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Campylobacter species have developed resistance to existing antibiotics. The development of alternative therapies is, therefore, a necessity. This study evaluates the susceptibility of Campylobacter strains to selected natural products (NPs) and frontline antibiotics. Two C. jejuni strains (ATCC&reg, 33560TM and MT947450) and two C. coli strains (ATCC&reg, 33559TM and MT947451) were used. The antimicrobial potential of the NPs, including plant extracts, essential oils, and pure phytochemicals, was evaluated by broth microdilution. The growth was measured by spectrophotometry and iodonitrotetrazolium chloride. Antibiotic resistance genes (tet(O) and gyrA) were characterized at the molecular level. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) ranged from 25 to 1600 &micro, g/mL. Cinnamon oil, (E)-Cinnamaldehyde, clove oil, eugenol, and baicalein had the lowest MIC and MBC values (25&ndash, 100 &micro, g/mL). MT947450 and MT947451 were sensitive to erythromycin and gentamicin but resistant to quinolones and tetracycline. Mutations in gyrA and tet(O) genes from resistant strains were confirmed by sequencing. The findings show that NPs are effective against drug-sensitive and drug-resistant Campylobacter strains. The resistance to antibiotics was confirmed at phenotypic and genotypic levels. This merits further studies to decipher the action mechanisms and synergistic activities of NPs.

In this study the effect of growth medium strength on the minimum inhibitory concentration (MIC) of different tannins and tannin extracts against Escherichia coli was systematically investigated for the first time. Three pure compounds (vescalagin, castalagin and gallic acid) and five extracts (chestnut, quebracho, mimosa, Colistizer and tannic acid) were studied. Broth microdilution was assayed and bacteria were grown using different growth medium strengths varying from half to double the concentration recommended by the producer. MICs were determined using the iodonitrotetrazolium chloride (INT) dye or turbidity measurements. It was observed that MIC values depend on the growth medium strength. With an increase in the growth medium concentration MIC values rose roughly linearly for all samples, while their relative order remained unchanged, indicating that a direct interaction of tannins with growth medium nutrients represents the likely source of their antimicrobial activity. Understanding the effect of growth medium strength can finally yield a plausible explanation for the observed variation in MIC values reported in the scientific literature as well as provide help in planning proper applications of tannins in the livestock production.

Antibiotic resistance in bacterial species is opening new avenues to search for alternative modes of antimicrobial treatment, medicinal plant extracts being one among them. The aim of this study was to access the possibility of medicinal plant extract from Shih in the manufacture of pharmaceutical preparations for oral hygiene specifically for the prevention and treatment of dental caries due to Streptococcus mutans. Antimicrobial effects of crude organic extract of Shih on S. mutans isolated from the saliva were examined by taking S. mutans with culture media only (−ve control); S. mutans treated with the antibiotic gentamicin (+ve control) and S. mutans treated with Shih. Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) were Determination by Iodonitrotetrazolium chloride (INT) colorimetric assay Time-kill dynamic assay was performed using broth microdilution method. The metabolic reason behind the bacteriostatic and bactericidal effect were studied by measuring the glucose utilization by the microbes, pH as a measure of acid production, nucleic acids quantitation to check the DNA status and inhibition of water-insoluble glucan synthesis were undertaken. Shih MIC for S. mutans was at 2.5 mg/ml and MBC was 4 mg/ml. S. mutans bacterial population started reclining within 60 min of incubation with Shih at MBC. Utilization of added glucose was very high at MIC due to bacteria overcoming the stress, whereas at MBC its utilization was less. Accordingly pH also became acidic to 2.9 with MIC and 4.03 with MBC. There was a great degree of inhibition in the formation of nucleic acids indicating this crude extract interferes with DNA replication. Inhibition of glucan synthesis was to the tune of 45% as compared to control. Thus we conclude that Shih has potentially effective antibacterial activity hence it can be proposed as a potentially effective antiplaque and anticariogenic agent in the form of mouth wash or gum paint. However, the cytotoxicity of the extract needs to be evaluated in in-vitro and in-vivo conditions before it is considered as a safe antiplaque and anticariogenic agent. Keywords: Artemisia herba-alba (Shih), Streptococcus mutans, Dental caries, Antimicrobial effect, Metabolic effects

The file attached to this record is the author's final peer reviewed version. Introduction: The antimicrobial activity of many Essential Oils (EOs) is well established, indicating that EOs may be a source of compounds for antimicrobial drug development. Thin Layer Chromatography-Direct Bioautography (TLC-DB) can quickly identify antimicrobial components in complex mixtures and can be applied to the screening of EOs for lead compounds. Objectives: This study aimed to identify antimicrobial components of oregano, rosewood and cumin EOs against antibiotic-sensitive and -resistant bacteria using TLC-DB and a multi-faceted approach of GC-MS, LC-MS and NMR techniques to characterise bioactive compounds. The study also aimed to quantify the antimicrobial activity of bioactive compounds in order to evaluate their potential for the development of therapies against antibiotic-resistant bacteria. Materials and Methods: EOs were eluted on TLC plates and sprayed with a suspension of Staphylococcus aureus, Enterococcus faecium, Escherichia coli or Pseudomonas aeruginosa (antibiotic-sensitive and -resistant isolates). Zones of inhibition, visualised with iodonitrotetrazolium chloride, were subject to GC-MS, LC-MS and NMR to characterise the bioactive compounds. Results: Seven compounds were identified from the three EOs using GC-MS, while LC-MS and NMR failed to detect the presence of any further non-volatile or heat labile compounds. Carvacrol was most antimicrobial compound identified, with minimum inhibitory concentrations ranging 0.99-31.62 mM. Conclusion: The identified antimicrobial compounds present in oregano, rosewood and cumin EOs including carvacrol may be candidates for the development of novel antimicrobial therapies against antibiotic-resistant bacteria.

The Escherichia coli LacZ gene is a widely used reporter for gene regulation studies in transgenic mice. It encodes bacterial β-galactosidase (Bact β-Gal), which causes insoluble precipitates when exposed to chromogenic homologues of galactose. We and others have recently reported that Bact β-Gal detection with Salmon-Gal (S-Gal) in combination with nitro blue tetrazolium chloride (NBT) is very sensitive and not prone to interference by acidic endogenous β-galactosidases. Unfortunately, as we show here, the method appears to be inadequate for evaluation of Bact β-Gal expression in keratinized epithelial appendages but not in other keratinized epithelia. NBT in the reaction mixture, just as other tetrazolium salts, inevitably causes unwanted staining artifacts in lingual filiform papillae, penile spines, and hair fibers by interacting with keratin sulfhydryl-rich regions. The methodological limitation can be overcome in part by pretreating the tissues before the S-Gal/NBT staining with an iodine–potassium iodide solution. Alternatively, the use of iodonitrotetrazolium chloride instead of NBT in the S-Gal reaction mixture provides enough color resolution to distinguish the specific Bact β-Gal staining in orange from the artifact staining in dark red. In summary, we provide evidence that S-Gal/NBT histochemistry has limitations, when staining keratinized epithelial appendages.

Schrankia leptocarpa DC. (Mimosaceae) is an African medicinal plant, traditionally used by plants practitioners in Benin to cure malaria, diarrhea and stomach ache. There is little ethnobotanical and almost no chemical information available for this species. Eight extracts from the entire plant of Schrankia leptocarpa were prepared using diethyl ether, acetone, cyclohexane, methylene chloride, ethyl acetate, methanol, butanol and ethanol/water (20:80). Phytochemical analysis were performed and antibacterial was evaluated against four reference bacteria, Staphylococcus aureus , Enteroccocus feacalis , Escherichia coli and Pseudomonas aeruginosa , and four patient isolated bacteria using iodonitrotetrazolium microtest. The antioxidant properties were investigated using 2,2-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion radical scavenging activity. All extracts showed antimicrobial activity ranging from 0.078 to 5 mg/ml against one or more bacteria. The most potent extract was the diethyl ether extract with a minimum inhibitory concentration value of 75 μg/ml on E. faecalis . Phytochemical screening showed a wide variety of phytoconstituents such as steroids, terpenes, phenolic acid, flavonoids, tannins and alkaloids. The DPPH radical scavenging activity indicated that the radical scavenging activity ranged from 1.35 to 3.47 μg/ml. The superoxide anion radical scavenging showed inhibitory percentage ranging from 32.04 to 86%. Higher activity was observed with ethyl acetate extract. The results provide an evidence for the traditional use of S. leptocarpa for the treatment of infective diseases. Keywords: Schrankia leptocarpa , antibacterial, antioxidant, 2,2-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion

Visando verificar a influência da utilização de diferentes métodos de quantificação de micélio extrarradicular ativo (MEA) e micélio extrarradicular total (MET) de fungos micorrízicos arbusculares (FMAs) e de diferentes hospedeiros nesta simbiose, foi realizado um experimento em casa de vegetação, com delineamento inteiramente casualizado e esquema fatorial: 3 espécies de FMAs (Glomus intraradices, G. etunicatum e G. clarum) e um controle sem FMA x 6 doses de P (0, 50, 100, 150, 200 e 250 mg kg-1 de substrato) x 2 plantas cítricas: tangerina cleópatra (Citrus reshni) e laranja caipira (Citrus sinensis), com 5 repetições por tratamento. Seis meses após o transplantio e inoculação das mudas, determinaram-se altura e diâmetro do caule, colheram-se as plantas e avaliaram-se matéria seca e nutrientes da parte aérea, porcentagem de colonização radicular e comprimento de MEA e MET pelos métodos de fluorescência induzida com diacetato de fluoresceína (FDA) e redução do iodonitrotetrazólio (INT). As doses crescentes de P proporcionaram redução da porcentagem de colonização radicular nos dois porta-enxertos estudados e aumentos nas características altura, diâmetro, matéria seca da parte aérea e quantidade total absorvida de macro e micronutrientes por ambos os porta-enxertos. Houve correlação negativa entre comprimento de MEA e quantidade total absorvida de macronutrientes (P, N, Ca e Mg) por laranja caipira e correlação positiva entre comprimento de MET e quantidade absorvida por tangerina cleópatra, sugerindo ser esta última mais micotrófica que a anterior, além de haver, possivelmente, outros mecanismos de absorção de nutrientes que não pelo MEA, cujos resultados variaram em função do método empregado. Different methods for quantification of active extraradicular mycelium (AEM) and total extraradicular mycelium (TEM) of arbuscular mycorrhizal fungi (AMF) were examined by conducting an experiment under greenhouse conditions, with a completely randomized factorial design and five replicates per treatment. The experiment was arranged in a 4 x 6 x 2 design and the factors were: 3 species of AMF (Glomus intraradices, G. etunicatum and G. clarum) and one control without AMF x 6 levels of P (0, 50, 100, 150, 200 and 250 mg kg-1) x 2 citrus species (Citrus reshni and Citrus sinensis). Six months after transplanting, height and diameter were evaluated and the plants were harvested. Shoot dry weight, total shoot macronutrients, percentage of root infection and the length of AEM and TEM by the fluorescein diacetate (FDA) and the iodonitrotetrazolium reduction (INT) methods were also evaluated. Increasing levels of P promoted decreases in percentage of root infection and increases in plant height, diameter, dry weight and macro and micronutrients absorbed by both citrus species. There was a negative correlation between AEM and total macronutrients absorbed (P, N, Ca and Mg) by Citrus sinensis and a positive correlation between TEM and those macronutrients by C. reshni indicating that the latter host may be more mycotrophic than the former and, possibly, that there are other mechanisms of nutrient absorption rather than by AEM, with results varying in relation to the method used.

Otimização de métodos de extração e avaliação de micélio extrarradicular ativo (MEA) e total (MET) constituem os principais passos para um estudo mais profundo da interação fungo micorrízico arbuscular (FMA) e hospedeiro, já que a absorção e translocação dos nutrientes do substrato ao hospedeiro são realizadas, respectivamente, por tais estruturas fúngicas. Métodos de extração de micélio extrarradicular de FMAs propostos por diversos autores foram reavaliados e mesclados, originando um método exclusivo e de fácil execução, juntamente com a calibração de métodos para avaliação e quantificação de MEA (fluorescência induzida com a hidrólise do diacetato de fluoresceína e redução de iodonitrotetrazólio), em substrato arenoso. Extraction and evaluation of active (AEM) and total (TEM) extraradicular mycelium is the first step in a comprehensive study of the interaction between host plant and arbuscular mycorrhizal fungi (AMF), since these hyphae are responsible for nutrient absorption and translocation from the soil to the host. Extraction methods for extraradicular mycelium in the literature were reviewed and reevaluated, and modifications were made, resulting in a new, simple technique. The evaluation and quantification method of AEM was tested using induced fluorescence following fluorescein diacetate (FDA) hydrolysis and iodonitrotetrazolium (INT) reduction in sandy soil.

International audience; Flavocytochrome b(558) (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22(phox) and gp91(phox). The latter subunit, also known as Nox2, has a cytosolic C-terminal "dehydrogenase domain" containing FAD/NADPH-binding sites. The N-terminal half of Nox2 contains six predicted transmembrane α-helices coordinating two hemes. We studied the role of the second transmembrane α-helix, which contains a "hot spot" for mutations found in rare X(+) and X(-) chronic granulomatous disease. By site-directed mutagenesis and transfection in X-CGD PLB-985 cells, we examined the functional and structural impact of seven missense mutations affecting five residues. P56L and C59F mutations drastically influence the level of Nox2 expression indicating that these residues are important for the structural stability of Nox2. A53D, R54G, R54M, and R54S mutations do not affect spectral properties of oxidized/reduced cytb, oxidase complex assembly, FAD binding, nor iodonitrotetrazolium (INT) reductase (diaphorase) activity but inhibit superoxide production. This suggests that Ala-53 and Arg-54 are essential in control of electron transfer from FAD. Surprisingly, the A57E mutation partially inhibits FAD binding, diaphorase activity, and oxidase assembly and affects the affinity of immunopurified A57E cytochrome b(558) for p67(phox). By competition experiments, we demonstrated that the second transmembrane helix impacts on the function of the first intracytosolic B-loop in the control of diaphorase activity of Nox2. Finally, by comparing INT reductase activity of immunopurified mutated and wild type cytb under aerobiosis versus anaerobiosis, we showed that INT reduction reflects the electron transfer from NADPH to FAD only in the absence of superoxide production.

The role of Leu 505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mim- icking the phenotype of a previously published X91- CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) re- ductase activities were partially maintained concom- itantly with a partial cytosolic factors translocation to the plasma membrane. This suggests that assembly and electron transfer from NADPH occurred par- tially in the Leu505Arg Nox2 mutant. Moreover, in a simplified CFS using purified mutant cytochrome b558 and recombinant p67 phox , p47 phox , and Rac1proteins, we found that the Km for NADPH and for NADH was about three times higher than those of purified WT cytochrome b558, indicating that the Leu505Arg mutation induces a slight decrease of the affinity for NADPH and NADH. In addition, oxidase activity can be extended by increasing the amount of p67 phox in the simplified CFS assay. However, the maximal reconstituted oxidase activity using WT pu- rified cytochrome b558 could not be reached using mutant cytochrome b558. In a three-dimensional model of the C-terminal tail of Nox2, Leu 505 appears to have a strategic position just at the entry of the NADPH binding site and at the end of the -helical loop (residues 484-504), a potential cytosolic factor binding region. The Leu505Arg mutation seems to affect the oxidase complex activation process through alteration of cytosolic factors binding and more particularly the p67 phox interaction with cyto- chrome b558, thus affecting NADPH access to its binding site. J. Leukoc. Biol. 81: 238-249; 2007.

International audience; Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.

The objective of this experiment was to quantify the extramatrical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus etunicatum (Becker & Gerdemann) grown on maize (Zea mays L. var. Piranão) provided with various levels of phosphate fertilizer and harvested at 30, 60 and 90 days after planting (DAP). Total extramatrical mycelium (TEM) was extracted from soil using a modified membrane filtration method, followed by quantification using a grid intersection technique. Active extramatrical mycelium (AEM) proportion was determined using an enzymatic method which measured dehydrogenase activity by following iodonitrotetrazolium reduction. At low levels of added P, there was relatively less TEM than at high levels of added P, but the AEM proportion at low soil P availability was significantly greater than at high soil P. O objetivo do trabalho foi quantificar o micélio extramatricial do fungo micorrízico arbuscular (AM) Glomus etunicatum (Becker & Gerdemann) e medir sua atividade 30, 60 e 90 dias após o plantio (DAP), utilizando-se a cultura do milho (Zea mays L. var. Piranão) com quatro doses de adubação fosfática. O micélio extramatricial total (TEM) foi extraído do solo pelo método da membrana de filtração modificado e foi quantificado pela técnica da grade de interseção. A proporção de micélio extramatricial ativo (AEM) foi determinada pelo método que mede a atividade das enzimas desidrogenases com coloração de iodonitrotetrazolio. Em baixas doses de P,o TEM foi menor do que a quantidade em altas doses de P, mas a proporção de AEM foi maior nas baixas doses de P.