Microbial screening methods for detection of antibiotic residues in slaughter animals

Monitoring of food products from animal origin for the presence of antimicrobial residues is preferably done using microbial screening methods because of their high cost-effectiveness. Traditionally applied methods fail to detect the maximum residue limits which were established when EU Council Regulation 2377/90 came into effect. Consequently, during the last decade this has led to the development of improved microbial screening methods. This review provides an overview of the efforts expended to bring antibiotic screening methods into compliance with EU legislation. It can be concluded that the current situation is still far from satisfactory

Comparing the Sensitivity of Algal, Cyanobacterial and Bacterial Bioassays to Different Groups of Antibiotics

Comparing the sensitivity of algal, cyanobacterial and bacterial bioassays to different groups of antibiotics van der Grinten, E.; Pikkemaat, M.G.; van den Brandhof, E.J.; Stroomberg, G.J.; Kraak, M.H.S. Published in: Chemosphere DOI: 10.1016/j.chemosphere.2010.04.011 Link to publication Citation for published version (APA): van der Grinten, E., Pikkemaat, M. G., van den Brandhof, E. J., Stroomberg, G. J., & Kraak, M. H. S. (2010). Comparing the sensitivity of algal, cyanobacterial and bacterial bioassays to different groups of antibiotics. Chemosphere, 80(1), 1-6. https://doi.org/10.1016/j.chemosphere.2010.04.011 General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. a b s t r a c t Antibiotics may affect both primary producers and decomposers, potentially disrupting ecosystem processes. Hence, it is essential to assess the impact of antibiotics on aquatic ecosystems. The aim of the present study was therefore to evaluate the potential of a recently developed test for detecting antibiotics in animal tissue, the Nouws Antibiotic Test (NAT), as a sensitive bioassay to assess the effects of antibiotics in water. To this purpose, we determined the toxicity of sulphamethoxazole, trimethoprim, flumequine, tylosin, streptomycin, and oxytetracycline, using the NAT adapted for water exposure. The sensitivity of the NAT was compared to that of bioassays with bacteria (Microtox), cyanobacteria and green algae. In the Microtox test with Vibrio fischeri as test organism, no effects were observed for any of the test compounds. For three of the six antibiotics tested, the cyanobacteria were more vulnerable than the green algae when using photosynthetic efficiency as an endpoint. The lowest EC50 values for four out of six tested antibiotics were obtained using the NAT bacterial bioassay. The bacterial plate system responded to antibiotics at concentrations in the lg L À1 and lower mg L À1 range and, moreover, each plate proved to be specifically sensitive to the antibiotics group it was designed for. It is concluded that the NAT bioassay adapted for water exposure is a sensitive test to determine the presence of antibiotics in water. The ability of this test to distinguish five major antibiotic groups is a very strong additional value

Generating segmental mutations in haloalkane dehalogenase:a novel part in the directed evolution toolbox

Directed evolution techniques allow us to genuinely mimic molecular evolution in vitro. To enhance this imitation of natural evolutionary processes on a laboratory scale in even more detail, we developed an in vitro method for the generation of random deletions and repeats. The pairwise fusion of two fragments of the same gene that are truncated by exonuclease BAL-31 either at the 3′ or 5′ side results in a deletion or a repeat at the fusion point. Although in principle the method randomly covers the whole gene, it can also be limited to a predefined area in the sequence by controlling the level of the initial truncation. To test the procedure and to illustrate its potential, we used haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) as a model enzyme, since the adaptation of this enzyme towards new substrates is known to occur via the generation of this type of mutation. The results show that the mutagenesis method presented here is an effective tool for accessing formerly unexplorable sequence space and can contribute to the success of future directed evolution experiments

Molecular dynamics simulations as a tool for improving protein stability

Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an α/β-hydrolase fold main domain and a cap domain composed of five α-helices. MD simulations of DhlA showed high mobility in a helix–loop–helix region in the cap domain, involving residues 184–211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5°C, whereas the Tm,app of the reduced mutant decreased by more than 8°C compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol–1 compared to the wild-type enzyme and also indicated that the helix–loop–helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.

Crystallographic and Kinetic Evidence of a Collision Complex Formed during Halide Import in Haloalkane Dehalogenase

Haloalkane dehalogenase (DhlA) converts haloalkanes to their corresponding alcohols and halide ions. The rate-limiting step in the reaction of DhlA is the release of the halide ion. The kinetics of halide release have been analyzed by measuring halide binding with stopped-flow fluorescence experiments. At high halide concentrations, halide import occurs predominantly via the rapid formation of a weak initial collision complex, followed by transport of the ion to the active site. To obtain more insight in this collision complex, we determined the X-ray structure of DhlA in the presence of bromide and investigated the kinetics of mutants that were constructed on the basis of this structure. The X-ray structure revealed one bromide ion firmly bound in the active site and two bromide ions weakly bound on the surface of the enzyme. One of the weakly bound ions is close to Thr197 and Phe294, near the entrance of the earlier proposed tunnel for substrate import. Kinetic analysis of bromide import by the Thr197Ala and Phe294Ala mutants of DhlA at high halide concentration showed that the rate constants for halide binding no longer displayed a wild-type-like parabolic increase with increasing bromide concentrations. This is in agreement with an elimination or a decrease in affinity of the surface-located halide-binding site. Likewise, chloride binding kinetics of the mutants indicated significant differences with wild-type enzyme. The results indicate that Thr197 and Phe294 are involved in the formation of an initial collision complex for halide import in DhlA and provide experimental evidence for the role of the tunnel in substrate and product transport.

Non-targeted workflow for identification of antimicrobial compounds in animal feed using bioassay-directed screening in combination with liquid chromatography-high resolution mass spectrometry

A non-targeted workflow is reported for the isolation and identification of antimicrobial active compounds using bioassay-directed screening and LC coupled to high-resolution MS. Suspect samples are extracted using a generic protocol and fractionated using two different LC conditions (A and B). The behaviour of the bioactive compound under these different conditions yields information about the physicochemical properties of the compound and introduces variations in co-eluting compounds in the fractions, which is essential for peak picking and identification. The fractions containing the active compound(s) obtained with conditions A and B are selected using a microbiological effect-based bioassay. The selected bioactive fractions from A and B are analysed using LC combined with high-resolution MS. Selection of relevant signals is automatically carried out by selecting all signals present in both bioactive fractions A and B, yielding tremendous data reduction. The method was assessed using two spiked feed samples and subsequently applied to two feed samples containing an unidentified compound showing microbial growth inhibition. In all cases, the identity of the compound causing microbiological inhibition was successfully confirmed

A strategy to determine the fate of active chemical compounds in soil; applied to antimicrobially active substances

Data on the fate of chemical substances in the environment after e.g. manure application is mandatory input for risk assessment in perspective of a more circular biobased economy. Such fate studies include a persistence study to determine a half-life value and a mobility study. It is recognized that not only the native substance should be considered, but that also degradation products should be included that might exert a similar effect as the native substance. We report a tiered fate study strategy that starts with a persistence study. For non-persistent substances a study is performed to determine if degradation products have a similar effect as the native compound. If so, a procedure using high resolution mass spectrometry is suggested to identify the potentially active degradation products. Based on the outcomes, substances are divided into three categories: (I) persistent, (II) degradable to inactive products or (III) degradable to active products. Even though the priority is with category I and III, for all substances and possible degradation products a mobility study is proposed. The fate strategy is successfully applied to ten antimicrobially active substances originating from the tetracyclines, sulfonamides, diaminopyrimidines, fluoroquinolones, macrolides and lincosamides. The fluoroquinolones, tetracyclines and trimethoprim were relatively persistent. The sulfonamides, macrolides and lincomycin (the latter also depending on soil type) degraded relatively quickly. Tylosin A proved to degrade to antimicrobially active degradation products which were tentitatively identified as tylosin C, tylosin A acid, tylosin B acid and tylosin C acid.</p

Combining an effect-based methodology with chemical analysis for antibiotics determination in wastewater and receiving freshwater and marine environment

Two different methodologies were combined to evaluate the risks that antibiotics can pose in the environment; i) an effect-based methodology based on microbial growth inhibition and ii) an analytical method based on liquid-chromatography coupled to mass spectrometry (LC-MS). The first approach was adapted and validated for the screening of four antibiotic families, specifically macrolides/β-lactams, quinolones, sulfonamides and tetracyclines. The LC-MS method was applied for the identification and quantification of target antibiotics; then, the obtained results were combined with ecotoxicological data from literature to determine the environmental risk. The two methodologies were used for the analysis of antibiotics in water samples (wastewater, river water and seawater) and biofluids (fish plasma and mollusk hemolymph) in two monitoring campaigns undertaken in the Ebro Delta and Mar Menor Lagoon (both in the Mediterranean coast of Spain). Both approaches highlighted macrolides (azithromycin) and quinolones (ciprofloxacin and ofloxacin) as the main antibiotics in wastewater treatment plant (WWTP) effluents with potential risk for the environment. However, no risk for the aquatic life was identified in the river, lagoon and seawater as antibiotic levels were much lower than those in WWTP effluents. Fish from Ebro River were the organisms presenting the highest antibiotic concentration when compared with bivalves (mussels) from the Mediterranean Sea and gastropods (marine snails) from the Mar Menor Lagoon. The effect-based methodology successfully determined antibiotic risk in wastewater, but its applicability was less clear in environmental waters such as seawater, due to its high detection limits. Improving sample preconcentration could increase the method sensibility. Overall, combination of both methodologies provides comprehensive insights in antibiotic occurrence and risk associated in areas under study.Versión del editor3,74