ABSTRACT Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6 h of incubation. Strains tpeL (-) induced strong cell rounding after 6 h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.

Abstract Colorectal carcinoma is considered the fourth leading cause of cancer deaths worldwide. Several microorganisms have been associated with carcinogenesis, including Enterococcus spp., Helicobacter pylori, enterotoxigenic Bacteroides fragilis, pathogenic E. coli strains and oral Fusobacterium. Here we qualitatively and quantitatively evaluated the presence of oral and intestinal microorganisms in the fecal microbiota of colorectal cancer patients and healthy controls. Seventeen patients (between 49 and 70 years-old) visiting the Cancer Institute of the Sao Paulo State were selected, 7 of whom were diagnosed with colorectal carcinoma. Bacterial detection was performed by qRT-PCR. Although all of the tested bacteria were detected in the majority of the fecal samples, quantitative differences between the Cancer Group and healthy controls were detected only for F. nucleatum and C. difficile. The three tested oral microorganisms were frequently observed, suggesting a need for furthers studies into a potential role for these bacteria during colorectal carcinoma pathogenesis. Despite the small number of patients included in this study, we were able to detect significantly more F. nucleatum and C. difficile in the Cancer Group patients compared to healthy controls, suggesting a possible role of these bacteria in colon carcinogenesis. This finding should be considered when screening for colorectal cancer.

Neste estudo foi avaliada a diversidade genética de 23 amostras de Fusobacterium nucleatum isoladas da cavidade bucal de 15 pacientes com doença periodontal, de oito cepas isoladas de sete indivíduos sadios, de nove isoladas de nove pacientes com AIDS e de duas isoladas de dois macacos Cebus apella. Pela ação da enzima EcoRI sobre o DNA bacteriano foram reconhecidos 28 ribotipos agrupados de A a J. Os isolados testados formaram 24 ribotipos os quais foram contidos nos grupos A, B, C, D, E e F, e as três cepas de referência e dois isolados clínicos de A. actinomycetemcomitans e E. coli CDC formaram quatro diferentes ribotipos contidos nos grupos G, H, I e J. Em adição, as nove cepas de F. nucleatum isoladas de pacientes com AIDS, seis pertenciam ao grupo C e três ao grupo D. Usando-se a ribotipagem foi possível distinguir F. nucleatum isolados de diferentes origens.

The genetic diversity of 23 oral Fusobacterium nucleatum isolated from 15 periodontal patients, eight from seven healthy subjects, nine from nine AIDS patients and two from two Cebus apella monkeys were analyzed. EcoRI restricted the bacterial DNA and 28 ribotypes grouped from A to J groups were obtained. Isolates formed 24 ribotypes which were contained into A, B, C, D, E and F groups, and three reference strains and two clinical isolates of A. actinomycetemcomitans, and E. coli CDC formed four different ribotypes into the G, H, I and J groups. Moreover, from nine F. nucleatum from AIDS patients, six were ribotyped as group C and three as group D. By using ribotyping we distinguished F. nucleatum recovered from different sources. It is possible that isolates from AIDS patients may contain some phenotypic or genotypic factor did not observed in this study.

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.

Bactérias não enterotoxigênicas do grupo Bacteroides fragilis e B. fragilis enterotoxigênicas foram isoladas e identificadas de crianças com e sem diarréia aguda aquosa. Neste estudo, 170 amostras fecais de 96 e 74 crianças, com e sem diarréia, respectivamente, foram analisadas. A produção de enterotoxina e a detecção do gene que media a produção da toxina foram determinadas por ensaios citotóxicos em células HT-29/C1 e por PCR, respectivamente. A espécie B. fragilis foi prevalente em ambos os grupos, e cepas de B. fragilis enterotoxigênicas foram isoladas de duas crianças com diarréia aquosa aguda. Maiores estudos são necessários para avaliar o papel de cada bactéria desse importante grupo bacteriano, incluindo-se o papel que as cepas enterotoxigênicas, desempenham no processo diarréico em crianças.

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.

Species of Clostridium are widely distributed in the environment, inhabiting both human and animal gastrointestinal tracts. Clostridium difficile is an important pathogen associated with outbreaks of pseudomembranous colitis and other intestinal disorders, such as diarrhea. In this study, the prevalence of Clostridium spp. and C. difficile, from hospitalized children with acute diarrhea, was examined. These children were admitted to 3 different hospitals for over 12 months. Eighteen (20%) and 19 (21%) stool specimens from children with (90) and without (91) diarrhea respectively, were positive to clostridia. Only 10 C. difficile strains were detected in 5.5% of the stool samples of children with diarrhea. None healthy children (without diarrhea) harbored C. difficile. From these 10 C. difficile, 9 were considered as toxigenic and genotyped as tcdA+/tcdB+ or tcdA-/tcdB+, and 1 strain as nontoxigenic (tcdA-/tdcB-). They were detected by the citotoxicity on VERO cells and by the multiplex-polymerase chain reaction. Thirty clinical fecal extracts produced minor alterations on VERO cells. The presence of C. difficile as a probable agent of acute diarrhea is suggested in several countries, but in this study, the presence of these organisms was not significant. More studies will be necessary to evaluate the role of clostridia or C. difficile in diarrhoeal processes in children.

Fusobacterium nucleatum é um organismo que cumpre o papel de ponte de ligação entre os colonizadores iniciais e tardios no biofilme dentário, e é considerado um possível periodontopatógeno. Em Odontologia, agentes antimicrobianos são usados para o tratamento e controle de doenças infecciosas associadas com a placa dental. Anti-sépticos têm sido usados em associação com antibióticos para reduzir infecções após cirurgias orais. Neste estudo, foi avaliada a influência de concentrações subinibitórias de clorexidina, triclosan, penicilina G e metronidazol, sobre a hidrofobicidade, aderência às células epiteliais bucais, e a ultra-estrutura de F. nucleatum. Todos os isolados foram susceptíveis a clorexidina, triclosan, e metronidazol. A maioria deles foi sensível à penicilina G. Também, a maioria dos isolados, foi hidrofílica quando cresceram com ou sem antimicrobianos. A aderência foi diminuída pela ação dos antimicrobianos usados. Os resultados sugerem que a aderência de F. nucleatum foi influenciada por adesinas devido a que estruturas como fímbrias ou cápsula não foram observadas pela microscopia eletrônica de transmissão.

Fusobacterium nucleatum is considered a bridge organism between earlier and later colonizers in dental biofilms and a putative periodontopathogen. In Dentistry, antimicrobial agents are used for treatment and control of infectious diseases associated with dental plaque. Antiseptics have been used in association with antibiotics to reduce infections after oral surgeries. In this study, the influence of subinhibitory concentrations (SC) of chlorhexidine, triclosan, penicillin G and metronidazole, on hydrophobicity, adherence to oral epithelial cells, and ultra-structure of F. nucleatum was examined. All isolates were susceptible to chlorhexidine, triclosan, and metronidazole; however, most of the isolates were susceptible to penicillin G, and all of them were hydrophilic when grown with or without antimicrobials. Adherence was decreased by all antimicrobials. Results suggest that adherence of F. nucleatum was influenced by adhesins because structures such as fimbries or capsule were not observed by transmission electronic microscope.

Cepas de A. actinomycetemcomitans, B. forsythus, P. gingivalis, C. rectus, E. corrodens, P. intermedia, F. nucleatum e T. denticola foram identificadas da placa subgengival de 50 pacientes com doença periodontal e 50 indivíduos sadios. Os produtos de PCR de cada espécie microbiana mostraram bandas específicas, favorecendo a identificação direta de bactérias periodontais de espécimes clínicos. Resultados negativos ou positivos idênticos entre PCR e a cultura ocorreram de 66% (A. actinomycetemcomitans) a 93% (F. nucleatum) das amostras. A detecção de A. actinomycetemcomitans, B. forsythus, C. rectus, E. corrodens, P. intermedia e T. denticola por PCR foi significativamente associada com a doença, mostrando valores altos de odds ratio (OR) para B. forsythus (2,97, 95% CI 1,88 - 4,70). Através da cultura foi observada a associação de A. actinomycetemcomitans, B. forsythus e P. intermedia com a periodontite, entretanto, P. gingivalis, C. rectus, E. corrodens e F. nucleatum não foram significativamente relacionados à doença.

A. actinomycetemcomitans, B. forsythus, P. gingivalis, C. rectus, E. corrodens, P. intermedia, F. nucleatum, and T. denticola were identified from subgingival plaque from 50 periodontal patients and 50 healthy subjects. PCR products from each species showed a specific band and could be used to identify periodontal organisms from clinical specimens. Identical negative or positive results between PCR and culture occurred in 66% (A. actinomycetemcomitans) to 93% (F. nucleatum) of the samples. PCR detection odds ratio values for A. actinomycetemcomitans, B. forsythus, C. rectus, E. corrodens, P. intermedia, and T. denticola were significantly associated with disease having a higher OR values for B. forsythus (2.97, 95% CI 1.88 - 4.70). Cultures showed that A. actinomycetemcomitans, B. forsythus and P. intermedia were associated with periodontitis, however, P. gingivalis, C. rectus, E. corrodens and F. nucleatum were not significantly associated with the disease.

Foi estudada a ação letal de 80 cepas de Fusobacterium nucleatum orais, isoladas de pacientes adultos com periodontite, indivíduos sadios e macacos Cebus apella, sobre camundongos isogênicos Balb/c. Os camundongos foram inoculados intraperitonealmente com cada inóculo bacteriano de aproximadamente 5 x 10(8) UFC/ml. Todos os isolados clínicos induziram a perda de peso e dos movimentos coordenados. Além disso, foram observadas alterações patológicas no figado, SNC, coração e rins, mostrando reações inflamatórias ou congestão vascular. 61,2% dos isolados de F. nucleatum, provenientes de pacientes periodontais, 51,7% dos indivíduos sadios e 60% dos macacos foram capazes de matar camundongos em 48 h. Os isolados clínicos foram significativamente mais patogênicos que F. nucleatum ATCC 10953 ou ATCC 25586. Também, B. fragilis ATCC 23745 mostrou-se letal contra os animais controle. Esses resultados sugerem que o LPS poderia estar envolvido nessa ação letal contra camundongos e poderia ter um importante papel na destruição dos tecidos ou na morte do animal.

The lethal action in Balb/c mice of 80 oral Fusobacterium nucleatum recovered from patients with adult periodontitis, healthy subjects or Cebus apella monkeys was studied. Mice were inoculated intraperitoneally with each bacterial inoculum of 5 x 10(8) CFU/ml. All the clinical isolates induced weight and coordinated movements loss. Pathological alterations in liver, CNS, heart, and kidney with inflammatory reactions or vascular congestion were observed. Of all the tested F. nucleatum isolates, 61.2% from periodontal patients, 57.1% from healthy subjects and 60% from monkeys, were capable of killing the mice in 48h. The clinical isolates were significantly more pathogenic than F. nucleatum ATCC 10953 or ATCC 25586. B. fragilis ATCC 23745 showed lethality against control mice. Our results suggest that LPS could be involved in lethal action against mice and it may play an important role in producing tissue damage or death of mice.