Avaliação in vitro da atividade antimicrobiana de extratos vegetais sobre microrganismos relacionados à lesão de mucosite oral

A mucosite oral é a complicação oral mais freqüente nos pacientes sob quimioterapia e/ou radioterapia. Vários microrganismos podem estar presentes nesta lesão o que dificulta o seu tratamento. A propriedade antimicrobiana de plantas tem sido estudada com o intuito de confirmar cientificamente sua ação, e o possível potencial no controle de doenças infecciosas, principalmente devido ao aumento de microrganismos resistentes aos antimicrobianos conhecidos. O estudo teve por objetivo observar a ação inibidora de extratos das plantas Arrabidaea chica, Bryophyllum calycinum, Mansoa alliacea, Azadirachta indica, Senna alata, Vatairea guianensis, Vismia guianensis, Ananas erectifolius, Psidium guajava, Euterpe oleracea e Symphonia globulifera sobre cepas de microrganismos frequentemente envolvidos em lesões de mucosite oral, tais como, Streptococcus mitis (ATCC 903), Streptococcus sanguis (ATCC 10557), Streptococcus mutans (ATCC 25175), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 9027), Candida albicans (ATCC 40175), Candida krusei (ATCC 40147) e Candida parapsilosis (ATCC 40038). A avaliação da atividade antimicrobiana e a determinação da Concentração Inibitória Mínima (CIM) foram realizadas através do método de disco-difusão em meio sólido. Os extratos brutos das plantas foram testados nas concentrações de 500, 250, 125, 62,50, 31,25 e 15,62 mg/ml utilizando como solvente o Dimetil-Sulfóxido (DMSO). Os extratos de anani e de pirarucu foram os que apresentaram maior espectro de ação, inibindo o crescimento de sete microrganismos dentre os oito testados. As menores CIM foram obtidas com os extratos de anani, lacre e mata pasto. O extrato de anani foi o mais ativo tendo demonstrado boa atividade antimicrobiana (CIM abaixo de 100 mg/mL) contra sete microrganismos (S. aureus, C. albicans, C. krusei, C. parapsilosis, S. mitis. S. sanguis e S. mutans, sendo inativo apenas para P. aeruginosa). O extrato de lacre demonstrou boa atividade frente a cinco microrganismos. Mata pasto teve boa atividade contra S. aureus, S. mitis e C. albicans. P. aeruginosa foi o microrganismo mais resistente sendo suscetível apenas para os extratos de pariri e pirarucu. Dentre os extratos avaliados, apenas o curauá não apresentou atividade sobre nenhum dos microrganismos testados. Os resultados obtidos demonstraram a capacidade antimicrobiana dos produtos vegetais testados. Entretanto, estudos futuros são necessários para esclarecer os seus mecanismos de ações e as possíveis interações com as drogas antimicrobianas, visando seu aproveitamento na terapêutica de doenças infecciosas.

Avaliação da atividade antimicrobiana de extratos de própolis em diferente maturação

A descoberta e síntese de antimicrobianos compõem um elemento de suma importância para a saúde, entretanto algumas dessas substâncias tornaram-se obsoletas devido ao surgimento de micro-organismos resistentes à terapêutica convencional. Dentro das formas de tratamento, os produtos naturais são uma fonte inesgotável de substâncias, entre elas a própolis, a qual é conhecida mundialmente devido a sua atividade antimicrobiana. O objetivo deste trabalho foi a análise microbiológica de seis amostras de própolis, coletadas de regiões de Prudentópolis- PR, em tempos distintos de depósito em colmeia, uma com até 40 dias de depósito (Própolis Nova) e outra com mais de 180 dias (Própolis Velha). A atividade antimicrobiana foi verificada frente a Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Micrococcus luteus e Enterococcus faecalis, através do ensaio de microdiluição em placa com teste colorimétrico usando resazurina. Ao fim dos testes obteve-se os valores variando entre 0,38 a 0,68 mg/ml para a CIM da própolis nova e 0,34 a 1,3 mg/ml para a velha, para os micro-organismos S. aureus e M. luteus tendo valores próximos para ambos assim como a CBM destes, entre 0,38 - 1,62 e 0,67 – 2,6 mg/ml, respectivamente. Da mesma forma, para o E. faecalis foram alcançados os valores de CIM entre 0,76 e 2,73 mg/ml para a própolis nova e 1,34 e 2,72 mg/ml para a própolis velha, bem como valores de CBM de 1,5 a 3,07 e 2,68 a 3,11 mg/ml, respectivamente, nas amostras 1V, 2N, 5V e 5N não se verificou CBM para as concentrações estudadas. Os micro-organismos gram-negativos não foram sensíveis ao própolis. Conclui-se que a própolis nova apresentou melhor ação antimicrobiana, principalmente contra S. aureus e M. luteus. No entanto, os dados também mostram que os valores de CIM e CBM foram bem próximos entre as diferentes própolis, fato que não evidencia razões para que a própolis velha seja descartada.

Ação antibacteriana de associações de antimicrobianos: nisina, óleos essenciais e compostos majotitários

Pós-graduação em Biologia Geral e Aplicada - IBB

Diversity of Staphylococcus species and prevalence of enterotoxin genes isolated from milk of healthy cows and cows with subclinical mastitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Staphylococcus sp na produção primária de leite: aspectos de saúde pública

Staphylococcus is one of the more important causes of the called Foodborne Disease(FD), being that from the 40 species described from genus, the more important is Staphylococcus aureus. During years believed that the S. aureus was the only specie from genus able to produce enterotoxins, responsable for the clinical frame in humans, but latest studies report the isolation of other species both positive coagulase (PC) as negative with enterotoxigenic potential. The symptoms of this intoxication appear after a short period of incubation (2-6 hours) and usually characterized by nausea, vomits, abdominal ache, diarrhea, and rarely is fatal. For the toxin to be formed in food is necessary that bacteria population to be at least 105 UFC/g, being that such toxins characterized by presenting great resistance front of gastrointestinal proteases and of homemade termical treatment. Among the main foods that might carry the microorganism, the milk and its derivatives have highlights. The contamination of the product might happen as from the milk from cows with clinical and/or subclinical mastitis, as the Staphylococcus genus is one of the main agents etiologic from this disease, equipments utensils badly sanitized equipments and utensils and from the manipulators. The control of these factors configures as fundamental condition for the achievement of a safe, quality product, which doesn’t offer risk to the consumers

Rapid turbidimetric assay to potency evaluation of tigecycline in lyophilized powder

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Antimicrobial and antileishmanial activities of diterpenoids isolated from the roots of salvia deserta

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Synergism of crude extracts and essential oils from medicinal plants with antimicrobial drugs

The emergence of resistant strains to conventional antimicrobial drugs has been constant as well as research aimed new alternatives of antibacterial agents. Therefore, considering that natural products have been an important potential source of new antimicrobial drugs, aim to verify the synergism by disk and time kill curve method between antimicrobials (extracts-Ext. and essential oils-EO) from four plant and eight antimicrobial drugs against Staphylococcus aureus and Escherichia coli strains from human specimens. The S. aures strains were highly susceptible with all plant antimicrobials (eg., 1.24 mg/ml with Vernonia polyanthes Ext. and 2.21 mg/ml with Eugenia uniflora EO for the Minimal Inhibitory Concentration-MIC). According disk method, the Bacharis dracunculifolia and V. polyanthes EO had synergism with all eight tested drugs while only Matricaria chamomilla Ext. showed synergism against S. aureus. The synergism was found with V. polyanthes and E. uniflora Ext. while M. chamomilla Ext. had antagonism against E. coli strains. By time kill curve, the bacterial growth inhibition was superior when drugs were tested alone and the synergism effect also was verified. The antagonism effect was detected only for E. coli strains and only with Ext. Results indicated the potential use of these products as coadjutants during treatment of infectious diseases.

Avaliação in vitro da eficácia de extratos de plantas medicinais do Cerrado frente Staphylococcus aureos isolado de difetentes fontes de propriedades leiteiras

The objective of this study is to evaluate the potencial microbial activity in-vitro from the extract of some endemic plants from Cerrado such as Baccharis dracunculifolia, Cochlospermum regium, Croton antisyphiliticus, Eugenia dysenterica and Lippia sidoides, against the agent Staphylococcus aureus isolated from bovine mastitic milk, osteo from cow’s teat, milker equipament, nasal cavitites and milker’s gullet. The extracts were prepared from aerial parts as well as the reticular systems of plants using the solvents methanol, hexane and chloroform at a concentration of 10%. To evaluate the antimicrobial activity, the technique of microdilution in broth was used for determining the Minimal Inibitory Concentration (MIC) followed by the determination of Minimal Bactericidal Concentration (MBC). The extracts from Baccharis dracunculifolia and Croton antisyphiliticus, followed by extracts from Lippia sidoides, reported respectively, presented better inhibitory activity against the multiplication of the bacteria Staphylococcus aureus. Furthermore, the results demonstrate that the isolated strains from the milk and nasal cavities of the milker showed strong resistance against gentamicin, active agent commonly applied to combat mastitis bovine. However, there was sensitivity against extracts from the reported plants, reinforcing the importance of the medicinal plants as a therapeutic resource and its aplicability.

Genetic transformation of Diaporthe phaseolorum, an endophytic fungus found in mangrove forests, mediated by Agrobacterium tumefaciens

We describe the genetic transformation of the mycelial tissue of Diaporthe phaseolorum, an endophytic fungus isolated from the mangrove species Laguncularia racemosa, using Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT uses both the hygromycin B resistant (hph) gene and green fluorescent protein as the selection agents. The T-DNA integration into the fungal genome was assessed by both PCR and Southern blotting. All transformants examined were mitotically stable. An analysis of the T-DNA flanking sequences by thermal asymmetric interlaced PCR (TAIL-PCR) demonstrated that the disrupted genes in the transformants had similarities with conserved domains in proteins involved in antibiotic biosynthesis pathways. A library of 520 transformants was generated, and 31 of these transformants had no antibiotic activity against Staphylococcus aureus, an important human pathogen. The protocol described here, using ATMT in D. phaseolorum, will be useful for the identification and analysis of fungal genes controlling pathogenicity and antibiotic pathways. Moreover, this protocol may be used as a reference for other species in the Diaporthe genus. This is the first report to describe Agrobacterium-mediated transformation of D. phaseolorum as a tool for insertional mutagenesis.

Up-regulation of chemokine C-C ligand 2 (CCL2) and C-X-C chemokine 8 (CXCL8) expression by monocytes in chronic idiopathic urticaria

The disturbed cytokinechemokine network could play an important role in the onset of diseases with inflammatory processes such as chronic idiopathic urticaria (CIU). Our main objectives were to evaluate the relation between proinflammatory chemokine serum levels from CIU patients and their response to autologous skin test (ASST) and basophil histamine release (BHR). We also aimed to assess the chemokine secretion by peripheral blood mononuclear cells (PBMC) upon polyclonal stimulus and to evaluate chemokine CC ligand 2/C-X-C chemokine 8 (CCL2/CXCL8) and Toll-like receptor-4 (TLR-4) expression in monocytes. We observed significantly higher serum levels of the CXCL8, CXCL9, CXCL10 and CCL2 in CIU patients compared to the healthy group, regardless of the BHR or ASST response. The basal secretion of CCL2 by PBMC or induced by Staphylococcus aureus enterotoxin A (SEA) was higher in CIU patients than in the control group, as well as for CXCL8 and CCL5 secretions upon phytohaemagglutinin stimulation. Also, up-regulation of CCL2 and CXCL8 mRNA expression was found in monocytes of patients upon SEA stimulation. The findings showed a high responsiveness of monocytes through CCL2/CXCL8 expression, contributing to the creation of a proinflammatory environment in CIU.

Signal propagation in Bayesian networks and its relationship with intrinsically multivariate predictive variables

A set of predictor variables is said to be intrinsically multivariate predictive (IMP) for a target variable if all properly contained subsets of the predictor set are poor predictors of the. target but the full set predicts the target with great accuracy. In a previous article, the main properties of IMP Boolean variables have been analytically described, including the introduction of the IMP score, a metric based on the coefficient of determination (CoD) as a measure of predictiveness with respect to the target variable. It was shown that the IMP score depends on four main properties: logic of connection, predictive power, covariance between predictors and marginal predictor probabilities (biases). This paper extends that work to a broader context, in an attempt to characterize properties of discrete Bayesian networks that contribute to the presence of variables (network nodes) with high IMP scores. We have found that there is a relationship between the IMP score of a node and its territory size, i.e., its position along a pathway with one source: nodes far from the source display larger IMP scores than those closer to the source, and longer pathways display larger maximum IMP scores. This appears to be a consequence of the fact that nodes with small territory have larger probability of having highly covariate predictors, which leads to smaller IMP scores. In addition, a larger number of XOR and NXOR predictive logic relationships has positive influence over the maximum IMP score found in the pathway. This work presents analytical results based on a simple structure network and an analysis involving random networks constructed by computational simulations. Finally, results from a real Bayesian network application are provided. (C) 2012 Elsevier Inc. All rights reserved.

Patologia ed epidemiologia molecolare delle infezioni associate all'impianto

Staphylococcus aureus and Staphylococcus epidermidis are leading pathogens of implant-related infections. This study aimed at investigating the diverse distribution of different bacterial pathogen factors in most prevalent S. aureus and S. epidermidis strain types causing orthopaedic implant infections. In this study the presence both of the ica genes, encoding for biofilm exopolysaccharide production, and the insertion sequence IS256, a mobile element frequently associated to transposons, was investigated in relationship with the prevalence of antibiotic resistance among Staphylococcus epidermidis strains. The investigation was conducted on 70 clinical isolates derived from orthopaedic implant infections. Among the clinical isolates investigated a dramatic high level of association was found between the presence of ica genes as well as of IS256 and multiple resistance to all the antibiotics tested. Noteworthy, a striking full association between the presence of IS256 and resistance to gentamicin was found, being none of the IS256-negative strain resistant to this antibiotic. This association is probably because of the link of the corresponding aminoglycoside-resistance genes, and IS256, often co-existing within the same staphylococcal transposon. Moreover we investigated the prevalence of aac(6’)-Ie-aph(2’’), aph (3’) IIIa, and ant(4’) genes, encoding for the three forms of aminoglycoside-modifying enzymes (AME), responsible for resistance to aminoglycoside antibiotics. All isolates were characterized by automated ribotyping, so that the presence of antibiotic resistance determinants was investigated in strains exhibiting different ribopatterns. Interestingly, combinations of coexisting AME genes appeared to be typical of specific ribopatterns. 200 S. aureus isolates, categorized into ribogroups by automated ribotyping, i.e. rDNA restriction fragment length polymorphism analysis, were screened for the presence of a panel of adhesins genes, accessory gene regulatory (agr) polymorphisms and toxins. For many ribogroups, characteristic tandem genes arrangements could be identified. Surprisingly, the isolates of the most prevalent cluster, enlisting 27 isolates, were susceptible to almost all antibiotics and never possessed the lukD/lukE gene, thus suggesting the role of factors other than antibiotic resistance and the here investigated toxins in driving the major epidemic clone to the larger success. Afterwards, .in the predominant S. aureus cluster, the bbp gene encoding bone sialoprotein-binding protein appeared a typical virulence trait, found in 93% of the isolates. Conversely, the bbp gene was identified in just 10% of the remaining isolates of the collection. In this cluster, co-presence of bbp with the cna gene encoding collagen adhesin was a pattern consistently observed. These findings indicate a crucial role of both these adhesins, able to bind the most abundant bone proteins, in the pathogenesis of orthopaedic implant infections, there where biomaterials interface bone tissues. Moreover a PCR screening for the ebpS gene, conducted on over two hundred S. aureus clinical isolates from implant related infections revealed the detection of six strains exhibiting an altered amplicon size, shorter than expected. In order to elucidate the sequence changes present in these gene variants, the trait comprised between the primers was analyzed in all six isolates bearing the modification and in four isolates exhibiting the regular amplicon size. From nucleotide translation, the corresponding encoded protein was found to lack an entire peptide segment of 60 amino acids. These variants, missing an entire hydrophobic region, could actually facilitate current structural studies, helping to assess whether the absent domain is strictly necessary for a functional adhesin conformation and its contribution to the topology of the protein. This study suggests that epidemic clones appear to pursue different survival strategies, where adhesins, when present, exhibit diverse importance as virulence factors. A practical message arising from the present study is that strategies for the prevention and treatment of implant orthopaedic infections should target adhesins conjointly present in epidemic clones. Furthermore, the choice of reference strains for testing the anti-infective properties of biomaterials should focus on a selection of the most prevalent clones as they exhibit distinct profiles of adhesins.

Staphylococcus hyicus bacteremia in a farmer

Bacteria known in animal infectious diseases can cause challenges in human diagnostic laboratories. We present pitfalls in the identification and susceptibility testing of Staphylococcus hyicus, a pathogen that typically causes exudative epidermitis in pigs. In this case, the coagulase-positive staphylococcus isolated from a septic patient was misidentified as Staphylococcus aureus.

Effect of ozone on periodontopathogenic species--an in vitro study

The in vitro study was aimed to determine the effect of ozone on periodontopathogenic microorganisms. Ozone was generated for 6 s-2 × 24 s (corresponding to 0.56 mg-2 × 2.24 mg of ozone) against 23 mainly anaerobic periodontopathogenic species. Agar diffusion test was used as a screening method. Then, the killing activity was tested in a serum-free environment and with 25% v/v inactivated serum. Further, the effect of ozone on bactericidal activity of native serum was analyzed against Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans. Agar diffusion test showed a high efficacy of ozone against microorganisms, especially against Porphyromonas gingivalis. This result was confirmed by the killing tests; most of the strains in a concentration of 10(5) were completely eliminated after twofold 18-s application of ozone. Only four of the six potentially "superinfecting" species (Staphylococcus aureus, Enterococcus faecalis, Enterobacter cloacae, Candida albicans) survived in part. Addition of heat-inactivated serum reduced the killing rate of ozone by 78% after 6-s and by 47% after twofold 18-s exposures; no strain was completely eradicated after any application of ozone. The bactericidal effect of native serum was enhanced after application of ozone; no effect was visible on the included A. actinomycetemcomitans strain which was found to be completely resistant to the bactericidal action of serum. In conclusion, (a) ozone has a strong antibacterial activity against putative periodontopathogenic microorganisms, and (b) the bactericidal effect is reduced in the presence of serum. Ozone may have potential as an adjunctive application to mechanical treatment in periodontitis patients.