Name: anti-virulent disruption of pathogenic biofilms using engineered quorum-quenching lactonases
Uploaded: 2016-01-01T13:00:00.000+00:00
Duration: 7 min 47 s
Description: Watch this Scientific Journal Video about Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases at JoVE.com

This work was supported by grants from the Academic Research Fund of the Ministry of Education, and the National Medical Research Council and the National Research Foundation, Singapore.

A의 biofilm 형성 1. 크리스탈 바이올렛 정량 baumannii가 S1 <ol><li> A의 5 ml의 문화를 성장 원성 국물에 baumannii가 S1 (LB) 16 시간 동안 진탕 배양기 (220 RPM)에서 30 ° C에서 (트립 톤 10g / L, 효모 추출물 5g / L). </li><li> A의 문화를 조정 원하는 OD 0.8 600 baumannii의 (S1). 96 웰 플레이트를 사용하여, 박테리아 배양 접종 : GKL 정제 효소 (40 ㎎ / ㎖) 10 ㎕를 함유 신선한 LB으로 (1 내지 100 희석); 새로운 문화의 최종 부피는 100 μL입니다. </li><li> 뿐만 아니라 제어 문화를 준비; 이 모든 효소가 포함되지 않습니다. 복제의 수를 수득 된 것과 유사한 조건을 반복한다. </li><li> 뚜껑 판을 덮고 밀봉 (10) L 플라스틱 용기에 넣습니다. 부드럽게 매체를 제거하기 전에 3 시간 동안 30 ℃에서 플레이트를 배양한다. </li><li> 잘 신선한 LB 배지의 또 다른 100 μl를 추가하고 30 ℃에서 21 시간 동안 접시를 품어. </li&gt; <li> 배양의 두 번째 기간 후, 부드럽게 모든 용지를 제거합니다. 200 μL 멸균 수와 플랑크톤 박테리아 세포를 씻으십시오. 세탁시 세포에 최소한의 방해가 있는지 확인합니다. </li><li> 각 웰에 1 % 크리스탈 바이올렛 용액 100 μl를 추가하고 실온에서 15 분 동안 품어. 200 μL 멸균 수로 잘 세척하여 크리스탈 바이올렛 솔루션을 제거합니다. 두 번 이상 세척을 반복합니다. </li><li> 각 웰에 33 % 아세트산 100 μl를 추가하고 부드러운 동요와 15 분 동안 품어; 이는 염료를 용해한다. </li><li> 600 nm에서의 크리스탈 바이올렛의 흡광도를 측정함으로써 바이오 필름 형성의 양을 정량. 크리스탈 바이올렛의 양은 형성된 바이오 필름의 양에 비례한다. </li></ol> A. 2. 공 초점 레이저 주사 현미경 baumannii가 S1 바이오 필름 <ol><li> A의 5 ml의 문화를 성장 16 시간 동안 진탕 배양기 (220 RPM)에서 30 ° C에서 LB에서 baumannii의 (S1). </li><li> 광고A의 바로 문화 원하는 OD 0.8 600 baumannii의 (S1). 35mm 유리 바닥 μ-접시를 사용하면, 세균 배양 접종 : 정제 GKL 효소 (40 ㎎ / ㎖)의 30 μl를 포함하는 신선한 LB에 (1 100 희석); 새로운 문화의 최종 부피는 1 ml의입니다. </li><li> 뚜껑 μ-접시를 덮고 밀봉 (10) L 플라스틱 용기에 넣습니다. 부드럽게 용지를 제거하기 전에 3 시간 동안 30 ° C에서 μ-접시를 품어. 1 ML의 총 부피에 데려 정제 GKL 효소와 신선한 매체의 30 μl를 추가합니다. 30 ° C에서 또 다른 21 시간 동안 품어. </li><li> 단계를 반복 2.3 및 30 ℃에서 또 다른 24 시간의 μ-접시를 품어. 그런 다음, 부드럽게 용지를 제거합니다. </li><li> μ-접시에 행크의 균형 소금 용액 (HBSS)에 용해 5 μg의 / ㎖ 알렉스 FLUO 488 - 복합 밀 배아 응집소 (WGA)의 500 μl를 추가하고 30 분 동안 37 ℃에서 배양; 이렇게 형성된 바이오 필름을 염색한다. T 염색 액을 제거하고 세척그는 μ-요리 HBSS 2 ㎖과 함께합니다. 세척 단계를 한 번 더 반복한다. </li><li> 3.7 % 포름 알데히드 HBSS에 용해시키고, 30 분 동안 37 ℃에서 배양의 500 μl를 추가; 이 μ-요리 상 생물막을 수정합니다. HBSS 2 ㎖로 한 번 μ-접시를 세척 한 후 완전히 솔루션을 제거합니다. μ 디시 생물막 고정 전에 CLSM 촬상 4 ℃에서 어둠 속에서 저장 될 수있다. </li><li> CLSM 이미징 및 분석을 위해, 스택 당 0.21 μm의 간격으로 이미지 당 97 스택을 생성하기 위해 63 배 배율을 사용합니다. </li></ol>

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The authors declare that they have no competing financial interests.

실험의 두 세트에서, A. baumannii의 S1은 박테리아 (15)에 의해 형성된 바이오 필름의 양을 줄일 수있다 높은 염 농도의 NaCl없이 LB 배지에서 배양 하였다. 이러한 인공물의 존재는 생물막의 형성 량뿐만 아니라 다른 처리 조건에 걸쳐 정수 담금질 효소의 영향을 과소 있었다. 촉매 비활성 효소의 사용은 효소 격리의 가능한 효과를 제거하기 위해 음성 대조군 중요하다. 비활성 효소 쿼럼 분자를 격리시키는 경우에도 1은 그림, 생물막 형성은 중단되지 않습니다. A. baumannii의 S1은 96 웰 플레이트의 웰에 공기와 액체의 계면 섬세 생물막 링형 구조를 형성한다. 따라서, 세균의 생물막 우발적 제거를 방지하기 위해 매체의 제거 및 세척 단계 동안 과도한 교반을 피하기 위해 중요하다. LB 미디어는 각 배양 후 제거플랑크톤 세포를 제거 할 수 있습니다. 또한, 96- 웰 플레이트에서 기류의 교란을 최소화하고 biofilm 형성 호의 마이크로 혐기성 환경을 만들기 위해 10 L 플라스틱 용기에 넣었다. 96 웰 플레이트에서 바이오 필름의 정량은 각 웰에 첨가 크리스탈 바이올렛의 양에 따라 달라집니다. 과량 크리스탈 바이올렛은 웰에 첨가되는 경우에는, 세척 단계의 수를 증가시킬 수있다. 크리스탈 바이올렛 염색 실험은 생물막 중단에 쿼럼 담금질 효율성의 상대 비교를 제공합니다. 크리스탈 바이올렛 염색이 바이오 필름의 형성을 측정하는 정량적이지만, 단지 정수 담금질 효소의 촉매 효율면에서 반 정량적이다. 정확한 통계적 비교를 위해 충분한 샘플 크기는 다른 처리 군에 중요하다. 아웃 라이어는 결과의 허위 진술을 방지하기 위해 제거해야합니다. 공 초점 이미지 및 세균성 생물막의 분석이다 USEF쿼럼 담금질 효소의 질적 효과를 평가하기위한 UL 도구입니다. 사용자 (활성 또는 비활성) 투여 정수 담금질 효소의 유형을 인식하는 경우에는 분석에 생물막을 선택하는 과정은, 바이어스 전위 채널 일 수있다. 바이어스의 가능성을 피하기 위해, 실험 효소 사용자로부터 정보를 제외하거나 선택을위한 임의의 방법을 사용하도록 설계 될 수있다. 편견 선택 전략은 CLSM에 의해 생물막 형태학의 더 나은 양적 비교를 할 수 있습니다. 그럼에도 불구하고, 이는 세균에 생물막 정수 담금질 효소의 결과를 평가하기위한 방법을 설명 첫 번째 연구이다. 크리스탈 바이올렛 염색 생물막 중단에 쿼럼 담금질 효소의 유틸리티를 보여 효소 &#39;MODI의 operandorum은 세포 수 및 독성 인자의 발현을 감소에만 국한되지 않습니다 것을 밝혔다. 한편, CLSM 분석에 의해 결정 형태 학적 변화도 가능 molecul에 대한 통찰력을 제공 할 수 있습니다이 효소의 AR 목표로하고있다. 현재 실험 세균 생물막에 효소를 예비 처리의 효과를 조사하기 위해 설계되었지만, 프로토콜은 세균 성장​​ 후 효소를 첨가하여 예비 성형 된 생물막에 효소의 효과를 평가하기 위해 혈청을 사용하여 생리 학적 조건 하에서 그것의 능력을 검사하기 위해 수정 될 수있다 문화 -like 조건. GKL 효소를 연구하는 데 사용되는 프로토콜은 생물막의 중단의 관계를 조사하기 위해 다른 정수 담금질 효소 및 병원균으로 확장 될 수있다.

감염성 질환에 대한 치료 옵션은 항생제 1 광범위한 면역 다제 내성 박테리아에 급격한 증가에 의해 복잡 하였다. 내성 세균 - 매개 감염에서 높은 이환율 및 사망률, 신약 개발 과정을 확대 및 / 또는 치료 옵션을 향상시키기 위해 더 항균 대안을 모색 할 필요가있다. 최근에, 항 병원성 접근법 따라서 저항 메커니즘 (2)의 위험을 완화, 비 살균 방법을 통해 독성을 예방 가능성 주어진 관심을 얻고있다. 쿼럼 센싱 박테리아 독성이 신호 현상의 중단에 &#39;마스터 스위치&#39;는 발병 3에 대한 유망한 항 독성 방법이다. 세균 집단 임계 밀도에 도달 한 후 독성의 발현은 세포 외 환경에서 정수 분자의 축적을 필요로한다. 로 현럼 분자는 자신의 동족 수용체와 결합하면 독성 인자의 활성화뿐만 아니라 항생제 내성 및 biofilm 형성 (4)과 관련된 유전자지도, 세포 내 매트릭스로 다시 확산. 일반적으로, 정족수 감지 중단에 차 대사 경로에 영향을주지 않고 쿼럼 분자와 수용체의 상호 작용을 억제하는 것을 포함한다. 따라서,이 세포 성장에 직접적인 의미가 없습니다. 체력이 저하되지 않기 때문에, 발전 및 치료에 대해 5 저항을 얻기위한 최소한의 박테리아 선택 압력이 존재한다. 또한, 정수 감지 중단은 항균제으로부터 보호를 제공하고, 면역 반응을 호스팅 생물막 형성의 경우와 같이, 고유 세균성 보호 메커니즘을 방해 할 수있다. 그것은 지구에 미생물의 99 %가 일 이내 거주 미생물에 중요한 생존의 장점을 부여, 복잡한 바이오 필름 같은 행렬에 존재하는 것으로 추정된다ESE 구조 6. 더욱 중요한 것은,이 고착 영역의 형성은 가장 지속적인 만성 병원 내 감염 (7)의 원인이다. Acinetobacter baumannii의 글로벌 병원 내 감염과 연관되어 있고 그 독성이 크게 정수 감지에 기인 주요 인간 병원체 중 하나 인 - 매개 biofilm 형성 8. 정원 담금질 효소는 그람 음성균 (9)에 의해 생성되는 N 아실 호모 세린 락톤 (AHLs)로 공지 된 화합물의 그룹을 대상으로하여 정수 - 매개 신호 전달을 방해에서 성공적으로 사용되어왔다. 여러 연구는 또한 생물막 10,11 독성 인자의 발현 및 세포 수의 감소를 통해 박테리아 발병을 차단하는이 효소의 용도에 확대. 불행하게도, 세균성 병원체에 의한 바이오 필름 형성에 대한 쿼럼 담금질 효소의 효과적인 사용의 만져서 알 수있는 데모의 부족이 남아있다. 그만큼다시 A를 방해, 쿼럼 억제제 (AHL 유사체), 대신 쿼럼 담금질 효소를 사용하려고왔다 baumannii의 생물막 형성 (12). 작은 분자 억제제를 사용하는이 방법은 유효한 방법이지만, 병진 용도에서의 생체 이용률을 유지하는 것은 도전이 될 수 있습니다. 효소는 치료 효과에 대한 생의학 장치의 표면에 고정화 향해 더 의무만큼 반대로, 촉매 정수 담금질 효소의 사용은 생체 이용률 문제를 회피 할 수있다. 여기, 우리는 크리스탈 바이올렛 염색 및 공 초점 레이저 주사 현미경 (CLSM)를 사용하여, Geobacillus의 kaustophilus (GKL) 세균 바이오 필름 ​​형성에 13에서 설계 쿼럼 담금질 lactonases의 효과에 대한 평가에 대해 설명합니다. 이 연구는 임상 적으로 A의 생물막 중단되는 최초의 성공적인 데모입니다 쿼럼 담금질 효소를 사용하여 baumannii의 S1 변형. 방법 설명본 연구에서 병원성 그람 음성 세균에 대한 후속 치료 개발 노력에 다른 쿼럼 담금질 효소의 효능을 평가하는 데 유용합니다.

<table><tbody><tr><td>Tryptone&amp;nbsp;</td><td>BD</td><td>211705</td><td></td></tr><tr><td>Yeast Extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>96-well plate</td><td>Costar</td><td>3596</td><td></td></tr><tr><td>Crystal Violet</td><td>Sigma-Aldrich</td><td>C6158</td><td></td></tr><tr><td>Acetic Acid</td><td>Lab-Scan</td><td>PLA00654X</td><td>Caution: Flammable</td></tr><tr><td>&amp;mu;-Dish</td><td>Ibidi</td><td>80136</td><td></td></tr><tr><td>Alex Fluo 488-conjugated WGA</td><td>Invitrogen</td><td>W11261</td><td></td></tr><tr><td>Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>Invitrogen</td><td>141475095</td><td></td></tr><tr><td>Formaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td>Caution: Corrosive&amp;nbsp;</td></tr><tr><td>Synergy HT Microplate Reader</td><td>BioTek</td><td></td><td></td></tr><tr><td>1X-81 Inverted Fluorescence Microscope</td><td>Olympus</td><td></td><td></td></tr></tbody></table>

anti-virulent disruption of pathogenic biofilms using engineered quorum-quenching lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

야생형 GKL 및 개선 GKL 이중 돌연변이 (E101G / R230C를) 크리스탈 바이올렛 정량 실험에서는 두 정수 담금질 효소 생물막 형성에 방해 가능성을 보여주기 위해 사용되었다. 두 효소 lactonase 대해 활성을 보여주기 위해 도시 한 3- 하이드 록시 데카 노일-L 호모 세린 락톤 (3-OH-10 C -HSL), A.에 의해 사용되는 주요 정수 분자 baumannii가 (S1) (14). 생물막 중단의 올바른 평가를 위해, (이전 AHL 리간드를 격리시키는하지 않음) 각각의 촉매 비활성 효소는 즉시 컨트롤 (GKL의 D266N 돌연변이와 GKL E101G / R230C / D266N 돌연변이)로 하였다. 야생형 A.를 사용 게다가 baumannii가 S1, 돌연변이 Δ abaI 변형 (AHL 합성 효소 결핍)의 생물막 형성 능력도 테스트했다. 쿼럼 담금질 효소, 야생형 GKL과 GKL E101G / R230C 돌연변이는 모두 크게 사전에 생물막 형성을 감소 할 수 있었다처리 A. baumannii가 S1 문화 (N = 10 0.0001 ≤ P 값) (그림 1). 그럼에도 불구하고, 두 효소 생물막 중단의 범위는 3-OH-C (10) -HSL에 대한 효능에 비례하지 않는다. GKL E101G / R230C 돌연변이의 이직률 (K 고양이) 3-OH-C (10) -HSL (14)에 대한 야생형 GKL에 비해 6 배 더 빠르게입니다. 그러나, 효소 간의 생물막 붕괴의 정도에 차이가 두 배이다. <img alt="그림 1" src="/files/ftp_upload/53243/53243fig1.jpg" /> 그림 1. A. baumannii의 바이오 필름 ​​파괴 분석. biofilm 형성는 크리스탈 바이올렛 염색에 의해 측정 하였다. 적색 열이 야생형 A. 의해 형성된 바이오 필름의 양을 나타낸다 쿼럼 담금질 lactonases의 추가없이 baumannii가와 Δ abaI 돌연변이. 블루 열은 AMO를 나타냅니다야생형 A.에 의해 형성된 바이오 필름의 UNT 비활성 GKL의 D266N 돌연변이, 야생형 GKL, 비활성 GKL E101G / R230C / D266N 돌연변이와 GKL E101G / R230C 돌연변이 : 네 가지 GKL 효소의 존재 baumannii가. ****, P-≤ 0.0001의 값입니다. 항균제와 화학 요법 (58)의 허가를 재현, 1,802에서 1,805 사이 (2014). <a href="https://www.jove.com/files/ftp_upload/53243/53243fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> A의 공 초점 영상 baumannii의 S1 생물막의 형성은 이러한 정착 도메인의 구조적 형태에 정수 담금질 효과의 질적 및 양적 측정을 제공하기 위해 사용되었다. 향상된 GKL E101G / R230C 변이체 및 촉매 비활성 효소는 비교를 위해 사용 하였다. 차분 화상의 콘트라스트 (DIC) 이미지 (개선 GKL E101G / R230C 변이체 치료는 생물막의 크기의 감소를 초래한다는 Figur 나타났다예 2). 형광 이미지의 분석은 또한 개선 GKL E101G / R230C 변이체 (표 1)으로 처리 할 때 표면적, 바이오 매스 및 생물막의 평균 두께 감소가 있다는 것을 밝혔다. <img alt="그림 2" src="/files/ftp_upload/53243/53243fig2.jpg" /> A.도 2의 대표적인 공 초점 레이저 주사 현미경 이미지 baumannii가 생물막. A. baumannii의 생물막 비활성 GKL E101G / R230C / D266N 돌연변이 (A)와 GKL E101G / R230C 변이체 (B)로 처리하고 염색 하였다 렉 플 루어 WGA를 488-결합. 생물막 (왼쪽)와 생물막 (오른쪽)의 형광 이미지의 DIC 이미지는 대표 XY (센터), YZ (오른쪽)과 XZ (아래) 부분에 대한 표시됩니다. 항균제와 화학 요법 &lt;허가를 재현강한&gt; (58), 1802에서 1805 사이 (2014). <a href="https://www.jove.com/files/ftp_upload/53243/53243fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <table fo:keep-together.within-page="1"><tbody><tr><td></td><td colspan="3"> SD의 ± 값 </td></tr><tr><td> 특성 </td><td> 어떤 치료하지 </td><td> 비활성 돌연변이와 치료 </td><td> E101G / R230C 돌연변이와 치료 </td></tr><tr><td> 바이오 매스 (μm의 3 / μm의 2) </td><td> 2.57 ± 1.65 </td><td> 3.39 ± 1.33 </td><td> 1.37 ** ± 0.20 </td></tr><tr><td> 평균 두께 (μm의) </td><td> 3.68 ± 2.51 </td><td> 3.41 ± 1.31 </td><td> 1.21 ** ± 0.21 </td></tr><tr><td> 표면적 (μm의) </td><td> 235,920.59 ± 79,456.46 </td><td> 209,872.6 ± 115,094.7 </td><td> 115,354.9 * ± 7,630.3 </td></tr><tr><td colspan="4"> N = 10 이미지 스택. **, P ≤ 0.001; * P ≤ 0.05, 비활성 E101G / R230C / D266N 돌연변이 치료와 비교. </td></tr></tbody></table> 표 1. A. baumannii의 생물막 구조 정량.

Watch this Scientific Journal Video about Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases at JoVE.com

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

엔지니어링 정원 담금질 Lactonases를 사용하여 병원성 바이오 필름의 안티 악성 중단

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

anti-virulent-disruption-pathogenic-biofilms-using-engineered-quorum

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.7K 조회수.  Yong Loo Lin School of Medicine. Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

비디오: Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Microtiter 디시 Biofilm 형성 분석

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

연구

면역학 및 감염병학

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

단일 합성 Curli 오페론을 생성하여 공학 점착성의 박테리아를

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

생체공학

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

바이오 필름 관련 타겟팅<em&gt; 황색 포도상 구균</em&gt; 사용 레사 주린 기반 약물 감수성 분석

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

유학을위한 방법론<em&gt; B. 서브 틸리</em작은 분자 바이오 필름 억제제를 특성화 모델로&gt; 바이오 필름

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

A Platform of Anti-biofilm Assays Suited to the Exploration of Natural Compound Libraries

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of antibiotic-tolerant infections. Biofilms have displayed an exceptionally high tolerance for chemotherapy, which is thought to be multifactorial. For instance, the matrix provides a physical barrier that decreases the penetration of antibiotics into the biofilm. Also, cells within the biofilms are phenotypically diverse. Likely, biofilm resilience arises from a combination of these and other, yet unknown, mechanisms. All of the currently existing antibiotics have been developed against single-cells (planktonic) bacteria. Therefore, so far, a very limited repertoire of molecules exists that can selectively act on mature biofilms. This situation has driven a progressive paradigm shift in drug discovery, in which searching for anti-biofilms has been urged to occupy a more prominent place. An additional challenge is that there are a very limited number of standardized methods for biofilm research, especially those that can be used for large-throughput screening of chemical libraries. Here, an experimental anti-biofilm platform for chemical screening is presented. It uses three assays to measure biofilm viability (with resazurin staining), total biomass (with crystal violet staining), and biofilm matrix (using a wheat germ agglutinin, WGA-fluorescence-based staining of the poly-N-acetyl-glucosamine, PNAG, fraction). All the assays were developed using Staphylococcus aureus as the model bacteria. Examples of how the platform can be used for primary screening as well as for functional characterization of identified anti-biofilm hits are presented. This experimental sequence further allows for the classification of the hits based upon the measured end-points. It also provides information on their mode of action, especially on long-term versus short-term chemotherapeutic effects. Thus, it is very advantageous for the quick identification of high-quality hit compounds that can serve as starting points for various biomedical applications.

Biofilm infections show high tolerance towards chemotherapy. No single assay captures the complexity of biofilms. Instead, complementary assays are needed. We present a screening platform (developed for S. aureus) that combines assays for viability, biomass, and biofilm matrix. It allows anti-biofilm drug discovery, including the assessment of long-term chemotherapeutic effects.

천연 화합물 라이브러리의 탐험에 적합 안티 바이오 필름 분석 실험의 플랫폼

Biofilms are regarded as one of the most challenging topics of modern biomedicine, and they are potentially responsible for over 80% of ...

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

소프트 한천 오버레이 기법의 사용은 세균 생산 억제 화합물에 대한 화면으로

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

COMSTAT 소프트웨어를 사용하여 체외 생물막 아키텍처에 대한 항균제의 효과 정량화

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

PCR-플레이트 딥웰 마이크로플레이트 장치를 이용한 더 큰 박테리아 바이오필름 바이오매스의 생성

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are ...

Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their high resistance to most clean-in-place (CIP) procedures frequently used in processing plants. Therefore, it is imperative to develop new biofilm control strategies for the dairy industry. This protocol is aimed at evaluating the efficacy of organic peroxyacids (peracetic, perpropionic, and perlactic acids and a commercial peracetic acid-based disinfectant) for eradicating dairy biofilms using a combination of static and dynamic methods. All the disinfectants were tested on the strongest biofilm-producing bacteria in either a single or a mixed biofilm using the minimum biofilm eradication concentration (MBEC) assay, a static high-throughput screening method. A contact time of 5 min with the disinfectants at the recommended concentrations successfully eradicated both the single and mixed biofilms. Studies are currently ongoing to confirm these observations using the Center for Disease Control (CDC) biofilm reactor, a dynamic method to mimic in situ conditions. This type of bioreactor enables the use of a stainless-steel surface, which constitutes most industrial equipment and surfaces. The preliminary results from the reactor appear to confirm the efficacy of organic peroxyacids against biofilms. The combined approach described in this study may be used to develop and test new biological or chemical formulations for controlling biofilms and eradicating microorganisms.

This protocol describes an approach combining static and dynamic methods to evaluate the efficacy of organic peroxyacids for eradicating biofilms in the dairy industry. This approach may also be used to test the effectiveness of new biological or chemical formulations for controlling biofilms.

정적 방법과 동적 방법을 결합한 접근법을 사용하여 낙농 생물막을 박멸하기 위한 유기 과산화산의 효능 평가

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their ...

생물학

<meta charset="utf8"></head><body>Quorum Sensing Inhibition에서 Thiophenesulfonamides의 생물학적 평가

Synthesis and Assay of Vibrio Quorum Sensing Inhibitors

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In Vibrio species, the master quorum sensing regulators LuxR/HapR control hundreds of quorum sensing genes, many of which influence virulence, metabolism, motility, and more. Thiophenesulfonamides are potent inhibitors of LuxR/HapR that bind the ligand pocket in these transcription factors and block downstream quorum sensing gene expression. This class of compounds served as the basis for the development of a set of simple, robust, and educational procedures for college students to assimilate their chemistry and biology skills using a CURE model: course-based undergraduate research experience. Optimized protocols are described that comprise three learning stages in an iterative and multi-disciplinary platform to engage students in a year-long CURE: (1) design and synthesize new small molecule inhibitors based on the thiophenesulfonamide core, (2) use structural modeling to predict binding affinity to the target, and (3) assay the compounds for efficacy in microbiological assays against specific Vibrio LuxR/HapR proteins. The described reporter assay performed in E. coli successfully predicts the efficacy of the compounds against target proteins in the native Vibrio species.

<meta charset="utf8"></head><body>저자 스포트라이트: 인간과 수생 생물의 비브리오증을 치료하기 위한 치료법의 발전

Thiophenesulfonamide compounds are potent and specific inhibitors of Vibrio quorum sensing regulators LuxR/HapR that block their activity in vivo, thus preventing transcription of genes for virulence, motility, and biofilms. This protocol details how these compounds are synthesized, modeled in silico, and assayed in vivo for activity against LuxR/HapR.

Vibrio Quorum Sensing Inhibitors의 합성 및 분석

Bacteria detect local population numbers using quorum sensing, a method of cell-cell communication broadly utilized to control bacterial behaviors. In ...

엔지니어링 정원 담금질 Lactonases를 사용하여 병원성 바이오 필름의 안티 악성 중단

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