Abstract 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.

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.

Pesticides in “PERA” orange samples (N = 57) from São Paulo City, Brazil were assessed and the pesticide intake contribution was estimated for chronic risk assessment. Seventy-six pesticides were evaluated by the gas chromatography multi-residue method, including isomers and metabolites (4.332 determinations). The mean recoveries at the limit of quantification level were in the range of 72-115% and the relative standard deviation for five replicate samples was 1-11%. The limits of detection and quantification ranged from 0.005 to 0.4 mg.kg−1 and from 0.01 to 0.8 mg.kg−1, respectively. Pesticides were found in 42.1% of the samples at levels ranging from 0.06 to 2.9 mg.kg−1. Of the contaminated samples, 3.5% contained residues (bifenthrin and clofentezine) above the maximum residue level and 12.3% contained unauthorized pesticides (azinphos-ethyl, parathion, myclobutanil, profenofos, and fenitrothion). The estimated risk characterization for orange intake by adults and children, respectively, ranged from 0.04 to 6.6% and from 0.1 to 26.5% of the acceptable daily intake. The detection of irregular residues emphasizes the need for better implementation of Good Agriculture Practices and greater control of formulated products. Other pesticides surveyed did not pose a health risk due to consumption.

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.

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.

Para viabilizar a futura realização de um Inquérito Nacional de Populações Expostas a Substâncias Químicas, executou-se um projeto-piloto para examinar a exequibilidade, testar e adequar metodologias e estabelecer parcerias. Foram realizados três estudos de corte transversal, respectivamente, com doadores de sangue residentes na Região Metropolitana de São Paulo-SP, conscritos do Exército Brasileiro e crianças escolares residentes no município do Rio de Janeiro-RJ, mediante aplicação de questionários e coleta de material biológico (sangue venoso e capilar, unha e cabelo) para análise de metais e compostos organoclorados persistentes. Concluiu-se que é viável a realização de um Inquérito Nacional, embora a estratégia de obtenção das amostras biológicas deva ser definida para cada subgrupo da população. Recomenda-se aproveitar as estruturas de serviços existentes para obtenção de amostras, utilizar matrizes tradicionais para maior comparabilidade e garantir sua realização periódica.

To enable the future establishment of a National Survey of Populations Exposed to Chemical Substances, apilot study was conducted to assess the feasibility, to test and adjust methodologies, and to establish partnerships. Three cross-sectional studies were performed: with blood donors residents in the Metropolitan Region of São Paulo, Brazilian army conscripts and schoolchildren living in the city of Rio de Janeiro, by applying a questionnaire and collecting biological material (capillary and venous blood, nail and hair) for the analysis of metals and persistent organochlorine compounds. We concluded that it is feasible to carry out a National Survey, but the strategy for obtaining the biological samples must be set for each sub-group of the population. Moreover, it is recommended to take advantage of the structures of existing services for obtaining samples to maintain the comparability and ensure their periodicity.

Para hacer viable la futura realización de una Encuesta Nacional de Poblaciones Expuestas a Sustancias Químicas, se ejecutó un proyecto piloto para examinar la posibilidad de ejecutar, probar y adecuar metodologías y establecer colaboraciones. Se realizaron tres estudios de corte transversal, respectivamente, con donantes de sangre residentes en la Región Metropolitana de São Paulo-SP, conscriptos del Ejército Brasileño y niños escolares residentes en el municipio de Rio de Janeiro-RJ, mediante aplicación de cuestionarios y colecta de material biológico (sangre venosa y capilar, uña y pelo) para análisis de metales y compuestos organoclorados persistentes. Se concluye que es viable la realización de una Encuesta Nacional, aunque la estrategia para la obtención de las muestras biológicas deba ser definida para cada subgrupo de la población. Se recomienda aprovechar las estructuras de servicios existentes para obtener las muestras, utilizar matrices tradicionales para una mejor comparación y garantizar su realización periódica.

Para viabilizar a futura realização de um Inquérito Nacional de Populações Expostas a Substâncias Químicas, executou-se um projeto-piloto para examinar a exequibilidade, testar e adequar metodologias e estabelecer parcerias. Foram realizados três estudos de corte transversal, respectivamente, com doadores de sangue residentes na Região Metropolitana de São Paulo-SP, conscritos do Exército Brasileiro e crianças escolares residentes no município do Rio de Janeiro-RJ, mediante aplicação de questionários e coleta de material biológico (sangue venoso e capilar, unha e cabelo) para análise de metais e compostos organoclorados persistentes. Concluiu-se que é viável a realização de um Inquérito Nacional, embora a estratégia de obtenção das amostras biológicas deva ser definida para cada subgrupo da população. Recomenda-se aproveitar as estruturas de serviços existentes para obtenção de amostras, utilizar matrizes tradicionais para maior comparabilidade e garantir sua realização periódica.

To enable the future establishment of a National Survey of Populations Exposed to Chemical Substances, apilot study was conducted to assess the feasibility, to test and adjust methodologies, and to establish partnerships. Three cross-sectional studies were performed: with blood donors residents in the Metropolitan Region of São Paulo, Brazilian army conscripts and schoolchildren living in the city of Rio de Janeiro, by applying a questionnaire and collecting biological material (capillary and venous blood, nail and hair) for the analysis of metals and persistent organochlorine compounds. We concluded that it is feasible to carry out a National Survey, but the strategy for obtaining the biological samples must be set for each sub-group of the population. Moreover, it is recommended to take advantage of the structures of existing services for obtaining samples to maintain the comparability and ensure their periodicity.

Para hacer viable la futura realización de una Encuesta Nacional de Poblaciones Expuestas a Sustancias Químicas, se ejecutó un proyecto piloto para examinar la posibilidad de ejecutar, probar y adecuar metodologías y establecer colaboraciones. Se realizaron tres estudios de corte transversal, respectivamente, con donantes de sangre residentes en la Región Metropolitana de São Paulo-SP, conscriptos del Ejército Brasileño y niños escolares residentes en el municipio de Rio de Janeiro-RJ, mediante aplicación de cuestionarios y colecta de material biológico (sangre venosa y capilar, uña y pelo) para análisis de metales y compuestos organoclorados persistentes. Se concluye que es viable la realización de una Encuesta Nacional, aunque la estrategia para la obtención de las muestras biológicas deba ser definida para cada subgrupo de la población. Se recomienda aprovechar las estructuras de servicios existentes para obtener las muestras, utilizar matrices tradicionales para una mejor comparación y garantizar su realización periódica.

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.

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.

Aggregatibacter actinomycetemcomitans é um importante agente etiológico da periodontite e produz infecções extra-bucais. Neste estudo, foram detectados os biótipos, o gene ltxA associado à produção de leucotoxina e o promotor ltx em A. actinomycetemcomitans de pacientes com e sem periodontite. A atividade leucotóxica sobre células HL-60 também foi avaliada. A atividade leucotóxica foi determinada através do método de exclusão do azul de tripam. A deleção de 530 bp no promotor ltx foi avaliada usando-se o par de iniciadores PRO. A. actinomycetemcomitans foi detectado por cultura e por PCR. Por cultura, A. actinomycetemcomitans foi detectado em nove pacientes com periodontite (18%) e em um indivíduo sadio (2%). Por PCR esse microrganismo foi detectado em 44% dos pacientes com periodontite e em 16% dos saudáveis. Verificou-se diferença estatística entre a detecção do promotor do operon ltx, por PCR, diretamente do biofilme subgengival e do DNA bacteriano. Somente uma amostra clínica apresentou A. actinomycetemcomitans altamente leukotóxico. O biótipo II foi o mais prevalente e não foi observada correlação biótipo-atividade leucotóxica. A expressão da leucotoxina por A. actinomycetemcomitans na doença periodontal e outras doenças infecciosas necessita ser avaliado.

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.

O objetivo desse estudo foi avaliar a ocorrência de bactérias entéricas e leveduras no biofilme subgengival de pacientes HIV-positivos com gengivite crônica ou periodontite necrosante. Os pacientes foram submetidos a exame clínico e radiográfico e de higiene bucal, sangramento à sondagem, condições gengivais e a perda de inserção. Os espécimes clínicos de sulcos gengivais ou bolsas periodontais foram inoculados em ágar Sabouraud dextrose com 100 mg/ml de cloranfenicol, água peptonada, caldo EVA, ágar EMB, ágar SS, ágar Bile esculina e ágar verde brilhante. O cultivo de leveduras foi realizado à temperatura ambiente, de 3-7 dias; das enterobactérias a 37ºC de 24-48 h. A identificação das leveduras foi realizada pela assimilação de carbono e nitrogênio, fermentação de açucares e formação de tubo germinativo. As bactérias de acordo com a morfologia celular e colonial e testes bioquímicos. Foram identificadas Candida albicans e sua prevalência foi maior em pacientes com contagens de CD4+ < 200/mm³, e sua ocorrência foi afetada pela extensão da destruição periodontal (P = 0,0345). Enterobacteriaceae e enterococos foram detectados em 32,5% dos pacientes com periodontite necrosante. As enterobactérias foram Enterobacter sakazakii, E. cloacae, Serratia liquefaciens, Klebsiella oxytoca e Enterococcus sp. Concluiu-se que bactérias patogênicas exógenas à cavidade bucal e C. albicans podem ser detectadas no biofilme subgengival de pacientes HIV-positivos com periodontite necrosante e gengivite.

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 37ºC, 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/mm³ 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.

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.

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.

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.

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.