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).

1. Preparation of Artificial Sputum Medium (ASM)
<ol>
 <li>Add 4 g DNA from fish sperm to 250 ml sterile water very slowly over a period of several hours. The DNA takes several hours to completely dissolve and can be stirred overnight at room temperature.</li>
 <li>Add 5 g mucin from porcine stomach (type II) slowly to 250 ml sterile water until the mucin has dissolved completely. The solution can be stirred overnight at 4 &deg;C.</li>
 <li>Dissolve 0.25 g of each essential and non-essential L-amino acid, with the exception of L-tyrosine and L-cysteine, in 100 ml sterile water. Dissolve 0.25 g of L-cysteine in 25 ml of 0.5 M potassium hydroxide (Mr 56.11 g/mol) and 0.25 g of L-tyrosine in 25 ml sterile water.</li>
 <li>Dissolve 5.9 mg diethylenetriaminepentaacetic acid (DTPA), 5 g NaCl and 2.2 g of KCl in 100 ml of sterile water.</li>
 <li>Combine the DNA, Mucin, L-amino acids, DTPA, NaCl and KCl in a 1 litre bottle.</li>
 <li>Add 5 ml of egg yolk emulsion and fill to approximately 850 ml with sterile water.</li>
 <li>Adjust pH to 6.9 with 1 M Tris (pH 8.5; Mr 121.14) and bring the volume to 1 litre with sterile water.</li>
 <li>Sterilize the ASM by filtration using a Vacuubrand ME 2 diaphragm vacuum pump and Millipore Steritop filter units with a pore and neck size of 0.22 &mu;m and 45 mm, respectively. Each Steritop filter unit can be re-used immediately up to three times; however, the filters need to be rinsed twice with sterile water before re-use. The filtration process is slow and can be performed over 2 days. Other versions of ASM have been developed that use the addition of antibiotics instead of filtration11 however, due to possible drug interactions, we do not recommend the method for this particular application.</li>
 <li>Unfiltered and filtered ASM should be stored at 4 &deg;C in the dark. Using fresh ASM is recommended however, it can be kept under these conditions for a maximum of one month.</li>
</ol>
2. Determination of the Planktonic Sessile Cell Minimum Metabolic Inhibitory Concentration (PSMIC)
<ol>
 <li>To determine the minimum metabolic inhibitory concentration (PSMIC) values for 15 planktonically grown P. aeruginosa isolates, the microdilution method should be performed, as described in the guidelines of the British Society for Antimicrobial Chemotherapy BSAC12. The antibiotic of choice, in this case tobramycin sulphate, is serially diluted in Luria-Bertani (LB) medium in a 96-well microtitre plate to provide an appropriate range of antibiotic concentrations.</li>
 <li>Dilute overnight cultures of P. aeruginosa in LB to an OD600 of 0.05 (&plusmn;0.01) and add 100 &mu;l volumes to the wells of the 96-well microtitre plate containing 100 &mu;l of the serially diluted antibiotic. In this case, the final concentrations of tobramycin sulphate ranged between 512 - 0.5 &mu;g/ml. Eight replicates of each antibiotic concentration should be performed.</li>
 <li>Negative control wells for each isolate should be set up, in which no antibiotic is added. Also, eight wells should contain only LB for use as a blank during downstream analysis (Section 2.6).</li>
 <li>Incubate the 96-well microtitre plates for 1 - 2 days at 37 &deg;C without shaking under aerobic or microaerophilic (5 % O2, 10 % CO2, and 85 % N2) conditions. Microaerophilic conditions are obtained using CampyGen gas generation packs in large anaerobic jars.</li>
 <li>Following incubation, bacterial growth is determined by measuring the absorbance of the bacterial culture in each well at a wavelength of 600 nm using a Fluostar Omega microplate reader and the MARS Data Analysis Software.</li>
 <li>Absorption from antibiotic-treated planktonic cultures (Aantibiotic treated planktonic cells) and absorption from the negative controls (Anegative control) should be corrected by subtraction of the background absorbance obtained from the wells containing LB only (Ablank). The percentage inhibition of viability is subsequently calculated as (mean Aantibiotic treated planktonic cells/mean Anegative control)x 100 %. The PSMIC90 is defined as the antibiotic concentration causing 90 % inhibition of planktonic bacterial growth.</li>
 <li>To determine bacterial viability after treatment with the antibiotic of choice, 10 &mu;l of 0.02 % (v/v) resazurin (diluted in distilled water) is added to each well and the plates are incubated under aerobic conditions for 1 - 2 h at 37 &deg;C, while shaking at 150 rpm. Viable cells will reduce the blue resazurin dye to the pink fluorescent resorufin form.</li>
 <li>Following incubation with resazurin, monitor the fluorescence of each well using an excitation wavelength of 540 nm and an emission wavelength of 590 nm in a Fluostar Omega microplate reader. The data should be analysed as described below.</li>
</ol>
3. Determination of Biofilm Sessile Cell Minimum Inhibitory Concentration (BSMIC)
<ol>
 <li>Overnight cultures of P. aeruginosa (in this case, 15 isolates of P. aeruginosa are used) should be diluted in LB to an OD600 of 0.05 (&plusmn;0.01), then further diluted 1:100 in fresh ASM (total volume 1.8 ml).</li>
 <li>The diluted cultures (1.8 ml) should be added to each well of a 24-well tissue culture treated plate. Three wells should contain ASM only for use as a blank during downstream analysis (Section 3.9).</li>
 <li>Secure the 24-well plates with laboratory parafilm and incubate for 3 days under aerobic or microaerophilic conditions at 37 &deg;C, while shaking at 75 rpm. Microaerophilic conditions should be obtained using CampyGen gas generation packs in large anaerobic jars.</li>
 <li>Dilute the antibiotic of choice, in this case tobramycin sulphate, to provide an appropriate concentration range in fresh ASM. In this instance, final concentrations ranged between 512 - 1 &mu;g/ml. Add each concentration of the antibiotic, in volumes of 200 &mu;l, to the appropriate wells of the 24-well plates. Four replicates of each antibiotic concentration should be performed. Biofilms not exposed to the antibiotic of choice were used as a negative control.</li>
 <li>Secure the 24-well plates with laboratory parafilm and incubate under aerobic or microaerophilic conditions for a further 24 h at 37 &deg;C, while shaking at 75 rpm.</li>
 <li>After incubation in the presence of the antibiotic of choice, disrupt the bacterial biofilms using 100 &mu;l of 100 mg/ml cellulase (diluted in 0.05 M citrate buffer [9.6 g/l Citrate.H20 in water and pH to 4.6 with NaOH]) and incubate the 24-well plates under aerobic conditions at 37 &deg;C, while shaking at 150 rpm for 1 h. If required, biofilms could be further disrupted by manual pipetting at this stage.</li>
 <li>To determine the metabolic activities of the bacterial cells released from the disrupted biofilms, 100 &mu;l of 0.02 % (v/v) resazurin (diluted in distilled water) should be added to each well of the 24-well plates and incubated for 1 - 2 h at 37 &deg;C, while shaking at 150 rpm.</li>
 <li>Following incubation with resazurin, measure the fluorescence of each well using an excitation wavelength of 540 nm and an emission wavelength of 590 nm in a Fluostar Omega microplate reader and the MARS Data Analysis Software.</li>
 <li>Fluorescence from the antibiotic-treated biofilms (Fantibiotic-treated biofilms) and fluorescence from the negative controls (Fnegative control) should be corrected by subtraction of the background fluorescence obtained from the wells containing ASM only (Fblank). The percentage inhibition of viability is subsequently calculated as (mean of Fantibiotic treated biofilms/mean Fnegative control) x 100 %. The BSMIC90 is defined as the antibiotic concentration causing 90 % inhibition of metabolic activity.</li>
</ol>
4. Representative Results
ASM biofilm formation is possible in small (2 ml) volumes and the biofilms are fully formed within 3 days (Figure 1A). This can be demonstrated by rigorously pipetting the biofilm, which should be difficult to disrupt. The microcolonies are comparable to those grown in larger volumes4 (Figure 1B). Figure 2 shows major differences between cells grown planktonically and in a biofilm as detected by electron microscopic image analysis. Biofilm cultures clearly show considerable levels of extracellular matrix surrounding the cells and individual structures within the biofilm are difficult to identify.
Several studies suggest that the biofilm lifestyle can affect antimicrobial susceptibility13, 14. Our small scale ASM assay can be used to determine the BSMIC of multiple antibiotics for multiple isolates at the same time. The workflow of the assay is shown in Figure 3. The effect of antibiotics on bacterial cell viability can be measured using the resazurin assay. Antibiotics, in this case tobramycin, can be added to the established biofilm and incubated for 24 h. After this the biofilm is disrupted and resazurin is added.
Metabolically active cells can reduce the resazurin dye resulting in a colour change from blue (resazurin) to pink (resorufin) 15. Figure 4A shows an example assay in which P. aeruginosa was incubated with different concentrations of tobramycin before biofilm disruption and addition of resazurin in a microtitre plate. The blue non-fluorescent colour indicates non-viable cells, whereas viable cells reduce the dye to the pink fluorescent form, resorufin. The SMIC can then be calculated by converting fluorescence into percentage remaining bacterial viability. Figure 4B shows the change in % viability with increasing tobramycin concentration. 10% viability was chosen as a cut-off in order to calculate the SMIC90.
Under aerobic conditions, the tobramycin SMIC90 values are higher for cells grown as a biofilm than those of planktonic cultures. Table 1 shows the variation in PSMIC90 and BSMIC90 for all isolates tested. Table 2 shows that under aerobic conditions, a dramatic increase in resistance to tobramycin (2 to &gt;32 fold increase in SMIC) was observed for most isolates when grown in ASM (biofilm mode) compared to LB (planktonic mode). In addition, biofilms grown under microaerophilic conditions exhibited an increased SMIC of between 2 and &gt;128-fold when compared to biofilms grown under aerobic conditions.
<img alt="Figure 1" src="/files/ftp_upload/3857/3857fig1.jpg" /> 
Figure 1. Biofilm formation of P. aeruginosa in ASM P. aeruginosa strain PAO1 forms macroscopically visible clumps (microcolonies) when grown in ASM. A, Biofilm formation in 30 ml ASM cultures (large-scale) after 7 days growth in screw cap glass Duran flasks . B, Biofilm formation in 2 ml ASM cultures (small-scale) after 3 days growth in 24-well polystyrene plates.
<img alt="Figure 2" src="/files/ftp_upload/3857/3857fig2.jpg" /> 
Figure 2. TEM micrographs of ASM biofilms A/C TEM micrograph (x;27,000) of PAO1 grown planktonically and in ASM, respectively, B/D TEM micrograph (x57,000) of PAO1grown planktonic and in ASM, respectively. Planktonically grown bacteria were cultivated overnight in LB broth. Biofilms were cultivated for 7 days in 30 ml ASM cultures. Black arrows refer to cells within the biofilm and stars refer to extracellular spaces. Scale bars = 1 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/3857/3857fig3.jpg" /> 
Figure 3. Workflow of the ASM biofilm antimicrobial susceptibility assay. 
<img alt="Figure 4" src="/files/ftp_upload/3857/3857fig4.jpg" /> 
Figure 4. Use of resazurin for the determination of antibiotic susceptibilities Bacterial cells were incubated with different concentrations of the antibiotic and the remaining metabolic activity was determined using resazurin. A, The blue non-fluorescent oxidized form of resazurin indicates non-viable cells and is reduced by metabolic active cells to pink fluorescent resorufin. B, Fluorescence intensity is converted into percentage of remaining bacterial viability. 10% viability was chosen as cut-off in order to calculate the MSMIC90. <a href="https://www.jove.com/files/ftp_upload/3857/3857fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<table border="1">
 <tbody>
 <tr>
 <td rowspan="2">Strains</td>
 <td colspan="2">PSMIC 90 (&mu;g/ml) 1</td>
 <td colspan="2">BSMIC 90 (&mu;g/ml) 1</td>
 </tr>
 <tr>
 <td>Aerobic</td>
 <td>Microaerophilic2</td>
 <td>Aerobic</td>
 <td>Microaerophilic 2</td>
 </tr>
 <tr>
 <td>PAO1</td>
 <td>4</td>
 <td>4</td>
 <td>8</td>
 <td>&gt;512</td>
 </tr>
 <tr>
 <td colspan="5">Liverpool Epidemic Strain (LES) isolates</td>
 </tr>
 <tr>
 <td>LESB5821</td>
 <td>8</td>
 <td>64</td>
 <td>64</td>
 <td>128</td>
 </tr>
 <tr>
 <td>LES40022</td>
 <td>32</td>
 <td>128</td>
 <td>8</td>
 <td>256</td>
 </tr>
 <tr>
 <td>LESB25</td>
 <td>16</td>
 <td>32</td>
 <td>256</td>
 <td>512</td>
 </tr>
 <tr>
 <td>LESB55</td>
 <td>16</td>
 <td>64</td>
 <td>64</td>
 <td>&gt;512</td>
 </tr>
 <tr>
 <td>LESB64</td>
 <td>16</td>
 <td>64</td>
 <td>&gt;512</td>
 <td>&gt;512</td>
 </tr>
 <tr>
 <td>LES43122</td>
 <td>4</td>
 <td>8</td>
 <td>32</td>
 <td>&gt;512</td>
 </tr>
 <tr>
 <td>LESB49</td>
 <td>16</td>
 <td>64</td>
 <td>64</td>
 <td>256</td>
 </tr>
 <tr>
 <td>LES109</td>
 <td>32</td>
 <td>128</td>
 <td>32 </td>
 <td>&gt;512</td>
 </tr>
 <tr>
 <td colspan="5">Non-LES isolates</td>
 </tr>
 <tr>
 <td>49461</td>
 <td>16</td>
 <td>32</td>
 <td>16</td>
 <td>&gt;512</td>
 </tr>
 <tr>
 <td>59032</td>
 <td>0.5</td>
 <td>2</td>
 <td>4</td>
 <td>&gt;512</td>
 </tr>
 <tr>
 <td>59073</td>
 <td>&gt;512</td>
 <td>&gt;512</td>
 <td>&gt;512</td>
 <td>&gt;512</td>
 </tr>
 <tr>
 <td>59076</td>
 <td>16</td>
 <td>32</td>
 <td>32</td>
 <td>&gt;512</td>
 </tr>
 <tr>
 <td>27</td>
 <td>8</td>
 <td>16</td>
 <td>4</td>
 <td>&gt;512</td>
 </tr>
 <tr>
 <td>45</td>
 <td>16</td>
 <td>32</td>
 <td>4</td>
 <td>&gt;512</td>
 </tr>
 </tbody>
</table>
Table 1. Susceptibility of P. aeruginosa to tobramycin.
1For determination of PSMICs and BSMICs tobramycin was used in 2-fold serial dilutions 
ranging from 512 - 0.5 &mu;g/ml (n = 8 for each concentration) and 512 - 1 &mu;g/ml (n = 4 for each 
concentration), respectively;; PSMICs were determined using the standard 
microdilution method1.
2Microaerophilic conditions were 5 % O2, 10 % CO2, and 85 % N2.
<table border="1">
 <tbody>
 <tr>
 <td rowspan="2">Strain</td>
 <td colspan="4">PSMIC90/BSMIC90fold change1</td>
 </tr>
 <tr>
 <td>PSMICaerobic &rarr; 
 PSMICmicroaerophilic</td>
 <td>BSMICaerobic &rarr; 
 BSMICmicroaerophilic</td>
 <td>PSMICaerobic &rarr; 
 BSMICaerobic</td>
 <td>PSMICmicroaerophilic &rarr; 
 BSMICmicroaerophilic</td>
 </tr>
 <tr>
 <td>PAO1</td>
 <td>0</td>
 <td>&gt;64</td>
 <td>2</td>
 <td>128</td>
 </tr>
 <tr>
 <td colspan="5">LES isolates</td>
 </tr>
 <tr>
 <td>LESB58</td>
 <td>8</td>
 <td>2</td>
 <td>8</td>
 <td>2</td>
 </tr>
 <tr>
 <td>LES400</td>
 <td>4</td>
 <td>32</td>
 <td>0.25</td>
 <td>2</td>
 </tr>
 <tr>
 <td>LESB25</td>
 <td>2</td>
 <td>2</td>
 <td>16</td>
 <td>16</td>
 </tr>
 <tr>
 <td>LESB55</td>
 <td>4</td>
 <td>&gt;8</td>
 <td>4</td>
 <td>&gt;8</td>
 </tr>
 <tr>
 <td>LESB64</td>
 <td>4</td>
 <td>ND</td>
 <td>&gt;32</td>
 <td>&gt;8</td>
 </tr>
 <tr>
 <td>LES431</td>
 <td>2</td>
 <td>&gt;16</td>
 <td>8</td>
 <td>&gt;64</td>
 </tr>
 <tr>
 <td>LESB49</td>
 <td>4</td>
 <td>4</td>
 <td>4</td>
 <td>4</td>
 </tr>
 <tr>
 <td>LES109</td>
 <td>4</td>
 <td>16</td>
 <td>0</td>
 <td>&gt;4</td>
 </tr>
 <tr>
 <td colspan="5">Non-LES isolates</td>
 </tr>
 <tr>
 <td>49461</td>
 <td>2</td>
 <td>&gt;32</td>
 <td>0</td>
 <td>&gt;16</td>
 </tr>
 <tr>
 <td>59032</td>
 <td>4</td>
 <td>&gt;128</td>
 <td>8</td>
 <td>&gt;256</td>
 </tr>
 <tr>
 <td>59073</td>
 <td>ND</td>
 <td>ND</td>
 <td>ND</td>
 <td>ND</td>
 </tr>
 <tr>
 <td>59076</td>
 <td>2</td>
 <td>&gt;16</td>
 <td>2</td>
 <td>&gt;16</td>
 </tr>
 <tr>
 <td>27</td>
 <td>2</td>
 <td>&gt;128</td>
 <td>0.5</td>
 <td>&gt;32</td>
 </tr>
 <tr>
 <td>45</td>
 <td>2</td>
 <td>&gt;128</td>
 <td>0.25</td>
 <td>&gt;16</td>
 </tr>
 </tbody>
</table>
Table 2. Fold change of PSMICs and BSMICs to tobramycin.
1ND, not determined; values in bold indicate SMIC fold changes &gt;10.

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J., Winstanley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluctuations+in+phenotypes+and+genotypes+within+populations+of+Pseudomonas+aeruginosa+in+the+cystic+fibrosis+lung+during+pulmonary+exacerbations.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusible+signal+factor-dependent+cell-cell+signaling+and+virulence+in+the+nosocomial+pathogen+Stenotrophomonas+maltophilia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newly+introduced+genomic+prophage+islands+are+critical+determinants+of+in+vivo+competitiveness+in+the+Liverpool+Epidemic+Strain+of+Pseudomonas+aeruginosa.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subtype+of+a+Pseudomonas+aeruginosa+cystic+fibrosis+epidemic+strain+exhibits+enhanced+virulence+in+a+murine+model+of+acute+respiratory+infection.">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 border="1">
 <tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DNA from fish sperm</td>
 <td>Sigma-Aldrich</td>
 <td>74782</td>
 </tr>
 <tr>
 <td>Mucin from porcine stomach, type II</td>
 <td>Sigma-Aldrich</td>
 <td>M2378</td>
 </tr>
 <tr>
 <td>L-Alanine</td>
 <td>Acros Organics</td>
 <td>102830250</td>
 </tr>
 <tr>
 <td>L-Arginine</td>
 <td>Sigma-Aldrich</td>
 <td>A5006</td>
 </tr>
 <tr>
 <td>L(+)-Asparagine monohydrate</td>
 <td>Acros Organics</td>
 <td>175271000</td>
 </tr>
 <tr>
 <td>L(+)-Aspartic acid</td>
 <td>Acros Organics</td>
 <td>105041000</td>
 </tr>
 <tr>
 <td>L-Cysteine</td>
 <td>Sigma-Adrich</td>
 <td>168149</td>
 </tr>
 <tr>
 <td>L(+)-Glutamic acid</td>
 <td>Acros Organics</td>
 <td>156211000</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Sigma-Aldrich</td>
 <td>G3126</td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>Acros Organics</td>
 <td>220911000</td>
 </tr>
 <tr>
 <td>L-Histidine</td>
 <td>Sigma-Adrich</td>
 <td>H8000</td>
 </tr>
 <tr>
 <td>L-Isoleucine</td>
 <td>Sigma-Aldrich</td>
 <td>I2752</td>
 </tr>
 <tr>
 <td>L-Leucine</td>
 <td>Sigma-Aldrich</td>
 <td>L8000</td>
 </tr>
 <tr>
 <td>L(+)-Lysine monohydrochloride</td>
 <td>Acros Organics</td>
 <td>125222500</td>
 </tr>
 <tr>
 <td>L-Methionine</td>
 <td>Sigma-Aldrich</td>
 <td>M9625</td>
 </tr>
 <tr>
 <td>L-Phenylalanine</td>
 <td>Acros Organics</td>
 <td>130310250</td>
 </tr>
 <tr>
 <td>L-Proline</td>
 <td>Sigma-Aldrich</td>
 <td>P0380</td>
 </tr>
 <tr>
 <td>L-Serine</td>
 <td>Acros Organics</td>
 <td>132660250</td>
 </tr>
 <tr>
 <td>L-Threonine</td>
 <td>Acros Organics</td>
 <td>138930250</td>
 </tr>
 <tr>
 <td>L(-)-Tryptophan</td>
 <td>Acros Organics</td>
 <td>140590250</td>
 </tr>
 <tr>
 <td>L-Tyrosine</td>
 <td>Acros Organics</td>
 <td>140641000</td>
 </tr>
 <tr>
 <td>L-Valine</td>
 <td>Sigma-Aldrich</td>
 <td>V0500</td>
 </tr>
 <tr>
 <td>Diethylenetriaminepentaacetic acid</td>
 <td>Sigma-Aldrich</td>
 <td>32318</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Fisher Scientific</td>
 <td>S/3160/60</td>
 </tr>
 <tr>
 <td>KCl</td>
 <td>BDH</td>
 <td>BDH0258</td>
 </tr>
 <tr>
 <td>KOH</td>
 <td>BDH</td>
 <td>BDH0262</td>
 </tr>
 <tr>
 <td>Egg yolk emulsion</td>
 <td>Sigma-Aldrich</td>
 <td>17148</td>
 </tr>
 <tr>
 <td>ME 2 diaphragm vacuum pump</td>
 <td>Vacuubrand</td>
 <td>696126</td>
 </tr>
 <tr>
 <td>Steritop filters (Pore size: 0.22 &mu;m, Neck size: 45 mm)</td>
 <td>Millipore</td>
 <td>SCGPT10RE</td>
 </tr>
 <tr>
 <td>Luria-Bertani medium</td>
 <td>Sigma</td>
 <td>L2897</td>
 </tr>
 <tr>
 <td>96-well microtitre plates</td>
 <td>Sarstedt</td>
 <td>82.1581</td>
 </tr>
 <tr>
 <td>24-well tissue culture-treated plates</td>
 <td>Iwaki</td>
 <td>3820-024</td>
 </tr>
 <tr>
 <td>CampyGen gas generation packs</td>
 <td>Oxoid </td>
 <td>CN0025 </td>
 </tr>
 <tr>
 <td>Microaerophilic chamber</td>
 <td>Oxoid </td>
 <td>HP0011 </td>
 </tr>
 <tr>
 <td>Tobramycin sulphate</td>
 <td>Sigma-Aldrich</td>
 <td>T1783</td>
 </tr>
 <tr>
 <td>Cellulase, from Aspergillus niger</td>
 <td>Sigma-Aldrich</td>
 <td>22178</td>
 </tr>
 <tr>
 <td>Resazurin</td>
 <td>Sigma-Aldrich</td>
 <td>199303</td>
 </tr>
 <tr>
 <td>Citrate.H20</td>
 <td>BDH</td>
 <td>BDH0288</td>
 </tr>
 <tr>
 <td>Fluostar omega microplate reader</td>
 <td>BMG-Labtech</td>
 <td>SPECTROstar Omega</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

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.

Watch this Scientific Journal Video about Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung at JoVE.com

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

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.

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 ...

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Immunology and Infection

32.4K Views.  University of Liverpool. 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.

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

A Method to Test the Efficacy of Handwashing for the Removal of Emerging Infectious Pathogens

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing methods in general. Additionally, little evidence exists comparing handwashing methods to determine which are most efficacious at removing infectious pathogens. Research is needed to provide evidence for the different approaches to handwashing that may be employed during infectious disease outbreaks. Here, a laboratory method to assess the efficacy of handwashing methods at removing microorganisms from hands and their persistence in rinse water is described. Volunteers' hands are first spiked with the test organism and then washed with each handwashing method of interest. Generally, surrogate microorganisms are used to protect human subjects from disease. The number of organisms remaining on volunteers' hands after washing is tested using a modified "glove juice" method: the hands are placed in gloves with an eluent and are scrubbed to suspend the microorganisms and make them available for analysis by membrane filtration (bacteria) or plaque assay (viruses/bacteriophages). Rinse water produced from the handwashing is directly collected for analysis. Handwashing efficacy is quantified by comparing the log reduction value between samples taken after handwashing to samples with no handwashing. Rinse water persistence is quantified by comparing rinse water samples from various handwashing methods to samples collected after handwashing with just water. While this method is limited by the need to use surrogate organisms to preserve the safety of human volunteers, it captures aspects of handwashing that are difficult to replicate in an in vitro study and fills research gaps on handwashing efficacy and the persistence of infectious organisms in rinse water.

Handwashing is widely recommended to prevent infectious disease transmission. However, there is little evidence on which handwashing methods are most efficacious at removing infectious disease pathogens. We developed a method to assess the efficacy of handwashing methods at removing microorganisms.

Handwashing is widely recommended to prevent infectious disease transmission. However, little comparable evidence exists on the efficacy of handwashing ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe infections, complications, and mortality worldwide with a huge impact on our society and on the health system. In particular, patients with compromised immune systems or pre-existing and chronic pathologies, such as cystic fibrosis (CF), are subjected to frequent antibiotic treatments to control the infections with the appearance and diffusion of multidrug resistant isolates. Therefore, there is an urgent need to address alternative therapies to counteract bacterial infections. Use of bacteriophages, the natural enemies of bacteria, can be a possible solution. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. Zebrafish embryos were infected with P. aeruginosa to demonstrate that phage therapy is effective against P. aeruginosa infections as it reduces lethality, bacterial burden and pro-inflammatory immune response in CF embryos.

Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos

Presented here is a protocol for Pseudomonas aeruginosa infection and phage therapy application in cystic fibrosis (CF) zebrafish embryos.

Antimicrobial resistance, a major consequence of diagnostic uncertainty and antimicrobial overprescription, is an increasingly recognized cause of severe ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

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