Resistência antimicrobiana em coliformes totais isolados de tanques de refrigeração de leite bovino

Milk that is adequate for consumption must be of hygienic quality, nutritional value, and should maintain its organoleptic properties. Isolation of fecal and/or total coliforms from bovine milk is considered an indicator of hygiene and good management practices, and can be used as a quality indicator. This study aimed to isolate, identify, and assess the resistance profile of coliforms isolated from collective bulk tanks and individual milk tanks. A total of 89 milk samples were collected from collective bulk tanks and, from these, 21 Klebsiella spp., one E. coli, and 29 Enterobacter spp. were isolated, whereas 102 milk samples from individual tanks showed isolation of one Klebsiella spp. and seven Enterobacter spp. In collective bulk tanks, at least 47% of Klebsiella spp. and Enterobacter spp. were resistant to cephalexin and 30% to ampicillin. From these, at least 24% showed multidrug resistance. Among the microorganisms isolated from the individual tanks, 85% or more were resistant to ampicillin. The ESBL phenotype and the blaTEM gene were detected in strains of Klebsiella spp. isolated from both tanks. It was concluded that contamination of milk with resistant total coliforms, and especially the storage of raw milk from several small producers in the collective bulk tank increase the risk of contamination.

Enterococcus faecium isolated from Lombo, a Portuguese traditional meat product: characterisation of antibacterial compounds and factors affecting bacteriocin production

Strain ST211CH, identified as a strain of Enterococcus faecium, isolated from Lombo produced a bacteriocin that inhibited the growth of Enterococcus spp., Listeria spp., Klebsiella spp., Lactobacillus spp., Pseudomonas spp., Staphylococcus spp. and Streptococcus spp. The mode of action of the bacteriocin named as bacteriocin ST211Ch was bactericidal against Enterococcus faecalis ATCC19443. As determined by Tricine-SDS-PAGE, the approximate molecular mass of the bacteriocin was 8.0 kDa. Loss in antimicrobial activity was recorded after treatment with proteolytic enzymes. Maximum activity of bacteriocin ST211Ch was measured in broth cultures of E. faecium strain ST211Ch after 24 h; thereafter, the activity was reduced. Bacteriocin ST211Ch remained active after exposure to various temperatures and pHs, as well as to Triton X-100, Tween-80, Tween-20, sodium dodecyl sulfate, NaCl, urea and EDTA. Effect of media components on production of bacteriocin ST211Ch was also studied. On the basis of PCR reactions targeting different bacteriocin genes, i.e. enterocins, curvacins and sakacins, no evidences for the presence of these genes in the total DNA of E. faecium strain ST211Ch was obtained. The bacterium most probably produced a bacteriocin different from those mentioned above. Based on the antimicrobial spectrum, stability and mode of action of bacteriocin ST211CH, E. faecium strain ST211Ch might be considered as a potential candidate with beneficial properties for use in biopreservation to control food spoilage bacteria.

Plasma levels of complement proteins from the alternative pathway in patients with age-related macular degeneration are independent of Complement Factor H Tyr(402)His polymorphism

Purpose: To investigate the influence of the Factor H (CFH) Tyr(402)His polymorphism on the plasma levels of the alternative pathway proteins CFH, C3, Factor B (FB), Factor D (FD), and Factor I (FI) and the inflammatory marker C-reactive protein (CRP) in 119 patients with age-related macular degeneration (AMD) and 152 unrelated control individuals. Methods: Patients with AMD and the control group were separated according to CFH polymorphism, age, and gender. Plasma complement proteins and CRP concentrations were determined with enzyme-linked immunosorbent assay, immunodiffusion, or nephelometry. Results: Significant differences in the concentrations of FD and FI were observed between the patients with AMD and the control individuals. We observed significantly reduced FD plasma levels in patients with AMD. We also identified a significant decrease in CFH plasma levels in female patients with AMD in relation to female controls. Plasma FI levels were significantly increased in patients with AMD compared to the control group. Regarding gender, a significant increase in FI plasma levels was observed in male patients. Finally, we found no significant correlation between the CFH Tyr(402)His polymorphism and the CFH, C3, FB, FD, FI, and CRP plasma levels. Conclusions: Patients with AMD present altered levels of FD and FI in a manner independent of this CFH polymorphism, and gender apparently contributes to the plasma levels of these two proteins in patients with AMD and control individuals.

Establishment of the bacterial fecal community during the first month of life in Brazilian newborns

OBJECTIVE: The establishment of the intestinal microbiota in newborns is a critical period with possible long-term consequences for human health. In this research, the development of the fecal microbiota of a group of exclusively breastfed neonates living in low socio-economic conditions in the city of Sao Paulo, Brazil, during the first month of life, was studied. METHODS: Fecal samples were collected from ten neonates on the second, seventh, and 30th days after birth. One of the neonates underwent antibiotic therapy. Molecular techniques were used for analysis; DNA was extracted from the samples, and 16S rRNA libraries were sequenced and phylogenetically analyzed after construction. A real-time polymerase chain reaction (PCR) was performed on the samples taken from the 30th day to amplify DNA from Bifidobacterium sp. RESULTS: The primary phylogenetic groups identified in the samples were Escherichia and Clostridium. Staphylococcus was identified at a low rate. Bifidobacterium sp. was detected in all of the samples collected on the 30th day. In the child who received antibiotics, a reduction in anaerobes and Escherichia, which was associated with an overgrowth of Klebsiella, was observed throughout the experimental period. CONCLUSION: The observed pattern of Escherichia predominance and reduced Staphylococcus colonization is in contrast with the patterns observed in neonates living in developed countries.

Performance and composition of bacterial communities in anaerobic fluidized bed reactors for hydrogen production: Effects of organic loading rate and alkalinity

This study evaluated the effects of the organic loading rate (OLR) and pH buffer addition on hydrogen production in two anaerobic fluidized bed reactors (AFBRs) operated simultaneously. The AFBRs were fed with glucose, and expanded clay was used as support material. The reactors were operated at a temperature of 30 degrees C, without the addition of a buffer (AFBR1) and with the addition of a pH buffer (AFBR2, sodium bicarbonate) for OLRs ranging from 19.0 to 140.6 kg COD m(-3) d(-1) (COD: chemical oxygen demand). The maximum hydrogen yields for AFBR1 and AFBR2 were 2.45 and 1.90 mol H-2 mol(-1) glucose (OLR of 84.3 kg COD m(-3) d(-1)), respectively. The highest hydrogen production rates were 0.95 and 0.76 L h(-1) L-1 for AFBR1 and AFBR2 (OLR of 140.6 kg COD m(-3) d(-1)), respectively. The operating conditions in AFBR1 favored the presence of such bacteria as Clostridium, while the bacteria in AFBR2 included Clostridium, Enterobacter, Klebsiella, Veillonellaceae, Chryseobacterium, Sporolactobacillus, and Burkholderiaceae. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Expanding the Clinical and Genetic Spectrum of Human CD40L Deficiency: The Occurrence of Paracoccidioidomycosis and Other Unusual Infections in Brazilian Patients

CD40 ligand (CD40L) deficiency or X-linked hyper-IgM syndrome (X-HIGM) is a well-described primary immunodeficiency in which Pneumocystis jiroveci pneumonia is a common clinical feature. We have identified an unusual high incidence of fungal infections and other not yet described infections in a cohort of 11 X-HIGM patients from nine unrelated Brazilian families. Among these, we describe the first case of paracoccidioidomycosis (PCM) in X-HIGM. The molecular genetic analysis of CD40L was performed by gene sequencing and evaluation of CD40L protein expression. Nine of these 11 patients (82%) had fungal infections. These included fungal species common to CD40L deficiency (P. jiroveci and Candida albicans) as well as Paracoccidioides brasiliensis. One patient presented with PCM at age 11 years and is now doing well at 18 years of age. Additionally, one patient presented with a simultaneous infection with Klebsiella and Acinetobacter, and one with condyloma caused by human papilloma virus. Molecular analysis revealed four previously described CD40L mutations, two novel missense mutations (c.433 T>G and c.476 G>C) resulting in the absence of CD40L protein expression by activated CD4(+) cells and one novel insertion (c.484_485insAA) within the TNFH domain leading to a frame shift and premature stop codon. These observations demonstrated that the susceptibility to fungal infections in X-HIGM extends beyond those typically associated with X-HIGM (P. jiroveci and C. albicans) and that these patients need to be monitored for those pathogens.

Cost-effectiveness of introducing the 10-valent pneumococcal conjugate vaccine into the universal immunisation of infants in Brazil

Background Cost-effectiveness studies have been increasingly part of decision processes for incorporating new vaccines into the Brazilian National Immunisation Program. This study aimed to evaluate the cost-effectiveness of 10-valent pneumococcal conjugate vaccine (PCV10) in the universal childhood immunisation programme in Brazil. Methods A decision-tree analytical model based on the ProVac Initiative pneumococcus model was used, following 25 successive cohorts from birth until 5 years of age. Two strategies were compared: (1) status quo and (2) universal childhood immunisation programme with PCV10. Epidemiological and cost estimates for pneumococcal disease were based on National Health Information Systems and literature. A 'top-down' costing approach was employed. Costs are reported in 2004 Brazilian reals. Costs and benefits were discounted at 3%. Results 25 years after implementing the PCV10 immunisation programme, 10 226 deaths, 360 657 disability-adjusted life years (DALYs), 433 808 hospitalisations and 5 117 109 outpatient visits would be avoided. The cost of the immunisation programme would be R$10 674 478 765, and the expected savings on direct medical costs and family costs would be R$1 036 958 639 and R$209 919 404, respectively. This resulted in an incremental cost-effectiveness ratio of R$778 145/death avoided and R$22 066/DALY avoided from the society perspective. Conclusion The PCV10 universal infant immunisation programme is a cost-effective intervention (1-3 GDP per capita/DALY avoided). Owing to the uncertain burden of disease data, as well as unclear long-term vaccine effects, surveillance systems to monitor the long-term effects of this programme will be essential.

Cross-reactivity of antipneumococcal surface protein C (PspC) antibodies with different strains and evaluation of inhibition of human complement factor H and secretory IgA binding via PspC

Pneumococcal surface protein C (PspC) is an important candidate for a cost-effective vaccine with broad coverage against pneumococcal diseases. Previous studies have shown that Streptococcus pneumoniae is able to bind to both human factor H (FH), an inhibitor of complement alternative pathway, and human secretory IgA (sIgA) via PspC. PspC was classified into 11 groups based on variations of the gene. In this work, we used three PspC fragments from different groups (PspC3, PspC5, and PspC8) to immunize mice for the production of antibodies. Immunization with PspC3 induced antibodies that recognized the majority of the clinical isolates as analyzed by Western blotting of whole-cell extracts and flow cytometry of intact bacteria, while anti-PspC5 antibodies showed cross-reactivity with the paralogue pneumococcal surface protein A (PspA), and anti-PspC8 antibodies reacted only with the PspC8-expressing strain. Most of the isolates tested showed strong binding to FH and weaker interaction with sIgA. Preincubation with anti-PspC3 and anti-PspC5 IgG led to some inhibition of binding of FH, and preincubation with anti-PspC3 partially inhibited sIgA binding in Western blotting. The analysis of intact bacteria through flow cytometry showed only a small decrease in FH binding after incubation of strain D39 with anti-PspC3 IgG, and one clinical isolate showed inhibition of sIgA binding by anti-PspC3 IgG. We conclude that although anti-PspC3 antibodies were able to recognize PspC variants from the majority of the strains tested, partial inhibition of FH and sIgA binding through anti-PspC3 antibodies in vitro could be observed for only a restricted number of isolates.

Cannabidiol reduces host immune response and prevents cognitive impairments in Wistar rats submitted to pneumococcal meningitis

Pneumococcal meningitis is a life-threatening disease characterized by an acute infection affecting the pia matter, arachnoid and subarachnoid space. The intense inflammatory response is associated with a significant mortality rate and neurologic sequelae, such as, seizures, sensory-motor deficits and impairment of learning and memory. The aim of this study was to evaluate the effects of acute and extended administration of cannabidiol on pro-inflammatory cytokines and behavioral parameters in adult Wistar rats submitted to pneumococcal meningitis. Male Wistar rats underwent a cisterna magna tap and received either 10 mu l of sterile saline as a placebo or an equivalent volume of S. pneumoniae suspension. Rats subjected to meningitis were treated by intraperitoneal injection with cannabidiol (2.5, 5, or 10 mg/kg once or daily for 9 days after meningitis induction) or a placebo. Six hours after meningitis induction, the rats that received one dose were killed and the hippocampus and frontal cortex were obtained to assess cytokines/chemokine and brain-derived neurotrophic factor levels. On the 10th day, the rats were submitted to the inhibitory avoidance task. After the task, the animals were killed and samples from the hippocampus and frontal cortex were obtained. The extended administration of cannabidiol at different doses reduced the TNF-alpha level in frontal cortex. Prolonged treatment with canabidiol, 10 mg/kg, prevented memory impairment in rats with pneumococcal meningitis. Although descriptive, our results demonstrate that cannabidiol has anti-inflammatory effects in pneumococcal meningitis and prevents cognitive sequel. (C) 2012 Elsevier B.V. All rights reserved.

Toll-like receptors 2, 3 and 4 and thymic stromal lymphopoietin expression in fatal asthma

Background Airway inflammation in asthma involves innate immune responses. Toll-like receptors (TLRs) and thymic stromal lymphopoietin (TSLP) are thought to be involved in airway inflammation, but their expression in asthmatics both large and small airways has not been investigated. Objective To analyse the expression of TLR2, TLR3, TLR4 and TSLP in large and small airways of asthmatics and compare their expression in smoking and non-smoking asthmatics; to investigate whether TLR expression is associated with eosinophilic or neutrophilic airway inflammation and with Mycoplasma pneumoniae and Chlamydophila pneumoniae infection. Methods Using immunohistochemistry and image analysis, we investigated TLR2, TLR3, TLR4 and TSLP expression in large and small airways of 24 victims of fatal asthma, FA, (13 non-smokers, 11 smokers) and nine deceased control subjects (DCtrl). TLRs were also measured in 18 mild asthmatics (MA) and 12 healthy controls (HCtrl). M. pneumoniae and C. pneumoniae in autopsy lung tissue were analysed using real-time polymerase chain reaction. Airway eosinophils and neutrophils were measured in all subjects. Results Fatal asthma patients had higher TLR2 in the epithelial and outer layers of large and small airways compared with DCtrls. Smoking asthmatics had lower TLR2 levels in the inner and outer layers of the small airways than non-smoking asthmatics. TSLP was increased in the epithelial and outer layers of the large airways of FA. FA patients had greater TLR3 expression in the outer layer of large airways and greater TLR4 expression in the outer layer of small airways. Eosinophilic airway inflammation was associated with TLR expression in the epithelium of FA. No bacterial DNA was detected in FA or DCtrls. MA and HCtrls had only a small difference in TLR3 expression. Conclusions and Clinical Relevance Increased expression of TLR 2, 3 and 4 and TSLP in fatal asthma may contribute to the acute inflammation surrounding asthma deaths.

Respiratory syncytial virus (RSV) pneumonia in a southern muriqui (Brachyteles arachnoides)

Background An adult male Brachyteles arachanoides, kept in captivity since 1990, was found dead without apparent clinical evidence. Methods Necropsy report, histopathology, immunohistochemistry, and ultrastructural examination were conducted. Results Pulmonary syncytial cells were positive for respiratory syncytial virus (RSV), and ultrastructural examination revealed viral particles inside macrophages compatible with the Paramyxoviridae family. Conclusions Muriquis are susceptible to RSV pneumonia followed by respiratory distress syndrome and death.

Effectiveness of the polysaccharide pneumococcal vaccine among HIV-infected persons in Brazil: a case control study

Abstract Background Polysaccharide pneumococcal vaccine is recommended for use in HIV-infected adults in Brazil but there is uncertainty about its effectiveness in this patient population. The main objective of this study was to assess the effectiveness of the 23-valent polysaccharide pneumococcal vaccine against invasive pneumococcal infection among HIV-infected adult patients in São Paulo, Brazil. Methods A case-control study of 79 cases and 242 controls matched on CD4+ cell count and health care setting was conducted. Among HIV-infected adults in São Paulo, Brazil, with and without S. pneumoniae recovered from a normally sterile site; prior receipt of 23 valent polysaccharide pneumococcal vaccine was determined by review of medical records and patient interview. Results After adjustment for confounding factors, the point estimate for the effectiveness of 23 valent polysaccharide vaccine among HIV-infected adults against all invasive pneumococcal infection was 18% (95% CI: <0 to 62%). Conclusion We were unable to demonstrate a statistically significant protective effect of 23 valent polysaccharide against invasive pneumococcal infection vaccine among HIV-infected adults in Brazil. While the vaccine is relatively inexpensive and safe, its effectiveness among HIV-infected adults in Brazil is uncertain.

Diferença de tempo de positividade: método útil no diagnóstico de infecção de corrente sanguínea relacionada com cateter?

INTRODUÇÃO: As infecções de corrente sanguínea relacionadas com cateter (ICSRCs) apresentam impacto significativo na morbidade e na mortalidade de pacientes internados, além de elevar custos hospitalares. A utilização de equipamentos automatizados no processamento de hemoculturas gerou uma alternativa para diagnóstico de ICSRC por meio da análise da diferença de tempo de positividade (DTP) entre hemoculturas pareadas (coletadas simultaneamente) de sangue periférico e sangue de cateter. Um diagnóstico acurado e rápido dessas infecções pode otimizar as condutas clínicas e terapêuticas, poupando a retirada precoce dos cateteres. OBJETIVOS: Avaliar na rotina a DTP como ferramenta auxiliar no diagnóstico de ICSRC e determinar os principais microrganismos isolados. MÉTODOS: Foram avaliadas retrospectivamente hemoculturas coletadas no complexo do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC/FMUSP) de maio a agosto de 2008. Somente amostras que apresentaram DTP maior que 120 minutos foram consideradas possíveis ICSRCs pelo critério laboratorial. RESULTADOS: A seção processou 11.017 hemoculturas aeróbias durante o período de estudo; somente 5% foram coletadas de forma pareada. Destas, 148 (28%) foram positivas, sendo 9% com crescimento somente em sangue periférico, 41% somente em sangue de cateter e 50% em ambas as amostras com 88% de homologia de microrganismos identificados. A DTP apresentou valores acima de 120 minutos em 50% dos casos e os microrganismos mais isolados foram Staphylococcus aureus (22%), Candida spp. (18%), Klebsiella spp. (7%) e Enterobacter spp. (7%). CONCLUSÃO: A determinação da DTP como ferramenta auxiliar no diagnóstico de ICSRC é viável e fácil de ser executada em laboratórios de rotina com automação, porém o processo de coleta das amostras pareadas deve ser rigidamente padronizado.

Community-acquired pneumonia in Chile: the clinical relevance in the detection of viruses and atypical bacteria

Background Adult community-acquired pneumonia (CAP) is a relevant worldwide cause of morbidity and mortality, however the aetiology often remains uncertain and the therapy is empirical. We applied conventional and molecular diagnostics to identify viruses and atypical bacteria associated with CAP in Chile. Methods We used sputum and blood cultures, IgG/IgM serology and molecular diagnostic techniques (PCR, reverse transcriptase PCR) for detection of classical and atypical bacteria (Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumoniae) and respiratory viruses (adenovirus, respiratory syncytial virus (RSV), human metapneumovirus, influenza virus, parainfluenzavirus, rhinovirus, coronavirus) in adults >18 years old presenting with CAP in Santiago from February 2005 to September 2007. Severity was qualified at admission by Fine's pneumonia severity index. Results Overall detection in 356 enrolled adults were 92 (26%) cases of a single bacterial pathogen, 80 (22%) cases of a single viral pathogen, 60 (17%) cases with mixed bacterial and viral infection and 124 (35%) cases with no identified pathogen. Streptococcus pneumoniae and RSV were the most common bacterial and viral pathogens identified. Infectious agent detection by PCR provided greater sensitivity than conventional techniques. To our surprise, no relationship was observed between clinical severity and sole or coinfections. Conclusions The use of molecular diagnostics expanded the detection of viruses and atypical bacteria in adults with CAP, as unique or coinfections. Clinical severity and outcome were independent of the aetiological agents detected.

Synthesis, structure and conformation of oligo- and polysaccharides

Carbohydrates are a complex group of biomolecules with a high structural diversity. Their almost omnipresent occurrence has generated a broad field of research in both biology and chemistry. This thesis focuses on three different aspects of carbohydrate chemistry, synthesis, structure elucidation and the conformational analysis of carbohydrates. The first paper describes the synthesis of a penta- and a tetrasaccharide related to the highly branched capsular polysaccharide from Streptococcus pneumoniae type 37. In the second paper, the structure of the O-antigenic repeating unit from the lipopolysaccharide of E. coli 396/C1 was determined along with indications of the structure of the biological repeating unit. In addition, its structural and immunological relationship with E. coli O126 is discussed. In the third paper, partially protected galactopyranosides were examined to clarify the origin of an intriguing 4JHO,H coupling, where a W-mediated coupling pathway was found to operate. In the fourth paper, the conformation of methyl a-cellobioside is studied with a combination of molecular dynamics simulations and NMR spectroscopy. In addition to the expected syn-conformation, detection and quantification of anti-ø and anti-ψ conformers was also possible.