Frequency of serovars and antimicrobial resistance in Shigella spp. from Brazil

A total of 296 Shigella spp. were received from State Public Health Laboratories, during the period from 1999 to 2004, by National Reference Laboratory for Cholera and Enteric Diseases (NRLCED) - IOC/Fiocruz, Rio de Janeiro, Brazil. The frequency of Shigella spp. was: S. flexneri (52.7%), S. sonnei (44.2%), S. boydii (2.3%), and S. dysenteriae (0.6%). The most frequent S. flexneri serovars were 2a and 1b. The highest incidence rates of Shigella isolation were observed in the Southeast (39%) and Northeast (34%) regions and the lowest rate in the South (3%) of Brazil. Strains were further analyzed for antimicrobial susceptibility by disk diffusion method as part of a surveillance program on antimicrobial resistance. The highest rates of antimicrobial resistance were to trimethoprim-sulfamethozaxole (90%), tetracycline (88%), ampicillin (56%), and chloramphenicol (35%). The patterns of antimicrobial resistance among Shigella isolates pose a major difficulty in the determination of an appropriate drug for shigellosis treatment. Continuous monitoring of antimicrobial susceptibilities of Shigella spp. through a surveillance system is thus essential for effective therapy and control measures against shigellosis.

Prevalence and genetic characterisation of HTLV-1 and 2 dual infections in patients with pulmonary tuberculosis in Central-West Brazil

Human T-cell lymphotropic virus (HTLV) may impact the clinical course of tuberculosis (TB). Both infections are highly endemic in Brazil. The aim of this study was to assess the prevalence of HTLV-1/2 in TB patients in Central-West Brazil and to perform a genetic characterisation of the respective isolates. Of the 402 patients, six (1.49%) were positive for anti-HTLV and five (1.24%; 95% confidence interval: 0.46-3.05) were infected with HTLV-1/2. Genetic characterisation demonstrated that the four HTLV-1 isolates belonged to the Transcontinental subgroup A of the Cosmopolitan subtype a and that the HTLV-2 isolate belonged to subtype a (HTLV-2a/c). The prevalence of HTLV infection observed in this study is higher than that observed in local blood donors and the HTLV-1 and 2 subtypes identified are consistent with those circulating in Brazil.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

The multifaceted resources and microevolution of the successful human and animal pathogen methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important bacterial pathogens based on its incidence and the severity of its associated infections. In addition, severe MRSA infections can occur in hospitalised patients or healthy individuals from the community. Studies have shown the infiltration of MRSA isolates of community origin into hospitals and variants of hospital-associated MRSA have caused infections in the community. These rapid epidemiological changes represent a challenge for the molecular characterisation of such bacteria as a hospital or community-acquired pathogen. To efficiently control the spread of MRSA, it is important to promptly detect the mecA gene, which is the determinant of methicillin resistance, using a polymerase chain reaction-based test or other rapidly and accurate methods that detect the mecA product penicillin-binding protein (PBP)2a or PBP2’. The recent emergence of MRSA isolates that harbour a mecA allotype, i.e., the mecC gene, infecting animals and humans has raised an additional and significant issue regarding MRSA laboratory detection. Antimicrobial drugs for MRSA therapy are becoming depleted and vancomycin is still the main choice in many cases. In this review, we present an overview of MRSA infections in community and healthcare settings with focus on recent changes in the global epidemiology, with special reference to the MRSA picture in Brazil.

Carbon stock and its compartments in a subtropical oxisol under long-term tillage and crop rotation systems

Soil organic matter (SOM) plays a crucial role in soil quality and can act as an atmospheric C-CO2 sink under conservationist management systems. This study aimed to evaluate the long-term effects (19 years) of tillage (CT-conventional tillage and NT-no tillage) and crop rotations (R0-monoculture system, R1-winter crop rotation, and R2- intensive crop rotation) on total, particulate and mineral-associated organic carbon (C) stocks of an originally degraded Red Oxisol in Cruz Alta, RS, Southern Brazil. The climate is humid subtropical Cfa 2a (Köppen classification), the mean annual precipitation 1,774 mm and mean annual temperature 19.2 ºC. The plots were divided into four segments, of which each was sampled in the layers 0-0.05, 0.05-0.10, 0.10-0.20, and 0.20-0.30 m. Sampling was performed manually by opening small trenches. The SOM pools were determined by physical fractionation. Soil C stocks had a linear relationship with annual crop C inputs, regardless of the tillage systems. Thus, soil disturbance had a minor effect on SOM turnover. In the 0-0.30 m layer, soil C sequestration ranged from 0 to 0.51 Mg ha-1 yr-1, using the CT R0 treatment as base-line; crop rotation systems had more influence on soil stock C than tillage systems. The mean C sequestration rate of the cropping systems was 0.13 Mg ha-1 yr-1 higher in NT than CT. This result was associated to the higher C input by crops due to the improvement in soil quality under long-term no-tillage. The particulate C fraction was a sensitive indicator of soil management quality, while mineral-associated organic C was the main pool of atmospheric C fixed in this clayey Oxisol. The C retention in this stable SOM fraction accounts for 81 and 89 % of total C sequestration in the treatments NT R1 and NT R2, respectively, in relation to the same cropping systems under CT. The highest C management index was observed in NT R2, confirming the capacity of this soil management practice to improve the soil C stock qualitatively in relation to CT R0. The results highlighted the diversification of crop rotation with cover crops as a crucial strategy for atmospheric C-CO2 sequestration and SOM quality improvement in highly weathered subtropical Oxisols.

Phytochemical investigation of Wissadula periplocifolia (L.) C. Presl and evaluation of its antibacterial activity

A phytochemical study on the aerial parts of Wissadula periplocifolia using chromatographic techniques has led to the isolation of sitosterol (1a), stigmasterol (1b), sitosterol 3-O-β-D-glucopyranoside (2a), stigmasterol 3-O-β-D-glucopyranoside (2b), phaeophytin A (3), 13²-hydroxy-(13²-S)-phaeophytin A (4), phaeophytin B (5), 17³-ethoxyphaeophorbide (6), 3,4-seco-urs-4(23),20(30)-dien-3-oic acid (7), 3-oxo-21β-H-hop-22(29)-ene (8), dammaradienone (9a), and taraxastenone (9b). The isolated compounds were characterised by spectroscopic analysis. A preliminary assay to evaluate the antibacterial activity of W. periplocifolia extracts and fractions showed that the dichloromethane, ethyl acetate, and n-butanol fractions were active against Enterococcus faecalis.

Anacardic acid derivatives from Brazilian propolis and their antibacterial activity

Propolis is a sticky, gummy, resinous substance collected by honeybees (Apis mellifera L.) from various plant sources, which has excellent medicinal properties. This paper describes the isolation and identification of triterpenoids and anacardic acid derivatives from Brazilian propolis and their antibacterial activity. Their structures were elucidated by ¹H and 13C NMR, including uni- and bidimensional techniques; in addition, comparisons were made with data from academic literature. These compounds were identified as: cardanols (1a + 1b), cardols (2a + 2b), monoene anacardic acid (3), a-amirine (4), b-amirine (5), cycloartenol (6), 24-methylene-cycloartenol (7) and lupeol (8). The determination of the position of the double bond after a reaction with Dimethyl disulfide (DMDS) is described for the phenol derivatives. The ethanolic extract was tested in vitro for antimicrobial activity by using the disc diffusion method and it showed significant results against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Shigella spp.

Granulocyte-macrophage colony-stimulating factor does not increase the potency or efficacy of a foot-and-mouth disease virus subunit vaccine

Foot-and-mouth disease (FMD) is one of the most feared diseases of livestock worldwide. Vaccination has been a very effective weapon in controlling the disease, however a number of concerns with the current vaccine including the inability of approved diagnostic tests to reliably distinguish vaccinated from infected animals and the need for high containment facilities for vaccine production, have limited its use during outbreaks in countries previously free of the disease. A number of FMD vaccine candidates have been tested and a replication-defective human adenovirus type 5 (Ad5) vector containing the FMDV capsid (P1-2A) and 3C protease coding regions has been shown to completely protect pigs against challenge with the homologous virus (FMDV A12 and A24). An Ad5-P1-2A+3C vaccine for FMDV O1 Campos (Ad5-O1C), however, only induced a low FMDV-specific neutralizing antibody response in swine potency tests. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been successfully used to stimulate the immune response in vaccine formulations against a number of diseases, including HIV, hepatitis C and B. To attempt to improve the FMDV-specific immune response induced by Ad5-O1C, we inoculated swine with Ad5-O1C and an Ad5 vector containing the gene for porcine GM-CSF (pGM-CSF). However, in the conditions used in this trial, pGM-CSF did not improve the immune response to Ad5-O1C and adversely affected the level of protection of swine challenged with homologous FMDV.

Serum neutralization with different types and subtypes of bovine herpesvirus 1 and 5

The serum neutralization (SN) test is the gold standard method to measure neutralizing antibodies to bovine herpesviruses. However, in view of the further subdivisions of bovine herpesviruses in types/subtypes, defining which virus to use at challenge in SN tests may be difficult. In view of that, this study was carried out to re-evaluate (SN) sensitivity with different types/subtypes of bovine herpesviruses types 1 (BoHV-1) and 5 (BoHV-5) as challenge viruses. Bovine sera (n=810) were collected from two distinct geographic regions and tested by SN with three type 1 viruses (BoHV-1.1 strains "Los Angeles" and "EVI123/98"; BoHV-1.2a strain "SV265/96") and three type 5 viruses (BoHV-5a strain "EVI88/95"; BoHV-5b strain "A663" and BoHV-5c "ISO97/95"). SN tests were performed with a 1 hour incubation of the serum-virus mixtures at 37ºC against 100 TCID50 of each of the viruses. SN sensitivity varied greatly depending on the challenge virus used in the test. The highest sensitivity (327 positive/810 total sera tested; 40.37%) was attained when the positive results to the six viruses were added together. No association could be found between any particular type or subtype of virus and the sensitivity of the test. When positive results to each single strain were considered, SN sensitivity varied from 41.7% to 81.7%, depending on the virus and the geographic region of origin of the sera. Variation was detected even when challenge viruses belonged to the same subtype, where disagreement between positive results reached 41%. These results indicate that one hour incubation SN tests against single viruses, as performed here, may display a significantly low sensitivity (p=0.05); performing SN tests against a number of different viruses may increase considerably SN sensitivity. Furthermore, the choice of virus used for challenge is critical in SN tests. In addition, sera from different geographic regions may give rise to disagreeing results with different strains of BoHV-1 and BoHV-5. This might be particularly relevant for control programs and in international trade, were maximum sensitivity should be targeted.

Expression of CYP2A3 mRNA and its regulation by 3-methylcholanthrene, pyrazole, and ß-ionone in rat tissues

Cytochrome P450 (CYP) 2A enzymes are involved in the metabolism of numerous drugs and hormones and activate different carcinogens. Human CYP2A6, mouse CYP2A5 and rat CYP2A3 are orthologous enzymes that present high similarity in their amino acid sequence and share substrate specificities. However, different from the human and mouse enzyme, CYP2A3 is not expressed in the rat liver. There are limited data about expression of CYP2A3 in extrahepatic tissues and its regulation by typical CYP inducers. Therefore, the objective of the present study was to analyze CYP2A3 mRNA expression in different rat tissues by RT-PCR, and to study the influence of 3-methylcholanthrene, pyrazole and ß-ionone treatment on its expression. Male Wistar rats were divided into four groups of 5 rats each, and were treated ip for 4 days with 3-methylcholanthrene (25 mg/kg body weight), pyrazole (150 mg/kg body weight), ß-ionone (1 g/kg body weight), or vehicle. Total RNA was extracted from tissues and CYP2A3 mRNA levels were analyzed by semiquantitative RT-PCR. CYP2A3 mRNA was constitutively expressed in the esophagus, lung and nasal epithelium, but not along the intestine, liver, or kidney. CYP2A3 mRNA levels were increased in the esophagus by treatment with 3-methylcholanthrene and pyrazole (17- and 7-fold, respectively), in lung by pyrazole and ß-ionone (3- and 4-fold, respectively, although not statistically significant), in the distal part of the intestine and kidney by 3-methylcholanthrene and pyrazole, and in the proximal part of the intestine by pyrazole. CYP2A3 mRNA was not induced in nasal epithelium, liver or in the middle part of the intestine. These data show that, in the rat, CYP2A3 is constitutively expressed in several extrahepatic tissues and its regulation occurs through a complex mechanism that is essentially tissue specific.

Relative contribution of expectancy and immediate arousal to the facilitatory effect of an auditory accessory stimulus

An auditory stimulus speeds up a digital response to a subsequent visual stimulus. This facilitatory effect has been related to the expectancy and the immediate arousal that would be caused by the accessory stimulus. The present study examined the relative contribution of these two influences. In a first and a third experiment a simple reaction time task was used. In a second and fourth experiment a go/no-go reaction time task was used. In each of these experiments, the accessory stimulus preceded the target stimulus by 200 ms for one group of male and female volunteers (G Fix). For another group of similar volunteers (G Var) the accessory stimulus preceded the target stimulus by 200 ms in 25% of the trials, by 1000 ms in 25% of the trials and was not followed by the target stimulus in 50% of the trials (Experiments 1a and 1b) or preceded the target stimulus by 200 ms in 6% of the trials and by 1000 ms in 94% of the trials (Experiments 2a and 2b). There was a facilitatory effect of the accessory stimulus for G Fix in the four experiments. There was also a facilitatory effect of the accessory stimulus at the 200-ms stimulus onset asynchrony for G Var in Experiments 1a and 1b but not in Experiments 2a and 2b. The facilitatory effects observed were larger in the go/no-go task than in the simple task. Taken together, these results suggest that expectancy is much more important than immediate arousal for the improvement of performance caused by an accessory stimulus.

Co-circulation of genotypes IA and IB of hepatitis A virus in Northeast Brazil

The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21% of the samples, respectively. Nucleotide sequencing was carried out in 46% of VP1/2A and in 53% of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0%. Phylogenetic analysis of the VP1/2A sequences showed that 65% belong to sub-genotype IA and 35% to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2% identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.

Effect of exercise training and carvedilol treatment on cardiac function and structure in mice with sympathetic hyperactivity-induced heart failure

The present investigation was undertaken to study the effect of β-blockers and exercise training on cardiac structure and function, respectively, as well as overall functional capacity in a genetic model of sympathetic hyperactivity-induced heart failure in mice (α2A/α2CArKO). α2A/α2CArKO and their wild-type controls were studied for 2 months, from 3 to 5 months of age. Mice were randomly assigned to control (N = 45), carvedilol-treated (N = 29) or exercise-trained (N = 33) groups. Eight weeks of carvedilol treatment (38 mg/kg per day by gavage) or exercise training (swimming sessions of 60 min, 5 days/week) were performed. Exercise capacity was estimated using a graded treadmill protocol and HR was measured by tail cuff. Fractional shortening was evaluated by echocardiography. Cardiac structure and gastrocnemius capillary density were evaluated by light microscopy. At 3 months of age, no significant difference in fractional shortening or exercise capacity was observed between wild-type and α2A/α2CArKO mice. At 5 months of age, all α2A/α2CArKO mice displayed exercise intolerance and baseline tachycardia associated with reduced fractional shortening and gastrocnemius capillary rarefaction. In addition, α2A/ α2CArKO mice presented cardiac myocyte hypertrophy and ventricular fibrosis. Exercise training and carvedilol similarly improved fractional shortening in α2A/α2CArKO mice. The effect of exercise training was mainly associated with improved exercise tolerance and increased gastrocnemius capillary density while β-blocker therapy reduced cardiac myocyte dimension and ventricular collagen to wild-type control levels. Taken together, these data provide direct evidence for the respective beneficial effects of exercise training and carvedilol in α2A/α2CArKO mice preventing cardiac dysfunction. The different mechanisms associated with beneficial effects of exercise training and carvedilol suggest future studies associating both therapies.

Histomorphometric analysis of the response of rat skeletal muscle to swimming, immobilization and rehabilitation

The objective of the present study was to determine to what extent, if any, swimming training applied before immobilization in a cast interferes with the rehabilitation process in rat muscles. Female Wistar rats, mean weight 260.52 ± 16.26 g, were divided into 4 groups of 6 rats each: control, 6 weeks under baseline conditions; trained, swimming training for 6 weeks; trained-immobilized, swimming training for 6 weeks and then immobilized for 1 week; trained-immobilized-rehabilitated, swimming training for 6 weeks, immobilized for 1 week and then remobilized with swimming for 2 weeks. The animals were then sacrificed and the soleus and tibialis anterior muscles were dissected, frozen in liquid nitrogen and processed histochemically (H&E and mATPase). Data were analyzed statistically by the mixed effects linear model (P < 0.05). Cytoarchitectural changes such as degenerative characteristics in the immobilized group and regenerative characteristics such as centralized nucleus, fiber size variation and cell fragmentation in the groups submitted to swimming were more significant in the soleus muscle. The diameters of the lesser soleus type 1 and type 2A fibers were significantly reduced in the trained-immobilized group compared to the trained group (P < 0.001). In the tibialis anterior, there was an increase in the number of type 2B fibers and a reduction in type 2A fibers when trained-immobilized rats were compared to trained rats (P < 0.001). In trained-immobilized-rehabilitated rats, there was a reduction in type 2B fibers and an increase in type 2A fibers compared to trained-immobilized rats (P < 0.009). We concluded that swimming training did not minimize the deleterious effects of immobilization on the muscles studied and that remobilization did not favor tissue re-adaptation.

The polymorphism of the serotonin-2A receptor T102C is associated with age

Epidemiological investigations suggest that T102C polymorphism of gene 5-HT2A may be associated with mean life span because diseases and behaviors related to this polymorphism, such as schizophrenia, suicide, aggression, and addiction, may potentially shorten mean life span. A sample of 687 individuals without previous neuropsychiatric disease was genotyped and separated into 3 groups according to their gender and age: 14-45 years old, 46-64 years old and 65-100 years old. Molecular genotyping was performed using the technique of polymerase chain reaction followed by restriction fragment length polymorphism using HpaII restriction enzyme. 5-HT2A genotype frequencies were: TT = 21.5% (148), CC = 16.6% (114) and TC = 61.9% (425) and allele frequencies were T = 52.5% and C = 46.5%. Significant differences were found between mean age of the TT genotype carriers (60.27 ± 12.60 years) and TC genotype carriers (56.80 ± 13.18 years) of T102C polymorphism of gene 5-HT2A (P = 0.026) as well as the age groups (P = 0.012). Carriers of genotype TT were older than the other two genotypes, whereas carriers of genotype CC had an intermediate age compared with TT and CC subjects. The present results demonstrate an association between T102C polymorphism of gene 5-HT2A and age. Our results suggest that T102C polymorphism of gene 5-HT2A is associated with mean life span, and thus this gene becomes a possible candidate for the group of adaptive genes to meat consumption proposed in the literature. Further studies should be conducted in order to elucidate this association.