This work was funded by National Nature Science Foundation of China for Young Scholars (No. 81300810), Shanghai Young Doctor Training Program (No. 20141057), and National Natural Science Foundation of China (81671982, 81271791 and 81571955). We would like to thank LetPub (www.letpub.com) for providing linguistic assistance during the preparation of this manuscript.

1. Biofilm Formation
<ol>
	<li>Biofilm formation in 96-well microplates
	<ol>
		<li>Retrieve the S. aureus strain USA300 and 3 biofilm-forming clinical strains (C1 - C3) stored at -80 &#176;C. 
		NOTE: The ability of the clinical strains to form biofilms was determined by the microtiter plate assay described previously15.</li>
		<li>Inoculate the bacterium in 5 mL tryptone soya broth (TSB) medium, and cultivate in an incubator with shaking at 37 &#176;C overnight to the stationary phase.</li>
		<li>Centrifuge the overnight bacterial culture at 4,000 x g for 10 min at 25 &#176;C and then discard the supernatant. Resuspend the pellets in phosphate buffered saline (PBS) to a final concentration of 2.0 x 109 CFU/mL. 
		NOTE: The concentration of the bacteria was estimated by measuring optical density and further determined by plate count16, revealing that 1 OD600 of suspension contained 1.5 x 108 CFU/mL.</li>
		<li>Dilute the bacterial suspension to 1:200 (1.0 x 107 CFU/mL) in TSB medium containing 0.5% glucose. Inoculate 200 &#181;L of bacterial suspension into each well of a cell-culture-treated polystyrene 96-well microplate.</li>
		<li>Incubate the microplate statically at 37 &#176;C for 24 h under a well oxygenated environment. 
		NOTE: The incubation time for mature biofilm formation may vary for different bacterial strains; this should be determined before the PDT experiment15.</li>
		<li>Discard the media in the wells and wash the microplate wells gently with PBS three times and then discard the supernatant. 
		NOTE: The step should be carried out very gently to avoid disturbing the formed biofilm.</li>
	</ol>
	</li>
	<li>Biofilm formation in dishes
	<ol>
		<li>Inoculate the S. aureus strain USA300 into 5 mL of TSB medium, and cultivate in an incubator with shaking at 37 &#176;C overnight to the stationary phase.</li>
		<li>Centrifuge the overnight bacterial culture at 4,000 x g for 10 min at 25 &#176;C and then discard the supernatant. Resuspend the pellets in PBS to a final concentration of 2.0 x 109 CFU/mL. Then, dilute the bacterial suspension to 1:200 (1.0 x 107 CFU/mL) in TSB medium containing 0.5% glucose. Inoculate 2 mL of bacterial suspension into a 35-mm optical quality glass bottom cell culture dish, and incubate statically at 37 &#176;C for 24 h. 
		NOTE: The concentration of the bacteria was estimated by measuring the optical density.</li>
		<li>Aspirate the media with a pipette, and then rinse the biofilms in the dish gently with PBS three times and then, discard the supernatant carefully. 
		NOTE: Avoid touching the pipette tip to the bottom of the dish. The step 2.2 should be performed immediately after this step to prevent drying of the formed biofilm.</li>
	</ol>
	</li>
</ol>
2. Light Irradiation
<ol>
	<li>Store 5-ALA in a 4 &#176;C refrigerator. Before the experiment, dilute 5-ALA with PBS to 10 mM. 
	NOTE: 5-ALA solution should be freshly prepared before the experiment.</li>
	<li>In the experimental group, add 200 &#181;L of 10 mM ALA to each well of the microplate or 2 mL to the culture dish. Cover the plate/dish with aluminum foil, and incubate for 1 h. Then, irradiate the plate/dish with a light-emitting diode (LED) with a light intensity of 100 mW/cm2 for 1 h to achieve a light dose of 360 J/cm2 at a major wavelength of 633 &#177; 10 nm17. 
	NOTE: In order to let the light energy be effectively and equally delivered to the biofilm in all of the wells/dishes, fix the distance from the peak of the light source to the well/dish at 6.0 cm, and limit the experimental region to the central irradiation area (10 cm x 8 cm). To ensure that the results are reproducible, the experiments should be performed at the same room temperature. 
	In the LED irradiation step, to avoid direct exposure of the plate to other light sources, such as sunlight, room lighting or lamplight, the LED was turned on before moving the plate/dish to the irradiation area, and the light was bright enough to finish the operation.</li>
	<li>Set up the control groups (three control groups were set up in our experiment).
	<ol>
		<li>For the first control group (ALA-LED-), add 200 &#181;L of PBS to each well of the microplate or 2 mL to the culture dish. Cover the plate/dish with aluminum foil, and incubate it for 2 h.</li>
		<li>For the second control group (ALA+LED-), add 200 &#181;L of 10 mM ALA to each well of the microplate or 2 mL to the culture dish. Cover the plate/dish with aluminum foil, and incubate it for 2 h.</li>
		<li>For the third control group (ALA-LED+), add 200 &#181;L of PBS to each well of the microplate or 2 mL to the culture dish. Cover the plate/dish with aluminum foil, and incubate it for 1 h. Then expose the plate to the LED with 360 J/cm2 light irradiation at a major wavelength of 633 &#177; 10 nm17. 
		NOTE: In the LED irradiation step, avoid direct exposure of the plate to other light sources, such as sunlight, room lighting, or lamplight.</li>
	</ol>
	</li>
</ol>
3. Determination of the Effectiveness of PDT Treatment
NOTE: To confirm the effect of ALA-PDT on the S. aureus biofilms, the viability of the cells with or without ALA-PDT was evaluated by CFU counting as well as by viability staining.
<ol>
	<li>Determination of the remaining viable bacterial cells
	<ol>
		<li>After ALA-PDT treatment, discard the media in the wells and wash the wells with PBS three times to remove all non-adherent cells for both experimental and control groups. 
		NOTE: This step should be carried out very gently.</li>
		<li>Scrape the adherent bacteria cells thoroughly from the wells with the pipette tip and collect the cells in conical tubes.</li>
		<li>Centrifuge the bacterial suspension at 4,000 x g for 10 min at 4 &#176;C, then discard the supernatant.</li>
		<li>Resuspend the bacteria in 1 mL of 0.25% pancreatin enzyme in PBS and incubate at 37 &#176;C for 1.5 h.</li>
		<li>Centrifuge at 4,000 x g for 10 min; discard the supernatant, then resuspend the pellet in 200 &#181;L of PBS.</li>
		<li>Make 1:10 serial dilutions of the cell solution with PBS; then, add 5 &#181;L of each serial dilution sample onto the tryptone soya agar (TSA) plate. Incubate the TSA plate at 37 &#176;C for 16 h; then, count (by naked eye) the number of bacterial colonies (CFU/mL).</li>
	</ol>
	</li>
	<li>Observation of S. aureus biofilms by CLSM
	<ol>
		<li>After light irradiation, wash the biofilms in the culture dish with PBS three times. 
		NOTE: This step should be carried out very gently.</li>
		<li>Add 1 mL of 1 &#181;M green-fluorescent nuclear and chromosome stain that is permeable to the prokaryotic cell membranes (e.g., SYTO9) and 1 mL of 1 &#181;M propidium iodide (PI) for 20 min to stain the biofilm as well as dead cells.</li>
		<li>Observe viable cells (green fluorescence, Ex/Em 485 nm/530 nm) and dead cells (red fluorescence, Ex/Em 485 nm/630nm) under a CLSM with a 63X 1.4-NA oil-immersion objective lens.</li>
		<li>Generate images using microscopy software.</li>
	</ol>
	</li>
</ol>

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F., McCarron, P. A., Tunney, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+photodynamic+therapy.">Antifungal photodynamic therapy.</a> Microbiol Res. 163 (1), 1-12 (2008).</li><li>Shi, H., Li, J., Zhang, H., Zhang, J., Sun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+5-aminolevulinic+acid+photodynamic+therapy+on+Candida+albicans+biofilms:+An+in+vitro+study.">Effect of 5-aminolevulinic acid photodynamic therapy on Candida albicans biofilms: An in vitro study.</a> Photodiagnosis Photodyn Ther. 15, 40-45 (2016).</li><li>Zhang, Q. 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The authors have nothing to disclose

PDT has been a well-studied therapy for the treatment of cancer since it was invented more than 100 years ago18. Over the last decade, PDT has been applied as an antimicrobial strategy and has shown effectiveness against some antibiotic-resistant pathogenic bacteria12. Compared to the planktonic state, bacterial biofilms appear to be more resistant to antibiotic treatment3, while the effect of ALA-PDT on biofilms has not been fully investigated yet.<...

S. aureus is an important Gram-positive pathogen that colonizes the skin and mucosa of human hosts. Its ability to form biofilms is considered an important aspect of its pathogenesis1. Bacterial biofilms are a community of bacteria embedded in a self-produced matrix, which is composed of extracellular polymeric substances, including polysaccharide, DNA, and protein. This matrix plays a significant role in the persistence of bacterial infections, contributing to a high degree of resistance to the human immune system and current anti-microbial therapies2. Antibiotics are still the major treatment for biofilm infections, although the effects of antibiotics on biofilms are limited. It has been shown previously that cells in biofilms are 10 - 1,000 times more resistant to antibiotics compared to their planktonic counterparts3. Thus, alternative strategies are needed to conquer this issue.
PDT, an alternative treatment for bacterial infections, uses the light of an appropriate wavelength to activate photosensitizers. This leads to the production of reactive oxygen species (ROS), which are lethal to target cells by disrupting the cell wall, inactivating enzymes, and damaging DNA4. This multi-target characteristic makes it difficult for bacteria to develop resistance to the PDT treatment.
The antimicrobial effect of PDT on bacterial and fungal biofilms, with multiple photosensitizers, such as toluidine blue, malachite green, methylene blue, chlorine e6, and porphyrins, has been studied in previous reports5,6,7,8,9,10,11,12,13. 5-ALA, a prodrug of the actual photosensitizer, PpIX, is characterized by its small molecular weight and rapid clearance12,14. These advantages give ALA-PDT major potential as a therapeutic application. Although the effect of ALA-PDT on planktonic bacteria has been studied by many groups12, the antimicrobial effect of ALA-PDT on bacterial biofilms has not yet been elucidated. Meanwhile, it is difficult to compare the results between previous studies. One of the reasons is that the different protocols are used by diverse groups. Thus, this protocol describes an in vitro model of an ALA-PDT system based on our previous work15. The effect of this model was confirmed by CFU calculation and viability staining with CLSM.

<table><tbody><tr><td>Tryptone Soya Broth (TSB)</td><td>OXOID</td><td>CM0129B</td><td></td></tr><tr><td>Tryptone Soya Agar (TSA)</td><td>OXOID</td><td>CM0131</td><td></td></tr><tr><td>SYTO9</td><td>Thermo Fisher Scientific</td><td>L7012</td><td>The LIVE/DEAD BacLight Bacterial Viability Kits&amp;nbsp;</td></tr><tr><td>Propidium iodide (PI)</td><td>Thermo Fisher Scientific</td><td>L7012</td><td>The LIVE/DEAD BacLight Bacterial Viability Kits&amp;nbsp;</td></tr><tr><td>Pancreatin</td><td>Sigma-Aldrich</td><td>P3292</td><td></td></tr><tr><td>5-aminolevulinic acid (ALA)</td><td>Fudan Zhangjiang Bio-Pharm</td><td>3.1</td><td></td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt; strain USA300</td><td>/</td><td>/</td><td>The source of USA 300 references &amp;ldquo;Tenover FC, Goering RV. J Antimicrob Chemother. 2009 Sep; 64(3):441-6&amp;rdquo;.</td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt; clinical strains (C1-C3)</td><td>/</td><td>/</td><td>All clinical strains were isolated from patients with chronic rhinosinusitis in the Department of Otorhinolaryngology-Head and Neck Surgery, Eye and ENT Hospital of Fudan University [Zhang QZ, Zhao KQ, Wu Y, et al. PLoS One. 2017 Mar; 12(3): e0174627].</td></tr><tr><td>96-well microplate</td><td>Corning Inc</td><td>3599</td><td>Clear Flat Bottom Polystyrene TC-Treated Microplates, Individually Wrapped, with Lid, Sterile</td></tr><tr><td>Fluorodish</td><td>NEST Biotechnology</td><td>801001</td><td>Glass bottom, Non-pyrogenic</td></tr><tr><td>Eppendorf Safe-Lock Tubes, 1.5 mL</td><td>Eppendorf</td><td>0030120086</td><td></td></tr><tr><td>Eppendorf microcentrifuge 5417</td><td>Eppendorf</td><td>Z365998 |&amp;nbsp;SIGMA</td><td></td></tr><tr><td>Incubator</td><td>Thermo Fisher Scientific</td><td>SHKE4000</td><td>MaxQ 4000 Benchtop Orbital Shakers</td></tr><tr><td>Light emitting diode (LED)</td><td>Wuhan Yage Optic and Electronic Technique CO</td><td>LED-IB</td><td></td></tr><tr><td>Leica TCS SP8 confocal laser-scanning microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Leica LAS AF software</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>IMARIS software</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

an in vitro model to study the effect of 5-aminolevulinic acid-mediated photodynamic therapy on staphylococcus aureus biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

The viability of the bacteria in the biofilms was decreased after ALA-PDT treatment when compared to the controls (ALA-LED-, ALA+LED-, and ALA-LED+) in both USA300 and the three clinical strains (Figure 1).
To confirm the results from the CFU assay and observe the antibacterial effect of ALA-PDT on the S. aureus biofilm in situ, the USA300 biofilms were visualized by CLSM with viab...

Watch this Scientific Journal Video about An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm at JoVE.com

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

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7.8K Views.  Fudan University. This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

Video: An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Immunology and Infection

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic inactivation of bacteria (aPDI). This process involves the administration of a photosensitizer (PS) activated by a suitable visible light source, producing exacerbated levels of reactive oxygen species (ROS) that damage crucial cellular biomolecules, ultimately causing bacterial cell death. It is crucial to create standardized, easy-to-use, and reproducible initial tests to assess and compare the effectiveness of light-induced phototoxicity of potential photosensitizers. This study introduces a simple and efficient in vitro technique for assessing photodynamic activity against planktonic bacterial cells. By employing a 96-well microplate format along with a large LED panel, the system facilitates the systematic evaluation of various compounds. Such a configuration allows for high-throughput screening of potential photosensitizers in a controlled and consistent environment, simplifying the process of identifying promising candidates for further development. This flexible platform serves as an important step in advancing the development of innovative photodynamic therapies for managing antibiotic-resistant infections.

This protocol describes a straightforward and economical method for evaluating the effectiveness of potential photosensitizers in antibacterial photodynamic inactivation (aPDI), using a 96-well plate format combined with an LED panel light source. This approach enables the simultaneous testing of multiple experimental conditions, including different concentrations, compounds, and bacterial strains.

The rise of multi-drug-resistant infections and the lack of new antibiotic classes have renewed interest in alternative therapies like photodynamic ...

Biochemistry

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

Medicine

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of infection where they use processes such as phagocytosis, production of reactive oxygen and nitrogen species (ROS, RNS, respectively), NETosis (neutrophil extracellular trap), and degranulation to kill microbes and resolve the infection. Interactions between neutrophils and planktonic microbes have been extensively studied. There have been emerging interests in studying infections caused by biofilms in recent years. Biofilms exhibit properties, including tolerance to killing by neutrophils, distinct from their planktonic-grown counterparts. With the successful establishment of both in vitro and in vivo biofilm models, interactions between these microbial communities with different immune cells can now be investigated. Here, techniques that use a combination of traditional biofilm models and well-established neutrophil activity assays are tailored specifically to study neutrophil and biofilm interactions. Wide-field fluorescence microscopy is used to monitor the localization of neutrophils in biofilms. These biofilms are grown in static conditions, followed by the addition of neutrophils derived from human peripheral blood. The samples are stained with appropriate dyes prior to visualization under the microscope. Additionally, the production of ROS, which is one of the many neutrophil responses against pathogens, is quantified in the presence of a biofilm. The addition of immune cells to this established system will expand the understanding of host-pathogen interactions while ensuring the use of standardized and optimized conditions to measure these processes accurately.

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

The present protocol describes the study of neutrophil-biofilm interactions. Staphylococcus aureus biofilms are established in vitro and incubated with peripheral blood-derived human neutrophils. The oxidative burst response from neutrophils is quantified, and the neutrophil localization within the biofilm is determined by microscopy.

Neutrophils are the first line of defense deployed by the immune system during microbial infection. In vivo, neutrophils are recruited to the site of ...

Optimized In Vivo Models for Intracellular S. aureus Infection Studies

Development and Assessment of Intracellular Infection Models for Staphylococcus aureus

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular persistence contributes to chronic and recurrent infections, complicating treatment and prolonging the disease. Consequently, there is a critical need for an intracellular infection model to better understand, prevent, and treat infections caused by S. aureus. This study indicated that antibiotics effectively eliminated extracellular bacteria but could not eradicate those that had entered the cells. Thus, a stable intracellular infection in vitro was established by RAW264.7 infected with S. aureus and co-culturing them with antibiotics. Subsequently, an intracellular infection model in mice was established by injecting peritoneal macrophages containing the intracellular infection. Vancomycin effectively cleared bacterial loads in mice challenged with planktonic S. aureus; however, it was ineffective against mice infected with equal or lower levels of intracellular bacteria within the peritoneal macrophages. This indicates that the intracellular infection model of S. aureus was successfully established, offering potential insights for the prevention and treatment of intracellular infections.

Staphylococcus aureus (S. aureus) has the capability to disseminate throughout the body, causing persistent and recurrent infections. To better understand these processes, this study establishes an intracellular infection model for S. aureus. This model will provide a crucial foundation for investigating the mechanisms behind intracellular infections.

S. aureus can invade and persist within host cells, including immune cells, which allows it to evade immune detection and clearance. This intracellular ...

In Vitro Biofilm Synthesis by Staphylococcus aureus

Source: Rana, P. S. et al., <a href="https://app.jove.com/v/63773">Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms.</a> J. Vis. Exp. (2022)
In this video, we demonstrate the preparation of in vitro biofilm by S. aureus in a poly-L-Lysine-coated multi-well plate. The biofilm-formed plate can be used for downstream experiments.

Source: Rana, P. S. et al., Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus ...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Summary

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Chapters in this video

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

An In Vitro Approach to Photodynamic Therapy

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms

Development and Assessment of Intracellular Infection Models for Staphylococcus aureus

In Vitro Biofilm Synthesis by Staphylococcus aureus