Framing the discussion of microorganisms as a facet of social equity in human health

What do “microbes” have to do with social equity? These microorganisms are integral to our health, that of our natural environment, and even the “health” of the environments we build. The loss, gain, and retention of microorganisms—their flow between humans and the environment—can greatly impact our health. It is well-known that inequalities in access to perinatal care, healthy foods, quality housing, and the natural environment can create and arise from social inequality. Here, we focus on the argument that access to beneficial microorganisms is a facet of public health, and health inequality may be compounded by inequitable microbial exposure

Live Cell Imaging of Germination and Outgrowth of Individual <em>Bacillus subtilis</em> Spores; the Effect of Heat Stress Quantitatively Analyzed with SporeTracker

<div><p>Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. <i>Bacillus</i> spp. spores can remain in a dormant, stress resistant state for a long period of time. Vegetative cells are formed by germination of spores followed by a more extended outgrowth phase. Spore germination and outgrowth progression are often very heterogeneous and therefore, predictions of microbial stability of food products are exceedingly difficult. Mechanistic details of the cause of this heterogeneity are necessary. In order to examine spore heterogeneity we made a novel closed air-containing chamber for live imaging. This chamber was used to analyze <i>Bacillus subtilis</i> spore germination, outgrowth, as well as subsequent vegetative growth. Typically, we examined around 90 starting spores/cells for ≥4 hours per experiment. Image analysis with the purposely built program “SporeTracker” allows for automated data processing from germination to outgrowth and vegetative doubling. In order to check the efficiency of the chamber, growth and division of <i>B. subtilis</i> vegetative cells were monitored. The observed generation times of vegetative cells were comparable to those obtained in well-aerated shake flask cultures. The influence of a heat stress of 85°C for 10 min on germination, outgrowth, and subsequent vegetative growth was investigated in detail. Compared to control samples fewer spores germinated (41.1% less) and fewer grew out (48.4% less) after the treatment. The heat treatment had a significant influence on the average time to the start of germination (increased) and the distribution and average of the duration of germination itself (increased). However, the distribution and the mean outgrowth time and the generation time of vegetative cells, emerging from untreated and thermally injured spores, were similar.</p> </div

Schematic picture of the top and side view of the designed closed air-containing chamber for live cell imaging.

<p>A chamber was prepared by attaching a Gene Frame® to a standard microscope slide and cover slip. A thin, semisolid matrix pad (160 µm) of 1% agarose – medium was made. The pad was loaded with exponentially growing vegetative cells or heat-activated spores. The cover slip was placed in upside down position onto the Gene Frame® (See Materials and Methods for details).</p

Heat-treated spores show a decrease in the overall number of spores that are able to germinate and grow out within 5 hours.

<p>Movies of heat- (85°C for 10 min) and un- treated spores (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058972#pone-0058972-g004" target="_blank">Fig. 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058972#pone-0058972-g005" target="_blank">5</a> for details) were analyzed with SporeTracker and the spores were scored (by additional manual inspection) for their ability to germinate and grow out. The total number of spores assessed in the control and stress condition was 218 and 325, respectively.</p

Mean generation time of <i>B. subtilis</i> 1A700 vegetative cells grown under the microscope and in shake flasks in different media.

a<p>Mean generation time is given including the standard deviation.</p>b<p>Generation times from individual starting vegetative cells growing into microcolonies under the microscope were calculated using SporeTracker (see Materials & Methods for details). The amount of individual starting cells analyzed and gathered from three independent biological replicates is given in brackets.</p>c<p>Amount of independent biological replicates is given in brackets.</p

Time-resolved images showing heterogeneous germination and outgrowth of <i>B. subtilis</i> 1A700 spores on minimal medium.

<p>Heat-activated spores (70°C for 30 min) were spotted on 100% defined minimal (MOPS-buffered) medium including AGFK and followed in time using phase-contrast microscopy. The spore marked in the square (Panel <b>A</b>) becomes phase-dark (germinates) within 60 min (Panel <b>B</b>), grows out, and forms a microcolony (Panel <b>F</b>). The spore marked in the circle (Panel <b>A</b>) becomes phase-dark (Panel <b>B</b>) but does not grow out within 5 hours (Panel <b>F</b>). The spore marked in the triangle (Panel <b>A</b>) remains phase-bright throughout the experiment (Panel <b>F</b>).</p

Mean values and standard deviation of different stages of germination and outgrowth of untreated and wet-heat-treated individual <i>B. subtilis</i> spores^a.

a<p>Spores of <i>B. subtilis</i> 1A700 were wet-heat-treated or not, then heat-activated and germinated in defined minimal (MOPS-buffered) medium including AGFK, and various germination and outgrowth parameters of individual spores were calculated as described in the Materials and Methods.</p>b<p>Mean time of different stages is given including the standard deviation. The amount of spores/cells analyzed from each stage and gathered from three independent biological replicates is given in brackets. The asterisk indicates that the mean of the distributions between the stress and control experiment are significantly different (<i>t</i>-test, <i>P</i><0.05). The dagger indicates that the variance of the distributions between the stress and control experiment are significantly different (<i>F</i>-test, <i>P</i><0.05).</p

Dual plot showing spore germination and outgrowth of one heat-activated <i>B. subtilis</i> 1A700 spore as analyzed with SporeTracker.

<p>Above: Phase-bright to phase-dark transition, marked with a small circle at 90% (start of germination) and 10% (end of germination) of the entire (pixel) intensity drop range (brightness). Below: various snapshots at different stages of germination and outgrowth. The exponential growth phase (appearing linear in the log₂ transformed plot of the measured area) is used to calculate the generation time. The burst of the cell out of the spore coat is accompanied by a relative short and significant increase in area (marked by the green circle). In SporeTracker, the cursor can be dragged across such a plot to observe live the various phases of spore germination and outgrowth and subsequent growth of the corresponding cell.</p

Time-resolved images showing growth and division of <i>B. subtilis</i> 1A700 vegetative cells using different media.

<p>Exponentially growing cells were spotted on 2.5% complex medium (TSB and LB shown in panel <b>A</b> and <b>B</b>, respectively) and 100% defined minimal (MOPS-buffered) medium (<b>C</b>), and followed in time using phase-contrast microscopy.</p

Evolution of longitudinal division in multicellular bacteria of the Neisseriaceae family

Rod-shaped bacteria typically elongate and divide by transverse fission. However, several bacterial species can form rod-shaped cells that divide longitudinally. Here, we study the evolution of cell shape and division mode within the family Neisseriaceae, which includes Gram-negative coccoid and rod-shaped species. In particular, bacteria of the genera Alysiella, Simonsiella and Conchiformibius, which can be found in the oral cavity of mammals, are multicellular and divide longitudinally. We use comparative genomics and ultrastructural microscopy to infer that longitudinal division within Neisseriaceae evolved from a rod-shaped ancestor. In multicellular longitudinally-dividing species, neighbouring cells within multicellular filaments are attached by their lateral peptidoglycan. In these bacteria, peptidoglycan insertion does not appear concentric, i.e. from the cell periphery to its centre, but as a medial sheet guillotining each cell. Finally, we identify genes and alleles associated with multicellularity and longitudinal division, including the acquisition of amidase-encoding gene amiC2, and amino acid changes in proteins including MreB and FtsA. Introduction of amiC2 and allelic substitution of mreB in a rod-shaped species that divides by transverse fission results in shorter cells with longer septa. Our work sheds light on the evolution of multicellularity and longitudinal division in bacteria, and suggests that members of the Neisseriaceae family may be good models to study these processes due to their morphological plasticity and genetic tractability