Name: use of artificial sputum medium to test antibiotic efficacy against pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung
Uploaded: 2012-06-05T12:00:00.000+00:00
Duration: 7 min 46 s
Description: 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

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="https://jove.unicartagenaproxy.elogim.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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Dis. 24, 677-687 (2005).</li><li>Bjarnsholt, T. <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+biofilms+in+the+respiratory+tract+of+cystic+fibrosis+patients.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+susceptibilities+of+Pseudomonas+aeruginosa+isolates+derived+from+patients+with+cystic+fibrosis+under+aerobic,+anaerobic,+and+biofilm+conditions.">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/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

Universidad de Cartagena UDEC

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

32.8K 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

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

科研

免疫与感染

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

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

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.

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

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

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

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

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. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

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.

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

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

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

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

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;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.

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

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

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

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

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.

伪多莫纳斯航空感染模型实时评估工具

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

<meta charset="utf8"></head><body>用于抗菌检测和微生物动力学分析的适应性 CF 模型

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

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

<meta charset="utf8"></head><body>开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

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.

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

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

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

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.

研究辅助供氧对囊性纤维化气道微生物组影响的模型的设计与开发

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

使用人工痰介质测试抗生素药效试验

摘要

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此视频中的章节

可视化痰生物膜发展影响使用腔室盖玻片型号

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

囊性纤维化患者痰液中铜绿假单胞菌的可视化

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

伪多莫纳斯航空感染模型实时评估工具

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

研究辅助供氧对囊性纤维化气道微生物组影响的模型的设计与开发

使用人工痰介质测试抗生素药效试验

摘要

探索更多视频

此视频中的章节

可视化痰生物膜发展影响使用腔室盖玻片型号

该WinCF模式 - 粘液的廉价和易于处理的缩影细支气管堵塞，研究肺部感染的微生物

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

伪多莫纳斯航空感染模型实时评估工具

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

研究辅助供氧对囊性纤维化气道微生物组影响的模型的设计与开发

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

囊性纤维化患者痰液中铜绿假单胞菌的可视化