Prevalence of resistance phenotypes in Staphylococcus aureus and coagulase-negative isolates of venous ulcers of primary healthcare patients

INTRODUCTION: In venous ulcers, the presence of Staphylococcus aureus and coagulase-negative staphylococcus resistance phenotypes can aggravate and limit the choices for treatment. METHODS: Staphylococcus isolated from 69 patients (98 ulcers) between October of 2009 and October of 2010 were tested. The macrolide, lincosamide, streptogramin B (MLS B) group resistance phenotype detection was performed using the D-test. Isolates resistant to cefoxitin and/or oxacillin (disk-diffusion) were subjected to the confirmatory test to detect minimum inhibitory concentration (MIC), using oxacillin strips (E-test®). RESULTS: The prevalence of S. aureus was 83%, and 15% of coagulase-negative staphylococcus (CoNS). In addition were detected 28% of methicillin-resistant Staphylococcus aureus (MRSA) and 47% of methicillin-resistant coagulase-negative staphylococcus (MRCoNS). Among the S. aureus, 69.6% were resistant to erythromycin, 69.6% to clindamycin, 69.6% to gentamicin, and 100% to ciprofloxacin. Considering the MRSA, 74% were highly resistant to oxacillin, MIC ≥ 256µg/mL, and the MLS Bc constitutive resistance predominated in 65.2%. Among the 20 isolates sensitive to clindamycin, 12 presented an inducible MLS B phenotype. Of the MRCoNS, 71.4%were resistant to erythromycin, ciprofloxacin and gentamicin. Considering the isolates positive for β-lactamases, the MIC breakpoint was between 0.5 and 2µg/mL. CONCLUSIONS: The results point to a high occurrence of multi-drug resistant bacteria in venous ulcers in primary healthcare patients, thus evidencing the need for preventive measures to avoid outbreaks caused by multi-drug resistant pathogens, and the importance of healthcare professionals being able to identifying colonized versus infected venous ulcers as an essential criteria to implementing systemic antibacterial therapy.

Analysis of phenotypes altered by temperature stress and hipermutability in Drosophila willistoni

Drosophila willistoni (Sturtevant, 1916) is a species of the willistoni group of Drosophila having wide distribution from the South of USA (Florida) and Mexico to the North of Argentina. It has been subject of many evolutionary studies within the group, due to its considerable ability to successfully occupy a wide range of environments and also because of its great genetic variability expressed by different markers. The D. willistoni 17A2 strain was collected in 1991 in the state of Rio Grande do Sul, Brazil (30°05'S, 51°39'W), and has been maintained since then at the Drosophila laboratory of UFRGS. Different to the other D. willistoni strains maintained in the laboratory, the 17A2 strain spontaneously produced mutant males white-like (white eyes) and sepia-like (brown eyes) in stocks held at 17°C. In order to discover if this strain is potentially hypermutable, we submitted it to temperature stress tests. Eighteen isofemale strains were used in our tests and, after the first generation, all the individuals produced in each strain were maintained at 29°C. Different phenotype alterations were observed in subsequent generations, similar to mutations already well characterized in D. melanogaster (white, sepia, blistered and curly). In addition, an uncommon phenotype alteration with an apparent fusion of the antennae was observed, but only in the isofemale line nº 31. This last alteration has not been previously described as a mutation in the D. melanogaster species. Our results indicate that the D. willistoni 17A2 strain is a candidate for hypermutability, which presents considerable cryptic genetic variability. Different factors may be operating for the formation of this effect, such as the mobilization of transposable elements, effect of inbreeding and alteration of the heat-shock proteins functions.

Analysis of the interacting components between larval Schistosoma mansoni and schistosome-susceptible and resistant Biomphalaria glabrata

Cell-free hemolymph (serum) and hemocytes from Schistosoma mansoni-susceptible (PR albino M-line) and resistant (10-R2) strains of Biomphalaria glabrata were compared by SDS-PAGE, immunoblotting and radioiodination. Whole serum of both snail strains is dominated by hemoglobin (Hb) (MW = 160 Kd). SDS-PAGE. of Hb-depleted serum indicated that the 10-R2 strain has dominant polypeptides in the 50 to 30 Kd range whereas PR albino snails have few low MW proteins. Antibodies raised to whole PR albino and 10-R2 serum, and the 160 Kd (Hb) band reacted similarly in immunoblot assays. Analysis of hemocytes revealed that 10-R2 snails have a surface-exposed protein at about 80 Kd which is not present on PR albino hemocytes. An examination of primary cultured sporocysts indicated the presence of four major surface proteins (40, 50, 55, 70 Kd) and two minor surface-exposed polypeptides (92, 170 Kd). Antibodies raised against live, intact sporocysts reacted almost exclusively with sporocyst-surface proteins when tested by immunoblotting.

Phenotypes of lung mononuclear phagocytes in HIV seronegative tuberculosis patients: evidence for new recruitment and cell activation

Mycobacterium tuberculosis preferentially resides in mononuclear phagocytes. The mechanisms by which mononuclear phagocytes keep M. tuberculosis in check or by which the microbe evades control to cause disease remain poorly understood. As an initial effort to delineate these mechanisms, we examined by immunostaining the phenotype of mononuclear phagocytes obtained from lungs of patients with active tuberculosis. From August 1994 to March 1995, consecutive patients who had an abnormal chest X-ray, no demostrable acid-fast bacilli in sputum specimens and required a diagnostic bronchoalveolar lavage (BAL) were enrolled. Of the 39 patients enrolled, 21 had microbiologically diagnosed tuberculosis. Thirteen of the 21 tuberculosis patients were either HIV seronegative (n = 12) or had no risk factor for HIV and constituted the tuberculosis group. For comparison, M. tuberculosis negative patients who had BAL samples taken during this time (n = 9) or normal healthy volunteers (n = 3) served as control group. Compared to the control group, the tuberculosis group had significantly higher proportion of cells expressing markers of young monocytes (UCHM1) and RFD7, a marker for phagocytic cells, and increased expression of HLA-DR, a marker of cell activation. In addition, tuberculosis group had significantly higher proportion of cells expressing dendritic cell marker (RFD1) and epithelioid cell marker (RFD9). These data suggest that despite recruitment of monocytes probably from the peripheral blood and local cell activation, host defense of the resident lung cells is insufficient to control M. tuberculosis.

Changes in nuclear phenotypes following cold shock in Panstrongylus megistus (Burmeister)

The nuclear phenotypes of Malpighian tubule epithelial cells of 5th instar male nymphs of the blood-sucking insect Panstrongylus megistus were studied immediately after a short (1 h) cold shock at 0ºC, and 10 and 30 days later. The objective was to compare the responses to a cold shock with those known to occur after hyperthermia in order to provide insight into the cellular effect of cold in this species. Nuclei which usually exhibited a conspicuous Y chromosome chromocenter were the most frequent phenotype in control and treated specimens. Phenotypes in which the heterochromatin was unravelled, or in which there was nuclear fusion or cell death were more abundant in the shocked specimens. Most of the changes detected have also been found in heat-shocked nymphs, except for nuclear fusion which generates giant nuclei and which appeared to be less effective or necessary than that elicited after heat shock. Since other studies showed that a short cold shock does not affect the survival of more than 14% of 5th instar nymphs of P. megistus with domestic habit and can induce tolerance to a prolonged cold shock, heat shock proteins proteins are probably the best candidates for effective protection of the cells and the insects from drastic damage caused by low temperature shocks.

Effect of sequential heat and cold shocks on nuclear phenotypes of the blood-sucking insect, Panstrongylus megistus (Burmeister) (Hemiptera, Reduviidae)

Thermal shocks induce changes in the nuclear phenotypes that correspond to survival (heterochromatin decondensation, nuclear fusion) or death (apoptosis, necrosis) responses in the Malpighian tubules of Panstrongylus megistus. Since thermal tolerance increased survival and molting rate in this species following sequential shocks, we investigated whether changes in nuclear phenotypes accompanied the insect survival response to sequential thermal shocks. Fifth instar nymphs were subjected to a single heat (35 or 40°C, 1 h) or cold (5 or 0°C, 1 h) shock and then subjected to a second shock for 12 h at 40 or 0°C, respectively, after 8, 18, 24 and 72 h at 28°C (control temperature). As with specimen survival, sequential heat and cold shocks induced changes in frequency of the mentioned nuclear phenotypes although their patterns differed. The heat shock tolerance involved decrease in apoptosis simultaneous to increase in cell survival responses. Sequential cold shocks did not involve cell/nuclear fusion and even elicited increase in necrosis with advancing time after shocks. The temperatures of 40 and 0ºC were more effective than the temperatures of 35 and 5ºC in eliciting the heat and cold shock tolerances, respectively, as shown by cytological analysis of the nuclear phenotypes. It is concluded that different sequential thermal shocks can trigger different mechanisms of cellular protection against stress in P. megistus, favoring the insect to adapt to various ecotopes.

Behavioural responses to human skin extracts and antennal phenotypes of sylvatic first filial generation and long rearing laboratory colony Rhodnius prolixus

Chagas disease is a major public health issue and is mainly spread by Triatominae insects (Hemiptera: Reduviidae). Rhodnius prolixus is the main vector species in Northern South America. Host-seeking behaviour in R. prolixus is mediated by different compounds that are produced by and emanate from the host or microbiota on the host's skin. We tested the behavioural responses of sylvatic first filial generation (F1) and colony insects to extracts of human skin with a dual choice olfactometer. In addition, we compared the antennal phenotypes in both populations. No statistical differences were found between the two populations at the behavioural level. Both showed a preference for face and feet extracts and this effect was abolished for face extracts after treatment with an antibacterial gel. The observation of the antennal phenotype showed that there were differences between both groups in the total length, total surface area and number and density of bristles. However, the number and density of chemoreceptive sensilla (basiconic and thin and thick-walled trichoids) and the total density of sensilla did not show statistically significant differences. These results demonstrate that colony insects, which have only been fed with living hens for the last 30 years, are attracted by human skin extracts in a similar way as F1 sylvatic insects.

Effect of soil interacting herbicides on soybean nodulation in Balcarce, Argentina

Two trials were performed in Balcarce, Argentina (37° 45' LS; 58° 18' LW) during 1993-94, to assess the effect of eight herbicides applied individually or in tank mixtures, on nodule number, nodule dry weight, seed yield and N percent in seed in soybean Asgrow 3205, inoculated with Bradyrhizobium japonicum CB 1809. Individual herbicides and doses in kg ha-1 of a.i. were metribuzin (0.48), acetochlor (0.90), metolachlor (1), flumioxazin (0.075), trifluralin (0.96), imazaquin (0.20), imazethapyr (0.10) and chlorimuron ethyl (0.0125). The mixtures were metribuzin+acetochlor (0.48+0.9), flumioxazin+acetochlor (0.075+0.9), imazaquin+acetochlor (0.2+0.9), metribuzin+metolachlor (0.48+1.92), and flumioxazin+ metolachlor (0.075+1.92). A control treatment without herbicides was included. Both trials were laid out as randomized complete blocks with four replicates, on a loam illitic thermic petrocalcic Paleudoll, 5.7% organic matter (OM), 25% clay, 30.4 cmol kg-1 CEC. Nodules were sampled at V2 (second node), V6 (sixth node) and R5 (beginning seed) growth stages. Herbicides did not significantly affect the beginning of nodulation or nodule number and mass at R5, not either grain yield or N accumulation. This indicates lack of interference between soil interacting herbicides and N fixation in the high organic matter, loam soils of SE Buenos Aires province, even though a tendency in less number and dry weight of nodules was evident at the two latter growth stages.

Image analysis and definition of nuclear phenotypes

Some basic topics concerned with the extraction of textural and geometric information from cell nucleus images as well as description and characterization of chromatin supraorganization and consequent classification of nuclear phenotypes are presented.

Normal expression of IFN-gammaR in four patients with uncommon mycobacterial infection phenotypes

Several primary immunodeficiency diseases affecting the interleukin 12/interferon gamma (IFN-gamma) pathway have been identified, most of them characterized by recurrent and protracted infections produced by intracellular microorganisms, particularly by several species of mycobacteria. In the present study we analyzed the expression of IFN-gamma receptor (IFN-gammaR) and signal transducer and activator of transcription 1 (STAT-1) in 4 children with Mycobacterium tuberculosis infection of uncommon clinical presentation. These molecules were evaluated by flow cytometry and Western blotting in B cells transformed with Epstein-Barr virus and mutations were scanned by single-strand conformational polymorphisms and DNA sequencing. The expression of IFN-gammaR1 was normal in all 4 patients. The genetic analysis of IFN-gammaR1 and IFN-gammaR2 coding sequences did not reveal any mutation. The expression of the STAT-1 molecule was similar in patients and healthy controls; however, when the phosphorylation of this transcription factor in response to IFN-gamma activation was evaluated by Western blot, a significant lower signal was evident in one patient. These data indicate that there are no alterations in the expression or function of the IFN-gammaR chains in these patients. However, the low level of STAT-1 phosphorylation found in one of these patients might be explained by a defect in one of the molecules involved in the signal transduction pathway after IFN-gamma interacts with its receptor. In the other three patients the inability to eliminate the mycobacteria may be due to a defect in another effector mechanism of the mononuclear phagocytes.

IGF2, LEPR, POMC, PPARG, and PPARGC1 gene variants are associated with obesity-related risk phenotypes in Brazilian children and adolescents

Association studies of genetic variants and obesity and/or obesity-related risk factors have yielded contradictory results. The aim of the present study was to determine the possible association of five single-nucleotide polymorphisms (SNPs) located in the IGF2, LEPR, POMC, PPARG, and PPARGC1genes with obesity or obesity-related risk phenotypes. This case-control study assessed overweight (n=192) and normal-weight (n=211) children and adolescents. The SNPs were analyzed using minisequencing assays, and variables and genotype distributions between the groups were compared using one-way analysis of variance and Pearson's chi-square or Fisher's exact tests. Logistic regression analysis adjusted for age and gender was used to calculate the odds ratios (ORs) for selected phenotype risks in each group. No difference in SNP distribution was observed between groups. In children, POMC rs28932472(C) was associated with lower diastolic blood pressure (P=0.001), higher low-density lipoprotein (LDL) cholesterol (P=0.014), and higher risk in overweight children of altered total cholesterol (OR=7.35, P=0.006). In adolescents, IGF2 rs680(A) was associated with higher glucose (P=0.012) and higher risk in overweight adolescents for altered insulin (OR=10.08, P=0.005) and homeostasis model of insulin resistance (HOMA-IR) (OR=6.34, P=0.010). PPARGrs1801282(G) conferred a higher risk of altered insulin (OR=12.31, P=0.003), and HOMA-IR (OR=7.47, P=0.005) in overweight adolescents. PARGC1 rs8192678(A) was associated with higher triacylglycerols (P=0.005), and LEPR rs1137101(A) was marginally associated with higher LDL cholesterol (P=0.017). LEPR rs1137101(A) conferred higher risk for altered insulin, and HOMA-IR in overweight adolescents. The associations observed in this population suggested increased risk for cardiovascular diseases and/or type 2 diabetes later in life for individuals carrying these alleles.

Virulence profile of ten Paracoccidioides brasiliensis isolates: association with morphologic and genetic patterns

Ten isolates of Paracoccidioides brasiliensis were examined for differences in virulence in outbred mice intravenously inoculated with the fungus, associated with mycelial morphology, and genetic patterns measured by random amplified polymorphic DNA (RAPD). Virulence was evaluated by viable yeast cell recovery from lungs and demonstration of histopathologic lesions in different organs. The results showed that the isolates presented four virulence degrees: high virulence, intermediate, low and non-virulence. RAPD clustered the isolates studied in two main groups with 56% of genetic similarity. Strains with low virulence, Pb265 or the non-virulent, Pb192, showed glabrous/cerebriform morphology and high genetic similarity (98.7%) when compared to the other isolates studied. The same was observed with Bt79 and Bt83 that shared 96% genetic similarity, cottony colonies and high virulence. The RAPD technique could only discriminate P. brasiliensis isolates according to glabrous/cerebriform or cottony colonies, and also high from low virulence strains. Isolates with intermediate virulence such as Pb18, Pb18B6, Bt32 and Bt56 showed variability in their similarity coefficient suggesting that RAPD was able to detect genetic variability in this fungal specie. Virulence profile of P. brasiliensis demonstrated that both mycelial morphologic extreme phenotypes may be associated with fungal virulence and their in vitro subculture time. Thus, RAPD technique analysis employed in association with virulence, morphologic and immunologic aspects might prove adequate to detect differences between P. brasiliensis isolates.

Study of antiretroviral mutants in HIV patients with treatment failures and the effect of risk factors in the virological failures

INTRODUCTION: Information about HIV phenotypes of resistant to available ART and the influence of different risk factors on virological failures (VF) in Costa Rican HIV positive patients prior or during HAART is unknown. MATERIALS AND METHODS: Eighty nine samples, 72 VF and 17 basal (before treatment) were analyzed by examining resistant mutants in reverse transcriptase (RT) and protease (PT) regions using Trugene or LIPA genotyping kits. Sixty eight control patients were selected and relevant information was collected in a questionnaire. RESULTS: Poor adherence, presence of resistant mutations and number of treatment's changes were the only significant factors found (p = 0.006, 0.04 and 0.01 respectively). From 66 sequenced samples, 78%, 50% and 50% showed resistance to NRTI (nucleoside reverse transcriptase inhibitors), NNRT (non-nucleoside reverse transcriptase inhibitors) and PI (protease inhibitors), respectively. The most frequent mutations were M41L, M184V, and T215FY in RT and L62PI, L10FIRV and M36I in PT. DISCUSSION: The most important factor related to treatment response in this study was adherence to treatment. Mutations in RT were related to the treatment failure while the ones found in PT were secondary mutations which have been previously described to influence the selection of primary resistance mutations in these regions. The study reveals the urgency to detect resistant mutations in VF to be considered by physicians for selection of treatment schedule, to analyze basal HIV patients for monitoring of the spread of resistant mutations and the importance to reinforce the adherence in the patients for overall treatment outcome.

Spontaneous morphogenetic juvenilization observed in laboratory populations of vector species of Chagas disease (Triatominae)

Reported are observations on spontaneous occurring morphogenetic juvenilization in laboratory populations of vector species of Chagas disease. Two general effects have been observed: arrested development and uncoordinated development. These are manifested by supernumerary nymphs (6th stage), intermediate nymphal-adult stages, badly deformed adults developed from 5th instar nymphs, uncoordinated development manifested by grotesque forms of adults, supernumerary adults unable to complete metamorphosis and complete supernumerary adults produced by 6th stage nymphs. The reoccurrence of insects with identical grades of juvenilization in the population is an indication that this is a genetic trait that might be inherited. The factors responsible for morphogenetic juvenilization cannot be transmitted through the juvenilized insects because they are sterile, than they were transmitted through normal insects probably as a recessive or a group recessive factors. The spontaneous morphogenetic juvenilization observed in laboratory populations has a striking similarity to juvenilizing effects induced by application of juvenile hormone analogues, described in the literature and also obtained in our laboratory in a study to be published. Thus it is suggested that both; the altered phenotypes occurring in wild populations and their "phenocopies" induced by the application of juvenile hormone analogues are products of gene controlled identical reactions.

Intestinal helminthiases in Ecuador: the relatíonship between prevalence, genetic, and socioeconomic factors

Prevalence of infection with the intestinal helminths, Ascaris lumbricoides, Trichuris trichiura, Ancylostoma duodenale and Strongyloides stercoralis was examinedin 632 residents of communities in Esmeraldas province of Ecuador. These communities were divided into two groups according to area of habitation which reflected different socioeconomic circumstances. Attempts were made to correlate infection status with race and ABO blood group phenotype. The racial groups included blacks, Chachi amerindians, and mixed-race mestizos. Greater prevalences of infection were seen in the area oflower socioeconomic status. No racial or blood group associations with helminth infection were seen controlling for socioeconomic status.