome sequence has opened up the possibility of investigating basic cellular mechanisms at the genomic level. Copper compounds have been extensively used in agriculture to control plant diseases. The copA and copB genes, identified by annotation of the Xac genome, encode homologues of proteins involved in copper resistance. A gene expression assay by Northern blotting revealed that copA and copB are expressed as a unique transcript specifically induced by copper. Synthesis of the gene products was also induced by copper, reaching a maximum level at 4 h after addition of copper to the culture medium. CopA was a cytosolic protein and CopB was detected in the cytoplasmic membrane. The gene encoding CopA was disrupted by the insertion of a transposon, leading to mutant strains that were unable to grow in culture medium containing copper, even at the lowest CUSO4 concentration tested (0.25 mM), whereas the wild-type strain was able to grow in the presence of 1 mM copper. Cell suspensions of the wild-type and mutant strains in different copper concentrations were inoculated in lemon leaves to analyse their ability to induce citrus canker symptoms. Cells of mutant strains showed higher sensitivity than the wild-type strain in the presence of copper, i.e. they were not able to induce citrus canker symptoms at high copper concentrations and exhibited a more retarded growth in planta.

Sensitivity of ant (Cephalotes) colonies and individuals to antibiotics implies feeding symbiosis with gut microorganisms

Ants in the tribe Cephalotini are exceptional in that they maintain microorganisms in their digestive tract. To understand what these microorganisms mean to the ants, we observed the feeding habits of Cephalotes pusillus and Cephalotes atratus, finding that in nature they feed on extrafloral nectars, homopteran secretions, and bird droppings. Feeding the antibiotic kanamycin to colonies of C. pusillus in the laboratory kills them. Ants desiccate or starve rather than feed on liquids to which the antibiotics gentamycin and netilmycin have been added, but feed and survive on liquids containing nystatin, penicillin, and ampicillin. We identified over 10 microorganisms from the intestine of C. pusillus with different antibiotic-resistance patterns. The bacteria are from the genera Corynebacterium, Brevibacterium, Sphingobacterium, Ochrobactrum, Myroides, Brevundimonas, Alcaligenes, Stenotrophomonas, Moraxella, and Pseudomonas. We hypothesize that the microorganisms provide nutrients to the ants by synthesizing amino acids from carbohydrates and nitrates. We do not know whether the ants collect the bacteria from the environment, but they transmit them to their young. They culture them in their digestive tract, eventually feeding on them.

Chromosome sensitivity to bleomycin in G2 lymphocytes from Down syndrome patients

Several studies have demonstrated that lymphocytes from patients with Down syndrome (DS) exhibit an increased frequency of chromosome aberrations when they are exposed to ionizing radiation or to chemicals at the G0 or G1 phases of the cell cycle, but not at G2 when compared to normal subjects. To determine the susceptibility of DS lymphocytes at G2 phase, bleomycin, a radiomimetic agent, was used to induce DNA breaks in blood cultures from 24 Down syndrome patients. All the patients with DS showed free trisomy 21 (47,XX + 21 or 47,XY + 21). Individuals that showed an average number of chromatid breaks per cell higher than 0.8 were considered sensitive to the drug. No control child showed susceptibility to bleomycin, and among the 24 patients with DS, only one was sensitive to the drug. No significant difference was observed between the two groups, regarding chromatid break frequencies in treated G2 lymphocytes. The distribution of bleomycin-induced breaks in each group of chromosomes was similar for DS and controls. No significant difference was found in the response to bleomycin between male and female subjects. Probably, the main factor involved in chromosome sensitivity of lymphocytes from patients with DS is the phase of the cell cycle in which the cell is treated.

Seed and seedling surface-sterilization for in vitro culture of tillandsia gardneri (Bromeliaceae)

Tillandsia gardneri is a bromeliad with ornamental value and a wide geographical distribution over Brazil. However, due to habitat loss and illegal overcollection in the wild it is included as a vulnerable species in the official list of endangered plants of the State of Rio Grande do Sul, Brazil. The development of a protocol for T. gardneri seed propagation in vitro may be useful for reintroducing plants in their natural habitats, and for germplasm conservation. A difficult problem encountered during the establishment of an in vitro culture is explants disinfection, especially when working with endangered species, from which explant availability is restricted. Thus, the establishment of a sterilization protocol is crucial for the initiation and success of a micropropagation system for T. gardneri. The objective of this study was to evaluate the effect of sodium hypochlorite concentration and exposure time in seed and seedling surface disinfection, tissue sensitivity and development. Sodium hypochlorite solutions (10 or 20%/5, 10 or 15 min; 25%/5 or 10 min; and 50%/5 min) were effective in eliminating seed superficial contaminants. There was no significant difference among the effective sterilization treatments in relation to seed germination (%), and seedling length and number of leaves, after 120 days in vitro. Also, no damage to seed and seedling tissues were observed. Surface sterilization of seedlings, for initiation of an in vitro culture, required higher concentrations of sodium hypochlorite (25%/15 min; 20 or 50%/5, 10 or 15 min; and 40%/5 and 10 min) for controlling fungal and yeast contamination, compared to seed sterilization. No significant differences among these treatments were found in relation to seedling length and number of leaves, after 60 days in vitro.

Assessment of central venous catheter-associated infections using semi-quantitative or quantitative culture methods

Semiquantitative (Maki) and quantitative (Brun- Buisson) culture techniques were employed in the diagnosis of catheter-related bloodstream infections (CRBSI) in patients who have a short-term central venous catheter (inserted for 30 days). The diagnosis of CRBSI was based on the results of semiquantitative and quantitative culture of material from the removed catheters. Catheter tips (118) from 100 patients were evaluated by both methods. Semiquantitative analysis revealed 34 catheters (28.8%) colonized by ≥15 colonyforming units (cfu), while quantitative cultures (34 catheters, 28.8%) showed the growth of ≥103 cfu/mL. Bacteremia was confirmed in four patients by isolating microorganisms of identical species from both catheters and blood samples. Using the semiquantitative culture technique on short-term central venous catheter tips, we have shown that with a cut-off level of ≥15 cfu, the technique had 100.0% sensitivity, specificity of 68.4%, 25.0% positive predictive value (PPV) and 100.0% negative predictive value (NPV), efficiency of 71.4% and a prevalence of 9.5%. The quantitative method, with a cut-off limit of ≥103 cfu/mL, gave identical values: the sensitivity was 100.0%, specificity 68.4%, positive predictive value (PPV) 25.0%, negative predictive value (NPV) 100.0%, efficiency 71.4% and prevalence 9.5%. We concluded that the semiquantitative and quantitative culture methods, evaluated in parallel, for the first time in Brazil, have similar sensitivity and specificity. Keywords: central venous catheter; semi-quantitative culture; quantitative culture; catheter-related bacteremia.

Diagnostic accuracy of semi-quantitative and quantitative culture techniques for the diagnosis of catheter-related infections in newborns and molecular typing of isolated microorganisms

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

The use of a mixture of somatic and culture filtrate antigens in the evaluation of the immune response to Paracoccidioides brasiliensis

Three Paracoccidioides brasiliensis antigens, namely a culture filtrate preparation, a somatic antigen and a mixture of equal parts of the two, were tested by two serological techniques against sera from patients with paracoccidioidomycosis, and in an in vivo delayed hypersensitivity model in mice. The antigen mixture was more sensitive than the two individual antigens for the evaluation of humoral and cellular immune response to P. brasiliensis, both in man and in experimental animals.

Comparison of Gross Pathology, Histopathology, and Mycobacterial Culture for the Diagnosis of Tuberculosis in Elk (Cervus elaphus)

Using the isolation of Mycobacterium bovis as the reference standard, this study evaluated the sensitivity, specificity and kappa statistic of gross pathology (abattoir postmortem inspection), histopathology, and parallel or series combinations of the two for the diagnosis of tuberculosis in 430 elk and red deer. Two histopathology interpretations were evaluated: histopathology I, where the presence of lesions compatible with tuberculosis was considered positive, and histopathology II, where lesions compatible with tuberculosis or a select group of additional possible diagnoses were considered positive. In the 73 animals from which M. bovis was isolated, gross lesions of tuberculosis were most often in the lung (48), the retropharyngeal lymph nodes (36), the mesenteric lymph nodes (35), and the mediastinal lymph nodes (16). Other mycobacterial isolates included: 11 M. paratuberculosis, 11 M. avium, and 28 rapidly growing species or M. terrae complex. The sensitivity estimates of gross pathology and histopathology I were 93% (95% confidence limits [CL] 84,97%) and 88% [CL 77,94%], respectively, and the specificity of both was 89% [CL 85,92%]). The sensitivity and specificity of histopathology II were 89% (CL 79,95%) and 77% (CL 72,81%), respectively. The highest sensitivity estimates (93- 95% [CL 84,98%]) were obtained by interpreting gross pathology and histopathology in parallel (where an animal had to be positive on at least one of the two, to be classified as combination positive). The highest specificity estimates (94-95% [CL 91-97%]) were generated when the two tests were interpreted in series (an animal had to be positive on both tests to be classified as combination positive). The presence of gross or microscopic lesions showed moderate to good agreement with the isolation of M. bovis (Kappa = 65-69%). The results show that post-mortem inspection, histopathology and culture do not necessarily recognize the same infected animals and that the spectra of animals identified by the tests overlaps.

Improved sensitivity of an interferon-gamma release assay (T-SPOT.TB™) in combination with tuberculin skin test for the diagnosis of latent tuberculosis in the presence of HIV co-infection

Background Interferon-gamma release assays (IGRA) are more specific than the tuberculin skin test (TST) for the diagnosis of Mycobacterium tuberculosis infection. Data on sensitivity are controversial in HIV infection. Methods IGRA (T-SPOT.TB) was performed using lymphocytes stored within 6 months before culture-confirmed tuberculosis was diagnosed in HIV-infected individuals in the Swiss HIV Cohort Study. Results 64 individuals (69% males, 45% of non-white ethnicity, median age 35 years (interquartile range [IQR] 31-42), 28% with prior AIDS) were analysed. Median CD4 cell count was 223 cells/μl (IQR 103-339), HIV-RNA was 4.7 log10 copies/mL (IQR 4.3-5.2). T-SPOT.TB resulted positive in 25 patients (39%), negative in 18 (28%) and indeterminate in 21 (33%), corresponding to a sensitivity of 39% (95% CI 27-51%) if all test results were considered, and 58% (95% CI 43-74%) if indeterminate results were excluded. Sensitivity of IGRA was independent of CD4 cell count (p = 0.698). Among 44 individuals with available TST, 22 (50%) had a positive TST. Agreement between TST and IGRA was 57% (kappa = 0.14, p = 0.177), and in 34% (10/29) both tests were positive. Combining TST and IGRA (at least one test positive) resulted in an improved sensitivity of 67% (95% CI 52-81%). In multivariate analysis, older age was associated with negative results of TST and T-SPOT.TB (OR 3.07, 95% CI 1,22-7.74, p = 0.017, per 10 years older). Conclusions T-SPOT.TB and TST have similar sensitivity to detect latent TB in HIV-infected individuals. Combining TST and IGRA may help clinicians to better select HIV-infected individuals with latent tuberculosis who qualify for preventive treatment.

Real-time broad-range PCR versus blood culture. A prospective pilot study in pediatric cancer patients with fever and neutropenia

MATERIALS AND METHODS: In a pilot study, results of real-time broad-range (16S rRNA) polymerase chain reaction (PCR) performed on 45 blood samples of pediatric cancer patients with fever and neutropenia were compared with blood culture results. RESULTS: The PCR assay used, having proven a high sensitivity in artificially spiked blood samples, was positive in only three of ten blood culture-positive samples, and it was positive in 10 of 35 (29%) culture-negative samples. CONCLUSION: This broad-range PCR assay, which may identify not-grown bacteria potentially contributing to fever, needs improvement in sensitivity, and different reasons for positive PCR in negative blood culture samples need to be assessed before clinical application.

Bovine primary chondrocyte culture in synthetic matrix metalloproteinase-sensitive poly(ethylene glycol)-based hydrogels as a scaffold for cartilage repair

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

Use of cardiac glycosides for treatment of cancer: Determinants of cancer cell sensitivity

Cardiac glycoside compounds have traditionally been used to treat congestive heart failure. Recently, reports have suggested that cardiac glycosides may also be useful for treatment of malignant disease. Our research with oleandrin, a cardiac glycoside component of Nerium oleander, has shown it to be a potent inducer of human but not murine tumor cell apoptosis. Determinants of tumor sensitivity to cardiac glycosides were therefore studied in order to understand the species selective cytotoxic effects as well as explore differential sensitivity amongst a variety of human tumor cell lines. ^ An initial model system involved a comparison of human (BRO) to murine (B16) melanoma cells. Human BRO cells were found to express both the sensitive α3 as well as the less sensitive α1 isoform subunits of Na+,K +-ATPase while mouse B16 cells expressed only the α1 isoform. Drug uptake and inhibition of Na+,K+-ATPase activity were also different between BRO and B16 cells. Partially purified human Na+,K+-ATPase enzyme was inhibited by cardiac glycosides at a concentration that was 1000-fold less than that required to inhibit mouse B16 enzyme to the same extent. In addition, uptake of oleandrin and ouabain was 3–4 fold greater in human than murine cells. These data indicate that differential expression of Na+,K+-ATPase isoform composition in BRO and B16 cells as well as drug uptake and total enzyme activity may all be important determinants of tumor cell sensitivity to cardiac glycosides. ^ In a second model system, two in vitro cell culture model systems were investigated. The first consisted of HFU251 (low expression of Na+,K+-ATPase) and U251 (high Na+ ,K+-ATPase expression) cell lines. Also investigated were human BRO cells that had undergone stable transfection with the α1 subunit resulting in an increase in total Na+,K+-ATPase expression. Data derived from these model systems have indicated that increased expression of Na+,K+-ATPase is associated with an increased resistance to cardiac glycosides. Over-expression of Na +,K+-ATPase in tumor cells resulted in an increase of total Na+,K+-ATPase activity and, in turn, a decreased inhibition of Na+,K+-ATPase activity by cardiac glycosides. However, of interest was the observation that increased enzyme expression was also associated with an elevated basal level of glutathione (GSH) within cells. Both increased Na+,K+-ATPase activity and elevated GSH content appear to contribute to a delayed as well as diminished release of cytochrome c and caspase activation. In addition, we have noted an increased colony forming ability in cells with a high level of Na+,K+-ATPase expression. This suggests that Na+,K+-ATPase is actively involved in tumor cell growth and survival. ^

International Experiences – is there an effect on intercultural sensitivity? : An explorative study conducted at Stockholm University

The presented research is an explorative study, aimed to identify differences in intercultural sensitivity of Swedish students with international experience, Swedish students with no international experiences and international students studying at Stockholm University. The research is placed within the field of higher education. International experiences are seen to be a key to intercultural competences and it is universities who need to prepare students for the demands of the globalized job market. The research is of qualitative nature and relevant data was collected by the means of semi- structured interviews. The data was coded according to qualitative thematic analysis and then analyzed and discussed in accordance to the researcher questions and existing literature in this field. The study revealed that there is no general difference in intercultural sensitivity between the three groups analyzed. However, international students show higher sensitivity in terms of respect of cultural differences and open-mindedness, while students with no international experiences show less sensitivity in terms of interaction involvement and suspending judgment. The identified themes offer valuable information on individual perceptions of cultural differences. This information can be used to improve measures and actions to increase intercultural sensitivity at Stockholm University.