Name: Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung (Video) | JoVE Journal
Uploaded: 2012-06-05T12:00:00.000+00:00
Duration: 7 min 46 s
Description: Learn to test antibiotic efficacy against Pseudomonas aeruginosa biofilms using artificial sputum medium that mimics cystic fibrosis lung conditions. This method reveals higher resistance than planktonic cultures.

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We acknowledge the support of the United Kingdom National Institute for Health Research, the Dr Hadwen Trust for Humane Research, the UK's leading medical research charity funding exclusively non-animal research techniques to replace animal experiments, and the Wellcome Trust (Grant 089215). We also acknowledge Novartis Pharmaceuticals UK Ltd (unrestricted educational grant).

<ol><li>Andrews, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BSAC+standardized+disc+susceptibility+testing+method+%28version+8%29%5BTitle%5D)+AND+%22J.+Antimicrob.+Chemother%22%5BJournal%5D)">BSAC standardized disc susceptibility testing method (version 8).</a> J. Antimicrob. Chemother. 64, 454-489 (2009).</li>
<li>Worlitzsch, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+reduced+mucus+oxygen+concentration+in+airway+Pseudomonas+infections+of+cystic+fibrosis+patients%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.</a> J. Clin. Invest. 109, 317-325 (2002).</li>
<li>Smith, A. L., Fiel, S. B., Mayer-Hamblett, N., Ramsey, B., Burns, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Susceptibility+testing+of+Pseudomonas+aeruginosa+isolates+and+clinical+response+to+parenteral+antibiotic+administration%3A+lack+of+association+in+cystic+fibrosis%5BTitle%5D)+AND+%22Chest%22%5BJournal%5D)">Susceptibility testing of Pseudomonas aeruginosa isolates and clinical response to parenteral antibiotic administration: lack of association in cystic fibrosis.</a> Chest. 123, 1495-1502 (2003).</li>
<li>Sriramulu, D. D., Lunsdorf, H., Lam, J. S., Romling, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microcolony+formation%3A+a+novel+biofilm+model+of+Pseudomonas+aeruginosa+for+the+cystic+fibrosis+lung%5BTitle%5D)+AND+%22J.+Med.+Microbiol%22%5BJournal%5D)">Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung.</a> J. Med. Microbiol. 54, 667-676 (2005).</li>
<li>Garbe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+JG024%2C+a+pseudomonas+aeruginosa+PB1-like+broad+host+range+phage+under+simulated+infection+conditions%5BTitle%5D)+AND+%22BMC+Microbiol%22%5BJournal%5D)">Characterization of JG024, a pseudomonas aeruginosa PB1-like broad host range phage under simulated infection conditions.</a> BMC Microbiol. 10, 301(2010).</li>
<li>Naughton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa+AES-1+exhibits+increased+virulence+gene+expression+during+chronic+infection+of+cystic+fibrosis+lung%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Pseudomonas aeruginosa AES-1 exhibits increased virulence gene expression during chronic infection of cystic fibrosis lung.</a> PLoS One. 6, e24526(2011).</li>
<li>Moskowitz, S. M., Foster, J. M., Emerson, J. C., Gibson, R. L., Burns, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+Pseudomonas+biofilm+susceptibilities+to+assign+simulated+antibiotic+regimens+for+cystic+fibrosis+airway+infection%5BTitle%5D)+AND+%22J.+Antimicrob.+Chemother%22%5BJournal%5D)">Use of Pseudomonas biofilm susceptibilities to assign simulated antibiotic regimens for cystic fibrosis airway infection.</a> J. Antimicrob. Chemother. 56, 879-886 (2005).</li>
<li>Field, T. R., White, A., Elborn, J. S., Tunney, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+oxygen+limitation+on+the+in+vitro+antimicrobial+susceptibility+of+clinical+isolates+of+Pseudomonas+aeruginosa+grown+planktonically+and+as+biofilms%5BTitle%5D)+AND+%22Eur.+J.+Clin.+Microbiol.+Infect.+Dis%22%5BJournal%5D)">Effect of oxygen limitation on the in vitro antimicrobial susceptibility of clinical isolates of Pseudomonas aeruginosa grown planktonically and as biofilms.</a> Eur. J. Clin. Microbiol. Infect. Dis. 24, 677-687 (2005).</li>
<li>Bjarnsholt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa+biofilms+in+the+respiratory+tract+of+cystic+fibrosis+patients%5BTitle%5D)+AND+%22Pediatr.+Pulmonol%22%5BJournal%5D)">Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.</a> Pediatr. Pulmonol. 44, 547-558 (2009).</li>
<li>Hill, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibiotic+susceptibilities+of+Pseudomonas+aeruginosa+isolates+derived+from+patients+with+cystic+fibrosis+under+aerobic%2C+anaerobic%2C+and+biofilm+conditions%5BTitle%5D)+AND+%22J.+Clin.+Microbiol%22%5BJournal%5D)">Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.</a> J. Clin. Microbiol. 43, 5085-5090 (2005).</li>
<li>Fung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+of+Pseudomonas+aeruginosa+in+a+mucin-containing+synthetic+growth+medium+mimicking+cystic+fibrosis+lung+sputum%5BTitle%5D)+AND+%22J.+Med.+Microbiol%22%5BJournal%5D)">Gene expression of Pseudomonas aeruginosa in a mucin-containing synthetic growth medium mimicking cystic fibrosis lung sputum.</a> J. Med. Microbiol. 59, 1089-1100 (2010).</li>
<li>Andrews, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BSAC+standardized+disc+susceptibility+testing+method+%28version+8%29%5BTitle%5D)+AND+%22The+Journal+of+antimicrobial+chemotherapy%22%5BJournal%5D)">BSAC standardized disc susceptibility testing method (version 8).</a> The Journal of antimicrobial chemotherapy. 64, 454-489 (2009).</li>
<li>Rybtke, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+implication+of+Pseudomonas+aeruginosa+biofilms+in+infections%5BTitle%5D)+AND+%22Inflamm+Allergy+Drug+Targets%22%5BJournal%5D)">The implication of Pseudomonas aeruginosa biofilms in infections.</a> Inflamm Allergy Drug Targets. 10, 141-157 (2011).</li>
<li>Moreau-Marquis, S., Stanton, B. A., O'Toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa+biofilm+formation+in+the+cystic+fibrosis+airway%5BTitle%5D)+AND+%22Pulm.+Pharmacol.+Ther%22%5BJournal%5D)">Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway.</a> Pulm. Pharmacol. Ther. 21, 595-599 (2008).</li>
<li>Ahmed, S. A., Gogal, R. M., Walsh, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+rapid+and+simple+non-radioactive+assay+to+monitor+and+determine+the+proliferation+of+lymphocytes%3A+an+alternative+to+%5B3H%5Dthymidine+incorporation+assay%5BTitle%5D)+AND+%22J.+Immunol.+Methods%22%5BJournal%5D)">A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay.</a> J. Immunol. Methods. 170, 211-224 (1994).</li>
<li>Fothergill, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+antibiotic+treatment+on+bacteriophage+production+by+a+cystic+fibrosis+epidemic+strain+of+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Antimicrob.+Agents+Chemother%22%5BJournal%5D)">Effect of antibiotic treatment on bacteriophage production by a cystic fibrosis epidemic strain of Pseudomonas aeruginosa.</a> Antimicrob. Agents Chemother. 55, 426-428 (2011).</li>
<li>Singh, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quorum-sensing+signals+indicate+that+cystic+fibrosis+lungs+are+infected+with+bacterial+biofilms%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms.</a> Nature. 407, 762-764 (2000).</li>
<li>Mowat, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+Aeruginosa+Population+Diversity+and+Turnover+in+Cystic+Fibrosis+Chronic+Infections%5BTitle%5D)+AND+%22Am.+J.+Respir.+Crit.+Care.+Med%22%5BJournal%5D)">Pseudomonas Aeruginosa Population Diversity and Turnover in Cystic Fibrosis Chronic Infections.</a> Am. J. Respir. Crit. Care. Med. , (2011).</li>
<li>Fothergill, J. L., Mowat, E., Ledson, M. J., Walshaw, M. J., Winstanley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluctuations+in+phenotypes+and+genotypes+within+populations+of+Pseudomonas+aeruginosa+in+the+cystic+fibrosis+lung+during+pulmonary+exacerbations%5BTitle%5D)+AND+%22J.+Med.+Microbiol%22%5BJournal%5D)">Fluctuations in phenotypes and genotypes within populations of Pseudomonas aeruginosa in the cystic fibrosis lung during pulmonary exacerbations.</a> J. Med. Microbiol. 59, 472-481 (2010).</li>
<li>Fouhy, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diffusible+signal+factor-dependent+cell-cell+signaling+and+virulence+in+the+nosocomial+pathogen+Stenotrophomonas+maltophilia%5BTitle%5D)+AND+%22J.+Bacteriol%22%5BJournal%5D)">Diffusible signal factor-dependent cell-cell signaling and virulence in the nosocomial pathogen Stenotrophomonas maltophilia.</a> J. Bacteriol. 189, 4964-4968 (2007).</li>
<li>Winstanley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Newly+introduced+genomic+prophage+islands+are+critical+determinants+of+in+vivo+competitiveness+in+the+Liverpool+Epidemic+Strain+of+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa.</a> Genome Res. 19, 12-23 (2009).</li>
<li>Carter, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+subtype+of+a+Pseudomonas+aeruginosa+cystic+fibrosis+epidemic+strain+exhibits+enhanced+virulence+in+a+murine+model+of+acute+respiratory+infection%5BTitle%5D)+AND+%22J.+Infect.+Dis%22%5BJournal%5D)">A subtype of a Pseudomonas aeruginosa cystic fibrosis epidemic strain exhibits enhanced virulence in a murine model of acute respiratory infection.</a> J. Infect. Dis. 202, 935-942 (2010).</li>
</ol>

No conflicts of interest declared.

In this study we used a novel in vitro model based on ASM to replicate P. aeruginosa biofilm conditions within the CF lung4. The model was modified successfully for small-scale, high-throughput testing of antimicrobial agents.
	The critical steps of this assay are:
	<ol>
		<li>Consistent preparation of the ASM media and maintaining sterility. We have devoted long hours to optimising the way in which each component is added to achieve reproducible results every time. Filtration of the ASM is slow but is preferable to autoclaving, which may damage the mucin component. We do not advise the addition of antibiotics, as suggested by others 11 because this may impose considerable selective pressures, drive mutations, induce prophage lysis 16 and significantly alter the expression of multiple bacterial genes.</li>
	 <li>The assay should be optimised according to the volume of ASM used. Shaking speeds are increased for smaller volumes and a shorter biofilm life-cycle is observed.</li>
	</ol>
	An obvious application of the small-scale ASM biofilm model is the more realistic determination of biofilm antimicrobial susceptibilities (BSMIC90). Anaerobic and microaerophilic niches are present in the CF lung and there is evidence that oxygen is limited deep within mature biofilms2, 17. Here we demonstrate that 10/14 clinical P. aeruginosa isolates from CF patient sputa exhibit a considerable (4 - &ge; 128 fold) decrease in sensitivity to tobramycin under microaerophilic conditions in ASM. The results of this study suggest that antibiotics, such as tobramycin, might be less effective against P. aeruginosa infections in the CF lung than indicated by conventional susceptibility testing methods. These results reflect previous studies on the antimicrobial susceptibility of biofilms 10. Small-scale ASM assays thus provide a simple high throughput platform for generating meaningful antibiotic susceptibility data to better inform therapeutic decisions. The assay is limited in the same way as conventional antibiotic susceptibility testing in that single colonies are picked for screening that may not be representative of the whole population. However, we believe that an approach (i) using non-surface attached biofilm growth and (ii) applicable to microaerophilic conditions, represents a clear alternative and a potential improvement to existing methods. We conclude that this assay is an appropriate model to study P. aeruginosa biofilm populations. Further testing in clinical settings would ascertain whether antibiotic susceptibilities based on biofilm-grown P. aeruginosa could lead to different antibiotic choices with potentially improved microbiological and clinical outcomes. Similar investigations using classic biofilm models have shown that BSMIC values lead to different recommendations for antibiotic treatment5,17.
	In addition to testing for the effectiveness of anti-infective agents, the ASM system represents a cheap, simple and reproducible alternative to animal models for studies such as those aimed at understanding the diversification of P. aeruginosa populations. We have observed extensive heterogeneity in natural populations of P. aeruginosa recovered from CF patient sputa 18, 19. Similar phenotypic and genotypic diversification can be observed during growth in ASM4 (and our unpublished data), making it an attractive in vitro model of the CF lung conditions. The relative simplicity of the ASM model makes it easy to design long-term adaptation experiments aimed, for example, at monitoring the effects of antibiotics or other stresses on P. aeruginosa population divergence. In addition, other bacterial pathogens can be grown in ASM. For example, Fouhy et al. 2007 have used ASM to study biofilm formation by S. maltophillia20.

<table><tbody><tr><td>DNA from fish sperm</td><td>Sigma-Aldrich</td><td>74782</td><td></td></tr><tr><td>Mucin from porcine stomach, type II</td><td>Sigma-Aldrich</td><td>M2378</td><td></td></tr><tr><td>L-Alanine</td><td>Acros Organics</td><td>102830250</td><td></td></tr><tr><td>L-Arginine</td><td>Sigma-Aldrich</td><td>A5006</td><td></td></tr><tr><td>L(+)-Asparagine monohydrate</td><td>Acros Organics</td><td>175271000</td><td></td></tr><tr><td>L(+)-Aspartic acid</td><td>Acros Organics</td><td>105041000</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma-Adrich</td><td>168149</td><td></td></tr><tr><td>L(+)-Glutamic acid</td><td>Acros Organics</td><td>156211000</td><td></td></tr><tr><td>L-Glutamine</td><td>Sigma-Aldrich</td><td>G3126</td><td></td></tr><tr><td>Glycine</td><td>Acros Organics</td><td>220911000</td><td></td></tr><tr><td>L-Histidine</td><td>Sigma-Adrich</td><td>H8000</td><td></td></tr><tr><td>L-Isoleucine</td><td>Sigma-Aldrich</td><td>I2752</td><td></td></tr><tr><td>L-Leucine</td><td>Sigma-Aldrich</td><td>L8000</td><td></td></tr><tr><td>L(+)-Lysine monohydrochloride</td><td>Acros Organics</td><td>125222500</td><td></td></tr><tr><td>L-Methionine</td><td>Sigma-Aldrich</td><td>M9625</td><td></td></tr><tr><td>L-Phenylalanine</td><td>Acros Organics</td><td>130310250</td><td></td></tr><tr><td>L-Proline</td><td>Sigma-Aldrich</td><td>P0380</td><td></td></tr><tr><td>L-Serine</td><td>Acros Organics</td><td>132660250</td><td></td></tr><tr><td>L-Threonine</td><td>Acros Organics</td><td>138930250</td><td></td></tr><tr><td>L(-)-Tryptophan</td><td>Acros Organics</td><td>140590250</td><td></td></tr><tr><td>L-Tyrosine</td><td>Acros Organics</td><td>140641000</td><td></td></tr><tr><td>L-Valine</td><td>Sigma-Aldrich</td><td>V0500</td><td></td></tr><tr><td>Diethylenetriaminepentaacetic acid</td><td>Sigma-Aldrich</td><td>32318</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S/3160/60</td><td></td></tr><tr><td>KCl</td><td>VWR</td><td>BDH0258</td><td></td></tr><tr><td>KOH</td><td>VWR</td><td>BDH0262</td><td></td></tr><tr><td>Egg yolk emulsion</td><td>Sigma-Aldrich</td><td>17148</td><td></td></tr><tr><td>ME 2 diaphragm vacuum pump</td><td>Vacuubrand</td><td>696126</td><td></td></tr><tr><td>Steritop filters (Pore size: 0.22 &amp;mu;m, Neck size: 45 mm)</td><td>EMD Millipore</td><td>SCGPT10RE</td><td></td></tr><tr><td>Luria-Bertani medium</td><td>Sigma-Aldrich</td><td>L2897</td><td></td></tr><tr><td>96-well microtitre plates</td><td>Sarstedt Ltd</td><td>82.1581</td><td></td></tr><tr><td>24-well tissue culture-treated plates</td><td>Iwaki, Asahi Techno Glass Corp.</td><td>3820-024</td><td></td></tr><tr><td>CampyGen gas generation packs</td><td>Oxford Labware</td><td>CN0025</td><td></td></tr><tr><td>Microaerophilic chamber</td><td>Oxford Labware</td><td>HP0011</td><td></td></tr><tr><td>Tobramycin sulphate</td><td>Sigma-Aldrich</td><td>T1783</td><td></td></tr><tr><td>Cellulase, from &lt;em&gt;Aspergillus niger&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>22178</td><td></td></tr><tr><td>Resazurin</td><td>Sigma-Aldrich</td><td>199303</td><td></td></tr><tr><td>Citrate.H&lt;sub&gt;2&lt;/sub&gt;0</td><td>VWR</td><td>BDH0288</td><td></td></tr><tr><td>Fluostar omega microplate reader</td><td>BMG-Labtech</td><td>SPECTROstar Omega</td><td></td></tr></tbody></table>

use of artificial sputum medium to test antibiotic efficacy against pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

The aim of this experiment is to assess the efficacy of antibiotics against pseudomonas Aerogen biofilms, under conditions, representative of chronic infections in the cystic fibrosis lung using a defined artificial sputum medium. Bacterial biofilms are grown in 24. Well microtiter plates.The biofilms are then exposed to antibiotics. Next, antibiotic treated cultures are disrupted using cellulase and incubated with a metabolic dye srin, which is reduced from blue to pink by viable cells. By measuring the absorbance of the wells, the biofilm Cecile cell minimum inhibitory concentration of B-S-M-I-C can be determined to quantify antibiotic susceptibility under near physiological conditions.The main advantage of this technique over existing methods, such as the determination of minimum inhibitory concentrations based on planktonic culture, is that the bacteria grown in a specialized media containing all the components of sputum, thus promoting biofilm formation. These biofilms are much more resistant to antibiotics. To make the medium first prepare two bottles containing 250 milliliters of sterile water in the first bottle.Gradually add the stir and then add four grams of fish sperm, DNA to 250 milliliters of sterile water. Stir the solution at room temperature and allow the DNA to dissolve to the second bottle. Gradually add five grams of type two mucin from porcine stomach and stir.Once the DNA and mucin solutions are ready, dissolve the amino acids in 100 milliliters of sterile water, with the exception of L tyrosine and L cysteine, which should be dissolved in 25 milliliters of water, or 25 milliliters of 0.5 molar potassium hydroxide respectively. Next, makes 5.9 milligrams of DTPA, five grams of sodium chloride and 2.2 grams of potassium chloride in 100 milliliters of water. Combine all the different solutions in a one liter bottle, then add five milliliters of egg yolk emulsion, and top the mixture up to 850 milliliters with water.Using one molar triss, adjust the pH to 6.9 and bring the final volume up to one liter with water to sterilize the medium, filter it using a vacuum brand ME two diaphragm vacuum pump, and a Millipore stereo top filter unit with a 45 millimeter neck and 0.2 micron pore size. The filtration process can be performed over two days. The A SM medium can be stored at four degrees Celsius for up to one month.Begin by diluting overnight pseudomona arosa cultures in LB medium to obtain an OD of 0.05. Then further dilute the bacteria at one to 100 in a SM medium plate, 1.8 milliliters per well of diluted bacterial culture in 21 wells of a 24 well tissue culture plate aliquot the same volume of a SM medium in the three remaining wells, which will serve as blank negative controls. Secure the 24 well plate with biofilm, and to allow biofilms to form, place it under micro error fillic conditions, which can be obtained using campi gen gas generation packs in large anaerobic jars.Incubate the culture on a shaker set at 75 RPM for three days at 37 degrees Celsius after three days, biofilms will appear as a gelatinous mass containing bacterial cells and cannot be disrupted by pipetting. Once the biofilms are formed at 200 microliters per well of antibiotic at serially diluted concentrations. Be sure to set up at least four replicates for each drug concentration to be tested and leave four wells containing biofilms untreated so that they can be used as negative controls.Then incubate the platers previously for a further 24 hours following the incubation with antibiotics. Use 100 microliters per well of cellulase to diluted to 100 milligrams per milliliter in citrate buffer to disrupt the biofilms, incubate the plates on a shaker set at 150 RPM under aerobic conditions at 37 degrees Celsius for one hour. Following this incubation manual, pipetting may be used to further disaggregate the biofilms if clumps of cells are visible in the medium.Next, add 100 microliters of rezin per well to quantify the viability of the cells released from the biofilms and incubate the cells for one to two hours. At 37 degrees Celsius, only viable cells are able to metabolize the blue zarin dye to its pink fluorescent form. Following the incubation, transfer the samples into a 96 well plate and measure the fluorescence of the wells using a five 40 nanometer excitation wavelength and a five 90 nanometer emission wavelength.In order to quantify bacterial viability and drug susceptibility, first, subtract the background fluorescence of the blank control from the other wells. Next, calculate the percentage of inhibition by dividing the mean fluorescence for each concentration by the mean fluorescence of the untreated controls and multiplying by 100. The B-S-M-Y-C is defined as the antibiotic concentration causing a 90%inhibition of metabolic activity as shown in these electron micrographs cells, grown in biofilms differ considerably from bacteria grown in planktonic cultures.In particular, large amounts of extracellular matrix shown in yellow surround the cells in the biofilm in green, but not in planktonic colonies. In addition, bacteria in biofilms are typically more resistant to antibiotics in this experiment. The minimum inhibitory concentration of tobramycin for pseudomonas aerogen planktonic culture in blue was determined to be approximately four micrograms per milliliter.In contrast, as shown in red, 100%of bacteria in biofilms survived up to 512 micrograms per milliliter of antibiotic. After watching this video, you should have a good understanding of how to culture bacteria in a defined artificial sputum medium. You should also be able to perform an antibiotic susceptibility test to determine the minimum inhibitory concentration required to inhibit bacterial growth in environment more realistic at cystic fibrosis lung.

use-artificial-sputum-medium-to-test-antibiotic-efficacy-against

Research

JoVE Journal

Immunology and Infection

Source: Vieira, J., et al. Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome. J. Vis. Exp. (2021).This video demonstrates the preparation of an artificial sputum medium that mimics the viscosity, nutrient composition, and pH of cystic fibrosis lung sputum. The resulting medium provides a physiologically relevant platform for ex vivo studies.

Preparation of Artificial Sputum Medium to Emulate Cystic Fibrosis Lung Sputum Ex Vivo

Source: Jackson, L., et al. Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients. J. Vis. Exp. (2020)This video demonstrates the in situ visualization of Pseudomonas aeruginosa within chemically cleared cystic fibrosis sputum using fluorescent labeling and confocal microscopy to reveal bacterial localization and biofilm structure.

Visualization of Pseudomonas aeruginosa in Sputum Samples from Cystic Fibrosis Patients

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This workflow can be used to perform antibiotic susceptibility testing using an established 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.

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

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities.  Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

This protocol describes a cystic fibrosis (CF) lung-relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

This study outlines a framework for developing an aggregate polymicrobial biofilm model and optimizing an RNA extraction protocol to assess Pseudomonas aeruginosa gene expression reflective of the cystic fibrosis lung environment. Applications include evaluating antimicrobial effects and studying antibiotic alternatives under conditions relevant to cystic fibrosis.

Cystic Fibrosis Aggregate Biofilm Model to Study Infection-relevant Gene Expression

Learn to test antibiotic efficacy against Pseudomonas aeruginosa biofilms using artificial sputum medium that mimics cystic fibrosis lung conditions. This method reveals higher resistance than planktonic cultures.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung (Video) | JoVE Journal

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

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