Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Auburn University 

In this article, a phage-based magnetoelastic sensor for the detection of Salmonella typhimurium is reported. Filamentous bacteriophage specific to S. typhimurium was used as a biorecognition element in order to ensure specific and selective binding of bacteria onto the sensor surface. Phage was immobilized onto the surface of the sensors by physical adsorption. The phage immobilized magnetoelastic sensors were exposed to S. typhimurium cultures with different concentrations ranging from 5x10(1) to 5x10(8) cfu/ml, and the corresponding changes in resonance frequency response of the sensor were studied. It was experimentally established that the sensitivity of the magnetoelastic sensors was higher for sensors with smaller physical dimensions. An increase in sensitivity from 159 Hz/decade for a 2 mm sensor to 770 Hz/decade for a 1 mm sensor was observed. Scanning electron microscopy (SEM) analysis of previously assayed biosensors provided visual verification of frequency changes that were caused by S. typhimurium binding to phage immobilized on the sensor surface. The detection limit on the order of 10(3) cfu/ml was obtained for a sensor with dimensions 1x0.2x0.015 mm.

Phage immobilized magnetoelastic sensor for the detection of Salmonella typhimurium.

Dans cet article, un capteur de magn&eacute;tolastiques phage-bas&eacute; pour la d&eacute;tection de Salmonella typhimurium est signal&eacute;. Bact&eacute;riophage filamenteux sp&eacute;cifique de Salmonella typhimurium a &eacute;t&eacute; utilis&eacute; comme un &eacute;l&eacute;ment de bioreconnaissance afin d&#39;assurer une liaison sp&eacute;cifique et s&eacute;lective des bact&eacute;ries sur la surface du capteur. Phage &eacute;tait immobilis&eacute; sur la surface des capteurs par adsorption physique. Les phage immobilis&eacute; magn&eacute;tolastiques capteurs ont &eacute;t&eacute; expos&eacute;s &agrave; des cultures de S. typhimurium avec diff&eacute;rentes concentrations allant de 5x10(1) &agrave; 5x10(8) UFC/ml, et les modifications correspondantes dans la r&eacute;ponse de fr&eacute;quence de r&eacute;sonance du capteur ont &eacute;t&eacute; &eacute;tudi&eacute;es. Il a &eacute;t&eacute; &eacute;tabli exp&eacute;rimentalement que la sensibilit&eacute; des capteurs magn&eacute;tolastiques &eacute;tait plus &eacute;lev&eacute;e pour les capteurs avec plus petites dimensions physiques. A observ&eacute; une augmentation de la sensibilit&eacute; de 159 Hz/d&eacute;cennie pour un capteur de 2 mm &agrave; 770 Hz/d&eacute;cennie pour un capteur de 1 mm. Scanning electron microscopy (SEM) analyse des biocapteurs pr&eacute;c&eacute;demment analys&eacute;es a fourni une v&eacute;rification visuelle des changements de fr&eacute;quences qui ont &eacute;t&eacute; caus&eacute;s par la liaison de S. typhimurium au phage immobilis&eacute; sur la surface du capteur. La limite de d&eacute;tection de l&#39;ordre de 10 UFC/ml a &eacute;t&eacute; obtenue pour un capteur avec dimensions 1x0.2x0.015 mm.

Phage immobilis&eacute; magn&eacute;tolastiques capteur pour la d&eacute;tection de Salmonella typhimurium.

In questo articolo, viene segnalato un fago-based magnetoelastic sensore per il rilevamento di Salmonella typhimurium. Filamentosi batteriofagi specifici per S. typhimurium &egrave; stato utilizzato come elemento di biorecognition al fine di garantire l&#39;associazione specifica e selettiva dei batteri sulla superficie del sensore. Fago &egrave; stato immobilizzato sulla superficie dei sensori di adsorbimento fisico. I sensori di magnetoelastic dei fagi immobilizzato sono stati esposti a culture di S. typhimurium con diverse concentrazioni che vanno da 5x10(1) a 5x10(8) cfu/ml, e sono stati studiati i cambiamenti corrispondenti nella risposta di frequenza di risonanza del sensore. &Egrave; stato sperimentalmente stabilito che la sensibilit&agrave; dei sensori magnetoelastic era superiore per sensori con pi&ugrave; piccole dimensioni fisiche. &Egrave; stato osservato un aumento della sensibilit&agrave; da 159 Hz/decennio per un sensore di 2 mm a 770 Hz/decennio per un sensore di 1 mm. Analisi di microscopia elettronica (SEM) di biosensori precedentemente dosati di scansione fornito verifica visiva dei cambiamenti di frequenza che sono stati causati dalla associazione S. typhimurium phage immobilizzato sulla superficie del sensore. Il limite di rilevabilit&agrave; dell&#39;ordine di 10 UFC/ml &egrave; stato ottenuto per un sensore con dimensioni 1x0.2x0.015 mm.

Dei fagi immobilizzato magnetoelastic sensore per il rilevamento di Salmonella typhimurium.

Staphylococcus aureus (S. aureus)-specific bacteriophage was used as a probe for detection of methicillin-resistant S. aureus (MRSA) in aqueous solution using a novel optical method. Biorecognition phage monolayers transferred to glass substrates using Langmuir-Blodgett (LB) technique were exposed individually to MRSA in solution at logarithmic concentrations ranging from 10(6) to 10(9)cfu/ml, and observed for real-time binding using a CytoViva optical light microscope system. Results indicate that LB monolayers possessed high levels of elasticity (K), measuring 22 and 29 mN/m for 10(9) and 10(11)pfu/ml phage concentrations, respectively. Near-instantaneous MRSA-phage binding produced 33+/-5%, 10+/-1%, 1.1+/-0.1%, and 0.09+/-0.01% coverage of the substrate that directly correlated to a decrease in MRSA concentrations of 10(9), 10(8), 10(7), and 10(6)cfu/ml. The exclusive selectivity of phage monolayers was verified with Salmonella enterica subsp. enterica serovar typhimurium (S. typhimurium) and Bacillus subtilis.

Real-time optical detection of methicillin-resistant Staphylococcus aureus using lytic phage probes.

Staphylocoque dor&eacute; (Staphylococcus aureus)-bact&eacute;riophage sp&eacute;cifique a &eacute;t&eacute; utilis&eacute; comme une sonde pour la d&eacute;tection de m&eacute;thicilline S. aureus (SARM) en solution aqueuse &agrave; l&#39;aide d&#39;une m&eacute;thode optique originale. Monocouches de phage bioreconnaissance transf&eacute;r&eacute;s &agrave; des substrats de verre &agrave; l&#39;aide de la technique de Langmuir-Blodgett (LB) ont &eacute;t&eacute; expos&eacute;s individuellement au SARM en solution &agrave; logarithmiques concentrations allant de 10 (6) &agrave; 10 (9) UFC/ml et observ&eacute;s pour la liaison en temps r&eacute;el &agrave; l&#39;aide d&#39;un syst&egrave;me de microscopie optique CytoViva. Les r&eacute;sultats indiquent que LB monocouches poss&egrave;dent des niveaux &eacute;lev&eacute;s d&#39;&eacute;lasticit&eacute; (K), 22 et 29 mN/m pour 10 9 et 10 (11) de la mesure des concentrations de phage UFP/ml, respectivement. Liaison quasi instantan&eacute;e de SARM-phage produit 33 +/-5 %, 10 &plusmn; 1 %, 1,1 +/-0,1 % et la couverture de 0.09+/-0.01% du substrat qui directement corr&eacute;l&eacute;e &agrave; une diminution des concentrations de SARM de 10 (9), 10 (8), 10 (7) et 10 (6) UFC/ml. La s&eacute;lectivit&eacute; exclusive de monocouches phage a &eacute;t&eacute; v&eacute;rifi&eacute;e avec Salmonella enterica subsp. enterica serovar typhimurium (Salmonella typhimurium) et de Bacillus subtilis.

Sondes de d&eacute;tection optique en temps r&eacute;el de Staphylococcus aureus m&eacute;thicilline-r&eacute;sistant en utilisant un phage lytique.

Staphylococcus aureus (S. aureus)-specifico batteriofago era usata come una sonda per il rilevamento di meticillino-resistenti Staphylococcus aureus (MRSA) in soluzione acquosa mediante un metodo ottico romanzo. Monostrati di fago Biorecognition trasferiti a substrati di vetro utilizzando la tecnica di Langmuir-Blodgett (LB) esposti singolarmente per MRSA in soluzione a concentrazioni logaritmiche che vanno da 10 a 10 (9) cfu/ml e fu osservate per l&#39;associazione in tempo reale utilizzando un sistema ottico del microscopio CytoViva. I risultati indicano che LB monostrati possedevano alti livelli di elasticit&agrave; (K), 22 e 29 mN/m per arrecavano e 10 (11) di misura concentrazioni dei fagi pfu/ml, rispettivamente. Associazione di MRSA-fago immediato prodotto 33 + 5%, 10 + /-1%, 1,1 + /-0,1% e la copertura 0.09+/-0.01% del substrato che direttamente correlate ad una diminuzione della concentrazione di MRSA di riesami, 10, paragrafo 8, 10 e 10 (6) cfu/ml. La selettivit&agrave; esclusiva di monostrati dei fagi &egrave; stata verificata con Salmonella enterica subsp. enterica sierotipo typhimurium (S. typhimurium) e Bacillus subtilis.

Sonde di rilevamento ottico in tempo reale di Staphylococcus aureus meticillino-resistente utilizzando dei fagi litici.

Stable, insoluble Langmuir monolayer films composed of Staphylococcus aureus-specific lytic bacteriophage were formed at an air-water interface and characterized. The phage monolayer was very strong, withstanding a surface pressure of âˆ¼40 mN/m at 20 Â°C. The surface pressure-area (Î -A) isotherm possessed a shoulder at âˆ¼7 Ã— 10(4)nm(2)/phage particle, attributed to a change in phage orientation at the air-water interface from horizontal to vertical capsid-down/tail-up orientation as surface pressure was increased. The Î -A-dependence was accurately described using the Volmer equation of state, assuming horizontal orientation to an air-water interface at low surface pressures with an excluded area per phage particle of 4.6 Ã— 10(4)nm(2). At high pressures phage particles followed the space-filling densely packed disks model with a specific area of 8.5 Ã— 10(3)nm(2)/phage particle. Lytic phage monolayers were transferred onto gold-coated silica substrates from the air-water interface at a constant surface pressure of 18 mN/m by Langmuir-Blodgett method, then dried and analyzed by scanning electron microscopy (SEM) and ellipsometry. Phage specific adsorption (Î“) in Langmuir-Blodgett (LB) films measured by SEM was consistent with that calculated independently from Î -A isotherms at the transfer surface pressure of 18 mN/m (Î“=23 phage particles/Î¼m(2)). The 50 nm-thickness of phage monolayer measured by ellipsometer agreed well with the horizontal phage average size estimated by SEM. Surface properties of phage Langmuir monolayer compare well with other monolayers formed from nano- and micro-particles at the air-water interface and similar to that of classic amphiphiles 1,2-diphytanoyl-sn-glycero-3-phosphocholine (phospholipid) and stearic acid.

Phage Langmuir monolayers and Langmuir-Blodgett films.

Films de monocouche de Langmuir stables, insolubles compos&eacute; de Staphylococcus aureus sp&eacute;cifiques bact&eacute;riophage lytique ont &eacute;t&eacute; form&eacute;es &agrave; l&#39;interface air-eau et caract&eacute;ris&eacute;s. La monocouche phage a &eacute;t&eacute; tr&egrave;s solide, r&eacute;sistant &agrave; une pression de surface de &sim;40 mN/m &agrave; 20 &deg; C. L&#39;isotherme de pression-superficie (&Pi;-A) poss&eacute;dait un &eacute;paulement &agrave; environ 7 &times; 4 nm 2/phage particule attribu&eacute; &agrave; un changement dans l&#39;orientation du phage &agrave; l&#39;interface air-eau d&#39;horizontal &agrave; orientation verticale de capside-down/queue-up comme pression de surface a &eacute;t&eacute; augment&eacute;e. La &Pi;-A-d&eacute;pendance a &eacute;t&eacute; d&eacute;crite avec pr&eacute;cision &agrave; l&#39;aide de l&#39;&eacute;quation d&#39;&Eacute;tat, en supposant une orientation horizontale &agrave; une interface air-eau &agrave; basse pression de surface avec une superficie exclue par particule phage de 4,6 &times; 10(4)nm(2) de Volmer. &Agrave; des pressions &eacute;lev&eacute;es particules phagiques a suivi le mod&egrave;le de disques dense remplissant l&#39;espace avec une zone sp&eacute;cifique de particule de 2/phage 8,5 &times; 3 nm. Monocouches de bact&eacute;riophages lytiques ont &eacute;t&eacute; transf&eacute;r&eacute;s sur des substrats de silice or-enduits de l&#39;interface air-eau &agrave; une pression de surface constante de 18 mN/m par m&eacute;thode de Langmuir-Blodgett, puis s&eacute;ch&eacute; et analys&eacute;s par electron microscope &agrave; balayage (SEM) et ellipsom&eacute;trie. Adsorption sp&eacute;cifique de phage (&Gamma;) dans les films de Langmuir-Blodgett (LB) mesur&eacute;e par SEM &eacute;tait conforme &agrave; celle calcul&eacute;e ind&eacute;pendamment de &Pi;-a isothermes &agrave; la pression de surface de transfert de 18 mN/m (&Gamma; = 23 phage particles/&mu;m(2)). Les 50 nm-&eacute;paisseur de monocouche phage mesur&eacute;e par ellipsom&egrave;tre concordent bien avec la taille moyenne de phage horizontal, estim&eacute;e par les propri&eacute;t&eacute;s de Surface SEM. du phage monocouche de Langmuir compare bien avec les autres monocouches form&eacute;s de nano - et micro-particules &agrave; l&#39;interface air-eau et semblables &agrave; celle de 1,2-diphytanoyl-sn-glycero-3-phosphocholine classique amphiphiles (phospholipides) et l&#39;acide st&eacute;arique.

Les monocouches de Langmuir phage et films de Langmuir-Blodgett.

Film di monostrati di Langmuir stabile, insolubile &egrave; composta da Staphylococcus aureus specifico batteriofago litico erano formate a un&#39;interfaccia aria-acqua e caratterizzata. Il monostrato dei fagi &egrave; stata molto forte, resistere ad una pressione superficiale di &sim;40 mN/m a 20 &deg; C. L&#39;isoterma di pressione-superficie (&Pi;-A) ha posseduto una spalla a &sim;7 &times; 4 particella nm 2/fago, attribuita ad un cambiamento nell&#39;orientamento dei fagi all&#39;interfaccia aria-acqua da orizzontale a orientamento verticale del capside-gi&ugrave;/coda-su come &egrave; stata aumentata la pressione di superficie. &Pi;-A-dipendenza &egrave; stato accuratamente descritto utilizzando l&#39;equazione di stato, assumendo un orientamento orizzontale a un&#39;interfaccia aria-acqua a basse pressioni superficiali con un&#39;area esclusa per particelle dei fagi di 4,6 &times; 10(4)nm(2) Volmer. Alle alte pressioni particelle dei fagi seguirono modello space-filling densamente dischi con una specifica area di particelle di legno 8,5 &times; 3 nm 2/fago. Monostrati dei fagi litici sono stati trasferiti su substrati di silicio rivestito in oro dall&#39;interfaccia aria-acqua ad una pressione di superficie costante di 18 mN/m dal metodo di Langmuir-Blodgett, poi essiccati e analizzato da analisi di microscopia elettronica (SEM) ed ellipsometry. Adsorbimento specifico dei fagi (&Gamma;) nel film di Langmuir-Blodgett (LB) misurata da SEM &egrave; stato coerenza con quello calcolato indipendentemente da isoterme &Pi;-alla pressione di superficie di trasferimento di 18 mN/m (&Gamma; = 23 particles/&mu;m(2)) dei fagi. 50 Nm-spessore del monostrato di fago misurata da ellipsometer concordato anche con la dimensione media dei fagi orizzontale stimata dalle propriet&agrave; SEM. superficie del fago monostrati di Langmuir confrontare bene con altri strati monomolecolari formate da nano - e micro-particelle all&#39;interfaccia aria-acqua e simile a quella del classico anfifili 1,2-diphytanoyl-sn-glycero-3-phosphocholine (fosfolipidi) e acido stearico.

Film Langmuir-Blodgett e monostrati di Langmuir dei fagi.

Discrimination of methicillin resistant (MRSA) and sensitive (MSSA) strains of Staphylococcus aureus, was achieved by the specially selected lytic bacteriophage with a wide host range of S. aureus strains and a penicillin-binding protein (PBP 2a) specific antibody. A quartz crystal microbalance with dissipation monitoring (QCM-D) was employed to analyze bacteria-phage interactions. The lytic phages were transformed into phage spheroids by exposure to water-chloroform interface. Phage spheroid monolayers were transferred onto QCM-D sensors by Langmuir-Blodgett (LB) technique. Biosensors were tested in the flow mode with bacterial water suspensions, while collecting frequency and energy dissipation changes. Bacteria-spheroid interactions resulted in decreased resonance frequency and an increase in dissipation energy for both MRSA and MSSA strains. Following the bacterial binding, these sensors were further exposed to a flow of the penicillin-binding protein (PBP 2a) specific antibody conjugated latex beads. Sensors tested with MRSA responded to PBP 2a antibody beads; while sensors examined with MSSA gave no response. This experimental difference establishes an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Both free and immobilized bacteriophages strongly inhibit bacterial growth on solid/air interfaces and in water suspensions. After lytic phages are transformed into spheroids, they retain their strong lytic activity and demonstrate high bacterial capture efficiency. The phage and phage spheroids can be used for screening and disinfection of antibiotic resistant bacteria. Other applications may include use on biosensors, bacteriophage therapy, and antimicrobial surfaces.

Detection and identification of methicillin resistant and sensitive strains of Staphylococcus aureus using tandem measurements.

Discrimination de r&eacute;sistant &agrave; la m&eacute;thicilline (SARM) et des souches sensibles (MSSA) de Staphylococcus aureus, a &eacute;t&eacute; atteint par le bact&eacute;riophage lytique sp&eacute;cialement s&eacute;lectionn&eacute; avec une gamme de S. aureus souches de gamme d&#39;h&ocirc;tes et un anticorps sp&eacute;cifique de la p&eacute;nicilline-liaison prot&eacute;ique (PBP 2 a). Une microbalance &agrave; cristal de quartz avec dissipation surveillance (QCM-D) a &eacute;t&eacute; utilis&eacute;e pour analyser les interactions bact&eacute;riophages-bact&eacute;ries. Les phages lytiques sont transform&eacute;es en sph&eacute;ro&iuml;des phage par l&#39;exposition &agrave; l&#39;interface eau-chloroforme. Monocouches sph&eacute;ro&iuml;de phage ont &eacute;t&eacute; transf&eacute;r&eacute;s sur des capteurs de QCM-D par la technique de Langmuir-Blodgett (LB). Biocapteurs ont &eacute;t&eacute; test&eacute;s sous le mode avec des suspensions bact&eacute;riennes de l&#39;eau, tout en collectant des changements de dissipation de fr&eacute;quence et de l&#39;&eacute;nergie. Interactions bact&eacute;ries-sph&eacute;ro&iuml;de a abouti &agrave; la fr&eacute;quence de r&eacute;sonance une diminution et une augmentation de l&#39;&eacute;nergie de dissipation pour les souches de SARM et de MSSA. Suite &agrave; la liaison bact&eacute;rienne, ces capteurs ont &eacute;t&eacute; davantage expos&eacute;s &agrave; un flux des prot&eacute;ine se liant &agrave; la p&eacute;nicilline (PBP 2 a) anticorps sp&eacute;cifique conjugu&eacute; billes de latex. Capteurs test&eacute; par SARM a r&eacute;pondu aux perles d&#39;anticorps PBP 2 a ; pendant l&#39;examen de capteurs avec MSSA a donn&eacute; aucune r&eacute;ponse. Cette diff&eacute;rence exp&eacute;rimentale &eacute;tablit une discrimination sans ambigu&iuml;t&eacute; entre r&eacute;sistants &agrave; la m&eacute;thicilline et sensibles S. aureus souches. Les bact&eacute;riophages libres et immobilis&eacute;es inhibent fortement la croissance bact&eacute;rienne sur des interfaces solide/air et dans des suspensions de l&#39;eau. Apr&egrave;s que les phages lytiques sont transform&eacute;s en sph&eacute;ro&iuml;des, ils conservent leur forte activit&eacute; lytique et d&eacute;montrent l&#39;efficacit&eacute; de captage bact&eacute;rienne &eacute;lev&eacute;e. Le phage et sph&eacute;ro&iuml;des phage peuvent &ecirc;tre utilis&eacute;s pour le d&eacute;pistage et la d&eacute;sinfection de bact&eacute;ries r&eacute;sistantes aux antibiotiques. Autres applications peuvent inclure l&#39;utilisation sur surfaces antimicrobiennes, th&eacute;rapie bact&eacute;riophage et biocapteurs.

D&eacute;tection et identification des souches sensibles et r&eacute;sistantes &agrave; la m&eacute;thicilline de Staphylococcus aureus en utilisant des mesures en tandem.

Discriminazione di meticillino-resistente (MRSA) e sensibile (MSSA) ceppi di Staphylococcus aureus, fu raggiunto da batteriofago litico appositamente selezionato con un host vasta gamma di S. aureus ceppi e un anticorpo specifico penicillina-binding protein di (PBP 2a). Una microbilancia al quarzo cristallo con dissipazione di monitoraggio (QCM-D) &egrave; stato impiegato per analizzare le interazioni dei fagi batteri. I fagi litici furono trasformati in sferoidi fago da esposizione all&#39;interfaccia acqua-cloroformio. Monostrati sferoide dei fagi sono stati trasferiti su sensori QCM-D da tecnica di Langmuir-Blodgett (LB). Biosensori sono stati testati in modalit&agrave; flusso con sospensioni batteriche dell&#39;acqua, mentre la raccolta modifiche di dissipazione di energia e frequenza. Interazioni batteri-sferoide portato a frequenza di risonanza &egrave; diminuito e un aumento di energia di dissipazione per i ceppi MRSA sia MSSA. Seguendo l&#39;associazione batterica, questi sensori sono stati ulteriormente esposti ad un flusso di penicillina-binding protein (PBP 2a) specifico anticorpo coniugato con lattice perline. Sensori testati con MRSA ha risposto alle perle di PBP 2a dell&#39;anticorpo; mentre sensori esaminati con MSSA ha dato alcuna risposta. Questa differenza sperimentale stabilisce una discriminazione univoca tra i ceppi meticillino resistente e sensibile S. aureus. Entrambi gratuiti e immobilizzati batteriofagi fortemente inibiscono la crescita batterica su interfacce solido/aria e nelle sospensioni di acqua. Dopo fagi litici vengono trasformati in sferoidi, essi conservano la loro forte attivit&agrave; litico e dimostrare acquisizione batterica ad alta efficienza. Il fago e sferoidi fago possono essere utilizzati per lo screening e la disinfezione dei batteri resistenti agli antibiotici. Altre applicazioni possono includere utilizzo su superfici antimicrobiche, terapia del batteriofago e biosensori.

Rajesh Guntupalli

Department of Anatomy,
Physiology and Pharmacology,
College of Veterinary Medicine,
Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine

Auburn University

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Rajesh Guntupalli

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Anatomy,Physiology and Pharmacology,College of Veterinary Medicine,Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine

Auburn University

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Department of Anatomy,
Physiology and Pharmacology,
College of Veterinary Medicine,
Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine