Determination of Polypeptide Antibiotic Residues in Food of Animal Origin by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

A novel UHPLC-MS/MS method for the determination of polypeptide antibiotic residues in animal muscle, milk, and eggs was developed and validated. Bacitracin A, colistin A, colistin B, polymyxin B1, and polymyxin B2 were extracted from the samples with a mixture of acetonitrile/water/ammonia solution 25%, 80/10/10 (v/v/v), and put through further evaporation, reconstitution, and filtration steps. The chromatographic separation was performed on a C18 column in gradient elution mode. Mass spectral acquisitions were performed in selective multiple reaction monitoring mode by a triple quadrupole mass spectrometer. The method was validated according to the criteria of Commission Decision 2002/657/EC. The method quantifies polypeptides in a linear range from 10 to 1000 µg kg-1, where the lowest concentration on the calibration curve refers to the limit of quantification (LOQ). The recoveries ranged from 70 to 99%, the repeatability was below 13%, and within-laboratory reproducibility was lower than 15%. The decision limit (CCα) and detection capability (CCß) values were calculated, and ruggedness and stability studies were performed, to fulfill the criteria for confirmatory methods. Moreover, the developed method may also be used for screening purposes by its labor efficiency.

A Novel Peptide Oligomer of Bacitracin Induces M1 Macrophage Polarization by Facilitating Ca2+ Influx.

Antimicrobial peptides (AMPs) are components of the innate immune system and form the first defense against pathogens for various organisms. In the present study, we assessed whether CSP32, a novel AMP oligomer of bacitracin isolated from a strain of Bacillus spp., regulates the polarization of murine macrophage-like RAW 264.7 cells. CSP32 stimulated phagocytosis while inducing the appearance of the typical M1 polarized macrophage phenotype; these M1 macrophages play a role in host defense against pathogens. Furthermore, our results showed that CSP32 enhanced the expression and production of pro-inflammatory mediators, such as cytokines and chemokines. In addition, the CSP32-stimulated inflammatory mediators were induced mainly by the mitogen-activated protein kinase/nuclear factor kappa B (MAPK/NF-κB) signaling pathway during M1 macrophage polarization. In particular, CSP32 markedly increased the numbers of Ca2+-positive macrophages while upregulating phospholipase C and activating protein kinase Cε. Furthermore, the inhibition of intracellular Ca2+ by BAPTA-AM, a Ca2+ chelator, significantly suppressed the CSP32-mediated phagocytosis, inflammatory mediator production, and NF-κB activation. In conclusion, our data suggested that CSP32-stimulated M1 macrophage polarization is dependent on the calcium signaling pathway and may result in enhanced immune capacities.

Analysis of Major Components of Bacitracin, Colistin and Virginiamycin in Feed Using Matrix Solid-phase Dispersion Extraction by Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry.

A quantitative LC-MS/MS method has been developed for simultaneous determination of bacitracin A, bacitracin B, colistin A, colistin B and virginiamycin M1 in feed. This rapid simple and effective extraction method was based on matrix solid-phase dispersion. Qualitative and quantitative analyses were performed by LC-ESI-MS/MS. CCß of polypeptide antibiotics upon the method ranged from 9.6 to 15.8 µg kg-1 and 19.4 to 27.5 µg kg-1, respectively. The limit of quantification of polypeptide antibiotics was 25 µg kg-1 in feed samples. The recoveries of polypeptide antibiotics spiked in feed samples at a concentration range of 25-100 µg kg-1 were found above 75.9-87.9% with relative standard deviations within days less than 15.7% and between days less than 20.6%. This rapid and reliable method can be used to efficiently separate, characterize and quantify the residues of polypeptide antibiotics in feed with advantages of simple pretreatment and environmental friendly.

Production of peptide antibiotics by Bacillus sp: GU 057 indigenously isolated from saline soil

A total of 112 soil samples were taken from differents areas of district D.I.Khan and Kohat (KPK) Pakistan and screened for production of antibiotics against the Micrococcus luteus and Staphylococcus aureus. Widest zone of inhibition (18mm) was produced by microorganism isolated from saline soil. The strain was later identified as Bacillus GU057 by standard biochemical assays. Maximum activity (18mm inhibition zone) was observed against Staphylococcus aureus after 48 hours of incubation at pH 8 and 4% concentration of glucose. The antibiotic was identified by autobiography as bacitracin. The Bacillus strain GU057 was confirmed as good peptide antibiotic producer and can effectively be indulged as biocontrol agent.

Systematic mutagenesis method for enhanced production of bacitracin by Bacillus licheniformis mutant strain UV-MN-HN-6

The purpose of the current study was intended to obtain the enhanced production of bacitracin by Bacillus licheniformis through random mutagenesis and optimization of various parameters. Several isolates of Bacillus licheniformis were isolated from local habitat and isolate designated as GP-35 produced maximum bacitracin production (14±0.72 IU ml-1). Bacitracin production of Bacillus licheniformis GP-35 was increased to 23±0.69 IU ml-1 after treatment with ultraviolet (UV) radiations. Similarly, treatment of vegetative cells of GP-35 with chemicals like N-methyl N'-nitro N-nitroso guanidine (MNNG) and Nitrous acid (HNO2) increased the bacitracin production to a level of 31±1.35 IU ml-1 and 27±0.89 IU ml-1 respectively. Treatment of isolate GP-35 with combined effect of UV and chemical treatment yield significantly higher titers of bacitracin with maximum bacitracin production of 41.6±0.92 IU ml-1. Production of bacitracin was further enhanced (59.1±1.35 IU ml-1) by optimization of different parameters like phosphate sources, organic acids as well as temperature and pH. An increase of 4.22 fold in the production of bacitracin after mutagenesis and optimization of various parameters was achieved in comparison to wild type. Mutant strain was highly stable and produced consistent yield of bacitracin even after 15 generations. On the basis of kinetic variables, notably Yp/s (IU/g substrate), Yp/x (IU/g cells), Yx/s (g/g), Yp/s, mutant strain B. licheniformis UV-MN-HN-6 was found to be a hyperproducer of bacitracin.

Isolation of bacillus subtilis MH-4 from soil and its potential of polypeptidic antibiotic production.

The genus Bacillus produces mainly polypeptide antibiotics such as bacitracin and polymyxin. Bacillus species were isolated from soil by soil sprinkle technique. And all were screened for the production of antibiotic. Bacillus subtilis MH-4 gave the maximum antimicrobial activity so finally selected for optimization. During optimization of culture conditions for Bacillus subtilis MH-4 best antibacterial activity was obtained at 96 hours of incubation period, at pH-8 and by using glycerol as carbon and L-glutamic acid as nitrogen source. Optimum temperature for antibiotic production was 37 degrees C. The antibiotic was confirmed to be bacitracin by paper chromatography. Antibiotic was further extracted successfully with 1-Butanol, and aqueous concentrate showed activity of 0.8 mg/ml. The antibiotic so produced was found to be narrow spectrum active against only Gram-positive bacteria.

Bacitracin reveals a role for multiple thiol isomerases in platelet function.

The platelet-specific integrin alphaIIb beta3 has endogenous thiol isomerase activity associated with the CXXC motifs within the beta subunit. Using a highly purified form of bacitracin, a thiol isomerase inhibitor, we now provide further evidence of the functional significance of this enzymatic activity in integrin activation. In addition, we demonstrate a role for multiple thiol isomerases in platelet function. This bacitracin prevented platelet aggregation to thrombin and collagen, and directly inhibited alphaIIb beta3 activation, as detected by PAC-1 binding. In parallel, bacitracin inhibited the endogenous thiol isomerase activity of purified alphaIIb beta3 with a 50% inhibitory concentration of 15.5 micromol/l. In order to determine whether the effects of bacitracin are solely mediated by inhibition of integrin enzymatic activity, we examined integrin-independent indices of platelet activation. We found bacitracin inhibited both platelet secretion (CD62P and CD63) and thromboxane (TxA2) production, with complete inhibition at different concentrations. Thus, we demonstrated a role for multiple thiol isomerases in platelet function. Taken together, these studies support a role for the endogenous integrin thiol isomerase activity in activation of alphaIIb beta3 and highlight the novel regulation of platelet function by other, as yet undefined thiol isomerases.

Fast separation of bacitracin on monolithic silica columns.

The development of isocratic and gradient stability indicating HPLC methods for bacitracin (Bc) and bacitracin zinc (BcZn), which are complex mixture of several related polypeptides, is described. The methods are based on a new type of reversed phase (RP-18e) monolithic silicagel stationary phase. Chromatographic experimental conditions used on conventional column with microparticles were adopted and further modified to achieve efficient separation of Bc. The influence of methanol and acetonitrile in combination with phosphate buffer was thoroughly studied to separate microbiologically active components A, B1, B2, B3 and their oxidative degradation products F, H1, H2 and H3. Chromatographic peaks of all the mentioned components were identified using compounds isolated previously by preparative HPLC. Applying isocratic or gradient approach, highly efficient separation was achieved together with drastically reduced analysis times (ca. 6 min) compared to all published HPLC methods up to date. With thus developed HPLC methods, it is possible to evaluate not only the main degradation product F, but for the first time also several other oxidative degradation products of Bc (H1, H2 and H3). Such methods are also suitable for routine quality control and stability testing. Validation of both isocratic and gradient methods confirmed the selectivity and efficiency comparable to that on microparticulate columns, yet contrary to conventional columns with highly reduced analysis time.

Screening for biologically active metabolites with endosymbiotic bacilli isolated from arthropods.

Endosymbiotic bacteria from the genus Bacillus were isolated from different compartments of the gut of various members of insects (Hexapoda) and millipedes (Diplopoda). They were grown in submerged culture and investigated by biological assays and HPLC-diode array analysis regarding their production of bioactive metabolites, which were isolated and determined in structure. Known compounds and yet unknown derivatives from the primary metabolism were detected, as well as antibacterially and antifungally acting peptide antibiotics.

Distribution and variation of bacitracin synthetase gene sequences in laboratory stock strains of Bacillus licheniformis.

Distribution and variation of bacitracin synthetase gene (bac) sequences in 22 laboratory stock strains of Bacillus licheniformis were studied by Southern hybridization of bac gene probes from B. licheniformis ATCC 10716 to genomic PstI or HindIII restriction fragments. Eleven strains gave hybridization signals. These hybridization patterns were classed into two types. Eight strains showed similar patterns to that of ATCC 10716 and all, except one, produced bacitracin. The three strains showed fairly different hybridization patterns from that of ATCC 10716, and one (ATCC 33632) of them produced bacitracin. None of the remaining 11 strains, including ATCC 14580 (type strain), gave any hybridization signals. All strains carrying bac gene sequences were erythromycin resistant. With one exception, all strains without bac gene sequences were erythromycin sensitive. These results show that B. licheniformis strains are divided into two groups with respect to presence of bac gene sequences and erythromycin resistance.

Simultaneous identification and quantitative determination of neomycin sulfate, polymixin B sulfate, zinc bacytracin and methyl and propyl hydroxybenzoates in ophthalmic ointment by TLC.

A thin layer chromatographic-densitometric method for identification and quantitation of neomycin sulfate, polymixin B sulfate, zinc bacytracin and auxiliary substances (methyl and propyl hydroxybenzoates) in ophthalmic ointment was developed. To separate these constituents the silica gel coated TLC plates and two mobile phases were used. The suitable mobile phases were: methanol-n-butanol-ammonia 25%-chloroform (14:4:9:12, v/v/v/v) for determination of antibiotics and n-pentane-glacial acetic acid (66:9, v/v) for methyl and propyl hydroxybenzoates. The antibiotic chromatograms were detected by using ninhydrin ethanol solution, while densitometric measurements were made at lambda = 550 nm. Hydroxybenzoates were identified by UV measurements at lambda = 260 nm. The constituents under consideration were well separated at sufficient detection level. The recovery for all constituents ranged from 98.08% to 104.95%.

Liquid chromatographic analysis of bacitracin methylene disalicylate in feed.

Because of its peptide structure, bacitracin is not chemically distinct from many matrixes such as feeds or residue samples. Thus, bacitracin must be isolated from the matrix components or chemically altered to form a distinct component. Because of the complexity of this problem, bacitracin is still analyzed almost exclusively by microbiological methods. However, advances in solid-phase extraction has made sample isolation from the matrix much more practical. In this investigation both strong-cation exchange and C8 columns were used to isolate bacitracin for liquid chromatographic (LC) analysis. Results of both LC and microbiological analyses are compared.

Total structures and antimicrobial activity of bacitracin minor components.

Total structures of 13 minor components of bacitracin (BC) were proposed, and their antimicrobial activities were investigated. The components of BC including bacitracins A (BC-A) and F (BC-F) were isolated by preparative HPLC and were hydrolyzed under acidic conditions. The resulting amino aids were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanineamide and were separated by HPLC to determine their absolute configurations. It was found that the N-terminal amino acids of BC-A and its related components were epimerized during the hydrolysis to yield their enantiomers. The formation of these artifactual amino acids suggests that our previously proposed structures of the BC minor components are incorrect; therefore, the structures were corrected based on these results. The structures of the BC minor components were the same as that of BCs-A and -F except that one to three of the L-isoleucines, including the N-terminal one, were replaced by L-valines. These structures were confirmed by tandem mass spectrometry under fast atom bombardment (FAB) conditions and Frit-FAB liquid chromatography/mass spectrometry. Based on the UV spectra of the BC components determined by photodiode array detection-HPLC analysis, a new systematic nonmenclature was proposed for the minor components. The isolated components were also used for the determination of their minimal inhibition concentrations and it was found that BC-A is 2 approximately 8 times more potent than the other minor components against strains of Micrococcus luteus and Staphylococcus aureus.

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.

Preparative high-performance liquid chromatographic separation and isolation of bacitracin components and their relationship to microbiological activity.

Bacitracin, a polypeptide antibiotic, is one of the most commonly used antibiotics in the world. The approved method of analysis for bacitracin is microbial. To correlate the microbiological method with a high-performance liquid chromatographic (HPLC) method, bacitracin was chromatographed using HPLC with ultraviolet detection and a YMC basic column. Adequate separation of the isomers was obtained to scale up this procedure to preparative HPLC using a Prep HPLC system and a 250 x 21 mm YMC basic column. The various fractions were separated, isolated and examined for microbial activity. The individual fractions could be precipitated by adding zinc or methylene disalicylic acid and lowering the pH. The crude fractions were recycled to ensure chromatographic purity. The chromatograms can accurately predict (in minutes) the microbiologically determined potency which usually takes 16-24 h to develop. The chromatographic procedure also provides information on the amounts of isomers and degradation products present in the sample, whereas the microbiological assay only provides activities or potencies of the antibiotic. The reported HPLC method also possesses some advantages over some other published HPLC methods in terms of accuracy and time of analysis.

Bio-flash chromatography--rapid, low-cost purification of peptides.

The use of wide pore 40/60 mu Sorbsil silicas bonded with a range of biocompatible phases now allows the biochemist to benefit from the advantages that flash chromatography has given to synthetic organic chemists. The technique of bio-flash chromatography allows rapid peptide and protein purification at low pressure (less than 15 psi) and at a fraction of the cost of high-pressure systems.

Isolation of bacitracins A and F by high-speed counter-current chromatography.

The major components of bacitracin were separated and purified using high-speed counter-current chromatography (HSCCC). A systematic search for optimum two-phase solvent systems resulted in two systems: chloroform-ethanol-methanol-water (5:3:3:4) and chloroform-ethanol-water (5:4:3). These were selected based on the determination of the partition coefficients of all the components and the settling time of the phases. HSCCC with these solvent systems separated two components, bacitracins A and F. Improvements in the flow-cell arrangement eliminated noise in detection, making in-line monitoring possible. A tandem mass spectrometric technique was used to characterize the isolated components.

Separation and isolation of the isomers of bacitracin by high-performance liquid chromatography and their relationship to microbial activity.

Bacitracin, a polypeptide antibiotic produced from strains of Bacillus licheniformis, is one of the most commonly used antibiotics in the world. Actually, the various products generally referred to as 'bacitracin' are mixtures of similar polypeptides which may differ by only one amino acid. The approved method of analysis for bacitracin is microbial. To correlate the microbiological method with an HPLC method, bacitracin was chromatographed using a YMC basic column with UV detection. Adequate separation of the isomers were obtained to scale up this procedure to preparative HPLC using a 250 x 21 mm YMC basic column. The various fractions were separated, isolated and examined for microbial activity. The chromatograms can accurately predict in minutes the microbiologically-determined potency which usually takes 16-24 h to develop. The chromatographic procedure also provides information on the amounts of isomers and degradation products present in the sample, whereas the microbiological assay only provides activities or potencies of the antibiotic. The reported HPLC method also possesses some advantages over some other published HPLC methods in terms of accuracy and time of analysis.

Improved high-speed counter-current chromatograph with three multilayer coils connected in series. II. Separation of various biological samples with a semi-preparative column.

The semipreparative capability of the newly developed high-speed counter-current chromatograph equipped with a set of three multilayer coils has been demonstrated in separations of a variety of biological samples including triterpenoic acids, indole auxins, bacitracin, flavonoids and tetracycline derivatives, each with a suitable two-phase solvent system. The sample quantities ranging from 50 to 500 mg were efficiently separated within a few hours. The separation of tetracycline derivatives was remarkably improved by adding ammonium acetate to the solvent system.

[Physico-chemical properties of the complexes of bacitracin with DNA and its effect on the process of transcription in vitro]. / Fiziko-khimicheskie svoistva kompleksov batsitratsina s DNK i vliianie ego na protsess transkriptsii in vitro.

The aim of the present paper was to study the action of one of the peptide antibiotics, bacitracin, as the regulator of gene activity at the transcription level. Therefore the commercial bacitracin has been fractionated into two main parts by paper chromotography. These two fractions have been identified as bacitracin A (biologically active) and bacitracin F (biologically inactive). The binding of both fractions to DNA has been studied. It has been shown that bacitracin A stabilizes DNA to a lesser degree than bacitracin F does. DNA-bacitracin complexes are formed in the major groove of the DNA helix by hydrogen bonds. The analysis of the the obtained experimental data allows us to suppose that bacitracin binding to DNA has a very specific character and that this antibiotic may act as the regulator of gene activity.