La tagatose-1,6-bisphosphate aldolase et la fructose-1,6-bisphosphate aldolase de classe I : mécanisme et stéréospécificité

La tagatose-1,6-biphosphate aldolase de Streptococcus pyogenes est une aldolase qui fait preuve d'un remarquable manque de spécificité vis à vis de ses substrats. En effet, elle catalyse le clivage réversible du tagatose-1,6-bisphosphate (TBP), mais également du fructose-1,6-bisphosphate (FBP), du sorbose-1,6-bisphosphate et du psicose-1,6-bisphosphate, quatre stéréoisomères, en dihydroxyacétone phosphate (DHAP) et en glycéraldéhyde-3-phosphate (G3P). Aldolase de classe I, qui donc catalyse sa réaction en formant un intermédiaire covalent obligatoire, ou base de Schiff, avec son susbtrat, la TBP aldolase de S. pyogenes partage 14 % d’identité avec l’enzyme modèle de cette famille, la FBP aldolase de muscle de mammifère. Bien que le mécanime catalytique de la FBP aldolase des mammifères ait été examiné en détails et qu’il soit approprié d’en tirer des renseignements quant à celui de la TBP aldolase, le manque singulier de stéréospécificité de cette dernière tant dans le sens du clivage que celui de la condensation n’est toujours pas éclairci. Afin de mettre à jour les caractéristiques du mécanisme enzymatique, une étude structurale de la TBP aldolase de S. pyogenes, un pathogène humain extrêmement versatile, a été entreprise. Elle a permis la résolution des structures de l’enzyme native et mutée, en complexe avec des subtrats et des inhibiteurs compétitifs, à des résolutions comprises entre 1.8 Å et 2.5 Å. Le trempage des cristaux de TBP aldolase native et mutante dans une solution saturante de FBP ou TBP a en outre permis de piéger un authentique intermédiaire covalent lié à la Lys205, la lysine catalytique. La determination des profils pH de la TBP aldolase native et mutée, entreprise afin d'évaluer l’influence du pH sur la réaction de clivage du FBP et TBP et ìdentifier le(s) résidu(s) impliqué(s), en conjonction avec les données structurales apportées par la cristallographie, ont permis d’identifier sans équivoque Glu163 comme résidu responsable du clivage. En effet, le mode de liaison sensiblement différent des ligands utilisés selon la stéréochimie en leur C3 et C4 permet à Glu163, équivalent à Glu187 dans la FBP aldolase de classe I, d’abstraire le proton sur l’hydroxyle du C4 et ainsi d’amorcer le clivage du lien C3-C4. L’étude du mécanimse inverse, celui de la condensation, grâce par exemple à la structure de l’enzyme native en complexe avec ses substrats à trois carbones le DHAP et le G3P, a en outre permis d’identifier un isomérisme du substrat G3P comme possible cause de la synthèse des isomères en C4 par cette enzyme. Ce résultat, ainsi que la decouverte d’un possible isomérisme cis-trans autour du lien C2-C3 de la base de Schiff formée avec le DHAP, identifié précedemment, permet de cerner presque complètement les particularités du mécanisme de cette enzyme et d’expliquer comment elle est capable de synthétiser les quatres stéréoisomères 3(S/R), 4(S/R). De plus, la résolution de ces structures a permis de mettre en évidence trois régions très mobiles de la protéine, ce qui pourrait être relié au rôle postulé de son isozyme chez S. pyogenes dans la régulation de l’expression génétique et de la virulence de la bactérie. Enfin, la résolution de la structure du mutant Lys229→Met de la FBP aldolase de muscle en complexe avec la forme cyclique du FBP, de même que des études cristallographiques sur le mutant équivalent Lys205→Met de la TBP aldolase de S. pyogenes et des expériences de calorimétrie ont permis d’identifier deux résidus particuliers, Ala31 et Asp33 chez la FBP aldolase, comme possible cause de la discrimination de cette enzyme contre les substrats 3(R) et 4(S), et ce par encombrement stérique des substrats cycliques. La cristallographie par rayons X et la cinétique enzymatique ont ainsi permis d'avancer dans l'élucidation du mécanisme et des propriétés structurales de cette enzyme aux caractéristiques particulières.

Risk factors, microbiological findings and outcomes of necrotizing fasciitis in New Zealand: a retrospective chart review

Background
The incidence and mortality from necrotizing fasciitis (NF) are increasing in New Zealand (NZ). Triggered by a media report that traditional Samoan tattooing was causing NF, we conducted a chart review to investigate the role of this and other predisposing and precipitating factors and to document NF microbiology, complications and interventions in NZ.

Methods
We conducted a retrospective review of 299 hospital charts of patients discharged with NF diagnosis codes in eight hospitals in NZ between 2000 and 2006. We documented and compared by ethnicity the prevalence of predisposing and precipitating conditions, bacteria isolated, complications and interventions used.

Results
Out of 299 charts, 247 fulfilled the case definition. NF was most common in elderly males. Diabetes was the most frequent co-morbid condition, followed by obesity. Nearly a quarter of patients were taking non-steroidal anti-inflammatory drugs (NSAID). Traditional Samoan tattooing was an uncommon cause. Streptococcus pyogenes and Staphylococcus aureus were the two commonly isolated bacteria. Methicillin-resistant Staphylococcus aureus was implicated in a relatively small number of cases. Shock, renal failure, coagulation abnormality and multi-organ dysfunction were common complications. More than 90% of patients underwent surgical debridement, 56% were admitted to an intensive care unit (ICU) and slightly less than half of all patients had blood product transfusion. One in six NF cases had amputations and 23.5% died.

Conclusion
This chart review found that the highest proportion of NF cases was elderly males with co-morbidities, particularly diabetes and obesity. Tattooing was an uncommon precipitating event. The role of NSAID needs further exploration. NF is a serious disease with severe complications, high case fatality and considerable use of health care resources.

Faringotonsilites em crianças: visão de uma amostra de pediatras e otorrinolaringologistas

As faringotonsilites agudas são infecções das vias aéreas superiores comuns na infância. OBJETIVO: Analisar opiniões e condutas de pediatras e otorrinolaringologistas do Estado de São Paulo em relação ao diagnóstico, tratamento e prevenção das faringotonsilites e suas complicações em crianças. MATERIAL E MÉTODOS: Selecionamos aleatoriamente 1370 pediatras e 1000 otorrinolaringologistas do Estado de São Paulo. Aos especialistas foi enviado questionário por correio. DESENHO do ESTUDO: Estudo transversal. RESULTADOS: 95,8% dos pediatras e 91,5% dos otorrinos não solicitam rotineiramente exames para diagnóstico laboratorial das faringotonsilites agudas na criança. Os antimicrobianos mais prescritos pelos pediatras nas faringotonsilites bacterianas foram: penicilina por via oral durante 10 dias (33,6%) e penicilina benzatina em dose única (19,7%). Os antimicrobianos mais prescritos pelos otorrinos para tratamento foram: penicilina por via oral durante 10 dias (35,4%) e penicilina por via oral durante 7 dias (25,7%). A medida de prevenção das faringotonsilites bacterianas considerada muito eficaz por mais da metade dos pediatras e otorrinos foi a cirurgia de tonsilectomia. A faringotonsilite de repetição foi o principal motivo para os otorrinos indicarem cirurgia de tonsilectomia aos escolares e adolescentes (49,3% e 53,4%, respectivamente). CONCLUSÕES: É necessário uniformizar condutas de pediatras e otorrinos para diagnóstico e tratamento das faringotonsilites em crianças.

Cervicovaginal aerobic microflora of women with spontaneous abortion or preterm delivery in Araraquara-Brazil

Rotina bacteriológica do conteúdo vaginal e cervical de 22 mulheres com histórico de aborto recente ou ruptura precoce das membranas foi realizada. Chlamydia trachomatis, Streptococcus pyogenes, Streptococcus agalactiae, Candida sp e Gardnerella vaginalis foram isolados em 54,5% (12) das pacientes. Apesar de Ureaplasma urealyticum ter sido frequentemente encontrado (45,5%), somente em 5 das 22 mulheres foi o único microrganismo presente nos materiais analisados. Esses resultados chamam a atenção para a importância de investigação quantitativa bem como qualitativa da microbiota genital em gestantes, tendo em vista ter consequências na gestação.

Evaluation of alternative methods for the disinfection of toothbrushes

The aim of this study was to evaluate alternative methods for the disinfection of toothbrushes considering that most of the previously proposed methods are expensive and cannot be easily implemented. Two-hundred toothbrushes with standardized dimensions and bristles were included in the study. The toothbrushes were divided into 20 experimental groups (n=10), according to microorganism considered and chemical agent used. The toothbrushes were contaminated in vitro by standardized suspensions of Streptococcus mutans, Streptococcus pyogenes, Staphylococcus aureus or Candida albicans. The following disinfectants were tested: 0.12% chlorhexidine digluconate, 50% white vinegar, a triclosan-containing dentifrice solution, and a perborate-based tablet solution. The disinfection method was immersion in the disinfectant for 10min. After the disinfection procedure, the number of remaining microbial cells was evaluated. The values of cfu/toothbrush of each group of microorganism after disinfection were compared by Kruskal-Wallis ANOVA and Dunn's test for multiple comparisons (5%). The chlorhexidine digluconate solution was the most effective disinfectant. The triclosan-based dentifrice solution promoted a significant reduction of all microorganisms' counts in relation to the control group. As to the disinfection with 50% vinegar, a significant reduction was observed for all the microorganisms, except for C. albicans. The sodium perborate solution was the less effective against the tested microorganisms. Solutions based on triclosan-containing dentifrice may be considered effective, nontoxic, cost-effective, and an easily applicable alternative for the disinfection of toothbrushes. The vinegar solution reduced the presence of S. aureus, S. mutans and S. pyogenes on toothbrushes.

Necrotizing soft tissue infection of the breast: Case report and literature review

Background: Necrotizing soft tissue infection (NSTI) is characterized by progressive infectious gangrene of the skin and subcutaneous tissue. Its treatment involves intensive care, broad-spectrum antibiotic therapy, and full debridement. Methods: We present two cases of NSTI of the breast, adding these cases to the 14 described in the literature, reviewing the characteristics and evolution of all cases. Case Report: On the fourth day after mastectomy, a 59-year-old woman with ulcerated breast cancer developed Type I NSTI caused by Pseudomonas aeruginosa, which had a favorable evolution after debridement and broad-spectrum antibiotics. The second patient was a 57-year-old woman submitted to a mastectomy and axillary dissection, who had recurrent seromas. On the 32nd post-operative day, after a seroma puncture, she developed Type II NSTI caused by β-hemolytic streptococci. She developed sepsis and died on the tenth day after debridement, intensive care, and broad-spectrum antibiotics. The cases are the first description of breast NSTI after mammary seroma aspiration and the first report of this condition caused by P. aeruginosa. Conclusion: Necrotizing soft tissue infection is rare in breast tissue. It frequently is of Type II, occurring mainly after procedures in patients with breast cancer. The surgeon's participation in controlling the focus of the infection is of fundamental importance, and just as important are broad-spectrum antibiotic therapy and support measures, such as maintenance of volume, correction of electrolytic disorders, and treatment of sepsis and septic shock. Once the infection has been brought under control, skin grafting or soft tissue flaps can be considered. The mortality rate in breast NSTI is 18.7%, all deaths being in patients with the fulminant Type II form. Surgical oncologists need to be alert to the possibility of this rare condition. © 2012, Mary Ann Liebert, Inc.

Estudo da atividade antimicrobiana de algumas árvores medicinais nativas com potencial de conservação/recuperação de florestas tropicais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Streptococcal acute pharyngitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification and characterization of ISXax2, a TN3-like transposable element potentially related with the virulence and pathogenicity of Xanthomonas citri subsp. citri

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Experimentally induced anachoresis in the periapical region after root canal filling

Anachoresis is the phenomenon through which blood-borne bacteria, dyes, pigments and other materials are attracted and fixed to circumscribed areas of inflammation. This study evaluated the occurrence of anachoresis in the periapical region of dogs submitted to root canal fillings. One hundred and four roots from four dogs were endodontically treated and root canals were filled with zinc-oxide-eugenol cement. Fifty percent were filled up to the dentinocemental junction and the others were overfilled. At 120 days after root canal treatment, experimental bacteremia was induced by intravenous inoculation of 105 CFU Streptococcus pyogenes. The dogs were sacrificed 48 hours and 30 days after the bacteremia. Culture and DNA amplification by PCR revealed the presence of the inoculated bacteria just in periapical tissues of dogs sacrificed 48 hours after bacteremia and not in animals sacrificed after 30 days. AP-PCR fingerprints of recovered colonies of S. pyogenes and the presence of genetic markers of resistance to antimicrobials were similar to the inoculated strain. Endodontically treated periapices seemed to be prone to the occurrence of anachoresis and there was no relationship between the phenomenon and the level of root canal filling.

Mastoiditis in children: a prospective, observational study comparing clinical presentation, microbiology, computed tomography, surgical findings and histology

The aim of this study was to obtain comprehensive data on clinical presentation, microbiology, computed tomography, surgical findings and histology in acute, sub-acute and chronic mastoiditis. We performed a prospective, observational study in children under 16 years of age presenting to our institution during the 2-year period beginning in April 2000. The children were examined and their condition treated in accordance with a standardized protocol elaborated by the paediatric, otolaryngology (ORL) and radiology departments. Thirty-eight patients were hospitalized (22 with acute mastoiditis, seven with sub-acute mastoiditis, nine with chronic mastoiditis). There were 30 complications present in 21 patients (55%). Streptococcus pyogenes was the most common pathogen (7/24 cases), followed by Streptococcus pneumoniae (4/24 cases). Mastoid surgery was performed in 29 patients. Histology of mastoid tissue revealed predominantly acute inflammation in two cases, mixed acute/chronic inflammation in 19 cases and predominantly chronic inflammation in seven cases. Radiologic data were evaluated retrospectively. Spiral, volume-based high-resolution (HR) computed tomography (CT) of the temporal bone had a sensitivity of 100%, specificity of 38%, positive predictive value (PPV) of 50% and negative predictive value (NPV) of 100% in detecting coalescence of mastoid trabeculae. Cranial CT with contrast had a sensitivity of 80%, specificity of 94%, PPV of 80% and NPV of 94% in identifying intra-cranial extension. Conclusion: histological evidence suggests that sub-acute/chronic infection underlies not only sub-acute and chronic mastoiditis, but most cases of acute mastoiditis as well. HR-CT of the temporal bone is effective in ruling out coalescence. Cranial CT is valuable in identifying intra-cranial extension. Cranial and HR-CT are recommended in the examination of children with mastoiditis.

Outbreaks of an ulcerative and haemorrhagic disease in Arctic char Salvelinus alpinus caused by Aeromonas salmonicida subsp. smithia

Arctic char Salvelinus alpinus farmed in different places in Austria and free of the viral diseases viral haemorrhagic septcaemia (VHS), infectious haematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) experienced disease and mortality. Diseased fish showed skin ulceration and pathological signs of sepsis. Aeromonas sp. was isolated as pure culture from the kidney of freshly euthanized diseased fish. Three independent isolates from outbreaks that occurred on 2 of the affected farms were analyzed phylogenetically by DNA sequence analysis of the rrs and gyrB genes and phenotypically with biochemical reactions. All 3 isolates were identified as Aeromonas salmonicida subsp. smithia. Analysis of virulence genes in these isolates revealed the presence of a Type III secretion system as well as several related virulence effector genes including aexT, encoding the Aeromonas exotoxin AexT, aopP and aopH. These genes are characteristic for virulent strains of typical and atypical subspecies of A. salmonicida.

T-cell epitope analysis using subtracted expression libraries (TEASEL): application to a 38-kDA autoantigen recognized by T cells from an insulin-dependent diabetic patient.

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases.

Hemoculturas positivas num serviço de pediatria: 2003-2012

Introdução: Em situações clínicas selecionadas é aconselhada investigação complementar da criança com febre, nomeadamente realização de hemocultura. Objetivos: Analisar as hemoculturas positivas por bactérias patogénicas num serviço de pediatria, nomeadamente agentes mais frequentes, sua evolução, respetivos antibiogramas e correlação com dados clínicos. Material e Métodos: Estudo retrospetivo de dados micro- biológicos das bactérias patogénicas isoladas em hemoculturas e dados clínicos de crianças com idade entre um mês e 17 anos, admitidas num serviço de pediatria, entre 2003 e 2012. Resultados: No período estudado, a percentagem anual de hemoculturas positivas por bactérias potencialmente patogénicas variou entre 0,8% e 2,9%. No total isolaram-se 158 bactérias patogénicas, sendo mais frequentes: Staphylococcus aureus (29,1%), Streptococcus pneumoniae (27,8%), Escherichia coli (10,1%), Enterococcus faecalis (8,2%), Neisseria meningitidis (5,7%) e Streptococcus pyogenes (5,7%). Nenhuma Neisseria meningitidis foi resistente à resistente à ampicilina, 9% dos Streptococcus pneumoniae tiveram resistência intermédia à penicilina, 8,7% dos Staphylococcus aureus tiveram resistência à meticilina e 6,3% das Escherichia coli tinham resistência à amoxicilina/ácido clavulânico. Sessenta e sete porcento das hemoculturas positivas por bactérias patogénicas correspondiam a crianças com idade inferior a 36 meses. Os diagnósticos mais relevantes foram: bacteriémia oculta, pneumonia, sépsis, meningite e pielonefrite. Ocorreu um óbito devido a choque sético (Streptococcus pneumoniae). Conclusão: Nos 10 anos analisados, as bactérias mais frequentes foram: Staphylococcus aureus, Streptococcus pneumoniae e Escherichia coli. Verificou-se diminuição da incidência da Neisseria meningitidis após 2005 e do Streptococcus pneumoniae após 2007. As suscetibilidades das diferentes bactérias patogénicas aos antimicrobianos mantiveram-se estáveis. Enfatiza-se a importância epidemiológica e clínica da monitorização de dados microbiológicos.

La tagatose-1,6-bisphosphate aldolase et la fructose-1,6-bisphosphate aldolase de classe I : mécanisme et stéréospécificité

La tagatose-1,6-biphosphate aldolase de Streptococcus pyogenes est une aldolase qui fait preuve d'un remarquable manque de spécificité vis à vis de ses substrats. En effet, elle catalyse le clivage réversible du tagatose-1,6-bisphosphate (TBP), mais également du fructose-1,6-bisphosphate (FBP), du sorbose-1,6-bisphosphate et du psicose-1,6-bisphosphate, quatre stéréoisomères, en dihydroxyacétone phosphate (DHAP) et en glycéraldéhyde-3-phosphate (G3P). Aldolase de classe I, qui donc catalyse sa réaction en formant un intermédiaire covalent obligatoire, ou base de Schiff, avec son susbtrat, la TBP aldolase de S. pyogenes partage 14 % d’identité avec l’enzyme modèle de cette famille, la FBP aldolase de muscle de mammifère. Bien que le mécanime catalytique de la FBP aldolase des mammifères ait été examiné en détails et qu’il soit approprié d’en tirer des renseignements quant à celui de la TBP aldolase, le manque singulier de stéréospécificité de cette dernière tant dans le sens du clivage que celui de la condensation n’est toujours pas éclairci. Afin de mettre à jour les caractéristiques du mécanisme enzymatique, une étude structurale de la TBP aldolase de S. pyogenes, un pathogène humain extrêmement versatile, a été entreprise. Elle a permis la résolution des structures de l’enzyme native et mutée, en complexe avec des subtrats et des inhibiteurs compétitifs, à des résolutions comprises entre 1.8 Å et 2.5 Å. Le trempage des cristaux de TBP aldolase native et mutante dans une solution saturante de FBP ou TBP a en outre permis de piéger un authentique intermédiaire covalent lié à la Lys205, la lysine catalytique. La determination des profils pH de la TBP aldolase native et mutée, entreprise afin d'évaluer l’influence du pH sur la réaction de clivage du FBP et TBP et ìdentifier le(s) résidu(s) impliqué(s), en conjonction avec les données structurales apportées par la cristallographie, ont permis d’identifier sans équivoque Glu163 comme résidu responsable du clivage. En effet, le mode de liaison sensiblement différent des ligands utilisés selon la stéréochimie en leur C3 et C4 permet à Glu163, équivalent à Glu187 dans la FBP aldolase de classe I, d’abstraire le proton sur l’hydroxyle du C4 et ainsi d’amorcer le clivage du lien C3-C4. L’étude du mécanimse inverse, celui de la condensation, grâce par exemple à la structure de l’enzyme native en complexe avec ses substrats à trois carbones le DHAP et le G3P, a en outre permis d’identifier un isomérisme du substrat G3P comme possible cause de la synthèse des isomères en C4 par cette enzyme. Ce résultat, ainsi que la decouverte d’un possible isomérisme cis-trans autour du lien C2-C3 de la base de Schiff formée avec le DHAP, identifié précedemment, permet de cerner presque complètement les particularités du mécanisme de cette enzyme et d’expliquer comment elle est capable de synthétiser les quatres stéréoisomères 3(S/R), 4(S/R). De plus, la résolution de ces structures a permis de mettre en évidence trois régions très mobiles de la protéine, ce qui pourrait être relié au rôle postulé de son isozyme chez S. pyogenes dans la régulation de l’expression génétique et de la virulence de la bactérie. Enfin, la résolution de la structure du mutant Lys229→Met de la FBP aldolase de muscle en complexe avec la forme cyclique du FBP, de même que des études cristallographiques sur le mutant équivalent Lys205→Met de la TBP aldolase de S. pyogenes et des expériences de calorimétrie ont permis d’identifier deux résidus particuliers, Ala31 et Asp33 chez la FBP aldolase, comme possible cause de la discrimination de cette enzyme contre les substrats 3(R) et 4(S), et ce par encombrement stérique des substrats cycliques. La cristallographie par rayons X et la cinétique enzymatique ont ainsi permis d'avancer dans l'élucidation du mécanisme et des propriétés structurales de cette enzyme aux caractéristiques particulières.