Laboratory evaluation of large-scale decontamination approaches.

AIMS: To evaluate the effectiveness of two spray-based decontamination methods for surface contamination reduction and to determine the potential for contamination spread by these methods. METHODS AND RESULTS: Material coupons (treated plywood and concrete) were contaminated with c. 1 × 10(7) spores of Bacillus atrophaeus by aerosol deposition. Decontaminants (pH-adjusted bleach or Spor-Klenz(®) RTU) were applied to coupons by either backpack sprayer or gas-powered sprayer. Contact time, reapplication frequency and rinse method were also varied. In addition to surface removal efficacy, partitioning of contamination between the rinsate and aerosol fractions was determined. Results indicated that pH-adjusted bleach was effective (≥6 logs reduction) when two applications and a 30 min contact time were administered, regardless of the decontaminant application method or material. Spor-Klenz(®) RTU was effective on wood, but achieved ≤3 logs reduction on concrete. A shortened application procedure with pH-adjusted bleach resulted in lower efficacy on wood, and a greater apparent potential for contamination spread. CONCLUSIONS: Consideration of material surface type is important when selecting a decontaminant. Also, achieving conditions that effectively inactivate surface biological contamination are critical to preventing the spread of contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: Results presented here are intended to help development of remediation plans following a biological contamination incident.

Effect of leader peptide mutations on biosynthesis of the lantibiotic Pep5.

Lantibiotics are lanthionine-containing antibiotic peptides which are synthesized from ribosomal prepeptides by post-translational modification. In order to elucidate the function of a conserved motif in the N-terminal leader sequence of lantibiotic prepeptides, three amino acids were exchanged in the leader peptide sequence of the lantibiotic Pep5. Exchanging Phe-19 for Ser and Glu-16 for Lys in the FDLEI-motif, reduced Pep5 production to 35 and 38% of the control whereas, after exchanging Asp-6 for Ser and Glu-16 for Lys in the FDLEI-motif, reduced Pep5 production to 35 and 38% of the control whereas, after exchanging ASp-6 for Lys, the production was decreased only to 82%. Proteolytic fragments of Pep5 or incorrectly modified Pep5 molecules, indicative of incorrect modifications, were not found in the culture supernatant. Thus, in contrast to the biosynthesis of the lantibiotic nisin, the FDLEI-motif is not essential for biosynthesis of Pep5 and has no influence on correct ring formation or processing, but seems to be important for optimal biosynthesis rates.

Kanchanamycins, new polyol macrolide antibiotics produced by Streptomyces olivaceus Tü 4018. I. Taxonomy, fermentation, isolation and biological activities.

The kanchanamycins, a group of novel 36-membered polyol macrolide antibiotics were detected in the culture filtrate and mycelium of Streptomyces olivaceus Tü 4018 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening. The compounds show antibacterial and antifungal activities, and are especially effective against Pseudomonas fluorescens. Besides the kanchanamycin complex, strain Tü 4018 produces the 42-membered macrolactones, oasomycin A and desertomycin A, as well as tryptophan-dehydrobutyrine diketopiperazine and daidzein.

Kanchanamycins, new polyol macrolide antibiotics produced by Streptomyces olivaceus Tii 4018. II. Structure elucidation.

Kanchanamycins are a new group of polyol macrolide antibiotics isolated from Streptomyces olivaceus Tü 4018. They all share a common bicyclic carbon skeleton formed by a 36-membered lactone ring and a 6-membered hemiacetal ring. A feature unusual for that class of macrolides is the terminal urea moiety observed in kanchanamycin A. The structures of the kanchanamycins were determined by electrospray MS and modern 2D NMR techniques. Due to substantial overlap of the signals intensive use of inverse detected heteronuclear correlation experiments (HSQC, HMBC, 2D-HSQC-TOCSY) was made.

Spirofungin, a new antifungal antibiotic from Streptomyces violaceusniger Tü 4113.

A new secondary metabolite was detected in the culture filtrate and extracts of Streptomyces violaceusniger Tü 4113 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening. The compound named spirofungin has a polyketide-spiroketal structure and shows various antifungal activities, particularly against yeasts.

Biosynthetic capacities of actinomycetes. 4. Echinoserine, a new member of the quinoxaline group, produced by Streptomyces tendae.

A new member of the quinoxaline group antibiotics has been detected by HPLC-diode-array screening. The main compound produced by Streptomyces tendae strain Tü 4031 showed a high degree of similarity in the UV-visible spectral region with echinomycin and their structural similarity was confirmed by structure elucidation using electron tandem mass spectrometry and 2D nuclear magnetic resonance. The new compound, named echinoserine, is a non-cyclic form of echinomycin, but it is not a biosynthetic precursor. Echinoserine is less antibiotically active than echinomycin.

Ethyl glucuronide: a marker of recent alcohol consumption with clinical and forensic implications.

A marker with a specific time spectrum of detection and both high sensitivity and specificity is required to diminish the clinically as well as forensically important gap on the time axis between short- and long-term markers of alcohol consumption like ethanol and CDT, GGT or MCV, respectively. Ethyl glucuronide (EtG) is a non-volatile, water-soluble, stable upon storage, direct metabolite of ethanol with a molecular weight of 222 g/mol that can be detected in body fluids for an extended time period after the complete elimination of alcohol from the body. We investigated 107 urine and 78 serum samples of a total of 107 inpatients in 4 groups: (1) 33 inpatients in acute alcohol withdrawal and long term treatment; (2/3) 29 and 15 addicted forensic psychiatric inpatients (#64 StGB, penal code); (4) 30 recently detoxified inpatients of a station for long term treatment by LC/MS-MS with the internal standard d5-EtG and additionally in the fourth group of patients also by gas chromatography/mass spectrometry (GC/MS). In 2 out of 33 inpatients of the first group, EtG could be determined 3 days after hospitalization; in an other subject, a relapse could be detected. In 2 out of 29 and in 1 out of 15 forensic inpatients of group 2 and 3, respectively--where neither clinical impression nor routine laboratory findings gave an indication for relapse--concentrations of EtG ranged between 0.1 and 18 mg/l in urine. For the serum samples of the 30 inpatients of group 4, we could demonstrate a total agreement for the results of the GC/MS and the LC/MS-MS method as to whether a sample was found to be positive or negative for EtG. We suggest that these results strengthen our earlier findings that ethyl glucuronide is a marker of alcohol consumption in general that can be detected for an extended time period after the complete elimination of alcohol from the body and a marker for relapse control with a specific time frame of detection intermediate between short- and long-term markers.

Development of a sensitive E-screen assay for quantitative analysis of estrogenic activity in municipal sewage plant effluents.

A simplified proliferation test with human estrogen receptor-positive MCF-7 breast cancer cells (E-screen assay) was optimized and validated for the sensitive quantitative determination of total estrogenic activity in effluent samples from municipal sewage plants. After solid phase extraction of 1 l sewage on either 0.2 g polystyrene copolymer (ENV+) or 1 g RP-C18 material and removal of the solvent, analysis of the extracts in the E-screen assay could be performed without any clean-up step. This was even possible with untreated sewage. Parallel extraction of four sewage samples on both different solid phase materials gave comparable quantitative results in the E-screen. A blank sample did not induce cell proliferation. As additive behaviour of the estrogenic response of single compounds was proven for two different mixtures each containing three xenoestrogens, total estrogenic activity in the sewage samples, expressed as 17 beta-estradiol equivalent concentration (EEQ), could be calculated comparing the EC50 values of the samples with those of the positive control 17 beta-estradiol. The detection limit of the E-screen method was 0.05 pmol EEQ/l (0.014 ng EEQ/l), the limit of quantification 0.25-0.5 pmol EEQ/l (0.07-0.14 ng EEQ/l). In total, extracts of nine effluent and one influent sample from five different municipal sewage plants in South Germany were analyzed in the E-screen. All samples strongly induced cell proliferation in a dose-dependent manner which was completely inhibited by coincubation with 5 nM of the estrogen receptor-antagonist ICI 182,780. The proliferative effect relative to the positive control 17 beta-estradiol (RPE) was between 30 and 101%. 17 beta-Estradiol equivalent concentrations were between 2.5 and 25 ng/l indicating a significant input of estrogenic substances via sewage treatment plants into rivers.

Determination of carbonyls using liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization.

A liquid chromatographic method for the determination of aldehydes and ketones based on mass spectrometric detection is described. Recently developed modular derivatizing agents are employed for analysis. These hydrazine reagents, e.g. 4-dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine (DMNTH), react with the carbonyl compounds with the formation of the respective hydrazones, which are separated by HPLC-MS with atmospheric pressure chemical ionization in the positive mode. Electrospray ionization may also be used for analysis. Particular focus is directed on various calibration approaches, including external calibration with standard solutions and internal calibration with a hydrazone standard of cyclobutanone, an aldehyde not likely to occur in real samples. A second approach for internal calibration is based on the 13C2-labelled acetaldehyde hydrazone standard. Different calibration approaches may then be used for the analysis of real samples. Limits of detection range from 2 x 10(-8) to 5 x 10(-8) mol L-1 for a series of hydrazones, including hydrazones of saturated aldehydes with alkyl chain lengths from 1 to 7 carbon atoms, and hydrazones of selected unsaturated and aromatic aldehydes as well as ketone hydrazones.

Oxidative decarboxylation of peptides catalyzed by flavoprotein EpiD. Determination of substrate specificity using peptide libraries and neutral loss mass spectrometry.

The flavoprotein EpiD catalyzes the COOH-terminal oxidative decarboxylation of the lantibiotic precursor peptide EpiA. Variations of the COOH-terminal heptapeptide S1FNSYCC7 of EpiA were used for determining the substrate specificity of EpiD. When Cys7 was replaced by serine, cysteine-amide, homocysteine, or a thioether amino acid residue, no reaction with EpiD was observed. Heptapeptide libraries with one variable amino acid residue at positions 1-7 of the peptide substrate S1FNSYCC7 were incubated with EpiD, and the reaction products were identified by neutral loss mass spectrometry. When the penultimate cysteine residue Cys6 of the substrate peptide was replaced with Ser, Thr, Ala, or Val, the reaction still occurred. Tyr5 could be replaced with other hydrophobic amino acid residues. Mass spectrometry was used to compare the kinetics of the reaction of EpiD with various peptides. Peptide sequencing of the reaction products was performed by tandem mass spectrometry, confirming that the last cysteine residue was modified. The removal of the acid COOH-terminal carboxyl group was confirmed by determination of the isoelectric points of the reaction products. To study the interaction between EpiA and EpiD, EpiA was coupled to N-hydroxysuccinimide-activated Sepharose HiTrap material; EpiD was only retarded under reducing conditions.

Ethyl glucuronide--a marker of alcohol consumption and a relapse marker with clinical and forensic implications.

Ethyl glucuronide (EtG) is a non-volatile, water-soluble, direct metabolite of ethanol that can be detected in body fluids and hair. We investigated urine and serum samples from three patient groups: (1) 33 in-patients in acute alcohol withdrawal; (2) 30 detoxified in-patients (treated for at least 4 weeks) from a 'motivation station'; and (3) 43 neuro-rehabilitation patients (non-alcoholics; most of them suffering from stroke, traumatic brain injury, Parkinson's disease etc.) using gas chromatography/mass spectrometry (GC/MS) with deuterium-labelled EtG as the internal standard and additionally in the second group of patients using liquid chromatography (LC/MS-MS). We found no correlation between the concentration of EtG in urine at hospitalization and the blood-ethanol concentration (r = 0.17), the time frame of detection (r = 0.5) or the total amount of clomethiazole required for the treatment of withdrawal symptoms (r = 0.28). In four out of 30 in-patients from the 'motivation station'--where neither clinical impression nor routine laboratory findings gave indications of relapse--concentrations of EtG in urine ranged between 4.2 and 196.6 mg/l. EtG concentrations in urine of between 2.89 and 23.49 mg/l were found in seven out of 43 neuro-rehabilitation patients using GC/MS. The GC/MS and the LC/MS-MS results showed a correlation of 0.98 with Pearson's correlation test and 1.0 with Spearman's correlation test. We suggest that EtG is a marker of alcohol consumption that can be detected for an extended time period after the complete elimination of alcohol from the body. When used as a relapse marker with a specific time frame of detection intermediate between short- and long-term markers, EtG fills a clinically as well as forensically important gap. Its specificity and sensitivity exceed those of all other known ethanol markers.

Electrospray mass spectrometry and tandem mass spectrometry of synthetic multicomponent peptide mixtures: determination of composition and purity.

Electrospray (ES) mass spectrometry and tandem mass spectrometry were used to evaluate the composition and purity of synthetic peptide mixtures ("peptide libraries") consisting of 48 peptides in equimolar amounts. For the structurally similar components of the mixtures little mass discrimination effects were found. The ion intensity distribution of the protonated molecular ions was found to be independent of the concentration. The peak heights in the ES mass spectra reflected the number of isobaric peptides in the mixture. By-products formed by incomplete removal of side chain protecting groups were detected fast and conveniently by tandem mass spectrometry. Daughter ion scans were used to identify common structural features of the peptides and the by-products. Parent ion and constant neutral loss scans allowed the detection of all peptides modified with the same protecting group.

Mass spectroscopic analysis of a novel enzymatic reaction. Oxidative decarboxylation of the lantibiotic precursor peptide EpiA catalyzed by the flavoprotein EpiD.

The epidermin biosynthetic reaction between the flavoprotein EpiD and the precursor peptide EpiA was investigated by reversed-phase chromatography and ion spray mass spectrometry. Several products with molecular masses 46 and 104 Da less than that of EpiA were observed; these results were confirmed by using an MBP-EpiD fusion protein as enzyme and the mutant peptides EpiAR-1Q and K-EpiA as substrates. The reaction was inhibited by Zn2+ ions. Modifications were localized in the C-terminal fragment of EpiA as shown by factor Xa cleavage of the products followed by mass spectrometry analysis. In addition, EpiD reacted with the precursor peptides and with proepidermin, indicating that the leader peptide is not necessary for the recognition of EpiA by EpiD. Sequence analysis of modified proepidermin revealed that at least the amino acids Ile(+1)-Tyr+20 are unmodified. The observed decrease in mass of 46 Da and the modification at the C terminus of EpiA is in agreement with the proposed enzymatic function of EpiD, the oxidative decarboxylation of the precursor peptide. In addition, the increased absorbance at 260 nm of the modified peptides indicates the presence of a thioenol group in the C-terminal proepidermin.

Identification of unusual amino acids in peptides using automated sequential Edman degradation coupled to direct detection by electrospray-ionization mass spectrometry.

The determination of the primary structure of peptides and proteins is routine in many laboratories; however, many of the obtained sequences are incomplete or can be misinterpreted when the samples contain unusual amino acids. Here we report the development of an automated peptide sequenator coupled to an electrospray-ionization (ESI) mass spectrometer (MS) that, in conjunction with minor modifications to the sequencing conditions and, in some cases, prior derivatization of amino acids, allows the detection of the phenylthiohydantoin (PTH) derivatives of a number of unusual amino acids. Using the coupled sequenator-ESI-MS system we were able to determine the complete sequence of the lantibiotic gallidermin, a partial sequence of the calcium-dependent peptide antibiotic CDA2 as well as the pool sequence of a mixture of synthetic peptides containing nonproteinogenic amino acids. In addition to the 20 proteinogenic amino acids, the procedure was able to detect PTH derivatives of hydroxyphenylglycine, 2,3-didehydroasparagine, 3-methylglutamic acid, oxytryptophan, ornithine, N-methylglycine, dihydroxyphenylalanine, and alpha-aminoisobutyric acid. Similarly, after a simple derivatization procedure, we were also able to correctly identify educts of 2,3-didehydroalanine, 2,3-didehydrobutyrine, lanthionine, and 3-methyllanthionine.

Composition and purity of combinatorial aryl ether collections analyzed by electrospray mass spectrometry.

Electrospray mass spectrometry (ESI-MS), tandem mass spectrometry and on-line RP-HPLC-ESI-MS were used to evaluate the composition and purity of three different aryl ether mixtures consisting of 10 and 45 aryl ethers synthesized on solid support by Williamson etherification. The libraries feature two potential pharmacophores connected with three different spacers and serve as models for a detailed component analysis. Individual members of the library and by-products were identified rapidly and conveniently by product ion scans. Compound collections obtained by two different synthetic methods, the split/combine approach and the premix method, showed different mass distributions in the ESI-MS spectra. Some components were not detected in direct ESI-MS measurements, but were found by MS/MS experiments. Precursor ion and constant neutral loss scans allowed the identification of components with common structural features.

Air monitoring of aldehydes by use of hydrazine reagents with a triazine backbone.

Two hydrazine reagents, 4- N, N-dimethylamino-6-(4'-methoxy-1'-naphthyl)-1,3,5-triazine-2-hydrazine (DMNTH) and N-methyl-4- N', N'-dimethylamino-6-(4'-methoxy-1'-naphthyl)-1,3,5-triazine-2-hydrazine (MDMNTH) have been synthesized and used for the determination of aldehydes in air samples. Test tubes with the reagents coated on silica gel were prepared and used for monitoring of carbonyls in air. After elution with acetonitrile the hydrazones formed were separated by reversed-phase liquid chromatography. Detection was performed by UV-visible and fluorescence spectroscopy. The results were validated by use of standard atmospheres of the carbonyls and of nitrogen dioxide and ozone, as potential interferents. In comparison with established hydrazine reagents, e.g. 2,4-dinitrophenylhydrazine (DNPH), the results from use of MDMNTH correlate well; lower recoveries were obtained by use of DMNTH. The limits of detection for the new reagents are superior to those for DNPH, because of the possibility of fluorescence detection.

Engineering of a novel thioether bridge and role of modified residues in the lantibiotic Pep5.

Pep5 is a 34-amino-acid antimicrobial peptide, produced by Staphylococcus epidermidis 5, that contains the thioether amino acids lanthionine and methyllanthionine, which form three intramolecular ring structures. In addition, two didehydrobutyrines are present in the central part of the lantibiotic and an oxobutyryl residue is located at the N terminus. All rare amino acids are introduced by posttranslational modifications of a ribosomally made precursor peptide. To elucidate the function of the modified residues for the antimicrobial action of Pep5, mutant peptides, in which single modified residues had been eliminated, were produced by site-directed mutagenesis. All of these peptides showed a reduced antimicrobial activity. In addition, those peptides from which the ring structures had been deleted became susceptible to proteolytic digest. This demonstrates that the ring structures serve as stabilizers of conformations essential for activity, e.g., amphiphilicity, as well as for protecting Pep5 against proteases of the producing strains. In addition, residues that could serve as precursors of new modified amino acids in lantibiotics were introduced into the Pep5 precursor peptide. This way, a novel methyllanthionine and a didehydroalanine were inserted into the flexible central part of Pep5, demonstrating that biosynthesis of modified amino acids is feasible by protein engineering and use of the lantibiotic modification system.

Nucleotide sequence of the lantibiotic Pep5 biosynthetic gene cluster and functional analysis of PepP and PepC. Evidence for a role of PepC in thioether formation.

The biosynthesis of Pep5, a lanthionine-containing antimicrobial peptide, is directed by the 20-kbp plasmid pED503. We identified a 7.9-kbp DNA-fragment within this plasmid which covers the information for Pep5 synthesis in the homologous host Staphylococcus epidermidis 5 which has been cured of pED503. This fragment contained, in addition to the previously described structural gene pepA and the immunity gene pepI [Reis, M., Eschbach-Bludau, M., Iglesias-Wind, M. I., Kupke, T. & Sahl, H.-G. (1994) Appl. Env. Microbiol. 60, 2876-2883], a gene pepT coding for a translocator of the ABC transporter family, a gene pepP coding for a serine protease and two genes pepB and pepC coding for putative modification enzymes; the gene arrangement is pepTIAPBC. We analyzed the biosynthetic genes with respect to their function in Pep5 biosynthesis. Deletion of PepT reduced Pep5 production to about 10%, indicating that it can be partially replaced by other host-encoded translocators. Inactivation of PepP by site-directed mutagenesis of the active-site His residue resulted in production of incorrectly processed Pep5 fragments with strongly reduced antimicrobial activity. Deletion of pepB and pepC leads to accumulation of Pep5 prepeptide in the cells without excretion of processed peptide. A pepC-deletion clone did not excrete correctly matured Pep5 but it did produce fragments from which serine and threonine were absent. Only one of these fragments contained a single lanthionine residue out of three expected while the remaining, unmodified cysteine residues could be detected by reaction with Ellman's reagent. These results demonstrate that PepC is a thioether-forming protein and strongly suggest that PepB is responsible for dehydration of serine and threonine.