우리는 공부하는 동안 그들의 도움을 메이오 클리닉 Mycobacteriology 연구소의 실험실 기술자에게 감사를 표합니다.

각 실험실은 inoculum의 준비 및 접시에 inoculum의 pipetting에 해당 biosafety 수준을 결정하기 위해 그들의 기관 생물 안전 관리 책임자와 공동으로 위험 평가를 수행하기 위해 필요합니다. microtiter 접시가 주사, 플라스틱 접착 도장과 봉인까지 우리 연구실에서는, 우리 3 실험실 (BSL3) 사례 biosafety 수준을 사용합니다. 이 번호판은 다음 열을도 밀폐되어 비닐 봉지 안에 배치됩니다. microtiter 판의 배양과 해석 읽기는 biosafety 레벨 2 실험실 (BSL2)에 실시됩니다. 1. 자료의 라벨링 <ol><li> 라벨 혈액 한천 플레이트 세 미들 7H10 접시, 미들 7H9 국물, 호수 트윈 유리 비드 튜브와 BSL2 해당 식별자 (예 : 환자 이름, 의료 기록 번호)와 대서양 표준시 microtiter 접시. </li><li> 라벨 혈액 한천 배지와 &quot;순도 검사&quot;로 도금 7H10. inoculum 정기적으로 식민지 카운트는 생물의 적절한 농도가 테스트에 사용하도록 권장합니다. 수행 콜로니 카운트는 &quot;1분의 1 콜로니 개수&quot;와 &quot;50분의 1 식민지 개수&quot;로 표시된 7H10 접시 두 가지가 필요합니다. </li></ol> 2. BSL3로 이동 기술자 준비 <ol><li> 적절한 개인 보호 장비는 BSL3에서 작업하는 데 필요한이며 솔리드 앞에 가운, 머리 커버, 장갑, 신발 커버, 그리고 인공 호흡기가 포함되어 있습니다. 각 기관들은 기관의 산업 위생 관리 책임자와 공동으로 자신의 호흡기 보호 계획을 수립해야합니다. 저희 연구실에서는, 우리는 전원이 공급되는 공기 정화 호흡기 (PAPRs) 및 장착 HEPA - 필터링, N95 절반 얼굴 호흡기를 포함하여 사용 가능한 여러 가지 호흡기를했습니다. </li></ol> 3. 생물 학적 안전 캐비닛의 준비 <ol><li> BSL3에서 표면을 소독하고 공기 패널에서 살균제 약 6인치로 적셔 종이 타월을 배치하여 생물 학적 안전 캐비닛 (BSC)를 준비합니다. </li><li> 폐기물 왼쪽 용기, 그리고 오른쪽에 작은 nephlometer 및 vortexer를 놓습니다. </li><li> Inoculating 루프, pipetors 및 도움말은 종이 타월 옆에 배치됩니다. </li><li> 유기체 (국물 또는 고체 매체에)와 종이 타월에 맥팔랜드 0.5 표준 랙에 넣으십시오. </li></ol> 4. nephelometer의 보정 <ol><li> nephelometer 호수 트윈 유리 구슬 튜브를 삽입하고 생리 트윈 공백을 사용하여 0으로 악기를 설정합니다. </li><li> nephelometer에 맥팔랜드 0.5 표준을 놓고 제조 업체의 지침에 따라 보정. </li></ol> 5. Inoculum의 준비 <ol><li> ≥ 0.5 맥팔랜드 표준 얻을 때까지 적절하게 표시된 호수 트윈 유리 비드 관에 멸균 루프를 사용하는 고체 배지에서 BSC, 스크 레이프 식민지에서 근무. </li><li> 시각 샘플을 검사하고, 맥팔랜드 표준 비교합니다. </li><li> 30-60초을위한 와동 샘플. </li><li> (타이머 설정) 15 분 정착 inoculum하실 수 있습니다. </li></ol> 6. microtiter 접시의 접종 <ol><li> 단계 4.1 및 4.2 맥팔랜드 0.5 보정 nephelometer를 사용하여 살린 트윈의 국물로 inoculum를 조정합니다. 이것은 30 분 (당 제조 사양) 이내에 완료해야합니다. </li><li> 200 μL pipetter과 살린 비드 트윈 튜브의 TOP 3분의 1 차에서 inoculum 100 μL를 제거하는 데 오랜 에어로졸 방지 pipet 팁을 사용합니다. 미들 7H9의 용기를 예방. 천천히 와동 20 초 동안 주사 7H9 국물. </li><li> 제조 업체의 패키지에서 대서양 표준시의 microtiter 플레이트를 제거합니다. 박 패키지가 손상된 경우 건조은 파란색 또는 경우 사용하지 마십시오. </li><li> 멸균 튜브에 멸균 물의 50 μL를 pipetting하여 1시 50분 희석의 준비에 나중에 사용하기 위해 1시 50분 희석 튜브를 준비합니다. </li><li> 신중하게 여물로 7H9 국물의 inoculum 잔. 에어로졸을 생성하지 마십시오. </li><li> 당신이 직면하고있는 레이블로 대서양 표준시의 microtiter 접시를 놓습니다. </li><li> 각각 잘 수있는 inoculum 100 μL를 추가하는 12 채널 pipetter을 사용합니다. </li><li> 식민지 카운트에 대한 dilutions 준비 : <ol type="a"><li> 1분의 1 희석는 &quot;1분의 1&quot;이라는 7H10 판 1 μl 루프를 줄이하여 잘 긍정적인 컨트롤 (H12)에서 준비가되어 있습니다. </li><li> 50분의 1 희석이 잘 긍정적인 컨트롤 (H12)에서 1 μl 루프를 inoculating 이상 준비 수관 50 μl와 튜브에 그것을 소용돌이로 준비가되어 있습니다. 번호판이 희석의 리크 1 μL은 &quot;50분의 1&quot;(으)로 분류. </li></ol></li><li> 적절하게 레이블이 붙은 혈액 한천과 7H10 플레이트 및 격리를위한 행진에 50 μL를 pipetting하여 여물에서 순도 접시의 예방. </li><li> 장소대서양 표준시의 microtiter 플레이트에 접착제 인감. 인감의 백색 다시를 사용하여 충분한 씰링을 보장하기 위해 각 자에 단단히 누르십시오. 주름을 피하십시오. </li><li> 열 인감 또는 테이프와 비닐 봉지와 가까이에 tuberculocidal 방역 및 장소 microtiter 플레이트로 적셔 종이 타월로 닦아하여 microtiter 접시를 소독. <ol type="a"><li> 비닐 봉투가가 기본 접시의 누출 또는 유출되어야 모든 국물이 들어 있는지 확인하기 위해 보조 컨테이너 역할을합니다. </li></ol></li></ol> 7. 자료와 생물 학적 안전 캐비닛의 살균 <ol><li> 에어로졸 생산을 방지하기 위해 폐기 용기에 넣어 부드럽게하기 전에 주사 여물 위에 또 다른 여물을 놓으십시오. </li><li> tuberculocidal 살균제로 닦고하여 nephlometer, vortexer, 접시, 튜브 등 모든 자료를 소독하고 BSC에서 제거하기 전에 제조 업체의 지시에 따라 소독을 확보하기 위해 적절한 시간을 기다립니다. 예를 들어, Prospray 우리가 사용하는 tuberculocidal 살균제는 mycobacteria 죽일 15 분 필요합니다. </li><li> autoclaving 전에 생물 학적 가방 날인의 폐기 컨테이너를 놓습니다. </li></ol> 8. 잠복 <ol><li> 플레이트는 아래 미디어 incubated되어야하며 치료는 어떤 액체를 흘리고 또는 물 튀기지 않도록 주의해야한다. </li><li> BSL2 연구실에 50-10%의 CO 2 5~10%의 CO 2와 35-37 ° C 배양기에서 대서양 표준시의 microtiter 플레이트 판, 7H10과 혈액 한천 플레이트를 품어. <ol class="lalpha"><li> 접시 잘 밀봉하여 내부 봉인된 비닐 봉지이기 때문에 저희 연구실에서는이 허용됩니다. </li></ol></li><li> 더 이상 2보다 microtiter 대서양 표준시 플레이트는 제조 업체의 지침에 따라 보육에 쌓아해야합니다. </li></ol> 9. 대표 결과 결과의 해석 : 실드 대서양 표준시 microtiter 접시 수동 지원 또는 Vizion (트렉 진단) 등의 자동 플레이트 리더와 함께 거울을 사용하여 읽을 수 있습니다. 접시는 즉시 접종 후 칠일로 검사하실 수 있습니다. 그들은 (예, 잘 하단의 세포의 보증금을 가지고) 긍정적인 있는지 확인하기 위해 위치 H11와 H12의 성장 제어 우물을 검사합니다. 성장 제어 우물이 긍정적하지 않는 경우, 접시를 reinc​​ubate 및 성장 제어 우물은 긍정 때까지 (예, 하루 10, 일 14) 정기적으로 다시 검사하십시오. <ol type="a"><li> 미러 읽기 : <ol type="i"><li> 장소는 플랫폼에 대서양 표준시 microtiter 접시를 봉인; </li><li> 배양 7 일 후 성장 제어 우물을 읽고, 이것은 부정, reinc​​ubate 대서양 표준시 microtiter 접시 경우; </li><li> 성장 컨트롤이 허용하는 경우, 다음 약물이 포함된 우물을 참조하십시오. 각 약물에 대한 내용은 성장 컨트롤과 함께 마약 우물을 비교하여 더 성장이없는 최초의 희석을 기록합니다. </li><li> 성장 탁도 또는 우물의 하단에있는 세포의 보증금으로 나타납니다. </li></ol></li><li> 악기 읽기 : 제조 업체의 지침을 따르십시오, 아래의 지침은 읽기가 Vizion 플랫폼과 할 것입니다 방법에 대한 예제입니다 <ol type="i"><li> 로그인 </li><li> &quot;MYCOTB&quot;대서양 표준시 플레이트 소프트웨어를 사용합니다. </li><li> 사용자가 직면하고있는 바코드와 함께 밀봉 플레이트를 놓습니다. 성장 탁도 또는 우물의 하단에있는 세포의 보증금으로 나타납니다. </li><li> 성장 컨트롤을 읽고, 이것은 부정, reinc​​ubate 대서양 표준시 microtiter 접시 경우, 긍정적인 성장을 표시하지 않는 각 약물의 첫 잘으로 화면을 감동하여 번호판을 읽으면. </li></ol></li></ol> 순도 번호판을 읽어 보시기 바랍니다. 혈액 한천 플레이트 48 시간 이후에는 성장을 보여주지해야하며 7H10 순도 판 하나의 유기체 유형의 합류 성장이 있어야합니다. 오염된 경우에는 대서양 표준시 microtiter 테스트는 순수한 문화에서 반복해야합니다. 물론 아래의 테이블을 사용하여 긍정적인 컨트롤에서 콜로니 수를 계산 : 콜로니 카운트 키 <table border="1"><tbody><tr><td> # 식민지는 &quot;1분의 1&quot;(으)로 분류 접시에 계산 </td><td> # 식민지는 &quot;50분의 1&quot;(으)로 분류 접시에 계산 </td><td> Inoculum 콜로니 개수 (cfu / ML) </td></tr><tr><td> &lt;50 </td><td> 0 </td><td> &lt;5 X 10 4 </td></tr><tr><td> 50-100 </td><td> 0-2 </td><td> 5 X 10월 4일에서 1일까지 X 10 5 </td></tr><tr><td> &gt; 100 </td><td> ≤ 10 </td><td> 1 X 10월 5일부터 5일까지 X 10 5 </td></tr><tr><td> &gt; 100 </td><td> &gt; 10 </td><td> &gt; 5 X 10 5 </td></tr></tbody></table> Mycobacterium tuberculo의 일부 변종의 패러 aminosalicylic 산성과 함께이 후행 수 있습니다언니. 이 알약을 모든 우물을보고하고 끝점으로 다음 희석 같은 크기 펠렛을 가지고 처음으로 잘 사용하여 해석될 수있다. 저항이 약물로 드문 경우입니다. <img src="/files/ftp_upload/3094/3094fig1.jpg" alt="그림 1"/> 그림 1. MYCOTB 플레이트를 사용하는 대표적인 결과. <table border="1"><tbody><tr><td> 약물 약어 </td><td> 마약 </td><td> 마이크 μ / ML </td></tr><tr><td> OFL </td><td> ofloxacin </td><td> 16 </td></tr><tr><td> MXF </td><td> moxifloxacin </td><td> 8 </td></tr><tr><td> RIF </td><td> rifampin </td><td> &gt; 16 </td></tr><tr><td> AMI </td><td> amikacin </td><td> 0.5 </td></tr><tr><td> STR </td><td> 스트렙토 마이신 </td><td> 16 </td></tr><tr><td> RFB </td><td> rifabutin </td><td> 8 </td></tr><tr><td> PAS </td><td> P - aminosalicylic 산성 </td><td> ≤ 0.5 </td></tr><tr><td> ETH </td><td> ethionamide </td><td> 5 </td></tr><tr><td> CYC </td><td> cycloserine </td><td> 4 </td></tr><tr><td> INH </td><td> isoniazid </td><td> 2 </td></tr><tr><td> KAN </td><td> kanamycin </td><td> 1.2 </td></tr><tr><td> EMB </td><td> ethambutol </td><td> 4.0 </td></tr></tbody></table>

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+Clinical+Mycobacteriology+Laboratory+in+the+Diagnosis+and+Management+of+Tuberculosis+in+Low+Prevalance+Settings.">The Role of the Clinical Mycobacteriology Laboratory in the Diagnosis and Management of Tuberculosis in Low Prevalance Settings.</a> J Clin Microbiol. , (2010).</li><li>Kam, K. M., Sloutsky, A., Yip, C. W., Bulled, N., Zingol, M., Espinal, M., Kim, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+critical+concentrations+of+second-line+anti-tuberculosis+drugs+with+clinical+and+microbiological+relevance.">Determination of critical concentrations of second-line anti-tuberculosis drugs with clinical and microbiological relevance.</a> Int J Tubercl Lung Dis. 14, 282-288 (2010).</li><li>Woods, G. L., Warren, N. G., Inderlied, C. B., Murray, P. R., Barron, E. J., Jorgensen, J. H., Landry, M. L., Pfaller, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility+Test+Methods:+Mycobacteria,+Nocardia,+and+Other+Actinomycetes.">Susceptibility Test Methods: Mycobacteria, Nocardia, and Other Actinomycetes.</a> Manual of Clinical Microbiology. , 1223-1247 (2007).</li><li>Bergmann, J. S., Woods, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+ESP+Culture+System+II+for+Testing+Susceptibilities+of+Mycobacterium+tuberculosis+Isolates+to+Four+Primary+Antituberculosis+Drugs.">Evaluation of the ESP Culture System II for Testing Susceptibilities of Mycobacterium tuberculosis Isolates to Four Primary Antituberculosis Drugs.</a> J Clin Microbiol. 36, 2940-2943 (1998).</li></ol>

긍정적인 성장 컨트롤을 가진 대서양 표준시의 microtiter 접시의 예제는 그림 1에 나와 있으며, 각각의 약물에 대한 끝점 MIC (μg / ML)이 돌고있다. 우리 연구실에서 수행 연구는 대서양 표준시의 microtiter 플레이트 방법은 Mycobacterium의 결핵의 자화율 시험을위한 황금 표준 한천 비중 방법에 해당하는 것을 지적했다. 대서양 표준시 microtiter 플레이트와 한천 비율 방법 14에서 21 일 동안 최종 해석 전통 한천 비율 방법과 비교하여 결과의​​ 빠른보고가 microtiter 대서양 표준시 microtiter 플레이트와 함께 얻은 것입니다, respectively.The 대서양 표준시 microtiter 플레이트 먼저 테스트의 장점을 가지고 있으며, 두번째 라인 모성 종자와 과도한 지연을 방지할 수 있습니다 동시에 마약. 대서양 표준시 microtiter 접시 또한 APM보다 설정과 읽기 훨씬 쉽게와 같은 VIZION 같이 수동으로 거울과 세미 자동화 시스템 모두는 접시를 읽는 데 사용할 수 있습니다. 결핵 약제 내성 검사에 대한 MIC 값이 있는지의 여부는 특히 &quot;경계선&quot;자화율있는 사람 격리를 위해, 전통적인 중요한 농도 값에 비해 의사들에게 유용한 새로운 정보를 제공할 수 있습니다.

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 대서양 표준시 microtiter 접시와 접착제 도장 </td><td> 트렉 진단, 클리블랜드 오하이오 </td><td> MYCOTB </td></tr><tr><td> 맥팔랜드 0.5 표준 </td><td> 트렉 진단, 클리블랜드, 오하이오 </td><td> E1041 </td></tr><tr><td> 미들 7H10 플레이트 </td><td> 어부 </td><td></td></tr><tr><td> 혈액 아가르 플레이트 </td><td> 어부 </td><td></td></tr><tr><td> 멸균은 μL 1 루프 </td><td> 어부 </td><td></td></tr><tr><td> 무균 루프 10 μL </td><td> 어부 </td><td></td></tr><tr><td> Sensititre 살린 - 트윈 유리 구슬 국물 </td><td> 트렉 진단, 클리블랜드, 오하이오 </td><td> 27,143 </td></tr><tr><td> 7H9 OADC 맛을 </td><td> 트렉 진단, 클리블랜드, 오하이오 </td><td> T3440 </td></tr><tr><td> 롱 Pipet 도움말 </td><td> 어부 </td><td></td></tr><tr><td> 무균 트러프 </td><td> 트렉 진단, 클리블랜드, 오하이오 </td><td></td></tr><tr><td> M. 결핵 </td><td> ATCC </td><td> 27,294 </td></tr><tr><td> Pipeters : 1000, 200, 20μL </td><td> 어부 </td><td></td></tr><tr><td> 팁 1000, 20, 20 μL </td><td> 어부 </td><td></td></tr><tr><td> 멀티채널 Pipetter </td><td> 어부 </td><td></td></tr><tr><td> 보육 50~10%의 CO 2 </td><td> 어부 </td><td></td></tr><tr><td> 비닐 봉투, 작은, CO 2 투과 </td><td> 폴리 플라스틱, 델러노, PA </td><td></td></tr><tr><td> 결핵 살균제 (예 : ProSpray C - 60) </td><td> Certol 국제 상업 도시, CO </td><td> PSC60/32 </td></tr><tr><td> 미러 박스 리더 </td><td> 어부 </td><td></td></tr></tbody></table>

antimicrobial susceptibility testing of mycobacterium tuberculosis complex for first and second line drugs by broth dilution in a microtiter plate format

항균 저항의 급속한 탐지 방지 Mycobacterium 결핵 (MTB)의 증가를 제어하기 위해 노력 중요합니다. MTB의 항균 자화율 테스트 (대서양 표준시)는 전통적으로 비례의 한천 방법으로 또는 / BACTEC (백톤 디킨슨, 스파크, MD), VersaTREK (트렉 진단, 클리블랜드, OH) 또는 BacT와 같은 악기에 macrobroth 테스트에 의해 수행되었습니다 알림 (bioMérieux, 하젤우드, MO). 고려하면서 한천 비례 방법은, AST의 &quot;황금&quot;표준은 집중적인 노동하고 마약없는 한천에 비해 약물이 포함된 한천에 식민지 카운트를 수행하여 저항의 계산이 필요합니다. 약물없는 매체에 비해 약물이 포함된 매체에 ≥ 1 % 성장이있다면, 유기체는 그 약물에 강한 것으로 간주됩니다. macrobroth 방법은 계측 및 첫 번째 라인 의약품에 대한 시험 브레이크 포인트 ( &quot;중요한&quot;) 약물 농도를 (isoniazid, ethambutol, rifampin, 그리고 pyrazinamide)가 필요합니다. 방법은 96 잘 microtiter 플레이트 형식으로 상업적으로 사용할 수 있습니다 여기에 설명된 [MYCOTB (트렉 진단)]과 amikacin (둘 다 첫 번째 (isoniazid, rifampin, ethambutol)와 두 번째 라인 의약품을 포함한 결핵의 치료에 사용되는 12 antimicrobials의 증가 농도를 포함, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, 패러 aminosalicylic 산, rifabutin 및 스트렙토 마이신). Pyrazinamide, 첫 번째 라인 약물은 산성 시험 조건에 대한 필요로 인해 microtiter 접시에 포함되지 않습니다. microtiter 시스템의 장점 모두 설정의 용이성 전통 한천 비중 (21 일)와 비교하여 빠른 시간에 차례 (14 일) 등이 있습니다. 또한, 접시는 국물 또는 고체 매체 중 하나를 사용하여 준비 inoculum에서 설정할 수 있습니다. microtiter 접시 형식이 새로운과 MTB가 실험실에서 고유의 안전 문제를 제시 있기 때문에,이 프로토콜은 안전하게 설치하는 방법을 설명하기 때문에, 부화 및 microtiter 접시를 참조하십시오.

Watch this Scientific Journal Video about Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format at J

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

Mycobacterium 결핵의 항균 자화율 시험에 도전하지만, 환자 치료를 위해 중요합니다. 이 microtiter 플레이트 형식 12 antimycobacterial 마약 테스트를 제공하며 적절한 치료에 도움이됩니다 최소 억제 농도 (MIC) 값을 제공합니다.

Microtiter 플레이트 형식의 맛을 희석하여 첫 번째와 두 번째 라인 의약품 Mycobacterium 결핵의 복합 항균 자화율 테스트

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

antimicrobial-susceptibility-testing-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

38.0K 조회수.  Mayo Clinic. Mycobacterium 결핵의 항균 자화율 시험에 도전하지만, 환자 치료를 위해 중요합니다. 이 microtiter 플레이트 형식 12 antimycobacterial 마약 테스트를 제공하며 적절한 치료에 도움이됩니다 최소 억제 농도 (MIC) 값을 제공합니다.

비디오: Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

모니터링 형성과 Antifungal 자화율 테스트를위한 96 자 Microtiter 플레이트 기반 방법 캔디다 albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

연구

면역학 및 감염병학

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

의 샘플 준비<em&gt; Mycobacterium 결핵</em핵 자기 공명 Metabolomic의 연구&gt; 압축을 풉니 다

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

의 성장 결핵은 Mycobacterium Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

다중 약물 내성을 보여<em&gt; 결핵균</em&gt; 증폭 마이크로 어레이

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

빠른 세균의 식별 및 항생제 감수성 테스트를위한 혈액 문화 펠렛의 제조

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Dalbavancin에 대한 참조 국물 Microdilution 방법<em&gt; 체외</em호기성 박테리아의 성장&gt; 감수성 테스트

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

에 대한 기능성 연구를위한 3D 인간의 폐 조직 모델<em&gt; 결핵균</em&gt; 감염

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

세포 내 타겟팅 억제제의 효능 및 세포 독성 검사를위한 시스템<em&gt; 결핵균</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

18FDG PET/CT 영상 공부 결핵균 감염 및 비 인간 영장류에서 치료 도구로 서의 분석

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

기자 효소 형광와 쥐에서 이미징 결핵균

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...