Multidrug resistance genes, including blaKPC and blaCTX-M-2, among Klebsiella pneumoniae isolated in Recife, Brazil

INTRODUCTION: The prevalence of cephalosporins and carbapenem-resistant Klebsiella pneumoniae strains is rising in Brazil, with potential serious consequences in terms of patients' outcomes and general care. METHODS: This study characterized 24 clinical isolates of K. pneumoniae from two hospitals in Recife, Brazil, through the antimicrobial susceptibility profile, analyses of β-lactamase genes (blaTEM, blaSHV,blaCTX-MblaKPC, blaVIM, blaIMP, and blaSPM), plasmidial profile and ERIC-PCR (Enterobacterial repetitive intergenic consensus-polymerase chain reaction). RESULTS: ERIC-PCR and plasmidial analysis grouped the isolates in 17 and 19 patterns, respectively. Six isolates from one hospital presented the same pattern by ERIC-PCR, indicating clonal dissemination. All isolates presented blaSHV, 62.5% presented blaCTX-M-2, 29% blaTEM, and 41.7% blaKPC. Metallo-β-lactamase genes blaand blawere not detected. Eleven isolates were identified carrying at least 3 β-lactamase studied genes, and 2 isolates carried blaSHVblaTEM, blaCTX-M-2 and blaKPC simultaneously. CONCLUSIONS: The accumulation of resistance genes in some strains, observed in this study, imposes limitations in the therapeutic options available for the treatment of infections caused by K. pneumoniae in Recife, Brazil. These results should alert the Brazilian medical authorities to establish rigorous methods for more efficiently control the dissemination of antimicrobial resistance genes in the hospital environment.

Phenotypic and molecular characterization of antimicrobial resistance and virulence factors in Pseudomonas aeruginosa clinical isolates from Recife, State of Pernambuco, Brazil

INTRODUCTION: The emergence of carbapenem resistance mechanisms in Pseudomonas aeruginosa has been outstanding due to the wide spectrum of antimicrobial degradation of these bacteria, reducing of therapeutic options. METHODS: Sixty-one clinical strains of P. aeruginosa isolated from five public hospitals in Recife, Pernambuco, Brazil, were examined between 2006 and 2010, aiming of evaluating the profiles of virulence, resistance to antimicrobials, presence of metallo-β-lactamase (MBL) genes, and clonal relationship among isolates. RESULTS: A high percentage of virulence factors (34.4% mucoid colonies; 70.5% pyocyanin; 93.4% gelatinase positives; and 72.1% hemolysin positive) and a high percentage of antimicrobial resistance rates (4.9% pan-resistant and 54.1% multi-drug resistant isolates) were observed. Among the 29 isolates resistant to imipenem and/or ceftazidime, 44.8% (13/29) were MBL producers by phenotypic evaluation, and of these, 46.2% (6/13) were positive for the blaSPM-1 gene. The blaIMP and blaVIM genes were not detected. The molecular typing revealed 21 molecular profiles of which seven were detected in distinct hospitals and periods. Among the six positive blaSPM-1 isolates, three presented the same clonal profile and were from the same hospital, whereas the other three presented different clonal profiles. CONCLUSIONS: These results revealed that P. aeruginosa is able to accumulate different resistance and virulence factors, making the treatment of infections difficult. The identification of blaSPM-1 genes and the dissemination of clones in different hospitals, indicate the need for stricter application of infection control measures in hospitals in Recife, Brazil, aiming at reducing costs and damages caused by P. aeruginosa infections.

Diagnosing lymphoma in a setting with a high burden of infection: a pediatric case of Epstein-Barr virus-associated aggressive B-cell lymphoma with t(8;14) (q23;q32) and extensive necrosis mimicking tuberculosis

The association of lymphoma with necrotic granuloma can pose diagnostic challenges and delay treatment, especially in settings with a high burden of infection. In these settings, the timely use of cytogenetic and molecular methods is most relevant. Here, we report a case of B-cell lymphoma with t (8;14) in a 5-year-old male child. The lymphoma was associated with necrotic granuloma and was initially misdiagnosed as tuberculosis. Polymerase chain reaction was used to detect clonal lymphoproliferation and to rule out Mycobacterium tuberculosis infection. Tumor cells harbored Epstein-Barr virus and expressed CD20, CD10, BCL6, and Ki67 (30%), leading to the diagnosis of B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma.

Color change using HSB color system of dental resin composites immersed in different common Amazon region beverages

The purpose of this study is to evaluate in vitro the color stability of composite resins when exposed to beverages with high coloring contents from the Amazon region. 240 samples from four different composite brands (Natural Look, Z350, 4Seasons and Opallis) of hue A3 were fabricated using an acrylic matrix. The samples were stored in distilled water at 37ºC for 24 hours. The initial color (T0) was registered using a Canon EOS Rebel XTi 10 mp camera, and then the samples were divided into four groups (n=15): G1 (coffee), G2 (açaí juice), G3 (energetic guaraná) and G4 (control - distilled water). The samples were exposed to solutions of DES (6hs) and RE (18hs) and placed in a double boiler under constant agitation, at 37ºC for 30 days. The samples were immersed in the coloring solutions for 15 minutes daily. After 7, 15 and 30 days, new photographic registers were made (T1, T2 and T3). The images were analyzed using Corel PHOTO-PAINT 12 software to identify the colors through the HSB system. The Kruskal-Wallis and t tests (p<0.05) demonstrated significant differences in color (hue, saturation and brightness). The results revealed that none of the tested composites showed color stability when exposed to coloring solutions, and that the Amazon region beverages (açaí juice and energetic guaraná) showed to be less coloring than coffee.

Nutrição mineral de seringueira IV. Toxicidade de Boro em Hevea brasiliensis

Com freqüência observa-se no Brasil nas áreas de seringueira sintomas visuais de deficiência de boro, quer na fase de formação das mudas em crescimento em viveiro, quer no jardim clonal e mesmo nos primeiros anos de desenvolvimento das plantas definitivas. Foi também constatado que estas deficiências estão associadas com adubação de N, P, K, Mg. Este fato levou os autores a determinar a resposta a plantulas de seringueira a níveis crescentes de boro no substrato (0,5; 1,0; 1,5; 2,0 e 2,5 ppm de B). Plantulas do clone Tjir 1 foram cultivadas em vasos contendo como substrato silica moida, acrescida de solução completa com os níveis de boro acima indicadas. Nove dias após o ensaio dos tratamentos o efeito do boro se fez notar. As plantas cresceram com menor intensidade e apareceram sintomas nas folhas, lembrando a carência de potássio. Houve queda acentuada de folhas. As concentrações de boro eram muito altas nas folhas variando entre 316 e 1300 ppm. No caule e nas raizes as concentrações eram menores de 21 a 85 ppm. Os autores concluem que a seringueira é muito sensível a toxicidade de boro, devendo ser dada uma atenção especial com este micronutriente nas plantações.

Plant tissue culture techniques

Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus) or organized tissues or organs put in culture, under controlled sterile conditions.

Coevolution of hosts and microorganisms: an analysis of the involvment of cytokines in host-parasite interactions

Parasites may employ particular strategies of eluding an immune response by taking advantage of those mechanisms that normally guarantee immunological self-tolerance. Much in the way as it occurs during the establishment of self-tolerance, live pathogens may induce clonal deletion, functional inactivation(anergy) and immunosupression. At this latter level, it appears that certain pathogens produce immunosupresive cytokine-like mediators or provoke like host the secrete cytokines that will compromise the anti-parasite immune response. It appears that immune responses that preferentially involve T helper l cells (secretors of interleukin-2-and interferon-y) tend to be protective, whereas T helper 2 cells (secretors of IL-4, IL5, IL-6, and IL-10), a population that antagonizes T helper cells, mediate disease susceptibility and are immunopathological reactions. Cytokines produced by T helper 2 cells mediate many symptoms of infection, including eosinophilia, mastocytosis, hyperimmunoglobulinemia, and elevated IgE levels. Administration of IL-2 and IFN-y has beneficial effects in many infections mediated by viruses, bacteria, and protozoa. The use of live vaccinia virus might be an avenue for the treatment of or vaccination against infection. We have found that a vaccinia virus expressing the gene for human IL-2, though attenuated, precipitates autoimmune disease in immunodeficient athymic mice. Thus, although T helper l cytokines may have desired immunostimulatory properties, they also may lead to unwarranted autoaggressive responses.

Multilocus enzyme electrophoresis study of Bacillus sphaericus

Multilocus enzyme electrophoresis (MLEE) has been used in the study of some Bacillus species. In this work we applied MLEE and numerical analysis in the study of the Bacillus sphaericus group. B. sphaericus can be distinguished from other entomopathogenic Bacillus by a unique allele (NP-4). Within the species, all insect pathogens were recovered in the same phenetic cluster and all of these strains have the same band position (electrophoresis migration) on the agarose gel (ADH-2). The entomopathogenic group of B. sphaericus seems to be a clonal population, having two widespread frequent genotypes (zymovar 59 and zymovar 119).

Characterization of subpopulations (clones and subclones) of the 21 SF strain of Trypanosoma cruzi after long lasting maintenance in the laboratory

Several studies have shown a clonal structure of Trypanosoma cruzi and its possible correlation with the behavioral heterogeneity of the parasite strains. In the present study, the 21 SF strain, that have been maintained in laboratory by successive passages in mice, for more than 15 years, showing a stability of biological and isoenzymic characteristics has been cloned, with the objective of establishing the characters of its clones and subclones. With the technique of isolation of a single parasite from the blood of infected mice, 5 clones and 14 subclones have been obtained. After four passages into mice, inoculum of 10(5) was obtained for each clone and subclone and inoculated into mice weighing 10 to 12 g. These were used for the study of the biological behavior of the clones: evolution of parasitemia, morphology of blood forms and host mortality. For isoenzymic characterization, the clones and subclones were analyzed for ALAT, ASAT, GPI and PGM enzymes. Results have shown that the 5 clones and the 14 subclones disclosed a biological behavior similar to the parental strain, with minor variability of the parasitemic profiles and also the same isoenzymic patterns. These results confirm the stability of the 21 SF strain and indicate a clonal homogeneity of its populations. This is compatible with the hypothesis that the T. cruzi strains represent an equilibrium of either homogenous or heterogeneous populations.

Genetic Variability and Microdistribution of Triatoma infestans Genotypes and Trypanosoma cruzi Clones in Arequipa Region (Peru)

The genetic variability of Triatoma infestans and Trypanosoma cruzi populations was studied by isoenzyme analysis in two distinct areas of Arequipa province (Peru); one, Santa Rita de Siguas, being an endemic area for Chagas' disease, the second, Arequipa, recently infected. Analysis of T. infestans genetic variability indicates, (i) temporal stability of genotypes found in Santa Rita de Siguas, (ii) high genetic differences between Arequipa and Santa Rita de Siguas populations suggesting minor contact between them, (iii) multiple origin of the T. infestans population in Arequipa, and (iv) poor dispersal capacity of T. infestans: the panmictic unit could be reduce to a house. Parasite isoenzyme analysis was performed in 29 Peruvian stocks of T. cruzi, mainly isolated from bugs taken in a single locality, Santa Rita de Siguas. The results show, (i) a high genetic polymorphism, (ii) nine different multilocus genotypes were detected and clustered in two different clades, (iii) most of the parasite isolates pertained to one of the clade and were genetically similar to those analyzed 12 years before. This sample allowed the study of the mating system of T. cruzi in strict sympatic conditions and gave more strength to the hypothesis of the clonal structure of T. cruzi populations

Genetic Diversity in Natural Populations of New World Leishmania

Our results have shown the wide diversity of parasites within New World Leishmania. Biochemical and molecular characterization of species within the genus has revealed that much of the population heterogeneity has a genetic basis. The source of genetic diversity among Leishmania appears to arise from predominantly asexual, clonal reproduction, although occasional bouts of sexual reproduction can not be ruled out. Genetic variation is extensive with some clones widely distributed and others seemingly unique and localized to a particular endemic focus. Epidemiological studies of leishmaniasis has been directed to the ecology and dynamics of transmission of Leishmania species/variants, particularly in localized areas. Future research using molecular techniques should aim to identify and follow Leishmania types in nature and correlate genetic typing with important clinical characteristics such as virulence, pathogenicity, drug resistance and antigenic variation. The epidemiological significance of such variation not only has important implications for the control of the leishmaniases, but would also help to elucidate the evolutionary biology of the causative agents.

The population structure of Trypanosoma cruzi: expanded analysis of 54 strains using eight polymorphic CA-repeat microsatellites

Recently we cloned and sequenced the first eight Trypanosoma cruzi polymorphic microsatellite loci and studied 31 clones and strains to obtain valuable information about the population structure of the parasite. We have now studied 23 further strains, increasing from 11 to 31 the number of strains obtained from patients with chronic Chagas disease. This expanded set of 54 strains and clones analyzed with the eight microsatellites markers confirmed the previously observed diploidy, clonal population organization and very high polymorphism of T. cruzi. Moreover, this new study disclosed two new features of the population genetic structure of T. cruzi. The first was the discovery that, similarly to what we had previously shown for strains isolated from insect vectors, mammals and humans with acute disease, isolates from patients in the chronic phase of Chagas disease could also be multiclonal, albeit at a reduced proportion. Second, when we used parsimony to display the genetic relationship among the clonal lineages in an unrooted Wagner network we observed, like before, a good correlation of the tree topography with the classification in three clusters on the basis of single locus analysis of the ribosomal RNA genes. However, a significant new finding was that now the strains belonging to cluster 2 split in two distant sub-clusters. This observation suggests that the evolutionary history of T. cruzi may be more complex than we previously thought.

Genetic diversity and genetic exchange in Trypanosoma cruzi: dual drug-resistant "progeny" from episomal transformants

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.

Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in Chagas disease patients

Integration of kDNA sequences within the genome of the host cell shown by PCR amplification with primers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southern hybridization with specific probes. The cells containing the integrated kDNA sequences were then perpetuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressed membrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronic Chagas disease. These antigens barely expressed in the membrane of uninfected, control macrophage clonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasic sera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizes auto-antigens in the membrane of T. cruzi kDNA transfected macrophage subclonal lines.

Population genetic analysis of Colombian Trypanosoma cruzi isolates revealed by enzyme electrophoretic profiles

Although Colombia presents an enormous biological diversity, few studies have been conducted on the population genetics of Trypanosoma cruzi. This study was carried out with 23 Colombian stocks of this protozoa analyzed for 13 isoenzymatic loci. The Hardy-Weinberg equilibrium, the genetic diversity and heterogeneity, the genetic relationships and the possible spatial structure of these 23 Colombian stocks of T. cruzi were estimated. The majority of results obtained are in agreement with a clonal population structure. Nevertheless, two aspects expected in a clonal structure were not discovered in the Colombian T. cruzi stocks. There was an absence of given zymodemes over-represented from a geographical point of view and the presumed temporal stabilizing selective phenomena was not observed either in the Colombian stocks sampled several times through the years of the study. Some hypotheses are discussed in order to explain the results found.