Name: methodologies for studying b. subtilis biofilms as a model for characterizing small molecule biofilm inhibitors
Uploaded: 2016-10-09T14:00:00
Description: Watch this Scientific Journal Video about Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors at JoVE.com

Electron microscope imaging was conducted at the Electron Microscopy Unit of the Weizmann Institute of Science, supported in part by the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging. This research was also supported by the ISF I-CORE grant 152/1, Mr. and Mrs. Dan Kane, Ms. Lois Rosen, by a Yeda-Sela research grant, by the Larson Charitable Foundation, by Ruth and Herman Albert Scholars Program for New Scientists, by the Ilse Katz Institute for Materials Sciences and Magnetic Resonance Research grant, by the Ministry of Health grant for alternative research methods, and by the France-Israel Cooperation - Maimonide-Israel Research Program. IKG is a recipient of the Rowland and Sylvia Career Development Chair.

1. Assessing the Effect of Small Molecule Inhibitors on Pellicle and Biofilm Colony Formation
<ol>
	<li>Prepare a 2x solution of defined biofilm-inducing MSgg medium25 without the calcium chloride and iron(III) chloride hexahydrate. After filter sterilization, add the calcium chloride. The medium is ready to use directly or it can be stored at 4 &#176;C in the dark.</li>
	<li>Prepare the 1x MSgg dilution on the day of the experiment.
	<ol>
		<li>Dilute the 2x MSgg medium to 1x with sterile distilled water (p...

Bacillus subtilis forms robust and highly structured biofilms both in liquid (pellicles) and on solid medium (colonies). Hence, it serves as an ideal model organism to characterize the mode of action of specific biofilm inhibitors. On solid media, cells form multicellular structures with distinctive features that are not evident in pellicles, like wrinkles radiating from the center to the edge. Thus, pellicles and colonies are complementary systems to study B. subtilis multicellularity.
<p class="jo...

Multi-cellular bacterial communities play significant roles in natural and anthropogenic environments, and can be beneficial or highly deleterious. These multi-cellular colonies are known as biofilms, wherein the individual cells are embedded in a self-produced extracellular polymeric substances (EPS) matrix. The EPS strongly adhere the cells to the surface they colonize. They serve as a shield against mechanical and chemical forces and create close contact between neighboring cells, facilitating cellular communication1. A biofilm can be viewed as a differentiated community, where cells use highly regulated, orchestrated processes to coordinate their activities within the community, as well as across species2-5. The transition from a planktonic, free-living mode of growth to a biofilm state is often associated with developmental processes. A good example is the Gram-positive soil bacterium Bacillus subtilis, and therefore an undomesticated strain serves as a robust model organism to study the developmental stages leading to biofilm formation. In this bacterium, motile cells organize themselves into conspicuous multicellular structures that carry out specialized tasks4. One group of cells, the matrix-producers secrete exopolysaccharides6, the amyloid protein TasA7,8, and the surface hydrophobicity protein BslA9,10; all of which participate in the assembly of the EPS11-13.
Given the abundance of biofilms in natural and anthropogenic niches and the putative fatal damage they can cause, there is a pressing need to find ways to prevent their formation. Small molecule inhibitors can aid in the discovery of new regulatory pathways, enzymes and structural proteins involved in biofilm formation, and thereby promote insights in the complex processes of multicellular community assembly. As B. subtilis is a well-studied model for biofilm formation14,15, it can be used to assess the effects of various biofilm inhibitors. This study tackles four fundamental methods that are key for evaluating the response of biofilms to small molecule inhibitors. First, to ensure that these inhibitors have a biofilm-specific target, the separation of the effect on planktonic growth from the effect on biofilm formation is critical. Most antibacterial agents target cells in their planktonic growth phase, but molecules that target the biofilm lifestyle are rare. Additionally, as molecules that do not affect planktonic growth are not toxic, they can reduce the selective pressure favoring antibiotic resistant mutants16. For example, when biofilms are treated with D-amino acids or certain other cell wall-interfering molecules, they are either disturbed or disassembled, but these inhibitors only mildly affect planktonic growth12,17. In contrast, many antibiotics dramatically impair planktonic growth, with little or no effect on biofilm formation17.
Second, establishing a consistent and robust experimental framework to study the effect of the small molecules is crucial. We observed that the active concentration range of small molecule inhibitors was sensitive to the pre-culture conditions and to the experimental setup used to study the effect of these small molecule inhibitors. Various reports, particularly those studying B. subtilis, revealed variations in the concentration range at which D-amino acids inhibit the formation of pellicles &#8212; the floating bacterial biofilms12,17-19. The results presented here suggest that the following factors account for differences in the active concentration range: the pre-culture conditions (logarithmic12,17 versus late-stationary20 growth phase), the growth medium used in the pre-culture condition (rich, undefined [Luria Broth, LB] versus defined [monosodium glutamate-glycerol, MSgg]), the inoculation ratio and especially the removal of the pre-culture medium before inoculation. The temperature of static pellicle growth showed a less important role in the activity range of the small molecule inhibitor D-leucine, a representative D-amino acid used in this study.
Finally, once the biofilms are treated with specific biofilm inhibitors, robust and informative methods are required to characterize the effects of these inhibitors on biofilm fitness. Here, two methods to independently characterize the effect of small molecule inhibitors are described in detail: (1) The effect on single cells within a biofilm colony and their resistance to antimicrobial agents. Cells in biofilms are typically more resistant to antibiotics when compared to free-living bacteria21-23. While this phenomenon is multifactorial, the ability of the EPS to reduce antibiotic penetration was often considered as an appealing explanation24. This method assesses the survival of pre-established biofilm cells after exposure to antibacterial substances. (2) The effect on the biofilm colony architecture, from the large to the small scale. Biofilm colonies are characterized by their three-dimensional structure and the presence of the EPS. Utilizing scanning electron microscopy, changes in the cell morphology, biofilm colony structure and the architecture and abundance of the EPS can be visualized from the large (mm) to the small scale (&#181;m).

<table border="0" cellpadding="0" cellspacing="0" width="897">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:301px;">Luria Broth, Lennox</td>
			<td style="width:305px;">Difco</td>
			<td style="width:127px;">240230</td>
			<td style="width:165px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Agar</td>
			<td>Difco</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">potassium phosphate monobasic&#160;</td>
			<td>Sigma, 136.09 g/mol</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">potassium phosphate dibasic&#160;</td>
			<td>Fisher Scientific, 174.18 g/mol</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-(N-morpholino)propanesulfonic acid</td>
			<td>Fisher Scientific, 209.27 g/mol</td>
			<td>BP308-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnesium chloride hexahydrate&#160;</td>
			<td>Merck, 203.30 g/mol&#160;</td>
			<td>1.05833.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride anhydrous</td>
			<td>J.T. Baker, 110.98 g/mol</td>
			<td>1311-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">manganese(II) chloride tetrahydrate</td>
			<td>Sigma, 197.91 g/mol</td>
			<td>31422-250G-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iron(III) chloride hexahydrate&#160;</td>
			<td>Sigma, 270.30 g/mo)</td>
			<td>F2877-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">zinc chloride anhydrous&#160;</td>
			<td>Acros Organics, 136.29 g/mol</td>
			<td>424592500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">thiamine hydrochloride</td>
			<td>Sigma, 337.27 g/mol</td>
			<td>T1270-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-tryptophan</td>
			<td>Fisher Scientific, 204.1 g/mol</td>
			<td>BP395-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-phenylalanine</td>
			<td>Sigma, 165.19 g/mol</td>
			<td>P5482-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-threonine</td>
			<td>Sigma, 119.12 g/mol</td>
			<td>T8625-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycerol anhydrous</td>
			<td>Bio-Lab Itd</td>
			<td>712022300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamic acid monosodium salts hydrate&#160;</td>
			<td>Sigma, 169.11 g/mol</td>
			<td>G1626-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-leucine</td>
			<td>Sigma, 169.11 g/mol</td>
			<td>855448-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol anhydrous</td>
			<td>Gadot</td>
			<td>830000054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">razor blade</td>
			<td>Eddison</td>
			<td>NA</td>
			<td style="width:165px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">circular cellulose filter papers</td>
			<td>Whatman, 90 mm</td>
			<td>1001-090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glutaraldehyde</td>
			<td>EMS (Electron Micoscopy Science), 25% in water</td>
			<td>16220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paraformaldehyde&#160;</td>
			<td>EMS, 16% in water</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium cacodylate</td>
			<td>Merck, 214.05 g/mol&#160;</td>
			<td>8.2067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">calcium chloride 2-hydrate</td>
			<td>Merck, 147.02 g/mol&#160;</td>
			<td>1172113</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stub-aluminium mount</td>
			<td>EMS, sloted head</td>
			<td>75230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">carbon adhesive tape</td>
			<td>EMS</td>
			<td>77825-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaker 37&#176;C</td>
			<td>New Brunswick Scientific Innowa42</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf table top centrifuge 5424</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:301px;">Digital Sonifier, Model 250, used with Double Step Microtip</td>
			<td>Branson</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator 30 &#176;C</td>
			<td>Binder</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator 23 &#176;C</td>
			<td>Binder</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter System, 500 ml, polystyrene</td>
			<td>Cornig Incorporated</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rotary Shaker - Orbitron Rotatory II</td>
			<td>Boekel</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S150 Sputter Coater&#160;</td>
			<td>Edwards</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CPD 030 Critical Point Dryer</td>
			<td>BAL-TEC</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Environmental Scanning Electron Microscope</td>
			<td>XL30 ESEM FEG Philips (FEI)</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

methodologies for studying b. subtilis biofilms as a model for characterizing small molecule biofilm inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

The pellicle assay is one method to study the highly regulated and dynamic processes of B. subtilis multicellularity. Besides this, the pellicle assay is suited to test a range of either pre-starter conditions or small molecule concentrations in a single cell-culture multidish plate in one experiment. However, B. subtilis pellicle formation is sensitive to the pre-culture conditions (e.g., growth medium of the pre-culture and its growth phase), the inoculation ratio and the removal of the pre-c...

Watch this Scientific Journal Video about Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors at JoVE.com

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

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