Ocorrência de microrganismos anaeróbios estritos no escarro de pacientes com bronquite crônica

No escarro de pacientes hospitalizados com bronquite crônica foram isolados microrganismos anaeróbios estritos - Fusobacterium, Veillonella, Bacteroides melaninogenicus, Peptrostreptococcus, Actinomyces ou difteróides anaeróbios. Como o isolamento de anaeróbios estritos foi realizado em alíquotas da diluição 1O-³ do escarro fluidificado e em 83% dos casos não houve isolamento simultâneo no material do orofaringe, considera-se como de origem brônquica os anaeróbios isolados e não como contaminação do escarro no orofaringe e cavidade oral.

"Cara inchada" of cattle, an infectious, apparently soil antibiotics-dependant periodontitis in Brazil

The objective of this review on the investigation of "cara inchada" in cattle (CI), pursued over the last 30 years, was to elucidate the pathogenicity of the disease and come to proper conclusions on its etiology. CI has been widely considered to be of nutritional origin, caused primarily by mineral deficiency or imbalance. However, the disease consists of a rapidly progressive periodontitis, affecting the periodontal tissues at the level of the premolars and molars during the period of tooth eruption generally starting in young calves. The disease led to great economic losses for farmers in central-western Brazil, after the occupation of new land for cattle raising in the 1960s and 1970s. The lateral enlargement of the maxillary bones of affected calves gave the disease the popular name of "cara inchada", i.e., swollen or enlarged face. The enlargement was found to be due to a chronic ossifying periostitis resulting from the purulent alveolitis of CI. Black-pigmented non-saccharolytic Bacteroides melaninogenicus, always together with Actinomyces (Corynebacterium) pyogenes, were isolated in large numbers from the periodontal lesions. B. melaninogenicus could be isolated in small numbers also from the marginal gingiva of a few healthy calves maintained on CI-free farms. "In vitro"-assays showed that streptomycin and actinomycin, as well as the supernatants of cultivates of actinomycetes from soils of CI-prone farms, applied in subinhibitory concentrations to the bacteria tested, enhanced significantly (up to 10 times) the adherence of the black-pigmented B.melaninogenicus to epithelial cells of the bovine gingiva. The antibiotics are apparently produced in large quantities by the increased number of soil actinomycetes, including the genus Streptomyces, that develop when soil microflora are modified by cultivating virgin forest or "Cerrado" (tree-savanna) for the first time for cattle grazing. The epidemiology of CI now provides strong evidence that the ingestion with the forage of such antibiotics could possibly be an important determinant factor for the onset and development of this infectious periodontitis. The antibiotic enhanced adherence of B.melaninogenicus to the sulcus-epithelium of the marginal gingiva, is thought to allow it to colonize, form a plaque and become pathogenic. There is experimental evidence that this determinant factor for the development of the periodontitis is present also in the milk of the mothers of CI-diseased calves. It has been shown that the bacteria isolated from the periodontal CI-lesions produce enzymes and endotoxins capable of destroying the periodontal tissues. The epidemiology of CI, with its decline in incidence and its disappearance after several years, could be explained by the fact that the former equilibrium of the microflora of the once undisturbed virgin soil has been reached again and that the number of antibiotic producing actinomycetes has been anew reduced. By this reasoning and all the data available, CI should be considered as a multifactorial infectious disease, caused primarily by the anaerobic black-pigmented non-saccharolytic Bacteroides melaninogenicus, always together with the micro-anaerobic Actinomyces pyogenes. Accordingly, the onset and development of the infectious periodontitis is apparently determined by ingestion with the forage of subinhibitory concentrations of antibiotics produced in recently cultivated virgin soils. This hypothesis is supported by the recent observation of renewed outbreaks of CI-periodontitis in former CI-prone areas, following fresh cultivation after many years. The infectious nature of CI is confirmed by trials in which virginiamycin was used efficiently for the oral treatment of CI-diseased cattle. Previously it has been shown, that spiramycin and virginiamycin, used as additives in mineral supplements, prevented CI-periodontitis.

Antimicrobial resistance and plasmid detection in strains of the Bacteroides fragilis group

Resistant populations of the Bacteroides fragilis group bacteria (two reference ones and two isolated from human and Callithrix penicillata marmoset) were obtained by the gradient plate technique, to clindamycin, penicillin G, metronidazole and mercuric chloride. All the four tested strains were originaly susceptible to the four antimicrobial drugs at the breakpoint used in this study. MICs determination for the four cultures gave constant values for each antimicrobial, on the several steps by the gradient plate technique. The intestinal human B. fragilis strains showed three DNA bands, that could be representative of only two plasmids in the closed covalently circular (CCC) form with molecular weights of approximately 25 and 2.5 Md. The results do not permit an association between the presence of plasmid in the human strain with the susceptibility to the studied drugs. The four strains were ß-lactamase negative in the two methods used, and no particular chromosomal genetic resistance marker was demonstred. The resistance (MIC) observed, after contact with penicillin G and mercuric chloride, were two-fold in the four tested strains

Susceptibility of clinical isolates of Bacteroides fragilis group strains to cefoxitin, cefoperazone and ticarcillin/clavulanate

A total of 40 strains of the B. fragilis group was isolated from clinical specimens in two hospital centers in Fortaleza from 1993 to 1997. The most frequently isolated species was Bacteroides fragilis (19 strains) and most isolates came from intra-abdominal and wound infections. The susceptibility profile was traced for cefoxitin, cefoperazone and ticarcillin-clavulanate by using the agar dilution reference method. All isolates were susceptible to ticarcillin-clavulanate (128/2mug/ml). Resistance rates of 15 and 70% were detected to cefoxitin (64mug/ml) and cefoperazone (64mug/ml), respectively. Such regional results permit a better orientation in choosing this group of antibiotics for prophylaxis and therapy especially in relation to cefoxitin, which is frequently used in the hospital centers studied.

Detection of non-enterotoxigenic and enterotoxigenic Bacteroides fragilis in stool samples from children in São Paulo, Brazil

Non-enterotoxigenic bacteria of the Bacteroides fragilis group and enterotoxigenic B. fragilis were identified from children with and without aqueous acute diarrhea. In this study, 170 stool samples from 96 children with and 74 without diarrhea were analyzed. Enterotoxin production and the toxin gene detection were detected by cytotoxicity assay on HT-29/C1 cells and by PCR, respectively. B. fragilis species was prevalent in both groups and enterotoxigenic B. fragilis strains were isolated from two children with diarrhea. More studies are important to evaluate the role of each bacteria of the B. fragilis group, including enterotoxigenic strains play in the diarrheal processes in children.

Virulence markers and antimicrobial susceptibility of bacteria of the Bacteroides fragilis group isolated from stool of children with diarrhea in São Paulo, Brazil

Bacteroides fragilis has been isolated from several human and non-human monomicrobial and mixed infections. In this study, some virulence markers and the antimicrobial susceptibility of bacteria of the B. fragilis group isolated from children's stools were evaluated. All the 64 isolates showed the following characteristics: capsulated, beta-hemolytic, hydrophilic, and serum-resistant. Only, 24 (37.5%) strains were resistant at 60ºC, for 30 min, and among them, 12 (18.75%) were resistant at 60ºC, for 60 min. Also, none strain was resistant at 100ºC. Four strains were able to hemagglutinate erythrocytes and D-mannose, D-galactose, D-arabinose, and D-xylose inhibited hemagglutination in 2 B. fragilis strains (p76a, p76b). The hemagglutination in the strain B. uniformis p3-2 was inhibited by D-xylose and D-galactose. The bft gene detection and the enterotoxin production were observed only in 13 EF-enterotoxigenic species. Fragilysin activity was confirmed on HT-29 cells. The antimicrobial determination confirmed that both imipenem and metronidazole were efficient against B. fragilis species; all the strains were resistant to lead and nickel. Plasmids of 2.9, 4.4, 4.8, and 8.9 kb were observed in 6 tested strains. These results show the values of the species identification from clinical infections, as well as of the periodic evaluation of the resistance patterns of the B. fragilis group at Brazilian medical institutions.

Survey of antimicrobial susceptibility patterns of the bacteria of the Bacteroides fragilis group isolated from the intestinal tract of children

The bacteria of the Bacteroides fragilis group are considered important clinical pathogens and they are the most common anaerobes isolated from human endogenous infections. In this study, the susceptibility patterns to antibiotics and metals of 114 species of the B. fragilis group isolated from children with and without diarrhea were determined. Susceptibility was assayed by using an agar dilution method with Wilkins-Chalgren agar. All B. fragilis strains were resistant to lead and nickel, but susceptible to metronidazole and imipenem. beta-lactamase production was detected by using biological and nitrocefin methods, respectively, in 50% and 90.6% of the isolates of children with diarrhea and in 60% and 90% of the isolates of children without diarrhea. Our results show an increase of antibiotics and metals resistance in this microbial group, and a periodic evaluation of the antimicrobial susceptibility is needed. In Brazil, the contamination for antibiotics or metal ions is often observed, and it is suggested an increase the antimicrobial resistance surveillance of this microbial group, mainly those isolated from children's diarrhea.

The redox potential interferes with the expression of laminin binding molecules in Bacteroides fragilis

The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.

Enterotoxigenic and nontoxigenic Bacteroides fragilis strains isolated in Brazil

The presence of enterotoxigenic Bacteroides fragilis and nontoxigenic B. fragilis (NTBF) among 109 strains isolated from 1980-2008 in Brazil were investigated by PCR. One strain, representing 0.9% of the total analyzed strains, harbored the bft gene which was identified as bft-1 isoform based on PCR-RFLP and sequencing. Forty-nine strains (44.9%) exhibited the NTBF pattern III which possesses the flanking region required for pathogenicity island acquisition in which the bftgene is codified. These data reinforce the potential of B. fragilis as an emerging enteropathogen in our country.

Antagonic effect of the inhibition of inducible nitric oxide on the mortality of mice acutely infected with Escherichia coli and Bacteroides fragilis

Sepsis, the leading cause of death in intensive care units, is associated with overproduction of nitric oxide (NO) due to inducible NO synthase (iNOS), responsible for some of the pathologic changes. Aminoguanidine (AG) is a selective iNOS inhibitor with reported inconsistent actions in sepsis. To investigate the influence of iNOS, we studied models of acute bacterial sepsis using acute challenges with aerobic (Escherichia coli) and anaerobic (Bacteroides fragilis) bacteria in the presence of AG. Six-week-old, 23 g, male and female BALB/c and C57Bl/6j mice, in equal proportions, were inoculated (ip) with bacteria in groups of 4 animals for each dose and each experiment in the absence or presence of AG (50 mg/kg, ip, starting 24 h before challenge and daily until day 6) and serum nitrate was measured by chemiluminescence. Both types of bacteria were lethal to mice, with an LD50 of 6 nephelometric units (U) for E. coli and 8 U for B. fragilis. Nitrate production peaked on the second day after E. coli inoculation with 8 and 6 U (P < 0.05), but was absent after non-lethal lower doses. After challenge with B. fragilis this early peak occurred at all tested doses after 24 h, including non-lethal ones (P < 0.05). AG-treated mice challenged with E. coli presented higher survival (P < 0.05) and increased LD50. AG-treated mice challenged with B. fragilis had lower LD50 and higher mortality. Control AG-treated animals presented no toxic effects. The opposite effect of iNOS blockade by AG in these models could be explained by restriction of oxygen for immune cells or an efficient action of NO in anaerobic localized infections. The antagonic role of NO production observed in our bacterial models could explain the reported discrepancy of NO action in sepsis.

Epidemiology and antimicrobial resistance of B. fragilis group organisms isolated from clinical specimen and human intestinal microbiota

Epidemiological aspects and the antimicrobial susceptibility profile of the Bacteroides fragilis group isolated from clinical and human intestinal specimens were examined in this study. B. fragilis group strains were isolated from 46 (37%) of 124 clinical specimens and the source of the samples was: Blood culture (3), intraabdominal infection (27), brain abscess (2), soft tissue infection (17), respiratory sinus (3), pleural aspirate (9), breast abscess (3), surgical infected wound (22), pelvic inflammatory disease (22), chronic otitis media (9) and miscellaneous (7). Intraabdominal and soft tissue infections were responsible for more than half of the clinical isolates. Susceptibility to penicillin, cefoxitin, tetracycline, metronidazole, chloramphenicol and clindamycin was examined. All isolates were susceptible to metronidazole and chloramphenicol. For clindamycin and cefoxitin the resistance rates observed were 21.7% and 10.9% respectively. Susceptibility profiles varied among the different species tested. A total of 37 species of B. fragilis group isolated from intestinal microbiota of individuals who had no antimicrobial therapy for at least 1 month before the sampling was also examined. All strains were also susceptible to chloramphenicol and motronidazole and the resistance rates to clindamycin and cefoxitin were 19.4% and 5.4% respectively. A few institutions, in Brazil, have monitored the antimicrobial susceptibility of B. fragilis group strains isolated from anaerobic infections. The resistance rates to cefoxitin and clindamycin and the variation in susceptibility patterns among the species isolated in this study emphasize the need for monitoring of susceptibility patterns of B. fragilis group organisms isolated, especially at our University Hospitals.

Peritonite infecciosa com quantitativos e qualitativos bacterianos conhecidos: estudo experimental em ratos

O objetivo deste estudo é o desenvolvimento de peritonite difusa com qualitativos e quantitativos bacterianos conhecidos. Foram analisados 150 ratos, adultos, machos, da raça Wistar, com peso médio de 150 gramas. Inocularam-se, percutaneamente, na cavidade peritoneal, suspensões constituídas de Escherichia coli e Bacteróides fragilis em concentrações conhecidas, na proporção de 1 ml para cada 100 gramas de peso. Os animais foram distribuídos em cinco grupos de trinta ratos. No grupo I (grupo-controle) inoculou-se solução de cloreto de sódio a 0,9%. Nos demais grupos a concentração do inóculo foi a seguinte: grupo II, com suspensão a 10 (9); grupo III, com suspensão a 10 (8): grupo IV, suspensão a 10 (7) e grupo V com suspensão a 10 (6). Sempre que se detectou o óbito, o animal era submetido à necropsia para avaliação da cavidade peritoneal e colheita de secreções para cultura. Os ratos sobreviventes foram aleatoriamente alocados em dois subgrupos. Os animais do subgrupo A foram sacrificados 24 horas após a inoculação e os do subgrupo B, 120 horas após a inoculação. Observou-se que os ratos do grupo I (controle) evoluíram sem o desenvolvimento de peritonite. Nos grupos II e III,100% dos ratos do subgrupo A e 95,83% dos ratos do subgrupo B desenvolveram peritonite aguda e óbito em menos de 24 horas. No grupo IV, somente 4,17% desenvolveram peritonite e foram a óbito em 72 horas, e no grupo V não ocorreu a formação de peritonite e não houve óbito. Os animais que foram a óbito dos grupos II e III, 96,67% mostraram alterações macroscópicas com exsudato peritoneal difuso, aderências peritoneais mas sem abscesso. Todos os animais com peritonite, desenvolveram derrame pleural bilateral. Nos animais que foram a óbito, nos grupos II e III, evidenciou-se a presença de Escherichia coli e Bacteroides fragilis como causadores das alterações peritoneais e pleurais. Este modelo mostrou que os animais que receberam altas concentrações bacterianas mostraram maior perda de peso, alterações clínicas de sepsis, peritonite difusa aguda, derrame pleural e óbito precoce.

Adherence and experimental infection of bacteria associated with periodontal infections of young cattle in Brazil ("Cara inchada")

In vitro- and in vivo-assays were conducted, to study the possible role of streptomycin- and actinomycin-producing soil actinomycetes for the pathogenesis of "Cara inchada" in cattle (CI). Adherence of Bacteroides spp. to epithelial cells of the bovine gingiva, known to be associated with the progressive lesions of CI, was significantly increased by the addition of streptomycin, actinomycin or antibiotic culture supernatants of the soil actinomycetes. Applications of these mixtures together with Actinomyces pyogenes to the marginal gingiva of the upper premolar teeth of about 1 month old Holstein Friesian calves did not lead to progressive lesions of CI. Only one calf exhibited a slight diarrhea and a temporary retraction of the gingiva at the site of application.

Modificação da microbiota associada às lesões peridentárias da "cara inchada" em bezerros transferidos para área indene

Observações sobre a epizootiologia da "cara inchada" dos bovinos (CI) indicam que animais clinicamente positivos se recuperam espontâneamente quando transferidos para área indene. No presente estudo, 13 bovinos com lesões peridentárias progressivas da "cara inchada" foram transferidos para área indene com a finalidade de se verificar a evolução clínica da doença e a composição da microbiota da bolsa peridentária em duas situações distintas: (1) nas lesões progressivas e (2) quando da recuperação clínica. O estudo bacteriológico semi-quantitativo e qualitativo foi realizado tendo como referência a percentagem de Bacteroides pigmentados de negro presentes nos cultivos. Nas lesões progressivas a percentagem média destes microrganismos foi de 71,3%. Após 4 a 7 meses da transferência os animais se recuperaram espontaneamente, observando-se uma melhora na condição nutricional, desaparecimento do abaulamento facial e do odor fétido bucal e cicatrização com epitelização das lesões peridentárias. Na avaliação da composição da micro-biota das bolsas peridentárias dos bezerros quando clinicamente recuperados, este mesmo grupo de micorganismos representou em média 1,7%. Os resultados revelaram a ocorrência de uma predominância de Bacteroides pigmentados de negro nas lesões peridentárias progressivas da "cara inchada"e sua remissão quantitativa percentual após a recuperação clínica dos animais, consubstanciando as evidências de sua natureza infecciosa primária.

A etiologia da "cara inchada", uma periodontite epizoótica dos bovinos

Uma versão condensada em português de um artigo de revisão sobre a periodontite da "cara inchada" dos bovinos, publicado em inglês, está apresentada com algumas informações adicionais. A doença foi responsável por grandes perdas de bovinos jovens, principalmente nas décadas de 1970 e l980 no Brazil Central. Em face da periodontite progressiva e a perdas de dentes, os animais não podem se alimentar convenientemente, tornam-se emaciados e podem morrer. A doença foi tida como uma deficiência ou desequilíbrio mineral. Mas as pesquisas de campo e de laboratório, realizadas durante 30 anos, mostraram que trata-se de doença infecciosa multifatorial a ser definida como Periodontite Epizoótica Bovina. Chegou-se à conclusão que os fatores principais para o seu desenvolvimento são: (1) a idade dos bovinos na fase de erupção dos dentes premolares e molares; (2) a presença de bactérias do grupo Bacteroides spp nos espaços subgengivais; e (3) a ingestão com a forragem de concentrações subinibitórias de antibióticos, sobretudo de estreptomicina, produzidos por actinomicetos cujo número é aumentado em solos virgens recém-cultivados na formação de pastagens após a derrubada da mata ou da vegetação de Cerrado; isto leva a um aumento da aderência dos bacteróides ao epitélio gengival e à destruição dos tecidos peridentários. Hoje em dia, a doença perdeu a sua importância e praticamente desapareceu, porque a microbiota do solo entrou novamente em equilíbrio e a abertura de grandes áreas virgens para a pecuária cessou. Porém, novos surtos podem ocorrer em áreas anteriormente positivas para a doença quando, na reforma de pastagens ou capineiras, houver um novo desequilíbrio da microbiota do solo. Outros antibióticos, como a espiramicina e virginiamicina, administrados por via oral ou adicionado a misturas minerais, podem controlar a periodontite.