S. aureus strains SF8300 WT and SF8300 &#916;fnbA/B were generously gifted by Prof. Binh Diep (University of California, San Francisco, USA). This work was supported by a grant of the FINOVI association (#AO13 FINOVI) under the aegis of the Foundation for the University of Lyon.

1. Culture of human epithelial cells
<ol>
	<li>Prepare complete culture medium with Dulbecco&#39;s modified Eagle medium (DMEM) high glucose with phenol red, supplemented with 10% fetal bovine serum (FBS) without antibiotics.</li>
	<li>Grow A549 epithelial cells in complete culture medium at 36 &#177; 1 &#176;C in 5% CO2. Ensure the use of an appropriately sized culture vessel to have enough cells for subsequent steps (see step 1.10). 
	NOTE: One 75 cm2 (T-75) flask is sufficient to seed two 24-well plates and subculture the cells.</li>
	<li>Two days before infection, prepare a single 24-well plate.</li>
	<li>Remove and discard the spent culture medium from the T-75 flask and wash the cells once with 10 mL of Dulbecco&#8242;s phosphate-buffered saline (DPBS).</li>
	<li>Add 5 mL of trypsin-EDTA and incubate the cells for 5 min at 36 &#177; 1 &#176;C in 5% CO2.</li>
	<li>Add 5 mL of complete culture medium and transfer the cells into a tube.</li>
	<li>Centrifuge the cells for 5 min at 300 &#215; g.</li>
	<li>Discard the supernatant and resuspend the cells in 10 mL of fresh complete culture medium.</li>
	<li>Count the cells with an automatic cell counter (or a counting chamber).</li>
	<li>Dilute the cells in complete culture medium to prepare 30 mL of cell suspension at a concentration of 2.0 &#215; 105 cells/mL.</li>
	<li>Add 1 mL of the cell suspension to each well of a 24-well plate, which corresponds to a cell density of approximately 1.0 &#215; 105 cell/cm&#178; for a well area of 2 cm&#178;.</li>
	<li>Incubate the cells for 48 h at 36 &#177; 1 &#176;C in 5% CO2 until they reach 100% confluence. 
	&#8203;NOTE: In addition to the conditions to be tested, three wells should be reserved for cell counting on the day of infection (see step 3.1.4). According to the number of conditions to be tested, Up to two 24-well plates can be prepared simultaneously. Volumes indicated in the protocol should be increased accordingly.</li>
</ol>
2. Culture of S. aureus strains
<ol>
	<li>Two days before infection, prepare complete infection medium with DMEM high glucose without phenol red, supplemented with 10% FBS without antibiotics.</li>
	<li>Thaw S. aureus strains to be tested on agar plates.</li>
	<li>Incubate the agar plates for 18-24 h at 36 &#177; 1 &#176;C.</li>
	<li>The day before inoculation, inoculate one colony of the S. aureus strain to be tested in 10 mL of complete infection medium.</li>
	<li>Incubate the bacteria for 18-24 h at 36 &#177; 1 &#176;C with shaking at 160 rpm. Use 50 mL tubes held at 45&#176; to avoid the bacteria settling. 
	&#8203;NOTE: Before starting with a new strain, it is recommended to verify its lysostaphin susceptibility in the same conditions of culture that will be used for further experiments (media, bacterial loads, and lysostaphin concentration and incubation time). It is also important to determine the bacterial load corresponding to an OD600nm of 0.5 because it could vary slightly from one strain to another. Culture conditions of bacterial strains could be adapted according to the experimental aim.</li>
</ol>
3. Infection assay with S. aureus
<ol>
	<li>Determination of cell density and viability
	<ol>
		<li>Remove and discard the spent culture medium from the three wells dedicated for counting A549 cells.</li>
		<li>Add 1 mL of complete infection medium containing 5 &#181;g/mL of Hoechst 33342 and 1 &#181;g/mL of propidium iodide. 
		NOTE: Hoechst 33342 is a known mutagen and should be handled with care. Propidium iodide, a potential mutagen, must be handled with care and disposed of safely according to applicable regulations.</li>
		<li>Incubate the cells for 30 min at 36 &#177; 1 &#176;C in 5% CO2.</li>
		<li>Count the cell number and calculate the cell viability using a wield-field fluorescence microscope. 
		NOTE: If a fluorescence microscope is not available, the cell density and viability can be calculated with trypan blue staining by using a cell counting chamber.</li>
	</ol>
	</li>
	<li>Preparation of the bacterial suspension
	<ol>
		<li>Dispense 25 mL of complete infection medium in a tube and pre-warm at 36 &#177; 1 &#176;C.</li>
		<li>Adjust the S. aureus suspension to anOD600nm of 0.5 in complete infection medium using a cell density meter.</li>
		<li>Prepare 20 mL of bacterial suspension for cell inoculation by diluting the 0.5 OD600nm in complete infection medium to achieve a multiplicity of infection (MOI) of 1 according to the number of cells per well. 
		NOTE: The MOI corresponds to the number of bacteria added per cell in each well. For example, to achieve an MOI of 1 with 1.0 &#215; 106 cells per well, prepare a bacterial suspension at 2.0 &#215; 106 CFU/mL so that 106 CFU can be added in a volume of 500 &#181;L (see step 3.3.3). The MOI can be adjusted according to the cell types and bacterial strains to be tested.</li>
		<li>Use an automatic spiral plater to determine the S. aureus load of the diluted bacterial suspension to be used for the cell inoculation step.</li>
		<li>Incubate the agar plates for 18-24 h at 36 &#177; 1 &#176;C.</li>
		<li>The next day, count the number of colonies with a colony counter to calculate the accurate MOI for each strain tested. 
		NOTE: If no automatic spiral plater is available, the bacterial load could be determined by serial dilution on an agar plate. See the bacteriological analytical manual for details14.</li>
	</ol>
	</li>
	<li>Cell inoculation
	<ol>
		<li>Observe every well of the 24-well plate by low magnification microscopy to ensure that the cells are healthy and growing as expected.</li>
		<li>Remove and discard the spent cell culture medium from the 24-well plate.</li>
		<li>Add 500 &#181;L of the bacterial suspension for inoculation to each well with 100% confluent cells.</li>
		<li>Incubate the cells for 2 h at 36 &#177; 1 &#176;C and 5% CO2. 
		NOTE: it is recommended to use three wells of the plate for each condition to be tested (triplicate) and to perform at least three independent experiments. The delay of incubation can be adapted according to the experimental aim.</li>
	</ol>
	</li>
	<li>Quantification of intracellular bacteria with improved enzyme protection assay (iEPA)
	<ol>
		<li>Prepare 7 mL of 4x lysis buffer with 3.5 mL of 2% Triton X-100 in sterile water and 3.5 mL of trypsin-EDTA.</li>
		<li>Prepare a lysostaphin stock solution at 10 mg/mL in acetate buffer and aliquot 25 &#181;L into cryovials. Store at -80 &#176;C for up to 6 months.</li>
		<li>Prepare 250 &#181;L of a fresh lysostaphin working solution at 1 mg/mL by mixing 25 &#181;L of the lysostaphin stock solution (10 mg/mL) and 225 &#181;L of 0.1 M Tris-HCl. Store at 4 &#176;C for up to 48 h.</li>
		<li>Prepare 6.25 mL of complete infection medium supplemented with lysostaphin by adding 6 mL of complete infection medium to 250 &#181;L of the lysostaphin working solution.</li>
		<li>Add 250 &#181;L of complete infection medium supplemented with lysostaphin into each well and gently agitate the plate by swiveling the plate by hand.</li>
		<li>Incubate the cells for 1 h at 36 &#177; 1 &#176;C in 5% CO2 to let the lysostaphin kill the extracellular bacteria.</li>
		<li>At the end of the incubation time, add 10 &#181;L of proteinase K at 20 mg/mL into each well to inactivate the lysostaphin.</li>
		<li>Incubate the cells for 2 min at room temperature.</li>
		<li>Add 250 &#181;L of 4x lysis buffer to lyse the cells by osmotic shock.</li>
		<li>Incubate the cells for 10 min at 36 &#177; 1 &#176;C.</li>
		<li>Mix thoroughly by pipetting up and down ten times all over the bottom of the well to ensure that the cells are fully lysed and homogenized.</li>
		<li>Use an automatic spiral plater to determine the S. aureus load of each well.</li>
		<li>Incubate the agar plates for 18-24 h at 36 &#177; 1 &#176;C.</li>
		<li>The next day, count the number of colonies with a colony counter to calculate the intracellular S. aureus load of each well.</li>
	</ol>
	</li>
	<li>Measurement of intracellular efficacy of antimicrobial compounds with enzyme protection assay (EPA)
	<ol>
		<li>Prepare 25 mL of 1x lysis buffer with 3.125 mL of 2% Triton X-100 in sterile water, 6.25 mL of trypsin-EDTA, and 15.625 mL of sterile water.</li>
		<li>Prepare 250 &#181;L of a fresh lysostaphin working solution at 1 mg/mL by mixing 25 &#181;L of a lysostaphin stock solution (10 mg/mL) and 225 &#181;L of 0.1 M Tris-HCl.</li>
		<li>Prepare 25 mL of complete infection medium supplemented with lysostaphin by adding 24.75 mL of complete infection medium to 250 &#181;L of the lysostaphin working solution.</li>
		<li>For each antimicrobial compound to be tested, prepare 3.1 mL of complete infection medium supplemented with lysostaphin and the antimicrobial compound at the concentration to be studied.</li>
		<li>Remove and discard the spent cell culture medium from the 24-well plate.</li>
		<li>Add 1 mL of complete infection medium supplemented with lysostaphin.</li>
		<li>Incubate the cells for 1 h at 36 &#177; 1 &#176;C in 5% CO2 to let the lysostaphin kill the extracellular bacteria.</li>
		<li>Remove and discard the medium supplemented with lysostaphin from the 24-well plate.</li>
		<li>Fill three wells with 1 mL of medium supplemented with lysostaphin plus the antimicrobial compound to be tested.</li>
		<li>Repeat step 3.5.9 for each antimicrobial compound to be tested.</li>
		<li>For the control condition, fill three wells with 1 mL of medium supplemented with lysostaphin without any antimicrobial compound.</li>
		<li>Incubate the cells for 24 h at 36 &#177; 1 &#176;C in 5% CO2.</li>
		<li>At the end of the incubation period, remove and discard the spent medium and gently wash each well three times with sterile DPBS with CaCl2 and MgCl2.</li>
		<li>Add 1 mL of 1x lysis buffer to each well to detach and lyse the cells by osmotic shock.</li>
		<li>Incubate the cells for 10 min at 36 &#177; 1 &#176;C.</li>
		<li>Mix thoroughly by pipetting up and down ten times all over the well to ensure that the cells are fully lysed and homogenized.</li>
		<li>Use an automatic spiral plater to determine the S. aureus load of each well.</li>
		<li>Incubate the agar plates for 18-24 h at 36 &#177; 1 &#176;C.</li>
		<li>The next day, count the number of colonies with a colony counter to calculate the intracellular S. aureus load of each well. 
		NOTE: The intracellular activity of each antimicrobial compound should be calculated according to the bacterial load of the control condition. It is also important to check the cytotoxicity of all antimicrobial compounds to prove that the differences observed between the control and the compounds are not due to cell death.</li>
	</ol>
	</li>
</ol>

<ol><li>Verhoeven, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+and+clinical+relevance+of+Staphylococcus+aureus+nasal+carriage%3A+an+update%5BTitle%5D)+AND+%22Expert+Review+of+Anti-Infective+Therapy%22%5BJournal%5D)">Detection and clinical relevance of Staphylococcus aureus nasal carriage: an update.</a> Expert Review of Anti-Infective Therapy. 12 (1), 75-89 (2014).</li>
<li>Gagnaire, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiology+and+clinical+relevance+of+Staphylococcus+aureus+intestinal+carriage%3A+a+systematic+review+and+meta-analysis%5BTitle%5D)+AND+%22Expert+Review+of+Anti-Infective+Therapy%22%5BJournal%5D)">Epidemiology and clinical relevance of Staphylococcus aureus intestinal carriage: a systematic review and meta-analysis.</a> Expert Review of Anti-Infective Therapy. 15 (8), 767-785 (2017).</li>
<li>Tong, S. Y. C., Davis, J. S., Eichenberger, E., Holland, T. L., Fowler, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staphylococcus+aureus+infections%3A+epidemiology%2C+pathophysiology%2C+clinical+manifestations%2C+and+management%5BTitle%5D)+AND+%22Clinical+Microbiology+Reviews%22%5BJournal%5D)">Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management.</a> Clinical Microbiology Reviews. 28 (3), 603-661 (2015).</li>
<li>Josse, J., Laurent, F., Diot, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staphylococcal+adhesion+and+host+cell+invasion%3A+fibronectin-binding+and+other+mechanisms%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Staphylococcal adhesion and host cell invasion: fibronectin-binding and other mechanisms.</a> Frontiers in Microbiology. 8, 2433(2017).</li>
<li>Hanssen, A. -M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization+of+Staphylococcus+aureus+in+tissue+from+the+nasal+vestibule+in+healthy+carriers%5BTitle%5D)+AND+%22BMC+Microbiology%22%5BJournal%5D)">Localization of Staphylococcus aureus in tissue from the nasal vestibule in healthy carriers.</a> BMC Microbiology. 17 (1), 89(2017).</li>
<li>Rigaill, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+intracellular+efficacy+of+antimicrobial+agents+used+for+Staphylococcus+aureus+decolonization+in+a+cell+model+mimicking+nasal+colonization%5BTitle%5D)+AND+%22Journal+of+Antimicrobial+Chemotherapy%22%5BJournal%5D)">Evaluation of the intracellular efficacy of antimicrobial agents used for Staphylococcus aureus decolonization in a cell model mimicking nasal colonization.</a> Journal of Antimicrobial Chemotherapy. 73 (11), 3044-3048 (2018).</li>
<li>Yang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+insights+into+Staphylococcus+aureus+deep+bone+infections%3A+the+Involvement+of+osteocytes%5BTitle%5D)+AND+%22mBio%22%5BJournal%5D)">Novel insights into Staphylococcus aureus deep bone infections: the Involvement of osteocytes.</a> mBio. 9 (2), 00415-00418 (2018).</li>
<li>Tuchscherr, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Staphylococcus+aureus+phenotype+switching%3A+an+effective+bacterial+strategy+to+escape+host+immune+response+and+establish+a+chronic+infection%5BTitle%5D)+AND+%22EMBO+Molecular+Medicine%22%5BJournal%5D)">Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection.</a> EMBO Molecular Medicine. 3 (3), 129-141 (2011).</li>
<li>Valour, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antimicrobial+activity+against+intraosteoblastic+Staphylococcus+aureus%5BTitle%5D)+AND+%22Antimicrobial+Agents+and+Chemotherapy%22%5BJournal%5D)">Antimicrobial activity against intraosteoblastic Staphylococcus aureus.</a> Antimicrobial Agents and Chemotherapy. 59 (4), 2029-2036 (2015).</li>
<li>Proctor, R. A., Prendergast, E., Mosher, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fibronectin+mediates+attachment+of+Staphylococcus+aureus+to+human+neutrophils%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Fibronectin mediates attachment of Staphylococcus aureus to human neutrophils.</a> Blood. 59 (4), 681-687 (1982).</li>
<li>Climo, M. W., Ehlert, K., Archer, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+and+suppression+of+lysostaphin+resistance+in+oxacillin-resistant+Staphylococcus+aureus%5BTitle%5D)+AND+%22Antimicrobial+Agents+and+Chemotherapy%22%5BJournal%5D)">Mechanism and suppression of lysostaphin resistance in oxacillin-resistant Staphylococcus aureus.</a> Antimicrobial Agents and Chemotherapy. 45 (5), 1431-1437 (2001).</li>
<li>Bur, S., Preissner, K. T., Herrmann, M., Bischoff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Staphylococcus+aureus+extracellular+adherence+protein+promotes+bacterial+internalisation+by+keratinocytes+independent+of+fibronectin-binding+proteins%5BTitle%5D)+AND+%22Journal+of+Investigative+Dermatology%22%5BJournal%5D)">The Staphylococcus aureus extracellular adherence protein promotes bacterial internalisation by keratinocytes independent of fibronectin-binding proteins.</a> Journal of Investigative Dermatology. 133 (8), 2004-2012 (2013).</li>
<li>Kim, J. -H., Chaurasia, A. K., Batool, N., Ko, K. S., Kim, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternative+enzyme+protection+assay+to+overcome+the+drawbacks+of+the+gentamicin+protection+assay+for+measuring+entry+and+intracellular+survival+of+Staphylococci%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">Alternative enzyme protection assay to overcome the drawbacks of the gentamicin protection assay for measuring entry and intracellular survival of Staphylococci.</a> Infection and Immunity. 87 (5), 00119(2019).</li>
<li>Maturin, L., Peeler, J. T. Aerobic plate count. Bacteriological Analytical Manual., Edition 8, Revision A. 3, Available from: 
 <a target="_blank" href="https://www.fda.gov/food/laboratory-methods-food/bam-chapter-3-aerobic-plate-count">https://www.fda.gov/food/laboratory-methods-food/bam-chapter-3-aerobic-plate-count</a> 3(2021).</li>
<li>Kolenda, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+the+activity+of+a+combination+of+three+bacteriophages+alone+or+in+association+with+antibiotics+on+Staphylococcus+aureus+embedded+in+biofilm+or+internalized+in+Osteoblasts%5BTitle%5D)+AND+%22Antimicrobial+Agents+and+Chemotherapy%22%5BJournal%5D)">Evaluation of the activity of a combination of three bacteriophages alone or in association with antibiotics on Staphylococcus aureus embedded in biofilm or internalized in Osteoblasts.</a> Antimicrobial Agents and Chemotherapy. 64 (3), 02231(2020).</li>
<li>Abad, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibiofilm+and+intraosteoblastic+activities+of+rifamycins+against+Staphylococcus+aureus%3A+promising+in+vitro+profile+of+rifabutin%5BTitle%5D)+AND+%22Journal+of+Antimicrobial+Chemotherapy%22%5BJournal%5D)">Antibiofilm and intraosteoblastic activities of rifamycins against Staphylococcus aureus: promising in vitro profile of rifabutin.</a> Journal of Antimicrobial Chemotherapy. 75 (6), 1466-1473 (2020).</li>
</ol>

The authors have no conflicts of interest to declare.

The assays described here are valuable for studying the extent of internalization and the intracellular survival of S. aureus in NPPCs, as well as the intracellular efficacy of antimicrobial compounds6,15,16. Some steps in both assay protocols can be critical. The health condition and the density of the cells must be perfectly controlled and consistent between independent experiments. The bacterial inoculum must be carefully standardized to obtain a real MOI close to the targeted theoretical MOI. In general, care must be taken not to detach any of the cells while pipetting. The washes to remove lysostaphin and antibiotics are critical steps in the EPA. The use of proteinase K has been found to improve this step when no antibiotic is used (see below). Last but not least, the cells should be fully detached in each well and thoroughly homogenized after the incubation with the lysis buffer to reliably quantify the S. aureus intracellular load.
In some instances, issues may be encountered, and several points must be checked first. In case of a lack of reproducibility, it must be kept in mind that S. aureus can form clumps, making quantification by absorbance inaccurate. The clumping of bacteria can be increased by centrifugation and washing steps if the culture medium is to be replaced (e.g., for eliminating a secreted protein). The bacterial suspension should be used rapidly because bacteria continue to grow at room temperature. The lysostaphin efficacy could decrease because of incorrect storage conditions, suboptimal pH for enzyme activity in the culture media, variability in the enzymatic activity between batches and providers, and lack of lysostaphin sensitivity of some strains in specific growth conditions. Phenol red could have a slight bacteriostatic effect, especially when the culture medium is relatively poor in nutrients compared to the typical broths used for growing bacteria. Thus, it is advisable to use a cell culture medium without phenol red, which also improves fluorescence microscopic observations by reducing the background noise.
Although this method is a valuable tool to study the intracellular fate of different strains, some limits of the method should be considered. The use of a very high MOI can overload the capability of internalization by NPPCs and level out the differences between the different strains tested. The extent of internalization of the most cytotoxic strains may be underestimated because lysostaphin (or antibiotics) rapidly destroys S. aureus that is released by damaged cells. Thus, experiments with extended durations (i.e., to study intracellular survival or intracellular activity of antibiotics) are easier to set up with strains with low cytotoxicity. Therefore, the incubation time and the MOI should be accurately adjusted according to the strain virulence, the cell type, and the experimental aim.
The method described here with the use of lysostaphin is more reliable than those based on gentamicin because, unlike lysostaphin, gentamicin tends to be internalized by host cells13. The other advantage is the possibility to inactivate the lysostaphin. Inhibition of lysostaphin activity was reported by Kim et al.13 with the use of EDTA to chelate zinc ions or 1,10-phenanthroline; however, intensive washes are still required to remove the enzyme before plating of the bacteria. Here, proteinase K enables rapid inactivation of lysostaphin. We observed that cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. By skipping the final washing step, the iEPA method greatly simplified technical handling and enabled the recovery of the internalized bacteria in loosely adherent or already detached cells. 
The more concentrated reagents and buffers used in iEPA also helped reduce pipetting effort and minimize the loss of cells. In addition, iEPA can be used with cells in suspension, as well as with organoids that are difficult to wash. In conclusion, enzyme protection assays enable the study of the extent of internalization and the intracellular fate of S. aureus, as well as the intracellular activity of antimicrobials drugs with different in vitro models. Improvements should be made to better characterize the relationship between internalization and cytotoxicity to better appreciate the importance of developing drugs capable of reaching S. aureus inside the cell.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62903">Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds</a>. One of the affiliations was updated.
The fifth affiliation was updated from:
D&#233;partement de Bact&#233;riologie, Institut des Agents Infectieux, Hospices Civils de Lyon, Lyon, France
to:
Department of Bacteriology, Institute for Infectious Agents, Hospices Civils de Lyon, Lyon, France&#160;

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62903">Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds</a>. One of the affiliations was updated.

Erratum: Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus is both a life-threatening pathogen and a commensal bacterium of the skin and the mucosa that colonizes two billion individuals around the world1. In humans, nasal carriers of S. aureus have an increased risk of infection with their own strain of carriage; however, the multifactorial determinants of S. aureus mucosal carriage are still unclear1,2. In addition to acute infections, patients can also develop chronic S. aureus infections that are often challenging to cure3. A better understanding of host-pathogen interactions during colonization and infection is crucial for developing novel therapeutic strategies and improving patient management.
In vitro, S. aureus can trigger its internalization into host cells expressing the &#945;5&#946;1 integrin4. The tripartite interaction between the staphylococcal fibronectin-binding proteins anchored to the cell wall of S. aureus, the fibronectin, and the &#946;1 integrin expressed at the host cell surface is well known as the main pathway of S. aureus internalization in NPPCs such as keratinocytes, osteoblasts, fibroblasts, and epithelial and endothelial cells4. Recent studies show that S. aureus can be found inside human cells during nasal colonization5,6 and infection7. However, the role of the intracellular reservoir in the pathogenesis of S. aureus infection remains unclear. The host cells could act as a shelter for S. aureus, which is protected from both the immune system8 and most antimicrobial compounds6,9.
The lysostaphin protection assay, described by Proctor10 earlier in the 1980s, enables the study of bacterial and host factors involved in the internalization of S. aureus isolates. Lysostaphin is a bacteriocin produced by Staphylococcus simulans, which&#160;exhibits potent activity against almost all S. aureus isolates, including antibiotic-resistant strains11. Lysostaphin has been used to destroy only extracellular S. aureus to enable the counting of only viable intracellular bacteria12. This technique has been widely used and has contributed to the discovery of several virulence factors of S. aureus. Gentamycin, alone and combined with lysostaphin, is also widely used to study intracellular bacteria.
However, a recent study showed that gentamycin enters eukaryotic cells and reaches internalized bacteria in a time- and concentration-dependent manner13. This study also demonstrated that lysostaphin does not enter eukaryotic cells, confirming that a lysostaphin-based enzyme protection assay (EPA) is the most accurate assay for quantifying intracellular S. aureus load by culture13. Regardless of which compound is used to destroy extracellular bacteria (e.g., lysostaphin or gentamycin), it should be removed by washing the cells before plating intracellular S. aureus on agar plates. Successive washes may result in the detachment of cells, especially poorly adherent cells (e.g., heavily infected cells), which would lead to an underestimation of the intracellular S. aureus load. This paper describes in detail how EPA can be used to quantify the intracellular S. aureus load&#160;and to measure the intracellular efficacy of antimicrobials compounds using an in vitro model. Of note, a simple method has been proposed to improve the reliability of intracellular load quantification by avoiding intensive washes.

<table><tbody><tr><td>24-well plate</td><td>CORNING-FALCON</td><td>353047</td><td></td></tr><tr><td>A549 cell line</td><td>ATCC</td><td>CCL-185</td><td></td></tr><tr><td>Acetate buffer solution pH 4.6</td><td>Fluka</td><td>31048</td><td>Used to prepare lysostpahine stock solution at 10 mg/mL in 20 mM sodium acetate.</td></tr><tr><td>AMBICIN (Recombinant lysostaphin)</td><td>AMBI</td><td>LSPN-50</td><td>Lyophilized recominant lysostaphin. Freeze at -80 &amp;deg;C for long-term storage.</td></tr><tr><td>COS - Colombia agar + 5% sheep blood</td><td>Biomerieux</td><td>43049</td><td>Any agar plate suitable for growing staphylococci can be used instead.</td></tr><tr><td>Densitometer WPA CO8000</td><td>Biochrom Ltd.</td><td>80-3000-45</td><td>Cell density meter</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium, high glucose with phenol red</td><td>Sigma-Aldrich</td><td>D6429</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium, high glucose without phenol red</td><td>Sigma-Aldrich</td><td>D1145</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Dulbecco&amp;prime;s Phosphate-buffered Saline with MgCl&lt;sub&gt;2&lt;/sub&gt; and CaCl&lt;sub&gt;2&lt;/sub&gt;, sterile-filtered</td><td>Sigma-Aldrich</td><td>D8662</td><td></td></tr><tr><td>Dulbecco&amp;prime;s Phosphate-buffered Saline, sterile-filtered</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Easyspiral dilute</td><td>Interscience</td><td>414000</td><td>Automatic diluter and spiral plater</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>HaCaT cell line</td><td>Cell lines service (CLS)</td><td>300493</td><td></td></tr><tr><td>Hoechst 33342, Trihydrochloride, Trihydrate, 10 mg/mL Solution in Water</td><td>Fisher scientific</td><td>11534886</td><td></td></tr><tr><td>Propidium iodide, 1.0 mg/mL solution in water</td><td>Invitrogen</td><td>P3566</td><td></td></tr><tr><td>Proteinase K, recombinant 20 mg/mL</td><td>Eurobio</td><td>GEXPRK01-B5</td><td>&gt; 30 U/mg, lot 901727</td></tr><tr><td>Scan 4000</td><td>Interscience</td><td>438000</td><td>Automatic colony counter</td></tr><tr><td>Sterile water</td><td>OTEC</td><td>600500</td><td></td></tr><tr><td>T-75 culture flask</td><td>CORNING-FALCON</td><td>353136</td><td></td></tr><tr><td>TC20 Automated cell counter</td><td>Biorad</td><td>1450102</td><td>Automatic cell counter</td></tr><tr><td>Tris 1 M pH 8.0</td><td>Invitrogen</td><td>AM9855G</td><td>Used to prepare lysostaphine working solution at 1 mg/mL in 0.1 M Tris-HCl.</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Trypsin - EDTA solution</td><td>Sigma-Aldrich</td><td>T3924</td><td>0.05% porcine trypsin and 0.02% EDTA in Hanks&amp;prime; Balanced Salt Solution with phenol red</td></tr><tr><td>Wide-field fluorescence microscope</td><td>Nikon</td><td>Ti2</td><td></td></tr></tbody></table>

improved enzyme protection assay to study staphylococcus aureus internalization and intracellular efficacy of antimicrobial compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

The results of S. aureus internalization by A549 epithelial cells are depicted in Figure 1A. A549 cells were inoculated with S. aureus SF8300 WT and SF8300 &#916;fnbA/B, which lacks fibronectin-binding proteins A and B, at an MOI of 1 for 2 h. To destroy extracellular S. aureus, lysostaphin was added to the culture medium, and the cells were incubated for 1 h. Next, lysostaphin was either removed by washing for EPA or inactivated with proteinase K for iEPA. Then, the cells were disrupted in lysis buffer, and the bacterial load was quantified by culture. By using EPA, the mean intracellular loads were 4.46 and 0.49 Log CFU/mL for SF8300 WT and SF8300 &#916;fnbA/B, respectively (Figure 1A, green bars). Using iEPA, the mean intracellular loads were 4.53 and 0.56 Log CFU/mL for SF8300 WT and SF8300 &#916;fnbA/B, respectively (Figure 1A,&#160;red bars). It is interesting to note that both EPA and iEPA showed similar results, which can be explained by the ease of performing the washes when the cells are in good condition and because the S. aureus-induced cytotoxicity is very low in these experimental settings (data not shown).
The results of intracellular activity of vancomycin, rifampicin, and levofloxacin against S. aureus are depicted in Figure 1B. To measure the intracellular activity of these antibiotics, HaCaT cells were inoculated with S. aureus ATCC 29213 at an MOI of 1 for 2 h. The cells were incubated with lysostaphin, with or without the antimicrobial compounds to be tested, for 24 h. Next, lysostaphin and the antimicrobial compounds were removed by washing. The cells were disrupted in lysis buffer, and the bacterial load was quantified by culture. The mean intracellular loads were 4.57, 4.51, 3.03, and 2.91 log CFU/mL for control, vancomycin (50 &#181;g/mL), rifampicin (7 &#181;g/mL), and levofloxacin (10 &#181;g/mL), respectively (Figure 1B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62903/62903fig01.jpg" /> 
Figure 1: Intracellular Staphylococcus aureus load in epithelial cells. (A) Enzyme protection assay (green bars) and improved enzyme protection assay (red bars) in A549 cells infected with S. aureus SF8300 WT and &#916;fnbA/B. (B) Intracellular activity of antimicrobial compounds in HaCaT cells infected with S. aureus ATCC 29213. Bars represent the mean values of three independent experiments performed in triplicate. Error bars represent the standard deviations. **** p &#60; 0.0001. Abbreviations: Ctrl = control; cfu = colony-forming units. <a href="https://www.jove.com/files/ftp_upload/62903/62903fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds at JoVE.com

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

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Research

JoVE Journal

Biology

2.7K Views.  University of Lyon, INSERM U1111, CNRS, UMR5308, ENS Lyon, UCBL1, University of St-Etienne, France. De enzymbeschermingstest maakt het mogelijk om de mate van Staph aureus internalisatie en het vermogen om te overleven in aanhangende cellen te bestuderen, evenals de werkzaamheid van antimicrobiële verbinding. De verbeterde enzymbeschermingstest vereenvoudigt de technische behandeling aanzienlijk en maakt het mogelijk om geïnternaliseerde bacteriën te herstellen uit zwak hechtende cellen en cellen in suspensie. De procedure wordt gedemonstreerd door Josselin Rigaill, arts en promovendus u...

Video: Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Immunology and Infection

Stress-induced Antibiotic Susceptibility Testing on a Chip

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step -&#160;bacterial growth -&#160;is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacterial culture protocols, defining microfluidic channels in polydimethylsiloxane&#160;(PDMS), cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and nonantibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.

We have developed a microfluidic platform for rapid antibiotic susceptibility testing. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria can survive these stressful conditions.

We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over ...

Bioengineering

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

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 ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

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 ...

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

Lysostaphin-Based Enzyme Protection Assay: An In Vitro Method to Quantify Intracellular Staphylococcus aureus Load by Enzyme-Mediated Selective Killing of Extracellular Bacteria

Source: Rigaill, J. et al. <a href="https://www.jove.com/v/62903">Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds.</a> J. Vis. Exp. (2021).
In this video, we demonstrate a lysostaphin-based in vitro enzyme protection assay to quantify the Staphylococcus aureus internalization in A549 cells. The assay helps in qualitative, quantitative, and characterization studies of the intracellular bacterial load in the host cells.

Source: Rigaill, J. et al. Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

A Peg Plate Biofilm Assay for Screening Antibacterial Agents

Source: Dalecki, A. G., et al. <a href="https://app.jove.com/v/53925">Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay.</a> J. Vis. Exp. (2016)
This video demonstrates an assay for screening antimicrobial compounds that efficiently eliminate biofilms using a peg-plate. The methodology monitors the biofilm&#39;s metabolic state after treatment with antimicrobial compounds, facilitating the determination of the minimum biofilm eradication concentration.

1. Biofilm Initiation

	Grow a culture of biofilm-producing organisms in a nutrient-rich medium. Inoculate&#160;Staphylococcus aureus&#160;strain Newman ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

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