<html> 1. Sangue Cultura Brodi Segnala come &quot;Positive&quot; <ol><li> Rimuovere il flacone cultura sangue segnalata dal cabinet monitoraggio incubazione continuo e posizionarlo in una cappa di sicurezza biologica. Nota: Le bottiglie possono contenere microrganismi pericolosi e precauzioni universali devono essere seguite. A causa del rischio di aerosol infettivi di campionamento, tutte le procedure di campionamento devono essere eseguite in una cappa a flusso laminare sicurezza biologica di classe II. </li></ol> 2. Gram Stain è preparato <ol><li> Preparare una colorazione di Gram dal segnalata brodo di coltura del sangue come da protocolli istituzionali locali. Nota: Quando microrganismi Gram-negativi sono identificati in microscopia brodo di coltura del sangue viene elaborata secondo il metodo seguente. Quando organismi Gram-positivi sono identificati, un metodo alternativo di targeting molecolare identificazione genetica e marcatori di resistenza viene applicata al brodo (non affrontato in questo articolo) 13. </li></ol>e &quot;&gt; 3. Trasferimento Segnalato Sangue brodo di coltura per una separazione tubo Serum <ol><li> Mescolare delicatamente il flacone cultura sangue invertendo 2-3x. </li><li> All&#39;interno dell&#39;armadio biosicurezza allegare una siringa da 10 ml per un dispositivo di trasferimento di sangue sicurezza. </li><li> Fissare il dispositivo di trasferimento di sangue al flacone di coltura sangue e prelevare 5 ml di brodo nella siringa. Trasferire il BC brodo aspirato in un tubo separazione del siero. </li></ol> 4. Concentrazione di Sangue brodo di coltura <ol><li> Centrifugare la provetta separazione del siero a 1.250 xg per 15 min che rimuove un grande volume di globuli rossi. </li><li> Aspirare e scartare il surnatante con una pipetta di trasferimento sterile facendo attenzione a lasciare circa 1 ml di buffy coat immediatamente sopra l&#39;interfaccia gel / liquido. Nota: Le bottiglie aerobici mostrano una netta separazione dei globuli rossi al fondo del tubo con l&#39;interfaccia gel / liquido apparire un colore bianco opaco. Al contrario, quandoapplicata al brodo di coltura anaerobica litico sangue dell&#39;interfaccia gel / liquido appare come un colore rosso intenso, grazie ai componenti di globuli rossi lisati rimanendo sospesi nel supernatante. </li></ol> 5. Ripetere centrifugazione Lavare Steps <ol><li> Mescolare delicatamente ultimi 1 ml di buffy coat fluido sopra l&#39;interfaccia gel con una pipetta sterile e poi trasferire l&#39;intero volume in una provetta da 1,5 ml microcentrifuga. Centrifugare la nuova provetta a 288 xg per 30 sec. </li><li> Trasferire il surnatante con una usa e getta 1 ml pipetta di plastica in una nuova provetta da 1,5 ml e scartare il tubo con il pellet. Nota: è fondamentale in questa fase che si ha cura di evitare il trasferimento del pellet. Ciò è particolarmente importante per un campione da lavorare dalla bottiglia BC anaerobica come il surnatante rimane pigmentata e il pellet non è sempre chiaramente visibile. </li></ol> 6. Lisi di cellule residue </p&gt; <ol><li> Centrifugare il campione a 18.407 xg per 1 min e poi aspirare (e scarti), il surnatante con una multa pipetta di trasferimento a punta di lasciare come poco liquido residuo possibile senza disturbare il pellet. </li><li> Risospendere il pellet residuo in 1 ml di DNAse sterile e RNAsi acqua gratuita pipettando su e giù. Nota: Alcuni autori hanno descritto l&#39;uso di soluzioni alcoliche in luogo di acqua sterile per risospendere il pellet che rende batteri non vitali. </li><li> Centrifugare la soluzione risospeso a 18.407 xg per 1 min. </li></ol> 7. Estrazione di proteine ​​batteriche <ol><li> Aspirare e scartare il surnatante, sempre con l&#39;aspirazione con una pipetta punta fine in modo che quanto più liquido possibile, viene rimosso. </li><li> Risospendere il pellet in 10 ml di acido formico (70% v / v). Nota: Acido formico può influenzare il corpo se viene inalato o se non viene a contatto con la pelle. Durante la preparazione o soluzioni manipolazione è recommended che il personale utilizza un aspiratore oltre a dispositivi di protezione individuale. </li><li> Mescolare bene con la pipetta per garantire una soluzione risospeso omogenea. Utilizzando la punta della pipetta per interrompere fisicamente il pellet può essere necessaria per garantire una soluzione più omogenea. </li></ol> 8. Preparazione di MALDI-TOF target Piatto <ol><li> Seminare una piastra segnale MALDI-TOF pulita con 2 ml di sospensione preparata per ogni posto di destinazione. Preparare tre siti bersaglio per ogni campione per superare il problema occasionale di riuscita legge. </li><li> Lasciare che la piastra segnale MALDI-TOF ad asciugare. La velocità di essiccazione può essere aumentata inserendo la piastra sul bordo della cappa per massimizzare il flusso d&#39;aria attraverso la piastra. </li><li> Sovrapporre ogni spot essiccato con 1 ml di soluzione di matrice (/ ml di acido α-ciano-4-idrossicinnamico 10 mg (HCCA), 50% di acetonitrile, 2,5% di acido trifluoroacetico). Nota: la soluzione Matrix può influenzare il corpo se viene inalato o se entra in Contact con la pelle. Durante la preparazione o la manipolazione di soluzioni, si raccomanda che il personale utilizza un aspiratore oltre a dispositivi di protezione individuale. </li><li> Lasciare la piastra segnale MALDI-TOF asciugare sul bordo della cappa come descritto sopra. Assicurare un preparato omogeneo riempie ciascuno dei punti di riferimento sul piatto. </li></ol> 9. Luogo MALDI-TOF piastra bersaglio nello spettrometro di massa <ol><li> Inserire la piastra segnale MALDI-TOF nello spettrometro di massa MALDI-TOF. Assicurarsi che la piastra è seduto contatto con le piastre a molla. Nota: non inserire la piastra di mira fino a quando tutti i punti di riferimento sono asciutti. </li><li> Assicurarsi che la guarnizione in gomma sia pulita di lanugine, polvere e peli di consentire possono essere create le condizioni di vuoto richieste. </li><li> Chiudere il coperchio fase MALDI-TOF e quindi premere il tasto &quot;in / out&quot; sulla parte anteriore dello strumento. Il coperchio sarà ora bloccare e consentire il vuoto necessario da creare. </li></ol> 10. MALDI-TOF MS Spectra Acquisition <ol><li> Aprire il MALDI biotipizzazione e software FlexControl. </li><li> All&#39;interno del software Typing selezionare &quot;nuova classificazione&quot; dal menu a discesa intitolato &quot;file&quot;. Assegnare un nome al progetto e quindi selezionare &quot;nuovo&quot;. Selezionare &quot;Avanti&quot; per completare l&#39;installazione all&#39;interno del software Typing. </li><li> All&#39;interno della finestra del software di controllo di identificare l&#39;immagine sullo schermo della piastra di mira. Evidenziare i punti di destinazione MALDI-TOF che sono in uso spostando il mouse sopra i punti inoculati tenendo premuto il tasto sinistro del mouse. </li><li> Utilizzare il pulsante destro del mouse per fare clic su un punto qualsiasi delle posizioni di destinazione selezionati e quindi selezionare &quot;aggiungi analiti&quot; dal menu a discesa. </li><li> Aggiungi cultura del sangue estremi identificativi alla colonna &quot;ID&quot; per ciascuno dei luoghi di destinazione e fare clic su &quot;Avanti&quot; una volta completato. </li><li> Selezionare &quot;tassonomia&quot; banca dati spettri commerciali e selezionare &quot;Avanti&quot;. Nota: Laboratori possono utilizzare ulteriori internamente o database commerciali per aumentare il numero di spettri di riferimento. </li><li> Fare clic su &quot;Fine&quot; e il MALDI-TOF MS avrà inizio la generazione di spettri. Nota: le impostazioni MALDI-TOF erano per le impostazioni del produttore (in modalità lineare positiva, 60 di frequenza laser Hz, 20 kV tensione di accelerazione, 16,7 kV di tensione IS2, 170 di ritardo estrazione ns, e 2,000-20,137 gamma di m / z). </li><li> Se il software di controllo MALDI non riesce a generare picchi, acquisizione di spettri manuale può essere eseguita (metodo non descritto). </li></ol> 11. Analisi post Interpretazione dei MALDI-TOF MS Scores <ol><li> Una volta gli spettri vengono acquisiti e l&#39;analisi è completa, selezionare il pulsante &quot;+&quot; accanto alla identificazione delle specie sul software Typing per espandere l&#39;elenco di identificazione per ogni target. </li><li> Studiare i primi 5 identificazioni, notando se la top 5 sono simili. Discordanti primi 5 generi con punteggi ≥ 1.7 dello stesso obiettivopunto sollevato la possibilità di brodi misti. Nota: la tecnologia MALDI-TOF ha scarsa sensibilità per il rilevamento di brodi misti. Ad esempio, quando un brodo misto contiene specie un punteggio elevato fiducia può essere identificato solo per una delle specie miste. </li><li> Quando un punteggio ≥ 2.0 è nota per la partita più alta in un luogo di destinazione, riportare la specie. </li><li> Quando il punteggio più alto in un punto bersaglio è ≥ 1.7, ma &lt;2,0, riportano solo i generi. Se criteri aggiuntivi dei primi 5 identificazioni sono concordanti, poi riferire a livello di specie. </li></ol> 12. Rimozione di MALDI-TOF target Piatto <ol><li> Quando tutti gli spettri sono complete premere il tasto &quot;in / out&quot; sulla macchina MALDI-TOF. </li><li> Aprire la piastra di mira ricettacolo MALDI-TOF e rimuovere la piastra bersaglio MALDI-TOF. </li><li> Se si utilizza una piastra segnale MALDI-TOF riutilizzabili, puliti tutti i punti di destinazione utilizzando alcool e conservare la piastra MALDI-TOF a temperatura ambiente una volta che è pulita e asciutta. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

E &#39;importante quando si applica MALDI-TOF MS alla cultura del sangue brodo che le operazioni di post centrifugazione vengono eseguite con sufficiente attenzione a non rimescolare i componenti separati. È particolarmente importante rimuovere i componenti di coltura sangue e proteine ​​cellulari umani, compresi emoglobina, che possono produrre picchi interferenti con gli spettri MALDI-TOF. Anche se MS produce consiglia di tagliare punteggio ≥ 2.0 per le specie e ≥ 1.7 per l&#39;id...

I pazienti con infezione sangue (BSI) a causa di batteri continuano ad avere alta mortalità in ospedale, che vanno 6-48% 1. La consegna di un adeguato antibiotica empirica promuove la sopravvivenza e nel sottogruppo di pazienti con sepsi grave, ogni ora di ritardo alla terapia appropriata è correlato alla diminuzione della sopravvivenza 2,3. Di conseguenza, un obiettivo chiave del laboratorio clinico è quello di rilevare rapidamente, identificare e comunicare la presenza di batteri nelle colture di sangue per informare le decisioni cliniche. È stato dimostrato che il laboratorio microbiologia ha la più grande influenza sulla terapia antimicrobica al momento di segnalare la colorazione di Gram 4 e recentemente, uno studio d&#39;osservazione ha dimostrato che il tempo desorbimento laser assistita dalla matrice / ionizzazione di volo spettrometria di massa (MALDI-TOF MS) eseguita direttamente sul brodi di coltura di sangue influenzare la prescrizione in oltre un terzo di BSI causate da batteri Gram-negativi 5. 6,7. La tecnologia è ormai ben consolidata ed è stato integrato in molti laboratori per l&#39;identificazione rapida e accurata dei microrganismi isolati su supporti 6,8 solida. L&#39;applicazione diretta di MALDI-TOF MS alla cultura del sangue (BC) brodo che hanno segnalato &quot;positivo&quot; per i microrganismi appello a entrambi i medici e responsabili di laboratorio a causa della possibilità di ottenere la tempestiva individuazione dei microrganismi a basso costo. L&#39;utilità clinica di applicazione diretta di MALDI-TOF MS alla cultura del sangue brodo è stato limitato dalla vasta gamma di sensibilità osservati rispetto ai metodi di coltura fenotipica standard di base di identificazione, con i rapporti di identificazione successo dei batteri Gram-negativi che vanno 47-98,9 9-11%. La variazione di sensibilità probabilesi riferisce alla composizione aC brodo, concentrazione batterica iniziale, variazione nei metodi di preparazione del campione e la gamma di microrganismi Gram-negativi riscontrati in popolazioni di studio 9. Rispetto a questi altri protocolli pubblicati il ​​metodo qui presentato evita l&#39;uso di etanolo, cloruro di ammonio o aggiuntivo (non a matrice) acetonitrile. Come risultato il pellet batterico rimanere vitali (fino al punto di estrazione delle proteine) consentendo potenziali metodi di prova sensibilità fenotipica da applicare direttamente a questi organismi in brodo. Inoltre, il metodo presentato ha dimostrato di essere poco costoso, affidabile e rapido con identificazione batterica disponibile entro 25 min delle emocolture Gram risultati della colorazione, con minime &#39;mani&#39; tempo 12. Questo metodo è un semplice protocollo spin-lisi in casa utilizzando estrazione acido formico applicato direttamente positivi brodi di coltura sangue per identificare Gram negativi bacteria con la tecnologia MALDI-TOF MS.

<table border="0" cellpadding="0" cellspacing="0" width="1319">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">BACTEC Plus Aerobic/F Medium</td>
			<td style="width:307px;">Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right" style="width:188px;">442192</td>
			<td style="width:551px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Lytic/10 Anaerobic /F Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442265</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BACTEC Peds Plus Medium</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">442194</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer - Blood transfer device</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">364880</td>
			<td>Single use sampling device reducing the risk of needlestick injury</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacutainer SST 5.0mL, Advance plus</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">367954</td>
			<td>Serum separating tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 10 mL</td>
			<td>Becton Dickinson; BD, Franklin Lakes, NJ, USA</td>
			<td align="right">302143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette</td>
			<td>Samco, USA</td>
			<td>222-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfer pipette (fine tipped)</td>
			<td>Samco, USA</td>
			<td>232-20S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge tube (2.0 mL)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>0030.120.094</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile water (DNAse and RNAse free)</td>
			<td>Life Technologies, Carlsbad, California, USA</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Sigma-Aldrich, St. Louis, Missouri, USA</td>
			<td>F0507</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Matrix solution&#160;</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td align="right">285074</td>
			<td>10 mg/ml &#945;-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 2.5% trifluoroacetic acid</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Benchtop microflex LT MALDI-TOF MS</td>
			<td>Bruker Daltonics, Bremen Germany</td>
			<td></td>
			<td>Utilizing BioTyper 3.1 (Build 65) and FlexControl 3.3 (Build 99) software</td>
		</tr>
	</tbody>
</table>

rapid identification of gram negative bacteria from blood culture broth using maldi-tof mass spectrometry

Un ruolo importante del laboratorio di microbiologia clinica è quello di fornire una rapida identificazione dei batteri che causano infezioni flusso sanguigno. L&#39;identificazione tradizionale richiede la sub-cultura del segnalato brodo di coltura di sangue con l&#39;identificazione disponibili solo dopo colonie su agar solido hanno maturato. MALDI-TOF MS è un metodo rapido affidabile per l&#39;identificazione della maggioranza dei batteri clinicamente rilevanti quando applicato colonie su supporti solidi. L&#39;applicazione di MALDI-TOF MS direttamente alla cultura del sangue brodo è un approccio interessante in quanto ha il potenziale di accelerare l&#39;identificazione delle specie di batteri e migliorare la gestione clinica. Tuttavia, un problema importante da superare è la rimozione pre-analisi di interferenti resine, proteine ​​e di emoglobina contenute nei campioni di sangue di cultura che, se non rimosso, interferiscono con la MS spettri e può portare a punteggi di identificazione discriminazione insufficienti o bassi. Inoltre è necessario concentrare Bacteria per sviluppare spettri di qualità sufficiente. Il metodo presentato descrive la concentrazione, la purificazione e l&#39;estrazione dei batteri Gram-negativi che consentano l&#39;identificazione precoce dei batteri da un segnalata cultura sangue brodo.

Il generata MALDI-TOF MS spettri vengono confrontati con il database di riferimento integrato di spettri. Un punteggio logaritmica viene assegnato per la fiducia del match tra l&#39;isolato di prova e il database di riferimento isolati, con la raccomandazione di un punteggio ≥ 1.7 richiesto per probabile identificazione a livello di generi (figura 1) e ≥ 2.0 per probabile l&#39;identificazione di specie (Figura 2). Relazione al livello di specie quando il punteggio ≥ 1.7 e le prim...

Watch this Scientific Journal Video about Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry at JoVE.com

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

L&#39;applicazione di desorbimento laser assistita da matrice tempo / ionizzazione di volo (MALDI-TOF) spettrometria di massa (MS) direttamente emocoltura brodo velocizza l&#39;identificazione dei batteri. Il metodo proposto è un metodo rapido e affidabile per l&#39;identificazione dei batteri Gram negativi direttamente dalla cultura sangue brodo.

Rapida identificazione dei batteri Gram-negativi dal sangue brodo di coltura Uso MALDI-TOF

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

rapid-identification-gram-negative-bacteria-from-blood-culture-broth

Research

JoVE Journal

Immunology and Infection

23.8K Views.  Westmead Hospital.  L'applicazione di desorbimento laser assistita da matrice tempo / ionizzazione di volo (MALDI-TOF) spettrometria di massa (MS) direttamente emocoltura brodo velocizza l'identificazione dei batteri. Il metodo proposto è un metodo rapido e affidabile per l'identificazione dei batteri Gram negativi direttamente dalla cultura sangue brodo. 

Video: Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Una procedura 1,5 ore per l&#39;identificazione di Enterococcus Specie Direttamente da emocolture

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Ricerca

Immunologia e infezioni

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Purificazione e visualizzazione di lipopolisaccaride da batteri Gram-negativi da Hot acquosa-fenolo di estrazione

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Rilevazione di batteri utilizzando DNAzimi fluorogenici

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

Whole-cell MALDI-TOF spettrometria di massa è un metodo accurato e rapido per analizzare diverse modalità di attivazione dei macrofagi

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

Isolamento di Double Negative Cellule T αβ dal rene

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

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

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

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

Preparazione di un Sangue cultura pellet per il Rapid batterica identificazione e test di sensibilità agli antibiotici

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

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

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

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

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

Un riferimento brodo di microdiluizione per Dalbavancina<em&gt; In Vitro</em&gt; Test della Sensibilità di batteri che crescono aerobico

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

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

Identificazione di batteri patogeni rari da 16S rRNA sequenziamento del gene e MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

Cultura dei macrofagi macrofagi colony-stimulating factor differenziata umana derivate da monociti

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Destrano migliora l'efficienza di trasduzione lentivirale di cellule NK primarie Murine ed umane

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...