Enteropatógenos detectados em crianças sadias em três comunidades de baixa renda, em Recife, Estado de Pernambuco, Brasil

Amostras de fezes de 646 crianças sadias entre 0-5 anos de idade, residentes em três comunidades com discretas diferenças nas condições econômicas e sanitárias, foram cultivadas oara isolamento de enteropatógenos (Escherichia coli, enteropatogênico clássico e invasor, Shigella e Salmonella) obtendo-se positivação para as bactérias pesquisadas em 82 (12,69%) das crianças. E. coli enteropatogênica clássica foi isolada com maior freqüência (6,04%) seguida por Shigella (4,1%) e Salmonella (2,17%). E. coli invasor só foi isolada em duas ocasiões. Evidencia-se através das análises bacteriológicas, um declínio significativo nos isolamentos das enterobactérias patogênicas, onde as condições econômico-sanitárias eram melhores. O percentual de 12,69% de portadores de enteropatógenos bacterianos, evidencia o papel desempenhado pelos assintomáticos na propagação e manutençãoa dos agentes de processos entéricos.

Circulation flow of enteropathogenia bacteria in institutionalized children. I. An epidemiologic study

The circulation flow and maintenance of enteropathogenic bacteria were studied from May 1982 to april 1983 in a population of institutionalized children and adult staff contacts in the city of Rio de Janeiro, Brazil. Subjects were assigned to three groups; A and B, included, respectively, 105 and 46 children with diarrhea who were admitted in the institution in different periods, and group C with 82 adult contacts. Faecal cultures were positive in 35.2%, 39.1% and 19.7% of subjects of groups A, B and C, respectively. It suggests that the transmission was probably fostered by the environment because of as high as 30% of faecal contamination was found in environmental samples. Higher rate of isolation and elevated antibodies levels pointed out that Escherichia coli (EPEC) was the prevalent agent. Shigella predominated in the serological tests. These findings suggest that the institution itself may play an important role in the epidemiology and transmission of enteric infections in the community.

Frequência de enterobactérias patogênicas em processos diarréicos infantis na cidade do Recife, Pernambuco, Brazil

Foram analisadas 326 amostras de fezes diarréicas provenientes de crianças entre 0 a 5 anos, internadas em dois hospitais de reidratação do Recife, Pernambuco. Foi introduzido o meio de Cary & Blair a 4 -C para transporte das fezes, não havendo diferença no percentual de isolamento quando o material permaneceu no meio de transporte entre 3 a 7 dias. Dos exames, 19,02% estavam positivos para um ou mais dos agentes bacterianos pesquisados, tendo sido encontrados 26 Salmonella de 3 espécies, 21 Escherichia coli enteroinvasiva, 10 Shigella de 3 sorotipos e 1 Yersisnia enterocolitica.

Antibacterial activity of Ocimum gratissimum L. essential oil

The essential oil (EO) of Ocimum gratissimum inhibited Staphylococcus aureus at a concentration of 0.75 mg/ml. The minimal inhibitory concentrations (MICs) for Shigella flexineri, Salmonella enteritidis, Escherichia coli, Klebsiella sp., and Proteus mirabilis were at concentrations ranging from 3 to 12 mg/ml. The endpoint was not reached for Pseudomonas aeruginosa (>=24 mg/ml). The MICs of the reference drugs used in this study were similar to those presented in other reports. The minimum bactericidal concentration of EO was within a twofold dilution of the MIC for this organism. The compound that showed antibacterial activity in the EO of O. gratissimum was identified as eugenol and structural findings were further supported by gas chromatography/mass spectra retention time data. The structure was supported by spectroscopic methods.

Monitoring of the dissemination of Salmonella in the chicken Frankfurt-sausage productionline of a sausage factory in the state of São Paulo, Brazil

Poultry meat and its derivatives are among the foodstuffs considered by environmental health authorities to present the highest risks to the public. A total of 185 samples were collected in five monthly batches, from different processing stages in a sausage plant that uses mechanically-deboned chicken meat (MDCM), and testedfor the presence of Salmonella. Enrichment was carried out in both Kauffman's tetrathionate broth and Rappaport-Vassiliadis broth and isolation on Salmonella-Shigella agar and brilliant-green agar. Live Salmonella bacteria were isolated from six samples of the raw meat and from the emulsion, in batches three, four, and five, but not from any sample in batches one or two. The six isolated strains were all classified as Salmonella Albany, which has not previously been reported in MDCM. Of the two enrichment broths, Rappaport-Vassiliadis gave the better results. The pattern of contamination suggests a probable common source, given that a new supplier was used in the third, fourth, and fifth months. It was also shown that the industrial cooking was effective in preventing Salmonella surviving in the final product.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Antibiotics shaping bacterial genome: deletion of an IS91 flanked virulence determinant upon exposure to suinhibitory antibiotic concentrations

The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin α-hemolysin. An Escherichia coli mutant lacking these proteins overexpresses the toxin α-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly-). Generation of Hly- clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the hha allele (hha::Tn5). Detailed analysis of different Hly- clones evidenced that recombination between partial IS91 sequences that flank the hly operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly- derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the hha ydgT mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS91 flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type E. coli strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly- clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS91 is flanking several virulence determinants in different enteric bacterial pathogenic strains from E. coli and Shigella. The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection.

Characterization of cytoplasmic antiviral signaling pathways

Summary Multicellular organisms have evolved the immune system to protect from pathogen such as viruses, bacteria, fungi or parasites. Detection of invading pathogens by the host innate immune system is crucial for mounting protective responses and depends on the recognition of microbial components by specific receptors. The results presented in this manuscript focus on the signaling pathways involved in the detection of viral infection by the sensing of viral nucleic acids. First, we describe a new regulatory mechanism controlling RNA-sensing antiviral pathways. Our results indicate that TRIF and Cardif, the crucial adaptor proteins for endosomal and cytoplasmic RNA detection signaling pathway, are processed and inactivated by caspases. The second aspect investigated here involves a signaling pathway triggered upon cytosolic DNA sensing. The interferon inducible protein DAI was recently described as a DNA sensor able to induce the activation of IRFs and NF-κΒ transcription factors leading to type I interferon production. Here we identify two RIP homotypic interaction motifs (RHIMs) in DAI and demonstrate that they mediate the recruitment of RIP1 and RIP3 and the subsequent NF-κΒ activation. Moreover, we observed that the mouse cytomegalovirus RHIM- containing protein M45 has the potential to block this signaling cascade by interfering with the formation of the DAI-RIP1/3 signaling complex. Finally, we report the generation and the initial characterization of NLRX1-deficient mice. NLRX1 is a member of the NOD-like receptor family localized to the mitochondria. The function of NLRX1 is still controversial: one study proposed that NLRX1 acts as an inhibitor of the RIG-like receptor (RLR) antiviral pathway by binding the adaptor protein Cardif, whereas another report implicated NLRX1 in the generation of reactive oxygen species (ROS) and the amplification of NF-κΒ and JNK triggered by TNF-α, poly(I:C) or Shigella infection. Collectively, our results indicate that NLRX1-deficiency does not affect RLR signaling nor TNF-α induced responses. Proteomics analysis identified UQCRC2, a subunit of the complex III of the mitochondrial respiratory chain, as a NLRX1 binding partner. This observation might reveal a possible functional link between NLRX1 and mitochondrial respiration and/or ROS generation. Résumé Au cours de l'évolution, les organismes multicellulaires ont développé le système immunitaire afin de se protéger contre les pathogènes. Une étape cruciale pour le déclenchement des réponses protectrices est la reconnaissance par les cellules du système immunitaire de molécules propres aux microbes grâce à des récepteurs spécifiques. Les résultats présentés dans cette thèse décrivent des nouveaux aspects concernant les voies de signalisation impliquées dans la détection des virus. Le premier projet décrit un mécanisme de régulation des voies activées par la détection d'ARN virale. Nos résultats montrent que TRIF et Cardif, des protéines adaptatrices des voies déclenchées par la reconnaissance de ces acides nucléiques au niveau des endosomes et du cytoplasme, sont clivés et inactivés par les caspases. Le projet suivant de notre recherche concerne une voie de signalisation activée par la détection d'ADN au niveau du cytoplasme. La protéine DAI a été récemment décrite comme un senseur pour cet ADN capable d'activer les facteurs de transcription IRF et NF-κΒ et d'induire ainsi la production des interférons de type I. Ici on démontre que DAI interagit avec RIP1 et RIP3 par le biais de domaines appelés RHIM et que ce complexe est responsable de l'activation de NF-κΒ. On a aussi identifié une protéine du cytomégalovirus de la souris, M45, qui contient ce même domaine et on a pu démontrer qu'elle a la capacité d'interférer avec la formation du complexe entre DAI et RIP1/RIP3 bloquant ainsi l'activation de NF-κΒ. Enfin on décrit ici la génération de souris déficientes pour le gène qui code pour la protéine NLRX1. Cette protéine fait partie de la famille des récepteurs NOD et est localisée dans la mitochondrie. Une étude a suggéré que NLRX1 agit comme un inhibiteur des voies antivirales activées par les récepteurs du type RIG-I (RLR) en interagissant avec la protéine adaptatrice Cardif. Une autre étude propose par contre que NLRX1 participe à la production des dérivés réactifs de l'oxygène et contribue ainsi à augmenter l'activation de NF- κΒ et JNK induite par le TNF-α ou le poly(I:C). Nos résultats montrent que l'absence de NLRX1 ne modifie ni la voie de signalisation RLR ni les réponses induites par le TNF-α. Des analyses ultérieures ont permis d'identifier comme partenaire d'interaction de NLRX1 la protéine UQCRC2, une des sous-unités qui composent le complexe III de la chaîne respiratoire mitochondriale. Cette observation pourrait indiquer un lien fonctionnel entre NLRX1 et la respiration mitochondriale ou la production des dérivés réactifs de l'oxygène au niveau de cette organelle.

The Plesiomonas shigelloides wbO1 gene cluster and the role of O1-antigen LPS in pathogenicity

The Plesiomonas shigelloides 302-73 strain (serotype O1) wb gene cluster encodes 15 proteins which are consistent with the chemical structure of the O1-antigen lypopolysaccharide (LPS) previously described for this strain. The P. shigelloides O1-antigen LPS export uses the Wzy-dependent pathway as correspond to heteropolysaccharides structures. By the isolation of two mutants lacking this O1-antigen LPS, we could establish that the presence of the O1-antigen LPS is crucial for to survive in serum mainly to become resistant to complement. Also, it is an important factor in the bacterial adhesion and invasion to some eukaryotic cells, and in the ability to form biofilms. This is the first report on the genetics from a P. shigelloides O-antigen LPS cluster (wb) not shared by Shigella like P. shigelloides O17, the only one reported until now.

TiO2 photocatalytic inactivation under simulated solar light of bacterial consortia in domestic wastewaters previously treated by UASB, duckweed and facultative ponds

In this work, TiO2 photocatalysis was used to disinfect domestic wastewaters previously treated by different biological treatment systems: Upward-flow Anaerobic Sludge Blanket (UASB), facultative pond, and duckweed pond. The microorganisms monitored were E. coli, total coliforms, Shigella species, and Salmonella species. Photocatalytic experiments were carried out using two light sources: a solar simulator (UV intensity: 68-70 W m-2) and black-light lamps (BLL UV intensity: 17-20 W m-2). Samples were taken after each treatment stage. Results indicate that bacterial photocatalytic inactivation is affected by characteristics of the effluent, including turbidity, concentration of organic matter, and bacterial concentration, which depend of the type of biological pretreatment previously used.

Anacardic acid derivatives from Brazilian propolis and their antibacterial activity

Propolis is a sticky, gummy, resinous substance collected by honeybees (Apis mellifera L.) from various plant sources, which has excellent medicinal properties. This paper describes the isolation and identification of triterpenoids and anacardic acid derivatives from Brazilian propolis and their antibacterial activity. Their structures were elucidated by ¹H and 13C NMR, including uni- and bidimensional techniques; in addition, comparisons were made with data from academic literature. These compounds were identified as: cardanols (1a + 1b), cardols (2a + 2b), monoene anacardic acid (3), a-amirine (4), b-amirine (5), cycloartenol (6), 24-methylene-cycloartenol (7) and lupeol (8). The determination of the position of the double bond after a reaction with Dimethyl disulfide (DMDS) is described for the phenol derivatives. The ethanolic extract was tested in vitro for antimicrobial activity by using the disc diffusion method and it showed significant results against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Shigella spp.

Enteropatógenos bacterianos em peixes criados em uma estação de reciclagem de nutrientes e no ecossistema relacionado

Avaliou-se a presença de enteropatógenos bacterianos em 72 amostras obtidas a partir de peixes criados em sistema de reciclagem de nutrientes, em estação experimental, no município de Petrópolis, RJ. Paralelamente, foram obtidas amostras do lodo utilizado como adubo orgânico e da cama de aves localizada na área interna dos tanques criatórios. A metodologia empregada incluiu o pré-enriquecimento em Caldo Lactosado e Água Peptonada Tamponada, seguido de enriquecimento em Água Peptonada Alcalina (pH 8,4-8,6) e subseqüente semeadura em Agar GSP para o isolamento de Aeromonas spp. e Plesiomonas shigelloides. Para os demais microrganismos, alíquotas de 1ml foram inoculadas nos meios de enriquecimento Caldo Rappaport-Vassiliadis e Caldo Tetrationato de Kauffmann com posterior semeadura em Agar Entérico Hektoen e Agar Salmonella-Shigella. Com a finalidade de monitorar o índice de coliformes fecais, visando conhecer a qualidade da água para este sistema, paralelamente à coleta de peixes foram avaliadas amostras de água dos tanques criatórios e de macrófitas. No cômputo geral foram isoladas 116 cepas de enteropatógenos bacterianos, destacando-se Aeromonas spp (67,2%) com 9 espécies (A. veronii biogrupo sobria, A. hydrophila, A. sobria, A. trota, A.eucrenophila, A. veronii biog. veronii, A. media, A. caviae e A jandaei) e Aeromonas spp., seguido de Edwardsiella tarda (16,4%), Plesiomonas shigelloides (12,9%) e Salmonella (3,4%). A análise da qualidade da água empregada no sistema revelou, de um modo geral, índices mais elevados de coliformes fecais nos tanques dos peixes (>1800/100 ml).

Microbiota cérvico-vaginal de ovelhas mestiças e sua susceptibilidade aos antibióticos

A criação de ovinos tem se desenvolvido nas últimas décadas, entretanto ainda são escassas informações sobre a composição e potencial patogênico da microbiota cérvico-vaginal de ovelhas. O presente estudo teve como objetivo conhecer os microrganismos constituintes da microbiota cérvico-vaginal de ovelhas, bem como sua susceptibilidade aos antimicrobianos. Foram realizadas coletas em 60 animais sadios, pertencentes a rebanhos de Petrolina e região. Foi realizado o isolamento bacteriano em ágar sangue e ágar MacConkey, sendo os microrganismos identificados de acordo com características morfológicas, tintoriais e bioquímicas. As amostras foram submetidas ao teste de difusão em disco para determinar o perfil de sensibilidade aos antimicrobianos: sulfametazina, enrofloxacina, doxiciclina, tetraciclina, penicilina, amoxicilina, cefalotina e lincomicina. Foram obtidos 94 isolados, sendo constatada uma maior frequência de Staphylococcus spp. (32,97%), Escherichia coli e Micrococcus spp., sendo observado ainda, isolados de Acinetobacter spp., Shigella spp., Enterobacter spp., Klebsiella spp. e Streptococcus spp. Os isolados apresentaram alta sensibilidade aos antimicrobianos testados sendo observado o menor percentual de sensibilidade para lincomicina. A presença de microrganismos oportunistas de potencial patogênico, na microbiota, como Staphylococcus spp e Escherichia coli, remete a uma análise criteriosa em relação ao diagnóstico de infecções genitais. Os isolados bacterianos obtidos neste estudo são sensíveis à maioria dos grupos de drogas antimicrobianas testadas, demonstrando o potencial de utilização desses princípios ativos, além da disponibilidade de escolha, visto a ausência de multirresistência.

Etiology of diarrheal infections in children of Porto Velho (Rondonia, Western Amazon region, Brazil)

In the present study, 470 children less than 72 months of age and presenting acute diarrhea were examined to identify associated enteropathogenic agents. Viruses were the pathogens most frequently found in stools of infants with diarrhea, including 111 cases of rotavirus (23.6% of the total diarrhea cases) and 30 cases of adenovirus (6.3%). The second group was diarrheogenic Escherichia coli (86 cases, 18.2%), followed by Salmonella sp (44 cases, 9.3%) and Shigella sp (24 cases, 5.1%). Using the PCR technique to differentiate the pathogenic categories of E. coli, it was possible to identify 29 cases (6.1%) of enteropathogenic E. coli (EPEC). Of these, 10 (2.1%) were typical EPEC and 19 (4.0%) atypical EPEC. In addition, there were 26 cases (5.5%) of enteroaggregative E. coli, 21 cases (4.4%) of enterotoxigenic E. coli, 7 cases (1.4%) of enteroinvasive E. coli (EIEC), and 3 cases (0.6%) of enterohemorrhagic E. coli. When comparing the frequencies of diarrheogenic E. coli, EPEC was the only category for which significant differences were found between diarrhea and control groups. A low frequency of EIEC was found, thus EIEC cannot be considered to be a potential etiology agent of diarrhea. Simultaneous infections with two pathogens were found in 39 diarrhea cases but not in controls, suggesting associations among potential enteropathogens in the etiology of diarrhea. The frequent association of diarrheogenic E. coli strains was significantly higher than the probability of their random association, suggesting the presence of facilitating factor(s).