Impact of antibiotic resistance on chemotherapy for pneumococcal infections

Over the past three decades, penicillin-resistant pneumococci have emerged worldwide. In addition, penicillin-resistant strains have also decreased susceptibility to other β-lactams (including cephalosporins) and these strains are often resistant to other antibiotic groups, making the treatment options much more difficult. Nevertheless, the present in vitro definitions of resistance to penicillin and cephalosporins in pneumococci could not be appropriated for all types of pneumococcal infections. Thus, current levels of resistance to penicillin and cephalosporin seem to have little, if any, clinical relevance in nonmeningeal infections (e.g., pneumonia or bacteremia). On the contrary, numerous clinical failures have been reported in patients with pneumococcal meningitis caused by strains with MICs ≥ 0.12 μg/ml, and penicillin should never be used in pneumococcal meningitis except when the strain is known to be fully susceptible to this drug. Today, therapy for pneumococcal meningitis should mainly be selected on the basis of susceptibility to cephalosporins, and most patients may currently be treated with high-dose cefotaxime (±) vancomycin, depending on the levels of resistance in the patient's geographic area. In this review, we present a practical approach, based on current levels of antibiotic resistance, for treating the most prevalent pneumococcal infections. However, it should be emphasized that the most appropriate antibiotic therapy for infections caused by resistant pneumococci remains controversial, and comparative, randomized studies are urgently needed to clarify the best antibiotic therapy for these infections

Epithelial-to-mesenchymal transition mediates docetaxel resistance and high risk of relapse in prostate cancer

Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel þ androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-k B transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44 þ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44 þ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evi-dence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer.

Inborn resistance of mice to myxoviruses: macrophages express phenotype in vitro.

A strain of avian influenza A virus was adapted to grow in mouse peritoneal macrophages in vitro. The adapted strain, called M-TUR, induced a marked cytopathic effect in macrophages from susceptible mice. Mice homozygous (A2G) or heterozygous (F1 hybrids between A2G and several susceptible strains) for the gene Mx, shown previously to induce a high level of resistance towards lethal challenge by a number of myxoviruses in vivo, yielded peritoneal macrophages which were not affected by M-TUR. Peritoneal macrophages could be classified as resistant or susceptible to M-TUR without sacrificing the cell donor. Backcrosses were arranged between (A2G X A/J)F1 and A/J mice. 64 backcross animals could be tested individually both for resistance of their macrophages in vitro after challenge with M-TUR, and for resistance of the whole animal in vivo after challenge with NWS (a neurotropic variant of human influenza A virus). Macrophages from 36 backcross mice were classified as susceptible, and all of these mice died after challenge. Macrophages from 28 mice were classified as resistant, and 26 mice survived challenge. We conclude that resistance of macrophages and resistance of the whole animal are two facets of the same phenomenon.

A novel source for miR-21 expression through the alternative polyadenylation of VMP1 gene transcripts

miR-21 is the most commonly over-expressed microRNA (miRNA) in cancer and a proven oncogene. Hsa-miR-21 is located on chromosome 17q23.2, immediately downstream of the vacuole membrane protein-1 (VMP1) gene, also known as TMEM49. VMP1 transcripts initiate ∼130 kb upstream of miR-21, are spliced, and polyadenylated only a few hundred base pairs upstream of the miR-21 hairpin. On the other hand, primary miR-21 transcripts (pri-miR-21) originate within the last introns of VMP1, but bypass VMP1 polyadenylation signals to include the miR-21 hairpin. Here, we report that VMP1 transcripts can also bypass these polyadenylation signals to include miR-21, thus providing a novel and independently regulated source of miR-21, termed VMP1–miR-21. Northern blotting, gene-specific RT-PCR, RNA pull-down and DNA branching assays support that VMP1–miR-21 is expressed at significant levels in a number of cancer cell lines and that it is processed by the Microprocessor complex to produce mature miR-21. VMP1 and pri-miR-21 are induced by common stimuli, such as phorbol-12-myristate-13-acetate (PMA) and androgens, but show differential responses to some stimuli such as epigenetic modifying agents. Collectively, these results indicate that miR-21 is a unique miRNA capable of being regulated by alternative polyadenylation and two independent gene promoters.

Minimal requirements for parameters to be used for the development of predictive models for microbial source tracking. The example of the pair somatic coliphages and phages infecting Bacteroides

Several methods and approaches for measuring parameters to determine fecal sources of pollution in water have been developed in recent years. No single microbial or chemical parameter has proved sufficient to determine the source of fecal pollution. Combinations of parameters involving at least one discriminating indicator and one universal fecal indicator offer the most promising solutions for qualitative and quantitative analyses. The universal (nondiscriminating) fecal indicator provides quantitative information regarding the fecal load. The discriminating indicator contributes to the identification of a specific source. The relative values of the parameters derived from both kinds of indicators could provide information regarding the contribution to the total fecal load from each origin. It is also essential that both parameters characteristically persist in the environment for similar periods. Numerical analysis, such as inductive learning methods, could be used to select the most suitable and the lowest number of parameters to develop predictive models. These combinations of parameters provide information on factors affecting the models, such as dilution, specific types of animal source, persistence of microbial tracers, and complex mixtures from different sources. The combined use of the enumeration of somatic coliphages and the enumeration of Bacteroides-phages using different host specific strains (one from humans and another from pigs), both selected using the suggested approach, provides a feasible model for quantitative and qualitative analyses of fecal source identification.

Potential impact of environmental bacteriophages in spreading antibiotic resistance genes

The idea that bacteriophage transduction plays a role in the horizontal transfer of antibiotic resistance genes is gaining momentum. Such transduction might be vital in horizontal transfer from environmental to human body-associated biomes and here we review many lines of evidence supporting this notion. It is well accepted that bacteriophages are the most abundant entities in most environments, where they have been shown to be quite persistent. This fact, together with the ability of many phages to infect bacteria belonging to different taxa, makes them suitable vehicles for gene transfer. Metagenomic studies confirm that substantial percentages of the bacteriophage particles present in most environments contain bacterial genes, including mobile genetic elements and antibiotic resistance genes. When specific genes of resistance to antibiotics are detected by real-time PCR in the bacteriophage populations of different environments, only tenfold lower numbers of these genes are observed, compared with those found in the corresponding bacterial populations. In addition, the antibiotic resistance genes from these bacteriophages are functional and generate resistance to the bacteria when these genes are transfected. Finally, reports about the transduction of antibiotic resistance genes are on the increase.

Epithelial-to-mesenchymal transition mediates docetaxel resistance and high risk of relapse in prostate cancer

Molecular characterization of radical prostatectomy specimens after systemic therapy may identify a gene expression profile for resistance to therapy. This study assessed tumor cells from patients with prostate cancer participating in a phase II neoadjuvant docetaxel and androgen deprivation trial to identify mediators of resistance. Transcriptional level of 93 genes from a docetaxel-resistant prostate cancer cell lines microarray study was analyzed by TaqMan low-density arrays in tumors from patients with high-risk localized prostate cancer (36 surgically treated, 28 with neoadjuvant docetaxel þ androgen deprivation). Gene expression was compared between groups and correlated with clinical outcome. VIM, AR and RELA were validated by immunohistochemistry. CD44 and ZEB1 expression was tested by immunofluorescence in cells and tumor samples. Parental and docetaxel-resistant castration-resistant prostate cancer cell lines were tested for epithelial-to-mesenchymal transition (EMT) markers before and after docetaxel exposure. Reversion of EMT phenotype was investigated as a docetaxel resistance reversion strategy. Expression of 63 (67.7%) genes differed between groups (P < 0.05), including genes related to androgen receptor, NF-k B transcription factor, and EMT. Increased expression of EMT markers correlated with radiologic relapse. Docetaxel-resistant cells had increased EMT and stem-like cell markers expression. ZEB1 siRNA transfection reverted docetaxel resistance and reduced CD44 expression in DU-145R and PC-3R. Before docetaxel exposure, a selected CD44 þ subpopulation of PC-3 cells exhibited EMT phenotype and intrinsic docetaxel resistance; ZEB1/CD44 þ subpopulations were found in tumor cell lines and primary tumors; this correlated with aggressive clinical behavior. This study identifies genes potentially related to chemotherapy resistance and supports evi-dence of the EMT role in docetaxel resistance and adverse clinical behavior in early prostate cancer.

Genetic linkage map of Phaseolus vulgaris and identification of QTLs responsible for resistance to Xanthomonas axonopodis pv. phaseoli

Common bean (Phaseolus vulgaris) cultivars with a high degree of resistance to Xanthomonas axonopodis pv. phaseoli (Xap) are not available in Brazil. Despite many studies, a low degree of resistance to Xap continues to exist due to its complex genetic inheritance, which is not well known. The objectives of this research were to complement a common bean genetic map based on the cross between a susceptible genotype 'HAB-52' and a resistant genotype 'BAC-6', and to map and analyze genomic regions (quantitative trait loci – QTLs) related to Xap resistance. Eleven linkage groups were determined using 143 RAPD markers, covering 1,234.5 cM of the genome. This map was used to detect QTLs associated with Xap resistance on leaves and pods. The averages of disease severity on leaves (represented by the transformed disease index – TDI) and pods (represented by the diameter of lesion on pods – DLP) were added to the data of the linkage map. Five TDI QTLs and only one LDP QTL were detected. The TDI QTLs were placed in the A, B, G and J linkage groups, with phenotypic variations ranging from 12.7 to 71.6%. The DLP QTL explained 12.9% of the phenotypic variation and was mapped in a distinct linkage group. These results indicate that there are different genes involved in the control of resistance on leaves and pods.

Resistance spectra of wheat cultivars and virulence diversity of Magnaporthe grisea isolates in Brazil

Seventy-two monoconidial isolates of Magnaporthe grisea were obtained from the States of Mato Grosso do Sul and Paraná. The isolates were inoculated on seedlings of 20 wheat (Triticum aestivum) cultivars under greenhouse conditions. The virulence diversity of M. grisea was assessed based on compatible and incompatible reactions of leaf blast on wheat cultivars. Fifty-four distinct virulence patterns were identified on test cultivars among the isolates collected from the two wheat growing States. Sixteen of these isolates corresponding to 22.2% showed similar virulence pattern. None of the wheat cultivars was resistant to all isolates of M. grisea, but the cultivars differed in degree of resistance as measured by the relative spectrum of resistance (RSR) and disease index (DI). Among the cultivars the RSR ranged from 0 to 53.3% and DI from 0.4662 to 0.9662 (0 to 1 scale). The wheat cultivar BR18 exhibited a broad resistance spectrum in relation to the rest of the tested cultivars to the isolates of M. grisea, and can be used in wheat resistance breeding.

Evaluation of genetic mixtures of sorghum lines for anthracnose resistance management

The main objective of this work was to evaluate the diversification of sorghum (Sorghum bicolor) populations as a way to manage resistance to the sorghum anthracnose fungus Colletotrichum graminicola. A total of 18 three-way hybrids were obtained by crossing six single cross male-sterile F1 hybrids, derived by crossing A (non restorer sterile cytoplasm) and B (non restorer normal cytoplasm) lines, with three fertile R (restorer) lines, previously evaluated for their differential reaction to the pathogen. Variation in the level of resistance was observed, as indicated by the values of the area under the disease progress curve (AUDPC) obtained for each hybrid. Lines contributed differently to the level of resistance of each hybrid. All hybrids in which CMSXS169R was the male progenitor were classified as highly resistant. Some hybrids had a level of resistance superior to the maximum levels of each line component individually.

Cyanobacteria from the Baltic Sea and Finnish lakes as an energy source and modulators of bioenergetic pathways

Cyanobacteria are a diverse group of oxygenic photosynthetic bacteria that inhabit in a wide range of environments. They are versatile and multifaceted organisms with great possibilities for different biotechnological applications. For example, cyanobacteria produce molecular hydrogen (H2), which is one of the most important alternatives for clean and sustainable energy. Apart from being beneficial, cyanobacteria also possess harmful characteristics and may become a source of threat to human health and other living organisms, as they are able to form surface blooms that are producing a variety of toxic or bioactive compounds. The University of Helsinki Culture Collection (UHCC) maintains around 1,000 cyanobacterial strains representing a large number of genera and species isolated from the Baltic Sea and Finnish lakes. The culture collection covers different life forms such as unicellular and filamentous, N2-fixing and non-N2-fixing strains, and planktonic and benthic cyanobacteria. In this thesis, the UHCC has been screened to identify potential strains for sustainable biohydrogen production and also for strains that produce compounds modifying the bioenergetic pathways of other cyanobacteria or terrestrial plants. Among the 400 cyanobacterial strains screened so far, ten were identified as high H2-producing strains. The enzyme systems involved in H2 metabolism of cyanobacteria were analyzed using the Southern hybridization approach. This revealed the presence of the enzyme nitrogenase in all strains tested, while none of them are likely to have contained alternative nitrogenases. All the strains tested, except for two Calothrix strains, XSPORK 36C and XSPORK 11A, were suggested to contain both uptake and bidirectional hydrogenases. Moreover, 55 methanol extracts of various cyanobacterial strains were screened to identify potent bioactive compounds affecting the photosynthetic apparatus of the model cyanobacterium, Synechocystis PCC 6803. The extract from Nostoc XPORK 14A was the only one that modified the photosynthetic machinery and dark respiration. The compound responsible for this effect was identified, purified, and named M22. M22 demonstrated a dual-action mechanism: production of reactive oxygen species (ROS) under illumination and an unknown mechanism that also prevailed in the dark. During summer, the Baltic Sea is occupied by toxic blooms of Nodularia spumigena (hereafter referred to as N. spumigena), which produces a hepatotoxin called nodularin. Long-term exposure of the terrestrial plant spinach to nodularin was studied. Such treatment resulted in inhibition of growth and chlorosis of the leaves. Moreover, the activity and amount of mitochondrial electron transfer complexes increased in the leaves exposed to nodularin-containing extract, indicating upregulation of respiratory reactions, whereas no marked changes were detected in the structure or function of the photosynthetic machinery. Nodularin-exposed plants suffered from oxidative stress, evidenced by oxidative modifications of various proteins. Plants initiated strategies to combat the stress by increasing the levels of alpha-tocopherol, mitochondrial alternative oxidase (AOX), and mitochondrial ascorbate peroxidase (mAPX).

Antimicrobial resistance of Staphylococcus spp. from small ruminant mastitis in Brazil

The study aimed to determine the antimicrobial resistance patterns and to identify molecular resistance markers in Staphylococcus spp. (n=210) isolated from small ruminant mastitis in Brazil. The antimicrobial resistance patterns were evaluated by the disk diffusion test and by detection of the presence of mecA, blaZ, ermA, ermB, ermC and msrA genes by PCR. The efflux pump test was performed using ethidium bromide and biofilm production was determined by Congo red agar test along with PCR for detection of the icaD gene. The isolates were most resistant to amoxicillin (50.0%), streptomycin (42.8%), tetracycline (40.4%), lincomycin (39.0%) and erythromycin (33.8%). Pan-susceptibility to all tested drugs was observed in 71 (33.8%) isolates and 41 Staphylococcus isolates were positive for the efflux pump. Although phenotypic resistance to oxacillin was observed in 12.8% of the isolates, none harbored the mecA gene. However, 45.7% of the isolates harbored blaZ indicating that beta-lactamase production was the main mechanism associated with staphylococci resistance to beta-lactams in the present study. The other determinants of resistance to antimicrobial agents ermA, ermB, ermC, and msrA were observed in 1.4%, 10.4%, 16.2%, and 0.9% of the isolates, respectively. In addition, the icaD gen was detected in 32.9% of the isolates. Seventy three isolates (54 from goats and 19 from sheep) were negative for all resistance genes tested and 69 isolates presented two or more resistance genes. Association among blaZ, ermA, ermB, ermC and efflux pump were observed in 17 isolates, 14 of which originated from goats and three from sheep. The data obtained in this study show the resistance of the isolates to beta-lactamics, which may be associated with the use of antimicrobial drugs without veterinary control.

Low level resistance of goosegrass (Eleusine indica) to glyphosate in Rio Grande do Sul-Brazil

In the last growing seasons, goosegrass (Eleusine spp.) control failures have been observed following application of glyphosate on Roundup Ready® soybean in Rio Grande do Sul (RS) - Brazil, suggesting this species' resistance to the herbicide. Thus, the objectives of this study were to identify the occurrence of goosegrass resistance to the herbicide glyphosate in RS; and to determine the predominant species of the genus Eleusine, as well as the LD50 and GR50 of the suspected resistant biotypes. Two experiments were conducted under greenhouse conditions: one to identify the biotypes resistant to glyphosate, and the other, a dose-response curve experiment, as well as a study of the botanical characteristics of the species. In the first experiment, 39 biotypes were tested, mainly Eleusine indica, collected with suspected resistance to glyphosate. The glyphosate dose was 2,160 g e.a. ha-1, and the control was evaluated at 28 days after treatment. All biotypes were effectively controlled,with the biotypes from the municipality of Boa Vista do Incra showing greater tolerance. Two biotypes suspected of resistance (12.1 and 12.3) and a susceptible biotype in a dose-response experiment were tested at the following doses: 0, 135, 270, 540, 1,080, 1,620, and 2,160 g e.a. ha-1. The results of this experiment showed that biotype 12.1 does not present resistance to glyphosate and biotype 12.3 has a low level resistance since it is effectively controlled by the herbicide at the maximum dose.

Reduction of seed production of red rice escapes in clearfield rice

Roguing is a practice used to reduce the seed source of red rice escapes to control in Clearfield-rice areas. However, there is great difficulty in performing it in large and heavily infested rice fields. This objective of this work was to evaluate the effects of the use of imazamox herbicide, applied in different rates and times, on plants of Clearfield-rice and red rice. Four experiments were conducted during the 2007/08 and 2008/09 growing seasons, in completely randomized block design and treatments arranged in factorial design, using three replications per treatment. The treatments had increasing rates of imazamox, application times and rice cultivars. The rice cultivars tested were IRGA 417, IRGA 422 CL, Avaxi CL and Puitá INTA CL. The variables evaluated were the number of panicles m-2, number of grains panicle-1, spikelet sterility in rice and red rice; and, rice grain yield and its components. The imazamox reduced the seed production of red rice escapes in a simulated situation of commercial Clearfield-rice area. The greater percentage reductions were obtained when this herbicide was applied at final formation of the panicle or panicle exertion of the red rice plant escapes to control. The Puitá INTA CL cultivar has high level of resistance to imazamox, independent of rate and application times tested, becoming the only alternative to the use of this practice.

Resistance of Raphanus raphanistrum to the herbicide metsulfuron-methyl

The ALS-inhibiting herbicides, especially metsulfuron-methyl, are widely used for weed control, mainly wheat and barley in southern Brazil. Raphanus raphanistrum is a major weed of winter crops. However, in recent years, R.raphanistrum, after being treated with metsulfuron, has shown no symptoms of toxicity, possibly due to herbicide resistance. Aiming to evaluate the existence of R.raphanistrum biotypes resistant to metsulfuron, an experiment was conducted in a greenhouse, in a completely randomized design with four replications. The plots consisted of pots with six plants. The treatments consisted of the interaction of resistant R. raphanistrum (biotype R) and susceptible R. raphanistrum (biotypes S) with ten doses of the herbicide (0.0; 0.6; 1.2; 2.4; 4.8; 9.6; 19.2; 38.4; 76.8 and 153.6 g i.a. ha-1). The application of the test herbicides occurred when the crop was at the stage of 3 to 4 true leaves. The variables analyzed were control and dry matter accumulation. Statistical analysis of dose-response curves was performed by non linear regression. Biotype S was susceptible to the herbicide even at doses below the recommended. Biotype R was insensitive to the herbicide obtaining values of resistance factor (F) higher than 85. The dose-response curve confirmed the existence of R. raphanistrum biotypes with high level of resistance to metsulfuron-methyl.