Micrococcus Flavus Research Articles

Getting Closer to the Real Bacterial Cell Wall Target: Biomolecular Interactions of Water‐Soluble Lipid II with Glycopeptide Antibiotics

A novel synthesized water-soluble variant of lipid II (LII) was used to evaluate the noncovalent interactions between a number of glycopeptide antibiotics and their receptor by bioaffinity electrospray ionization mass spectrometry (ESI-MS). The water-soluble variant of lipid II is an improved design, compared to the traditionally used tripeptide N,N'-diacetyl-L-lysyl-D-alanyl-D-alanine (KAA), of the target molecule on the bacterial cell wall. A representative group of glycopeptide antibiotics was selected for this study to evaluate the validity of the novel cell-wall-mimicking target LII. Structure-function relationships of various glycopeptide antibiotics were investigated by means of 1) bioaffinity mass spectrometry to evaluate solution-phase molecular interactions with both LII and KAA, 2) fluorescence leakage experiments to study the interactions with the membrane-embedded lipid II, and 3) minimum inhibitory concentrations against the indicator strain Micrococcus flavus. Our results with the novel LII molecule reveal that some antibiotics interact differently with KAA and LII. Additionally, our data cast doubt on the hypothesis that antibiotic selfdimerization assists in the in-vivo efficacy. Finally, the water-soluble lipid II proved to be a better model of the bacterial cell wall.

Fatty acids with antibacterial activity from the cyanobacterium Oscillatoria redekei HUB 051

Bioassay-guided fractionation of the n-hexane extract prepared from the biomass of the cyanobacterium Oscillatoria redekei syn. Limnothrix redekei HUB 051 by silica gel and RP-18 column chromatography followed by HPLC resulted in the isolation of a mixture of two unsaturated hydroxy fatty acids. Their further separation using normal phase HPLC resulted in the identification of α-dimorphecolic acid, a 9-hydroxy-10E, 12Z-octadecadienoic acid (9-HODE) and of coriolic acid, a 13-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE). In agar plate diffusion test these fatty acids inhibited the growth of the Gram-positive bacteria Bacillus subtilis SBUG 14, Micrococcus flavus SBUG 16 and Staphylococcus aureus SBUG 11 and ATCC 25923, but no activity was observed against multiresistant Staphylococcus aureus strains. This is the first report about antibacterial activity of these hydroxylated unsaturated fatty acids and about the occurrence of coriolic acid in cyanobacteria.

Primary Structure of Inorganic Polyphosphate/ATP-NAD Kinase fromMicrococcus flavus, and Occurrence of Substrate Inorganic Polyphosphate for the Enzyme

The gene encoding an inorganic polyphosphate/ATP-NAD kinase was cloned from Micrococcus flavus, and its primary structure was analyzed. Alignment of the primary structure with those of other characterized NAD kinases revealed candidate amino acid residues, mainly charged ones, that would be related to inorganic polyphosphate use. The alignment also showed that the primary structure found carried a protruding C-terminal polypeptide. Although the C-terminal polypeptide was demonstrated to be dispensable for the kinase activities, and was proposed to be removed in M. flavus, the entire primary structure including the C-terminal polypeptide was homologous with that of the ATP synthase beta chain. The inorganic polyphosphate used by the inorganic polyphosphate/ATP-NAD kinase as a phosphoryl donor was isolated from cells of M. flavus, suggesting that the ability of the enzyme to use inorganic polyphosphate is of physiological significance and is not an evolutionary trait alone.

The Antibacterial Activity of Topical Retinoids: The Case of Retinaldehyde

Background: Retinoids such as retinoic acid (RA), retinol (ROL) and retinaldehyde (RAL) are currently used in many formulations and indications ranging form acne to skin aging. Most if not all their pharmacological activities occur through binding to nuclear receptors with subsequent modulation of the activities of several genes. Little attention has been given to the many other potential actions on the surface of the skin. Aim: To analyse the potential anti-infective activities of topical ROL, RAL and RA. Methods: Microbial minimal inhibitory concentrations (MIC) of ROL, RAL and RA were determined by a microdilution method on reference strains including Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus flavus, Propionibacterium acnes, Micrococcus luteus, Enterococcus faecium,Staphylococcus hominis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and 133 clinical strains including methicillin-resistant S. aureus, methicillin-sensitive S. aureus, coagulase-negative Staphylococcus, Streptococcus group B, Enterococcus faecalis, vancomycin-resitant E. faecalis, vancomycin-resistant E. faecium and Pseudomonas/Klebsiella. In two clinical trials in healthy human volunteers, skin bacterial densities were evaluated in samples obtained with the cylinder scrub method: (1) 2 and 5 h after a single application of 0.05% RAL or vehicle on the forearm and (2) in a single-blind randomized study where 0.05% RAL or vehicle were applied daily for 2 weeks on the forehead of 22 volunteers. Paired results from treated (or vehicle) and untreated areas were analysed. Results: Of the three retinoids tested, only RAL showed a significant in vitro antibacterial activity; this activity was found against reference strains of gram-positive bacteria like S. aeureus, Micrococcus spp. or P. acnes. No activity was found against gram-negative bacteria. These results on reference strains were confirmed on 133 clinical isolates. MIC₅₀ and MIC₉₀ values for RAL were 8 and 16 mg/l, respectively, for methicillin-sensitive S. aureus and 4 and 8 mg/l for methicillin-resistant S. aureus. The two in vivo studies showed that areas treated with RAL had a significant decrease in the bacterial counts. In the forehead study, the median decrease was 10² log/cm² for P. acnes and 10^1.8 log/cm² for staphylococci. No resistant bacteria were found after 2 weeks of topical use. Preliminary results suggest that the antibacterial effect of RAL is due, in part, to the aldehyde group in the lateral chain, since non-retinoid pseudo-analogues of the chain, like citral and hexenal, showed a similar antibacterial activity. Conclusion: We have shown that RAL differs from parent natural retinoids such as ROL and RA in demonstrating significant antibacterial activities upon topical use. This activity is likely due to the aldehyde group in the isoprenoic lateral chain, which illustrates the potential bifunctional properties of some retinoids.

Membrane activity of the peptide antibiotic clavanin and the importance of its glycine residues.

The peptide antibiotic clavanin A (VFQFLGKIIHHVGNFVHGFSHVF-NH(2)) is rich in histidine and glycine residues. In this study the antimicrobial activity and membrane activity of wild-type clavanin A and seven Gly --> Ala mutants thereof were investigated. Clavanin A effectively killed the test microorganism Micrococcus flavus and permeabilized its cytoplasmic membrane in the micromolar concentration range, suggesting that the membrane is the target for this molecule. Consistent with this suggestion, it was observed that clavanin A efficiently inserted into different phospholipid monolayers mainly via hydrophobic interactions. Bilayer permeabilization was observed for both low and high molecular mass fluorophores enclosed in unilamellar vesicles and occurred at the same concentration as the antimicrobial activity. It is therefore suggested that the loss of barrier function does not involve specific receptors in the target membrane. Circular dichroism spectroscopy indicated that under membrane mimicking conditions a random coil --> helical transition was induced for all clavanin derivatives tested. Observed differences in peptide-membrane interaction and biological activity between the various clavanin derivatives demonstrated the functional importance of Gly at the positions 6 and 13. These two glycines may act as flexible hinges that facilitate the hydrophobic N-terminal end of clavanin to deeply insert into the bilayer. On the contrary, no such role is evident for Gly 18, as its substitution by Ala actually stimulated membrane interaction and biological activity. This study suggests that the combined hydrophobicity, overall state of charge, and conformational flexibility of the peptide determine the (membrane) activity of clavanin A and its Gly --> Ala mutants.

Formation and Efficacy of Vancomycin Group Glycopeptide Antibiotic Stereoisomers Studied by Capillary Electrophoresis and Bioaffinity Mass Spectrometry

The conformational stability of vancomycin group antibiotics (i.e., vancomycin and avoparcin) in aqueous solution has been studied. These complex glycopeptide antibiotics contain many chiral centers allowing the potential formation of stereoisomers. Using capillary electrophoresis these stereoisomers could be separated and detected by UV and/or mass spectrometry. Fresh aqueous samples of both vancomycin and avoparcin already contained a plethora of stereoisomers. Thermal degradation of the antibiotics was studied as well. For vancomycin thermal degradation led primarily to the formation of CDP-I and aglycons. In the case of avoparcin thermal degradation led mainly to the interconversion between stereoisomers. These antibiotic stereoisomers may exhibit different antibacterial efficacy. Solution-phase association constants of fresh and heated samples of these antibiotics and their bacterial cell wall mimicking receptors were determined by bioaffinity mass spectrometry and revealed that the heated samples exhibited, in general, a lower affinity. Minimum inhibitory concentrations (Micrococcus flavus) were determined and confirmed the decrease in antibacterial efficacy upon heating.

Multilayered antimicrobial polyethylene films applied to the packaging of ground beef

AbstractA natural antimicrobial agent, grapefruit seed extract (GFSE), was incorporated in a 0.5% or 1.0% concentration on the food‐contact surface of multilayered polyethylene (PE) film by a co‐extrusion or solution‐coating process. The fabricated films were tested in their antimicrobial activity against several food spoilage microorganisms, and then applied to the packaging of ground beef. During the storage of the packaged beef at 3°C, changes in microbial counts, pH, TBA value and meat colour were monitored in order to see the effect of the packaging film on meat quality changes. Coating the PE film with GFSE with the aid of a polyamide binder resulted in a more effective level of antimicrobial activity on the agar plate medium than did its incorporation by a co‐extrusion process. The film co‐extruded with a 1.0% GFSE layer showed antimicrobial activity only against Micrococcus flavus ATCC 10240, while the film coated with 1.0% GFSE showed activity against several microorganisms, such as Escherichia coli IFO 3301, Staphylococcus aureus IFO 3060 and Bacillus subtilis IFO 12113. Both types of GFSE‐incorporated multilayer PE films contributed to a reduction of the growth rates of aerobic and coliform bacteria on the ground beef, when compared to plain PE film. The film coated with a higher concentration (1.0%) of GFSE had a more pronounced effect than did the other films in inhibiting spoilage bacterial growth. They could also slow down chemical changes of the packaged beef. The GFSE‐coated film was more effective in preserving beef quality, but the level of GFSE addition did not affect the quality changes of the beef. Copyright © 2001 John Wiley & Sons, Ltd.

Identification and Partial Characterization of Lacticin BH5, a Bacteriocin Produced by Lactococcus lactis BH5 Isolated from Kimchi

Strain BH5 was isolated from naturally fermented Kimchi and identified as a bacteriocin producer that has bactericidal activity against Micrococcus flavus ATCC 10240. Strain BH5 was identified tentatively as Lactococcus lactis by API test. Lactococcus lactis BH5 showed a broad spectrum of activity against most of the nonpathogenic and pathogenic microorganisms tested by the modified deferred method. The activity of lacticin BH5, named tentatively as the bacteriocin produced by L. lactis BH5, was detected at the mid-log growth phase, reached its maximum during the early stationary phase, and decreased after the late stationary phase. Lacticin BH5 also showed a relatively broad spectrum of activity against nonpathogenic and pathogenic microorganisms as tested by the spot-on-lawn method. Its antimicrobial activity on sensitive indicator cells was completely destroyed by protease XIV. The inhibitory activities of lacticin BH5 were detected during treatments up to 100°C for 30 min. Lacticin BH5 was very stable over a pH range of 2.0 to 9.0 and was stable with all the organic solvents examined. It demonstrated a typical bactericidal mode of inhibition against M. flavus ATCC 10240. The apparent molecular mass of lacticin BH5 was estimated to be in the region of 3 to 3.5 kDa, by the direct detection of bactericidal activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Synthesis and antimicrobial activity of new adamantane derivatives II

A series of new derivatives of adamantane was synthesised. The new compound 4-(adamant-1-ylethylenoxycarbonyl)phthalanhydride obtained from 1-adamantaneethanol and trimellitic anhydride chloride, as well as 4-(adamant-1-yl-methylenoxycarbonyl)phthalanhydride, appeared very useful for preparation of a number of N-substituted phthalimides. Antimicrobial activity of newly obtained derivatives such as, for example, 4-(adamant-1-ylethylenoxycarbonyl)- N-( l-phenylalanyl)phthalimide or 4-(adamant-1-ylmethylenoxycarbonyl)- N-( l-leucyl)-phthalimide was tested against Staphylococcus aureus, Bacillus sp., Micrococcus flavus and Enterococcus faecium. The minimal inhibitory concentrations for these compounds against Bacillus cereus were 15 and 8 μg/ml, respectively.

Inorganic Polyphosphate/ATP-NAD Kinase of Micrococcus flavus and Mycobacterium tuberculosis H37Rv

An enzyme with both inorganic polyphosphate [poly(P)]- and ATP-dependent NAD kinase activities was isolated from Micrococcus flavus. The enzyme was a dimer consisting of 34 kDa subunits, and was named poly(P)/ATP-NAD kinase. Internal amino acid sequences of the enzyme showed homologies with some function-unknown proteins released on the GenBank database. Among such proteins, hypothetical Rv1695 protein (Accession No. Z98268-16), which was encoded by a gene named “Rv1695” on genomic DNA of Mycobacterium tuberculosis H37Rv, was proposed to be poly(P)-dependent NAD kinase. By cloning and expression in Escherichia coli, Rv1695 was shown to encode poly(P)/ATP-NAD kinase and named ppnk. The ppnk product, recombinant-poly(P)/ATP-NAD kinase (Ppnk) was purified and characterized. The enzyme was a tetramaer consisting of 35 kDa subunits when expressed in E. coli. Poly(P)/ATP-NAD kinases of M. flavus and Ppnk of M. tuberculosis H37Rv specifically and completely phosphorylated NAD by utilizing commercially available poly(P)s and nucleoside triphosphates as phosphoryl donors.

Synthesis and antimicrobial activity of new adamantane derivatives I.

A series of fourteen derivatives of adamantane was synthesised. The new compound 4-(adamant-1-ylmethoxycarbonyl)phthalanhydride obtained from 1-adamantane-methanol and trimellitic anhydride chloride appeared very useful for preparation of a number of N-substituted phthalimides. Antimicrobial activity of the newly obtained derivatives such as, for example, 4-(adamant-1-ylmethoxycarbonyl)-N-(5-carboxypentamethylene)p hthalimide or 4-(adamant-1-ylmethoxycarbonyl)-N-(L-alanyl)phthalimide was tested against Staphylococcus aureus, Bacillus sp., Micrococcus flavus and Enterococcus faecium. The minimal inhibitory concentration (MIC) for these compounds against S. aureus were 0.022 and 0.05 microg/ml, respectively.

Antibacterial and binding characteristics of bovine, ovine and caprine lactoferrins: a comparative study

A cation-exchange chromatographic membrane was used for the rapid isolation of caprine and ovine lactoferrin (LF) from cheese whey. Caprine LF was isolated by one-step cation-exchange chromatography, but the isolation of ovine LF needed a further reversed-phase HPLC purification step. The antibacterial activity of these LFs, determined towards Micrococcus flavus and Escherichia coli, was compared with that of bovine LF. Of the apo-LFs the caprine protein had the highest activity. In all cases the holo-LFs showed low or negligible activity. The antibacterial properties of cationic peptides obtained by peptic hydrolysis of caprine LF were studied against E. coli and M. flavus, and the activity of these peptides was compared with that of bovine lactoferricin. In addition, the binding characteristics of bovine, ovine and caprine LF to bacterial cells of E. coli were investigated with an enzyme-linked binding assay using horseradish peroxidase-bovine LF as a conjugate. Ovine and bovine LF strongly inhibited the binding of the LF complex to the bacterial surface. The iron-free forms of these LFs showed a greater ability for binding to the bacterial cells than the iron-saturated forms. However, several cationic peptides and caprine LF did not inhibit the binding of the LF conjugate although they exhibited a marked antibacterial effect. The results of binding of the LF complex in the presence of LFs from different species and of membrane-active peptides are discussed in relation to the antibacterial activity of these proteins and peptides.

Cyanobacteria—a potential source of new biologically active substances

Hydrophilic and lipophilic extracts of twelve cyanobacterial strains, isolated from fresh and brackish water, and two waterblooms, collected during the summer from the Baltic Sea, were investigated for their antibiotic activities against seven microorganisms. No inhibitory effects were found against the three Gram-negative bacteria Escherichia coli, Proteus mirabilis and Serratia marcescens and the yeast Candida maltosa. Of all cyanobacterial samples, extracts from seven species inhibited the growth of at least one of the Gram-positive bacteria Micrococcus flavus, Staphylococcus aureus and Bacillus subtilis. M. flavus proved to be the most sensitive bacterium in the agar diffusion test system. In particular, the hexane and dichlormethane extracts showed antimicrobial effects. But only one water extract, prepared from material of a natural waterbloom, was found to be active.

A chelating agent possessing cytotoxicity and antimicrobial activity: 7-Morpholinomethyl-8-hydroxyquinoline

7-Morpholinomethyl-8-hydroxyquinoline (MO-8HQ), which like 8-hydroxyquinoline (8HQ) readily forms a chelate, was synthesized and found to possess cytotoxicity and antimicrobial activity. Both 8HQ and MO-8HQ were cytotoxic to human carcinoma cell lines at micromolar concentrations. MO-8HQ also inhibited DNA synthesis of tumor cells at micromolar concentrations, suggesting that MO-8HQ might chelate metals necessary for the enzymatic catalysis of DNA biosynthesis. MO-8HQ was more active against Gram positive bacteria than Gram negative bacteria and its potency correlated with iron chelation. An “unsaturated” chelate with a MO-8HQ to Fe ratio of 2:1 exhibited greater antibacterial activity than MO-8HQ alone. Among the organisms tested, Micrococcus flavus was most susceptible with a MIC of 3.9 μg ml . MO-8HQ also exhibited anti-fungal activity at 7–15 μg ml . MO-8HQ:Fe chelate markedly increased the susceptibility of Escherichia coli to deoxycholate. Addition of Ca 2+ or Mg 2+ reversed the sensitivity of bacteria to deoxycholate as well as to rifampicin. It is suggested that MO-8HQ exerts its biological activity as a membrane-active agent through metal ion chelation.

Construction of a single-copy integration vector and its use to study gene expression in Bacillus licheniformis.

A versatile system consisting of an integrational vector and a bacitracin (Bt)-producing beta-galactosidase (beta-Gal)-negative (Lac-) Bacillus licheniformis TLH strain was constructed to quantify promoter activity and to study gene regulation in a single-copy set-up. The vector pTLH utilizes the promoterless Escherichia coli lacZ gene derived from pQF52 and contains the pBR322 origin of replication and a kanamycin-resistance gene for selection in both B. licheniformis and E. coli. The vector also contains an inner part of the first gene of the Bt synthetase (bts) operon which enables its integration into the bts of B. licheniformis by Campbell-type recombination. This recombination event can be easily tested on a Micrococcus flavus lawn where loss of Bt production, i.e. no clearing zone on the lawn, is indicative of the proper integration. The Lac- B. licheniformis TLH strain was developed by elimination of the natural beta-Gal activity of B. licheniformis strain ATCC 10716 UM12 using NTG mutagenesis.

Influence of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling capacity.

Three mutants of the antibiotic nisin Z, in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling properties. 1H-NMR analyses showed that all three mutants harbor an unmodified serine residue at position 33, instead of the usual dehydroalanine. Apparently, the nature of the residue preceding the serine to be dehydrated, strongly affects the efficiency of modification. Cleavage of [Glu32,Ser33]nisin Z by endoproteinase Glu-C yielded [Glu32]nisin Z(1-32)-peptide, which has a net charge difference of -2 relative to wild-type nisin Z. The activity of [Lys32,Ser33]nisin Z against Micrococcus flavus was similar to that of wild-type nisin, while [Trp32,Ser33]nisin Z, [Glu32,Ser33]nisin Z and [Glu32]nisin Z(1-32)-peptide exhibited 3-5-fold reduced activity, indicating that negative charges in the C-terminal part of nisin Z are detrimental for activity. All variants showed significant loss of activity against Streptococcus thermophilus. The potency of the nisin variants to act as signaling molecules for auto-induction of biosynthesis was significantly reduced. To obtain mutant production, extracellular addition of (mutant) nisin Z to the lactococcal expression strains was essential.

5500596 Local coil array for magnetic resonance imaging of the lower extremities

The volatile composition of eight Stachys species has been studied. The investigated taxa were St. alopecuros (L.) Bentham., St. scardica (Griseb.) Hayek, St. cretica L. ssp. cretica, St. germanica L. ssp. heldreichii (Boiss.) Hayek, St. recta L., St. spinulosa L., St. euboica Rech. and St. menthifolia Vis., growing wild in Greece. The essential oils were obtained by hydrodistillation in a modified Clevenger-type apparatus, and their analyses were performed by GC and GC–MS. Identification of the substances was made by comparison of mass spectra and retention indices with literature records. Sesquiterpene hydrocarbons were shown to be the main group of constituents of all taxa. Furthermore, the obtained essential oils were tested against the following six bacteria: Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 35210), Bacillus subtilis (ATCC 10907), Bacillus cereus (clinical isolates), Micrococcus flavus (ATCC 10240), Staphylococcus epidermidis (ATCC 2228), as well as against the following five fungi: Aspergillus niger (ATCC 6275), Penicillium ochrochloron (ATCC 9112), Epidermophyton floccosum (clinical isolates), Candida albicans (clinical isolates) and Trichophyton mentagrophytes (clinical isolates). The tested essential oils showed better activity against bacterial species than against fungi. Pseudomonas aeruginosa was the most resistant strain, as none of the essential oils was active against this strain. The essential oil of St. scardica has been proven most active against both bacteria and fungi.

Planetary quarantine in the solar system. Survival rates of some terrestrial organisms under simulated space conditions by proton irradiation

We have been studying the survival rates of some species of terrestrial unicellular and multicellular organism (viruses, bacteria, yeasts, fungi, algae, etc.) under simulated interstellar conditions, in connection with planetary quarantine. The interstellar environment in the solar system has been simulated by low temperature, high vacuum (77 K, 4 × 10 −8 torr), and proton irradiation from a Van de Graaff generator. After exposure to a barrage of protons corresponding to about 250 years of irradiation in solar space, tobacco mosaic virus, Bacillus subtilis spores, Staphylococcus aureus, Micrococcus flavus, Aspergillus niger spores, and Clostridium mangenoti spores showed survival rates of 82, 45, 74, 13, 28, and 25%, respectively.

Bacitracin production by a new strain of Bacillus subtilis. Extraction, purification, and characterization.

A new strain of Bacillus subtilis C 126 was isolated from sugar cane fermentation and produced an antibiotic that inhibited the growth of Micrococcus flavus. The production of the antibiotic in culture medium followed to extraction with n-butanol, thin layer chromatography, and microbiological tests indicated that a polypeptide antibiotic was produced. The fraction obtained by Sephadex G-25 column and analyzed by HPLC indicated that bacitracin complex was produced.

Viable but nonculturable bacteria in drinking water

Klebsiella pneumoniae, Enterobacter aerogenes, Agrobacterium tumefaciens, Streptococcus faecalis, Micrococcus flavus, Bacillus subtilis, and Pseudomonas strains L2 and 719 were tested for the ability to grow and maintain viability in drinking water. Microcosms were employed in the study to monitor growth and survival by plate counts, acridine orange direct counts (AODC), and direct viable counts (DVC). Plate counts dropped below the detection limit within 7 days for all strains except those of Bacillus and Pseudomonas. In all cases, the AODC did not change. The DVC also did not change except that the DVC, on average, were ca. 10-fold lower than the AODC.