This work has been supported by Elizabeth E. Kennedy Research Award.

생산자 균주의 1 준비 <ol><li> 킬 폐렴 구균 균주는 -80 ° C 냉동고 주식에서 5 % 양 혈액 트립 틱 소이 한천 접시에 박테리오신 생산을위한 시험한다. NOTE : 억제를 판정하는 균주의 초기 성장을위한 혈액 플레이트의 사용은 크게 분석의 재현성을 증가시킨다. 성장 조건은 특정 유기체에 대해 수정해야 할 수도 있지만, 또한, 박테리오신을 생산하는 다른 비 - 폐렴 구균 균주를 사용할 수있다. 우리는 성공적으로 오버레이 분석에서 연쇄상 구균 mitis 및 락토 코커스 락 티스의를 사용하고 있습니다. </li><li> 5 % CO 2와 37 ° C에서 밤새 플레이트 (O / N)를 품어. </li><li> 생산자 균주의 배양 후, TSA 25ml를 함유 플레이트에 카탈라제 3,000 4,000 단위를 피펫 팅하여 트립신 콩 한천 (TSA) 판을 제조하고 멸균 유리 비드를 이용하여 용액을 카탈라아제 확산. 약 10 분 아래에 접시를 건조 시키생물 안전 캐비닛. </li><li> 피펫 팁에 박테리아의 가시적 양을 수집합니다. 건조 TSA 플레이트에 피펫 팁을 찔러. 참고 : 하나의 생산 균주 이상은 하나의 TSA 플레이트에 찔 수 있습니다. 결과 후광이 겹치지 않도록 그것은 찢고 밖으로 공간으로 중요합니다. <ol><li> 가능한 경우, 양성 대조군 및 음성 대조군으로서 오버레이에 사용되는 균주로서 프로듀서 박테리오신 알려진 것을 포함한다. 참고 : 양성 및 음성 컨트롤은 이전에 게시 된 아르 요청 10시 사용할 수 있습니다. </li></ol></li><li> 6 시간 동안 5 % CO 2와 37 ° C에 여러 찌르기를 포함하는 TSA 플레이트를 품어. </li></ol> 오버레이 스트레인의 2 만들기 글리세롤 주식 <ol><li> 오버레이 분석의 일 이전에 (글리세롤의 추가 후 중간 지수 단계로 성장하고 -80 ° C에서 보관 문화) 글리세롤 주식을 준비합니다. 폐렴 구균 STR 5-10 식민지를 접종아인은 0.5 % 효모 추출물 (THY)와 토드 휴잇 매체 감도 또는 페로몬 분비 검사를한다. 참고 : BlpC 유도 기자의 건설 이전에 2를 설명하고 균주의 요청에 따라 사용할 수 있습니다. </li><li> 흔들림없이 37 ° C의 물을 욕조에 단단히 밀폐 뚜껑 THY의 5 ㎖의 유리 튜브에 5 ~ 10 식민지를 품어. </li><li> 그들은 0.5 OD에게 0.3의 620에 도달 할 때까지 37 ° C에서 문화를 품다. 참고 :이 사용되는 균주에 따라 약 4-5 시간이 소요됩니다. <ol><li> 부드럽게 튜브에게 OD를 측정하기 전에 몇 번 반전. </li></ol></li><li> 실험 당일 수행 될 수 없거나 균주는 글리세롤을 보유하고 나중에 실험을 재개, 향후 여러 번 사용할 경우. 글리세롤 축적량 들어, 20 %의 최종 농도로 배양하여 글리세롤을 추가한다. -80 ° C에서 1.5 ML의 microcentrifuge 튜브 및 저장소로 나누어지는 문화. 참고 : 샘플의 할 수있다-80 ° C에서 달 tored. 글리세롤 주식을하지 않는 경우, 오버레이 분석을 위해 직접 샘플을 사용합니다. </li></ol> 3 오버레이 분석 <ol><li> 직전, 응용 프로그램을 오버레이 오버레이 구성 요소를 혼합한다. 15 ML 원뿔 관에서 THY 5 ㎖, 카탈라아제의 3,000 ~ 4,000 U, 그리고 그 중 하나 일 또는 실온에서 해동 된 문화의 글리세롤 주식을 성장시켰다 오버레이 균주 배양액의 200 μl를 추가합니다. 기자 균주를 사용하는 경우, 오버레이 혼합물에 X 갤런의 50 μL (40 ㎎ / ㎖)를 추가합니다. 접시 당 하나의 원뿔 튜브를 사용합니다. </li><li> 이전 용융 TSA의 추가로 실온에서 오버레이 혼합 평형. </li><li> 인큐베이터에서 칼에 찔린 균주 TSA 플레이트를 제거합니다. 전자 레인지에 고체 TSA 녹여 사용할 준비가 될 때까지 55 ° C의 물을 욕조에 보관하십시오. 1.5 % 한천로 용융 TSA 3 ㎖를 추가 혼합물을 오버레이 55 ° C로 냉각. <ol><li> 피펫 직전 오버레이 혼합물을 제조하고, 혼합물은 아주 고화 시작신속. 참고 : 오버레이 도금하기 전에 응고되기 시작하면, 그 결과는 해석 할 수없는 것입니다. </li></ol></li><li> 피펫 팅에 의해 및 5 ㎖의 피펫이 혼합물에 거품을 소개하지 않도록주의 여러 번 아래로 오버레이 혼합물을 섞는다. 천천히 칼에 찔린 TSA 플레이트에 직접 오버레이 혼합물을 피펫. 한천이 굳어 할 수 있도록 몇 분 동안 벤치 탑에있는 플레이트에 보관하십시오. <ol><li> 오버레이 혼합물을 배포 할 수있는 판을 돌리지 마십시오,이 오버레이에 찔 문화에서 성장을 끌어. </li></ol></li><li> 조심스럽게 5 % CO 2 O / N (약 16 ~ 18 시간)을 포함 37 ° C 배양기에 오버레이 TSA 플레이트를 배치합니다. </li><li> 하룻밤 성장 후 번호판을 관​​찰한다. 판 억제 또는 페로몬 신호가 발생한 지역을 제외하고 오버레이 균주의 성장 불투명 나타납니다. 이들은 각각 찔 변형 주위를 청소 또는 청색의 영역으로 표시됩니다. 참고 : 비록 지표 성과청색 또는 정화 직경 ements은 정량적 평가를 위해 결정될 수있다 직경의 차이는 또한 접시에 찔린 박테리아의 양에 따라 달라질 수있다. 오버레이 분석은 정량적 평가는 신중하게 해석되어야한다, 질적 분석이다. 음성 대조군에 비해 눈에 띄는 개간 활동으로 간주된다. </li></ol>

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The authors have nothing to disclose.

이 오버레이 분석은 박테리오신 생산자 활동의 범위를 결정하고 폐렴 구균 균주 사이에서 발생할 수있는 경쟁적인 상호 작용을 보여주기 위해 빠른 방법이다. 오버레이 분석은 하나의 플레이트에 대한 여러 박테리오신 생산 균주 선별을 위해 사용하도록 구성 될 수있다. 우리는 성공적으로 96 웰 플레이트의 절반에서 SBA 판을 접종 48 핀 복제기를 사용하여이 분석에 큰 부담 모음을 상영했다​​....

폐렴 구균, 비 인두의 polymicrobial 지역 사회의 일반적인 식민지 개척자는, 세균 생성 된 항균 펩타이드 (박테리오신)의 생산을 통해 다른 폐렴 구균과 밀접하게 관련 종과 경쟁 할 수있다. 현재까지 조사마다 완전히 순서가 폐렴 구균 균주는 B의 acteriocin- 리터의 이케 페이지의 eptide 궤적, BLP의 버전이 포함되어 있습니다. 폐렴 구균에 의하여 생산 박테리오신은 양의 시험 관내 결과 및 생체 내 경쟁 1-4에 중요한 것으로 입증되었다. 경쟁적인 역학 특정 펩티드 페로몬에 응답하여 동족 내성 단백질과 함께 다양한 박테리오신의 발현에 의해 영향을 받는다. BLP 궤적 유도 펩티드 페로몬, BlpC는, 센서 키나아제, BlpH에 결합되는 정족수 인식 시스템에 의해 제어하고, 그 결과 신호 캐스케이드를 개시한다BLP 궤적 5,6의 전사. BlpC의 사 대립 유전자 변형이 있습니다 자사의 동족 BlpH 6 각 바인드. 셀 자체 박테리오신의 효과 살아남 들어 동족 내성 단백질을 인코딩하는 유전자는 유전자 박테리오신 각각 같은 오페론에 전사된다. 변형 방지에 필요한 특정 내성 유전자를 결여하는 경우 경쟁자 균주는 외인성 페로몬에 응답 BLP 궤적을 상향 조절 할 수없는 경우, 또는 죽이는 발생한다. 수송 복합체, BlpAB이 분비 및 처리 펩타이드 페로몬과 박테리오신 펩티드 2,4 필요합니다. 최근에는 폐렴 구균 균주의 상당한 부분들이없는 그들과 박테리오신 1,2 페로몬 분비 렌더링 blpA 유전자의 보존 된 돌연변이를 의미하는 &quot;사기꾼&quot;가 있다고 밝혀졌다. 이 균주는 울음 소리에 의해 분비되는 외인성 BlpC 2에 응답 할 수 있습니다면역 단백질의 생산을 허용 보링 균주. 상청액으로부터 박테리오신의 조질 제제는 순도를 달성하는 생화학 방법의 단계를 사용하여 제조자 균주로부터 제조 될 수 있지만,이 방법은 균주의 큰 컬렉션을 스크리닝 유용한 아니며 박테리오신 발현 수준은 국물 성장 배양 낮은 경우 2,7- 9. 오버레이 분석은 시험 관내에 존재할 수있는 균주간에 경쟁 역학 조사 신속한 방법으로 사용된다. 그렇게하려면, 폐렴 구균 균주는 미리 접종 한천 플레이트 상과 BLP 궤적의 유도에 충분한 로컬 박테리아 농도를 달성하기 위해 증가시킨다. 두 번째 변형은 다음 다음 감도를 테스트 할 수있는 사전 접종 균주에 걸쳐 적용되는 용융 부드러운 한천 솔루션에 접종한다. (억제 활성 무관) BLP 궤적 행위 평가하기 위해 균주 번째 일련의 오버레이 할 수이전에 설명 BlpC 기자 균주를 REE. 이 균주는 폐렴 구균에 의해 분비되는 가장 일반적인 세 BlpC 페로몬에 반응 세 가지 blpH 대립 유전자 중 하나를 포함하도록 구성되었다. 리포터 균주 BlpH 의존 프로모터의 제어하에 있고 자기 자극을 방지 10,11 blpC 유전자에서 삭제를 수행하는 통합의 lacZ 유전자를 가지고있다.

<table border="0" cellpadding="0" cellspacing="0" width="753">
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			<td height="42" style="height:42px;width:212px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:351px;">Comments/Description</td>
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			<td height="42" style="height:42px;width:212px;">Trypticase Soy Agar with 5% Sheep Blood</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">B21261X&#160;</td>
			<td>For growth from freezer cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Catalase</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C100-500mg</td>
			<td>4,741 Units per plate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Todd Hewitt Broth + Yeast Extract (THY)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">DF0492-17-6&#160;</td>
			<td>30 g of THB, 5 g yeast extract, 1 L ddH20 autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">Trypticase Soy Broth + Agar plates (TSA)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">B11768&#160;</td>
			<td>30 g of TS, 15 g agar, 1 L ddH20 autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:212px;">5-Bromo-4-chloro-3-indolyl &#946;-D-galactopyranoside (X-gal)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">B4252-50MG</td>
			<td>Dissolve in dimethylformamide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Petri dishes</td>
			<td style="width:71px;">Fisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Spectrophometer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td>Measures the OD620 of cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">37 &#176;C water bath</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">37 &#176;C incubator with 5% CO2</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">15 ml conical tube</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

using the overlay assay to qualitatively measure bacterial production of and sensitivity to pneumococcal bacteriocins

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.

오버레이 분석에서 두 가지 결과가 있습니다. 하룻밤 배양 한 후 칼에 찔린 균주의 성장을 할 수 있어야한다. 그림 1A에서 오버레이 변형 박테리오신이 생산되고에 민감하다. 통관의 영역은 칼에 부담을 둘러싼 시각화 할 수 있습니다. 또한, 오버레이 혼합물에 찔 균주의 소용돌이가 가능하며,도 1a에서 볼 수있다. 그림 1B에서 오버레이 변형은 자상을 둘러싼 ?...

Watch this Scientific Journal Video about Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins at JoVE.com

Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.&#160; Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.

오버레이 분석을 사용하여 성적으로 폐렴 구균 박테리오신에 대한 세균의 생산 및 감도를 측정하는

Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms.  We have ...

using-overlay-assay-to-qualitatively-measure-bacterial-production

Research

JoVE Journal

Immunology and Infection

13.4K 조회수.  University of Michigan. Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species.  Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. 

비디오: Using the Overlay Assay to Qualitatively Measure Bacterial Production of and Sensitivity to Pneumococcal Bacteriocins

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

마우스의 이미지 세균 감염에 루시페라제를 사용하여

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

연구

면역학 및 감염병학

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Mycobacteria의 가능성을 측정하는 기능 전체 혈액 분석은 리포터 - 진 BCG 또는 M. TB (BCG 태그 사용 럭스 / M. TB 럭스)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS로 인한 세균 경쟁을 모니터링 할 비주얼 분석

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

호스트 세포 RN​​A의 합성에 바이러스 감염의 효과를 측정하는 클릭 화학을 사용하여

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

를 사용하여 폐렴 구균 식민지에서 생균제의 효과를 조사<em&gt; 체외</em&gt; 준수의 분석

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

의 캡슐 혈청 형<em&gt; 폐렴 구균</em&gt; Quellung 반응을 이용하여

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

의 캡슐 혈청 형<em&gt; 폐렴 구균</em&gt; 라텍스 응집에 의해

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

방법은 세균 Pyomelanin 생산을 억제하고 산화 스트레스에 감도에 대응 증가를 확인하는 방법

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

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.