Accumulation of manganese in Neisseria gonorrhoeae correlates with resistance to oxidative killing by superoxide anion and is independent of superoxide dismutase activity

As a facultative aerobe with a high iron requirement and a highly active aerobic respiratory chain, Neisseria gonorrhoeae requires defence systems to respond to toxic oxygen species such as superoxide. It has been shown that supplementation of media with 100 muM Mn(II) considerably enhanced the resistance of this bacterium to oxidative killing by superoxide. This protection was not associated with the superoxide dismutase enzymes of N. gonorrhoeae. In contrast to previous studies, which suggested that some strains of N. gonorrhoeae might not contain a superoxide dismutase, we identified a sodB gene by genome analysis and confirmed its presence in all strains examined by Southern blotting, but found no evidence for sodA or sodC. A sodB mutant showed very similar susceptibility to superoxide killing to that of wild-type cells, indicating that the Fe-dependent SOD B did not have a major role in resistance to oxidative killing under the conditions tested. The absence of a sodA gene indicated that the Mn-dependent protection against oxidative killing was independent of Mn-dependent SOD A. As a sodB mutant also showed Mn-dependent resistance to oxidative killing, then it is concluded that this resistance is independent of superoxide dismutase enzymes. Resistance to oxidative killing was correlated with accumulation of Mn(II) by the bacterium. We hypothesize that this bacterium uses Mn(II) as a chemical quenching agent in a similar way to the already established process in Lactobacillus plantarum. A search for putative Mn(II) uptake systems identified an ABC cassette-type system (MntABC) with a periplasmic-binding protein (MntC). An mntC mutant was shown to have lowered accumulation of Mn(II) and was also highly susceptible to oxidative killing, even in the presence of added Mn(II). Taken together, these data show that N. gonorrhoeae possesses a Mn(II) uptake system that is critical for resistance to oxidative stress.

Post-antibiotic growth suppression of linezolid against Gram-positive bacteria

The in vitro post-antibiotic effects (PAEs) of eight different concentrations of linezolid against Gram-positive cocci were investigated and the results analysed using the sigmoid E-max model for mathematically modelling the PAE. Mean maximal linezolid PAEs against strains of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium and Streptococcus pneumoniae were 2.2, 1.8, 2.8, 2.0 and 3.0 h, respectively. Resistance to methicillin (for the staphylococci), vancomycin (for the enterococci) and penicillin (for the pneumococci) had no effect on the duration of the PAE. Results of PAE testing support twice-daily dosing of linezolid in humans.

Identification and characterization of pptA: a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

Prevalência elevada do Alloiococcus otitidis na otite média com efusão através da PCR simultânea

A etiologia da otite média com efusão (OME) não é completamente conhecida, mas agentes infecciosos podem contribuir para sua patogênese. O conhecimento sobre a epidemiologia bacteriana da OME em áreas geográficas distintas é essencial para a implementação de tratamentos racionais, quando indicados. OBJETIVO: Determinar a prevalência do Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis e Alloiococcus otitidis nas efusões da orelha média de crianças com otite média recorrente (OMR) e otite média com efusão crônica (OMEC) que foram submetidas à miringotomia e comparar os resultados obtidos por cultura e PCR. FORMA DE ESTUDO: Estudo clínico com coorte transversal. MATERIAL E MÉTODO: 128 efusões obtidas por timpanocentese de 75 crianças entre 11 meses e 10 anos de idade foram analisadas por cultura e PCR simultânea. RESULTADOS: Cultivaram-se bactérias em 25,1% das amostras e os patógenos principais foram encontrados em 19,6%. O A.otitidis não foi isolado em cultura. A PCR identificou bactérias em 85,9%, com os seguintes resultados individuais: A.otitidis, 52,3%; H.influenzae, 39,1%; S.pneumoniae, 12,5% e M.catarrhalis, 10,2%. A PCR foi significativamente mais sensível que a cultura (P<0,01). O S.pneumoniae foi mais encontrado em OMR do que em OMEC (P=0,038). CONCLUSÕES: A prevalência das bactérias na OME em um grupo de crianças brasileiras é semelhante àquelas relatadas em outros países, sendo o H.influenzae o mais encontrado dentre os patógenos principais da orelha média. O S.pneumoniae foi mais freqüente em OMR do que em OMEC. A PCR é mais sensível na detecção de bactérias na efusão da orelha média, comparada com cultura, e é essencial para a identificação do A.otitidis. O elevado percentual de detecção do A.otitidis sugere mais investigações sobre sua atuação no início e no prolongamento de doenças da orelha média.

Achados otomicroscópicos e histológicos da miringoesclerose induzida em ratos: estudo crítico de um modelo experimental

A miringoesclerose é caracterizada por hialinização da lâmina própria da membrana timpânica. Trabalhos experimentais têm utilizado a otomicroscopia ou a histologia para estudar a miringoesclerose em animais, porém sem correlacionar precisamente estes dois métodos de avaliação. OBJETIVO: Este estudo procura avaliar a acurácia da otomicroscopia no diagnóstico de miringoesclerose em modelo experimental com ratos. FORMA DE ESTUDO: experimental. MATERIAL E MÉTODO: Foi induzida miringoesclerose por inoculação transtimpânica de Streptococcus pneumoniae em 25 ratos Wistar, que foram examinados quinzenalmente por otomicroscopia e sacrificados após oito semanas, tendo suas membranas timpânicas examinadas por histologia. RESULTADOS: Da confrontação das alterações otomicroscópicas com os achados histológicos, houve uma sensibilidade de 80% e especificidade de 75%. CONCLUSÃO: Pelos resultados obtidos neste estudo, a otomicroscopia não representou um bom método para avaliar a miringoesclerose neste modelo experimental.

Synthesis, Antimicrobial and Antiproliferative Activity of Novel Silver(I) Tris(pyrazolyl)methanesulfonateand 1,3,5-Triaza-7-phosphadamantane Complexes

Five new silver(I) complexes of formulas [Ag(Tpms)] (1), [Ag(Tpms)-(PPh3)] (2), [Ag(Tpms)(PCy3)] (3), [Ag(PTA)][BF4] (4), and [Ag(Tpms)(PTA)] (5) {Tpms = tris(pyrazol-1-yl)methanesulfonate, PPh3 = triphenylphosphane, PCy3 = tricyclohexylphosphane, PTA = 1,3,5-triaza-7-phosphaadamantane) have been synthesized and fully characterized by elemental analyses, H-1, C-13, and P-31 NMR, electrospray ionization mass spectrometry (ESI-MS), and IR spectroscopic techniques. The single crystal X-ray diffraction study of 3 shows the Tpms ligand acting in the N-3-facially coordinating mode, while in 2 and 5 a N2O-coordination is found, with the SO3 group bonded to silver and a pendant free pyrazolyl ring. Features of the tilting in the coordinated pyrazolyl rings in these cases suggest that this inequivalence is related with the cone angles of the phosphanes. A detailed study of antimycobacterial and antiproliferative properties of all compounds has been carried out. They were screened for their in vitro antimicrobial activities against the standard strains Enterococcus faecalis (ATCC 29922), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (SF37), Streptococcus sanguinis (SK36), Streptococcus mutans (UA1S9), Escherichia coli (ATCC 25922), and the fungus Candida albicans (ATCC 24443). Complexes 1-5 have been found to display effective antimicrobial activity against the series of bacteria and fungi, and some of them are potential candidates for antiseptic or disinfectant drugs. Interaction of Ag complexes with deoxyribonucleic acid (DNA) has been studied by fluorescence spectroscopic techniques, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNA EB system on addition of Ag complexes shows that the fluorescence quenching of DNA EB complex occurs and compound 3 is particularly active. Complexes 1-5 exhibit pronounced antiproliferative activity against human malignant melanoma (A375) with an activity often higher than that of AgNO3, which has been used as a control, following the same order of activity inhibition on DNA, i.e., 3 > 2 > 1 > 5 > AgNO3 >> 4.

Impacto da vacinação contra o Haemophilus influenzae b na redução de meningites, Goiás

OBJETIVO: Avaliar o impacto da vacinação contra o Haemophilus influenzae b na incidência de meningites em crianças menores de cinco anos de idade. MÉTODOS: Utilizou-se o delineamento tipo "antes-depois" para comparar as taxas de incidência de meningites por Haemophilus influenzae b nos períodos pré-vacinação (julho/95-junho/99) e pós-vacinação (julho/99-junho/2001) no Estado de Goiás. A definição de caso de meningite bacteriana seguiu os critérios da Organização Mundial de Saúde. As taxas de meningite por Streptococcus pneumoniae e Neisseria. meningitidis foram utilizadas para efeito de comparação. Para análise estatística foram utilizados o teste de chi2 e o t de Student. Valores de p<0,05 foram considerados estatisticamente significantes. RESULTADOS: Foi detectada meningite bacteriana aguda em 979 crianças no período de estudo. A incidência de meningite por Haemophilus influenzae b diminuiu de 10,8x10(5) no período pré-vacinal para 2,3x10(5) no segundo ano pós-vacina, significando 78% de redução no risco, principalmente na faixa etária de 7-23 meses (p<0,05). Foram prevenidos 65 casos de meningite por Haemophilus influenzae b. Observou-se aumento na incidência de meningite por S. pneumoniae. Foi observada falha vacinal em um caso. CONCLUSÕES: Expressivo declínio da incidência de meningite por Haemophilus influenzae b foi detectado, precocemente, logo após o primeiro ano de introdução da vacina contra o Haemophilus influenzae b. Assim, se faz necessária a vigilância contínua com instrumental de alta acurácia para: (i) detectar re-emergência do Haemophilus influenzae b; (ii) avaliar possibilidade de falha vacinal; (iii) identificar mudanças no padrão dos sorotipos do H. influenzae.

Hospitalization rates for pneumococcal disease in Brazil, 2004 - 2006

OBJECTIVE: To estimate hospitalization rates for pneumococcal disease based on the Brazilian Hospital Information System (SIH). METHODS: Descriptive study based on the Hospital Information System of Brazilian National Health System data from January 2004 to December 2006: number of hospitalizations and deaths for pneumococcal meningitis, pneumococcal sepsis, pneumococcal pneumonia and Streptococcus pneumoniae as the cause of diseases reported in Brazil. Data from the 2003 Brazilian National Household Survey were used to estimate events in the private sector. Pneumococcal meningitis cases and deaths reported to the Notifiable Diseases Information System during the study period were also analyzed. RESULTS: Pneumococcal disease accounted for 34,217 hospitalizations in the Brazilian National Health System (0.1% of all hospitalizations in the public sector). Pneumococcal pneumonia accounted for 64.8% of these hospitalizations. The age distribution of the estimated hospitalization rates for pneumococcal disease showed a "U"-shape curve with the highest rates seen in children under one (110 to 136.9 per 100,000 children annually). The highest hospital case-fatality rates were seen among the elderly, and for sepsis and meningitis. CONCLUSIONS: PD is a major public health problem in Brazil. The analysis based on the SIH can provide an important input to pneumococcal disease surveillance and the impact assessment of immunization programs.

Estudo da etiologia das pneumopatias agudas da infância através da aspiração pulmonar transtorácica, São Paulo, Brasil

No período de abril de 1979 a julho de 1980 foram estudadas 45 crianças, de ambos os sexos, na faixa etária de 2 meses a 5 anos de idade, selecionadas entre as atendidas no Instituto da Criança "Pedro Alcântara" do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, e acometidas de pneumopatias agudas. Dos 45 pacientes estudados, a maioria (46,7%) encontrava se na faixa de 2-12 meses de idade e, quanto ao estado nutricional, 42,2% eutróficos e 57,8% desnutridos, variando essa desnutrição do grau I ao grau III. Empregando amostras obtidas por aspiração pulmonar e examinando-as pelo método de coloração de Gram e cultura, foi possível identificar o agente etiológico de 26 (57,7%) pneumonias bacterianas nos 45 casos estudados. A identificação direta pelo método de Gram mostrou-se útil como orientação inicial para antibioticoterapia em 44,4% dos casos. Houve nítida predominância do Streptococcus pneumoniae como agente etiológico bacteriano nas pneumopatias agudas da criança nas várias faixas etárias estudadas seguido do Haemophilus influenzae e o Staphylococcus aureus. O estudo comparativo da cultura do aspirado pulmonar e da hemocultura permitiu maior precisão diagnostica (56,7%), revelando-se a hemocultura positiva em apenas 30,0% dos casos. Não foi constatada nenhuma participação de germes anaeróbios nas pneumopatias da amostragem estudada.

Acute respiratory infections in children from a deprived urban population from Uruguay

To obtain base line data on incidence, duration, clinical characteristics and etiology of acute respiratory infections (ARI), 276 children from deprived families living in Montevideo were followed during 32 months. The target population was divided into two groups for the analysis of the results: children aged less than 12 months and those older than this age. During the follow-up period 1.056 ARI episodes were recorded. ARI incidence was 5.2 per child/year. It was 87% higher in infants than in the older group, as was the duration of the episodes. Most of the diseases were mild. Tachypnea and retractions were seldom observed, but 12 children were refered to the hospital, and 2 infants died. Viral etiology was identified in 15.3% of the episodes. RSV was the predominant agent producing annual outbreaks. Moderate to heavy colonization of the upper respiratory tract by Streptococcus pneumoniae (32.3%) and Hemophilus sp. (18.9%) was recorded during ARI episodes. This community-based study furnish original data on ARI in Uruguay. It enabled to asses the impact of these infections on childhood.

Etiology and severity of community acquired pneumonia in children from Uruguay: a 4-year study

The 4-year study (1987-1990) covered the major clinical-epidemiological characteristics of pneumonia in children as diagnosed at the emergency service of the Children's Hospital, as well as etiologies, and factors involved in the most severe cases. Etiology was determined in 47.7% of the 541 pneumonia cases, involving 283 pathogens of which 38.6% were viruses and 12.6% bacteria. Viral and mixed etiologies were more frequent in children under 12 months of age. Bacteria predominated in ages between 6 and 23 months. Among the viruses, respiratory syncytial virus predominated (66%). The bacterial pneumonias accounted for 12.2% of the recognized etiologies. The most important bacterial agents were S. pneumoniae (64%) and H. influenzae (19%). H. influenzae and mixed infections had a relevant participation during the 1988 season, pointing to annual variations in the relative participation of pathogens and its possible implication in severity of diseases. Correlation of severity and increased percentage of etiological diagnosis was assessed: patients with respiratory rates over 70 rpm, or pleural effusion and/or extensive pulmonary parenchyma compromise yielded higher positive laboratory results. Various individual and family risk factors were recognized when comparing pneumonia children with healthy controls.

Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for detection of pneumococcal polysaccharide antigens in pleural fluid effusion samples.: Comparison with bacterial culture, counterimmunoelectrophoresis and latex agglutination

A dot-enzyme-linked immunosorbent assay (Dot-ELISA) for pneumococcal antigen detection was standardized in view of the need for a rapid and accurate immunodiagnosis of acute pneumococcal pneumonia. A total of 442 pleural fluid effusion samples (PFES) from children with clinical and laboratory diagnoses of acute bacterial pneumonia, plus 38 control PFES from tuberculosis patients and 20 negative control serum samples from healthy children were evaluated by Dot-ELISA. The samples were previously treated with 0.1 M EDTA pH 7.5 at 90°C for 10 min and dotted on nitrocellulose membrane. Pneumococcal omniserum diluted at 1:200 was employed in this assay for antigen detection. When compared with standard bacterial culture, counterimmunoelectrophoresis and latex agglutination techniques, the Dot-ELISA results showed relative indices of 0.940 to sensitivity, 0.830 to specificity and 0.760 to agreement. Pneumococcal omniserum proved to be an optimal polyvalent antiserum for the detection of pneumococcal antigen by Dot-ELISA. Dot-ELISA proved to be a practical alternative technique for the diagnosis of pneumococcal pneumonia.

Bacterial antigen detection in cerebrospinal fluid by the latex agglutination test

Eighty purulent cerebrospinal fluid (CSF) samples from patients with clinical evidence of meningitis were studied using the Directigen latex agglutination (LA) kit to determine the presence of bacterial antigen in CSF. The results showed a better diagnostic performance of the LA test than bacterioscopy by Gram stain, culture and counterimmunoelectrophoresis (CIE), as far as Neisseria meningitidis groups B and C, and Haemophilus influenzae type b are concerned, and a better performance than bacterioscopy and culture considering Streptococcus pneumoniae. Comparison of the results with those of culture showed that the LA test had the highest sensitivity for the Neisseria meningitidis group C. Comparing the results with those of CIE, the highest levels of sensitivity were detected for N. meningitidis groups B and C. Regarding specificity, fair values were obtained for all organisms tested. The degree of K agreement when the LA test was compared with CIE exhibited better K indices of agreement for N. meningitidis groups B and C.

Massive shift in the pneumococcal nasopharyngeal flora after the 7-valent conjugate vaccine: epidemiological studies and testing pathogenic potential in animal models

Dissertation presented to obtain the PhD degree in Biology/Molecular Biology by Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica

Empiema Torácico. Revisão Baseada em Três Casos Clínicos

O derrame parapneumónico caracteriza-se pela necessidade de um processo invasivo para a sua resolução e o empiema pela presença de pus na cavidade pleural. Em ambos os casos, o diagnóstico por TAC e o tratamento precoces resultando em menores morbilidade e mortalidade. São indicação para um tratamento invasivo os derrames loculados, os que ocupam mais de 50% do tórax, os que revelam coloração por Gram e exame cultural positivos, ou derrames com pH inferior a 7,20, glucose inferior a 60 mg/dl, e nível de DHL superior a três vezes o limite normal no soro. Estas características resultam da evolução através de três estádios dos derrames incorrectamente tratados: 1) exsudativo; 2) fibrino-purulento; 3) fibrótico. Dependendo do estádio evolutivo, a abordagem terapêutica varia entra toracentese terapêutica, colocação de drenagem torácica com ou sem instilação de fibrinolíticos, cirurgia toracoscópica vídeo-assistida e decorticação pulmonar. Os autores fazem uma revisão do estudo destas situações baseados em três casos clínicos com apresentações muito díspares: uma doente com empiema por Streptococcus pyogenes que faleceu rapidamente por hemoptise maciça; um doente com empiema resultante de pneumonia aguda ocorrida durante um voo de avião; uma doente com empiema e bacteriemia por Streptococcus pneumoniae conduzindo a diagnóstico até então desconhecido de infecção por VIH.