Antimicrobial assay

Antimicrobial assays represent a valuable method by which marine plant, animal, and microbial extracts can be tested for inhibitory effects against cooccurring and potentially deleterious microbes (e.g., pathogens, competitors, or fouling microbes). The ability of secondary metabolites to inhibit ecologically relevant microbes can then be used to assess the potential function of marine 

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In our laboratories the following organisms have been selected as representative and qualitative antimicrobial assays are carried out using the agar well diffusion assay procedure (5) ... 

Llrlodenine from Fractions 33-40 - Crystallization of the residue of these fractions (118 mg) from chloroform yielded 85 mg of yellow needles, mp 280-281°. The melting point, IR, and UV data were consistent with that reported for the yellow alkaloid, llrlodenine, previously reported from the heartwood (9). Direct comparison (melting point, mixture melting point, IR, and UV) with an authentic sample of llrlodenine confirmed the identity. Antimicrobial assay showed llrlodenine to be the active component present in these fractions (Table II). 

Hits from the screen were used to select related compounds for further testing from the Lilly collection of compounds [33], The HPLC assay was used to measure MurD inhibition and standard antimicrobial assays were carried out with S. pneumoniae, Haemophilus influenzae 76, Staphylococcus aureus 027, and Moraxella catarrhalis BC-1. A series of indole and phenoxypropyl amine derivatives with activity against MurD, were discovered that exhibited broad-spectrum antibacterial activity. 

While this approach has limitations related to the peptidic nature of the linker such as stability to harsh reaction conditions and requirement of specific functional groups to be coupled with the linker arms, its application to particular libraries and chemistries may be useful. Its biological utility was assessed in a bead-based antimicrobial assay on bacterial cells [54], which produced good correlations between the MIC (minimal inhibitory concentration) values for the compounds released in situ in the culture medium from the beads or tested as standard solutions. 

No in vitro antimicrobial assay systems that correctly predict the in vivo effect have yet been established for antifungal research. As an alternative approach, an in vivo assay using a mouse Candida albicans infection model was adopted for use in finding new antifungal compounds. FR109615 was isolated from the... 

Fig. (1). Peptidomics strategies used to study Drosophila immunity. (A) Using antimicrobial assays (antibacterial and antifungal), the bioactive peptides were isolated from the blood of bacteria-challenged Drosophila. MS was used for molecular mass assignment, to identify post-translational modifications, and for primary structure elucidation (B) Identification of peptidic immune effectors through differential display analysis (DD) by MALDI-MS and micro/nano RP-HPLC coupled (online) or not (off-line) to ESI-MS. When the HPLC was performed off-line to the mass spectrometer, fractions were individually analyzed by MALDI-MS. The identification and the structural characterization were performed either by molecular mass assignment and/or sequencing by ESI-MS/MS.

For antimicrobial assays, there are several common methods employed. Due to its ease of operation, the most common method used is the disk diffusion method, which involves the application of a material onto a filter paper disk, and then the disk is placed onto solid medium previously seeded with the test microorganism of interest. Sometimes, the sample is dissolved in an appropriate solvent before application onto the paper disk. This method is very common in the evaluation of antibiotics and is the method adopted by the National Committee for Clinical Laboratory Standards (NCCLS). The method depends on the aqueous solubility of the antibiotics in order to facilitate diffusion through the solid medium. Essentials oils, however, are generally hydrophobic, do not readily diffuse through an aqueous medium and, therefore, the prevalence of false negatives or reduced activity might then be anticipated. 

Despite the PPG showing a weak activity, it has developed further antimicrobial assays. Pardo and cols, reported [34] the MIC= 400 pg/mL and MBC= 800 pg/mL of verbascoside. Moreover, Avila and cols. [33] studied its mode of action in vitro. There is no evidence of inclusion of [3H]-leueine into the cell, whereas [3H]-thymidine and [3H]-uridine were not observed. That resulted in the conclusion that the mode of action of verbascoside is through the inhibition of protein production, since leucine is an important metabolite in protein synthesis. 

Three fluoride derivatives (192-194) from 191 were prepared given the potential effects of the introduction of fluorine into organic compounds. Fig. (51). The results obtained in the antimicrobial assay showed that the effect of scutione (191) and its fluoride derivatives 192-194 was limited to Gram-positive bacteria (Table 9), and unexpectedly scutione was more active than the fluoride derivatives. 

This work was conducted in cooperation with Mika Matsumoto (Shimojima), Ms. S. Ohhira, and M. Ueda. We are grateful to Dr. M. Murata of Ochanomizu University for kindly providing the microorganisms, and to Dr. N. Tominaga of Ochanomizu University for her helpful discussions on the antimicrobial assay. 

From this survey of the cited literature we can notice that problems concerning the standardization of antimicrobial assays are not completely solved. Often, incomparability of results obtained by the different researchers using different methods greatly diminishes quality data and makes compilation of databases useless. 

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