Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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The shufflon of IncI1 plasmids is rearranged constantly during different growth conditions
Brouwer, Mike ; Jurburg, Stephanie ; Harders, Frank ; Kant, Arie ; Mevius, Dik ; Roberts, Adam P. ; Bossers, Alex - \ 2019
Wageningen University & Research
PRJEB30618 - ERP113093 - Escherichia coli
One of the factors that can affect conjugation of IncI1 plasmids, amongst others, is the genetic region known as the shufflon. This multiple inversion system modifies the pilus tip proteins used during conjugation, thus affecting the affinity for different recipient cells. Although recombination is known to occur in in vitro conditions, little is known about the regulation and the extend of recombination that occurs. To measure the recombination of the shufflon, we have amplified the entire shufflon region and sequenced the amplicons using nanopore long-read sequencing. This method was effective to determine the order of the segments of the shufflon and allow for the analysis of the shufflon variants that are present in a heterogeneous pool of templates. Analysis was performed over different growth phases and after addition of cefotaxime. Furthermore, analysis was performed in different E. coli host cells to determine if recombination is likely to be influenced. Recombination of the shufflon was constantly ongoing in all conditions that were measured, although no differences in the amount of different shufflon variants or the rate at which novel variants were formed could be found. As previously reported, some variants were abundant in the population while others were scarce. This leads to the conclusion that the shufflon is continuously recombining at a constant rate, or the method used here was not sensitive enough to detect differences in this rate. For one of the plasmids, the host cell appears to have an effect on the specific shufflon variants that were formed which were not predominant in another host, indicating that host factors may be involved. As previously reported, the pilV-A and pilV-A’ ORFs are formed at higher frequencies than other pilV ORFs. These results demonstrate that the recombination that occurs within the shufflon is not random. While any regulation of the shufflon affected by these in vitro conditions could not be revealed, the method of amplifying large regions for long-read sequencing for the analysis of multiple inversion systems proved effective.
Bicistronic Design-Based Continuous and High-Level Membrane Protein Production in Escherichia coli
Claassens, Nico J. ; Finger-Bou, Max ; Scholten, Bart ; Muis, Frederieke ; Groot, Jonas J. De; Gier, Jan Willem De; Vos, Willem M. De; Oost, John Van Der - \ 2019
ACS synthetic biology 8 (2019)7. - ISSN 2161-5063 - p. 1685 - 1690.
bicistronic design - Escherichia coli - membrane proteins - protein production - translational coupling

Escherichia coli has been widely used as a platform microorganism for both membrane protein production and cell factory engineering. The current methods to produce membrane proteins in this organism require the induction of target gene expression and often result in unstable, low yields. Here, we present a method combining a constitutive promoter with a library of bicistronic design (BCD) elements, which enables inducer-free, tuned translation initiation for optimal protein production. Our system mediates stable, constitutive production of bacterial membrane proteins at yields that outperform those obtained with E. coli Lemo21(DE3), the current gold standard for bacterial membrane protein production. We envisage that the continuous, fine-tunable, and high-level production of membrane proteins by our method will greatly facilitate their study and their utilization in engineering cell factories.

Monitoring antimicrobial resistance trends in commensal Escherichia coli from livestock, the Netherlands, 1998 to 2016
Hesp, Ayla ; Veldman, Kees ; Goot, Jeanet van der; Mevius, Dik ; Schaik, G. van - \ 2019
Eurosurveillance 24 (2019)25. - ISSN 1025-496X
AMR - antimicrobial resistance - Escherichia coli - monitoring - quantitative - surveillance - trend analysis

BackgroundMonitoring of antimicrobial resistance (AMR) in animals is essential for public health surveillance. To enhance interpretation of monitoring data, evaluation and optimisation of AMR trend analysis is needed.AimsTo quantify and evaluate trends in AMR in commensal Escherichia coli, using data from the Dutch national AMR monitoring programme in livestock (1998-2016).MethodsFaecal samples were collected at slaughter from broilers, pigs and veal calves. Minimum inhibitory concentration values were obtained by broth microdilution for E. coli for 15 antimicrobials of eight antimicrobial classes. A Poisson regression model was applied to resistant isolate counts, with explanatory variables representing time before and after 2009 (reference year); for veal calves, sampling changed from 2012 represented by an extra explanatory variable.ResultsResistant counts increased significantly from 1998-2009 in broilers and pigs, except for tetracyclines and sulfamethoxazole in broilers and chloramphenicol and aminoglycosides in pigs. Since 2009, resistant counts decreased for all antimicrobials in broilers and for all but the phenicols in pigs. In veal calves, for most antimicrobials no significant decrease in resistant counts could be determined for 2009-16, except for sulfamethoxazole and nalidixic acid. Within animal species, antimicrobial-specific trends were similar.ConclusionsUsing Dutch monitoring data from 1998-2016, this study quantified AMR trends in broilers and slaughter pigs and showed significant trend changes in the reference year 2009. We showed that monitoring in commensal E. coli is useful to quantify trends and detect trend changes in AMR. This model is applicable to similar data from other European countries.

Nucleic acid lateral flow assays using a conjugate of a DNA binding protein and carbon nanoparticles
Aktas, Gülsen Betül ; Wichers, Jan H. ; Skouridou, Vasso ; Amerongen, Aart van; Masip, Lluis - \ 2019
Microchimica acta 186 (2019)7. - ISSN 0026-3672
Enzyme conjugate - Escherichia coli - Immunochromatographic test - Nucleic acid lateral flow assay - Signal enhancement - Single-chain Cro

Nucleic acid lateral flow assays (NALFA) are often performed with gold nanoparticles. These are typically associated with ligand-labeled PCR amplicons via affinity interactions of adsorbed/conjugated proteins. Otherwise, they are conjugated to specific ssDNA sequences that hybridize to the target sequence. To avoid the need to generate ssDNA and to reduce the costs associated with primer labeling and antibody use, NALFA assays were developed that allow the direct detection of PCR amplicons using conjugates of a DNA binding protein with carbon nanoparticles (CNPs). The target gene encoding 16S ribosomal RNA of Escherichia coli was amplified by PCR using a single fluorophore-labeled forward primer and a reverse primer extended with the binding sequence of the bacteriophage lambda Cro repressor protein. Three different detection approaches were evaluated: (a) scCro/CNPs conjugate (black color), (b) HRP-scCro enzyme conjugate (red color), and (c) HRP-scCro/CNPs conjugate for dual color development. The limits of detection were between 6.9 and 10.4 ng of PCR product for all three approaches. These correspond to 3.0 to 4.5 × 103 CFU·mL−1. The single-step scCro/CNP approach proved to be the fastest one to perform and gave no false-positive signals. It also showed a broad dynamic range even though the signal intensities were lower compared to the enzyme-amplified tests. However, the latter ones produced some background signal. In our perception, the application of scCro in lateral flow assays to bind dsDNA appears to be an excellent alternative to the use of small tags that have to be chemically linked to synthetic primers. Finally, the approach is generic because any primer sequence can be extended with the specific scCro binding sequence. [Figure not available: see fulltext.].

Substantial extracellular metabolic differences found between phylogenetically closely related probiotic and pathogenic strains of Escherichia coli
Hooft, Justin J.J. Van Der; Goldstone, Robert J. ; Harris, Susan ; Burgess, Karl E.V. ; Smith, David G.E. - \ 2019
Frontiers in Microbiology 10 (2019)FEB. - ISSN 1664-302X
Escherichia coli - Extracellular metabolome - Mass spectrometry - Metabolomics - Nissle 1917 - Pathogenic - Probiotic

Since its first isolation a century ago, the gut inhabitant Escherichia coli strain Nissle 1917 has been shown to have probiotic activities; however, it is yet not fully elucidated which differential factors play key roles in its beneficial interactions with the host. To date, no metabolomics studies have been reported investigating the potential role of small molecules in functional strain differentiation of Nissle from its genetically close neighbors. Here, we present results of liquid chromatography coupled to high-resolution mass spectrometry characterization of extracellular metabolomes of E. coli strains as a proxy of their bioactivity potential. We found that phylogroup B2 strains exported a more diverse arsenal of metabolites than strains of other phylogroups. Zooming into the phylogroup B2 metabolome identified consistent substantial differences between metabolic output of E. coli Nissle and other strains, particularly in metabolites associated to the Argimine biosynthesis pathway. Nissle was found to release higher levels of Ornithine and Citrulline whilst depleting greater amounts of Arginine from the medium. Moreover, a novel Nissle-specific metabolite not reported before in bacteria, 5-(Carbamoylamino)-2-hydroxypentanoic acid (Citrulline/Arginic Acid related) was observed. Finally, Nissle, CFT073 and NCTC12241/ATCC25922 shared the excretion of N5-Acetylornithine, whereas other strains released N2-Acetylornithine or no N-Acetylornithine at all. Thus, we found substantial metabolic differences in phylogenetically very similar E. coli strains, an observation which suggests that it is justified to further investigate roles of small molecules as potential modulators of the gut environment by probiotic, commensal, and pathogenic strains, including E. coli Nissle 1917.

Diversity of plasmids and genes encoding resistance to extended spectrum cephalosporins in commensal Escherichia coli From Dutch livestock in 2007-2017
Ceccarelli, Daniela ; Kant, Arie ; Essen-Zandbergen, Alieda Van; Dierikx, Cindy ; Hordijk, Joost ; Wit, Ben ; Mevius, Dik J. ; Veldman, Kees T. - \ 2019
Frontiers in Microbiology 10 (2019)FEB. - ISSN 1664-302X
ESBL - Escherichia coli - Livestock - PAmpC - Plasmid

Extended-spectrum β-lactamase (ESBL) and plasmid-mediated AmpC β-lactamase (pAmpC) genes confer resistance to extended spectrum cephalosporin's. The spread of these genes is mostly facilitated by plasmid-mediated horizontal transfer. National surveillance activities to detect ESBL/pAmpC-producers in commensal bacteria from livestock are in place in the Netherlands since several years. This study aimed at reporting gene and plasmid diversity of commensal ESBL/pAmpC-producing Escherichia coli isolated from healthy animals during surveillance activities between 2007 and 2017. A collection of 2304 extended-spectrum cephalosporin-resistant (ESC-R) E. coli isolated from feces of broilers, dairy cattle, slaughter pigs, turkeys, ducks, and veal calves was investigated and ESBL/pAmpC genes were determined. Gene location of a selection of 473 E. coli isolates was determined and typing of plasmids linked to the ESBL/pAmpC genes was performed. Twenty-two different ESBL/pAmpC genes were identified with bla CTX-M-1 being the most prevalent gene in livestock (43.7%), followed by bla CMY -2 and bla SHV -12 , independent of the animal source. Prevalence of typically human associated bla CTX-M-15 was highest in cattle. Less than 10% E. coli isolates owed their ESC-R phenotype to promoter mutations of the chromosomal ampC gene. Majority (92%) of ESBL/pAmpC genes analyzed were plasmid located, with IncI1α being the most represented plasmid family in isolates from all animals, followed by IncF (veal calves, dairy cattle and slaughter pigs), IncK (broilers and laying hens), IncX1 in broilers, and emerging IncX3 in broilers and dairy cattle. Prevalence and molecular diversity of ESC-R E. coli isolated from livestock over an 11-year period revealed a composite scenario of gene-plasmid combinations.

Selective breeding for high natural antibody level increases resistance to avian pathogenic Escherichia coli (APEC) in chickens
Berghof, T.V.L. ; Matthijs, M.G.R. ; Arts, J.A.J. ; Bovenhuis, H. ; Dwars, R.M. ; Poel, J.J. van der; Visker, M.H.P.W. ; Parmentier, H.K. - \ 2019
Developmental and Comparative Immunology 93 (2019). - ISSN 0145-305X - p. 45 - 57.
APEC - Breeding - Chicken - Disease resistance - Escherichia coli - Natural antibody

Keyhole limpet hemocyanin (KLH)-binding natural antibody (NAb) titers in chickens are heritable, and higher levels have previously been associated with a higher survival. This suggests that selective breeding for higher NAb levels might increase survival by means of improved general disease resistance. Chickens were divergently selected and bred for total NAb levels binding KLH at 16 weeks of age for six generations, resulting in a High NAb selection line and a Low NAb selection line. To for test differences in disease resistance, chickens were challenged with avian pathogenic Escherichia coli (APEC) in two separate experiments. Chickens at 8 days of age received one of four intratracheal inoculations of 0.2 mL phosphate buffered saline (PBS): 1) mock inoculate, 2) with 0.2 mL PBS containing 108.20 colony-forming units (CFU)/mL APEC, 3) with 0.2 mL PBS containing 106.64 CFU/mL APEC, and 4) with 0.2 mL PBS containing 107.55 CFU/mL APEC. Mortality was recorded during 7 days post inoculation. Overall, 50–60% reduced mortality was observed in the High line compared to the Low line for all APEC doses. In addition, morbidity was determined of the surviving chickens at 15 days of age. The High line had lower morbidity scores compared to the Low line. We conclude that selective breeding for high KLH-binding NAb levels at 16 weeks of age increase APEC resistance in early life. This study and previous studies support the hypothesis that KLH-binding NAb might be used as an indicator trait for to selective breed for general disease resistance in an antigen non-specific fashion.

The impact of socio-economic development and climate change on E. coli loads and concentrations in Kabul River, Pakistan
Iqbal, Muhammad Shahid ; Islam, M.M.M. ; Hofstra, Nynke - \ 2019
Science of the Total Environment 650 (2019). - ISSN 0048-9697 - p. 1935 - 1943.
Bacterial modelling - Escherichia coli - Global change - Hydrological modelling - Scenario analysis

Microbial pollution is a major problem worldwide. High concentrations of Escherichia coli have been found in Kabul River in Pakistan. E. coli concentrations vary under different socio-economic conditions, such as population and livestock densities, urbanisation, sanitation and treatment of wastewater and manure, and climate-change aspects, such as floods and droughts. In this paper, we assess potential future E. coli loads and concentrations in the Kabul River using the Soil and Water Assessment Tool with scenarios that are based on the most recent Shared Socio-economic Pathways and Representative Concentration Pathways (SSPs and RCPs) developed for the Intergovernmental Panel on Climate Change (IPCC). Scenario_1 considers moderate population and livestock density growth, planned urbanisation and strongly improved wastewater and manure treatment (based on SSP1, “Sustainability”), and moderate climate change (RCP4.5, moderate greenhouse gas (GHG) emissions). Scenario_2 considers strong population and livestock density growth, moderate urbanisation, slightly improved wastewater treatment, no manure treatment (based on SSP3, “Regional rivalry”) and strong climate change (RCP8.5, high GHG emissions). Simulated E. coli responses to Scenario_2 suggest a mid-century increase in loads by 111% and a late century increase of 201% compared to baseline loads. Similarly, simulated E. coli loads are reduced by 60% for the mid-century and 78% for the late century compared to the baseline loads. When additional treatment is simulated in Scenario_1, the loads are reduced even further by 94%, 92% and 99.3% compared to the baseline concentrations when additional tertiary treatment, manure treatment or both have been applied respectively. This study is one of the first to apply combined socio-economic development and climate change scenario analysis with an E. coli concentration model to better understand how these concentrations may change in the future. The scenario analysis shows that reducing E. coli concentrations in Pakistan's rivers is possible, but requires strongly improved waste water treatment and manure management measures.

Data from: Unraveling the causes of adaptive benefits of synonymous mutations in TEM-1 β-lactamase
Zwart, M.P. ; Schenk, M.F. ; Hwang, S. ; Koopmanschap-Memelink, A.B. ; Lange, N. de; Pol, Lion van de; Nga, Tran T.T. ; Szendro, Ivan G. ; Krug, Joachim ; Visser, J.A.G.M. de - \ 2018
betalactamase - TEM-1 - resistance gene - synonymous mutations - Cefotaxime - fitness landscape - epistasis - bulk competition - Escherichia coli
While synonymous mutations were long thought to be without phenotypic consequences, there is growing evidence they can affect gene expression, protein folding and ultimately the fitness of an organism. In only a few cases have the mechanisms by which synonymous mutations affect the phenotype been elucidated. We previously identified 48 mutations in TEM-1 β-lactamase that increased resistance of Escherichia coli to cefotaxime, 10 of which were synonymous. To better understand the molecular mechanisms underlying the beneficial effect of synonymous mutations, we made a series of measurements for a panel containing the 10 synonymous together with 10 non-synonymous mutations as a reference. Whereas messenger levels were unaffected, we found that total and functional TEM protein levels were higher for 5 out of 10 synonymous mutations. These observations suggest that some of these mutations act on translation or a downstream process. Similar effects were observed for some small-benefit non-synonymous mutations, suggesting a similar causal mechanism. For the synonymous mutations, we found that the cost of resistance scales with TEM protein levels. A resistance landscape for four synonymous mutations revealed strong epistasis: None of the combinations of mutations exceeded the resistance of the largest-effect mutation and there were synthetically neutral combinations. By considering combined effects of these mutations, we could infer that functional TEM protein level is a multi-dimensional phenotype. These results suggest that synonymous mutations may have beneficial effects by increasing the expression of an enzyme with low substrate activity, which may be realized via multiple, yet unknown, post-transcriptional mechanisms
High prevalence of intra-familial co-colonization by extended-spectrum cephalosporin resistant Enterobacteriaceae in preschool children and their parents in Dutch households
Liakopoulos, Apostolos ; Bunt, Gerrita van den; Geurts, Yvon ; Bootsma, Martin C.J. ; Toleman, Mark ; Ceccarelli, Daniela ; Pelt, Wilfrid van; Mevius, Dik J. - \ 2018
Frontiers in Microbiology 9 (2018)FEB. - ISSN 1664-302X
Co-carriage - ESBL/AmpC - Escherichia coli - Household - Insertion sequence - Netherlands - Plasmid
Extended-spectrum cephalosporin-resistant (ESCR) Enterobacteriaceae pose a serious infection control challenge for public health. The emergence of the ESCR phenotype is mostly facilitated by plasmid-mediated horizontal extended-spectrum β-lactamases (ESBLs) and AmpC gene transfer within Enterobacteriaceae. Current data regarding the plasmid contribution to this emergence within the Dutch human population is limited. Hence, the aim of this study was to gain insight into the role of plasmids in the dissemination of ESBL/AmpC genes inside Dutch households with preschool children and precisely delineate co-colonization. In 87 ESCR Enterobacteriaceae from fecal samples of parents and preschool children within 66 Dutch households, genomic localization, plasmid type and insertion sequences linked to ESBL/AmpC genes were determined. Chromosomal location of ESBL/AmpC genes was confirmed when needed. An epidemiologically relevant subset of the isolates based on household co-carriage was assessed by Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis for genetic relatedness. The narrow-host range I1a and F plasmids were the major facilitators of ESBL/AmpC-gene dissemination. Interestingly, we documented a relatively high occurrence of chromosomal integration of typically plasmid-encoded ESBL/AmpC-genes. A high diversity of non-epidemic Escherichia coli sequence types (STs) was revealed; the predominant STs belonged to the pandemic lineages of extraintestinal pathogenic E. coli ST131 and ST69. Intra-familiar co-carriage by identical ESCR Enterobacteriaceae was documented in 7 households compared to 14 based on sole gene typing, as previously reported. Co-carriage was more frequent than expected based on pure chance, suggesting clonal transmission between children and parents within the household.
Competitive exclusion reduces transmission and excretion of extended-spectrum-β-lactamase-producing Escherichia coli in broilers
Ceccarelli, Daniela ; Essen-Zandbergen, Alieda van; Smid, Bregtje ; Veldman, Kees T. ; Boender, Gert Jan ; Fischer, Egil A.J. ; Mevius, Dik J. ; Goot, Jeanet A. van der - \ 2017
Applied and Environmental Microbiology 83 (2017)11. - ISSN 0099-2240 - 13 p.
Broiler chicken - Competitive exclusion - ESBL - Escherichia coli - Excretion - Intervention - Poultry - Transmission - β-lactamases

Extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (pAmpC) are enzymes able to hydrolyze a large variety of β-lactam antibiotics, including third-generation cephalosporins and monobactams. Broilers and broiler meat products can be highly contaminated with ESBL- and pAmpCproducing Escherichia coli strains, also known as extended-spectrum cephalosporin (ESC)-resistant E. coli strains, and can be a source for human infections. As few data on interventions to reduce the presence of ESC-resistant E. coli in broilers are available, we used transmission experiments to examine the role of competitive exclusion (CE) on reducing transmission and excretion in broilers. A broiler model to study the transmission of ESC-resistant E. coli was set up. Day-old chickens were challenged with an ESBL-producing E. coli strain isolated from healthy broilers in the Netherlands. Challenged and not challenged chicks were housed together in pairs or in groups, and ESBL-producing E. coli transmission was monitored via selective culturing of cloacal swab specimens. We observed a statistically significant reduction in both the transmission and excretion of ESBL-producing E. coli in chicks treated with the probiotic flora before E. coli challenge compared to the transmission and excretion in untreated controls. In conclusion, our results support the use of competitive exclusion as an intervention strategy to control ESC-resistant E. coli in the field.

Ethyl acetate production by the elusive alcohol acetyltransferase from yeast
Kruis, Alex ; Levisson, Mark ; Mars, Astrid E. ; Ploeg, Max van der; Garcés Daza, Fernando ; Ellena, Valeria ; Kengen, Servé W.M. ; Oost, John van der; Weusthuis, Ruud A. - \ 2017
Metabolic Engineering 41 (2017). - ISSN 1096-7176 - p. 92 - 101.
Alcohol acetyltransferase - Escherichia coli - Ethyl acetate - Saccharomyces cerevisiae - yeast - α/β hydrolase
Ethyl acetate is an industrially relevant ester that is currently produced exclusively through unsustainable processes. Many yeasts are able to produce ethyl acetate, but the main responsible enzyme has remained elusive, hampering the engineering of novel production strains. Here we describe the discovery of a new enzyme (Eat1) from the yeast Wickerhamomyces anomalus that resulted in high ethyl acetate production when expressed in Saccharomyces cerevisiae and Escherichia coli. Purified Eat1 showed alcohol acetyltransferase activity with ethanol and acetyl-CoA. Homologs of eat1 are responsible for most ethyl acetate synthesis in known ethyl acetate-producing yeasts, including S. cerevisiae, and are only distantly related to known alcohol acetyltransferases. Eat1 is therefore proposed to compose a novel alcohol acetyltransferase family within the α/β hydrolase superfamily. The discovery of this novel enzyme family is a crucial step towards the development of biobased ethyl acetate production and will also help in selecting improved S. cerevisiae brewing strains.
Dynamics of CMY-2 producing E. coli in a broiler parent flock
Dame-Korevaar, Anita ; Fischer, Egil A.J. ; Stegeman, Arjan ; Mevius, Dik ; Essen-Zandbergen, Alieda van; Velkers, Francisca ; Goot, Jeanet van der - \ 2017
Veterinary Microbiology 203 (2017). - ISSN 0378-1135 - p. 211 - 214.
Antibiotic resistance - Broiler parent stock - CMY - Escherichia coli - Poultry

Extended-spectrum β-lactamase and plasmid mediated AmpC β-lactamase (ESBL/pAmpC) producing bacteria are resistant to Extended Spectrum Cephalosporins (ESC), and are present in all levels of the broiler production chain. We determined the prevalence, concentration, and persistence of ESBL/pAmpC-Escherichia coli in a broiler parent flock during the rearing and laying period. One-day old chickens were housed in four separate pens. Until week 33 no antibiotics or coccidiostatics were used. During rearing 57 chickens in each pen (n = 228), and in the laying period two groups of 33 chickens were individually sampled (n = 66). Environmental samples were taken from week 16 onwards. ESBL/pAmpC-E. coli presence was determined by selective culturing. In the samples of week 16–19 the concentration of ESBL/pAmpC-E. coli was determined. All ESC-resistant isolates found were positive for pAmpC gene blaCMY-2 located on IncA/C plasmids, in several E. coli MLST types. CMY-2-E. coli prevalence decreased from 91% (95%CI 86–94%) at day 7 (week 1) to 0% (95%CI 0–5%) in week 21. However, CMY-2-E. coli remained present in the environmental samples during the whole study. CMY-2-E. coli concentration varied between detection limit (<10^3) and 2·10^4 cfu/g faeces. The sharp reduction of CMY-2-E. coli in this broiler parent flock in absence of antibiotics suggests a selective disadvantage of blaCMY-2 on IncA/C plasmids on animal level. The underlying mechanism should be studied further as this may provide new insights on how to reduce ESBL/pAmpC prevalence and transmission in the broiler production chain.

Intramammary immunization with ultraviolet-killed Escherichia coli shows partial protection against late gestation intramammary challenge with a homologous strain
Pomeroy, B. ; Gurjar, A. ; Sipka, A. ; Klaessig, S. ; Salmon, S. ; Quesnell, R. ; Schukken, Y.H. - \ 2016
Journal of Dairy Science 99 (2016)11. - ISSN 0022-0302 - p. 9014 - 9026.
Escherichia coli - late gestation - mastitis - vaccination

The objective of this study was to evaluate the efficacy of intramammary immunization with UV-killed Escherichia coli ECC-Z on prevention of intramammary colonization after a challenge with a dose of the homologous E. coli ECC-Z live bacteria. A total of 10 cows were included in a study to evaluate the efficacy of intramammary immunization. All 10 cows received an intramammary immunization of 100 cfu of UV-killed E. coli ECC-Z bacteria into one hind quarter at the time of dry off. Approximately 2 wk before the anticipated calving date, both hind quarters of all cows were challenged with 100 cfu of live E. coli ECC-Z bacteria. Five of the cows were vaccinated parenterally with a commercial J5 bacterin, and 5 cows served as controls with no parenteral vaccination. The cows were then followed over time and infection risk, clinical scores, somatic cell count, and milk production were observed over time. The results of these 10 cows showed partial protection of intramammary immunization on the outcome of a subsequent homologous intramammary challenge. Immunization resulted in a lower probability of infection, a lower bacteria count, lower somatic cell counts and milk conductivity, a lower clinical mastitis score, and increased milk production compared with unimmunized control quarters. Once the analysis was corrected for immunization, parenteral J5 vaccination had no significant effect on any of the measured parameters. These results provide the first evidence that intramammary immunization may improve the outcome of an intramammary E. coli infection in late gestation and onset of mastitis immediately following parturition. Unlike systemic vaccination, which generally does not reduce the intramammary infection risk, the intramammary immunization did show a 5-times reduced odds of an established intramammary infection after challenge. Cytokine profiles indicated a local return of proinflammatory response after challenge as the data showed a more pronounced increase in in IFN-γ with a subsequent negative feedback due to a spike in the level of IL-10 in immunized quarters relative to nonimmunized quarters. Although these results are preliminary and obtained on only 10 cows, the results provide insight into the biological benefits of triggering mucosal immunity in the mammary gland.

Diurnal differences in milk composition and its influence on in vitro growth of Staphylococcus aureus and Escherichia coli in bovine quarter milk
Eisenberg, S.W.F. ; Boerhout, E.M. ; Ravesloot, L. ; Daemen, A.J.J.M. ; Benedictus, L. ; Rutten, V.P.M.G. ; Koets, A.P. - \ 2016
Journal of Dairy Science 99 (2016)7. - ISSN 0022-0302 - p. 5690 - 5700.
Bovine - Escherichia coli - Milk composition - Staphylococcus aureus

In experimental intramammary inoculation studies, it has been observed that mastitis susceptibility is influenced, among others, by cow factors. To identify milk characteristics leading to these differences, quarter milk samples of morning and evening milk were collected and analyzed for their composition (protein, fat, lactose, urea, lactoferrin, lactoperoxidase, and β-lactoglobulin concentrations), somatic cell count, and antibodies against Staphylococcus aureus. Furthermore, in vitro growth of S. aureus and Escherichia coli in fresh quarter milk samples was determined. All measured parameters differed significantly between quarters and also between morning and evening milk with the exception of lactose levels. In addition, quantitative growth of S. aureus and E. coli was significantly different in morning milk compared with evening milk. Mixed model analysis revealed that replication of S. aureus was negatively associated with the presence of fat, S. aureus-specific IgG1 antibodies, contamination of the milk sample and morning milk. Replication of E. coli was negatively associated with fat concentrations, and positively associated with morning milk. The significant difference between morning and evening milk supports the theory that changes in milk composition influence bacterial growth. Although all determined milk components differed significantly between quarters and in time no significant association with bacterial growth could be identified with the exception of fat for both studied species and IgG1 titers for S. aureus. The negative association of fat with bacterial growth was assumed to occur due to activation of lipolysis by milk handling and can most likely be neglected for in vivo relevance. The fact that S. aureus-specific IgG1 titers were negatively associated with S. aureus growth in vitro encourages the ongoing effort to develop a vaccine against S. aureus-induced mastitis.

Compaction of isolated Escherichia coli nucleoids : Polymer and H-NS protein synergetics
Wegner, Anna S. ; Wintraecken, Kathelijne ; Spurio, Roberto ; Woldringh, Conrad L. ; Vries, Renko de; Odijk, Theo - \ 2016
Journal of Structural Biology 194 (2016)1. - ISSN 1047-8477 - p. 129 - 137.
Dynamic light scattering - EMSA - Escherichia coli - H-NS protein - Nucleoid - Polyethylene glycol - Polymer physics - Supercoiling

Escherichia coli nucleoids were compacted by the inert polymer polyethylene glycol (PEG) in the presence of the H-NS protein. The protein by itself appears to have little impact on the size of the nucleoids as determined by fluorescent microscopy. However, it has a significant impact on the nucleoidal collapse by PEG. This is quantitatively explained by assuming the H-NS protein enhances the effective diameter of the DNA helix leading to an increase in the depletion forces induced by the PEG. Ultimately, however, the free energy of the nucleoid itself turns out to be independent of the H-NS concentration. This is because the enhancement of the supercoil excluded volume is negligible. The experiments on the nucleoids are corroborated by dynamic light scattering and EMSA analyses performed on DNA plasmids in the presence of PEG and H-NS.

Metabolic engineering of the mixed-acid fermentation pathway of Escherichia coli for anaerobic production of glutamate and itaconate
Vuoristo, Kiira S. ; Mars, Astrid E. ; Sangra, Jose Vidal ; Springer, Jan ; Eggink, Gerrit ; Sanders, Johan P.M. ; Weusthuis, Ruud A. - \ 2015
AMB Express 5 (2015)1. - ISSN 2191-0855 - 11 p.
Anaerobic fermentation - Escherichia coli - Ethanol - Glutamic acid - Itaconic acid - Metabolic engineering - Redox balance

Itaconic acid, an unsaturated C5-dicarboxylic acid, is a biobased building block for the polymer industry. The purpose of this study was to establish proof of principle for an anaerobic fermentation process for the production of itaconic acid by modification of the mixed acid fermentation pathway of E. coli. E. coli BW25113 (DE3) and the phosphate acetyltransferase (pta) and lactate dehydrogenase (ldhA) deficient strain E. coli BW25113 (DE3) Δpta-ΔldhA were used to study anaerobic itaconate production in E. coli. Heterologous expression of the gene encoding cis-aconitate decarboxylase (cadA) from A. terreus in E. coli BW25113 (DE3) did not result in itaconate production under anaerobic conditions, but 0.08 mM of itaconate was formed when the genes encoding citrate synthase (gltA) and aconitase (acnA) from Corynebacterium glutamicum were also expressed. The same amount was produced when cadA was expressed in E. coli BW25113 (DE3) Δpta-ΔldhA. The titre increased 8 times to 0.66 mM (1.2 % Cmol) when E. coli BW25113 (DE3) Δpta-ΔldhA also expressed gltA and acnA. In addition, this strain produced 8.5 mM (13 % Cmol) of glutamate. The use of a nitrogen-limited growth medium reduced the accumulation of glutamate by nearly 50 % compared to the normal medium, and also resulted in a more than 3-fold increase of the itaconate titre to 2.9 mM. These results demonstrated that E. coli has potential to produce itaconate and glutamate under anaerobic conditions, closing the redox balance by co-production of succinate or ethanol with H2 and CO2.

Carriage of extended-spectrum β-lactamases in pig farmers is associated with occurrence in pigs
Dohmen, W. ; Bonten, M.J.M. ; Bos, M.E.H. ; Marm, S. van; Scharringa, J. ; Wagenaar, J.A. ; Heederik, D.J.J. - \ 2015
Clinical Microbiology and Infection 21 (2015)10. - ISSN 1198-743X - p. 917 - 923.
Animal reservoirs - Animals - Antimicrobial resistance - CTX-M - Epidemiology - Escherichia coli - Zoonosis

Livestock may serve as a reservoir for extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE). The objectives of this study were to determine the prevalence of carriage with ESBL-PE in pig farmers, family members and employees, and its association with carriage in pigs. Rectal swabs were taken from 2388 pigs (398 pooled samples) on 40 pig farms and faecal samples were obtained from 142 humans living or working on 34 of these farms. Presence of ESBL-PE was determined by selective plating (agar). ESBL genes were analysed by PCR or microarray analysis, and gene sequencing. Genotypes and plasmids were determined by multilocus sequence typing and PCR-based replicon typing for selected isolates. ESBL genes were detected in Escherichia coli from eight humans (6%) (blaCTX-M-1, n = 6; blaTEM-52, n = 1 and blaCTX-M-14, n = 1) on six farms. In 157 pig isolates (107 pooled samples) on 18 farms (45%) ESBL genes were detected (blaCTX-M-1, n = 12; blaTEM-52, n = 6; and blaCTX-M-14, n = 3). Human and pig isolates within the same farm harboured similar ESBL gene types and had identical sequence and plasmid types on two farms (e.g. E. coli ST-453, blaCTX-M-1, IncI1), suggesting clonal transmission. For the remaining farms, sequence types, but not plasmid types, differed. Human ESBL carriage was associated with average number of hours working on the farm per week (OR = 1.04, 95% CI 1.02-1.06) and presence of ESBLs in pigs (OR = 12.5, 95% CI 1.4-111.7). Daily exposure to pigs carrying ESBL-PE is associated with ESBL carriage in humans.

Comparative genomic hybridization of Streptococcus suis strains - part 2
Greeff, Astrid de - \ 2010
Wageningen UR
E-MEXP-2533 - Escherichia coli - Streptococcus suis
Comparative genome hybridization was used to study genomic diversity among S. suis strains in more detail. All strains were hybridized to an oligo-array based on the genome of P1/7 with P1/7 as an internal reference in all experiments.
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