A comparison of virulence patterns and in vivo fitness between hospital- and community-acquired methicillin-resistant Staphylococcus aureus related to the USA400 clone.

Methicillin-resistant Staphylococcus aureus (MRSA) isolates genetically related to the CA-MRSA clone MW2/USA400 (ST1-SCCmecIV lineage) from the United States have emerged in hospitals in Rio de Janeiro and are associated with nosocomial bloodstream infections. To understand the virulence mechanisms involved in the adaptability of ST1 isolates as a hospital pathogen in Rio de Janeiro, we compared the virulence traits and fitness properties of the Brazilian isolates with those displayed by the CA-MRSA isolates from the United States. Similar to the USA400 from the United States, all the Brazilian isolates tested carried the genes encoding SEH and LukDE. In contrast, none of the Brazilian isolates carried the lukSF PVL, sea, sec, and sek genes. Competition experiments in mice demonstrated a significant increase in the fitness for the CA-MRSA isolates MW2 and USA400-0051 from the United States compared to other isolates. In the foreign body animal model, 83 % more North-American bacterial cells were recovered compared to the Brazilian ST1 isolates. Differences in gene expression of important virulence factors were detected. Transcription of rnaIII and psmα3 was increased about two-fold in the isolates from the United States, and sasG about two-fold in the Brazilian isolates. Thus, it is possible that the virulence attenuation observed among the Brazilian hospital isolates, associated with the acquisition of multiple resistant determinants, are consequences of microevolutionary events that contributed to the necessary fitness adjustment of this lineage, allowing a typically community-acquired MRSA (MW2/USA400) to emerge as a successful hospital pathogen (Brazilian ST1-SCCmecIV).

Efficiency of superstimulatory protocol P-36 associated with the administration of eCG and LH in Nelore cows.

Recent work with P-36 demonstrates that the replacement of the last two doses of Follicle-Stimulating Hormone (FSH) with equine chorionic gonadotropin (eCG) increases embryo yields. However, it is unclear if the positive effect of eCG is related to its FSH-like activity, LH-like activity, or both. This study aimed to verify the replacement of eCG with pLH on the last day of superstimulatory treatment. Twenty-five Nelore cows were allocated to four groups: P-36 (control), P-36/eCG, P-36/LH2, and P-36/LH4. All animals underwent four treatments in a crossover design. The control group cows were superstimulated with decreasing doses of porcine Follicle-Stimulating Hormone (pFSH, 133 mg, im). In the P-36/eCG, P-36/LH2, and P-36/LH4 groups, the last two doses of pFSH were replaced in the former group by two doses of eCG (200 IU each dose, im) and in the latter two groups by two doses of pLH (1 and 2 mg each dose, im), respectively. Donors received fixed-time artificial insemination 12 and 24 hours after pLH. Embryo flushing was performed on D16. Data were analyzed by ANOVA (Proc Mixed, SAS). There was a trend of decreasing ovulation rate when comparing groups LH2 and eCG (P = 0.06). However, there was no significant difference in the mean number of viable embryos among groups P-36 (3.3 ± 0.7), P-36/eCG (4.5 ± 0.5), P-36/LH2 (3.7 ± 0.8), and P-36/LH4 (4.2 ± 1.0). It is concluded that the replacement of eCG by pLH on the last day of superstimulatory treatment can be performed with no significant variation in the production of viable embryos.

Improvement of embryo production by the replacement of the last two doses of porcine follicle-stimulating hormone with equine chorionic gonadotropin in Sindhi donors.

The aim of this study was to evaluate the superovulatory (SOV) response of Sindhi (Bos indicus) donors submitted to an ovarian follicular superstimulatory protocol replacing the last two doses of pFSH by eCG. Forty-eight SOV treatments were performed in a crossover design in 19 nulliparous and primiparous females that were randomly divided into two groups: FSH (n=24), which consisted of eight pFSH injections, or FSH/eCG (n=24), which consisted of six pFSH injections followed by two eCG injections. Each female underwent two or three SOV treatments that consisted of an i.m. injection of 2mg estradiol benzoate and the insertion of an intravaginal progesterone-releasing device on Day 0. On Day 4, superstimulatory treatments were initiated and 100mg pFSH was divided into twice daily decreasing doses over a 4-day period. In the FSH/eCG group, the last two doses of pFSH were replaced by two doses of eCG (150 IU eCG each). At the time of the fifth and sixth injections of FSH, 0.150 mg PGF(2α) was injected i.m. The intravaginal progesterone-releasing device was removed at the time of the last FSH or eCG injection and ovulation was induced with 0.2 mg GnRH 18 h later. All females were artificially inseminated with frozen-thawed semen from the same bull 6 and 18 h after GnRH treatment. Seven days after GnRH treatment, embryos/ova were recovered and classified. Follicular superstimulatory (number of follicles ≥6mm at the time of the last FSH or eCG injection) and SOV (CL number) responses were determined by transrectal ultrasonography. Data were analyzed using generalized linear models and results were presented as least squares means±standard error. The FSH/eCG group had higher superstimulatory (33.8±3.9 compared to 23.8±2.6 follicles; P=0.03) and SOV (16.8±2.9 compared to 10.8±2.1 CL; P=0.10) responses. Although the number of total ova/embryos was not different between groups (8.2±1.8 compared to 5.9±1.4 for FSH/eCG and FSH groups, respectively; P=0.25), the number (5.8±1.3 compared to 2.6±0.7; P=0.02) and percentage (75.6±5.7 compared to 53.2±9.7%; P=0.05) of transferable embryos was greater for the FSH/eCG females. Therefore, there was improvement in follicular superstimulatory and SOV responses and embryo quality in FSH/eCG-treated females.

Virulence characteristics of genetically related isolates of group B streptococci from bovines and humans.

The present study had the objective of evaluating the pathogenic potential of the genetically related strains of Streptococcus agalactiae no. 80427 (human origin) and no. 87159 (bovine origin), and comparing the results with two other strains isolated from bovine mastitis (no. 87244) and invasive human infection (no. 90356), with no genetic or epidemiologic relationship between them or with the first 2 isolates. Virulence genes hylB (hyaluronidase) and lmb (laminin-binding protein) were detected in the 4 strains, and genes bac (beta protein) and bca (alpha protein) were only detected in human strains. The protein profile obtained using SDS-PAGE did not indicate any differences between the 4 strains. No significant difference was detected between human and bovine strains in the assays of adherence to and invasion of 16HBe cells, as well as in the resistance assay for intracellular bacterial survival in macrophages. However, the strain 87159 exhibited a greater survival in the killing test with whole human blood and was more virulent in newborn mice than the 80427 strain. The strain 87244 was not virulent in mice. These data suggest that isolates of human and bovine origins may express similar virulence attributes, leading to a possible, however limited, dissemination.

Genetic relatedness between group B streptococci originating from bovine mastitis and a human group B Streptococcus type V cluster displaying an identical pulsed-field gel electrophoresis pattern.

Twenty isolates of group B streptococcus (GBS) were recovered from the milk of cows with bovine mastitis on three farms located in the south and south-east of Brazil between 1987 and 1988. These isolates were characterised by molecular methods and compared with a collection of 103 human GBS isolates from colonised and infected patients in the same region between 1980 and 2003. Some of the bovine isolates shared identical or similar pulsed-field gel electrophoresis (PFGE) patterns with a PFGE clone of human GBS type V. In addition, these bovine and human isolates also possessed the same ribotype. Multilocus sequence typing (MLST) of representative isolates confirmed the genetic relationship between the human and bovine GBS isolates with identical PFGE patterns, which clustered in the same ST-26 clonal complex. These data support the hypothesis that some bovine GBS strains are related closely to human isolates and may infect humans, or vice versa. Further comparative genomic analyses of GBS isolates from bovine and human origins are required to investigate this hypothesis further.

Pulsed-field gel electrophoresis of human group B streptococci isolated in Brazil.

The present study addresses epidemiological aspects of Brazilian human group B streptococci (GBS). GBS (103 isolates) were serotyped with specific rabbit anticapsular antibodies by double diffusion in agarose gels. They represented 3 serotypes: 26 II, 41 III, and 36 V. Thereafter, the strains were characterized by pulsed-field gel electrophoresis (PFGE) of DNA treated with SmaI. DNA restriction band sizes were compared and displayed 54 PFGE profiles that were arranged into 18 patterns. Of the predominant patterns detected for the 41 type III isolates 4 were observed in 15 strains from individuals with infections whereas only 3 were identified in 22 streptococci from healthy carriers. Such differences did not separate types II and V streptococci from carriers and patients. The PFGE method is a sensitive, precise, and powerful tool for discriminating streptococcal strains for epidemiological purposes.

1H and 13C NMR spectra of 3,8-dimethoxyfuro[3,2-g]coumarin and maculine from Esenbeckia grandiflora Martius (Rutaceae).

One- and two-dimensional NMR experiments were used for the unambiguous assignment of the (1)H and (13)C NMR chemical shifts of the furoquinoline alkaloid maculine (1) and the new furanocoumarin 3,8-dimethoxyfuro[3,2-g]coumarin (2).

Total assignment of 1H and 13C NMR spectra of the alkaloid 3,3-diisopentenyl-N-methyl-2,4-quinoldione and novel reaction derivatives.

One- and two-dimensional NMR experiments were used for the unambiguous assignment of the 1H and 13C NMR chemical shifts of 3,3-diisopentenyl-N-methyl-2,4-quinoldione and five novel reaction derivatives.

Lavagem traqueobrônquica por sondagem nasotraqueal em bezerros / Tracheobronchial lavage in calves using a nasotracheal technique

Avaliou-se a técnica de lavagem traqueobrônquica por sondagem nasotraqueal e caracterizou-se a população celular em 10 bezerros clinicamente sadios. Após a contenção dos animais em decúbito lateral e auxílio de sonda guia, foi introduzida uma sonda de menor diâmetro até a bifurcação da traquéia, para produzir tosse e obter o lavado traqueobrônquico. A média de células totais nas amostras de lavado foi de 133.750 células/ml. A citologia, foram observados na contagem diferencial: 77,2 por cento macrófagos, 14,9 por cento células epiteliais cilíndricas, 6,0 por cento neutrófilos e 1,8 por cento linfócitos. Das células epiteliais cilíndricas, 79,0 por cento eram do tipo ciliadas e 21,0 por cento não-ciliadas. A média de contagem de macrófagos binucleados foi de 78,5 células/lâmina, a de macrófagos trinucleados de 20,5/lâmina e a de células gigantes 28,5/lâmina. Concluiu-se que o método de colheita por sondagem nasotraqueal é eficiente para caracterizar a citologia do lavado traqueobrônquico de bezerros clinicamente sadios.

Study of pulmonary experimental paracoccidioidomycosis by analysis of bronchoalveolar lavage cells: resistant vs. susceptible mice.

Adult Swiss (susceptible) and BALB/c (non-susceptible) mice were inoculated by the intravenous route with 1 x 10(6) yeast cells of Paracoccidioides brasiliensis, strain 18. Immunologic parameters, histopathology and features of the bronchoalveolar lavage (BAL) were evaluated at week 2, 4, 8 and 16 post-infection. The pulmonary infection was progressive in Swiss mice and regressive in Balb/c mice. The numbers of total cells, lymphocytes and polymorphonuclear neutrophils increased in BAL, as well as the percentages of giant cells, and CD4 and CD8 positive cells. The ultrastructural study of BAL cells revealed a predominance of macrophages and a frequency of 13.2% of type II pneumocytes. As the infection progressed, the number of fungal cells and spreading macrophages, as well as the stimulated release of H2O2 by macrophages, increased. The animals exhibited an exacerbation of the humoral immune response and a depression of cellular immunity during the infection. There was a good correlation between the intensity and the pattern of the pulmonary histopathology and the cellular findings in the BAL. The present model reproduces some anatomoclinical patterns of the human disease and shows that BAL may be a useful tool in monitoring the pulmonary infection caused by P. brasiliensis.

Linamarin- the toxic compound of cassava

Cassava is a widely grown root crop which accumulates two cyanogenic glucosides, linamarin and lotaustralin. Linamarin accounts for more than 80 per cent of the cassava cyanogenic glucosides. It is a beta-glucoside of acetone cyanohydrin and ethyl-methyl-ketone-cyanohydrin. Linamarin beta-linkage can only be broken under high pressure, high temperature and use of mineral acids, while its enzymatic break occurs easily. Linamarase, an endogenous cassava enzyme, can break this beta-linkage. The enzymatic reaction occurs under optimum conditions at 25ºC, at pH 5.5 to 6.0. Linamarin is present in all parts of the cassava plant, being more concentrated on the root and leaves. If the enzyme and substrate are joined, a good detoxification can occur. All the cassava plant species are known to contain cyanide. Toxicity caused by free cyanide (CN-) has already been reported, while toxicity caused by glucoside has not. The lethal dose of CN- is 1 mg/kg of live weight; hence, cassava root classification into toxic and non-toxic depending on the amount of cyanide in the root. Should the cyanide content be high enough to exceed such a dose, the root is regarded as toxic. Values from 15 to 400 ppm (mg CN- of fresh weight) of hydrocyanic acid in cassava roots have been mentioned in the literature. However, more frequent values in the interval 30 to 150 ppm have been observed. Processed cassava food consumed in Brazil is safe in regard to cyanide toxicity.

Estreptococos do grupo C isolados no Brasil: incidência das espécies, perfil de seuscetibilidade à penicilina e eritromicina e comportamento frente ao teste da bacitracina / Group C streptococci: susceptilities to penicilin, erythromycin and bacitracin

Uma coleção de 27 amostras de estreptococos do grupo C de Lancefield tiveram suas espécies determinadas através de testes bioquímicos. As amostras foram também examinadas quanto à suscetibilidade à penicilina, eritromicina e bacitracina (discos contendo 0,04UI). Uma das amostras era padrão do sorogrupo e as demais foram isoladas no Brasil. Das 1127 amostras estudadas, 112 foram obtidas de material clínico humano, sendo 105 classificadas como S. equisimilis, 3 como S. anginosus, 3 como S. zooepidemicus e 1 como S. dysgalactiae. Dentre as 15 amostras de origem animal, 6 amostras eram de S. equisimilis e 9 de S. zoopidemicus. Quinze das amostras humanas foram sensíveis à bacitracina pelo método de difusão em disco. A concentração mínima inibitória (CMI) da penicilina para as 112 amostras humanas variou de 0,0075 a 0,12 ug/ml e de 0,0075 a 0,03 ug/ml para as 15 amostras animais. O valor da CMI mais encontrado nas amostras de ambas as origens foi de 0,015 ug/ml. Quatorze das 127 amostras foram resistentes à eritromicina sendo que 10 destas eram humanas (S. equisimilis) e 4 animais (3 de S. equisimilis e 1 de S. zooepidemicus). Até 6% dos estreptococos do grupo C são sensíveis à bacitracina (resiltados falso-positivos) e isto representa uma percentagem aceitável. Se estudos adicionais confirmarem nossas observações, esforços para melhorar a identificação de amostras de grupo C deveriam ser feitos para evitar uma incorreta identificação presuntiva como estreptococos do grupo A nos laboratórios de rotina. Mais atenção deveria ser dada para a suscetibilidade à eritromicina uma vez que 11% das nossas amostras foram resistentes a este antimicrobiano, enquanto que os dados da literatura relatam uma resistência em torno de 2%.

Sputum cytology in the diagnosis of pulmonary paracoccidioidomycosis.

The presence of Paracoccidioides brasiliensis was determined in sputum samples from 50 patients with paracoccidioidomycosis using four different techniques: (a) cell-block preparations stained with silver methenamine, (b) direct microbiologic examination, (c) smears stained with Shorr, and (d) smears stained with silver methenamine. Overall, cell-block preparations and smears stained with silver methenamine proved to be the most sensitive techniques, followed by smears stained with Shorr and direct microbiologic examination in decreasing order of sensitivity. Sputum cytology tended to be less positive in patients with interstitial pulmonary lesions as determined by chest X-ray than in patients with alveolar lesions. In addition to its high sensitivity, cell-block preparation technique allows storage of blocks and slides for further studies.

Cytochemical evaluation of gamma-glutamyl-transpeptidase activity in cervical smears.

The cervicovaginal smears of 43 patients attending an outpatient service for early cancer detection were cytochemically studied for the presence of gamma-glutamyl-transpeptidase (GGT) in epithelial cells. This was done in order to evaluate such an enzyme phenotype as a marker for cancer development. The results showed that 70% of the 38 patients with a cytologic diagnosis of "inflammatory" or preneoplastic/neoplastic conditions had GGT-positive cells in their smears. None of the five cytologically normal cases showed any epithelial cells with GGT activity. Although most of the GGT-positive cells were metaplastic, some morphologically normal, dysplastic or neoplastic cells also expressed the enzyme. The data suggest that cytochemically detectable transpeptidase activity appears whenever alterations of the normal epithelial microenvironment occurs, but is not necessarily linked to the carcinogenic process. Therefore, cytochemically GGT-positive cells should not be used as an indicator of neoplastic transformation of the cervical epithelium.

A comparative study of the immunoantigenicity of eight Paracoccidioides brasiliensis isolates

Para se detectar diferenças imuno-antigênicas entre 8 amostras de P. brasiliensis isoladas de diferentes áreas endêmicas (Botucatu: Pb 1, 2 e 3; Säo Paulo: Pb: 18,192 e 265; Venezuela: Pb 9 e 73), estudaram-se: 1. A reatividade antigênica de cada amostra nas reaçöes de imunofluorescência indireta (II) e de imunodifusäo dupla em gel de ágar (ID) contra painel de 20 soros controles positivos para paracoccidioidomicose; 2. A capacidade de induzir resposta imune humoral (medida por imunodifusäo) e celular (medida pelo teste de coxim plantar) em camundongos imunizados com antígenos de cada amostra. Observamos: 1. As amostras Pb 265 e Pb 9 mostraram-se mais reativas na II; 2. Os antígenos das amostras Pb 192 e Pb 73 foram significativamente mais reativas na ID; 3. Estes dados demonstram diferenças de antigenicidade entre estas amostras; 4. A amostra Pb 18 mostrou baixo poder indutor de resposta imune celular e alta capacidade de induçäo de resposta imune humoral em camundongos imunizados, revelando dissociaçäo de sua imunogenicidade. Estas diferenças podem indicar a existência de cepas distintas do fungo ou refletir modificaçöes do parasita no hospedeiro ou durante seu cultivo