Novel co-culture plate enables growth dynamic-based assessment of contact-independent microbial interactions

<div><p>Interactions between microbes are central to the dynamics of microbial communities. Understanding these interactions is essential for the characterization of communities, yet challenging to accomplish in practice. There are limited available tools for characterizing diffusion-mediated, contact-independent microbial interactions. A practical and widely implemented technique in such characterization involves the simultaneous co-culture of distinct bacterial species and subsequent analysis of relative abundance in the total population. However, distinguishing between species can be logistically challenging. In this paper, we present a low-cost, vertical membrane, co-culture plate to quantify contact-independent interactions between distinct bacterial populations in co-culture via real-time optical density measurements. These measurements can be used to facilitate the analysis of the interaction between microbes that are physically separated by a semipermeable membrane yet able to exchange diffusible molecules. We show that diffusion across the membrane occurs at a sufficient rate to enable effective interaction between physically separate cultures. Two bacterial species commonly found in the cystic fibrotic lung, <i>Pseudomonas aeruginosa</i> and <i>Burkholderia cenocepacia</i>, were co-cultured to demonstrate how this plate may be implemented to study microbial interactions. We have demonstrated that this novel co-culture device is able to reliably generate real-time measurements of optical density data that can be used to characterize interactions between microbial species.</p></div

Growth curves of <i>P</i>. <i>aeruginosa</i> (PA) and <i>B</i>. <i>cenocepacia</i> (BC) in co-culture.

<p>The culture conditions can be seen in the legend on the right side of this figure. All wells are started with 100% LB. The purple lines are gathered from the co-culture of PA and BC. The isolated PA and BC cultures are the solid blue and orange lines respectively. The black lines are controls from the side of the wells that were not inoculated for the isolated PA and BC cultures. The black line slightly increases (Blank 1) as a result of pyoverdine (produced by PA) that partially absorbs at 600 nm. This result is discussed further in the Supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182163#pone.0182163.s003" target="_blank">S2 Fig</a>). The growth curves from each of the two competing PA and BC cultures (dashed blue and orange lines respectively) are nearly identical (similar to blue and purple in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182163#pone.0182163.g003" target="_blank">Fig 3</a>) and thus are averaged to simplify the plot. The maximum and minimum values of the generated growth curves, conducted in triplicate (n = 3), are displayed as shaded regions around the plotted averages.</p

Real-time diffusion of metabolites across a membrane.

<p>One side of each co-culture chamber was inoculated with <i>E</i>. <i>coli</i>, as seen in the pictorial legend on the right, each box represents a chamber with a black dashed line representing the membrane. The terms ‘pre-mixed’ and ‘gradient’ describe the initial media conditions. The gradient condition was loaded with LB on one side and 1x DPBS on the other. The pre-mixed condition was loaded with LB that was diluted in half with DPBS to simulate complete diffusion of LB across the membrane. These two conditions were tested with four initial concentrations of LB, 25%, 50%, 75%, and 100%, all diluted using 1x DPBS. The final pre-mixed concentration of the medium for each well was 12.5%, 25%, 37.5%, and 50% LB. The maximum and minimum values of the generated growth curves, conducted in triplicate (n = 3), are displayed as shaded regions around the plotted averages. This experiment was cultured as described in the methods for 24 hours.</p

Comparison of isolated versus competing cultures.

<p>(A) The green (100% LB) and orange (50% LB) lines are the isolated culture condition that have <i>E</i>. <i>coli</i> cultured on only one side of the membrane with blank media on the other. The OD for the side of the well that is not inoculated is plotted in black (it maintains the original OD; there is no growth, as expected). In this condition, <i>E</i>. <i>coli</i> has access to all of the nutrients on both sides of the membrane, but cell growth is physically constrained to one side. The blue (100% LB) and purple (50% LB) dashed lines are the competing culture condition that have <i>E</i>. <i>coli</i> cultured on both sides of the membrane. For the competing cultures, the growth curves from both sides are plotted individually. In this condition, each <i>E</i>. <i>coli</i> population must compete for the available nutrients. The maximum and minimum values of the generated growth curves, conducted in triplicate, are displayed as shaded regions around the plotted averages. (B) The biomass produced from the 50% LB isolated (solid orange) and competing (dashed purple) conditions at 10 hours is approximated by the CFU count of each culture. The CFU counts for the isolated condition as displayed are divided in half to compare to the competing condition, discussed further in the text. These data are the result of four experiments. The boxplot whiskers represent +/- 2.7σ from the mean.</p

Co-culture plate design.

<p>The co-culture plate consists of eight individual co-culture chambers. Each chamber consists of two wells that are able to hold liquid cultures that are physically separated by a semi-permeable membrane that allows for diffusion-mediated interactions. A representative isometric mechanical drawing of a single co-culture chamber is shown in (A), note it is composed of two wells. For a better view of the wells, a cross-sectional view is shown in (B); the semi-permeable membrane is labelled. The co-culture plate, composed of eight co-culture chambers, has the same profile as a standard 96-well plate. Each well on the co-culture plate aligns with two wells of a 96-well plate and the culture volume is 2 mL per well (4 mL total per chamber) (C). An SEM image captures <i>E</i>. <i>coli</i> cells fixed on the surface of a polycarbonate membrane with 0.1 μm pores (D); the scale bar is 2 μm.</p

A non-encapsulated mutant of <i>C</i>. <i>jejuni</i> is defective in weight loss, diarrhea, and shedding of organism in stool. Panel A.

<p>Weights following infection with either wildtype or 81–176 <i>kpsM</i> in dZD C57Bl/6J male mice. * WT vs <i>kpsM</i>, P<0.05. Diarrhea noted by D. <b>Panel B.</b> qPCR detection of <i>Campylobacter jejuni</i> (<i>cadF</i>) following infection in dZD mice. * WT vs <i>kpsM</i>, P<0.05. (N = 4/group).</p

Investigation of Encephalopathy Caused by Shiga Toxin 2c-Producing <em>Escherichia coli</em> Infection in Mice

<div><p>A large outbreak of Shiga toxin (Stx)-producing enteroaggregative <i>Escherichia coli</i> (EAEC) O104:H4 occurred in northern Germany. From this outbreak, at least 900 patients developed hemolytic uremic syndrome (HUS), resulting in more than 50 deaths. Thirty percent of the HUS patients showed encephalopathy. We previously established a mouse model with encephalopathy associated with blood brain barrier (BBB) damage after oral infection with the Shiga toxin (Stx) 2c-producing <i>Escherichia coli</i> O157: H- strain E32511 (E32511). In this model, we detected high expression of the Stx receptor synthase enzyme, glycosphingolipid globotriaosylceramide (Gb3) synthase, in endothelial cells (ECs) and neurons in the reticular formation of the medulla oblongata by <i>in situ</i> hybridization. Caspase-3 was activated in neurons in the reticular formation of the medulla oblongata and the anterior horn of the spinal cord. Astrocytes (ASTs) were activated in the medulla oblongata and spinal cord, and a decrease in aquaporin 4 around the ECs suggested that BBB integrity was compromised directly by Stx2c or through the activation of ASTs. We also report the effectiveness of azithromycin (AZM) in our model. Moreover, AZM strongly inhibited the release of Stx2c from E32511 <i>in vitro</i>.</p> </div

Bacterial count at sub-minimum inhibitory concentration (MIC) of AZM and other antimicrobial agents against E32511. AZM, azithromycin; FOM, fosfomycin; NFLX, norfloxacin; KM, kanamycin; OFLX, orfloxacin; CPFX, ciprofloxacin.

<p>AZM, azithromycin; FOM, fosfomycin; NFLX, norfloxacin; KM, kanamycin; OFLX, orfloxacin; CPFX, ciprofloxacin.</p

Microglial activation in i.p. LPS-injected and E32511 model mice.

<p>(A) Microglia in the medulla oblongata and the spinal cord were not activated in the control (uninfected) and E32511-infected mice, but were activated in i.p. LPS-injected mice. Scale bars: 2 µm. (B) The number of activated microglia significantly increased in the LPS-injected mice but not in the control and E32511-infected mice (LPS vs. control, <i>p</i><0.0001; LPS vs. E32511, <i>p</i><0.0001 and control vs. E32511, <i>p</i> = 1). Microglia were observed and counted manually from 5 non-overlapping fields. Statistical difference was measured by student's t test. **<i>p</i><0.001.</p

Intestinal morphometry.

<p>Intestinal sections from dZD fed mice stained for H&E or PAS. <b>Panel A:</b> ileum. <b>Panel B:</b> colon.</p