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Dimerization of Proline Dehydrogenase from Thermus thermophilus Is Crucial for Its Thermostability
Huijbers, Mieke M.E. ; Wu, Jenny W. ; Westphal, Adrie H. ; Berkel, Willem J.H. van - \ 2019
Biotechnology Journal 14 (2019)5. - ISSN 1860-6768
flavoprotein - protein oligomerization - thermostability - Thermus thermophilus - triosephosphate isomerase barrel

Thermus thermophilus proline dehydrogenase (TtProDH) catalyzes the first step in proline catabolism. The thermostable flavoenzyme consists of a distorted triosephosphate isomerase (TIM) barrel and three N-terminal helices: αA, αB, and αC. Using maltose-binding protein (MBP) fused constructs, it has been recently demonstrated that helix αC is crucial for TtProDH catalysis and for tetramerization through positioning of helix α8. Here, the structural features that determine the thermostability of TtProDH are reported. Selective disruption of two ion pairs in the dimerization interface of several MBP-TtProDH variants result in the formation of monomers. The newly created monomers have improved catalytic properties but their melting temperatures are decreased by more than 20 °C. Sequence comparison suggests that one of the ion-pairs involved in dimerization is unique for ProDHs from Thermus species. In summary, intermolecular ion-pairs improve the thermostability of TtProDH and a trade-off is made between thermostability and catalytic activity.

Hydrocarbon Synthesis via Photoenzymatic Decarboxylation of Carboxylic Acids
Zhang, Wuyuan ; Ma, Ming ; Huijbers, Mieke M.E. ; Filonenko, Georgy A. ; Pidko, Evgeny A. ; Schie, Morten van; Boer, Sabrina de; Burek, Bastien O. ; Bloh, Jonathan Z. ; Berkel, Willem J.H. van; Smith, Wilson A. ; Hollmann, Frank - \ 2019
Journal of the American Chemical Society 141 (2019)7. - ISSN 0002-7863 - p. 3116 - 3120.

A recently discovered photodecarboxylase from Chlorella variabilis NC64A ( CvFAP) bears the promise for the efficient and selective synthesis of hydrocarbons from carboxylic acids. CvFAP, however, exhibits a clear preference for long-chain fatty acids thereby limiting its broad applicability. In this contribution, we demonstrate that the decoy molecule approach enables conversion of a broad range of carboxylic acids by filling up the vacant substrate access channel of the photodecarboxylase. These results not only demonstrate a practical application of a unique, photoactivated enzyme but also pave the way to selective production of short-chain alkanes from waste carboxylic acids under mild reaction conditions.

Longitudinal study of ESBL Escherichia coli carriage on an organic broiler farm
Hoek, Angela H.A.M. van; Veenman, Christiaan ; Florijn, Alice ; Huijbers, Patricia M.C. ; Graat, Elisabeth A.M. ; Greeff, Sabine de; Dierikx, Cindy M. ; Duijkeren, Engeline van - \ 2018
Journal of Antimicrobial Chemotherapy 73 (2018)12. - ISSN 0305-7453 - p. 3298 - 3304.

Objectives: To determine the molecular characteristics of ESBL-producing Escherichia coli (ESBL-E) collected during a longitudinal study on an organic broiler farm in order to investigate clonal expansion and horizontal gene transfer. Methods: Isolates were obtained from a longitudinal study performed previously on an organic broiler fattening farm. Samples from individually followed-up broilers, the broiler house, the transport van and persons that took the samples, taken at several timepoints (days 1, 3, 4, 7, 10, 42 and 70) within a production round and during the consecutive one (days 1, 2, 3 and 70), had been investigated for the occurrence of ESBL-E. In the current study, ESBL genes and MLST STs of these ESBL-E were determined. Plasmids were characterized and subtyped. Results: On arrival in round_1, ESBL-E of ST88 predominated, while on days 3, 4, 7 and 10 ST10 was most often found and at slaughter age ST155 and ST1551 prevailed. A shift in STs was also observed in round_2. None of the 35 individually selected broilers followed up in round_1 was positive for the same ESBL-E ST at all sampling times. All isolates carried CTX-M-1 group genes, confirmed as blaCTX-M-1 in 158 isolates. Further analysis of 36 isolates of different STs showed blaCTX-M-1 on IncI1/ST3 plasmids. Conclusions: The rapid dissemination of ESBL-E on this broiler farm was not due to the spread of one specific E. coli clone, but most likely the result of horizontal transfer of an IncI1/ST3 plasmid carrying blaCTX-M-1 resulting in a shift in the predominant ESBL-E population in broilers.

Functional impact of the n-terminal arm of proline dehydrogenase from thermus thermophilus
Huijbers, Mieke M.E. ; Alen, Ilona van; Wu, Jenny W. ; Barendregt, Arjan ; Heck, Albert J.R. ; Berkel, Willem J.H. Van - \ 2018
Molecules 23 (2018)1. - ISSN 1420-3049
Flavoprotein - Proline dehydrogenase - Protein engineering - Protein oligomerization - Solubility tag - Suicide inhibition - TIM-barrel
Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to ∆1-pyrroline-5-carboxylate. Thermus thermophilus ProDH (TtProDH) contains in addition to its flavin-binding domain an N-terminal arm, consisting of helices αA, αB, and αC. Here, we report the biochemical properties of the helical arm truncated TtProDH variants ∆A, ∆AB, and ∆ABC, produced with maltose-binding protein as solubility tag. All three truncated variants show similar spectral properties as TtProDH, indicative of a conserved flavin-binding pocket. ∆A and ∆AB are highly active tetramers that rapidly react with the suicide inhibitor N-propargylglycine. Removal of the entire N-terminal arm (∆ABC) results in barely active dimers that are incapable of forming a flavin adduct with N-propargylglycine. Characterization of V32D, Y35F, and V36D variants of ∆AB established that a hydrophobic patch between helix αC and helix α8 is critical for TtProDH catalysis and tetramer stabilization.
Cyanodermella asteris sp. nov. (Ostropales) from the inflorescence axis of Aster tataricus
Jahn, Linda ; Schafhauser, Thomas ; Pan, Stefan ; Weber, Tilmann ; Wohlleben, Wolfgang ; Fewer, David P. ; Sivonen, Kaarina ; Flor, Liane ; Pée, Karl Heinz van; Caradec, Thibault ; Jacques, Philippe ; Huijbers, Mieke M.E. ; Berkel, Willem J.H. van; Ludwig-Müller, Jutta - \ 2017
Mycotaxon 132 (2017)1. - ISSN 0093-4666 - p. 107 - 123.
Ascomycota - Asteraceae - Lecanoromycetes - Pezizomycotina

An endophytic fungus isolated from the inflorescence axis of Aster tataricus is proposed as a new species. Phylogenetic analyses based on sequences from the ribosomal DNA cluster (the ITS1+5.8S+ITS2, 18S, and 28S regions) and the RPB2 gene revealed a relationship between the unknown fungus and the Stictidaceae lineage of the Ostropales. The new species, Cyanodermella asteris, grows in standard fungal growth media as a fluffy, pink filamentous fungus. Asexual and sexual sporulation has not yet been observed on media or in the plant.

Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor
Huijbers, Mieke M.E. ; Martínez-Júlvez, Marta ; Westphal, Adrie H. ; Delgado-Arciniega, Estela ; Medina, Milagros ; Berkel, Willem J.H. Van - \ 2017
Scientific Reports 7 (2017). - ISSN 2045-2322
Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ΔABC, which lacks helices αA, αB and αC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered.
Trimming proline dehydrogenase : protein and cofactor minimization
Huijbers, Mieke M.E. - \ 2017
Wageningen University. Promotor(en): Willem van Berkel. - Wageningen : Wageningen University - ISBN 9789463430517 - 181
proline - thermus thermophilus - enzymes - amino acids - binding proteins - catalysts - proline - thermus thermophilus - enzymen - aminozuren - bindende eiwitten - katalysatoren

Proline is one of the proteinogenic amino acids and one of the most abundant amino acids in the cell. Next to serving as one of the non-essential amino acids, proline also has a central role in metabolism. In Chapter 1, the different functions of this imino acid are described, as well as the proline metabolic enzymes. The focus is on the enzyme proline dehydrogenase (ProDH), which catalyzes the flavin-dependent conversion of L-proline to Δ1-pyrroline-5-carboxylate (P5C). Malfunctioning of this enzyme has severe implications for human health and has been associated with tumorigenesis and schizophrenia.

This thesis deals with the engineering and biochemical characterization of Thermus thermophilus ProDH (TtProDH) in order to gain more insight into the structure-function relationship of this thermo-resistant flavoenzyme. TtProDH is a membrane-associated protein and recombinant soluble forms of the enzyme have only been obtained in limited amounts. Chapter 2 describes the heterologous production of TtProDH in Escherichia coli. Using maltose-binding protein (MBP) as solubility tag, high yields of active holoenzyme are obtained. The MBP-tag can be efficiently removed from the fusion protein with trypsin, yielding native TtProDH. This enzyme is thermotolerant as well as solvent tolerant; however, both fused and clipped TtProDH are prone to aggregation. In Chapter 3, we show that the hydrophobic N-terminal helix of TtProDH is responsible for this non-native self-association. Phe10 and Leu12, located at the protein surface, were replaced by glutamates, generating the F10E/L12E (EE) variant of MBP-TtProDH. This more polar variant exclusively forms tetramers and exhibits excellent catalytic features. Specific removal of the MBP-tag of the EE variant is less easy than for WT, as trypsinolysis of the fusion enzyme leads to degradation of TtProDH. Since the MBP tag does not influence the spectral and catalytic properties of the enzyme, further experiments were performed with MBP-tagged variants of TtProDH.

ProDH has a distorted (βα)8 TIM-barrel fold which is conserved throughout the PutA/ProDH family. In contrast, the N-terminal sequence of ProDH is poorly conserved. TtProDH contains, next to the distorted TIM-barrel, three N-terminal helices, αA, αB and αC, of which the function is not well understood. In Chapter 4, we describe the characterization of helical arm-truncated variants, lacking respectively one (ΔA), two (ΔAB), or three (ΔABC) N-terminal helices. All three variants show flavin properties that are highly similar to EE, indicating no changes in the microenvironment of the flavin isoalloxazine ring. ΔA and ΔAB are highly active tetramers, whereas removal of the complete N-terminal arm (ΔABC) results in poorly active dimers. Furthermore, EE, ΔA and ΔAB rapidly react with the suicide inhibitor N-propargylglycine, while ΔABC is not capable of forming a flavin adduct with N-propargylglycine. This indicates that helix αC has a crucial role in both the oligomerization and activity of TtProDH. Closer examination revealed an ionic interaction as well as a hydrophobic patch between helices αC and α8, the latter helix being crucial for substrate recognition. To investigate the functional role of helix αC in further detail, additional enzyme variants were created that disrupt the interactions between both helices. While disrupting the ionic interaction had minor effects, disrupting the hydrophobic patch leads to dimer formation, loss of activity and decreased reactivity with N-propargylglycine. This supports that helix αC is crucial for TtProDH catalysis and tetramerization through positioning of helix α8.

The quaternary structure of TtProDH was investigated in more detail in Chapter 5. Two ionic interactions at the dimeric interface were selectively disrupted by changing Asp205 and Glu207 of TtProDH variants EE, ΔA, ΔAB and ΔABC into lysines. These KK-variants form monomers (except for EE KK, which forms dimers) and have improved catalytic properties at moderate temperatures compared to their non-KK counterparts. However, their melting temperatures are decreased by more than 20 °C. This indicates that a trade-off is made between thermostability and catalytic activity.

In Chapter 6, we studied the cofactor binding of TtProDH. Flavoenzymes contain either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. To study the cofactor binding of TtProDH, we produced MBP-TtProDH EE in its apoform using a riboflavin auxotrophic E. coli strain. Reconstitution of the enzyme with either FAD or FMN revealed that MBP-TtProDH has no preference for FAD as cofactor. Kinetic parameters of both holo-FAD and holo-FMN are similar, as are the dissociation constants for FAD and FMN release. We show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In addition, we obtained the crystal structure TtProDH ΔABC, which shows no electron density for an AMP moiety of the cofactor. This indicates the presence of mainly FMN in the enzyme. The capability of TtProDH to display equal properties with both cofactors is unique for flavoenzymes, and classification of TtProDH as an FAD-containing enzyme should be reconsidered.

In Chapter 7, we discuss the novel findings described in this thesis and put them in a broader perspective. We have created a minimalist ProDH that is an excellent catalyst, but is deprived of all structural features that are unnecessary for in vitro functioning. Our results expand the knowledge on the structure-function relationship of ProDHs, and give insight into enzyme functionality from an industrial perspective. We also discuss how this knowledge might be used in future studies for a better understanding of the properties of eukaryotic ProDHs, with a special interest in the human enzyme.

A more polar N-terminal helix releases MBP-tagged Thermus thermophilus proline dehydrogenase from tetramer-polymer self-association
Huijbers, Mieke M.E. ; Berkel, Willem J.H. van - \ 2016
Journal of Molecular Catalysis. B, Enzymatic 134 (2016). - ISSN 1381-1177 - p. 340 - 346.
Flavoprotein - Molecular self-association - Proline dehydrogenase - Protein oligomerization - Thermus thermophilus

Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme involved in the biosynthesis of . l-glutamate. ProDH is of interest for biocatalysis because the protein might be applied in multi-enzyme reactions for the synthesis of structurally complex molecules. We recently demonstrated that the thermotolerant ProDH from . Thermus thermophilus (TtProDH) is overproduced in . Escherichia coli when using maltose-binding protein (MBP) as a solubility tag. However, MBP-TtProDH and MBP-clipped TtProDH are prone to aggregation through non-native self-association. Here we provide evidence that the hydrophobic N-terminal helix of TtProDH is responsible for the self-association process. The more polar MBP-tagged F10E/L12E variant exclusively forms tetramers and exhibits excellent catalytic features over a wide range of temperatures. Understanding the hydrodynamic and catalytic properties of thermostable enzymes is important for the development of industrial biocatalysts as well as for pharmaceutical applications.

Transmission dynamics of extended-spectrum β-lactamase and AmpC β-lactamase-producing Escherichia coli in a broiler flock without antibiotic use
Huijbers, Patricia M.C. ; Graat, E.A.M. ; Hoek, Angela H.A.M. van; Veenman, Christiaan ; Jong, Mart C.M. de; Duijkeren, Engeline van - \ 2016
Preventive Veterinary Medicine 131 (2016). - ISSN 0167-5877 - p. 12 - 19.
Antibiotic resistance - ESBL - Organic farm - Phylogenetic group - Reproduction ratio

Extended-spectrum and AmpC β-lactamase-producing Escherichia coli (ESBL/AmpC-EC) are found throughout the broiler production pyramid. Transmission of resistance between broilers and humans could occur at any point, representing a potential public health issue. Insight in farm transmission dynamics could provide a basis for control, leading to fewer contaminated broilers. The aim was quantifying transmission rates and routes of ESBL/AmpC-EC, and specific phylogenetic groups, in an organic broiler flock without antibiotic use. In each of two consecutive production rounds, 80 randomly chosen broilers were followed individually. Cloacal swabs from these, 20 other randomly chosen broilers, and 11 environmental samples were taken at several moments from arrival till slaughter. ESBL/AmpC-EC were isolated by selective pre-enrichment, and ESBL/AmpC-genes and E. coli phylogenetic groups were determined. Transmission parameters (β) were estimated using a Generalised Linear Model with a susceptible-infectious-susceptible model. Effect of direct broiler contact as compared to contact through the environment and previous carriage c.q. infectious status and their interaction were included as explanatory variables. Multiplying β by the length of the infectious period gives the reproduction ratio (R). On day 1, prevalence was 28.8% (95%CI 19.2–40.0%) and 0.0% (95%CI 0.0–4.5%) among individually followed broilers, in round 1 and 2 respectively. In round 2, the environment was positive before arrival of day-old chicks. After 3 days, almost 100% of broilers and environmental samples were positive in both rounds. Most samples were positive for CTX-M-1 group genes, and A1 and B1 were predominant phylogenetic groups. From day 3 there was a shift towards more phylogenetic groups. R was 1.70 (95%CI 0.55–5.25) for total ESBL/AmpC-EC. Risk for broilers to become infectious was lower if previously infectious (βpreviously infectious = 0.02 vs. βnot previously infectious = 3.41; P <0.0001). For phylogenetic groups separately, R was 0.88 (95%CI 0.38–2.07), 0.51 (95%CI 0.27–0.98), 0.99 (95%CI 0.65–1.51) for A1, B1 and rest (i.e. A0, B2, D1, D2) groups, respectively. The interaction effect for A1 and B1 was reflected in the fact that when broilers were previous infectious, the environment was relatively more important for transmission of the A1 group, while this was direct contact between broilers for the B1 group. Positive day-old chicks and the environment both play a role in introduction and transmission of ESBL/AmpC-EC in flocks. These results suggest that, even without selective pressure from antibiotics, total ESBL/AmpC-EC persistence, and resulting endemic situation, seem to be caused by shifts in carriage of different phylogenetic groups. It implies that contaminated broilers enter the slaughterhouse.

The cyclochlorotine mycotoxin is produced by the nonribosomal peptide synthetase CctN in Talaromyces islandicus ('Penicillium islandicum')
Schafhauser, Thomas ; Kirchner, Norbert ; Kulik, Andreas ; Huijbers, Mieke M.E. ; Flor, Liane ; Caradec, Thibault ; Fewer, David P. ; Gross, Harald ; Jacques, Philippe ; Jahn, Linda ; Jokela, Jouni ; Leclère, Valérie ; Ludwig-Müller, Jutta ; Sivonen, Kaarina ; Berkel, Willem J.H. van; Weber, Tilmann ; Wohlleben, Wolfgang ; Pée, Karl Heinz van - \ 2016
Environmental Microbiology 18 (2016)11. - ISSN 1462-2912 - p. 3728 - 3741.

Talaromyces islandicus ('Penicillium islandicum') is a widespread foodborne mold that produces numerous secondary metabolites, among them potent mycotoxins belonging to different chemical classes. A notable metabolite is the hepatotoxic and carcinogenic pentapeptide cyclochlorotine that contains the unusual amino acids β-phenylalanine, 2-aminobutyrate and 3,4-dichloroproline. Although the chemical structure has been known for over five decades, nothing is known about the biosynthetic pathway of cyclochlorotine. Bioinformatic analysis of the recently sequenced genome of T. islandicus identified a wealth of gene clusters potentially coding for the synthesis of secondary metabolites. Here, we show by RNA interference-mediated gene silencing that a nonribosomal peptide synthetase, CctN, is responsible for the synthesis of cyclochlorotine. Moreover, we identified novel cyclochlorotine chemical variants, whose production also depended on cctN expression. Surprisingly, the halogenase required for cyclochlorotine biosynthesis is not encoded in the cct cluster. Nonetheless, our findings enabled us to propose a detailed model for cyclochlorotine biosynthesis. In addition, comparative genomics revealed that cct-like clusters are present in all of the sequenced Talaromyces strains indicating a high prevalence of cyclochlorotine production ability.

Transmission of antibiotic resistance from animals to humans : Broilers as a reservoir of ESBL-producing bacteria
Huijbers, P.M.C. - \ 2016
Wageningen University. Promotor(en): Mart de Jong; Lisette Graat; E. van Duijkeren. - Wageningen : Wageningen University - ISBN 9789462576216 - 156
broilers - man - disease transmission - antibiotic resistance - bacteria - enterobacteriaceae - poultry farming - epidemiology - vleeskuikens - mens - ziekteoverdracht - antibioticaresistentie - bacteriën - enterobacteriaceae - pluimveehouderij - epidemiologie

Huijbers, P.M.C. (2016). Transmission of antibiotic resistance from animals to humans: Broilers as a reservoir of ESBL-producing bacteria. PhD thesis, Wageningen University, Wageningen, the Netherlands.

Antibiotic resistance in animals becomes a public health issue when there is transmission of antibiotic resistant bacteria, or their resistance genes, from animals to humans. β-lactam antibiotics are critically important for the treatment of human bacterial infections. Resistance to this class of antibiotics, mediated by extended-spectrum β-lactamases (ESBL) has emerged. Broilers might contribute to transmission to humans due to the high prevalence of ESBL-producing Enterobacteriaceae among their intestinal biome, compared to other livestock species, companion animals, and wildlife. Transmission to humans might occur via the food chain, by direct contact or via the environment. The aim was to investigate transmission of antibiotic resistant bacteria between animals and humans, and more specifically transmission of ESBL-producing E. coli between broilers, and between broilers and humans in varying degrees of contact with these animals. Systematically collected and categorised evidence from literature showed that clinically relevant antibiotic resistant bacteria were present in the natural environment, that is in soil, water, air and wildlife. It was therefore hypothesised that humans in areas with high broiler densities might have an increased risk for carriage of ESBL-producing Enterobacteriaceae. This hypothesis was rejected, as the observed risk was lower for these individuals. The situation might be different for individuals living on broiler farms as ESBL-producing E. coli were detected on all investigated farms. Among broilers, the within farm prevalence approached 100%, and there was no difference between conventional and organic farms at five weeks, i.e. just before slaughter on conventional farms. On organic farms, the prevalence decreased to 80.0% at 70 days, i.e. slaughter age. Not only transmission to humans via the farm environment, but close physical contact with broilers might, therefore, lead to increased risk for carriage. Prevalence among farmers, their family members and employees on both conventional (19.1%) and organic (18.5%) broiler farms was higher compared to humans in the general population (5.1%). Moreover, people in close contact with live broilers showed the highest risk (27.1 vs. 14.3%). Evidence for clonal transmission of ESBL-producing E. coli between humans and broilers was found on conventional farms, and horizontal gene transfer was suspected on both conventional and organic farms. Even without selection pressure from antibiotics ESBL-producing E. coli were able to transmit and persist in an organic broiler flock, which shows that broilers form a reservoir of antibiotic resistance genes. This leads to an increased risk of carriage of humans on farms through direct contact with broilers and possibly via the direct farm environment. As only a very small percentage of the general population is exposed to live broilers, direct contact with broilers does not appear to be important for carriage in the general human population.

Letter to the editor: Inoculation of mice with avian blaCTX-M-1- or blaCMY-2-carrying Escherichia coli strains does not lead to long-term colonization
Duijkeren, Engeline van; Overbeek, Wendy M. van; Huijbers, Patricia M.C. ; Giessen, Arjen W. van de; Hoek, Angela H.A.M. van - \ 2015
Journal of Antimicrobial Chemotherapy 70 (2015)11. - ISSN 0305-7453 - p. 3164 - 3165.
Role of the Environment in the Transmission of Antimicrobial Resistance to Humans: A Review
Huijbers, P.M.C. ; Blaak, H. ; Jong, M.C.M. de; Graat, E.A.M. ; Vandenbroucke-Grauls, C.M.J.E. ; Roda Husman, A.M. de - \ 2015
Environmental Science and Technology 49 (2015)20. - ISSN 0013-936X - p. 11993 - 12004.
To establish a possible role for the natural environment in the transmission of clinically relevant AMR bacteria to humans, a literature review was conducted to systematically collect and categorize evidence for human exposure to extended-spectrum ß-lactamase-producing Enterobacteriaceae, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus spp. in the environment. In total, 239 datasets adhered to inclusion criteria. AMR bacteria were detected at exposure-relevant sites (35/38), including recreational areas, drinking water, ambient air, and shellfish, and in fresh produce (8/16). More datasets were available for environmental compartments (139/157), including wildlife, water, soil, and air/dust. Quantitative data from exposure-relevant sites (6/35) and environmental compartments (11/139) were scarce. AMR bacteria were detected in the contamination sources (66/66) wastewater and manure, and molecular data supporting their transmission from wastewater to the environment (1/66) were found. The abundance of AMR bacteria at exposure-relevant sites suggests risk for human exposure. Of publications pertaining to both environmental and human isolates, however, only one compared isolates from samples that had a clear spatial and temporal relationship, and no direct evidence was found for transmission to humans through the environment. To what extent the environment, compared to the clinical and veterinary domains, contributes to human exposure needs to be quantified. AMR bacteria in the environment, including sites relevant for human exposure, originate from contamination sources. Intervention strategies targeted at these sources could therefore limit emission of AMR bacteria to the environment
Draft genome sequence of Talaromyces (Penicillium) islandicus WF-38-12, a neglected mold with significant biotechnological potential - Genome announcement
Schafhauser, T. ; Wibberg, D. ; Rueckert, C. ; Winkler, A. ; Flor, L. ; Pée, K.H. van; Fewer, D.P. ; Sivonen, K. ; Jahn, L. ; Ludwig-Müller, J. ; Caradec, T. ; Jacques, P. ; Huijbers, M.M.E. ; Berkel, W.J.H. van; Weber, T. ; Wohlleben, W. ; Kalinowsky, J. - \ 2015
Journal of Biotechnology 211 (2015). - ISSN 0168-1656 - p. 101 - 102.
Talaromyces (Penicillium) islandicus is a common mold found in stored rice or cereals. It has a highly versatile metabolism characterized by the secretion of numerous biopolymer degrading enzymes, mycotoxins, and anthraquinones that altogether offer a broad range of potential industrial applications. Here, we report the draft genome sequence of Talaromyces islandicus, which provides the basis of a biotechnological usage of this species.
High yields of active Thermus thermophilus proline dehydrogenase are obtained using maltose-binding protein as a solubility tag.
Huijbers, M.M.E. ; Berkel, W.J.H. van - \ 2015
Biotechnology Journal 10 (2015)3. - ISSN 1860-6768 - p. 395 - 403.
multifunctional puta flavoprotein - escherichia-coli - purification - domain - stress - biosynthesis - oxidase - crystallization - overexpression - identification
Proline dehydrogenase (ProDH) catalyzes the FAD-dependent oxidation of proline to ¿1-pyrroline-5-carboxylate, the first step of proline catabolism in many organisms. Next to being involved in a number of physiological processes, ProDH is of interest for practical applications because the proline imino acid can serve as a building block for a wide range of peptides and antibiotics. ProDH is a membrane-associated protein and recombinant soluble forms of the enzyme have only been obtained in limited amounts. We here report on the heterologous production of ProDH from Thermus thermophilus (TtProDH) in Escherichia coli. Using maltose-binding protein as solubility tag, high yields of active holoenzyme are obtained. Native TtProDH can be produced from cleaving the purified fusion protein with trypsin. Size-exclusion chromatography shows that fused and clipped TtProDH form oligomers. Thermal stability and co-solvent tolerance indicate the conformational robustness of TtProDH. These properties together with the high yield make TtProDH attractive for industrial applications.
Transmission dynamics of ESBL/AmpC-producing E. coli on a broiler farm
Huijbers, P.M.C. ; Hoek, A.H.A.M. van; Jong, M. de; Duijkeren, E. van - \ 2015
In: Proceedings of the 3rd International One Health Congress,. -
Background Transmission of ESBL/AmpC-producing E. coli between broilers and humans resulting from close contact between humans, broilers and their environment on farms has been suggested. To further elucidate the role of broilers in the carriage of these bacteria among humans, more information on farm transmission dynamics is necessary. The aim of this study was to quantify transmission rates and routes within an organic broiler flock, and to determine whether carriage of ESBL/AmpC-producing E. coli is persistent in broilers. Methods A longitudinal study was conducted on an organic broiler farm. Eighty broilers were randomly selected and tagged for individual tracking. Cloaca swabs from these broilers, 20 randomly chosen untagged broilers, and 11 environmental samples from the broiler house were taken seven times from arrival on the farm to slaughter age (day 1, 3, 4, 7, 10, 42, and 70). ESBL/AmpC-producing E. coli were isolated by selective enrichment and culture on selective plates. ESBL/AmpC-production was confirmed by combination disc diffusion test. At each sampling time the prevalence of positive broilers and environmental samples was calculated. Information about the ESBL/AmpC-status of tagged broilers, randomly chosen broilers, and environmental samples at each sampling time was combined to calculate the transmission rate parameter and the reproduction ratio (R0) using a susceptible-infectious-susceptible (SIS) model. Results The prevalence of ESBL/AmpC-producing E. coli on day 1 of the production round was 28.8% (95%CI 19.2-40.0%) in tagged broilers, and 45.5% (16.8-76.6%) in environmental samples. After 3 days, almost 100% of broiler and environmental samples were positive, which is indicative of the rapid transmission of these bacteria in the broiler house. Preliminary analysis of the R0 indicates that this is significantly larger than 1, indicating that ESBL/AmpC-producing E. coli are able to transmit and persist in broiler flocks. Carriage was intermittent in all broilers, except one who was positive at all sampling times. At the end of the production round, 39.4% (95%CI 27.6-52.2%) of tagged broilers, and 27.3% (95%CI 6.0-61.0%) of environmental samples were contaminated with ESBL/AmpC-producing E. coli, indicating that these bacteria could enter the food chain. Conclusions This study shows that ESBL/AmpC-producing E. coli are able to transmit and persist in broiler flocks in the absence of antimicrobials, resulting in an endemic situation. Ongoing molecular characterisation of broiler and environmental isolates will yield important information about the contribution of different routes in the transmission of ESBL/AmpC-producing E. coli on broiler farms and provide a basis for intervention.
Methicillin-resistant Staphylococcus aureus and extended-spectrum and AmpC b-lactamase-producing Escherichia coli in broilers and in people living and/or working on organic broiler farms
Huijbers, P.M.C. ; Hoek, A.H.A.M. van; Graat, E.A.M. ; Haenen, A.P.J. ; Florijn, A. ; Hengeveld, P.D. ; Duijkeren, E. van - \ 2015
Veterinary Microbiology 176 (2015)1-2. - ISSN 0378-1135 - p. 120 - 125.
livestock-associated mrsa - risk-factors - prevalence - netherlands - humans
The aim of this study was to estimate the prevalence of methicillin-resistantStaphylococcus aureus (MRSA) and extended-spectrum and AmpC b-lactamase (ESBL/AmpC)-producing Escherichia coli among broilers, and humans living and/or working on organic broiler farms; further characterise isolates; and compare these results with those from conventional farms. In the Netherlands, only 9 certified organic broiler farms were present. On 8 of these farms, 60 throat swabs and 20 cloacal swabs were taken per farm for MRSA and ESBL/AmpC-E. coli detection, respectively, at an average age of both 34 (T1) and 68 (T2) days. Faecal swabs and questionnaires were returned by 27 out of 36 humans. For selected ESBL/AmpC-producing E. coli isolates, phylogenetic groups, b-lactamase genes, plasmid families, and sequence types were determined. MRSA was not detected in broiler and human samples. ESBL/AmpC-producing E. coli were isolated from broilers on 7/8 farms at T1 and on all farms at T2. Furthermore, 3 farmers at T1, and 2 farmers and 1 family member at T2 were positive. Genes found in broilers and humans were almost exclusively blaCTX-M-1 and blaCMY-2. Given the high overall human ESBL/AmpC-prevalence (18.5%), which is similar to conventional farms, contact with live broilers is assumed a risk factor for carriage. Farm and sample-level prevalence at T1 are consistent with those from conventional farms. At T2, just before slaughter, sample-level prevalence of ESBL/AmpC-E. coli appears to have decreased (94.3%vs. 80%), which could have important consequences for contamination of retail meat.
Extended-spectrum and AmpC B-lactamase-producing Escherichia coli in broilers and peoplelivingand/or working on broiler farms: prevalence, risk factors and molecular characteristics
Huijbers, P.M.C. ; Graat, E.A.M. ; Haenen, A.P.J. ; Santen, M.G. van; Essen-Zandbergen, A. van; Mevius, D.J. ; Duijkeren, E. van; Hoek, A.H.A.M. van - \ 2014
Journal of Antimicrobial Chemotherapy 69 (2014)10. - ISSN 0305-7453 - p. 2669 - 2675.
livestock-associated mrsa - enterobacteriaceae - netherlands - humans - identification - plasmids - poultry - genes - meat - pcr
OBJECTIVES: The objectives of this study were to: estimate the prevalence of extended-spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase-producing Escherichia coli carriage among broiler farmers, their family members and employees; identify and quantify risk factors for carriage, with an emphasis on contact with live broilers; and compare isolates from humans and broilers within farms with respect to molecular characteristics to gain insight into transmission routes. METHODS: A cross-sectional prevalence study was conducted on 50 randomly selected Dutch broiler farms. Cloacal swabs were taken from 20 randomly chosen broilers. Faecal swabs were returned by 141 individuals living and/or working on 47 farms. ESBL/AmpC-producing E. coli were isolated and, for selected isolates, phylogenetic groups, plasmids and sequence types were determined. Questionnaires were used for risk factor analysis. RESULTS: All sampled farms were positive, with 96.4% positive pooled broiler samples. The human prevalence was 19.1%, with 14.3% and 27.1% among individuals having a low and a high degree of contact with live broilers, respectively. Five pairs of human-broiler isolates had identical genes, plasmid families and E. coli sequence types, showing clonal transmission. Furthermore, similar ESBL/AmpC genes on the same plasmid families in different E. coli sequence types in humans and broilers hinted at horizontal gene transfer. CONCLUSIONS: The prevalence among people on broiler farms was higher than in previous studies involving patients and the general population. Furthermore, an increased risk of carriage was shown among individuals having a high degree of contact with live broilers. The (relative) contribution of transmission routes that might play a role in the dissemination of ESBL/AmpC-encoding resistance genes to humans on broiler farms should be pursued in future studies.
Flavin dependent monooxygenases
Huijbers, M.M.E. ; Montersino, S. ; Westphal, A.H. ; Tischler, D. ; Berkel, W.J.H. van - \ 2014
Archives of Biochemistry and Biophysics 544 (2014). - ISSN 0003-9861 - p. 2 - 17.
flavoprotein tryptophan 2-monooxygenase - baeyer-villiger monooxygenases - site-directed mutagenesis - p-hydroxyphenylacetate 3-hydroxylase - para-hydroxybenzoate hydroxylase - sphingobium-xenophagum bayram - l-phenylalanine oxidase - l-lactate monooxygenase - rh
Flavin-dependent monooxygenases catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. As such, they are involved in key biological processes ranging from catabolism, detoxification and biosynthesis, to light emission and axon guidance. Based on fold and function, flavin-dependent monooxygenases can be distributed into eight groups. Groups A and B comprise enzymes that rely on NAD(P)H as external electron donor. Groups C–F are two-protein systems, composed of a monooxygenase and a flavin reductase. Groups G and H comprise internal monooxygenases that reduce the flavin cofactor through substrate oxidation. Recently, many new flavin-dependent monooxygenases have been discovered. In addition to posing basic enzymological questions, these proteins attract attention of pharmaceutical and fine-chemical industries, given their importance as regio- and enantioselective biocatalysts. In this review we present an update of the classification of flavin-dependent monooxygenases and summarize the latest advances in our understanding of their catalytic and structural properties
Geographic coupling of juvenile and adult habitat shapes spatial population dynamics of a coral reef fish
Huijbers, C.M. ; Nagelekerken, I. ; Debrot, A.O. ; Jongejans, E. - \ 2013
Ecology 94 (2013)8. - ISSN 0012-9658 - p. 1859 - 1870.
larval dispersal - marine reserves - menidia-menidia - seagrass beds - life-history - connectivity - nurseries - mangroves - estuarine - chemistry
Marine spatial population dynamics are often addressed with a focus on larval dispersal, without taking into account movement behavior of individuals in later life stages. Processes occurring during demersal life stages may also drive spatial population dynamics if habitat quality is perceived differently by animals belonging to different life stages. In this study, we used a dual approach to understand how stage-structured habitat use and dispersal ability of adults shape the population of a marine fish species. Our study area and focal species provided us with the unique opportunity to study a closed island population. A spatial simulation model was used to estimate dispersal distances along a coral reef that surrounds the island, while contributions of different nursery bays were determined based on otolith stable isotope signatures of adult reef fish. The model showed that adult dispersal away from reef areas near nursery bays is limited. The results further show that different bays contributed unequally to the adult population on the coral reef, with productivity of juveniles in bay nursery habitat determining the degree of mixing among local populations on the reef and with one highly productive area contributing most to the island's reef fish population. The contribution of the coral reef as a nursery habitat was minimal, even though it had a much larger surface area. These findings indicate that the geographic distribution of nursery areas and their productivity are important drivers for the spatial distribution patterns of adults on coral reefs. We suggest that limited dispersal of adults on reefs can lead to a source–sink structure in the adult stage, where reefs close to nurseries replenish more isolated reef areas. Understanding these spatial population dynamics of the demersal phase of marine animals is of major importance for the design and placement of marine reserves, as nursery areas contribute differently to maintain adult populations.
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