מחקר זה נתמך על ידי מענקים בארה&quot;ב שירות בריאות ציבור AI69233 וAI073843 מהמכון הלאומי לאלרגיה ומחלות זיהומיות. EPS הוא עמית בורוז Wellcome בפתוגנזה של מחלות זיהומיות. קמ&quot;ש מומן על ידי התאית ומולקולרית מיקרוביולוגיה הדרכת מענק תכנית 5 T32 A107611-10.

1. טיהור של המוגלובין מדם טרי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לרכוש דם אדם טרי בתוספת נוגדי קרישה. לשמור על דם על קרח או ב 4 ° C בכל הטיהור. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה דם במשך 20 דקות ב1500 x גרם. תאי הדם האדומים (RBCs) יהיו בתחתית של התחתית. בזהירות לשאוב supernatant והעדינות resuspend גלול ב 0.9% (w / v) פתרון NaCl קר כקרח. חזור צנטריפוגה ולשטוף 3 פעמים. </li><li style=";text-align:right;direction:rtl"> Resuspend הגלולה בנפח 1 מתוך קרים כקרח 10 מ&quot;מ טריס-HCl (pH 8.0). זה יניע תמוגה של RBCs עקב לחץ האוסמוטי. הוסף ל~ טולואן נפח סופי של 20%. </li><li style=";text-align:right;direction:rtl"> לדגור על 4 מעלות צלזיוס בצלייה למשך לילה. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה lysate ב20000 XG לשעה 1. לאסוף את שבר hemolysate האמצע עוזב טולואן (צף על גבי) וגלולה ללא פגע. השתמש פיפטה-צוואר ארוך כדי לאסוף את שבר האמצע. </li><li style=";text-align:right;direction:rtl"> עובר דרך מסנן מזרק 0.44 מיקרומטר. אם הפתרון מכיל חלקיקיםעניין ולא יכול להיות מועבר דרך המסנן, חזור על שלב 1.5. </li><li style=";text-align:right;direction:rtl"> לטהר המוגלובין (HB) עם כרומטוגרפיה נוזלית ביצועים גבוהי עמודה (HPLC) אניון חליפין (ריאן, 1000 8 מיקרומטר PL-סקסופון, 150 מ&quot;מ × 4.6 מ&quot;מ). השלב הנייד הוא 10 mM טריס-HCl (pH 8.0) ושלב ב &#39;נייד הם 10 mM טריס-HCl (pH 8.0) + 0.5 M NaCl. 0% -100% שיפוע של B ממס נדרס 2 דקות בשיעור 2.0 מ&quot;ל / דקת זרימה. Elution מנוטר על בסיס קליטה (λ: 410 ננומטר ו 280 ננומטר). לאסוף רק השבריר מתאפיין בצבע אדום בהיר ושיא ספיגה בולט (איור 1). </li><li style=";text-align:right;direction:rtl"> דיאליזת elution נגד פוספט נאגר מלוח (PBS) למשך הלילה ולאחר מכן לכמה שעות שוב. לעקר על ידי עובר דרך מסנן מזרק מיקרומטר 0.22. </li><li style=";text-align:right;direction:rtl"> כדי למדוד את ריכוז המוגלובין, להכין פתרוני המוגלובין סטנדרטיים של ריכוזים ידועים בPBS. לקבוע את הריכוז של המוגלובין במדגם על ידי ערבוב את הפתרונים הסטנדרטיים (ראה טבלה עם ריאגנטיםed) או פתרון לדוגמא עם ריאגנט של 2x דרבקין (שהוכן מאבקה) ביחס 1:1. לדוגמה, לערבב פתרון Hb 100 μl עם מגיב 100 μl 2x של דרבקין בצלחות 96-כן. דגירה במשך 15 דקות וספיגה במידה 540 ננומטר. להתוות עקומת סטנדרט ולקבוע את ריכוז המוגלובין במדגם. חמש עד 15 תשואות מ&quot;ג / מ&quot;ל ​​הן טיפוסיות. </li><li style=";text-align:right;direction:rtl"> הפעל מיקרוגרם 15-20 המטוהרים המוגלובין על 15% SDS-PAGE בשני עותקים. כתם אחד מהג&#39;לי; להעביר את החלבונים מג&#39;ל אחר על גבי קרום ניטרוצלולוזה וimmunoblot למוגלובין (איור 2). </li><li style=";text-align:right;direction:rtl"> להקפיא ולאחסן 1 aliquots מ&quot;ל של המוגלובין בחנקן נוזלי. </li></ol> 2. הכנת הברזל פוגע תקשורת צמיחה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן Roswell Park Memorial מכון (RPMI) מרק על ידי המסת אבקת RPMI במים, מוסיף סודה לשתייה כפי שהומלץ על ידי היצרן וחומצות cassamino% 1 (CA) (W / V). לעקר על ידי עובר דרך מסנן 0.2 מיקרומטר והחנות refrigמקורר. </li><li style=";text-align:right;direction:rtl"> הכן מתכת מדולדלת RPMI (NRPMI) על ידי הוספת 7% (w / v) Chelex 100 וערבוב במשך הלילה על צלחת ומערבבים. הסר Chelex 100 על ידי עובר דרך מסנן וחנות בקירור 0.2 מיקרומטר. תוספת תקשורת עם מתכות שאינן ברזל חיוניים: 25 מיקרומטר ZnCl 2, 25 מיקרומטר MnCl 2, 100 מ&quot;מימ CaCl 2 ו1 המ&quot;מ MgCl 2 הוכן מראש כפתרון x סטרילי 1000. השתמש בכלי פלסטיק חד פעמים לצעד זה כדי למנוע זיהום מאספקת ברזל מחדש שמישה. </li></ol> 3. צמיחת סטפילוקוקוס אוראוס באמצעות המוגלובין כמקור ברזל Sole <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הרצף ס סטפילוקוקוס לבידוד באגר סויה tryptic (TSA) ממניות קפוא. לדגור על 37 מעלות צלזיוס במשך 20-24 שעות. </li><li style=";text-align:right;direction:rtl"> Resuspend ethylenediamine-N, ניצבת בין bis (חומצה 2-hydroxyphenylacetic) (EDDHA) באתנול נטול מים עד 100 מ&quot;מ. EDDHA אינו נכנס פתרון אבל מעוקרת על ידי אתנול. </li><li style=";text-align:right;direction:rtl"> הוסף לEDDHA RPMI לריכוז סופי של 0.5 מ&quot;מ. להתירEDDHA לפזר לפחות 30 דקות לפני שתמשיך לשלב הבא. עקב וריאציה תצווה לתצווה, ריכוז EDDHA סופי ייתכן שיהיה הצורך הוריד ל0.25 מ&quot;מ כדי לאפשר התפתחות חיידקים. </li><li style=";text-align:right;direction:rtl"> לחסן מושבות בודדות של ס סטפילוקוקוס לתוך 5 מ&quot;ל של RPMI מכיל EDDHA ב15 צינורות חרוטי פקק מ&quot;ל. לדגור על 37 ° C עם הרעד ב180 סיבובים לדקה (סל&quot;ד) ל16-20 שעות. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה תרבויות הלילה עבור 5 דקות ב7500 XG וresuspend בגלולה המכילה NRPMI EDDHA 0.5 מ&quot;מ. לנרמל OD 600 ל ~ 3. </li><li style=";text-align:right;direction:rtl"> הכן NRPMI המכיל 2.5 מיקרוגרם למ&quot;ל והמוגלובין EDDHA 0.1-1.0 מ&quot;מ. בשל שונות בין הקבוצות, ריכוז EDDHA נדרש Chelate ברזל חופשי בNRPMI עשוי להשתנות. </li><li style=";text-align:right;direction:rtl"> 10 μl תת השעיה של חיידקים מצעד 3.5 לתוך 1 מ&quot;ל NRPMI + + EDDHA המוגלובין בצינור 15 מיליליטר פקק חרוטים. </li><li style=";text-align:right;direction:rtl"> דגירת התרבויות על 37 מעלות צלזיוס למשך עד 48 שעות עם הרעד ב180 סל&quot;ד או בתוף מתגלגל. </li><li&gt; כל 6-12 שעות תיקח 600 קריאות OD על ידי הסרת 50 μl של התרבות ולערבב אותו עם 150 μl של PBS בצלחות 96-כן. </li></ol>

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ברזל הוא חומר מזין חיוני הנדרש על ידי אורגניזמים מכל הממלכות של 15 חיים. בבעלי חוליות, ברזל מבודד את להימנע מרעילות הנגרמת על ידי רכיב זה. תפיסה זו גם מסתירה ברזל מפלישת חיידקים בתהליך המכונה חסינות תזונתית 16. בתגובה, פתוגנים התפתחו אסטרטגיות עוקפות חסינות ...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב / חומרים </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> טור חילופי אניון HPLC </td><td> ריאן </td><td> PL1551-3802 </td><td></td></tr><tr><td> המגיב של דרבקין </td><td> סיגמא </td><td> D5941-6VL </td><td></td></tr><tr><td> סטנדרטי המוגלובין </td><td> פואנטה מדעית </td><td> H7506-STD </td><td></td></tr><tr><td> RPMI </td><td> HyClone </td><td> SH30011.02 </td><td></td></tr><tr><td> צורת נתרן Chelex 100 </td><td> סיגמא </td><td> C7901 </td><td></td></tr><tr><td> EDDHA </td><td> LGC התקנים GmbH </td><td> ANC 001 </td><td></td></tr><tr><td> המוגלובין נוגדן </td><td> סנט קרוז ביוטכנולוגיה, Inc </td><td> SC-21005 </td><td> &lt;/ Td&gt; </tr><tr><td> אגר סויה Tryptic </td><td> BD </td><td> 236920 </td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

ס aureus הוא חיידק הפתוגני שדורש ברזל כדי למלא תפקידים מטבוליים חיוניים ובגורמים למחלות. המאגר העשיר ביותר של ברזל בתוך המארח האנושי הוא heme, שהוא cofactor של המוגלובין. לרכישת ברזל מהמוגלובין, ס סטפילוקוקוס מנצל מערכת מורכבת ידועה כגורם המשטח ברזל המוסדר (ISD) 1 המערכת. מרכיבי ההמוגלובין ISD המערכת הראשון לאגד המארח, ואז לחלץ ולייבא heme, ולבסוף לשחרר ברזל ממקור צמחי בציטופלסמה החיידקים 2,3. מסלול זה כבר נתח באמצעות מספר רב של מחקרים במבחנת 4-9. יתר על כן, תרומתה של מערכת ISD לזיהום הודגמה שוב ושוב במודלי עכבר 8,10-14. הקמת התרומה של מערכת ISD לרכישת ברזל המוגלובין-נגזר וצמיחה הוכיחה להיות יותר מאתגר. מבחני צמיחה באמצעות ההמוגלובין כמקור ברזל בלעדי מסובכים בy חוסר היציבות של המוגלובין הזמין מסחרי, מזהם ברזל חופשי במדיום הגידול, והרעילות הקשורה chelators ברזל. כאן אנו מציגים שיטה המתגברת על מגבלות אלה. המוגלובין באיכות הגבוהה מוכן מדם טרי ומאוחסן בחנקן נוזלי. המוגלובין מטוהר הוא בתוספת ברזל לימוצה בינוני מחקה את סביבת ברזל העניים נתקלה ידי פתוגנים בתוך מארח החוליות. על דרך רעבה ס סטפילוקוקוס של ברזל חופשי ולהשלים עם צורת מניפולציות מינימאליות של המוגלובין נזרז את הצמיחה באופן שתלוי לחלוטין ביכולת לקשור המוגלובין, לחלץ heme, יעבור heme דרך מעטפת תא החיידק ולבזות heme בציטופלסמה. assay זה יהיה שימושי לחוקרים המבקשים להבהיר את המנגנונים של רכישת הברזל hemoglobin-/heme-derived בס סטפילוקוקוס ואולי חיידקים פתוגנים אחר.

יש לנו מטוהר ההמוגלובין אנושי מhemolysate עם HPLC (צעד הפרוטוקול 1.7). איור 1 הקלטות ספיגת eluate ב 280 ואורכי גל 410 ננומטר. חלק 5 נאסף ומקטעים אחרים שנזרקו. תשואות של 5-15 מיליגרם של המוגלובין למ&quot;ל של eluate נרכשות בדרך כלל. המוגלובין מטוהר נותח על ידי SDS-PAGE במשוכפל וג&#39;לים היה...

Watch this Scientific Journal Video about Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source at JoVE.com

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

כאן אנו מתארים assay צמיחה<em&gt; סטפילוקוקוס אוראוס</em&gt; באמצעות ההמוגלובין כמקור היחידי של ברזל תזונתי זמין. בדיקה זו קובעת את התפקיד של גורמי חיידקים המעורבים ברכישת ברזל המוגלובין נגזר.

צמיחת סטפילוקוקוס אוראוס באמצעות ההמוגלובין אנושי כמקור ברזל

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

staphylococcus-aureus-growth-using-human-hemoglobin-as-an-iron-source

Research

JoVE Journal

Immunology and Infection

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

16.4K Views.  Vanderbilt University Medical School. כאן אנו מתארים assay צמיחה סטפילוקוקוס אוראוס באמצעות ההמוגלובין כמקור היחידי של ברזל תזונתי זמין. בדיקה זו קובעת את התפקיד של גורמי חיידקים המעורבים ברכישת ברזל המוגלובין נגזר.

Video: Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

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.

השימוש זרימה טפטוף ו כורים דיסק סיבוב עבור Staphylococcus aureus Biofilm ניתוח

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.

זיהום תת עורי של סטפילוקוקוס אוראוס עמיד Methicillin (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 ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

חסכונית שיטת מעקב אחר מקור מיקרוביאלית באמצעות וירוסים אדם ובעלי חיים ספציפיים

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

אנדוקרדיטיס דגם ניסיוני של Methicillin עמיד<em&gt; Staphylococcus aureus</em&gt; (MRSA) ב עכברוש

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

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.

חקר אינטראקציות של<em&gt; סטפילוקוקוס</em&gt; עם נויטרופילים ידי cytometry זרימה מיקרוסקופית וזמן לשגות

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.

מיקוד Associated Biofilm<em&gt; Staphylococcus aureus</em&gt; Assay רגישות בסמים בהתבסס Resazurin שימוש

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.

genotyping של<em&gt; Staphylococcus aureus</em&gt; על ידי ריבוזומלי 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, ...

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.

מתודולוגיה לחקר ההעברה האופקית ג&#39;ין<em&gt; Staphylococcus aureus</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.

דגם העכבר כדי להעריך מולדת התגובה החיסונית לזיהום Staphylococcus aureus

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

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

ככמת את רעילות הציטוהילווקיוס האורמית נגד פולימורציטים אנושיות

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes ...