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.

Development of HPLC with fluorescent detection using NBD-F for the quantification of colistin sulfate in rat plasma and its pharmacokinetic applications.

Colistin sulfate, composed of a mixture of colistin A sulfate (CLA) and colistin B sulfate (CLB), is available for treating life-threatening infections caused by multidrug-resistant Gram-negative bacteria. In this study, the CLA and CLB were quantified separately. Colistin sulfate was extracted from rat plasma with a solid-phase extraction C18 cartridge and reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), and the fluorescent derivatives were subjected to reversed-phase high-performance liquid chromatography analysis and used to investigate the pharmacokinetics of CLA and CLB in rat plasma. The recovery rates of CLA and CLB were 41.2 ± 4.4 and 45.5 ± 3.1%, respectively. The recovery rate calculated from the total area of CLA and CLB was 43.9 ± 3.6%. When 2 mm NBD-F and 10 mm boric acid buffer (pH 9.5) were added to colistin sulfate, the highest recovery rate was obtained. The best heating time was 5 min at 60°C. The lower limits of quantification for CLA, CLB and the total area of CLA and CLB were 0.05, 0.05 and 0.1 µg/mL; the coefficients of variations were 13.5, 14.5 and 14.1%, respectively. This method was found to have acceptable linearity, precision and accuracy, and has been successfully applied to a pharmacokinetic study in rat plasma.

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.

Pharmacokinetics of the Individual Major Components of Polymyxin B and Colistin in Rats.

The pharmacokinetics of polymyxin B1, polymyxin B2, colistin A, and colistin B were investigated in a rat model following intravenous administration (0.8 mg/kg) of each individual component. Plasma and urine concentrations were determined by LC-MS/MS, and plasma protein binding was measured by ultracentrifugation. Total and unbound pharmacokinetic parameters for each component were calculated using noncompartmental analysis. All of the polymyxin components had a similar clearance, volume of distribution, elimination half-life, and urinary recovery. The area under the concentration-time curve for polymyxins B1 and B2 was greater than those of colistins A and B. Colistin A (56.6 ± 9.25%) and colistin B (41.7 ± 12.4%) displayed lower plasma protein binding in rat plasma compared to polymyxin B1 (82.3 ± 4.30%) and polymyxin B2 (68.4 ± 3.50%). These differences in plasma protein binding potentially equate to significant differences in unbound pharmacokinetics, highlighting the need for more stringent standardization of the composition of commercial products currently available for clinical use.

Characterization of the colistin (polymyxin E1 and E2) biosynthetic gene cluster.

Colistin is a mixture of polymyxin E1 and E2, bactericidal pentacationic lipopeptides used to treat infections caused by Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Industrial production of colistin is obtained by a fermentation process of the natural producer Paenibacillus polymyxa var colistinus. NonRibosomal peptide synthetases (NRPS) coding the biosynthesis of polymyxins A, B and P have been recently described, rendering thereof the improvement of their production possible. However, the colistin biosynthesis pathway was not published so far. In this study, a Paenibacillus alvei has been identified by biochemical (Api 50 CH system) and molecular (16S rDNA sequencing) methods. Its culture supernatant displayed inhibitory activity against Gram-negative bacteria (P. aeruginosa, K. pneumoniae, Salmonella spp.). Two polymyxins, E1 and E2, were recovered from the supernatant and were characterized by high resolution LC-MS. A genomic library (960 clones) was constructed to identify the gene cluster responsible for biosynthesis of polymyxins. Selection of the clones harbouring the sequences of interest was obtained by a simple PCR-based screening. We used primers targeting NRPS sequences leading to the incorporation of amino acids present in polymyxins E. The sequences from three clones of interest were assembled on 50.4 kb. Thus, five open reading frames corresponding to a new NRPS gene cluster of 41 kb were identified. In silico, analyses revealed the presence of three NRPS implicated in the biosynthesis of polymyxins E. This work provides insightful information on colistin biosynthesis and might contribute to future drug developments in this group of antibiotics.

Study on anaerobic digestion treatment of hazardous colistin sulphate contained pharmaceutical sludge.

Pharmaceutical sludge is considered as a hazardous substance with high treatment and disposal fees. Anaerobic digestion could not only transform the hazardous substance into activated sludge, but also generate valuable biogas. This research had two objectives. First: studying the feasibility of anaerobic digestion and determining the biochemical methane potential (BMP) of pharmaceutical sludge under different Inoculum to substrate TS ratios (ISRs) of 0, 0.65, 2.58 and 10.32 in mesophilic condition of 37±1°C. Secondly, investigating the removal efficiency of colistin sulphate during anaerobic digestion. The results showed that the use of anaerobic digestion to treat the pharmaceutical sludge is feasible and that it can completely eliminate the colistin sulphate. The highest biogas production from pharmaceutical sludge is 499.46 mL/g TS at an ISR of 10.32.

Multicomponent antibiotic substances produced by fermentation: implications for regulatory authorities, critically ill patients and generics.

Teicoplanin and polymyxin E (colistin) are antibiotics consisting of multiple, closely related subcomponents, produced by fermentation. The principal components comprise a complex mixture of chemically related, active substances (teicoplanin A(2-1)-A(2-5) and polymyxin E(1-2), respectively), which might be required to be present in specific ratios to ensure optimal antibacterial and clinical efficacy. These subcomponents differ in their fatty acid and amino acid composition and, as such, the lipophilic and protein binding characteristics differ between components. This has therapeutic implications for critically ill patients, as the volume of distribution of the teicoplanin A2 and polymyxin E analogues at the onset of an intravenous infusion may impact on expected pharmacokinetics and influence outcome.

Effect of sub-minimum inhibitory concentrations of ciprofloxacin, amikacin and colistin on biofilm formation and virulence factors of Escherichia coli planktonic and biofilm forms isolated from human urine

The aim of this study was to determine the effect of subinhibitory concentrations (sub-MICs) of ciprofloxacin, amikacin and colistin on biofilm formation, motility, curli fimbriae formation by planktonic and biofilm cells of E. coli strains isolated from the urine of patients with various urinary system infections. Quantification of biofilm formation was carried out using a microtiter plate assay and a spectrophotometric method. Bacterial enumeration was used to assess the viability of bacteria in the biofilm. Curli expression was determined by using YESCA agar supplemented with congo red. Using motility agar the ability to move was examined. All the antibiotics used at sub-MICs reduced biofilm formation in vitro, decreased the survival of bacteria, but had no effect on the motility of planktonic as well as biofilm cells. The inhibitory effect of sub-MICs of antimicrobial agents on curli fimbriae formation was dependent on the form in which the bacteria occurred, incubation time and antibiotic used. Our results clearly show that all the three antibiotics tested reduce biofilm production, interfere with curli expression but do not influence motility. This study suggests that ciprofloxacin, amikacin and colistin may be useful in the treatment of biofilm-associated infections caused by E. coli strains

Analysis of colistin A and B in fishery products by ultra performance liquid chromatography with positive electrospray ionization tandem mass spectrometry.

A rapid and simple method for the determination of colistin A and B in fishery products by reversed phase ultra performance liquid chromatography with positive electrospray ionization tandem spectrometry (UPLC-ESI-MS/MS) method was described. The samples were extracted with 1.0 mol/L of hydrochloric acid in methanol-water and then purified on the PLS solid phase extraction columns. Then the eluate was evaporated to less than 1 mL under a gentle stream of nitrogen at 40 °C and formic acid-acetonitrile-water (0.2/10/90, v/v/v) was added to adjust volume to 1 mL final volume. An aliquot (10 µL) was injected onto the LC column for analysis with the mobile phase of 0.2% formic acid in acetonitrile and 0.2% formic acid in water at 0.20 mL min⁻¹. Multiple reaction monitoring was performed using precursor-product ion combinations. Calibration curves were linear from 200 ng/mL to 2000 ng/mL for colistin A and B. Mean recoveries were between 72.9% and 82.9%. The LOD was 10.0 µg/kg and LOQ was 40.0 µg/kg. The intra-day assay precision values for QC samples were between 2.17% and 9.00%, and inter-day values were between 2.80% and 6.97%. The method has merits of simplicity, sensitivity and rapidity, and it can be used for the determination of colistin A and B in fishery products.

Strategies to evade false positives in the in situ analysis of peptide antibiotics in SDS-PAGE gels.

In situ activity assay is one of the promising techniques for the characterization of peptide antibiotics. This assay was carried out for the peptide purified from a new bacterial isolate Paenibacillus alvei and commercial peptide antibiotic polymixin E. Towards this, the routine and new protocols were tried. Interestingly, the unexpected result of these experiments - the "switch over activity" has led us to have further investigations. Here, we have addressed the potential problem in the methodology of in situ assay and demonstrated a fool proof protocol to evade the false positive results.

Polymyxin E production by P. amylolyticus.

AIMS: To investigate antibiotic production by bacteria isolated from the hindgut of Tipula abdominalis, the aquatic crane fly. METHODS AND RESULTS: A group of five isolates with 99.1% 16S rRNA sequence similarity to Paenibacillus amylolyticus were identified as antibacterial producers using the cross-streak method against both Gram-positive and Gram-negative bacteria. For one isolate, P. amylolyticus C27, biochemical tests were performed to confirm 16S rRNA identification and the antibacterials were purified using chromatographic methods. Postsource decay (PSD) mass spectroscopy (MS) was used to identify the antimicrobials, which were found to be polymyxins E(1) and E(2). Investigation of the remaining four isolates using PSD MS revealed they all produce polymyxins E(1) and E(2) as well. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Although variants of the polymyxin antibiotics are known to be produced by several species within the Paenibacillus genus, this first investigation of antibacterial production by bacteria isolated from the hindgut of T. abdominalis describes a novel source for polymyxin E production as well as the first report of antibiotic production by P. amylolyticus.

Isolation and structural characterization of colistin components.

Preparative-scale separation of colistin sulphate bulk sample was carried out on a preparative poly(styrene-divinylbenzene) stationary phase. Isocratic elution with acetonitrile-sodium sulphate solution (0.7% m/v; pH adjusted to 2.5 with TFA) - water (16:50:34, % v/v/v) was carried out at a flow rate of 4.0 ml min(-1). Six colistin components were isolated and characterized using 1H and 13C NMR. The molecular weights were confirmed by mass spectrometry. The structures of 2 components were determined for the first time. Polymyxin E7 was identified as having the same composition as polymyxin E1, except that the fatty acid moiety was 7-methyloctanoic acid. Isoleucine polymyxin E8 was characterized as having the same composition as isoleucine polymyxin E1 with 7-methylnonanoic acid as the fatty acid moiety.