Name: Amikacin Protection Assay for Quantification and Visualization of Escherichia coli Cell Invasion (Video) | JoVE Journal
Uploaded: 2026-07-03T12:40:00.000+00:00
Duration: 9 min 22 s
Description: Learn an amikacin-based protection assay for quantifying and visualizing adherent-invasive Escherichia coli invasion of epithelial cells. This method overcomes gentamicin resistance limitations in IBD patient isolates.

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This work was supported by the Howard Hughes Medical Institute Emerging Pathogens Initiative (K.O., J.N.), Welch Foundation grant I-1561 (K.O.), Once Upon a Time Foundation (K.O.), NIH grant R35 GM134945 (K.O.), NIH grant NIDDK K08-DK-123316 (J.N.), Burroughs Wellcome Fund Career Awards for Medical Scientists - 1020904 (J.N.) and NIH T32 AI007520 (S.F.).

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L., Boudeau, J., Barnich, N., Perruchot, M. H., Colombel, J. F., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherent+invasive+Escherichia+coli+strains+from+patients+with+Crohn's+disease+survive+and+replicate+within+macrophages+without+inducing+host+cell+death.">Adherent invasive Escherichia coli strains from patients with Crohn's disease survive and replicate within macrophages without inducing host cell death.</a> Infect Immun. 69 (9), 5529-5537 (2001).</li><li>Bringer, M. A., Billard, E., Glasser, A. L., Colombel, J. F., Darfeuille-Michaud, A. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+Crohn's+disease-associated+AIEC+within+macrophages+is+dependent+on+TNF-alpha+secretion.">Replication of Crohn's disease-associated AIEC within macrophages is dependent on TNF-alpha secretion.</a> Lab Invest. 92 (3), 411-419 (2012).</li><li>Ristuccia, A. M., Cunha, B. A. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+amikacin.">An overview of amikacin.</a> Ther Drug Monit. 7 (1), 12-25 (1985).</li><li>Sambuy, Y., De Angelis, I., Ranaldi, G., Scarino, M. L., Stammati, A., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caco-2+cell+line+as+a+model+of+the+intestinal+barrier:+influence+of+cell+and+culture-related+factors+on+Caco-2+cell+functional+characteristics.">The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics.</a> Cell Biol Toxicol. 21 (1), 1-26 (2005).</li><li>Vllasaliu, D., Falcone, F. H., Stolnik, S., Garnett, M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membrane+influences+intestinal+epithelial+cell+growth+and+presents+a+barrier+to+the+movement+of+macromolecules.">Basement membrane influences intestinal epithelial cell growth and presents a barrier to the movement of macromolecules.</a> Exp Cell Res. 323 (1), 218-231 (2014).</li><li>Wine, E., Ossa, J. C., Gray-Owen, S. D., Sherman, P. M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherent-invasive+Escherichia+coli,+strain+LF82+disrupts+apical+junctional+complexes+in+polarized+epithelia.">Adherent-invasive Escherichia coli, strain LF82 disrupts apical junctional complexes in polarized epithelia.</a> BMC Microbiol. 9, 180 (2009).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+cell+membranes.">Permeabilization of cell membranes.</a> Methods Mol Biol. 588, 63-66 (2010).</li><li>Delmas, J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+adaptation+of+adherent-invasive+Escherichia+coli+to+exposure+to+bile+salts.">Metabolic adaptation of adherent-invasive Escherichia coli to exposure to bile salts.</a> Sci Rep. 9 (1), 2175 (2019).</li><li>Zhang, S., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+metabolites+fuel+the+growth+and+virulence+of+E.+coli+linked+to+Crohn's+disease.">Mucosal metabolites fuel the growth and virulence of E. coli linked to Crohn's disease.</a> JCI Insight. 7 (10), (2022).</li><li>Viennois, E., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+emulsifiers+directly+impact+adherent-invasive+E.+coli+gene+expression+to+drive+chronic+intestinal+inflammation.">Dietary emulsifiers directly impact adherent-invasive E. coli gene expression to drive chronic intestinal inflammation.</a> Cell Rep. 33 (1), 108229 (2020).</li><li>Chassaing, B., Etienne-Mesmin, L., Bonnet, R., Darfeuille-Michaud, A. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+salts+induce+long+polar+fimbriae+expression+favouring+Crohn's+disease-associated+adherent-invasive+Escherichia+coli+interaction+with+Peyer's+patches.">Bile salts induce long polar fimbriae expression favouring Crohn's disease-associated adherent-invasive Escherichia coli interaction with Peyer's patches.</a> Environ Microbiol. 15 (2), 355-371 (2013).</li><li>Gotoh, K., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bile+acid-induced+virulence+gene+expression+of+Vibrio+parahaemolyticus+reveals+a+novel+therapeutic+potential+for+bile+acid+sequestrants.">Bile acid-induced virulence gene expression of Vibrio parahaemolyticus reveals a novel therapeutic potential for bile acid sequestrants.</a> PLoS One. 5 (10), e13365 (2010).</li><li>Bajunaid, W., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+T3SS+of+Shigella:+Expression,+structure,+function,+and+role+in+vacuole+escape.">The T3SS of Shigella: Expression, structure, function, and role in vacuole escape.</a> Microorganisms. 8 (12), (2020).</li><li>Poore, K., Lenneman, B. 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A., Sateriale, A. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+mechanisms+of+epithelial+cell+structure+manipulation+by+intestinal+pathogens.">Convergent mechanisms of epithelial cell structure manipulation by intestinal pathogens.</a> PLoS Pathog. 21 (7), e1013367 (2025).</li><li>Jaishankar, J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+Vibrio+parahaemolyticus+LPS+to+mediate+evasion+of+host+immune+response+during+infection.">A critical role for Vibrio parahaemolyticus LPS to mediate evasion of host immune response during infection.</a> Proc Natl Acad Sci U S A. 122 (33), e2426547122 (2025).</li><li>Cieza, R. J., Hu, J., Ross, B. N., Sbrana, E., Torres, A. 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The authors declare no conflicts of interest.

The amikacin protection assay is a modified approach for assessing&#160;E. coli&#160;invasion into epithelial cells. Several steps are critical for the success of this assay, including collagen coating of the plates, maintaining monolayer integrity, and efficient cell lysis. Caco-2 cells are an adherent cell line that can attach to tissue-culture-treated plastic and form monolayers20. However, during infection, the use of serum-free media and repeated washing can influence cell detachment, leading to an under-representation of bacterial invasion21. To troubleshoot this, coating plates with extracellular matrix components, such as type IV collagen, can promote cell attachment and the formation of a stable, confluent monolayer during infection21. This is important as the AIEC strain LF82 has been shown to disrupt tight junctions during invasion, and incomplete monolayer formation can bypass this step, leading to skewed results22. Lastly, efficient cell lysis is another key step that affects bacterial recovery, as incomplete lysis could mask invasiveness, confounding assay outcomes23. To circumvent this, it is important to thoroughly resuspend the cell lysate after the incubation with Triton-X-100.
In addition to the technical challenges that could contribute to invasion assay variability, the percent of inoculum recovered post-infection for AIEC strain LF82 has been shown to range between 0.61 and 17.25%8. This variability could be influenced by the absence of certain environmental cues, such as bile salts, mucin, and other intestinal metabolites in this in vitro assay that are known to contribute to AIEC virulence24,25,26. Specifically, bile salts have been shown to induce the expression of long polar fimbriae, which increase AIEC attachment and translocation across M cells27. Other gastrointestinal pathogens, such as Vibrio and Shigella, are known to regulate their type III secretion systems in response to bile to mediate epithelial cell invasion28,29. Subculturing with bile salts to induce virulence factor expression is standard in gentamicin protection protocols for other pathogens, and growing AIEC with these environmental cues may stabilize invasion and yield more consistent profiles30,31.
Another limitation of this assay is that bacterial invasion is assessed at a single time-point, which, although standard in the field, likely oversimplifies the invasion process12. Bacterial invasion is a dynamic process involving adherence, internalization, intracellular replication, and escape from the host cell32. Because novel genes contributing to AIEC invasion have been challenging to identify, the use of a single time-point assay could underrepresent the number of genes contributing to this process12. Time-course invasion assays, therefore, have been used to assess how specific genes contribute to invasion and to study AIEC replication in I-407 and Hep-2 cells8,33. While the use of a time course invasion assay is valuable for characterizing the contribution of individual genes to invasion, it is labor-intensive, particularly when screening many clinical isolates. For this reason, a single time point is used to assess invasion.
In addition, the intracellular lifestyle of AIEC has not been very well characterized. Most studies use percent invasion as the primary readout to detect changes in CFU of various virulence factor deletion mutants14,34. Visualization of infected host cells can provide unique insights into AIEC biology that are missed when using only CFU enumeration35. Therefore, to comprehensively evaluate the role of different genes in cell invasion, performing immunofluorescence microscopy of infected cells should be done in tandem with CFU enumeration. This is important as it can lead to identifying genes that function in endosomal survival and escape, target the host&#39;s cytoskeleton, or exhibit differences in bacterial burden per cell, which could be overlooked using CFU recovery alone. It is important to note that using the immunofluorescence staining protocol described here to detect intracellular bacteria does not distinguish between truly internalized bacteria and those adhered to the cell surface. A differential staining approach, in which extracellular bacteria are labeled with a distinct secondary antibody prior to permeabilization and intracellular bacteria are stained following permeabilization, would address this limitation and could be implemented in future studies to more definitively distinguish these populations36.
The significance of this method largely lies in the ability to screen multidrug-resistant E. coli isolates from IBD patients that undergo diverse antibiotic treatment11. Gentamicin is used to treat patients with early-onset IBD and can be administered to treat other bacterial infections37. The emergence of gentamicin-resistant isolates would preclude the use of the standard assay, making amikacin a more reliable alternative11. Using the protocols outlined in this paper will allow for a more thorough characterization of AIEC infection biology and will help identify new genes that can contribute to invasion. These methods are important in the context of bacterial pathogenesis and host-pathogen interaction research, as they enable the discovery of novel virulence factors, the characterization of the bacterial intracellular life cycle, and the interrogation of host pathways targeted during infection. Additionally, they are broadly applicable to study other invasive pathogens using in vitro epithelial cell models.

Inflammatory bowel disease (IBD), which includes Crohn&#8217;s disease (CD)&#160; and ulcerative colitis,&#160;is a chronic gastrointestinal inflammatory condition that affects millions of people worldwide1. Despite the rising global incidence of IBD, its precise etiology remains unclear, owing to the complex interplay of genetic, environmental, microbial, and immunological factors2,3. A signature feature of IBD is altered gut microbial composition, which is often driven by environmental factors, such as a high-sugar, high-fat diet common in industrialized countries4,5. The microbiota of IBD patients is frequently characterized by reduced microbial diversity, enrichment of pathogenic Gammaproteobacteria, and depletion of protective Firmicutes6,7. In patients with&#160;CD,&#160;Escherichia coli&#160;(E. coli) is enriched in the gut microbiota, associates with and invades the intestinal mucosa, and positively correlates with active and severe disease8,9. This emerging pathotype of&#160;E. coli&#160;associated with CD is termed adherent-invasive Escherichia coli (AIEC).
AIEC are gram-negative opportunistic pathogens that colonize the ileal mucosa of CD patients at a greater propensity than healthy controls10. AIEC strains isolated from CD patients are genetically diverse, often spanning all E. coli phylogroups that include both commensal and pathogenic E. coli11. Unlike pathogens such as Shigella and Salmonella, which rely on the use of type III secretion systems to facilitate invasion of host cells, AIEC lack these conserved virulence systems12. Therefore, our understanding of AIEC virulence largely relies on its&#160;in vitro&#160;phenotype, which is characterized by its ability to adhere to and invade epithelial cells and survive within macrophages10.&#160;
Epithelial cell invasion by AIEC is mediated by multiple virulence factors, including the type 1 pilus and long polar fimbriae. FimH, located at the tip of the type 1 pilus, mediates adhesion to mannose residues on the heavily glycosylated host receptor CEACAM613. This binding promotes AIEC entry into enterocytes and supports its intracellular replication14. AIEC also adheres to microfold (M) cells, specialized epithelial cells located above Peyer&#39;s patches through interactions between its long polar fimbriae and the M cell surface protein glycoprotein 215,16. This adhesion enables AIEC to translocate across M cells, reach underlying macrophages, and trigger the expression of pro-inflammatory cytokines such as TNF-&#945;, which not only promotes inflammation but also supports bacterial replication within macrophages17,18. These pathogenic mechanisms are hypothesized to perpetuate chronic inflammation in the host, as observed in CD.
AIEC are defined by their in vitro phenotype, making the gentamicin protection assay indispensable for strain classification and virulence factor discovery8. This approach can be complicated by gentamicin-resistant isolates, which can result in their misclassification as invasive11. To circumvent this issue, alternative antibiotics that effectively kill gentamicin-resistant isolates without penetrating host cell membranes were tested. Through this approach, amikacin was identified as a suitable substitute for gentamicin, offering comparable efficacy and compatibility with standard invasion assays, as well as minimal cytotoxicity, as evidenced by trypan blue staining in previous studies and further validated here using a lactate dehydrogenase release assay (Supplementary Figure 1)11. Amikacin is a semisynthetic aminoglycoside derived from kanamycin that inhibits bacterial growth by disrupting protein synthesis19. Amikacin is particularly effective against multi-drug resistant gram-negative bacteria, including Pseudomonas, Acinetobacter, and Escherichia species19. Notably, structural modifications to the kanamycin backbone render amikacin less susceptible to bacterial aminoglycoside-inactivating enzymes19. As a result, amikacin represents a robust alternative to gentamicin for targeting multi-drug-resistant bacteria in invasion assays. The following protocols describe how to assess the invasion phenotype of E. coli using both the reference strain NRG857c and clinical isolates with the amikacin protection assay.

<table><tbody><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Tissue culture flask</td><td>Fisher Scientific&amp;nbsp;</td><td>FB012937</td><td></td></tr><tr><td>Amikacin disulfate&amp;nbsp;</td><td>Research Products International&amp;nbsp;</td><td>A20540-25.0</td><td></td></tr><tr><td>Antibiotic-Antimycotic (100x)</td><td>Gibco</td><td>15240062</td><td></td></tr><tr><td>Anti-E.coli antibody</td><td>Abcam</td><td>ab137967</td><td></td></tr><tr><td>BioLite microwell plates - 6 well</td><td>Fisher Scientific&amp;nbsp;</td><td>12-556-004</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>Collagen I, Rat tail, 100 mg</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Countess cell counting chamber slide</td><td>Invitrogen</td><td>C10312</td><td></td></tr><tr><td>DMEM, high glucose</td><td>Gibco</td><td>11965118</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Corning</td><td>35-011-CV</td><td></td></tr><tr><td>Galcial acetic acid</td><td>Sigma-Aldrich</td><td>A6283</td><td></td></tr><tr><td>Glycine ultrapure</td><td>USBiological</td><td>G8165</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488</td><td>Invitrogen</td><td>A-11008</td><td></td></tr><tr><td>Hoechst 33342, trihydrochloride trihydrate</td><td>Invitrogen</td><td>H3570</td><td></td></tr><tr><td>Liofilchem&amp;nbsp;MTS amikacin [AK] 0.016-256 &amp;mu;g/mL</td><td>Fisher Scientific&amp;nbsp;</td><td>22-777-704</td><td>Amikacin MIC Test Strips</td></tr><tr><td>Paraformaldehyde solution, 4% in PBS</td><td>Fisher Scientific&amp;nbsp;</td><td>J19943.K2</td><td></td></tr><tr><td>PBS, 1x</td><td>Corning</td><td>21-040-CV</td><td></td></tr><tr><td>Poly-L-lysine coated sterile German glass coverslips for cell culture</td><td>Neuvitro</td><td>NC1327375</td><td></td></tr><tr><td>ProLong gold antifade mountant&amp;nbsp;</td><td>Invitrogen</td><td>P36934</td><td></td></tr><tr><td>Rhodamine-Phalloidin</td><td>Invitrogen</td><td>R415</td><td>Red-orange 540/565 nm</td></tr><tr><td>Surface treated sterile tissue culture plates</td><td>Fisher Scientific&amp;nbsp;</td><td>FB012929</td><td>24-well plates</td></tr><tr><td>Triton X-100 (Electrophoresis)</td><td>Fisher Scientific&amp;nbsp;</td><td>BP151-100</td><td></td></tr><tr><td>Trypan blue</td><td>Invitrogen</td><td>T10282</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Gibco</td><td>25200056</td><td></td></tr><tr><td>Tween-20</td><td>Fisher Scientific&amp;nbsp;</td><td>BP337-100</td><td></td></tr></tbody></table>

amikacin protection assay for quantification and visualization of escherichia coli cell invasion

Amikacin Protection Assay for Quantification and Visualization of Escherichia coli Cell Invasion

Epithelial cell invasion is a key phenotype for many enteric pathogens, including Salmonella, Shigella, and Vibrio. An emerging invasive pathogen is adherent-invasive Escherichia coli (AIEC), which is implicated in the pathogenesis of inflammatory bowel disease (IBD). AIEC strains lack virulence factors commonly associated with other invasive pathogens, making genetic identification challenging. As a result, AIEC strains are classified phenotypically based on their ability to invade epithelial cells using a field-standard gentamicin protection assay. However, since AIEC strains are often isolated from IBD patients who receive various antibiotics as part of their treatment regimen, the rise of gentamicin-resistant isolates has made this assay less reliable for AIEC classification. To circumvent this issue, an alternative amikacin-based protection assay was developed to quantify and visualize AIEC cell invasion. This assay utilizes amikacin, a broad-spectrum semisynthetic aminoglycoside that effectively targets strains resistant to other aminoglycosides while remaining extracellular to eukaryotic host cells. Thus, extracellular bacteria are killed during infection while intracellular bacteria are &#34;protected&#34;. This forms the basis of a quantitative assay to compare strain invasiveness, measured as the percent invasion score using colony-forming-units (CFU) recovered post-infection. This assay can also be used as a qualitative measurement of invasion by immunostaining infected epithelial cells and visualizing these cells through confocal microscopy. This method serves as a diagnostic tool to screen IBD patients for AIEC that can also be applied to other multidrug-resistant pathogens to assess cell invasion, a proxy for virulence and pathogenicity.

To evaluate the effectiveness of amikacin treatment in eliminating extracellular bacteria and to visualize intracellular invasion, we performed infection experiments using Caco-2 cell monolayers were performed. As shown in Figure 1, treatment with either gentamicin or amikacin following infection with E. coli NRG857c (positive control) or E. coli DH5α (negative control) resulted in similar invasion profiles. These findings indicate that amikacin is as effective as gentamicin in eliminating extracellular bacteria and accurately reflects intracellular bacterial burden, supporting its use as an alternative antibiotic for invasion assays. A recovery range of ~4-10% recovery of NRG857c and &lt;1% for E. coli DH5α is considered acceptable for accurate interpretation of invasion phenotypes relative to mutant strains or other clinical isolates, consistent with previous observations11. If the percent invasion scores for the positive and negative controls are outside of this expected recovery range, the results should be interpreted with caution, and the assay should be repeated.
To demonstrate that amikacin can effectively target gentamicin-resistant clinical E. coli isolates, gentamicin and amikacin protection assays were performed in parallel, and the invasion profiles of the isolates were examined. As shown in Figure 2, treatment of gentamicin-resistant isolates with gentamicin resulted in highly variable recovery values, ranging from 21.52% to 2788.50%. In contrast, treatment with amikacin dramatically reduced recovery across all isolates, highlighting its effectiveness and enabling more accurate and reliable invasion measurements. 
Finally, to assess the intracellular phenotype of AIEC during invasion, Caco-2 cells were infected with E. coli NRG857c. Figure 3 shows a representative image of an infected Caco-2 cell with bacteria (green) stained by immunofluorescence, host actin (red), and DNA (blue) visualized by counterstaining with rhodamine-phalloidin and Hoechst, respectively. At 4 h post-infection, NRG857c is observed replicating in the cytoplasm, localizing near the nucleus and within actin boundaries, which confirms the intracellular nature of these bacteria.  
<img alt="Bar graph comparing bacterial strain invasion percentages with Gentamicin and Amikacin treatment." class="xfigimg" src="/files/ftp_upload/71108/71108fig1.jpg" data-altupdatedat="1787025648000" data-alt="Figure 1"> 
Figure 1: Addition of gentamicin or amikacin to Caco-2 cells infected with AIEC strain NRG857c result in comparable invasion profiles. Caco-2 cell monolayers were infected with AIEC strain NRG857c and E. coli DH5α at an MOI of 10. After 3 hours of infection, cells were treated for 1 h with gentamicin (100 µg/mL) or amikacin (240 µg/mL) to eliminate extracellular bacteria. Cells were then lysed with 1% Triton-X-100, and homogenates were serially diluted and plated on LB agar for CFU enumeration. Invasion was expressed as the percentage of recovered bacteria relative to the initial inoculum. Data represent three independent experiments (n = 3); error bars indicate standard deviation of the mean. <a href="https://www.jove.com/files/ftp_upload/71108/71108fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Gentamicin and Amikacin strain invasion bar chart; antibiotic effect comparison on bacterial strains." class="xfigimg" src="/files/ftp_upload/71108/71108fig2.jpg" data-altupdatedat="1787025648000" data-alt="Figure 2"> 
Figure 2: Amikacin effectively targets gentamicin-resistant E. coli strains. Caco-2 cell monolayers were infected with E. coli strains isolated from IBD patients at an MOI of 10. After 3 h of infection, cells were treated for 1 h with gentamicin (100 µg/mL) or amikacin (240 µg/mL) to eliminate extracellular bacteria. Cells were then lysed with 1% Triton-X-100, and homogenates were serially diluted and plated on LB agar for CFU enumeration. Invasion was expressed as the percentage of recovered bacteria relative to the initial inoculum. Data represent three independent experiments (n = 3); error bars indicate standard deviation of the mean. <a href="https://www.jove.com/files/ftp_upload/71108/71108fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy of cells; nucleus blue, cytoskeleton red, proteins green; cellular imaging." class="xfigimg" src="/files/ftp_upload/71108/71108fig3.jpg" data-altupdatedat="1787025648000" data-alt="Figure 3"> 
Figure 3: Visualization of epithelial cell invasion by E. coli. Representative confocal micrographs of Caco-2 cells infected E. coli NRG857c. Caco-2 cells were infected for 3 h followed by 1 h incubation with amikacin (240 µg/mL). Host actin was stained with rhodamine-Phallodin (red), and host and bacterial DNA were stained with Hoechst (blue). E. coli (green) was detected using an anti-rabbit E. coli primary antibody and a goat anti-rabbit Alexa Fluor 488 secondary antibody. Images were acquired on a 63× objective with a 3× zoom using a Leica confocal microscope. Scale bars = 10 µm. <a href="https://www.jove.com/files/ftp_upload/71108/71108fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bacterial infection process diagram; centrifugation, colony counting, CFU/mL calculation, microscopy." class="xfigimg" src="/files/ftp_upload/71108/71108fig4v2.jpg" data-altupdatedat="1787025648000" data-alt="Figure 4"> 
Figure 4: Flow chart of the study. (A) AIEC strains were grown overnight, washed, and added to Caco-2 monolayers at an MOI of 10. After 3 h of infection and 1 h of amikacin treatment, intracellular bacteria were recovered and quantified by plating serial dilutions in triplicate. (B) Infected monolayers were fixed, stained with antibodies, and imaged using fluorescence microscopy. <a href="https://www.jove.com/files/ftp_upload/71108/71108fig4largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Amikacin treatment results in minimal cytotoxicity in epithelial cells. Caco-2 cells were treated with gentamicin (100 µg/mL) or amikacin (240 µg/mL) for 1 h. Cytotoxicity was determined by measuring the lactate dehydrogenase released into the culture medium. Error bars indicate standard deviation of the mean (n = 3).<a href="https://www.jove.com/files/ftp_upload/71108/Supplementary%20Figure%201%20-%20JoVE.pdf" target="_blank">Please click here to download this file.</a>

Antibiotic resistance amongst adherent-invasive Escherichia coli strains poses challenges for assessing bacterial invasion using the traditional gentamicin protection assay. An improved method to evaluate bacterial invasion quantitatively by enumerating colony-forming units and qualitatively through visualization of infected cells by immunofluorescence confocal microscopy is described.

Epithelial cell invasion is a key phenotype for many enteric pathogens, including Salmonella, Shigella, and Vibrio. An emerging invasive pathogen is ...

We focus on characterizing how adherent invasive E.coli invade intestinal epithelial cells and identifying bacterial factors that contribute to this process. Gentamycin-resistant isolates limit traditional invasion assays. This amikacin-based protocol allows for accurate quantification of intracellular bacteria.To begin, gather all reagents and media required for the invasion assay. Next, retrieve the frozen glycerol stock of adherent-invasive Escharicia colli, or AIEC, strain from the 80 degrees Celsius freezer. Using a sterile wooden stick or pipette tip, gently scrape bacteria from the stalk and streak it onto a Luria-Bertani, or LB, auger plate to obtain single colonies.Incubate the plate at 37 degrees Celsius overnight. The next day, using a sterile wooden stick or pipette tip, pick a single colony from the auger plate. Inoculate the colony into 3 milliliters of LB broth and incubate the culture at 37 degrees Celsius at 200 revolutions per minute overnight.Then perform a 1 to 10 dilution of the overnight culture in 1 milliliter of LB broth. Measure the optical density at 600 nanometers of the diluted culture using a spectrophotometer. Transfer 100 microliters of the overnight culture into a 1.5 milliliter microcentrifuge tube and centrifuge at 6, 000 G for 10 minutes at 4 degrees Celsius.Aspirate the supernatant carefully without disturbing the cell pellet and re-suspend the pellet in 1 milliliter of serum and antibiotic-antimycotic-free media. Calculate the required volume of bacterial suspension to achieve a multiplicity of infection or MOI of 10 using the provided formula. To prepare the infection media, transfer the calculated volume of bacterial suspension into a sterile 1.5 milliliter microcentrifuge tube and make up the volume to 1 milliliter with serum and antibiotic-antimycotic-free media.Mix gently by pipetting up and down several times. Prepare a 96-well plate to determine the colony forming units per milliliter of the diluted overnight bacterial culture by performing tenfold serial dilutions in sterile 1X PBS. Transfer 200 microliters of the diluted overnight culture into the first well of the plate.Pipette 20 microliters of the culture into the next row containing 180 microliters of 1X PBS. Mix by pipetting up and down 10 times and repeat the dilution process until reaching 10 to the power of 6 using a fresh pipette tip each time. Now plate 10 microliters of spot dilutions in triplicate from the 10 to the power of 2 to 10 to the power of 6 dilutions onto LB auger plates.Allow the spots to absorb into the auger near a flame. Incubate the plates overnight at 27 to 30 degrees Celsius to prevent colony overgrowth and obtain clear individual colonies. Remove the healthy confluent Caco-2 epithelial cell monolayers cultured in a collagen coated 24-well culture plate from the incubator and aspirate the complete media from each well.Pipette one milliliter of serum and antibiotic-antimycotic-free media into each well. Aspirate the media to remove traces of media and repeat the wash one more time for a total of two washes. Now add 1 milliliter of the previously prepared infection media containing AIEC into each well of the 24-well plate containing the Caco-2 cells.Centrifuge the 24-well plate at 500G for 15 minutes at room temperature to synchronize bacterial contact with the epithelial monolayer and incubate the plate at 37 degrees Celsius with 5%carbon dioxide for three hours to allow bacterial invasion to occur. Prepare antibiotic-containing media by adding 240 micrograms per milliliter of amikacin to pre-warmed serum and antibiotic-antimycotic-free media. After the infection period, aspirate the media from the Caco-2 cell monolayers and wash each well with 1 milliliter of serum and antibiotic-antimycotic-free media.Aspirate the media and repeat the wash once more for a total of two washes. Then pipette one milliliter of 240 micrograms per milliliter amikacin containing media into each well. In parallel, treat another set of cells with 100 micrograms per milliliter gentamicin for comparison and incubate the cells at 37 degrees Celsius with 5%carbon dioxide for 1 hour to eliminate extracellular bacteria.After incubation, aspirate the media from each well. Add 1 milliliter of PBS to wash away adherent bacteria. Aspirate the PBS and repeat the wash one more time for a total of two washes.Now add 200 microliters of 1%Triton X100 diluted in PBS into each well and incubate on an orbital shaker at 90 revolutions per minute for 10 minutes to lyse epithelial cells. Pipette up and down approximately 30 times per well to detach the cells from the plate. Next, perform tenfold serial dilutions of the cell lysates in a 96-well plate using PBS.Plate 10 microliters of spot dilutions in triplicate from the undiluted to 10 to the power of 3 dilutions onto LB auger plates. After the spots dry, incubate the plates overnight at 27 to 30 degrees Celsius to obtain clear individual colonies. Following incubation, remove the plates and count well-isolated colonies from the appropriate spot dilutions containing more than seven colonies per spot across the triplicates.Finally, calculate the inoculum and retrieved colony-forming units per milliliter and percent invasion using the equations. Treatment with either gentamicin or amikacin following infection of Caco-2 cell monolayers with the invasive AIEC strain NRG857c, or the non-invasive Escherichia coli DH5 alpha control strain resulted in similar invasion profiles. These findings indicated that amikacin was as effective as gentamicin in eliminating extracellular bacteria and accurately reflected intracellular bacterial burden.Treatment of gentamicin-resistant clinical Escherichia coli isolates obtained from patients with inflammatory bowel disease with gentamicin resulted in highly variable bacterial recovery values ranging from around 21 to 2, 788%In contrast, amikacin treatment dramatically reduced bacterial recovery across all isolates, demonstrating its effectiveness and enabling more reliable invasion measurements. This protocol enables accurate quantification of intracellular bacteria and assessment of bacterial invasion in epithelial cells. Additional methods such as immunofluorescence microscopy can be used to further validate bacterial invasion and validate intracellular localization.Future studies can identify novel virulence factors and investigate host pathways that are targeted during adherent invasive E.coli infection.

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