我们承认，英国国家健康研究学会，博士Hadwen信托人文研究，英国领先的医学研究慈善机构的资金完全非动物研究技术，以取代动物实验，和威康信托基金会（格兰特089215）的支持。我们也承认，诺华制药公司（英国）有限公司（无限制教育补助金）。

1。制备人工痰中等（ASM）的<ol><li> 4克鱼精子DNA加入250毫升无菌水，过几个小时内非常缓慢。的DNA需要几个小时才能完全溶解，并可以在室温下搅拌过夜。 </li><li>从猪胃（II型）慢慢到250毫升的无菌水加入5Ğ粘液，直到粘蛋白完全溶解。该解决方案可在4搅拌过夜°C。 </li><li> L-酪氨酸，L-半胱氨酸的异常，在100毫升无菌水溶解0.25克，每必要和非必要的L-氨基酸。溶于25毫升0.5 M的氢氧化钾（M R 56.11克/摩尔）和L-酪氨酸0.25克25毫升无菌水0.25ĞL-半胱氨酸。 </li><li>溶于5.9毫克二乙三胺五乙酸（DTPA），5 g氯化钠，氯化钾2.2克，100毫升无菌水。 </li><li>结合的DNA，粘蛋白，L-氨基酸，胺，氯化钠，氯化钾，在1升瓶。 </li><li>加入5毫升蛋黄émulsion和填充到约850毫升无菌水。 </li><li>调整pH值至6.9，用1 M的Tris（pH值8.5; 121.14 M R），并用无菌水使体积为1升。 </li><li> 1 VACUUBRAND ME 2隔膜真空泵，微孔过滤Steritop单位使用0.22微米和45毫米的孔隙​​和颈部大小，分别过滤消毒的ASM。可以重新每个Steritop过滤装置，立即使用了三次，但过滤器必须用无菌水冲洗，再利用前的两倍。过滤过程是缓慢的，可超过2天进行。其他版本的ASM已经开发使用添加抗生素，而不是过滤11然而，由于药物的相互作用可能，我们不建议这个特定的应用程序的方法。 </li><li>过滤和过滤的ASM，应保存在4°C，在黑暗中。建议使用新鲜的ASM然而，在这些条件下可以保存最多的一个月。 </l我&gt; </ol> 2。测定浮游固着细胞的最低代谢抑菌浓度（PSMIC） <ol><li>为了确定代谢的最低抑制浓度为15 planktonically增长的体育 （PSMIC） 假单胞菌株，应稀释法，如抗菌化疗BSAC传输12英国社会的指导方针。抗生素的选择，在这种情况下，妥布霉素硫酸，连续稀释卢里亚-Bertani（LB）培养基中提供适当的抗生素浓度范围在96孔微孔板。 </li><li>稀释隔夜文化体育绿脓杆菌在LB 0.05外径600（±0.01），添加100μL量的96孔的微孔板板含有100μL连续稀释抗生素的水井。在这种情况下，硫酸妥布霉素最终浓度介于512 - 0.5微克/毫升。八复制的每个antibiotic浓度应执行。 </li><li>每个隔离应设立阴性对照井，没有添加抗生素。此外，八口井应该包含只使用空白作为一个在下游分析（2.6节）的LB。 </li><li>在37°C孵育96以及酶标板1 - 2天，无晃动好氧或微需氧（10％的CO 2，O 2 5％和85％N2）条件下。微需氧条件得到大型厌氧罐中使用CampyGen气代包。 </li><li>孵化之后，通过测量使用Fluostar欧米茄酶标仪和火星的数据分析软件的600纳米波长的吸光度在每口井的细菌培养，确定细菌的生长。 </li><li>从抗生素治疗浮游文化（一抗生素治疗的浮游细胞 ）和阴性对照（ 阴性对照 ）吸收的吸收，应予以纠正减去从井的LB（ 空白 ）获得的背景吸收的离子。其后的生存能力的抑制率（指抗生素治疗的浮游细胞 /平均阴性对照 ）×100％计算。 PSMIC 90被定义为造成90％的浮游细菌生长的抑制浓度抗生素。 </li><li>以确定细菌的生存能力后选择抗生素治疗，10微升0.02％（V / V），刃天青（蒸馏水稀释），每孔加入板块在有氧条件下孵育1 - 2小时37° C，而在150转摇。活菌粉红色荧光的试卤灵形式将减少刃天青蓝色染料。 </li><li>孵化与刃天青，监测每口井使用540纳米的激发波长和发射波长在Fluostar欧米茄酶标仪590 nm的荧光。所述是应分析数据低。 </li></ol> 3。生物膜固着细胞的最低抑菌浓度测定（BSMIC） <ol><li>隔夜文化体育绿脓杆菌 （在这种情况下，用于15株绿脓杆菌 ）应在LB稀释到0.05 OD 600（±0.01），然后再稀释1:100新鲜的ASM（总容积1.8毫升）。 </li><li>应该被添加到每个井的24孔组织培养处理板（1.8毫升）稀释文化。 3口井应该包含ASM只使用空白作为一个在下游分析（3.9节）。 </li><li>争取3天需氧或微需氧条件下的实验室封口膜和孵育24孔板，在37°C，而在75转摇。微需氧条件下，应使用在大型的厌氧罐气体CampyGen代包。 </li><li>稀释抗生素的选择，在这种情况下，妥布霉素硫酸，提供适当的浓度范围内新鲜ASM的。在这种情况下，终浓度为512 - 1微克/毫升之间。体积200μL，加入每个浓度的抗生素，在24孔板相应的井。四个每个抗生素浓度应执行的复制。生物膜接触到抗生素的选择，被用来作为阴性对照。 </li><li>需氧或微需氧条件下为24小时，在37°C下安全实验室封口膜和孵育24孔板，同时摇转75。 </li><li>孵化后在选择抗生素的存在，扰乱使用细菌生物膜100毫克/毫升纤维素（0.05 M的柠檬酸缓冲液稀释100μL[9.6克/升Citrate.H 2 0在水和pH值用NaOH 4.6] ）和有氧条件下孵化的24孔板，在37°C，而在150 rpm下振荡1小时。如果需要，生物膜可以进一步打乱手动移液器在这个阶段。 </li><li>要确定的代谢活动释放破坏生物膜细菌细胞，刃天青（蒸馏水稀释）100微升0.02％（V / V）应添加到每个井的24孔板，在37°C孵育1 - 2小时，同时震动150转。 </li><li>孵化与刃天青，测量每口井使用540纳米的激发波长和发射波长590纳米在Fluostar欧米茄酶标仪和火星的数据分析软件的荧光。 </li><li>抗生素治疗生物膜（ 抗生素治疗生物膜 ）和阴性对照（ 阴性对照 ）的荧光的荧光应包含的ASM井获得的背景荧光纠正减法（六空白 ）。其后的生存能力的抑制率（平均抗生素治疗生物膜的F /均值F 阴性对照 ）×100％计算。 BSMIC 90定义为antibio抽动浓度造成90％的代谢活性的抑制。 </li></ol> 4。代表结果 ASM生物膜的形成是可能的小卷（2毫升），生物膜得到充分形成3天之内（ 图1A）。这可以证明，通过严格的吹打生物膜，这应该是很难破坏。 microcolonies是与生长量较大（ 图1B）。图2显示了增长planktonically和生物膜的电子显微图像分析检测细胞之间的重大分歧。生物膜文化清楚地表明，外周围的基质细胞和生物膜内的个体结构相当的水平难以确定。 一些研究表明，生物膜的生活方式可以影响药敏13，14。我们的小规模的ASM检测可以用来DETE的rmine BSMIC的同时多株多种抗生素。检测的工作流程如图3所示。抗生素对细菌细胞活力的影响，可以采用刃天青检测。抗生素，在这种情况下，妥布霉素，可以添加到既定的生物膜和培养24小时。在此之后，生物膜被破坏和刃天青添加。 代谢活跃的细胞，可以减少刃天青染料从蓝色的颜色变化（刃天青）粉色（试卤灵）15。 图4A显示了一个例子法在体育绿脓杆菌培养生物膜破坏和刃天青此外，在微孔板前不同浓度妥布霉素。蓝色的非荧光色表示非活菌，而活菌粉红色荧光的形式，试卤灵减少染料。中芯国际，然后可以通过荧光转换成百分比计算其余细菌的生存能力。 图4B显示％的可行性与妥布霉素浓度增加的变化。 10％的生存能力被选为截断，以计算中芯国际90。 在有氧条件下，妥布霉素中芯国际90值较高的生物膜比浮游文化作为一种细胞生长。表1显示，所有菌株在PSMIC 90和BSMIC 90的变化。表2表明，在有氧条件下，在阻力急剧增加，妥布霉素（2&gt;中芯国际的32倍）观察到的大多数菌株生长在ASM（生物膜模式）时相比，LB（浮游生物模式）。此外，微需氧条件下生长的生物膜展出时增加2至128倍的中芯国际相比，在有氧条件下生长的生物膜。 <img alt="图1" src="/files/ftp_upload/3857/3857fig1.jpg" /> 图1。在ASM 体育绿脓杆菌生物膜形成绿脓杆菌株PAO1 一个 30毫升的ASM文化，形成生物膜（大型）经过7天 ​​的螺丝帽玻璃杜兰瓶，B，在2毫升的ASM文化的形成生物膜的生长（形成肉眼可见团块（microcolonies）的ASM中成长。小规模的），后3天，在24孔聚苯乙烯板的增长。 <img alt="图2" src="/files/ftp_upload/3857/3857fig2.jpg" /> 图2。 TEM照片的ASM生物膜的A / C的TEM照片（27,000）PAO1种植planktonically在ASM，分别为B / D型的TEM照片（X57，000）的PAO1grown浮游和ASM中，分别。 planktonically生长的细菌，在LB培养基中培养过夜。生物膜培养7天，30毫升的ASM文化。黑色箭头指细胞内的生物膜和恒星是指外空间。比例尺= 1微米。 <img alt="图3" src="/files/ftp_upload/3857/3857fig3.jpg" /> 图3。 ASM的生物膜药敏检测的工作流程。 <img alt="图4" src="/files/ftp_upload/3857/3857fig4.jpg" /> 图4。刃天青为使用抗生素敏感性测定细菌细胞培养，用不同浓度的抗生素和余下的代谢活性测定用刃天青。，蓝色荧光非氧化刃天青的形式表示非活细胞代谢减少粉红色荧光的试卤灵，B的活性细胞，荧光强度转换成百分比剩余细菌的生存能力。 10％的存活率，选择作为切断以计算MSMIC90， <a href="http://www.jove.com/files/ftp_upload/3857/3857fig4large.jpg" target="_blank">点击这里查看LAR</a>蒙古包的身影。 </span&gt; <table border="1"><tbody><tr><td rowspan="2"> 株 </td><td colspan="2"> PSMIC 90（微克/毫升）1 </td><td colspan="2"> BSMIC 90（微克/毫升）1 </td></tr><tr><td> 有氧 </td><td> 微需氧2 </td><td> 有氧 </td><td> 微需氧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"> 利物浦流行株（LES）的隔离 </td></tr><tr><td> LESB58 21 </td><td> 8 </td><td> 64 </td><td> 64 </td><td> 128 </td></tr><tr><td> LES400 22 </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> LES431 22 </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"> 非涡株 ​​， </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 &lt;/ TD&gt; </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> 表1。药敏绿脓杆菌妥布霉素。 测定的PSMICs和BSMICs妥布霉素1 2倍系列稀释 范围从512 - 0.5微克/毫升（每个浓度= 8），512 - 1微克/毫升（n = 4时为每个 浓度），分别;; PSMICs人使用标准的确定 稀释法1。 O 2的 5％，10％的CO 2，85％N 2微需氧条件。 <table border="1"><tbody><tr><td rowspan="2"> 应变 </td><td colspan="4"> 90 / PSMIC BSMIC 90倍的变化1 </td></tr><tr><td> PSMIC 有氧 → PSMIC 微需氧 </td><td> BSMIC 有氧 → BSMIC 微需氧 </td><td> PSMIC 有氧 → BSMIC 有氧 </td><td> PSMIC 微需氧 → BSMIC 微需氧 </td></tr><tr><td> PAO1 </td><td> 0 </td><td> &gt; 64 </td><td> 2 </td><td> 128 </td></tr><tr><td colspan="5"> 就业辅导组隔离 </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&gt; <td> 4 </td><td> &gt; 8 </td></tr><tr><td> LESB64 </td><td> 4 </td><td> ND折价</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"> 非涡株 ​​， </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折价</td><td> ND折价</td><td> ND折价</td><td> ND折价</td></tr><tr><td> 59076 </td><td> 2 </td><td> &gt; 16 </TD&gt; <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> 表2。倍数变化的PSMICs和妥布霉素BSMICs的。 1钕，没有确定值，大胆表明中芯国际的褶皱变化&gt; 10。

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Microbiol. 43, 5085-5090 (2005).</li><li>Fung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+of+Pseudomonas+aeruginosa+in+a+mucin-containing+synthetic+growth+medium+mimicking+cystic+fibrosis+lung+sputum.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BSAC+standardized+disc+susceptibility+testing+method+(version+8).">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+implication+of+Pseudomonas+aeruginosa+biofilms+in+infections.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+biofilm+formation+in+the+cystic+fibrosis+airway.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+rapid+and+simple+non-radioactive+assay+to+monitor+and+determine+the+proliferation+of+lymphocytes:+an+alternative+to+[3H]thymidine+incorporation+assay.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+antibiotic+treatment+on+bacteriophage+production+by+a+cystic+fibrosis+epidemic+strain+of+Pseudomonas+aeruginosa.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum-sensing+signals+indicate+that+cystic+fibrosis+lungs+are+infected+with+bacterial+biofilms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+Aeruginosa+Population+Diversity+and+Turnover+in+Cystic+Fibrosis+Chronic+Infections.">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/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>

没有利益冲突的声明。

在这项研究中，我们采用体外模型基于一种新的ASM上复制体育在CF肺4 绿脓杆菌生物膜条件。该模型已成功修改为小规模，高通量检测的抗菌药物。 此法的关键步骤是： <ol><li>一致的ASM媒体和保持无菌准备。我们致力于优化，其中每个组件被添加的方式实现可重复性的结果每次长时间。过滤的ASM是缓慢的，但最好是消毒，这可能破坏黏液的组成部分。我们不建议抗生素此外，其他11建议，因为这可能会施加相当的选择性压力，驱动突变，诱发噬菌体裂解16多个细菌基因的表达显着改变。 </li><li>该法应根据体积Ø优化f的ASM使用。摇动的速度增加量较小，观察生物膜生命周期较短。 </li></ol>明显的应用是一个小规模的ASM生物膜模型的生物膜抗菌（BSMIC 90） ​​的敏感性更现实的决心。厌氧和微需氧龛是在目前的CF肺部，并有证据显示，被限制在2生物膜成熟，17深氧气。在这里，我们表明，临床体育 10/14从CF患者痰铜绿假单胞菌株 ASM中的微需氧条件下表现出了相当大（4 - ≥128倍）妥布霉素的敏感性下降。这项研究的结果表明，妥布霉素如抗生素，可能不太对体育的有效绿脓杆菌，比常规药敏试验方法在CF肺部感染。这些结果反映了10生物膜药敏以往的研究。小规模的ASM检测，从而提供一个简单的高通量平台，产生有意义的药敏数据，以更好地告知治疗的决定。该法被限制在以同样的方式作为常规药敏试验，单菌落筛选，可能无法代表整个人口的回升。然而，我们认为，办法（一）使用非表面附着生物膜的生长和（二）适用于微需氧条件，代表一个明确的选择和一个潜在的改善现有方法。我们得出这样的结论：该法是一个合适的模型来研究体育绿脓杆菌生物膜人口。在临床上的进一步测试将确定是否生物膜生长的体育基础的抗生素敏感性绿脓杆菌可能导致不同的微生物学和临床疗效与可能改善抗生素的选择。使用经典的生物膜模型的类似调查显示，导致BSMIC值抗生素治疗5,17不同的建议。 此外抗感染药物的有效性测试，ASM系统是一种廉价，动物模型，简单和可重复性的研究，如那些旨在了解体育多元化的替代绿脓杆菌人口。我们已经看到在自然种群广泛的异质性体育绿脓杆菌恢复CF病人痰18，19。在ASM 4（未发表资料）增长过程中，可以观察到类似的表型和基因型多样化，使得它在体外的CF肺部疾病模型的吸引力。 ASM模型相对简单，可以很容易地设计长期适应实验旨在监测P.抗生素或其他压力的影响，例如， 绿脓杆菌人口的分歧。此外，可以种植在其他细菌病原体ASM的。 Fouhy 等。例如，2007年用于ASM研究与生物膜形成maltophillia 20。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>从鱼精子的DNA </td><td> Sigma-Aldrich公司</td><td> 74782 </td></tr><tr><td> II型猪胃粘蛋白从</td><td> Sigma-Aldrich公司</td><td> M2378 </td></tr><tr><td> L-丙氨酸</td><td> Acros Organics公司</td><td> 102830250 </td></tr><tr><td> L-精氨酸</td><td> Sigma-Aldrich公司</td><td> A5006 </td></tr><tr><td>的L（+）-天冬酰胺一水</td><td> Acros Organics公司</td><td> 175271000 </td></tr><tr><td> L（+）-天门冬氨酸</td><td> Acros Organics公司</td><td> 105041000 </td></tr><tr><td> L-半胱氨酸</td><td> Sigma-爱秩序的</td><td> 168149 </td></tR&gt; <tr><td>的L（+）-谷氨酸</td><td> Acros Organics公司</td><td> 156211000 </td></tr><tr><td> L-谷氨酰胺</td><td> Sigma-Aldrich公司</td><td> G3126 </td></tr><tr><td>甘氨酸</td><td> Acros Organics公司</td><td> 220911000 </td></tr><tr><td> L-组氨酸</td><td> Sigma-爱秩序的</td><td> H8000 </td></tr><tr><td> L-异亮氨酸</td><td> Sigma-Aldrich公司</td><td> I2752 </td></tr><tr><td> L-亮氨酸</td><td> Sigma-Aldrich公司</td><td> L8000 </td></tr><tr><td> L（+）-赖氨酸盐酸盐</td><td> Acros Organics公司</td><td> 125222500 </td></tr><tr><td> L-蛋氨酸</td><td> Sigma-Aldrich公司</td><td> M9625 </td></tr><tr><td> L-苯丙氨酸</td><td> Acros Organics公司</td><td> 130310250 </td></tr><tr><td> L-脯氨酸</td><td> Sigma-Aldrich公司</td><td> P0380 </td></tr><tr><td> L-丝氨酸</td><td> Acros Organics公司</td><td> 132660250 </td></tr><tr><td> L-苏氨酸</td><td> Acros Organics公司</td><td> 138930250 </td></tr><tr><td> L（ - ）-色氨酸</td><td> Acros Organics公司</td><td> 140590250 </td></tr><tr><td> L-酪氨酸</td><td> Acros Organics公司</td><td> 140641000 </td></tr><tr><td> L-缬氨酸</td><td> Sigma-Aldrich公司</td><td> V0500 </td></tr><tr><td>二乙酸</td><td> Sigma-Aldrich公司</td><td> 32318 </td></tr><tr><td>氯化钠</td><td> Fisher Scientific则</td><td> S/3160/60 </td></tr><tr><td>氯化钾</td><td> BDH问题</td><td> BDH0258 </td></tr><tr><td&gt;氢氧化钾</td><td> BDH问题</td><td> BDH0262 </td></tr><tr><td>蛋黄乳液</td><td> Sigma-Aldrich公司</td><td> 17148 </td></tr><tr><td> ME 2隔膜真空泵</td><td> VACUUBRAND </td><td> 696126 </td></tr><tr><td> steritop过滤器（孔径：0.22微米，颈部尺寸：45毫米） </td><td> Millipore公司</td><td> SCGPT10RE </td></tr><tr><td>卢里亚-Bertani中等</td><td>西格玛</td><td> L2897 </td></tr><tr><td> 96孔酶标板</td><td>萨尔斯塔特</td><td> 82.1581 </td></tr><tr><td> 24孔组织培养处理板</td><td>磐</td><td> 3820-024 </td></tr><tr><td> CampyGen天然气发电包</td><td> OXOID </td><td> CN0025 </td></tr><tr><td>微需氧室</td><td> OXOID </td><td> HP0011 </td></tr><tr><td>妥布霉素硫酸</td><td> Sigma-Aldrich公司</td><td> T1783 </td></tr><tr><td>纤维素酶， 黑曲霉 </td><td> Sigma-Aldrich公司</td><td> 22178 </td></tr><tr><td>刃天青</td><td> Sigma-Aldrich公司</td><td> 199303 </td></tr><tr><td> Citrate.H 2 0 </td><td> BDH问题</td><td> BDH0288 </td></tr><tr><td> fluostar欧米茄酶标仪</td><td> BMG公司的LABTECH </td><td> SPECTROstar欧米茄</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

有越来越多的关注， 在体外药敏试验的相关性应用于体育株时从囊性纤维化（CF）患者的铜绿 。现有的方法依赖于单一或少数菌株生长的需氧和planktonically。预定截止权衡，以确定是否细菌敏感或耐药的任何特定的抗生素1。然而，在慢性肺部感染的CF， 体育绿脓杆菌人口生物膜中存在，并有证据表明，环境主要是微需氧2。在肺和那些在诊断测试中的细菌之间的情况形成了鲜明的差异已成为问题的可靠性，甚至相关的这些测试3。 人工的痰介质（ASM）的体育是一种文化中的CF病人痰的组成部分，包括氨基酸，黏蛋白及游离DNA。 绿脓杆菌&lt;/ EM&gt;增长过程中的CF感染，形成自我聚集的生物膜结构和人口的分歧4,5,6，在增长的ASM模仿。本研究的目的是要制定一个酶标板法研究P.药敏绿脓杆菌在ASM，这是适用于微需氧和有氧条件下生长的基础。 一个ASM法在微孔板格式。 体育绿脓杆菌生物膜被允许开发，连续3天在不同浓度的抗菌药物孵化前24小时。生物膜破坏后，细胞活力测定与刃天青染色。此法被用来确定为15个不同的体育妥布霉素柄细胞的最低抑菌浓度（中芯国际）需氧和微需氧的条件下，中芯国际值绿脓杆菌株相比，那些与标准肉汤增长得到。虽然一些证据增加浮游同行相比，他们的成长中的ASM株的MIC值，细菌在微需氧的条件，这表明大大增加抗&gt; 128倍，对妥布霉素，在ASM系统测试时被发现的最大区别相比，在有氧条件下进行检测。 当前药敏试验方法和临床疗效之间缺乏关联3当前方法的有效性提出质疑。几个在体外模型已被用于先前研究体育绿脓杆菌生物膜7，8。然而，这些方法依赖于表面附着生物膜，而ASM生物膜像那些在CF肺9观察。此外，粘液减少氧浓度已经证明，以改变对体育的行为绿脓杆菌 2，影响抗生素的敏感性10。因此如胶似漆Ğ微需氧条件下，ASM可以提供一个更现实的环境中学习药敏。

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

目前的诊断药敏试验依靠浮游株生长在营养丰富，有氧条件下的。在这里，我们采用了一种替代人工痰媒介研究的肺囊肿性纤维化的代表都需氧和微需氧条件下绿脓杆菌生物膜药敏。

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

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

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

Research

JoVE Journal

Immunology and Infection

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

32.4K 次观看.  University of Liverpool. 目前的诊断药敏试验依靠浮游株生长在营养丰富，有氧条件下的。在这里，我们采用了一种替代人工痰媒介研究的肺囊肿性纤维化的代表都需氧和微需氧条件下绿脓杆菌生物膜药敏。

视频: 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).

铜绿假单胞菌和酿酒酵母流细胞生物膜

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

科研

免疫与感染

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.

RNA的分离绿脓杆菌殖民小鼠胃肠道

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.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

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.

绿脓杆菌引起的肺损伤模型

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.

在免疫鼠害的鲁棒性肺炎模型评价抗菌功效反对<i&gt;秒。肺炎</i&gt;<i&gt;小时。流感</i&gt;<i&gt; K。肺炎</i&gt;<em&gt;点。铜绿假单胞菌</em&gt;或<em&gt;一个。鲍曼不动杆菌</em

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.

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

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.

一种测试洗手方法去除新发感染性病原体的方法

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.

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

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.

噬菌体纤维化 斑马鱼胚 胎中伪多多多阿韦诺萨感染的噬菌体治疗应用

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.