Development of a collection of bacteria causing meningitis in Rio de Janeiro from 1990 to 1991

From March 1990 to December 1992, the National Institute for Quality Control of Health-INCQS Research Collection received 1476 bacterial samples isolated from human cerebrospinal fluid of patients suspect of meningitis in Rio de Janeiro, from the São Sebastião State Institute of Infectious Diseases (IEISS). Neisseria meningitidis was found in most of these materials, followed in smaller number by Haemophilus sp. and Streptococcus pneumoniae. The great majority of N. meningitidis strains was serogroup B, followed by serogroup C and a few strains of serogroup W135. More than 50 of the isolated bacterial agents came from the predominant 0-4 years age group. The majority of the strains were from patients in the region known as "Baixada Fluminense" (Low Lands). The aim of the work presented here is to obtain samples of meningitis cases in at least 70 of the State of Rio de Janeiro and develop a collaborative research between INCQS-FIOCRUZ and the IEISS, in order to set up a collection of strains for future studies. However, despite work being carried out in a rather satisfactory way, difficulties still arise and have to be overcome, to survey data.

Penicillin tolerance among Beta-hemolytic streptococci and production of the group carbohydrates, hemolysins, hyaluronidases and deoxyribonucleases

Penicillin tolerance among 67 strains of beta-hemolytic streptococci was examined by determining the ratio of the minimal bactericidal concentration to the minimal inhibitory concentration as 32 or greater. Tolerance was demonstrated in 15 group A strains and in 11,7, and 4 of groups B, C and G, respectively. Thereafter the effects of a subminimal inhibitory concentration (1/2MIC) of penicillin on the bacterial products of four tolerant and four nontolerant strains (two of each Lancefield group) were analyzed and compared. The antibiotic caused a marked increase in the expression of the group carbo-hydrates for strains of group B. Penicillin was found to reduce the cell-bound hemolysin activities of the four tolerant strains and to increase the activity of the other (free) form of nontolerant groups A, C and G hemolysins. Penicillin caused an increase in the extracellular hyaluronidase activities of one group A and groups B, C and G streptococci. With added antibiotic the production of deoxyribonuclease by tolerant groups A, C and G was greatly enhanced and that of the group B streptococcus was arrested.

Microbial flora variations in the respiratory tract of mice

A stable microbial system in the respiratory tract acts as an important defense mechanism against pathogenic microorganisms. Perturbations in this system may allow pathogens to establish. In an ecological environment such as the respiratory tract, there are many diverse factors that play a role in the establishment of the indigenous flora. In the present work we studied the normal microbial flora of different areas of the respiratory tract of mice and their evolution from the time the mice were born. Our interest was to know which were the dominant groups of microorganisms in each area, which were the first capable of colonizing and which dominated over time to be used as probiotic microorganisms. Our results show that Gram negative facultatively anaerobic bacilli and strict anaerobic microorganisms were the last ones to appear in the bronchia, while aerobic and Gram positive cocci were present in all the areas of the respiratory tract. The number of facultative aerobes and strict anaerobes were similar in the nasal passage, pharynx instilled and trachea, but lower in bronchia. The dominant species were Streptococcus viridans and Staphylococcus saprophyticcus, followed by S. epidermidis, Lactobacilli and S. cohnii I which were present on every studied days but at different proportions. This paper is the first part of a research topic investigating the protective effect of the indigenous flora against pathogens using the mice as an experimental model.

Detection of pathogenic bacteria in skin lesions of patients with chiclero's ulcer: reluctant response to antimonial treatment

We investigated the bacterial flora present in skin lesions of patients with chiclero's ulcer from the Yucatan peninsula of Mexico using conventional culture methods (11 patients), and an immunocolorimetric detection of pathogenic Streptococcus pyogenes (15 patients). Prevalence of bacteria isolated by culture methods was 90.9% (10/11). We cultured, from chiclero's ulcers (60%), pathogenic bacterial such as Staphylococcus aureus (20%), S. pyogenes (1.6%), Pseudomonas aeruginosa (1.6%), Morganella morganii (1.6%), and opportunist pathogenic bacteria such as Klebsiella spp. (20.0%), Enterobacter spp. (20%), and Enterococcus spp. (20%). We also cultured coagulase-negative staphylococci in 40% (4/10) of the remaining patients. Micrococcus spp. and coagulase-negative staphylococci constituted the bacterial genuses more frequently isolated in the normal skin of patients with chiclero's ulcer and healthy individuals used as controls. We also undertook another study to find out the presence of S. pyogenes by an immunocolorimetric assay. This study indicated that 60% (9/15) of the ulcerated lesions, but not normal controls, were contaminated with S. pyogenes. Importantly, individuals with purulent secretion and holding concomitant infections with S. pyogenes, S. aureus, P. aeruginosa, M. morganii, and E. durans took longer to heal Leishmania (L.) mexicana infections treated with antimonial drugs. Our results suggest the need to eliminate bacterial purulent infections, by antibiotic treatment, before starting antimonial administration to patients with chiclero's ulcer.

Screening of Amazonian plants from the Adolpho Ducke forest reserve, Manaus, state of Amazonas, Brazil, for antimicrobial activity

Tropical forests are species-rich reserves for the discovery and development of antimicrobial drugs. The aim of this work is to investigate the in vitro antimicrobial potential of Amazon plants found within the National Institute on Amazon Research's Adolpho Ducke forest reserve, located in Manaus, state of Amazonas, Brazil. 75 methanol, chloroform and water extracts representing 12 plant species were tested for antimicrobial activity towards strains of Mycobacterium smegmatis, Escherichia coli, Streptococcus sanguis, Streptococcus oralis, Staphylococcus aureus and Candida albicans using the gel-diffusion method. Active extracts were further evaluated to establish minimum inhibitory concentrations (MIC) and antimicrobial profiles using bioautography on normal-phase thin-layer chromatography plates. Diclinanona calycina presented extracts with good antimicrobial activity and S. oralis and M. smegmatis were the most sensitive bacteria. D. calycina and Lacmellea gracilis presented extracts with the lowest MIC (48.8 µg/ml). D. calycina methanol and chloroform leaf extracts presented the best overall antimicrobial activity. All test organisms were sensitive to D. calycina branch chloroform extract in the bioautography assay. This is the first evaluation of the biological activity of these plant species and significant in vitro antimicrobial activity was detected in extracts and components from two species, D. calycina and L. gracilis.

The utility of the polymerase chain reaction assay for aetiologic definition of unspecified bacterial meningitis cases

Most patients with acute suppurative meningitis are otherwise healthy individuals with regard to immune mechanisms against invasive bacterial disease. This medical emergency is among the most dramatic and potentially ravaging diseases that affect humans, particularly young children. The illness often strikes suddenly, and can either result in death or leave the survivors with significant neurological dysfunctions. The demonstration of a bacterial aetiology is necessary for decisions regarding treatment and prophylaxis. Conventional bacteriological methods frequently fail to identify an agent, as a result of administration of antibiotics or delayed lumbar punctures. We investigated the major aetiologic sources of unspecified bacterial meningitis cases (G00.9, ISCD-10) by polymerase chain reaction (PCR)-based identification of Neisseria meningitidis (crgA), Streptococcus pneumoniae (ply) and Haemophilus influenzae (bexA) in cerebrospinal fluid samples. The multiplex PCR detected N. meningitidis in 92%, S. pneumoniae in 4% and H. influenzae in 1% of the 192 clinical samples assayed; 3% were negative for all three DNA targets. Bacterial DNA detection was found to be a valuable adjunct to enhance bacterial meningitis surveillance when the yield of specimens by culture is reduced. The implementation of PCR assays as a diagnostic procedure in Public Health Laboratories is perceived to be a significant advance in the investigation of bacterial meningitis.

The microbiological signature of human cutaneous leishmaniasis lesions exhibits restricted bacterial diversity compared to healthy skin

Localised cutaneous leishmaniasis (LCL) is the most common form of cutaneous leishmaniasis characterised by single or multiple painless chronic ulcers, which commonly presents with secondary bacterial infection. Previous culture-based studies have found staphylococci, streptococci, and opportunistic pathogenic bacteria in LCL lesions, but there have been no comparisons to normal skin. In addition, this approach has strong bias for determining bacterial composition. The present study tested the hypothesis that bacterial communities in LCL lesions differ from those found on healthy skin (HS). Using a high throughput amplicon sequencing approach, which allows for better populational evaluation due to greater depth coverage and the Quantitative Insights Into Microbial Ecology pipeline, we compared the microbiological signature of LCL lesions with that of contralateral HS from the same individuals.Streptococcus, Staphylococcus,Fusobacterium and other strict or facultative anaerobic bacteria composed the LCL microbiome. Aerobic and facultative anaerobic bacteria found in HS, including environmental bacteria, were significantly decreased in LCL lesions (p < 0.01). This paper presents the first comprehensive microbiome identification from LCL lesions with next generation sequence methodology and shows a marked reduction of bacterial diversity in the lesions.

Pesquisa do estreptococo do Grupo B em gestantes da Zona Leste de São Paulo

O presente trabalho trata-se de estudo transversal e retrospectivo, realizado com trinta mulheres usuárias de uma Unidade Básica de Saúde (UBS) da Zona Leste de São Paulo, por meio de entrevista e consulta de prontuários, com o objetivo de verificar como ocorre a pesquisa do estreptococo do grupo B em gestantes. As participantes da amostra realizaram pré-natais na UBS etiveram seus bebês no período de janeiro de 2009 a dezembro de 2010. Realizaram a cultura do EGB 23 mulheres (76,7% do total), 82,6% com resultados negativos e 17,4%, positivos; 43,5% delas realizaram o exame entre 35 e 37 semanas de gestação; 23,5% não realizaram o exame, a maior parte por ausência de solicitação. Foi possível verificar que ocorreram falhas no rastreamento do EGB durante o período selecionado.

Atividade in vitro do extrato de própolis contra agentes bacterianos da mastite bovina

Este trabalho foi desenvolvido com o objetivo de avaliar a atividade in vitro do extrato alcoólico de própolis, contra agentes da mastite bovina, comparando-o aos principais antimicrobianos utilizados no tratamento convencional. Foram utilizados 36 isolados coagulase-positivos de Staphylococcus sp. e 27 isolados de Streptococcus sp.; 94,4% dos Staphylococcus sp. e 85,2% dos Streptococcus sp. foram susceptíveis ao extrato de própolis.

Tannin-tolerant bacteria from crossbred Holstein x Zebu cows

The objective of this work was to isolate and characterize tannin-tolerant ruminal bacteria from crossbred Holstein x Zebu cows fed a chopped mixture of elephant grass (Pennisetum purpureum), young stems of "angico-vermelho" (Parapiptadenia rigida), and banana tree (Musa sp.) leaves. A total of 117 bacteria strains were isolated from enrichment cultures of rumen microflora in medium containing tannin extracts. Of these, 11 isolates were able to tolerate up to 3 g L-1 of tannins. Classical characterization procedures indicated that different morphological and physiological groups were represented. Restriction fragments profiles using Alu1 and Taq1 of 1,450 bp PCR products from the 16S rRNA gene grouped the 11 isolates into types I to VI. Sequencing of 16S rRNA PCR products was used for identification. From the 11 strains studied, seven were not identifiable by the methods used in this work, two were strains of Butyrivibrio fibrisolvens, and two of Streptococcus bovis.

Flavonoides glicosilados de Erythroxylum pulchrum a. st.-hil. (Erythroxylaceae)

The phytochemical investigation of Erythroxylum pulchrum St. Hil. (Erythroxylaceae) led to the isolation of three known flavonoid glycosides quercetin-3-O-α-L-rhaminoside, ombuin-3-ruthinoside and ombuin-3-ruthinoside-5-glucoside. These flavonoids are being described for the first time in this E. pulchrum. The structures of the compounds were determined by analysis of IR, MS and NMR data, as well as by comparison with literature data. The methanolic extract of leaves from E. pulchrum inhibited the growth of the Bacillus subtilis CCT 0516, Escherichia coli ATCC 2536, Pseudomonas aeruginosa ATCC 8027, P. aeruginosa ATCC 25619, Staphylococcus aureus ATCC 6538, S. aureus ATCC 25925, Streptococcus sanguinis ATCC 15300, S. salivarius ATCC 7073, S. mutans ATCC 25175 and Streptococcus ATCC. S. aureus ATCC 25925 was the most sensitive among the other S. sanguinis while S. salivarius proved the most resistant.

Salacia crassifolia (Celastraceae): CHEMICAL CONSTITUENTS AND ANTIMICROBIAL ACTIVITY

The phytochemical study of hexane extract from leaves of Salacia crassifolia resulted in the isolation of 3β-palmitoxy-urs-12-ene, 3-oxofriedelane, 3β-hydroxyfriedelane, 3-oxo-28-hydroxyfriedelane, 3-oxo-29-hydroxyfriedelane, 28,29-dihydroxyfriedelan-3-one, 3,4-seco-friedelan-3-oic acid, 3β-hydroxy-olean-9(11):12-diene and the mixture of α-amirin and β-amirin. β-sitosterol, the polymer gutta-percha, squalene and eicosanoic acid were also isolated. The chemical structures of these constituents were established by IR, 1H and 13C NMR spectral data. Crude extracts and the triterpenes were tested against Entamoeba histolytica, Giardia lamblia and Trichomonas vaginalis and no activity was observed under the in vitro assay conditions. The hexane, chloroform, ethyl acetate and ethanol crude extracts, and the constituent 3,4-seco-friedelan-3-oic acid and 28,29-dihydroxyfriedelan-3-one showed in vitro antimicrobial activity against Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Streptococcus sanguinis and Candida albicans.

Associação de glutamina e probióticos no trofismo mucoso do cólon na peritonite experimental

OBJETIVO: O objetivo deste estudo foi investigar o efeito da associação da glutamina e probióticos sobre a mucosa intestinal em ratos submetidos à peritonite experimental. MÉTODO: 16 ratos Wistar (250-350g) com peritonite experimental criada pelo método da punção dupla do ceco foram randomizados para receber diariamente no pós-operatório em conjunto com a dieta, a adição por gavagem de 0,500g de glutamina e leite reconstituído, contendo probióticos (10(6) unidades formadoras de colônias/g de Bifidobacterium lactis BL e Streptococcus thermophilus) (grupo glutamina-probióticos; n=8) ou 0,495g de caseína e leite reconstituído sem probióticos (grupo controle; n=8). O conteúdo das duas dietas foi isonitrogenado e isocalórico. Todos os animais foram sacrificados 120 horas após a peritonite experimental. A profundidade de criptas e espessura de parede da mucosa do cólon foram medidas em biopsias realizadas 2 cm acima da reflexão peritoneal. O restante da mucosa colônica foi pesado e nela mensurou-se o conteúdo de DNA. RESULTADOS: Os animais que receberam glutamina e probióticos apresentaram mucosa mais pesada (0,49±0,12 vs. 0,42±0,07g; p=0,02), maior conteúdo de DNA (0,31±0,07 vs. 0,22±0,05 mg/g de tecido; p<0,01) e criptas mais profundas (272±51 vs. 311±39µ; p=0,04) que o grupo controle. CONCLUSÃO: A associação da glutamina e probióticos confere um maior trofismo na mucosa colônica em ratos submetidos à peritonite experimental.

Antibioticoprofilaxia com ampicilina na rotura prematura das membranas: estudo randomizado e duplo cego

Objetivos: avaliar se o uso profilático da ampicilina pode evitar ou reduzir a morbidade infecciosa materna e perinatal decorrente da ruptura prematura das membranas (RPM) e prolongar a gestação em pacientes portadoras dessa complicação. Métodos: estudo prospectivo, randomizado e duplo-cego, avaliando 121 gestantes portadoras de RPM, divididas em dois grupos. O grupo tratado (61 gestantes) recebeu ampicilina e o grupo controle (60 gestantes) recebeu placebo, nas mesmas condições de horário, tempo de uso, embalagem e cor das cápsulas. Como parâmetros de infecção materna considerou-se a morbidade febril materna (índice térmico), presença de corioamnionite e/ou endometrite. Os parâmetros neonatais avaliados foram o índice de Apgar (1° e 5° minutos), colonização bacteriana do conduto auditivo e hemocultura. Para a análise estatística foram utilizados os testes: exato de Fisher, Wilcoxon e o chi². Resultados: o uso da ampicilina não prolongou a gestação, não reduziu a morbidade febril puerperal e nem as taxas de corioamnionite e/ou endometrite. Quanto à morbidade infecciosa perinatal também não foi possível demonstrar nenhuma redução decorrente do uso da ampicilina nem influência sobre as condições de nascimento. Estes dados foram consistentes em casos de RPM com até 72 horas de evolução, pois o limitado número de casos com tempo maior de evolução não permitiu uma análise estatística isenta de erro tipo II. Conclusões: com base nos resultados desse trabalho foi possível concluir que o uso profilático de ampicilina em gestantes com RPM com até 72 horas de evolução não reduz a morbidade infecciosa materna nem perinatal. No entanto, a presença de Streptococcus agalactiae do grupo B em hemocultura de recém-nascido do grupo controle indicou a necessidade imperiosa de antibioticoterapia para as gestantes colonizadas por esse microrganismo.

Morbidade Materna e Morbimortalidade Perinatal Associada à Infecção Ascendente na Rotura Prematura das Membranas

Objetivo: avaliar as repercussões da infecção ascendente sobre a mãe, o feto e o recém-nascido (RN) nos casos de rotura prematura das membranas (RPM). Métodos: estudo prospectivo, avaliando 50 gestantes portadoras de RPM e seus RN. A corioamnionite clínica foi rastreada por meio de critérios clínicos (curva térmica, dor abdominal à palpação e/ou amolecimento uterino, odor e características da secreção vaginal) e subsidiários (leucograma e proteína C reativa). Por sua vez, a corioamnionite histológica foi avaliada com estudo macroscópico e microscópico da placenta, membranas e cordão umbilical. No estudo microscópico, utilizou-se a microscopia óptica com coloração pela hematoxilina-eosina. Os RN foram avaliados pela mensuração do peso e índice de Apgar no 1o e 5o minuto. O leucograma e a cultura do material colhido do ouvido e aspirado gástrico complementaram o estudo. Para análise estatística foram utilizados os testes exato de Fisher e t de Student, com nível de significância de 5% (p<0,05). Resultados: a taxa de corioamnionite clínica foi de 29,4% (15/50), ao passo que a de corioamnionite histológica foi de 40% (20/50). Todos os casos de corioamnionite clínica apresentaram período de latência (PL) superior a 24 horas. Os RN apresentaram sinais de infecção em 31,4% (16/51), todos com PL maior que 24 horas. Os principais microrganismos isolados do conduto auditivo e aspirado gástrico dos RN foram: Klebsiella pneumoniae, Staphylococcus aureus, cocos Gram positivos e Streptococcus agalactiae - Grupo B de Lancefield (SGB). Os RN infectados apresentaram menor escore de Apgar no 1o e 5o minuto, peso ao nascer inferior e maior morbidade e mortalidade perinatal quando comparados com os RN não infectados. Conclusões: baseados na análise dos resultados obtidos no presente estudo, foi possível concluir que o período de latência prolongado aumenta a chance de infecção ascendente, que, por sua vez, proporciona maior probabilidade de parto prematuro, aumentando portanto a morbidade materna (corioamnionite clínica), bem como a morbidade e mortalidade perinatal.