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Livestock farming has exerted intense environmental pressure on our planet. The high emissions to the environment and the high demands of resources for the production process have encouraged the search for decarbonization and circularity in the livestock sector. In this context, the objective of this study was to evaluate and compare the environmental performance of two different uses for biogas generated in the anaerobic digestion of animal waste, either for electricity generation or biomethane. For this purpose, a life cycle assessment approach was applied to evaluate the potential of anaerobic digestion as a management technology for three different livestock wastes, related to beef cattle, dairy, and sheep in the Brazilian animal production context. The results suggest that the treatment scenarios focusing on biomethane generation were able to mitigate the highest percentage of damages (77 to 108%) in the global warming category when compared to the scenarios without the use of anaerobic digestion (3.00·10 2 to 3.71·10 3 kgCO 2 eq ) or in the perspective of electricity generation (mitigation of 74 to 96%). In terms of freshwater eutrophication, the generation of electricity (- 2.17·10 -2 to 2.31·10 -3 kg P eq ) is more favorable than the purification of biogas to biomethane (- 1.73·10 -2 to 2.44·10 -3 kg P eq ), due to the loss of methane in the upgrading process. In terms of terrestrial ecotoxicity, all scenarios are very similar, with negative values (- 1.19·10 1 to - 7.17·10 2 kg 1,4-DCB) due to the benefit of nutrient recovery, especially nitrogen, associated with the use of digestate as fertilizer, which was one of the critical points in all scenarios. Based on these results, it is evident that proper management of all stages of the treatment life cycle is the key to decarbonization and circularity in livestock waste management. The biogas use does not present different effects on the environmental performance of the scenarios studied, demonstrating that the purpose should be chosen according to the needs of each plant or management system., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We determined the frequency and mutational spectrum of BRCA1 and BRCA2 in a series of patients at high risk for developing breast cancer from Brazil. A total of 1267 patients were referred for BRCA genetic testing, and no obligation of fulfilling criteria of mutation probability methods for molecular screening was applied. Germline deleterious mutations in BRCA1/2 (i.e., pathogenic/likely pathogenic variants) were identified in 156 out of 1267 patients (12%). We confirm recurrent mutations in BRCA1/2, but we also report three novel mutations in BRCA2, not previously reported in any public databases or other studies. Variants of unknown significance (VUS) represent only 2% in this dataset and most of them were detected in BRCA2. The overall mutation prevalence in BRCA1/2 was higher in patients diagnosed with cancer at age > 35 years old, and with family history of cancer. The present data expand our knowledge of BRCA1/2 germline mutational spectrum, and it is a valuable clinical resource for genetic counseling and cancer management programs in the country., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The recent manuscript entitled "Relationship between clinical features and intestinal microbiota in Chinese patients with ulcerative colitis" reported a difference in the intestinal microbiota of patients with ulcerative colitis according to the severity of the colitis. The influence of the intestinal microbiota on the development and progress of gastrointestinal disorders is well established. Besides the diversity in the microbiome, the presence of virulence factors and toxins by commensal bacteria may affect an extensive variety of cellular processes, contributing to the induction of a proinflammatory environment., Competing Interests: Conflict-of-interest statement: All the authors report no relevant conflicts of interest for this article., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

The cytochrome c-oxidase (COX) enzyme, also known as mitochondrial complex IV (MT-C4D), is a transmembrane protein complex found in mitochondria. COX deficiency is one of the most frequent causes of electron transport chain defects in humans. Therefore, high energy demand organs and tissues are affected in patients with mutations in the COX15 gene, with variable phenotypic expressiveness. We describe the case of a male newborn with hypertrophic cardiomyopathy and serum and cerebrospinal fluid hyperlacticaemia, whose exome sequencing revealed two variants in a compound heterozygous state: c.232G > A; p.(Gly78Arg), classified as likely pathogenic, and c.452C > G; p.(Ser151Ter), as pathogenic; the former never previously described in the literature., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

The use of molecular diagnostics for pathogen detection in epidemiological studies have allowed us to get a wider view of the pathogens associated with diarrhea, but the presence of enteropathogens in asymptomatic individuals has raised several challenges in understanding the etiology of diarrhea, and the use of these platforms in clinical diagnosis as well. To characterize the presence of the most relevant bacterial enteropathogens in diarrheal episodes, we evaluated here the prevalence of diarrheagenic E. coli pathotypes, Salmonella spp., and Yersinia enterocolitica in stool samples of children with and without diarrhea using real-time quantitative PCR (qPCR). We found that the presence of genetic markers associated with bacterial pathogens was significantly higher in stool samples from the diarrhea group compared to the control ( P < 0.001). Bacterial loads in samples positive for eae and aggR markers were also determined. Compared to samples from asymptomatic children, a significantly higher number of copies of the eae gene were found in diarrhea samples. Also, the presence of genetic markers associated with STEC strains with clinical significance was evaluated in eae -positive samples by high-throughput real-time PCR. The data presented herein demonstrated that asymptomatic children of an urban area in Brazil might be enteropathogen reservoirs, especially for STEC., (Copyright © 2020 Merino, Nakano, Delannoy, Fach, Alberca, Farfan, Piazza and Avila-Campos.)

Leigh syndrome is an early onset progressive disorder caused by defects in mitochondrial oxidative phosphorylation. Pathogenic variants in nuclear and mitochondrial genes are associated with the syndrome. Homozygous pathogenic variants in the C12orf65 gene impair the mitochondrial oxidative phosphorylation system. We describe a new case of Leigh syndrome caused by a novel pathogenic variant of the C12orf65 gene resulting in the lack of the Gly-Gly-Gln (GGQ) domain in the predicted protein, and review clinical and molecular data from previously reported patients. Our study supports that the phenotype caused by C12orf65 gene variants is heterogeneous and varies from spastic paraparesis to Leigh syndrome. Loss-of-function variants are more likely to cause the disease, and variants affecting the GGQ domain tend to be associated with more severe phenotypes, reinforcing a possible genotype-phenotype correlation.

: The objective is to report a patient with congenital afibrinogenemia and vascular abnormalities and also review the clinical and molecular issues. The female proband, diagnosed with congenital afibrinogenemia, was admitted at a hospital due to a hemorrhagic shock. Angiotomography revealed ectasias from ascending branch to the abdominal aorta, with multiple calcifications and atheroma. Clinical exome identified a homozygous novel pathogenic variant in FGG gene. In our review the main symptom, at diagnosis, was umbilical cord bleeding and the degree of clinical involvement varied from asymptomatic to severe. The FGA gene was the most affected and possible hot spots were observed. Variants considered as loss of function were the most frequent. The association of vascular abnormalities in a patient with congenital afibrinogenemia alerts for a closer follow-up of vascular issues in these patients.

Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are strongly associated with periodontitis, and their evaluations are relevant to understand their role in the etiology and progression of periodontal diseases. In this study, the qualitative and quantitative detection of A. actinomycetemcomitans and F. nucleatum, as well as their genetic diversity, were evaluated in individuals with gingivitis, chronic periodontitis and periodontally healthy. In addition, the biotyping, serotyping, and prevalence of the ltx and cdt genes in A. actinomycetemcomitans were also determined. Subgingival biofilms obtained from gingivitis (70), periodontitis (75) and healthy (95) individuals were analyzed by cultures and PCR. Bacterial typing and presence of ltx and cdt genes in A. actinomycetemcomitans were also verified. DNA from A. actinomycetemcomitans and F. nucleatum was detected respectively, in 65.7% and 57.1% of gingivitis, 80% and 68% of periodontitis, and 57.8% and 37.8% of healthy. A. actinomycetemcomitans from gingivitis were biotypes I, II, IV, V, and X, and serotypes a, c, and e. In periodontitis, biotypes II, VI, and X, and serotypes a, b, and c were found. In healthy subjects, biotypes II and X, and serotypes b and c were found. The LTX and ltxA were observed in strains from gingivitis and periodontitis pockets. Subsequently, our data also showed no direct relationship between ltxA gene expression and leukotoxin gene 530-bp presence. On the other hand, cdt gene predominated during the inflammatory disease process. Our results strongly support a role of A. actinomycetemcomitans and F. nucleatum in advanced stage of periodontal disease., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Keutel syndrome is caused by mutations in the matrix gamma-carboxyglutamic acid ( MGP ) gene (OMIM 154870) and is inherited in an autosomal recessive fashion. It is characterized by brachydactyly, pulmonary artery stenosis, a distinctive facial phenotype, and cartilage calcification. To date, only 36 cases have been reported worldwide. We describe clinical and molecular findings of the first Brazilian patient with Keutel syndrome. Keutel syndrome was suspected based on clinical and morphological evaluation, so we sequenced the MGP gene using the TruSight One Sequencing Panel (Illumina). The obtained MGP gene sequence was then validated by Sanger sequencing. We identified a novel pathogenic homozygous variant of the MGP gene (c.2T>C; p.Met1Thr) confirming Keutel syndrome. Proper diagnosis of this syndrome is important for clinical management and is an indication for genetic counseling. Keutel syndrome should be suspected in patients with cartilage calcifications and brachydactyly when associated with a distinctive facial phenotype and pulmonary artery stenosis.

Objective: In this study, the presence of the prtC and fimA genes involved in the pathogenicity of oral Porphyromonas spp. isolated from dogs with periodontitis and healthy, as well as their genetic diversity was investigated., Design: Thirty-two Beagle dogs, 24 with periodontitis and 8 healthy were evaluated. Subgingival samples from only one gingival site of both groups were collected. Bacteria grown in anaerobiosis were identified by RAPID ID 32A kits. From each strain the respective DNA was obtained and used to genotyping by conventional PCR and AP-PCR., Results: Dogs with periodontitis harbored 28 P. gulae, 2 P. creviocaricanis, 1 P. cangingivalis and 7 P. macacae; and from healthy dogs, 11 P. gulae and 5 P. circumdentaria. In P. gulae isolated from periodontal dogs the gene prtC was observed in 19 (67.85%) and in 7 (63.63%) from healthy dogs. P. gulae strains from periodontal dogs harbored either the gene fimA I or fimA II; while strains from healthy dogs harbored the gene fimA I, fimA II, fimA III or fimA IV, as well as 1 P. circumdentaria the gene fimA II. By AP-PCR strains were grouped in different clusters suggesting heterogeneity of these microorganisms., Conclusions: The results presented herein inform that Porphyromonas spp. isolated from dogs with and without periodontitis harbored the prtC and fimA genes and it could be a role in the establishment of the infectious process., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 6h of incubation. Strains tpeL (-) induced strong cell rounding after 6h 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., (Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

The administration of antimicrobial agents leads to an ecological imbalance of the host-microorganisms relationship, and it causes a rapid and significant reduction in the microbial diversity. The aim of the current study was to evaluate the impact of antibiotic therapy on intestinal microbiota of children between 3 and 12 years of age. The fecal samples were collected from hospitalized children (n = 31) and from healthy untreated children (n = 30). The presence of bacteria and their quantities were assessed by culture-based methods and quantitative polymerase chain reaction (qPCR). By culture method, in the children receiving antibiotics, a low recovery of Bifidobacterium spp. (54.8%), Bacteroides spp./Parabacteroides spp. (54.8%), Clostridium spp. (35.5%), and Escherichia coli (74.2%) was observed compared with the children without antibiotic therapy (100%, 80%, 63.3%, and 86.6%, respectively). By qPCR, the children receiving antibiotics showed a lower copy number for all microorganisms, except to Lactobacillus spp. (p = 0.0092). In comparison to the nontreated children, the antibiotic-treated children showed a significantly lower copy number of Bifidobacterium spp. (p = 0.0002), Clostridium perfringens (p < 0.0001), E. coli (p = 0.0268), Methanobrevibacter smithii (p = 0.0444), and phylum Firmicutes (p = 0.0009). In conclusion, our results obtained through qualitative and quantitative analyses, demonstrate that antibiotic therapy affect the intestinal microbiome of children.

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.

Enterotoxigenic Bacteroides fragilis (ETBF) is an important part of the human and animal intestinal microbiota and is commonly associated with diarrhea. ETBF strains produce an enterotoxin encoded by the bft gene located in the B. fragilis pathogenicity island (BfPAI). Non-enterotoxigenic B. fragilis (NTBF) strains lack the BfPAI and usually show two different genetic patterns, II and III, based on the absence or presence of a BfPAI-flanking region, respectively. The incidence of ETBF and NTBF strains in fecal samples isolated from children without acute diarrhea or any other intestinal disorders was determined. All 84 fecal samples evaluated were B. fragilis-positive by PCR, four of them harbored the bft gene, 27 contained the NTBF pattern III DNA sequence, and 52 were considered to be NTBF pattern II samples. One sample was positive for both ETBF and NTBF pattern III DNA sequences. All 19 B. fragilis strains isolated by the culture method were bft-negative, 9 belonged to pattern III and 10 to pattern II. We present an updated overview of the ETBF and NTBF incidence in the fecal microbiota of children from Sao Paulo City, Brazil.

The sialidase activity and genetic diversity of 22 Clostridium perfringens strains isolated from chickens with necrotic enteritis were determined. Sialidase activity was detected in 86.4 % of the strains. All C. perfringens showed a high value of similarity (>96 %), and they were grouped into seven clusters clearly separated from the other reference bacterial strains. From these clusters four patterns were defined in accordance with their phenotypic (sialidase production and antibiotic resistance profile) and genotypic (presence of nanI and nanJ genes) characteristics. Our results showed heterogeneity among strains, but they were genotypically similar, and it is suggested further studies are needed to better understand the pathogenesis of necrotic enteritis.

Objectives: Primary teeth work as guides for the eruption of permanent dentition, contribute for the development of the jaws, chewing process, preparing food for digestion, and nutrient assimilation. Treatment of pulp necrosis in primary teeth is complex due to anatomical and physiological characteristics and high number of bacterial species present in endodontic infections. The bacterial presence alone or in association in necrotic pulp and fistula samples from primary teeth of boys and girls was evaluated., Material and Methods: Necrotic pulp (103) and fistula (7) samples from deciduous teeth with deep caries of 110 children were evaluated. Bacterial morphotypes and species from all clinical samples were determined., Results: A predominance of gram-positive cocci (81.8%) and gram-negative coccobacilli (49.1%) was observed. In 88 out of 103 pulp samples, a high prevalence of Enterococcus spp. (50%), Porphyromonas gingivalis (49%), Fusobacterium nucleatum (25%) and Prevotella nigrescens (11.4%) was observed. Porphyromonas gingivalis was detected in three out of seven fistula samples, Enterococcus spp. in two out of seven samples, and F. nucleatum, P. nigrescens and D. pneumosintes in one out of seven samples., Conclusions: Our results show that Enterococcus spp. and P. gingivalis were prevalent in necrotic pulp from deciduous teeth in boys from 2 to 5 years old, and that care of the oral cavity of children up to five years of age is important.

The presence of gene 16S rRNA and genes encoding toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA/cdtB) of Clostridium difficile in stool samples from children with (110) and without (150) diarrhea was determined by using a TaqMan system. Fifty-seven (21.9%) out of 260 stool samples harbored the 16S rRNA gene. The genetic profile of tcdA+/tcdB- and cdtA+/cdtB+ was verified in one C. difficile-positive diarrhea sample and of tcdA+/tcdB+ in three C. difficile-positive nondiarrhea samples. The presence of tcdA+/tcdB+ in stools obtained from children without diarrhea, suggests that they were asymptomatic carriers of toxigenic strains.

Neuraminidase was produced by 32.1% and 28.5% of Porphyromonas from dogs with and without periodontitis, respectively; and by 31.8% of bacteria from humans. The presence of neuraminidase in Porphyromonas spp. suggests that this enzyme can be involved with the pathogenesis of the periodontal disease, and the use of this assay to detect the neuraminidase production in oral Porphyromonas species is suggested., (© 2013.)

Aggregatibacter actinomycetemcomitans is strongly implicated in the pathogenesis of periodontitis. In this study, the phenotypic and genotypic features of A. actinomycetemcomitans and the presence of genes involved in toxicity were determined. Sixty-five patients with periodontal pocket and 48 healthy subjects were evaluated. Biotyping, adherence and invasion, neuraminidase and biofilm production, presence of capsule and fimbria, as well as the presence of flp-1, apaH, ltx, and cdt genes were determined. Biotype II was the most prevalent. Sixty-six strains were adherent and 33 of them were able to invade KB cells. Sixty strains produced neuraminidase, and 55 strains biofilms. Strains showed capsule but not fimbriae. Forty-six strains were cytotoxic, and most strains harbored the apaH and flp-1 genes. LTX promoter and the ltxA gene were observed in all strains from periodontal patients. The cdtA gene was observed in 50 (71.4%) strains, cdtB in 48 (68.6%) strains, cdtC in 60 (85.7%), and cdtABC in 40 (57.1%) strains. The presence of A. actinomycetemcomitans harboring the cdtC gene from healthy subjects may represent a transitory microorganism in the oral microbiota. More studies are necessary to understand the real role of this microorganism in the pathogenesis of periodontal disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The occurrence of Porphyromonas gulae, Porphyromonas macacae, Fusobacterium nucleatum and Fusobacterium canifelinum in subgingival plaque from dogs with and without periodontitis as well as their antimicrobial susceptibility were evaluated. From 50 dogs with periodontitis were identified 38 P. gulae, 8 P. macacae, 26 F. nucleatum and 15 F. canifelinum, and from 50 dogs without periodontitis were identified 15 P. gulae, 12 F. nucleatum and 11 F. canifelinum. All strains were susceptible to most of the antibiotics tested, however, different resistance rates to clarithromycin, erythromycin and metronidazole among strains were observed. The role of P. gulae, P. macacae, F. nucleatum and F. canifelinum in periodontal disease of household pets needs to be defined to a better prevention and treatment of the canine periodontitis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A rapid PCR approach was developed to detect Porphyromonas gulae strains from subgingival samples of dogs with and with periodontitis. The presence of P. gulae was observed in 92% and 56%, respectively, in dogs with and without periodontitis. The new primer pair was specific to detect this microorganism, and this technique could be used to evaluate a correlation between periodontitis and P. gulae in companion animals., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: This study examined the antimicrobial resistance profile and the prevalence of resistance genes in Bacteroides spp. and Parabacteroides distasonis strains isolated from children's intestinal microbiota., Methods: The susceptibility of these bacteria to 10 antimicrobials was determined using an agar dilution method. β-lactamase activity was assessed by hydrolysis of the chromogenic cephalosporin of 114 Bacteriodales strains isolated from the fecal samples of 39 children, and the presence of resistance genes was tested using a PCR assay., Results: All strains were susceptible to imipenem and metronidazole. The following resistance rates were observed: amoxicillin (93%), amoxicillin/clavulanic acid (47.3%), ampicillin (96.4%), cephalexin (99%), cefoxitin (23%), penicillin (99%), clindamycin (34.2%) and tetracycline (53.5%). P-lactamase production was verified in 92% of the evaluated strains. The presence of the cfiA, cepA, ermF, tetQ and nim genes was observed in 62.3%, 76.3%, 27%, 79.8% and 7.8% of the strains, respectively., Conclusions: Our results indicate an increase in the resistance to several antibiotics in intestinal Bacteroides spp. and Parabacteroides distasonis and demonstrate that these microorganisms harbor antimicrobial resistance genes that may be transferred to other susceptible intestinal strains.

In this study the presence of periodontopathic pathogens in atheromatous plaques removed from coronary arteries of patients with chronic periodontitis and periodontally healthy subjects by PCR was detected. Our results indicate a significant association between the presence of Porphyromonas gingivalis and atheromas, and the periodontal bacteria in oral biofilm may find a way to reach arteries., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18%) of the periodontal patients and one (2%) healthy subject. However, by PCR, this organism was detected in 44% of the periodontal patients and in 16% of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases.

The purpose of this study was to determine the prevalence of enteric bacteria and yeasts in biofilm of 80 HIV-positive patients with plaque-associated gingivitis or necrotizing periodontitis. Patients were subjected to extra, intra oral and radiographic examinations. The oral hygiene, bleeding on probing, gingival conditions, and attachment loss were evaluated. Clinical specimens were collected from gingival crevices or periodontal pockets, transferred to VMGA III, diluted and transferred to Sabouraud Dextrose agar with 100 μg/ml of chloramphenicol, peptone water, EVA broth, EMB agar, SS agar, Bile esculin agar and Brilliant green agar. Isolation of yeasts was carried out at room temperature, for 3-7 days; and for the isolation of enteric microorganisms plates were incubated at 37°C, for 24-48 h. The yeasts identification was performed according to the carbon and nitrogen assimilation, fermentation of carbohydrates and germ tube formation. Bacteria were identified according to their colonial and cellular morphologies and biochemical tests. Yeasts were identified as Candida albicans and its occurrence was more common in patients with CD4+ below 200/mm(3) and was affected by the extension of periodontal involvement (P = 0.0345). Enteric bacteria recovered from clinical specimens were identified as Enterobacter sakazakii, Enterobacter cloacae, Serratia liquefaciens, Klebsiella oxytoca and Enterococcus sp. Enterobacteriaceae and enterococci were detected in 32.5% of clinical samples from patients with necrotizing periodontitis. In conclusion, non-oral pathogenic bacteria and C. albicans were more prevalent in periodontal sites of HIV-positive patients with necrotizing periodontitis and chronic gingivitis.

Viral and bacterial associations appear to be implicated in the development of periodontal infections. Little information is available describing the periodontopathic agents in root canals with necrotic pulp. In this study, the occurrence and the combinations among herpes simplex virus type 1 (HSV-1) and Dialister pneumosintes, Tannerella forsythia, and Treponema denticola in patients with chronic periodontitis and necrotic pulp were evaluated. Clinical samples from healthy subjects and patients with periodontal or pulp infections were analyzed using a nested polymerase chain reaction PCR to detect HSV and PCR to detect the 3 periodontal bacteria. The presence of Tannerella forsythia and Treponema denticola was observed in healthy, periodontitis, and necrotic pulp patients. HSV was observed in periodontitis and necrotic pulp patients, and no healthy subject harbored D. pneumosintes or HSV. The occurrence of Tannerella forsythia was not statistically significant in patients with necrotic pulp (P = 0.704). Periodontal bacteria were observed varying from 10.3% to 20.7% in periodontitis and necrotic pulp patients. The presence of Treponema denticola - HSV association was predominant in patients showing necrotic pulp (24.1%); however, HSV alone was observed in one patient with periodontitis and in another patient with necrotic pulp. The presence of double association among bacteria or bacteria - HSV could indicate a role in both periodontitis and necrotic pulp, and Tannerella forsythia - Treponema denticola - HSV and Tannerella forsythia - D. pneumosintes - Treponema denticola - HSV associations might be important in periodontitis.

Bacteroides fragilis is the most common anaerobic bacterium isolated from human intestinal tract infections. Before B. fragilis interacts with the intestinal epithelial cells, it is exposed to bile salts at physiological concentrations of 0.1-1.3%. The aim of this study was to determine how pre-treatment with bile salts affected B. fragilis cells and their interaction with intestinal epithelial cells. B. fragilis NCTC9343 was treated with conjugated bile salts (BSC) or non-conjugated bile salts (BSM). Cellular ultrastructure was assessed by electron microscopy, gene expression was quantified by comparative quantitative real-time RT-PCR. Adhesion to the HT-29 human intestinal cell line and to PVC microtitre plates (biofilm formation) was determined. Exposure to 0.15% BSC or BSM resulted in overproduction of fimbria-like appendages and outer membrane vesicles, and increased expression of genes encoding RND-type efflux pumps and the major outer membrane protein, OmpA. Bile salt-treated bacteria had increased resistance to structurally unrelated antimicrobial agents and showed a significant increase in bacterial co-aggregation, adhesion to intestinal epithelial cells and biofilm formation. These data suggest that bile salts could enhance intestinal colonization by B. fragilis via several mechanisms, and could therefore be significant to host-pathogen interactions.

Bacteroides fragilis is considered an important clinical pathogen and the most common anaerobe isolated from human and animal clinical specimens; enterotoxigenic strains produce diarrhea. The presence of enterotoxigenic (ETBF) and nonenterotoxigenic B. fragilis in stool samples from calves with or without acute diarrhea and the antimicrobial susceptibility of the strains were evaluated. The stool samples were plated onto a selective B. fragilis-bile-esculin agar, and incubated anaerobically (10% CO(2)/90% N(2)), at 37 degrees C, for 72 h. Species of the B. fragilis group were identified by using the API 32-A kit. Enterotoxigenic strains were detected by PCR and the cytotoxic assay. From 54 diarrhea and 54 nondiarrhea stools, 124 and 92 members of the B. fragilis group, respectively, were recovered. Only two ETBF strains were isolated from two different diarrhea samples and the bft gene was detected in both. Moreover, the bft gene was detected in DNA from four different diarrheal stools samples but no ETBF strain was recovered. All the bacteria were susceptible to chloramphenicol, imipenem, moxifloxacin, piperacillin/tazobactam, metronidazole and tigecycline. Most of the isolates from both calves with and without diarrhea were resistant to all metals. Our results are of concern, and suggest the need to increase the surveillance of antibiotic and metal resistance of this microbial group isolated from animal production such as calves.

Enterotoxigenic Bacteroides fragilis (ETBF) strains are associated with diarrhea disease in farm animals and young children. In this study, the bft gene subtyping from ETBF strains recovered from one immunodeficient and two immunocompetent children with diarrhea were determined. Thirteen ETBF strains were isolated and by using a multiplex-PCR their bft subtypes were determined. All 13 ETBF strains harbored the bft-1 subtype and by AP-PCR they were clustered in the same group I. This study shows that ETBF strains can be present in acute diarrhea and that bft-1 subtype is often present in these organisms. However, further studies are needed to evaluate the role of this bft-1 subtype in the pathogenesis of diarrhea.

In this study, a novel, simple and rapid hemagglutination assay by using a peanut lectin to detect a neuraminidase activity in strains of the Bacteroides fragilis group was developed. One hundred and fourteen species of the B. fragilis group isolated from children with and without diarrhea and 15 reference strains were evaluated. Neuraminidase production was determined by using the method above described and its inhibition was observed by using galactose. The neuraminidase production was observed in 54 (84.37%) diarrhea and in 43 (86%) non-diarrhea strains. HA titers were ranged from 2 to 32. This neuraminidase assays based on PNA hemagglutination is highly sensitive, reproducible and could be used as a tool to detect the sialidase activity in anaerobic bacteria, particularly, in species of the B. fragilis group.

Enterotoxigenic Bacteroides fragilis (ETBF) strains produce a metalloprotease toxin (BFT) related to diarrheal disease in animals, young children, and adults. Three different isoforms of the enterotoxin, designated BFT-1, BFT-2, and BFT-3, have been identified and sequenced. In the present study, the pathogenicity of the ETBF strains carrying bft-1 or bft-2 was evaluated. Each toxin gene subtype of ETBF (bft-1 or bft-2) was intragastrically monoassociated to germ-free mice during 10 days and histopathological data from intestines and liver compared with those from mice monoassociated to a non-enterotoxigenic B. fragilis. Histopathological alterations were observed in all groups of animals related to ETBF. These alterations were characterized mainly by ulceration, edema, and inflammatory infiltration in intestine. However, these lesions were slightly more severe in mice monoassociated with bft-2 subtype. No alteration or lesion was observed in animals associated with the non-enterotoxigenic B. fragilis. In conclusion, strains harboring bft-1 or bft-2 gene subtypes were able to induce histopathological alterations in intestine of a gnotobiotic mice model and it could explain the effect produced for the enterotoxin.

The effects of subinhibitory concentrations of clindamycin on the morphological, biochemical and genetic characteristics of species of the Bacteroides fragilis group isolated from children with diarrhea were determined. The minimal inhibitory and subinhibitory concentrations for clindamycin were determined. Minimal inhibitory concentration values ranging from 0.25 to 512 microg mL(-1) were observed. Cultures grown with clindamycin were used to determine the macroscopic morphological characteristics, cellular viability, ultrastructural characteristics and DNA integrity. Clindamycin did not alter colonial morphology, but after 6 h elongated cells were observed. Also, extracellular vesicles and electron-lucent areas inside the cytoplasm were observed. Bacteria treated with clindamycin also showed fragmentation of DNA as determined by electrophoresis. The alterations produced by clindamycin might be indicative of a possible modification of the structures involved in bacterial pathogenesis.

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.

Twenty Bacteroides fragilis group species isolated from children with and without diarrhea were analyzed. Antibiotic susceptibility was performed using an agar dilution method; beta-lactamase production was determined using a nitrocefin method, and plasmids were extracted using a commercial Miniprep System. MIC values ranged from 16 to 256 microg/ml for penicillin, 4-128 microg/ml for amoxicillin/clavulanic acid, 0.25-256 microg/ml for clindamycin, and 16-256 microg/ml for penicillin. beta-Lactamase was detected in all isolates. Only five isolates harbored plasmids varying from 7.8 to 1.8 kb. Loss of 6.4- and 3.8-kb plasmids in B. fragilis C68c was related to antibiotic resistance. Low molecular weight plasmids of 2.8-1.8 kb were stable. PCR amplification of cfiA and cepA genes was observed using total DNA, and the cfiA gene was also amplified from the 6.4-kb plasmid.

Clostridium difficile is an important pathogen associated with outbreaks of pseudomembranous colitis and other intestinal disorders such as diarrhea. In this study, 181 stool samples from children with and without acute diarrhea were analysed. Eighteen children with acute diarrhea were positive to C. ramosum, C. difficile, C. limosum, C. clostridioforme, C. septicum, C. butyricum, C. innocuum and Clostridium sp. Nineteen children without diarrhea harbored C. ramosum, C. septicum, C. barattii, C. butyricum, C. innocuum, C. sphenoides, C. bifermentans, C. clostridioforme and C. paraputrificum. No patient with diarrhea harbored C. barattii, C. bifermentans, C. paraputrificum and C. sphenoides. In addition, ten C. difficile strains were detected in 5 (5.5%) of the children with diarrhea. Also, no children from control group harbored C. difficile, C. limosum and Clostridium sp. Most of the tested strains were resistant to all the used antimicrobial. Nine C. difficile were toxigenic on VERO cells and by multiplex PCR, six strains showed both toxin A and B genes and three strains showed only toxin B gene. In this study, the presence of C. difficile was not significant, and it is suggested the need of more studies to evaluate the role of clostridia or C. difficile play in the childhood diarrhea and these organisms must be looked for routinely and a periodic evaluation of antimicrobial susceptibility should be performed.

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 60 masculine C, for 30 min, and among them, 12 (18.75%) were resistant at 60 masculine C, for 60 min. Also, none strain was resistant at 100 masculine C. 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.

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.

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.