A portable fluorescent microsphere-based lateral flow immunosensor for the simultaneous detection of colistin and bacitracin in milk.

The polypeptide antibiotics colistin (COL) and bacitracin (Baci) are extensively used as veterinary drugs and feedstock additives in the livestock industry, which inevitably causes residues in animal-origin food, which can accelerate human tolerance to antibiotics. In this study, a portable lateral flow immunoassay (LFIA) for the simultaneous determination of COL and Baci residues in milk was developed. The replacement of gold nanoparticles used in the traditional LFIA with fluorescent microspheres (FMs) to label monoclonal antibodies (mAbs) allowed qualitative and quantitative analyses within a few minutes. Based on the principle of competitive binding to FM-labelled mAbs between analytes in samples and fixed antigens on the membrane, the assay provided qualitative cut-off values of 100 and 50 ng mL-1 for Baci and COL in milk samples. Furthermore, a strip reader-based semi-quantitative detection system could detect lower limits of 7.85 and 1.89 ng mL-1 for Baci and COL, respectively. In conclusion, the proposed multiplex LFIA immunosensor provides an auxiliary analytical tool for the rapid and simultaneous screening of COL and Baci in large cohorts of samples.

UVC- and UVC/H2O2-Driven nonribosomal peptide antibiotics degradation: application to zinc bacitracin as a complex emerging contaminant.

Zinc bacitracin (Zn-Bc) belongs to the group of nonribosomal peptide antibiotics (NRPA), comprising a mixture of non-biodegradable congeners characterized by complex structures containing cyclic, polycyclic, and branched chains. However, reports on the use of AOPs for the degradation of NRPA are non-existent. In this context, the present work investigated the photodegradation of Zn-Bc in aqueous solution by direct photolysis and the UVC/H2O2 process. The effects of the specific UVC photon emission rate and initial H2O2 concentration were studied following a Doehlert-design response surface approach. The results showed that all congeners photolyzed at the highest UVC doses in the absence of hydrogen peroxide, with a calculated quantum yield of 0.0141 mol Zn-Bc mol photons-1. However, no TOC removal was observed after 120 minutes of irradiation, suggesting the disruption of the peptide bonds in the antibiotic molecules without significant changes in the amino acid residues. The addition of H2O2 substantially accelerated Zn-Bc photodegradation, resulting in a remarkable removal of up to 71% of TOC. Most importantly, the antimicrobial activity against Staphylococcus aureus could be completely removed by both treatments. These findings point out that the UVC/H2O2 process can be straightly engineered for the treatment of metalloantibiotics-containing wastewater in pharmaceutical facilities.

Simultaneous detection of bacitracin and polymyxin B in livestock products using liquid chromatography with tandem mass spectrometry.

With the overarching aim to develop a simple and reliable method for the quantitative analysis of polypeptide antibiotics in various livestock products, the content of bacitracin, and polymyxin B in pork, beef, chicken, milk, and eggs was analyzed using colistin sulfate as an internal standard. The extracted samples were eluted via solid-phase extraction using 2% formic acid in acetonitrile/methanol (1:1, v/v). The two polypeptides were identified and quantified based on the intensities of mass fragments from the respective triply charged precursor ions (bacitracin: 474.97 amu and polymyxin B: 402 amu) at the defined retention time windows using liquid chromatography with electrospray ionization tandem mass spectrometry in time-scheduled multiple reaction monitoring mode. The calibration curves showed good linearity over the concentration range 50-2500 ng/mL with determination coefficients ≥ 0.991. The mean recoveries were in the range 80.3-88.8% with relative standard deviations <13% for all samples. The limits of quantitation ranged from 30-250 ng/g. The developed method was applied to market samples, but the target analytes were not detected in any of the samples. The developed method is reliable for the simultaneous detection of bacitracin and polymyxin B in pork, beef, chicken, milk, and eggs.

Optimal conditions for determination of bacitracin, bacitracin zinc and bacitracin methylene disalicylate in animal feed by ultra-performance liquid tandem mass spectrometry.

An integrated method combining solid-phase extraction (SPE) with ultra-performance liquid tandem mass spectrometry (UPLC-MS/MS) has been established for quantifying bacitracin (BTC), bacitracin zinc (BZ), and bacitracin methylene disalicylate (BMD) in animal feed. A pretreatment procedure that can effectively, quickly, and simultaneously extract and purify BTC, BZ, or BMD in feed was developed for the first time through the optimization of extraction and SPE conditions. After extraction with acetonitrile + methanol + 15 % ammonia solution (1:1:1, v:v:v) and dilution with EDTA solution (1.5 mmol/L, pH 7.0), a SPE procedure was carried out with C18 cartridge. Following LC-MS/MS analysis utilized a Waters Peptide BEH C18 column with a gradient elution of 0.1 % formic acid in water/acetonitrile with. This method demonstrated a strong linear correlation (R2 > 0.9980) across a 0.01-1.0 mg/L concentration span, based on a matrix-matched standard curve. Satisfactory recoveries of BTC (bacitracin A, B1, B2, and B3), BZ, and BMD in different feeds were obtained from 80.7 % to 108.4 %, with relative standard deviations below 15.7 %. Low limits of quantification ranging within 7.2-20 µg/kg were achieved for bacitracin A, B1, B2, and B3. This method provided an effective and reliable detection method to prevent the addition of BTC and different BTC formulations in feeds.

Role of three-body recombination for charge reduction in MALDI process.

Ions in Matrix-Assisted Laser Desorption/Ionization (MALDI) are predominantly singly charged for small analyte molecules. With the estimated high number density and low temperature of electrons, the three-body recombination mechanism is attractive and should be considered as an important cause for the charge reduction in the plume. Theoretical calculations indicate that the rate coefficient of the three-body recombination is about 50 times higher than that of the two-body recombination if the analyte molecule has insufficient degrees of freedom. Experimental results show that, for small analyte molecules, the ratio of AH2(2+)/AH(+) is close to the theoretical 5% value from the three-body recombination modeling and this ratio declines with the increasing electron and matrix molecule number density caused by greater laser irradiance. The ratio of [A + 2](+)/[A + 1](+) is higher than the theoretical isotopic value, and the excess [A + 2](+) could exclusively result from the three-body recombination collisions. Further evidence demonstrates that [A + 2](+)/[A + 1](+) increases with electron number density, which is in correspondence with the model. All of these theoretical and experimental results indicate that three-body recombination is an essential charge reduction mechanism for small molecules in the MALDI plume.

A novel cell-associated protection assay demonstrates the ability of certain antibiotics to protect ocular surface cell lines from subsequent clinical Staphylococcus aureus challenge.

In vivo effectiveness of topical antibiotics may depend on their ability to associate with epithelial cells to provide continued protection, but this contribution is not measured by standard antibiotic susceptibility tests. We report a new in vitro method that measures the ability of test antibiotics azithromycin (AZM), erythromycin (ERY), tetracycline (TET), and bacitracin (BAC) to associate with mammalian cells and to protect these cells from destruction by bacteria. Mammalian cell lines were grown to confluence using antibiotic-free medium and then incubated in medium containing a single antibiotic (0 to 512 µg/ml). After incubation, the cells were challenged with Staphylococcus aureus ocular isolates, without antibiotics added to the culture medium. Epithelial cell layer integrity was assessed by gentian violet staining, and the minimum cell layer protective concentration (MCPC) of an antibiotic sufficient to protect the mammalian cells from S. aureus was determined. Staining was also quantified and analyzed. Bacterial viability was determined by culture turbidity and growth on agar plates. Preincubation of Chang and human corneal limbal epithelial cells with AZM, ERY, and TET at ≥64 µg/ml provided protection against AZM-susceptible S. aureus strains, with increasing protection at higher concentrations. TET toxicity was demonstrated at >64 µg/ml, whereas AZM displayed toxicity to one cell line at 512 µg/ml. BAC failed to show consistent protection at any dose, despite bacterial susceptibility to BAC as determined by traditional antibiotic susceptibility testing. A range of antibiotic effectiveness was displayed in this cell association assay, providing data that may be considered in addition to traditional testing when determining therapeutic dosing regimens.

Spectrophotometric determination of bacitracin in bulk drug as dabsyl derivative in a range of visible light.

A fast spectrophotometric method has been developed for bacitracin identification and determination after condensation reaction with dabsyl chloride. In addition, determination of dye stability of sulfonamide derivative and identification of the molar ratio of reagents was done at various time-points. The developed method has a good linearity with very broad spectrum, correlation coefficient of r = 0.9972, good precision (RSD = 1.54 +/- 0.11%), and recovery at three different levels of concentration was found between 98.33% and 103.47%. Usefulness of the method was demonstrated by positive results obtained during determination of bacitracin concentration in bulk drug.

Development of a liquid chromatography-tandem mass spectrometry method to determine colistin, bacitracin and virginiamycin M1 at cross-contamination levels in animal feed.

Cross-contamination of animal feed with antibiotics may occur during manufacturing in feed mills, because shared production lines can be used for medicated and non-medicated feed, but may also occur during transport, storage and at the farm level. This is a major issue in the current context where antimicrobial usage must be controlled in order to maintain their effectiveness. A LC-MS/MS method was developed for the determination of colistin, bacitracin A and virginiamycin M1 in feed for pigs, poultry and rabbits at concentrations similar to those encountered in cross-contamination. After investigating various issues related to colistin behaviour and matrix effects, we successfully validated this method according to the requirements of European regulations in terms of linearity, trueness, precision, limit of quantification and limit of decision. Trueness ranged 88.6-107.8% and precision ranged 12.6-21.2%. We then applied this method to the analysis of medicated pig feed to check the performance of the method on "real" samples of medicated feed. We subsequently analysed non-medicated pig, and rabbit feed samples, collected directly on farms, to check the rate of cross-contamination. No samples were contaminated by colistin, bacitracin, or virginiamycin.

Colloidal gold-based immunochromatographic strip assay for the rapid detection of bacitracin zinc in milk.

In this study, a murine monoclonal antibody (mAb) of 6D2-G10 against bacitracin zinc (BAC) was produced and applied to an immunochromatographic strip (ICS) for the initial detection of BAC in milk. The ICS with a cut-off value of 25 ng/mL could be perceived by the naked eye within 10 min. With the assist of the strip reader, the limit of detection (LOD) was measured as 0.82 ng/mL, the half-maximal inhibitory concentration (IC50) was recorded as 3.16 ng/mL, and the linear detection range was from 0.97 to 10.30 ng/mL. The recoveries ranged from 87.7% to 96.0% with the highest coefficient of variation (CV) of 9.1% in the intra-assay and from 84.3% to 90.2% with the highest CV of 10.7% in the inter-assay. In short, the established ICS provided a serviceable analytical tool for qualitatively and quantitatively monitoring BAC in milk.

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.

Comparative recovery of Streptococcus mutans on five isolation media, including a new simple selective medium.

For the isolation of Streptococcus mutans, several selective media have been developed, of which Mitis-Salivarius Sucrose Bacitracin agar (MSB) is the most widely used (Gold et al., 1973). Recently, the Trypticase Yeast-Extract Cystine agar medium (TYC, de Stoppelaar et al., 1967) was modified into a selective medium for S. mutans, called Trypticase Yeast-Extract Cystine Sucrose Bacitracin (TYCSB, van Palenstein Helderman et al., 1983). The aim of this study was to compare the recovery of S. mutans from clinical samples on Mitis-Salivarius agar (MS), MSB, TYC, and TYCSB. Further, a new simple selective medium for S. mutans was introduced. This medium, called TSY20B, was supposed to have the same qualities as TYCSB, but its preparation is less laborious. One hundred eighty-five plaque and saliva samples from 37 subjects were plated on MS, MSB, TYC, and TYCSB, and 285 samples from 23 subjects were plated on TYCSB and TSY20B. All plates were incubated at 37 degrees C in a 91% N2, 5% CO2, 4% H2 atmosphere for five days. The S. mutans counts on TYC and TYCSB were significantly higher than on MS or MSB by almost a factor of 10. Seventy-seven percent of the samples gave higher S. mutans counts on TYCSB than on MSB. Especially, samples with high S. mutans d/g numbers gave lower S. mutans counts on MSB. These data clearly indicate that MSB agar is inhibitory for S. mutans and should not be used. An additional advantage of TYCSB over MSB agar is the possibility of distinguishing S. mutans serotypes d/g from other serotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatography determination of Zn-bacitracin in animal feed by post-column derivatization and fluorescence detection.

A sensitive and selective method is presented for the determination of Zn-Bacitracin in adulterated animal feed by reversed-phase ion-pair high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde prior to fluorescence detection. The calibration function was estimated to be between 8.0 and 65.0 mg l(-1) of Zn-BC. The detection and quantification limits of the chromatographic method were 2.5 and 7.5 mg 1(-1), respectively. Using the extraction procedure of Zn-Bacitracin from the feedstuff that we recently proposed and applying this new chromatographic method, it was possible to detect this antibiotic at levels below 5 mg kg(-1) in different kinds of feedstuffs with a standard deviation less than 6.0%.

Peptide separation by pH-zone-refining countercurrent chromatography.

Peptides without protecting groups have been successfully separated by pH-zone-refining countercurrent chromatography (CCC) using an ion-pair reagent, di-(2-ethylhexyl)phosphoric acid (DEHPA), as a modifier in the stationary phase. Preliminary studies indicated that two parameters, i.e., the DEHPA concentration in the stationary phase and hydrophobicity of the solvent system should be adjusted according to the hydrophobicity of the analytes. Hydrophobic and hydrophilic groups of dipeptides were each separated under optimized conditions. The method was successfully applied to gram-quantity separations of bacitracin complex and bovine insulin.

New color reaction for determination of bacitracin in ophthalmic ointments.

A colorimetric method was developed for the rapid analysis of bacitracin in ophthalmic ointments. The method involves the oxidation of alpha-aminocarboxylic acid with sodium hypobromite in an alkaline medium and condensation of the resulting aldehyde with phloroglucinol in concentrated hydrochloric acid to yield a pink color which gives an absorbance maximum at 505 nm. The relationship between absorbance and the quantity of bacitracin reacted obeyed Beer's law over the 15-100-mug/ml concentration range studied, and the produced color was stable for several hours. Furthermore, the method, which can be applied directly to the aqueous dissolution sample, gave results comparable to the official microbiological analytical procedure. The standard deviation is equal to plus or minus 1.81%.

High-performance liquid chromatographic analysis of neomycin in petrolatum-based ointments and in veterinary formulations.

A high-performance liquid chromatographic (HPLC) method has been developed for the assay of neomycin in petrolatum-based ophthalmic and topical ointments and in veterinary formulations. Neomycin assay interferences from drugs, such as bacitracin and polymyxin B and inactive components, e.g. wax, were eliminated by a methanol wash and/or a partitioning method. The extracted neomycin was derivatized with 2,4-dinitrofluorobenzene followed by normal-phase HPLC with detection at 254 nm. The average recovery of neomycin from spiked samples was approximately 100% with a relative standard deviation of less than 1%.

Analysis of lipophilic peptides and therapeutic drugs: on-line-nonaqueous capillary electrophoresis-mass spectrometry.

This minireview addresses the usefulness of nonaqueous capillary electrophoresis-mass spectrometry (NACE-MS), mainly in the analysis of lipophilic peptides such as gramicidin S and bacitracin, and therapeutic drugs such as pyrazoloacridine, the H2-antagonist mifentidine, tamoxifen, and their metabolites. The beneficial effects of NACE-MS in typical bioanalytical applications are analyzed case by case. A suitable and widely applicable NACE-MS analysis is identified, which is an electrolyte buffer containing ammonium acetate (5-50 mM) and/or acetic acid (up to 100 mM) with varying composition of organic solvents. Either acetonitrile or methanol or a mixture of the two are mostly utilized in the nonaqueous media. Primary considerations in developing NACE-MS are also discussed.

Structural characterization of bacitracin components by Frit-fast atom bombardment (FAB) liquid chromatography/mass spectrometry (LC/MS).

The structural characterization of minor components of bacitracin (BC) complex was carried out using a technique of liquid chromatography/mass spectrometry (LC/MS). Satisfactory total ion current chromatogram of BC complex and excellent mass spectra of many components were given by Frit-fast atom bombardment (FAB) LC/MS analytical system, and the structures of 13 minor components could be proposed. The 13 minor components were classified into two groups, bacitracin A (BC-A) related components and bacitracin F (BC-F) related components depending on their common N-terminal moieties. The structures of BC-A related components and BC-F related components were the same as those of BC-A and BC-F, respectively, except that one to three of isoleucine and leucine residues are replaced by valines. The BC-F related components were degradation products of BC-A related components through the same degradation process as that of BC-A.

Flow microcalorimetric assay of antibiotics--I. Polymyxin B sulphate and its combinations with neomycin sulphate and zinc bacitracin on interaction with Bordetella bronchiseptica (NCTC 8344).

A flow microcalorimetric assay for polymyxin B sulphate has been developed which has a better reproducibility (relative standard deviation less than 3%) and sensitivity (0.35 micrograms ml-1) than conventional microbiological assays, and requires an assay time of ca. 4.5 h. The combinations with zinc bacitracin, with neomycin sulphate, and with both zinc bacitracin and neomycin sulphate indicate antagonism between these antibiotics upon interaction with Bordetella bronchiseptica (NCTC 8344). The combinations of all three antibiotics assayed were: (1) equimolar proportions; and (2) those proportions present in the commercial preparation TrisepR (ICI, Macclesfield, UK).

Flow microcalorimetric assay of antibiotics--II. Neomycin sulphate and its combinations with polymyxin B sulphate and zinc bacitracin on interaction with Bacillus pumilus (NCTC 8241).

A flow microcalorimetric assay for Neomycin has been developed which is monitored through interaction of the antibiotic with Bacillus pumilus as the test organism. The assay has better reproducibility (relative standard deviation 2.3%) and is more sensitive than conventional microbiological bioassay (0.5-2 micrograms ml-1). The effects of combinations with zinc bacitracin, with polymyxin B sulphate, and with both zinc bacitracin and polymyxin B sulphate (both in equimolar proportions), and in those proportions present in the commercial preparation TrisepR (ICI, Macclesfield, UK) have also been investigated. Synergy was observed for the combinations of Neomycin with the other two antibiotics in binary mixtures at the relative proportions found in TrisepR. The addition of all three antibiotics at the levels used in TrisepR did not show synergy. However, addition of all three antibiotics at equimolar concentrations did show synergy. It is suggested that microcalorimetry may be useful in in vitro experiments for exploring the relative proportions required for maximal effect in antibiotic combinations.

Flow microcalorimetric assay of antibiotics--III. Zinc bacitracin and its combinations with polymyxin B sulphate and neomycin sulphate on interaction with Micrococcus luteus.

A flow microcalorimetric assay for zinc bacitracin has been developed which has better reproducibility (relative standard deviation less than 2%) and sensitivity (0.02 micrograms ml-1) than conventional microbiological assays, and requires an assay time of between 7.5-9 h. The assay is not suitable for zinc bacitracin determinations in the presence of equimolar concentrations of polymyxin B sulphate or neomycin sulphate, or of these antibiotics in the proportions in which they occur in the commercial preparation Trisep (ICI, Macclesfield, UK).