Эта работа выполнена при финансовой поддержке американского Национального института здоровья [грант R01AI-39108 для ASB] и Американской ассоциации сердца [гранты SDG 0630219N и AID 09GRNT2180065 в YQX].

1. Подготовка MRSA штаммов инфекции <ol><li> Инокулировать loopful культуры MRSA из запаса труб при температуре -80 ° С до бараньей крови Trypticase соевый агар (TSA) пластины (см. таблицу конкретных реагентов и оборудования), и инкубировать при температуре 37 ° С в течение ночи. </li><li> Проверьте чистоту культуры на тарелку кровяной агар (как фенотип колонии), чтобы убедиться в отсутствии загрязнения. </li><li> Выберите один из колонии овец пластины крови TSA и привить колонии в 5 мл соевого Trypticase бульона (TSB) в 15 мл оснастки вершинами трубы. </li><li> Инкубировать при температуре 37 ° С в течение ночи при встряхивании на 200 оборотов в минуту. </li></ol> 2. Подготовка хирургия Катетеры Вырезать полиэтиленовых труб (PE10,. Becton Dickinson, порядок не 427 401) до 10 см длиной, и расплавить один конец, нажимая кончиком стерильной щипцами. Цель запечатывания одного конца катетера, чтобы избежать кровотечения во время катетеризации. 3. Предварительно сПодготовка urgery и анестезии <ol><li> Место Sprague-Dawley крыс (Харлан, Индианаполис, штат Индиана женщины, 250-300 граммов) в камеру, содержащую isofluorane-кислородной газовой смеси (50%: 50%) до анестезии вступает в силу (например, мышцы находятся в расслабленном состоянии и педали рефлексы отсутствуют ), а также поддерживать животных в состоянии наркоза во время операции на газовой смеси. </li><li> Очистите область шеи от подбородка до чуть ниже грудины с Бетадин и 70% этанола. </li></ol> 4. Хирургические процедуры <ol><li> Использование стерильных всей операции. Сделайте разрез (1-1,5 см) по вертикали только через скин-слоя шейки над грудиной. </li><li> Используя тупой диссекции, разделить лицевой панели подвергать правой сонной артерии с помощью 2 пар зубчатых изогнутый пинцет. </li><li> Осторожно потяните вверх из артерии шеи полости, а за два 10 см длины шва шелком по артерии и галстук от артерии подвергаются головной конец, поставить клип на артеры, чтобы предотвратить кровотечение. </li><li> Сделайте небольшое отверстие в верхней части артерии с помощью катетера интродьюсер (Becton Dickinson, порядка нет. 406 999), вставить катетер с пинцетом через отверстие в артерии, снять клип и надавите катетер по направлению к сердцу, пока сопротивление выполняется. </li><li> Галстук свободно шва вокруг хвостовой конец артерии и обеспечить катетер на месте шва шелком, когда катетер находится в месте, определенном: а) длина катетера (4-5 см), II), устойчивость к дальнейшему развитию и III), пульсация катетер с сердцебиением. Оставьте катетер на месте до конца эксперимента. </li><li> Сокращение избыточных шва и катетер концов, и убедитесь, что нет кровотечения из катетера. Заправьте концы под кожу шеи и закрыть с кожей клипов. </li><li> Положите крысу в клетку в теплом месте, пока не оправилась от наркоза, а также обеспечить пищей и водой. Проверьте крысы часто во время ипосле выздоровления от наркоза. </li><li> Эта операция является &quot;Категория Е&quot; процедуры. Процедура производится боль беспросветной анальгетиками, так как инфекции MRSA сопровождающих страдания животных. </li></ol> 5. MRSA инфекция <ol><li> Инфекция может быть выполнена от 1 до 7 дней после операции, но поддерживать постоянный в течение эксперимента. </li><li> Чистый хвост крысы с 70% этанола, и ввести 0,5 мл MRSA в нужном клеток (10 4 - 10 6 КОЕ / животное большинства штаммов золотистого стафилококка). С 27 G 1/2 дюйма иглы внутривенно из хвостовой вены Внимание : MRSA культура относится к биобезопасности 2-го уровня (BSL2) и имеет умеренную потенциальную опасность для персонала и окружающей среды. </li><li> Держите давление на сайте, пока гемостаза происходит перед возвращением крысы в ​​клетке. </li></ol> 6. Жертва Крысы и тканей культуры Целевая <ol><li> Жертвоприношение крыс ф инъекции натрия фенобарбитала (200 мг/ Кг) после 1 до 6 дней после заражения. </li><li> Положите крысу на спину и протрите грудь на 70% этанола. </li><li> Сделать V-образный разрез на груди под грудиной, а также сокращение хрящи ребер с каждой стороны от грудины раскрыть сердце. </li><li> Потяните вверх сердце мягко, и клип в сердце ткани близко к аорте и анализировать до освобождения сердца. </li><li> Поместите сердце в стерильную чашку Петри с 4x4 дюйм в марлю, сделать разрез через левого желудочка внутренней стене и открыть левосторонний камеры. </li><li> Проверьте визуально для катетера. Проверьте и удалите растительность из клапана с ножницами и щипцами. </li><li> Взвесьте и гомогенизации растительности, и сделать серийных разведений PBS с количественной культуры. </li></ol> 7. Представитель Результаты Сразу после установки катетера в артерию и нажимает катетер 4-5 см по направлению к сердцу, сопротивление будет experienКНИ. Если нет сопротивления почувствовал, катетер не может успешно вставлена ​​в левой части сердца камеры, которые могут повлиять на размещение катетера. Правильное размещение катетера, опубликованных ранее 9 показано на рисунке 1. Часть образцов MRSA инфекции должны быть количественные культурный убедитесь, что точное количество жизнеспособных бактерий в заражении и чистоту инфекции образца. Кроме того, организм оправился от растительности должна быть такой же, как те, которые используются в посевной. В таблице 1 приведен пример вирулентности S. золотистого напряжения в модели крысы эндокардит, который был опубликован ранее 9. Все животные сталкиваются с проблемой посевного материала из 10 5 10 6 КОЕ и принес от 3 ​​до 6 дней после инфекции, разработанных endocardits с высоким С. золотистого плотности в сердечной растительности, а также почек и селезенки ( <stРонг&gt; Таблица 1). Крысы с стерильных культур клапан считается неинфицированных. <img alt="Рисунок 1" src="/files/ftp_upload/3863/3863fig1.jpg" /> Рисунок 1. Катетер находится в правильном месте (левая сторона сердца камеры), а также многочисленные растительности видны всем аортального клапана 9. <table border="1"><tbody><tr><td> Посевной (количество Animas) </td><td colspan="3"> Средний войти 10 КОЕ / г ткани ± стандартное отклонение в: </td></tr><tr><td></td><td> Растительность </td><td> Почка </td><td> Селезенка </td></tr><tr><td> 10 6 КОЕ / животных (9) </td><td> 10,36 ± 0,85 </td><td> 7,30 ± 0,64 </td><td> 6,70 ± 0,57 </td></tr><tr><td> 10 5 КОЕ / животных (8) </td><td> 9,93 ± 0,53 </td><td> 7,14 ± 0,53 </td><td> 6,44 ± 0,63</td></tr><tr><td> 10 4 КОЕ / животных (7) </td><td> 3,46 ± 0,50 * </td><td> 1,81 ± 0,74 * </td><td> 1,58 ± 0,59 * </td></tr></tbody></table> SD, стандартное отклонение. р &lt;0,001 по отношению к 10 5 или 10 6 КОЕ-вызов животных. Таблица 1. С. золотистого плотности в сердечной растительности с различными посевного материала в модели крысы эндокардит 9.

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Нет конфликта интересов объявлены.

Крыса эндокардит является важным и хорошо изученных животных моделей для прижизненного исследования патогенеза и противомикробные препараты для лечения бактериальных инфекций 9-11. Кроме того, модель крысы эндокардит представляет собой композицию острые и подострые инфек?...

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experimental endocarditis model of methicillin resistant staphylococcus aureus (mrsa) in rat

Эндоваскулярная инфекций, в том числе эндокардит, являются опасными для жизни инфекционных синдромах 1-3. Золотистый стафилококк является наиболее распространенным мире причиной такого синдрома с недопустимо высокой заболеваемости и смертности, даже с соответствующими антимикробных агентов лечения 4-6. Рост инфекций, вызванных метициллин-резистентные S. стафилококк (MRSA), высокие темпы ванкомицин клинических неудач лечения и растущих проблем линезолид и даптомицина сопротивление все еще ​​более осложняется при лечении пациентов с такими инфекциями, и привело к высокой стоимости медицинских 7, 8. Кроме того, следует подчеркнуть, что последние исследования с антибиотиками результаты лечения были основаны в клинических условиях, и, следовательно, вполне может оказывать влияние множество факторов варьируется от пациента к пациенту. Таким образом, соответствующие животной модели эндоваскулярной инфекции, в котором множество факторов аналогичные от животного к животному большерешающее значение для расследования микробного патогенеза, а также эффективность новых антимикробных препаратов. Эндокардита у крыс является хорошо установленным экспериментальным модель животного, близкое человека родной эндокардитом клапана. Эта модель была использована для изучения роли частности стафилококковой факторов вирулентности и эффективности антибиотиков для лечения стафилококкового эндокардита. В этом докладе мы описываем экспериментальную модель эндокардита, вызванного MRSA, которые могут быть использованы для исследования бактериальной патогенеза и ответ на лечение антибиотиками.

Watch this Scientific Journal Video about Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat at JoVE.com

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Экспериментальная модель эндокардит крысы из-за метициллин-резистентные<em&gt; С. золотистый</em&gt;.

Экспериментальная модель Эндокардит метициллин устойчивый<em&gt; Золотистый стафилококк</em&gt; (MRSA) у крыс

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

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Immunology and Infection

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

15.1K Views.  Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. Экспериментальная модель эндокардит крысы из-за метициллин-резистентные С. золотистый.

Video: Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

Использование потоков для слива воды и вращающихся дисков для реакторов Золотистый стафилококк Биопленка Анализ

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

Подкожный Заражение метициллину Staphylococcus Aureus устойчивы (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

Преодоление невосприимчивости в экспериментальный аутоиммунный энцефаломиелит (ЭАЭ) штаммов мыши по усыновлению и передачи Антигенная вызов

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

Золотистый стафилококк рост использования человеческого гемоглобина в качестве источника железа

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

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Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

Изучение взаимодействий<em&gt; Золотистый стафилококк</em&gt; С нейтрофилами с помощью проточной цитометрии и микроскопии Длительная

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

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.

Ориентация биопленки Associated<em&gt; Стафилококк</em&gt; Использование ресазурин На основе лекарственной чувствительности анализа

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

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

генотипирование<em&gt; Стафилококк</em&gt; рибосомальным Spacer ПЦР (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Методика изучения горизонтальных генного переноса в<em&gt; Стафилококк</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Мыши модель для оценки врожденный иммунный ответ инфекции стафилококк

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...