an assay to determine polymicrobial biofilm initiation on virus-infected cells

An Assay to Determine Polymicrobial Biofilm Initiation on Virus-Infected Cells

To carry out a polymicrobial biofilm assay, seed 96-well plates with 200 microliters of 2 times 10 to the 5th HeLa cells per milliliter. Rock the plates at 37 degrees Celsius for 30 to 45 minutes, before incubating in 5% carbon dioxide at 37 degrees Celsius for 18 hours. Use 1x optimum to wash the monolayers. Then, seed with a single strain of HSV. Test only one strain at a time.
Incubate the plates at 37 degrees Celsius in 5% carbon dioxide for three hours. Using 100 microliters of 37 degrees Celsius PBS with magnesium and calcium, wash the infected monolayers. Then, use 37 degrees Celsius HBSS to replace the PBS. Leave 25 microliters of the HBSS in each well.
Next, add GT, and/or S. aureus working suspensions, and incubate the plates at 37 degrees Celsius in 5% carbon dioxide for 30 minutes. Following incubation, aspirate one column of the plate at a time. Immediately refill the wells using 300 microliters of PBS with magnesium and calcium.
After repeating this wash step twice, add 200 microliters at a 1 to 50 dilution of filter-sterilized RIPA lysis buffer to each well.
When working with other strains of bacteria or fungi, make sure that the concentration of RIPA used is sufficient for cell lysis, but still permissive for microbial viability testing.
Rapidly triturate the HeLa cell lysate, and pipette 50 microliters onto mannitol salts and/or fungicidal medium. Then, using a glass rod bent at a 90-degree angle, spread the lysate over the surface of the plate.
After incubating the plates for 18 hours, manually count the number of colonies per plate. Controls consist of S. aureus and/or C. albicans adherence to HSV-uninfected HeLa cells.

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Universidad de Cartagena UDEC

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Immunology

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1. HSV Strains and Handling
NOTE: Recombinant non-spreading HSV-1(KOS) gL86 and HSV-2 (KOS) 333gJ-&#160;with beta-galactosidase reporter activity is used for the assay.
<ol>
	<li>Use virus from a single lot and store at -80 &#176;C at a 1:1 ratio of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) with 20% fetal bovine serum (FBS) and skim milk until use. Before viral lot storage, determine virus concentration by&#160;o-nitrophenyl-&#946;-D-galactopyranoside (ONPG) and 5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal) assay.</li>
	<li>Determine virus viability and multiplicity of infection (MOI) by X-Gal staining for each experimental assay run using reporter virus entry assay, as previously described (Figure 1).</li>
	<li>Dilute virus (Opt-MEM) to desired MOI. Fix monolayers (paraformaldehyde; 0.5 ml/well) before staining. Place virus viability controls in a separate microtiter plate in parallel with polymicrobial assay plates.</li>
</ol>
2. HeLa 299 Cell Handling
<ol>
	<li>Grow at 37 &#176;C, 5% CO2&#160;in DMEM with 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum (FBS), gentamicin (50 &#181;g/ml) and L-glutamine. Passage cells at 80% confluence with Trypsin solution (ethylenediaminetetraacetic acid, 0.53 M EDTA; 0.05% Trypsin; 5 ml/flask).</li>
</ol>
3.&#160;C. albicans&#160;Handling
NOTE:&#160;C. albicans&#160;obtained from a clinical laboratory source is stored at -80 &#176;C in Remmel skim milk 2x medium.
<ol>
	<li>Culture frozen stock onto Sabouraud Dextrose Medium (37 &#176;C). After 24 hr subculture the&#160;C. albicans&#160;onto Fungisel medium (37 &#176;C; 48 hr) for use.</li>
	<li>Generate germ tube (GT) forms (pick representative colonies; 3 ml FBS; 3 hr; 37 &#176;C; absorbance600 (Abs600), 0.3). After incubation, wash GT (Hanks&#39;&#160;Balanced Salt Solution, HBSS; 2x; 4,000 x g). Add washed GT to warmed HBSS (37 &#176;C; 0.32 Abs600). GT forms should be 99% of cells observed as determined by hemocytometer count.</li>
	<li>Make yeast form (YF) stock suspensions by picking representative Fungisel colonies (HBSS, 3 ml; 0.32 Abs600). Count the number of YF forms/ml microscopically using a hemocytometer. YF forms should be 99% of cells observed as determined by hemocytometer count.</li>
	<li>Make working fungal stock (250 &#181;l of GT or YF stock in 25 ml HBSS; 37 &#176;C; 105&#160;colony forming unit, (CFU/ml)</li>
</ol>
4.&#160;S. aureus&#160;Handling
<ol>
	<li>Store&#160;S. aureus&#160;American Type Culture Collection, ATCC 25923 (-80 &#176;C; Remmel skim milk 2x). Culture onto sheep blood agar (5%; 37 &#176;C; 24 hr). Pick representative colonies and transfer to mannitol salts medium within 2 days for stock (37 &#176;C; 18 hr).</li>
	<li>Make&#160;S. aureus&#160;stock suspension (3 ml HBSS; 1.32 Abs600 ;108&#160;CFU/ml)
	<ol>
		<li>Make working&#160;S. aureus&#160;stock (100 &#181;l of the stock in 25 ml HBSS; 105&#160;CFU/ml).</li>
	</ol>
	</li>
</ol>
5.&#160;Candida&#160;and&#160;S. aureus&#160;Suspensions
<ol>
	<li>Make mixed&#160;C. albicans&#160;and&#160;S. aureus&#160;suspension (250 &#181;l YF or GT stock and 100 &#181;l&#160;S. aureus&#160;stock in 25 ml HBSS).</li>
</ol>
6. Polymicrobial Biofilm Assay
<ol>
	<li>Seed 96 well plates with 200 &#181;l of 2 x 105&#160;HeLa cells/ml (85% confluence level). Rock plates (30 - 45 min; 37 &#176;C) before incubation (37 &#176;C; 5% CO2&#160;incubator; 18 hr). Wash monolayers (1x; Opt-MEM) then seed with HSV (HSV-1 (KOS) gL86 or HSV-2 (KOS) 33 gJ-&#160;in 100 &#181;l Opt-MEM ; MOI 50 and 10). Incubate plates (3 hr; 37 &#176;C; 5% CO2). Use only one viral strain per day.</li>
	<li>Wash infected monolayers (1x; phosphate-buffered saline (PBS) with Mg+2&#160;and Ca+2; 100 &#181;l). Replace PBS with warm HBSS leaving 25 &#181;l in each well.
	<ol>
		<li>Add YF, GT and/or&#160;S. aureus&#160;working suspensions (100 &#181;l; target to cell ratio =5:1; n=16) as indicated in&#160;Table 1. Incubate plates (static; 30 min; 37 &#176;C; 5% CO2).</li>
	</ol>
	</li>
	<li>After incubation, aspirate one column at a time immediately refilling with 300 &#181;l PBS with Mg+2&#160;and Ca+2. Repeat this step twice then add radio-immunoprecipitation assay lysis buffer (RIPA; filter sterilized; 200 &#181;l of a 1:50 dilution).</li>
	<li>Rapidly triturate the HeLa cell lysate then place 50 &#181;l onto mannitol salts (MS) and/or Fungisel (F) media (Figure 2). Spread the lysate using a glass rod bent at a 90&#176; angle. Incubate the plates (18 hr at 37 &#176;C). Manually count the number of colonies per plate. Controls consist of&#160;S. aureus&#160;and/or&#160;C. albicans&#160;adherence to HSV-uninfected HeLa cells.</li>
</ol>
<img alt="Equation1" src="/files/ftp_upload/22008/22008_Table_1.jpg" style="margin: auto;" />
Table 1. General Template Determination of Microbial Adherence in Polymicrobial Interactions.&#160;GT=&#160;Candida albicans&#160;germ tube phenotype; YF=&#160;C. albicans&#160;yeast form phenotype; MSSA= methicillin-sensitive&#160;Staphylococcus aureus; MS= mannitol salts medium; F= Fungisel medium.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C.albicans</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Sabouraud Dextrose</td>
			<td>BD</td>
			<td>211584</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungisel Agar</td>
			<td>Dot Scientific</td>
			<td>7205A</td>
			<td></td>
		</tr>
		<tr>
			<td>S.aureus</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol Salt Agar</td>
			<td>Troy Biologicals</td>
			<td>7143B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep blood agar</td>
			<td>Troy Biologicals</td>
			<td>221239</td>
			<td></td>
		</tr>
		<tr>
			<td>Hela cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1xDMEM (Dubelcco&#39;s Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate</td>
			<td>Corning</td>
			<td>10-017-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin 50mg/ml</td>
			<td>Sigma</td>
			<td>1397</td>
			<td>50&#181;g/ml final concentration in the complete DMEM</td>
		</tr>
		<tr>
			<td>Trypsin EDTA (0.05% Trypsin, 0.53M EDTA)Solution 1X</td>
			<td>Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11150</td>
			<td>10% final concentration in the complete DMEM</td>
		</tr>
		<tr>
			<td>Other medium and reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ONPG</td>
			<td>Thermo Scientific</td>
			<td>34055</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Pure X gal</td>
			<td>Invitrogen</td>
			<td>15520-018</td>
			<td></td>
		</tr>
		<tr>
			<td>1x HBSS (Hanks&#39; Balanced Salt Solution)</td>
			<td>Corning</td>
			<td>20-021-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>1XPBS</td>
			<td>Dot Scientific</td>
			<td>30042-500</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA Lysis</td>
			<td>Life Technologies</td>
			<td>89901</td>
			<td></td>
		</tr>
		<tr>
			<td>Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well plates</td>
			<td>Evergreen Scientific</td>
			<td>222-8030-01F</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture tubes 100x13</td>
			<td>Thomas Scientific</td>
			<td>9187L61</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Plotkin, B. J. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/54162">Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi.</a> J. Vis. Exp. (2016)
This video illustrates the initiation of polymicrobial biofilm formation on virus-infected cells by bacteria and fungi. Herpes simplex virus infection impedes bacterial attachment to the cells, preventing bacterial-fungal co-localization. In contrast, uninfected cells display bacterial-fungal co-localization and initiation of biofilm formation.

<img alt="Figure 1" src="/files/ftp_upload/22008/22008_Figure1.jpg" /> 
Figure 1. X-gal Staining Pictures of Dosage Dependent HSV-1 Infection in HeLa Cells.&#160;HeLa cells infected with HSV-1 at various MOI and X-Gal stained. (A) HeLa cells in well of 96 well plate with X-Gal stained mock-infected HeLa cell control; 20x initial magnification; (B) HeLa cells in well of 96 well plate with X-Gal stained HeLa cell infected with HSV-1 at an multiplicity of infection (MOI) of 10; 20x initial magnification; (C) HeLa cells in well of 96 well plate with X-Gal stained HeLa cell infected with HSV-1 at an multiplicity of infection (MOI) of 50; 20x initial magnification; scale bar applies to all.
<img alt="Figure 2" src="/files/ftp_upload/22008/22008_Figure2.jpg" /> 
Figure 2.&#160;Effect of HSV-1 (panels A, C, E) and HSV-2 (panels B, D, F) at Multiplicities of Infection (MOI) of 50 and 10 on Adherence of&#160;S. aureus&#160;and/or&#160;C. albicans&#160;to HeLa Cells. (A)&#160;S. aureus&#160;(Sa) binding to HSV-1 infected cells in the presence of&#160;C. albicans&#160;germ tubes (GT) or yeast forms (YF); (B)&#160;S. aureus&#160;(Sa) binding to HSV-2 infected cells in the presence of&#160;C. albicans&#160;germ tubes (GT) or yeast forms (YF); (C)&#160;C. albicans&#160;germ tubes (GT) binding to HSV-1 infected cells in the presence of&#160;S. aureus&#160;(Sa);&#160;(D)&#160;C. albicans&#160;germ tubes (GT) binding to HSV-2 infected cells in the presence of&#160;S. aureus&#160;(Sa); (E)&#160;C. albicans&#160;yeast forms (YF) binding to HSV-1 infected cells in the presence of&#160;S. aureus&#160;(Sa); (F)&#160;C. albicans&#160;yeast forms (YF) binding to HSV-2 infected cells in the presence of&#160;S. aureus&#160;(Sa). All data points are Mean +/- SEM, n= 16 normalized to virus-free control.&#160;*&#160;= significantly different (p&#60; 0.05) from uninfected HeLa cell control. # = significantly different (p&#60; 0.05) from paired point indicated by bracket; scale bar applies to all.

1. HSV Strains and Handling
NOTE: Recombinant non-spreading HSV-1(KOS) gL86 and HSV-2 (KOS) 333gJ-&#160;with beta-galactosidase reporter activity is used ...

Take a multi-well plate containing mammalian cell monolayers.
Seed test wells with Herpes simplex virus. Control wells lack the virus.
The virus attaches to cell-surface heparan sulfate and is internalized via endocytosis.
Virus infection triggers heparanase secretion, which cleaves heparan sulfate and unmasks specific fungal receptors.
Co-incubate the cells with Staphylococcus aureus and Candida albicans germ tubes.
In virus-infected cells, heparan sulfate depletion inhibits bacterial interaction while promoting fungal receptor interactions, preventing bacterial-fungal co-localization.
In control cells, bacteria interact with heparan sulfate, while fungi bind to available receptors.
This microbial co-localization initiates adherence &#8212; the first step in biofilm formation.
Post-incubation, remove unbound microbes. Overlay with a lysis buffer, releasing bacteria and fungi into the media.
Spread the cell lysate on plates containing growth media. Incubate for colony formation.
A decrease in bacterial colonies with a corresponding increase in fungal colonies indicates virus-induced inhibition of bacterial-fungal co-localization and biofilm initiation.

27 ビュー. ウイルス感染細胞における多微生物バイオフィルムの開始を決定するためのアッセイ

ビデオ: ウイルス感染細胞における多微生物バイオフィルムの開始を決定するためのアッセイ - 実験

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

規制カンジダバイオ フィルムの成長する赤い光で毎日光線

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

JoVE Journal

免疫・感染学

Assessing Biofilm Dispersal in Murine Wounds

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring otitis media, and many more. Microbial cells within these infections are protected by an extracellular polymeric substance (EPS), which can prevent antibiotics and host immune cells from clearing the infection. To overcome this obstacle, investigators have begun developing dispersal agents as potential therapeutics. These agents target various components within the biofilm EPS, weakening the structure, and initiating dispersal of the bacteria, which can theoretically improve antibiotic potency and immune clearance. To determine the efficacy of dispersal agents for wound infections, we have developed protocols that measure biofilm dispersal both ex vivo and in vivo. We use a mouse surgical excision model that has been well-described to create biofilm-associated chronic wound infections. To monitor dispersal in vivo, we infect the wounds with bacterial strains that express luciferase. Once mature infections have established, we irrigate the wounds with a solution containing enzymes that degrade components of the biofilm EPS. We then monitor the location and intensity of the luminescent signal in the wound and filtering organs to provide information about the level of dispersal achieved. For ex vivo analysis of biofilm dispersal, infected wound tissue is submerged in biofilm-degrading enzyme solution, after which the bacterial load remaining in the tissue, versus the bacterial load in solution, is assessed. Both protocols have strengths and weaknesses and can be optimized to help accurately determine the efficacy of dispersal treatments.

Here, we describe ex vivo and in vivo methods for assessing bacterial dispersal from a wound infection in mice. This protocol can be utilized to test the efficacy of topical antimicrobial and anti-biofilm therapies, or to assess the dispersal capacity of different bacterial strains or species.

ネズミの創傷におけるバイオフィルム分散の評価

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring ...

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation on implanted devices or catheters, which is associated with increased virulence and mortality. Biofilms produced by different Candida species exhibit enhanced resistance against various antifungal drugs. Therefore, there is a need to develop effective immunotherapies or adjunctive treatments against Candida biofilms. While the role of cellular immunity is well established in anti-Candida protection, the role of humoral immunity has been studied less.
It has been hypothesized that inhibition of biofilm formation and maturation is one of the major functions of protective antibodies, and Candida albicans germ tube antibodies (CAGTA) have been shown to suppress in vitro growth and biofilm formation of C. albicans earlier. This paper outlines a detailed protocol for evaluating the role of antibodies on biofilms formed by C. tropicalis. The methodology for this protocol involves C. tropicalis biofilm formation in 96-well microtiter plates, which were then incubated in the presence or absence of antigen-specific antibodies, followed by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) assay for measuring the metabolic activity of fungal cells in the biofilm.
The specificity was confirmed by using appropriate serum controls, including Sap2-specific antibody-depleted serum. The results demonstrate that antibodies present in the serum of immunized animals can inhibit Candida biofilm maturation in vitro. In summary, this paper provides important insights regarding the potential of antibodies in developing novel immunotherapies and synergistic or adjunctive treatments against biofilms during invasive candidiasis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

A Soluble Tetrazolium-Based Reduction Assay to Evaluate the Effect of Antibodies on Candida tropicalis Biofilms

A 96-well microtiter plate-based protocol using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-carboxanilide-2H-tetrazolium (XTT) reduction assay is described herein, to study antibodies' effects on biofilms formed by C. tropicalis. This in vitro protocol can be used to check the effect of potential new antifungal compounds on the metabolic activity of Candida species cells in biofilms.

カンジダ・トロピカリス・バイオフィルムに対する抗体の効果を評価するための可溶性テトラゾリウムベースの還元アッセイ

Candida species are the fourth-most common cause of systemic nosocomial infections. Systemic or invasive candidiasis frequently involves biofilm formation ...

An Assay to Determine Polymicrobial Biofilm Initiation on Virus-Infected Cells

記録

An Assay to Determine Polymicrobial Biofilm Initiation on Virus-Infected Cells