Colin J. Ingham

The micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms

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 × 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 β-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.

Mixed-Culture Transcriptome Analysis Reveals the Molecular Basis of Mixed-Culture Growth in Streptococcus thermophilus and Lactobacillus bulgaricus

ABSTRACT 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 others 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.

Population Heterogeneity of Lactobacillus plantarum WCFS1 Microcolonies in Response to and Recovery from Acid Stress

ABSTRACT 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.

MEMS and the microbe

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.

Quantitative Analysis of Population Heterogeneity of the Adaptive Salt Stress Response and Growth Capacity of Bacillus cereus ATCC 14579

ABSTRACT 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.

Growth and Multiplexed Analysis of Microorganisms on a Subdivided, Highly Porous, Inorganic Chip Manufactured from Anopore

ABSTRACT A highly porous inorganic material (Anopore) was shown to be an effective support for culturing and imaging a wide range of microorganisms. An inert barrier grid was printed on the rigid surface of Anopore to create a “living chip” of 336 miniaturized compartments (200/cm2) with broad applications in microbial culture.

Mutually facilitated dispersal between the nonmotile fungus Aspergillus fumigatus and the swarming bacterium Paenibacillus vortex

In the heterogeneous environment surrounding plant roots (the rhizosphere), microorganisms both compete and cooperate. Here, we show that two very different inhabitants of the rhizosphere, the nonmotile fungus Aspergillus fumigatus and the swarming bacterium Paenibacillus vortex, can facilitate each others dispersal. A. fumigatus conidia (nonmotile asexual fungal spores) can be transported by P. vortex swarms over distances of at least 30 cm and at rates of up to 10.8 mm h−1. Moreover, conidia can be rescued and transported by P. vortex from niches of adverse growth conditions. Potential benefit to the bacteria may be in crossing otherwise impenetrable barriers in the soil: fungal mycelia seem to act as bridges to allow P. vortex to cross air gaps in agar plates. Transport of conidia was inhibited by proteolytic treatment of conidia or the addition of purified P. vortex flagella, suggesting specific contacts between flagella and proteins on the conidial surface. Conidia were transported by P. vortex into locations where antibiotics inhibited bacteria growth, and therefore, growth and sporulation of A. fumigatus were not limited by bacterial competition. Conidia from other fungi, similar in size to those fungi from A. fumigatus, were not transported as efficiently by P. vortex. Conidia from a range of fungi were not transported by another closely related rhizosphere bacterium, Paenibacillus polymyxa, or the more distantly related Proteus mirabilis, despite both being efficient swarmers.

Two Homologous Agr-Like Quorum-Sensing Systems Cooperatively Control Adherence, Cell Morphology, and Cell Viability Properties in Lactobacillus plantarum WCFS1

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.

A Multi-Platform Flow Device for Microbial (Co-) Cultivation and Microscopic Analysis

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.

Rapid susceptibility testing and microcolony analysis of Candida spp. cultured and imaged on porous aluminum oxide.

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.