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Microcolony Imaging of Aspergillus fumigatus Treated with Echinocandins Reveals Both Fungistatic and Fungicidal Activities
Ingham, C.J. ; Schneeberger, P.M. - \ 2012
PLoS ONE 7 (2012)4. - ISSN 1932-6203
in-vitro - caspofungin - pharmacodynamics - microorganisms - antifungal - micafungin - culture - growth - chip
Background: The echinocandins are lipopeptides that can be employed as antifungal drugs that inhibit the synthesis of 1,3-beta-glucans within the fungal cell wall. Anidulafungin and caspofungin are echinocandins used in the treatment of Candida infections and have activity against other fungi including Aspergillus fumigatus. The echinocandins are generally considered fungistatic against Aspergillus species. Methods: Culture of A. fumigatus from conidia to microcolonies on a support of porous aluminium oxide (PAO), combined with fluorescence microscopy and scanning electron microscopy, was used to investigate the effects of anidulafungin and caspofungin. The PAO was an effective matrix for conidial germination and microcolony growth. Additionally, PAO supports could be moved between agar plates containing different concentrations of echinocandins to change dosage and to investigate the recovery of fungal microcolonies from these drugs. Culture on PAO combined with microscopy and image analysis permits quantitative studies on microcolony growth with the flexibility of adding or removing antifungal agents, dyes, fixatives or osmotic stresses during growth with minimal disturbance of fungal microcolonies. Significance: Anidulafungin and caspofungin reduced but did not halt growth at the microcony level; additionally both drugs killed individual cells, particularly at concentrations around the MIC. Intact but not lysed cells showed rapid recovery when the drugs were removed. The classification of these drugs as either fungistatic or fungicidal is simplistic. Microcolony analysis on PAO appears to be a valuable tool to investigate the action of antifungal agents.
Rapid Susceptibility Testing and Microcolony Analysis of Candida spp. Cultured and Imaged on Porous Aluminum Oxide
Ingham, C.J. ; Boonstra, S. ; Levels, S. ; Lange, H.J. ; Meis, J.F. ; Schneeberger, P.M. - \ 2012
PLoS ONE 7 (2012)3. - ISSN 1932-6203
antifungal drug-resistance - flow-cytometry - interpretive breakpoints - voriconazole - growth - fun-1 - microorganisms - fluconazole - albicans - support
Background: Acquired resistance to antifungal agents now supports the introduction of susceptibility testing for species-drug combinations for which this was previously thought unnecessary. For pathogenic yeasts, conventional phenotypic testing needs at least 24 h. Culture on a porous aluminum oxide (PAO) support combined with microscopy offers a route to more rapid results. Methods: Microcolonies of Candida species grown on PAO were stained with the fluorogenic dyes Fun-1 and Calcofluor White and then imaged by fluorescence microscopy. Images were captured by a charge-coupled device camera and processed by publicly available software. By this method, the growth of yeasts could be detected and quantified within 2 h. Microcolony imaging was then used to assess the susceptibility of the yeasts to amphotericin B, anidulafungin and caspofungin (3.5 h culture), and voriconazole and itraconazole (7 h culture). Significance: Overall, the results showed good agreement with EUCAST (86.5% agreement; n = 170) and E-test (85.9% agreement; n = 170). The closest agreement to standard tests was found when testing susceptibility to amphotericin B and echinocandins (88.2 to 91.2%) and the least good for the triazoles (79.4 to 82.4%). Furthermore, large datasets on population variation could be rapidly obtained. An analysis of microcolonies revealed subtle effects of antimycotics on resistant strains and below the MIC of sensitive strains, particularly an increase in population heterogeneity and cell density-dependent effects of triazoles. Additionally, the method could be adapted to strain identification via germ tube extension. We suggest PAO culture is a rapid and versatile method that may be usefully adapted to clinical mycology and has research applications.
A Multi-Platform Flow Device for Microbial (Co-) Cultivation and Microscopic Analysis
Hesselman, M.C. ; Odoni, D.I. ; Ryback, B.M. ; Groot, S. de; Heck, R.G.A. van; Keijsers, J. ; Kolkman, P. ; Nieuwenhuijse, D. ; Nuland, Y.M. ; Sebus, E. ; Spee, R. ; Vries, H. de; Wapenaar, M.T. ; Ingham, C.J. ; Schroen, K. ; Martins Dos Santos, V.A.P. ; Spaans, S.K. ; Hugenholtz, F. ; Passel, M.W.J. van - \ 2012
PLoS ONE 7 (2012)5. - ISSN 1932-6203
culture - microorganisms - population - resistance - membranes - bacteria - chamber - support - arrays
Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms
Where bio meets nano: The many uses for nanoporous aluminium oxide in biotechnology
Ingham, C.J. ; Maat, J. ter; Vos, W.M. de - \ 2012
Biotechnology Advances 30 (2012)5. - ISSN 0734-9750 - p. 1089 - 1099.
porous anodic alumina - ionization mass-spectrometry - enhanced raman-scattering - hexagonal pore arrays - population heterogeneity - laser desorption/ionization - surface modification - optical-detection - membrane filters - multilayer film
Porous aluminum oxide (PAO) is a ceramic formed by an anodization process of pure aluminum that enables the controllable assembly of exceptionally dense and regular nanopores in a planar membrane. As a consequence, PAO has a high porosity, nanopores with high aspect ratio, biocompatibility and the potential for high sensitivity imaging and diverse surface modifications. These properties have made this unusual material attractive to a disparate set of applications. This review examines how the structure and properties of PAO connect with its present and potential uses within research and biotechnology. The role of PAO is covered in areas including microbiology, mammalian cell culture, sensitive detection methods, microarrays and other molecular assays, and in creating new nanostructures with further uses within biology
|Microbiologische kweekmethoden: ook hier geldt de wet van Moore
Ingham, C.J. ; Vos, W.M. de - \ 2011
In: Microcanon / Smit, H., Reijners, W., van Doorn, J., van der Oost, J., Willemsen, P., Diemen : NVVM - ISBN 9789085713272 - 288 p.
Volgens de wet van Moore verdubbelt het aantal transistors op een computerchip elke twee jaar dankzij de technologische vooruitgang. Ook in de microbiologie zien we trends die voldoen aan de wet van Moore of die deze zelfs overtreffen. Bijvoorbeeld op het gebied van DNA-onderzoek met de steeds snellere methoden voor DNA-sequentieanalyse. Toch leveren snellere methoden op een bepaald gebied niet altijd het maximale resultaat op, omdat andere factoren dat belemmeren. Vooral het kweken van micro-organismen wordt wel eens beschouwd als ouderwets en sterk achterblijvend bij de moleculaire technieken. De vraag is of dit wel terecht is
Organic modification and subsequent biofunctionalization of porous anodic alumina using terminal alkynes
Maat, J. ter; Regeling, R. ; Ingham, C.J. ; Weijers, C.A.G.M. ; Giesbers, M. ; Vos, W.M. de; Zuilhof, H. - \ 2011
Langmuir 27 (2011)22. - ISSN 0743-7463 - p. 13606 - 13617.
ultraviolet difference spectroscopy - normal-alkanoic acids - coated glass slides - candida-albicans - cell-wall - molecular assemblies - peanut agglutinin - carbohydrate interactions - carbon nanotubes - oxide membranes
Porous anodic alumina (PAA) is a well-defined material that has found many applications. The range of applications toward sensing and recognition can be greatly expanded if the alumina surface is covalently modified with an organic monolayer. Here, we present a new method for the organic modification of PAA based on the reaction of terminal alkynes with the alumina surface. The reaction results in the the formation of a monolayer within several hours at 80 °C and is dependent on both oxygen and light. Characterization with X-ray photoelectron spectroscopy and infrared spectroscopy indicates formation of a well-defined monolayer in which the adsorbed species is an oxidation product of the 1-alkyne, namely, its a-hydroxy carboxylate. The obtained monolayers are fairly stable in water and at elevated temperatures, as was shown by monitoring the water contact angle. Modification with 1,15-hexadecadiyne resulted in a surface that has alkyne end groups available for further reaction, as was demonstrated by the subsequent reaction of N-(11-azido-3,6,9-trioxaundecyl)trifluoroacetamide with the modified surface. Biofunctionalization was explored by coupling 11-azidoundecyl lactoside to the surface and studying the subsequent adsorption of the lectin peanut agglutinin (PNA) and the yeast Candida albicans , respectively. Selective and reversible binding of PNA to the lactosylated surfaces was demonstrated. Moreover, PNA adsorption was higher on surfaces that exposed the ß-lactoside than on those that displayed the a anomer, which was attributed to surface-associated steric hindrance. Likewise, the lactosylated surfaces showed increased colonization of C. albicans compared to unmodified surfaces, presumably due to interactions involving the cell wall ß-glucan. Thus, this study provides a new modification method for PAA surfaces and shows that it can be used to induce selective adsorption of proteins and microorganisms
|Snelle soatest in ontwikkeling - nanotechnologie maakt goedkoper onderzoek mogelijk
Bijlsma, E. ; Zuilhof, H. ; Ingham, C.J. - \ 2011
Spitsnieuws (2011). - 1 p.
Binnen een dag weten of je een soa hebt: over een paar jaar is het mogelijk. Onderzoekers van de Universiteit van Wageningen ontwikkelden met behulp van nanotechnologie een methode om sneller, goedkoper en nauwkeuriger te testen op seksueel overdraagbare aandoeningen.
Mixed-culture transcriptome analysis reveals the molecular basis of mixed-culture growth in Streptococcus thermophilus and Lactobacillus bulgaricus
Sieuwerts, S. ; Molenaar, D. ; Hijum, S.A.F.T. van; Beerthuyzen, M. ; Stevens, M.J.A. ; Janssen, P.W. ; Ingham, C.J. ; Bok, F.A.M. de; Vos, W.M. de; Hylckama Vlieg, J.E.T. van - \ 2010
Applied and Environmental Microbiology 76 (2010)33. - ISSN 0099-2240 - p. 7775 - 7784.
lactic-acid bacteria - microarray data - milk - metabolism - plantarum - delbrueckii - pathways - yogurt - identification - proteinases
Many food fermentations are performed using mixed cultures of lactic acid bacteria. Interactions between strains are of key importance for the performance of these fermentations. Yogurt fermentation by Streptococcus thermophilus and Lactobacillus bulgaricus (basonym, Lactobacillus delbrueckii subsp. bulgaricus) is one of the best-described mixed-culture fermentations. These species are believed to stimulate each other's growth by the exchange of metabolites such as folic acid and carbon dioxide. Recently, postgenomic studies revealed that an upregulation of biosynthesis pathways for nucleotides and sulfur-containing amino acids is part of the global physiological response to mixed-culture growth in S. thermophilus, but an in-depth molecular analysis of mixed-culture growth of both strains remains to be established. We report here the application of mixed-culture transcriptome profiling and a systematic analysis of the effect of interaction-related compounds on growth, which allowed us to unravel the molecular responses associated with batch mixed-culture growth in milk of S. thermophilus CNRZ1066 and L. bulgaricus ATCC BAA-365. The results indicate that interactions between these bacteria are primarily related to purine, amino acid, and long-chain fatty acid metabolism. The results support a model in which formic acid, folic acid, and fatty acids are provided by S. thermophilus. Proteolysis by L. bulgaricus supplies both strains with amino acids but is insufficient to meet the biosynthetic demands for sulfur and branched-chain amino acids, as becomes clear from the upregulation of genes associated with these amino acids in mixed culture. Moreover, genes involved in iron uptake in S. thermophilus are affected by mixed-culture growth, and genes coding for exopolysaccharide production were upregulated in both organisms in mixed culture compared to monocultures. The confirmation of previously identified responses in S. thermophilus using a different strain combination demonstrates their generic value. In addition, the postgenomic analysis of the responses of L. bulgaricus to mixed-culture growth allows a deeper understanding of the ecology and interactions of this important industrial food fermentation process
High-resolution microcontact printing and transfer of massive arrays of microorganisms on planar and compartmentalized nanoporous aluminium oxide
Ingham, C.J. ; Bomer, J. ; Sprenkels, A. ; Berg, A. van der; Vos, W.M. de; Hylckama, J. van - \ 2010
Lab on a Chip 10 (2010)11. - ISSN 1473-0197 - p. 1410 - 1416.
chip - cells - growth
Handling microorganisms in high throughput and their deployment into miniaturized platforms presents significant challenges. Contact printing can be used to create dense arrays of viable microorganisms. Such "living arrays", potentially with multiple identical replicates, are useful in the selection of improved industrial microorganisms, screening antimicrobials, clinical diagnostics, strain storage, and for research into microbial genetics. A high throughput method to print microorganisms at high density was devised, employing a microscope and a stamp with a massive array of PDMS pins. Viable bacteria (Lactobacillus plantarum, Esherichia coli), yeast (Candida albicans) and fungal spores (Aspergillus fumigatus) were deposited onto porous aluminium oxide (PAO) using arrays of pins with areas from 5 x 5 to 20 x 20 mum. Printing onto PAO with up to 8100 pins of 20 x 20 mum area with 3 replicates was achieved. Printing with up to 200 pins onto PAO culture chips (divided into 40 x 40 mum culture areas) allowed inoculation followed by effective segregation of microcolonies during outgrowth. Additionally, it was possible to print mixtures of C. albicans and spores of A. fumigatus with a degree of selectivity by capture onto a chemically modified PAO surface. High resolution printing of microorganisms within segregated compartments and on functionalized PAO surfaces has significant advantages over what is possible on semi-solid surfaces such as agar
Method for taking a plurality of samples
Sprenkels, A.J. ; Bomer, J.G. ; Ingham, C.J. ; Hylckama Vlieg, J.E.T. van; Vos, W.M. de; Berg, A. van den; Haaft, P. ten - \ 2009
Octrooinummer: WO2009078724, verleend: 2009-06-25.
The invention provides a method for taking a plurality of samples (200) from a substrate (40) with sample material (20) by using an optical microscope (1000) and a stamp (50). At least part of the stamp (50) is transparent and the stamp (50) is arranged in front of the objective (41). The elevations (58) of the stamp (50) are contacted with sample material (20) in a substrate (40).The samples (200) may be replicated to other substrates.
Swarming and complex pattern formation in Paenibacillus vortex studied by imaging and tracking cells
Ingham, C.J. ; Jacob, E. Ben - \ 2008
BMC Microbiology 8 (2008). - ISSN 1471-2180 - 16 p.
expanding bacterial colonies - adaptive self-organization - myxococcus-xanthus - bacillus-subtilis - proteus-mirabilis - growth - communication - motility - model - evolution
Background: Swarming motility allows microorganisms to move rapidly over surfaces. The Grampositive bacterium Paenibacillus vortex exhibits advanced cooperative motility on agar plates resulting in intricate colonial patterns with geometries that are highly sensitive to the environment. The cellular mechanisms that underpin the complex multicellular organization of such a simple organism are not well understood. Results: Swarming by P. vortex was studied by real- time light microscopy, by in situ scanning electron microscopy and by tracking the spread of antibiotic- resistant cells within antibioticsensitive colonies. When swarming, P. vortex was found to be peritrichously flagellated. Swarming by the curved cells of P. vortex occurred on an extremely wide range of media and agar concentrations (0.3 to 2.2% w/ v). At high agar concentrations (> 1% w/ v) rotating colonies formed that could be detached from the main mass of cells by withdrawal of cells into the latter. On lower percentage agars, cells moved in an extended network composed of interconnected snakes with short-term collision avoidance and sensitivity to extracts from swarming cells. P. vortex formed single Petri dish-wide supercolonies with a colony-wide exchange of motile cells. Swarming cells were coupled by rapidly forming, reversible and non-rigid connections to form a loose raft, apparently connected via flagella. Inhibitors of swarming ( p-Nitrophenylglycerol and Congo Red) were identified. Mitomycin C was used to trigger filamentation without inhibiting growth or swarming; this facilitated dissection of the detail of swarming. Mitomycin C treatment resulted in malcoordinated swarming and abortive side branch formation and a strong tendency by a subpopulation of the cells to form minimal rotating aggregates of only a few cells. Conclusion: P. vortex creates complex macroscopic colonies within which there is considerable reflux and movement and interaction of cells. Cell shape, flagellation, the aversion of cell masses to fuse and temporary connections between proximate cells to form rafts were all features of the swarming and rotation of cell aggregates. Vigorous vortex formation was social, i. e. required > 1 cell. This is the first detailed examination of the swarming behaviour of this bacterium at the cellular level.
Population Heterogeneity of Lactobacillus plantarum WCFS1 Microcolonies in Response to and Recovery from Acid Stress
Ingham, C.J. ; Beerthuyzen, M. ; Vlieg, J.E.T.V.H. - \ 2008
Applied and Environmental Microbiology 74 (2008)24. - ISSN 0099-2240 - p. 7750 - 7758.
individual bacterial-cells - lag times - listeria-monocytogenes - antibiotic tolerance - escherichia-coli - bacillus-cereus - image-analysis - low ph - growth - filamentation
Within an isogenic microbial population in a homogenous environment, individual bacteria can still exhibit differences in phenotype. Phenotypic heterogeneity can facilitate the survival of subpopulations under stress. As the gram-positive bacterium Lactobacillus plantarum grows, it acidifies the growth medium to a low pH. We have examined the growth of L. plantarum microcolonies after rapid pH downshift (pH 2 to 4), which prevents growth in liquid culture. This acidification was achieved by transferring cells from liquid broth onto a porous ceramic support, placed on a base of low-pH MRS medium solidified using Gelrite. We found a subpopulation of cells that displayed phenotypic heterogeneity and continued to grow at pH 3, which resulted in microcolonies dominated by viable but elongated (filamentous) cells lacking septation, as determined by scanning electron microscopy and staining cell membranes with the lipophilic dye FM4-64. Recovery of pH-stressed cells from these colonies was studied by inoculation onto MRS- Gelrite-covered slides at pH 6.5, and outgrowth was monitored by microscopy. The heterogeneity of the population, calculated from the microcolony areas, decreased with recovery from pH 3 over a period of a few hours. Filamentous cells did not have an advantage in outgrowth during recovery. Specific regions within single filamentous cells were more able to form rapidly dividing cells, i.e., there was heterogeneity even within single recovering cells.
Rapid drug susceptibility testing of mycobacteria by culture on a highly porous ceramic support
Ingham, C.J. ; Ayad, A.B. ; Nolsen, K. ; Mulder, B. - \ 2008
The International Journal of Tubercolosis and Lung Disease 12 (2008)6. - ISSN 1027-3719 - p. 645 - 650.
flow-cytometry - tuberculosis - assay - diagnosis - resistance - infection - anopore - growth - gene - tb
BACKGROUND: Phenotypic, culture-based methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis are relatively simple and may be particularly appropriate for resource-limited settings where tuberculosis (TB) is most prevalent. However, these methods can be slow and generate significant amounts of infectious waste. Low-cost digital imaging and a unique porous ceramic support for cell culture (Anopore) may offer opportunities to improve this situation. OBJECTIVE: To testa rapid DST method based on fluorescence microscopy of mycobacteria grown for a few generations on Anopore. DESIGN: Mycobacteria were cultured with and without drugs, and the resulting microcolonies were heat-killed and stained with the fluorogenic dye Syto16. Micros-copy, image-capture with a charge-coupled device camera and digital processing were used to quantify the inhibition of growth by drugs. Rapid DST for rifampicin and isoniazid was performed for clinical isolates. RESULTS: Mycobacteria could be cultured, killed, stained and imaged on Anopore. For DST, the Anopore method gave an accurate result in 3 days. CONCLUSION: This is an unprecedented speed for culture-based DST for this group of organisms and results in minimal infectious waste (<20 000 colony forming units). Analysis of mycobacteria by fluorescence and electron microscopy on Anopore also opens up research possibilities.
MEMS and the microbe
Ingham, C.J. ; Vlieg, J.E.T.V.H. - \ 2008
Lab on a Chip 8 (2008)10. - ISSN 1473-0197 - p. 1604 - 1616.
complex pattern-formation - escherichia-coli - natural-environment - bacillus-subtilis - synthetic biology - protein-synthesis - in-vitro - l-forms - bacteria - cells
In recent years, relatively simple MEMS fabrications have helped accelerate our knowledge of the microbial cell. Current progress and challenges in the application of lab-on-a-chip devices to the viable microbe are reviewed. Furthermore, the degree to which microbiologists are becoming the engineers and are tailoring microbial cells and protocells as potential components for bioMEMS devices is highlighted. We conclude this is a highly productive time for microbiologists and microengineers to unite their shared interest in the micron scale world.
Two homologous Agr-like quorum-sensing systems cooperatively control adherence, cell morphology, and cell viability properties in Lactobacillus plantarum WCFS1
Fujii, T. ; Ingham, C.J. ; Nakayama, J. ; Beerthuyzen, M.M. ; Kunuki, R. ; Molenaar, D. ; Sturme, M.H.J. ; Vaughan, E.E. ; Kleerebezem, M. ; Vos, W.M. de - \ 2008
Journal of Bacteriology 190 (2008)23. - ISSN 0021-9193 - p. 7655 - 7665.
gram-positive bacteria - staphylococcus-aureus - bacillus-subtilis - biofilm formation - escherichia-coli - transcriptional regulators - enterococcus-faecalis - streptococcus-mutans - signal-transduction - lactococcus-lactis
A two-component regulatory system of Lactobacillus plantarum, encoded by genes designated lamK and lamR (hpk10 and rrp10), was studied. The lamK and lamR genes encode proteins which are highly homologous to the quorum-sensing histidine kinase LamC and the response regulator LamA, respectively. Transcription analysis of the lamKR operon and the lamBDCA operon and liquid chromatography-mass spectrometry analysis of production of the LamD558 autoinducing peptide were performed for DeltalamA, DeltalamR, DeltalamA DeltalamR deletion mutants and a wild-type strain. The results suggested that lamA and lamR are cooperating genes. In addition, typical phenotypes of the DeltalamA mutant, such as reduced adherence to glass surfaces and filamentous cell morphology, were enhanced in the DeltalamA DeltalamR mutant. Microarray analysis suggested that the same cell wall polysaccharide synthesis genes, stress response-related genes, and cell wall protein-encoding genes were affected in the DeltalamA and DeltalamA DeltalamR mutants. However, the regulation ratio was more significant for the DeltalamA DeltalamR mutant, indicating the cooperative effect of LamA and LamR
From the cover: the micro-petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms
Ingham, C.J. ; Sprenkels, A. ; Bomer, J. ; Molenaar, D. ; Berg, Aad van den; Hylckama Vlieg, J.E.T. van; Vos, W.M. de - \ 2007
Proceedings of the National Academy of Sciences of the United States of America 104 (2007)46. - ISSN 0027-8424 - p. 18217 - 18222.
sp-nov. - bacteria - microfluidics - galactosidase - platform - future - cells
A miniaturized, disposable microbial culture chip has been fabricated by microengineering a highly porous ceramic sheet with up to one million growth compartments. This versatile culture format, with discrete compartments as small as 7 x 7 µm, allowed the growth of segregated microbial samples at an unprecedented density. The chip has been used for four complementary applications in microbiology. (i) As a fast viable counting system that showed a dynamic range of over 10,000, a low degree of bias, and a high culturing efficiency. (ii) In high-throughput screening, with the recovery of 1 fluorescent microcolony in 10,000. (iii) In screening for an enzyme-based, nondominant phenotype by the targeted recovery of Escherichia coli transformed with the plasmid pUC18, based on expression of the lacZ reporter gene without antibiotic-resistance selection. The ease of rapid, successive changes in the environment of the organisms on the chip, needed for detection of -galactosidase activity, highlights an advantageous feature that was also used to screen a metagenomic library for the same activity. (iv) In high-throughput screening of >200,000 isolates from Rhine water based on metabolism of a fluorogenic organophosphate compound, resulting in the recovery of 22 microcolonies with the desired phenotype. These isolates were predicted, on the basis of rRNA sequence, to include six new species. These four applications suggest that the potential for such simple, readily manufactured chips to impact microbial culture is extensive and may facilitate the full automation and multiplexing of microbial culturing, screening, counting, and selection.
|Quantification of the adaptive salt stress response of Bacillus cereus
Besten, H.M.W. den; Ingham, C.J. ; Moezelaar, R. ; Zwietering, M.H. ; Abee, T. - \ 2007
In: Proceedings 5th International Conference Predictive Modelling in Foods IC PMF 2007, Athens, Greece, 16-19 September 2007. - Athens : Agricultural University of Athens - ISBN 9789608931374 - p. 181 - 184.
|Direct imaging based quantification of the growth dynamics of salt-stressed Bacillus cereus
Besten, H.M.W. den; Ingham, C.J. ; Moezelaar, R. ; Zwietering, M.H. ; Abee, T. - \ 2007
In: Proceedings 5th International Conference Predictive Modelling in Foods IC PMF 2007. - Athens : Agricultural University of Athens - ISBN 9789608931374 - p. 55 - 58.
Quantitative analysis of population heterogeneity of the adaptive salt stress response and growth capacity of Bacillus cereus ATCC 14579
Besten, H.M.W. den; Ingham, C.J. ; Hylckama Vlieg, J.E.T. van; Beerthuyzen, M.M. ; Zwietering, M.H. ; Abee, T. - \ 2007
Applied and Environmental Microbiology 73 (2007)15. - ISSN 0099-2240 - p. 4797 - 4804.
escherichia-coli o157-h7 - listeria-monocytogenes - lag times - single-cell - bacterial-populations - clostridium-botulinum - individual cells - image-analysis - inoculum size - heat
Bacterial populations can display heterogeneity with respect to both the adaptive stress response and growth capacity of individual cells. The growth dynamics of Bacillus cereus ATCC 14579 during mild and severe salt stress exposure were investigated for the population as a whole in liquid culture. To quantitatively assess the population heterogeneity of the stress response and growth capacity at a single-cell level, a direct imaging method was applied to monitor cells from the initial inoculum to the microcolony stage. Highly porous Anopore strips were used as a support for the culturing and imaging of microcolonies at different time points. The growth kinetics of cells grown in liquid culture were comparable to those of microcolonies grown upon Anopore strips, even in the presence of mild and severe salt stress. Exposure to mild salt stress resulted in growth that was characterized by a remarkably low variability of microcolony sizes, and the distributions of the log10-transformed microcolony areas could be fitted by the normal distribution. Under severe salt stress conditions, the microcolony sizes were highly heterogeneous, and this was apparently caused by the presence of both a nongrowing and growing population. After discriminating these two subpopulations, it was shown that the variability of microcolony sizes of the growing population was comparable to that of non-salt-stressed and mildly salt-stressed populations. Quantification of population heterogeneity during stress exposure may contribute to an optimized application of preservation factors for controlling growth of spoilage and pathogenic bacteria to ensure the quality and safety of minimally processed foods.
Rapid antibiotic sensitivity testing and trimethoprim-mediated filamentation of clinical isolates of the Enterobacteriaceae assayed on a novel porous culture support
Ingham, C.J. ; Ende, M. van den; Wever, P.C. ; Schneeberger, P.M. - \ 2006
Journal of Medical Microbiology 55 (2006)11. - ISSN 0022-2615 - p. 1511 - 1519.
membrane filters - sos response - sulfadiazine - bacteria - anopore - growth - microorganisms - morphology - cloacae
A porous inorganic material (Anopore) was employed as a microbial culture and microcolony imaging support. Rapid Anopore-based antibiotic sensitivity testing (AST) methods were developed to assess the growth of clinical isolates, with the primary focus on testing the response of the Enterobacteriaceae to trimethoprim, but with the method supporting a wider applicability in terms of strains and antibiotics. It was possible to detect the growth of Enterobacter aerogenes after 25 min culture and to distinguish a trimethoprim-sensitive from a trimethoprim-resistant strain with 40 min incubation. MIC90 determinations were made on Anopore; these were in good agreement with the results from the Vitek 2 and E-test methods. The Anopore method correctly identified sensitive (40/40) and resistant (17/17) strains of the Enterobacteriaceae and other Gram-negative rods within only 2-3 h culture. Additionally, a trimethoprim-resistant subpopulation (10% of population) could be detected by microcolony formation within 2 h, and a smaller subpopulation (1%) after 3-5 h. These results suggest that this is a viable approach for the rapid AST of purified strains, and that it may be able to deal with mixed populations. The microscopic examination of microcolonies during AST is an advantage of this method which revealed additional information. Filamentation triggered by trimethoprim was discovered in many species of the Enterobacteriaceae for which this phenomenon has not previously been reported. Filamentation was characterized by heterogeneity in terms of cell length, and also uneven nucleic acid distribution and flattening of damaged cells. The development and application of Anopore-based AST within clinical diagnostics is discussed.