Genotypic characterization of virulence factors in Escherichia coli strains from patients with cystitis

Adhesins (P-fimbriae, S-fimbriae, type 1 fimbriae and afimbrial adhesin), toxins (α-hemolysin and cytotoxic necrotizing factor type 1), iron acquisition systems (aerobactin) and host defense avoidance mechanisms (capsule or lipopolysaccharide) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. In this work, 162 Uropathogenic Escherichia coli (UPEC) strains from patients with cystitis were genotypically characterized by polymerase chain reaction (PCR) assay. We developed three multiplex PCR assays for virulence-related genes papC, papE/F, papG alleles, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, and kpsMTII, all of them previously identified in UPEC strains. The PCR assay results identified 158 fimH (97.5%), 86 kpsMTII (53.1%), 53 papC/papEF/papG (32.7%), 45 sfa (27.8%), 42 iucD (25.9%), 41 hly (25.3%), 36 usp (22.2%), 30 cnf-1(18.5%) and 10 afa (6.2%) strains. No strain was positive for cdtB. In this work, we also demonstrated that adhesins may be multiple within a single strain and that several virulence genes can occur combined in association.

Chagas' Disease and HIV Co-infection: Genotypic Characterization of the Trypanosoma cruzi Strain

In the past few years, new aspects of the immunopathology of Chagas' disease have been described in immunosuppressed patients, such as fatal central nervous system lesions related to the reactivation of the parasite. This article is the first description of the genotypic characterization, at the strain level, of Trypanosoma cruzi isolated from a patient with Chagas` disease/AIDS co-infection. The presence of four hypodense lesions was observed in the cranial compute tomographic scan. The diagnosis of AIDS was assessed by the detection of anti-HIV antibodies using enzyme-linked immunosorbent assay (ELISA) and Western blot techniques. The CD4+ lymphocyte counts were maintained under 200 cells/mm3 during one year demonstrating the severity of the state of immunosuppression. Chagas' disease was confirmed by serological and parasitological methods. Trypomastigote forms were visualized in a thick blood smear. The parasite isolated is genotypically similar to the CL strain. The paper reinforces that cerebral Chagas' disease can be considered as another potential opportunistic infection in AIDS resulting from the reactivation of a dormant T. cruzi infection acquired years earlier.

MOLECULAR IDENTIFICATION AND ANTIMICROBIAL RESISTANCE PATTERN OF SEVEN CLINICAL ISOLATES OF Nocardia spp. IN BRAZIL

Nocardia is a ubiquitous microorganism related to pyogranulomatous infection, which is difficult to treat in humans and animals. The occurrence of the disease is on the rise in many countries due to an increase in immunosuppressive diseases and treatments. This report of cases from Brazil presents the genotypic characterization and the antimicrobial susceptibility pattern using the disk-diffusion method and inhibitory minimal concentration with E-test® strips. In summary, this report focuses on infections in young adult men, of which three cases were cutaneous, two pulmonary, one neurological and one systemic. The pulmonary, neurological and systemic cases were attributed to immunosuppressive diseases or treatments. Sequencing analysis of the 16S rRNA segments (1491 bp) identified four isolates of Nocardia farcinica, two isolates of Nocardia nova and one isolate of Nocardia asiatica. N. farcinica was involved in two cutaneous, one systemic and other pulmonary cases; N. nova was involved in one neurological and one pulmonary case; and Nocardia asiatica in one cutaneous case. The disk-diffusion antimicrobial susceptibility test showed that the most effective antimicrobials were amikacin (100%), amoxicillin/clavulanate (100%), cephalexin (100%) and ceftiofur (100%), while isolates had presented most resistance to gentamicin (43%), sulfamethoxazole/trimethoprim (43%) and ampicillin (29%). However, on the inhibitory minimal concentration test (MIC test), only one of the four isolates of Nocardia farcinica was resistant to sulfamethoxazole/trimethoprim.

ISOLATION AND GENOTYPING OF Toxoplasma gondii IN SERONEGATIVE URBAN RATS AND PRESENCE OF ANTIBODIES IN COMMUNICATING DOGS IN BRAZIL

The role of rodents in the epidemiology of toxoplasmosis was investigated inLondrina, Paraná State, Brazil. One hundred and eighty-one Rattus rattus and one Mus musculus were caught in 37 places. Blood and tissues were collected and submitted to the indirect fluorescence antibody test (IFAT) and the bioassay. Serum samples from 61 contacting dogs were also collected. Sixteen rats (8.8%) were positive for Toxoplasma gondii, but just two of them were positive by serology and bioassay test. Antibodies were found in nine (4.9%) rats. Tissues of nine rats bioassayed were positive and four isolates were obtained. Restriction fragment length polymorphism (RFLP) analysis was performed using 12 markers (SAG1, SAG2, SAG2-alt, C22-8, C29-2, L358, PK1, BTUB, GRA6, SAG3, Apico, CS3). Genotyping revealed that the four strains isolated from this study have been isolated before in cats and chickens from Brazil. None of the isolates was identified like clonal archetypal T-types I, II, and III. The rats presented lower serologic Toxoplasma gondii prevalence (8.8%) compared to contacting dogs (70.5%).

Molecular analysis and dimorphism of azole-susceptible and resistant Candida albicans isolates

INTRODUCTION: Candida albicans is responsible for superficial or systemic infections known as candidiasis, which may be found in infected tissue as unicellular budding yeasts, hyphae, or pseudohyphae. In this study, the effects of both fluconazole and itraconazole antifungal agents on the hyphal formation and genotypic characterization of C. albicans isolates classified as either susceptible or resistant were investigated. METHODS: The hyphal production of five C. albicans isolates under the action of antifungal agents was investigated by culturing yeast on growth medium and on hyphal induction medium. The genotypic characterization was carried out for 13 isolates of C. albicans using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method. RESULTS: The dimorphism analysis showed that the hyphal formation was higher in resistant than in the susceptible isolates to both azoles. The RAPD-PCR method identified the formation of two different groups. In group A, four resistant and two susceptible isolates were clustered, and in group B, one resistant and six susceptible isolates were clustered. CONCLUSIONS: Considering that hyphal formation was higher in resistant isolates in the presence of azole drugs, we confirmed that the hyphal production is closely related to susceptibility to azoles. These drugs may affect the morphogenesis of C. albicans depending on their susceptibility to these drugs. In relation to RAPD-PCR, most resistant isolates classified in group A and susceptible isolates in group B demonstrated that this method presented a similar standard between the two groups, suggesting that by this technique, a strong correlation between genotypes and fluconazole-resistant samples may be found.

Should Trypanosoma cruzi be called "cruzi" complex? A review of the parasite diversity and the potential of selecting population after in Vitro culturing and mice infection

Morpho-biological diversity of Trypanosoma cruzi has been known since Chagas' first works in 1909. Several further studies confirmed the morphological differences among the parasite strains, which were isolated from different reservoirs and vectors, as well as from human beings. In the early sixties, antigenic differences were found in the parasite strains from various sources. These differences, coupled to the observation of regional variations of the disease, led to the proposal of the term cruzi complex to designate the taxon T. cruzi. Since then this protozoan has been typed in distinct biodemes, zymodemes and lineages which were consensually grouped into T. cruzi I, T. cruzi II and into non-grouped strains. T. cruzi genotypic characterization, initially carried out by schizodeme analysis and more recently by various other techniques, has shown a great diversity of the parasite strains. In fact, T. cruzi is formed by groups of heterogeneous sub-population, which present specific characteristics, including distinct histotropism. The interaction of the different infecting clones of the cruzi complex and the human host will determine the morbidity of the disease.

Trypanosoma cruzi strains isolated from human, vector, and animal reservoir in the same endemic region in Mexico and typed as T. cruzi I, discrete typing unit 1 exhibit considerable biological diversity

In this study, three strains of Trypanosoma cruzi were isolated at the same time and in the same endemic region in Mexico from a human patient with chronic chagasic cardiomyopathy (RyC-H); vector (Triatoma barberi) (RyC-V); and rodent reservoir (Peromyscus peromyscus) (RyC-R). The three strains were characterized by multilocus enzyme electrophoresis, random amplified polymorphic DNA, and by pathological profiles in experimental animals (biodemes). Based on the analysis of genetic markers the three parasite strains were typed as belonging to T. cruzi I major group, discrete typing unit 1. The pathological profile of RyC-H and RyC-V strains indicated medium virulence and low mortality and, accordingly, the strains should be considered as belonging to biodeme Type III. On the other hand, the parasites from RyC-R strain induced more severe inflammatory processes and high mortality (> 40%) and were considered as belonging to biodeme Type II. The relationship between genotypes and biological characteristics in T. cruzi strains is still debated and not clearly understood. An expert committee recommended in 1999 that Biodeme Type III would correspond to T. cruzi I group, whereas Biodeme Type II, to T. cruzi II group. Our findings suggest that, at least for Mexican isolates, this correlation does not stand and that biological characteristics such as pathogenicity and virulence could be determined by factors different from those identified in the genotypic characterization

Biodiversity of rhizobia associated with cowpea cultivars in soils of the lower half of the São Francisco River Valley

The biodiversity of rhizobium in soils of the São Francisco Valley is unknown and can be studied using cowpea as trap plants. The objective of this study was to verify the diversity of diazotrophic bacteria that nodulate cowpea in soils of the lower half of the São Francisco River Valley by morphological and genotypic characterization. Seven soil samples (A1, A2, A3, A4, C1, C2 and MC) were collected to capture bacteria associated to five cowpea cultivars (IPA 206, BRS Pujante, BRS Marataoã, Canapu Roxo, and Sempre Verde), in a 5x7 factorial design with three replications. Thirty days after plant emergence, the nodules were collected and the bacteria isolated and analyzed in relation to their growth characteristics in YMA medium. The 581 isolates were grouped in 49 morphologic groups. Of this total, 62.3 % formed colonies in up to three days, 33.4 % grew from the 6th day on, and 4.3 % began to grow 4 to 5 days after incubation. Regarding the formation of acids and alkalis, 63 % acidified the medium, 12 % made it alkaline and 25 % maintained the medium at neutral pH. The highest diversity was observed in the A3 sample and in isolates associated with the cultivars Canapu Roxo and BRS Pujante. Thirty-eight representative isolates were chosen for the genotypic characterization, clustered in four groups based on the restriction analysis of 16s rDNA. This grouping was strongly correlated with the sampling site; 13 rhizobium isolates had an electrophoretic profile distinct from the standard rhizobium strains used in this study.

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Characterization of a new coagulase-negative Staphylococcus species (Staphylococcus pettenkoferi) isolated from blood cultures from a hospitalized patient in Porto Alegre, Brazil

INTRODUCTION: Staphylococcus pettenkoferi was originally isolated and described by Trülzsch et al (2002). In this study, we characterized two isolates of this newly described species. METHODS: Blood cultures were initially processed using the BacT/ALERT® device, and the isolates were initially characterized using the Vitek2 identification system. RESULTS: The initial characterization revealed slow-growing Gram-positive cocci that formed opaque colonies on sheep blood agar. Other phenotypic/genotypic tests were performed. CONCLUSIONS: We would like to emphasize that this new staphylococcus species is phenotypically similar to other CoNS, especially S. auricularis. This could potentially lead to misidentification of these uncommon species.

Genetic characterization of Trypanosoma cruzi natural clones from the state of Paraíba, Brazil

Eighteen Trypanosoma cruzi stocks from the state of Paraíba, Brazil, isolated from man, wild mammals, and triatomine bugs were studied by multilocus enzyme electrophoresis and random primed amplified polymorphic DNA. Despite the low number of stocks, a notable genetic, genotypic, and phylogenetic diversity was recorded. The presence of the two main phylogenetic subdivisions, T. cruzi I and II, was recorded. The strong linkage disequilibrium observed in the population under survey suggests that T. cruzi undergoes predominant clonal evolution in this area too, although this result should be confirmed by a broader sample. The pattern of clonal variation does not suggests a recent origin by founder effect with a limited number of different genotypes.

Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopetide resistance by conjugative transfer of VanA elements.

Genetic characterization of morphologically variant strains of Paracoccidioides brasiliensis

Molecular characterization of Paracoccidioides brasiliensis variant strains that had been preserved under mineral oil for decades was carried out by random amplified polymorphic DNA analysis (RAPD). On P. brasiliensis variants in the transitional phase and strains with typical morphology, RAPD produced reproducible polymorphic amplification products that differentiated them. A dendrogram based on the generated RAPD patterns placed the 14 P. brasiliensis strains into five groups with similarity coefficients of 72%. A high correlation between the genotypic and phenotypic characteristics of the strains was observed. A 750 bp-RAPD fragment found only in the wild-type phenotype strains was cloned and sequenced. Genetic similarity analysis using BLASTx suggested that this RAPD marker represents a putative domain of a hypothetical flavin-binding monooxygenase (FMO)-like protein of Neurospora crassa.

Pathogenicity, molecular characterization, and cercosporin content of Brazilian isolates of Cercospora kikuchii

Cercospora kikuchii, involved with the defoliation of soybean (Glycine max) plants, is normally associated with Septoria glycines in late season. Seventy-two isolates from different regions in Brazil, obtained mainly from purple stained seeds, showed phenotypic variation. Cercosporin content and rate of colony growth was higly variable among isolates. A strong correlation between cercosporin content and virulence was identified. Genetic variation among and within populations was evaluated based on 86 RAPD loci. The RAPD analysis clustered all isolates into seven groups. No relationship was observed between RAPD groups and geographic origin or cercosporin content. The sequence of the internal spacer regions (ITS1-5.8S-ITS2) from 13 isolates chosen according to the previous RAPD and clustering analysis showed high similarity (97%-100%) to the GenBank sequences of C. kikuchii (AY266160, AY266161, AY152577 and AF291708). It is clear from this work that Brazilian isolates of C. kikuchii from different geographic regions, are variable in relation to virulence, RAPD profiles and cercosporin content. Cercosporin content could be a good parameter for choosing an adequate isolate for screening resistant or tolerant cultivars. Considering that this pathogen is easily seed-borne, findings are expected to show the same haplotypes in different regions. Migration could be favoured by infected seeds as demonstrated by RAPD analysis.

Genotype characterization of Haematobia irritans from different Brazilian geographic regions based on randomly amplified polymorphic DNA (RAPD) analysis

Blood-sucking diptera are important parasites in bovine production systems, especially regarding confinement conditions. Haematobia irritans, the horn fly, is one of the most troublesome species within bovine production systems, due to the intense stress imposed to the animals. An important aspect while studying the variability within a species is the study of the geographic structure of its populations and, attempting to find out the genetic flow of Brazilian populations of horn fly, the RAPD technique, which is suited for this purpose, has been used. The use of molecular markers generated from RAPD made it possible to identify the geographic origin of samples from different Brazilian geographic regions, as well as to estimate the genotypic flow among the different Brazilian populations of the horn fly.