The work reported herein was supported by grants from Auburn University AUDFS and USAF CRADA 07-277-60MDG-01. The views expressed in this article are those of the authors, and do not reflect the official policy or position of the United States Air Force, Department of Defense, or the U.S. Government.

1. Setting the Stage
<ol>
	<li>Obtain type strain S. aureus ATCC 12600, S. aureus ATCC 27690 and Bacillus subtilis ATCC 6051. Methicillin-resistant strains of S. aureus - MRSA1, MRSA 2, MRSA 5, MRSA 13, MRSA 26, MRSA 34, MRSA 45, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882; The lytic phage 12600.</li>
	<li>Obtain PBP 2a antibody conjugated latex beads.</li>
	<li>...

<ol>
	<li>Guntupalli, R., Sorokulova, I., Krumnow, A., Pustovyy, O., Olsen, E., Vodyanoy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+optical+detection+of+methicillin-resistant+Staphylococcus+aureus+using+lytic+phage+probes.">Real-time optical detection of methicillin-resistant Staphylococcus aureus using lytic phage probes.</a> Biosens. Bioelectron. 24, 151-154 (2008).</li><li>Guntupalli, R., Sorokulova, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+identification+of+methicillin+resistant+and+sensitive+strains+of+Staphylococcus+aureus+using+tandem+measurements.">Detection and identification of methicillin resistant and sensitive strains of Staphylococcus aureus using tandem measurements.</a> J. Microbiol. Methods. 90, 182-191 (2012).</li><li>Guntupalli, R., Sorokulova, I., Long, R., Olsen, E., Neely, W., Vodyanoy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage+Langmuir+monolayers+and+Langmuir-Blodgett+films.">Phage Langmuir monolayers and Langmuir-Blodgett films.</a> Colloids and Surfaces, B: Biointerfaces. 82, 182-189 (2011).</li><li>Barie, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic-resistant+gram-positive+cocci:+implications+for+surgical+practice.">Antibiotic-resistant gram-positive cocci: implications for surgical practice.</a> World. J. Surg. 22, 118-126 (1998).</li><li>Byun, D. E., Kim, S. H., Shin, J. H., Suh, S. P., Ryang, D. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methicillin+resistant'+Staphylococcus+aureus+infections+during+1978-79:+clinical+and+bacteriologic+observations.">Methicillin resistant' Staphylococcus aureus infections during 1978-79: clinical and bacteriologic observations.</a> J. Antimicrob. Chemother. 7, 649-655 (1981).</li><li>Knopf, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+infections+caused+by+multiresistant+pathogens.+Clinical+management+exemplified+by+multiresistant+Staphylococcus+aureus.">Nosocomial infections caused by multiresistant pathogens. Clinical management exemplified by multiresistant Staphylococcus aureus.</a> Urologe A. 36, 248-254 (1997).</li><li>Knapp, C. C., Ludwig, M. D., Washington, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+differential+inoculum+disk+diffusion+method+and+Vitek+GPS-SA+card+for+detection+of+oxacillin-resistant+staphylococci.">Evaluation of differential inoculum disk diffusion method and Vitek GPS-SA card for detection of oxacillin-resistant staphylococci.</a> J. Clin. Microbiol. 32, 433-436 (1994).</li><li>Struelens, M. J., Nonhoff, C., Van, D. A., Philippe Mertens, R., Serruys, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+rapid+ATB+Staph+for+5-hour+antimicrobial+susceptibility+testing+of+Staphylococcus+aureus.">Evaluation of rapid ATB Staph for 5-hour antimicrobial susceptibility testing of Staphylococcus aureus.</a> J. Clin. Microbiol. 33, 2395-2399 (1995).</li><li>Woods, G. L., LaTemple, D., Cruz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+MicroScan+rapid+gram-positive+panels+for+detection+of+oxacillin-resistant+staphylococci.">Evaluation of MicroScan rapid gram-positive panels for detection of oxacillin-resistant staphylococci.</a> J. Clin. Microbiol. 32, 1058-1059 (1994).</li><li>Knapp, C. C., Ludwig, M. D., Washington, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+BBL+crystal+MRSA+ID+system.">Evaluation of BBL crystal MRSA ID system.</a> J. Clin. Microbiol. 32, 2588-2589 (1994).</li><li>Qadri, S. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rod+shape+determination+by+the+Bacillus+subtilis+class+B+penicillin-binding+proteins+encoded+by+pbpA+and+pbpH.">Rod shape determination by the Bacillus subtilis class B penicillin-binding proteins encoded by pbpA and pbpH.</a> J. Bacteriol. 185, 4717-4726 (2003).</li><li>Grieco, S. H. H., Lee, S., Dunbar, W. S., MacGillivray, R. T. A., Curtis, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+filamentous+phage+yield+during+computer-controlled+fermentation.">Maximizing filamentous phage yield during computer-controlled fermentation.</a> Bioprocess and Biosystems Engineering. 32, 773-779 (2009).</li><li>Olsen, E. V., Pathirana, S. T., Samoylov, A. M., Barbaree, J. M., Chin, B. A., Neely, W. C., Vodyanoy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+and+selective+biosensor+for+Salmonella+and+its+detection+in+the+environment.">Specific and selective biosensor for Salmonella and its detection in the environment.</a> J. Microbiol. Methods. 53, 273-285 (2003).</li><li>Pathirana, S. T., Barbaree, J., Chin, B. A., Hartell, M. G., Neely, W. C., Vodyanoy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+biosensor+for+Salmonella.">Rapid and sensitive biosensor for Salmonella.</a> Biosens. 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Phys. 155, 206-222 (1959).</li><li>Hook, F., Rodahl, M., Brzezinski, P., Kasemo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+Dissipation+Kinetics+for+Protein+and+Antibody-Antigen+Adsorption+under+Shear+Oscillation+on+a+Quartz+Crystal+Microbalance.">Energy Dissipation Kinetics for Protein and Antibody-Antigen Adsorption under Shear Oscillation on a Quartz Crystal Microbalance.</a> Langmuir. 14, 729-734 (1998).</li><li>Griffith, J., Manning, M., Dunn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamentous+bacteriophage+contract+into+hollow+spherical+particles+upon+exposure+to+a+chloroform-water+interface.">Filamentous bacteriophage contract into hollow spherical particles upon exposure to a chloroform-water interface.</a> Cell. 23, 747-753 (1981).</li><li>Hosseinidoust, Z., Van de Ven, T. G. M., Tufenkji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Capture+Efficiency+and+Antimicrobial+Activity+of+Phage-Functionalized+Model+Surfaces.">Bacterial Capture Efficiency and Antimicrobial Activity of Phage-Functionalized Model Surfaces.</a> Langmuir. 27, 5472-5480 (2011).</li><li>Schofield, D. A., Molineux, I. J., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent'+Reporter+Phage+for+the+Detection+of+Category+A+Bacterial+Pathogens.">Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens.</a> J. Vis. Exp. (53), e2740 (2011).</li><li>Voinova, M. V., Jonson, M., Kasemo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Missing+mass"+effect+in+biosensor's+QCM+applications.">Missing mass" effect in biosensor's QCM applications.</a> Biosens. Bioelectron. 17, 835-841 (2002).</li><li>Gervals, L., Gel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+of+biotinylated+bacteriophages+on+biosensor+surfaces.">Immobilization of biotinylated bacteriophages on biosensor surfaces.</a> Sensors and Actuators. 125, 615-621 (2007).</li><li>Nanduri, V., Sorokulova, I. B., Samoylov, A. M., Simonian, A. L., Petrenko, V. 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It is well known that phages can be used as biosensor probes for bacterial pathogens 28. It is demonstrated in this work that phage together with PBP 2a antibodies can be utilized to resolve the old problem: rapid discrimination antibiotic resistant and sensitive strains.
It was found however those normal unmodified staphylococcal phages are not suitable for bacteria detection with QCM devices, even though they bind bacteria. The phage tail is so long that acoustic waves cannot "rea...

Methicillin resistant strains of Staphylococcus aureus have been suggested as a factor in essential infections and nosocomial outbreaks 4-8. Common ways of the recognition of methicillin resistance, such as the disk diffusion oxacillin agar screen test, or broth microdilution, rely on tailored culture conditions to enhance the expression of resistance. Alterations include the utilization of oxacillin, incubation at 30 or 35 &deg;C rather than 37 &deg;C, and the inclusion of NaCl to the growth medium. Furthermore, for correct detection by these types of techniques, a long incubation period of 24 hr instead of 16 to 18 hr is required. Rapid techniques with appropriate (&gt;96%) level of sensitivity for identification of methicillin resistance include automated microdilution techniques such as the Vitek GPS-SA card, the Rapid ATB Staph system, and the Rapid Microscan Panel system which produce results after 3-11 hr 9-11. The Crystal MRSA ID system is a rapid method based upon recognition of growth of S. aureus in the presence of 2% NaCl and 4 mg of oxacillin per liter with an oxygen-sensitive fluorescence sensor. Claimed sensitivities range between 91 to 100% after 4 hr of incubation 12-14. These phenotypic methods are limited in their accuracies by the impact of prevalent strains that express heterogeneous resistance. Therefore, the best widely accepted methods for the recognition of methicillin resistance is PCR or DNA hybridization of the mecA gene 15. However this technique requires purified DNA and is extremely sensitive to various admixtures (impurities), which include cell debris16.
Furthermore, these techniques need a long time to perform. Strategies to the recognition of the mecA gene product, protein PBP 2a, could be utilized to determine resistance and may be more reliable compared to standard test techniques 17.
It had been earlier shown that bacteriophage 12600 can be utilized as a recognition probe for Staphylococcus aureus strains including those having methicillin resistance 1,2,18. In this work we proposed a novel technique in the specific recognition and detection of MRSA, such as the recognition of bacteria along with conformation of MRSA in real time. For this specific purpose a S. aureus bacteriophage with a wide spectrum of hosts (including MRSA strains) combined with monoclonal antibody against protein (PBP 2a) have been used. PBP 2a is a cell wall protein and it is the cause of antibiotic resistivity of MRSA. However PBP 2a antibody is not specific for S. aureus since some other bacteria have antibiotic binding proteins with sequence similarity to PBP 2a 19,20. Consequently in this work, S. aureus bacteriophage and antibodies against PBP 2a protein have been used. To be able to develop a biosensor to specifically detect and identify MRSA a device with a two-step action has been utilized. The initial step used a S. aureus bacteriophage monolayer as a sensor probe, while the second step employed PBP 2a specific antibodies. Therefore, step one will recognize S. aureus bacteria, as the other one will be sensitive to the antibiotic-binding protein. When signals received from two steps are positive, it indicates the specific detection of MRSA.

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		 </tr>
		 <tr>
			<td>Phosphate buffered saline (PBS) </td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>P4417 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>spectrophotometric-grade chloroform </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO </td>
			<td>154733 </td>
			<td>(99.8% A.C.S.) </td>
		 </tr>
		 <tr>
			<td>Hexane-Anhydrous </td>
			<td>Sigma-Aldrich, 
				St. Louis, MO</td>
			<td>29609-0</td>
			<td>(95%)</td>
		 </tr>
		 <tr>
			<td>Ethyl Alcohol </td>
			<td>Pharmco products Inc. Brookfield, CT </td>
			<td>64-17-5</td>
			<td>190 Proof</td>
		 </tr>	
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		 </tr>
		 <tr>
			<td>PBP 2a antibody conjugated latex beads</td>
			<td>Denka Seiken Co., Ltd, Tokyo, Japan</td>
			<td>The MRSA-Screen test</td>
			<td></td>
		 </tr>
		 <tr>
			<td>S. aureus ATCC 12600, S. aureus ATCC 27690 and Bacillus subtilis ATCC 6051 from </td>
			<td>American Type Culture Collection (Manassas, VA);</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>MRSA1, MRSA 2, MRSA 5, MRSA 13, MRSA 26, MRSA 34, MRSA 45, B. anthracis Sterne, Salmonella typhimurium LT2, Shigella flexneri, Yersinia enterocolotica, Proteus mirabilis, Klebsiella pneumoniae 13882; The lytic phage 12600 </td>
			<td>The culture collection of Auburn University, Auburn, AL</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Centrifuge </td>
			<td>Beckman Coulter</td>
			<td>Optima L-90K
				Ultra Centrifuge</td>
			<td></td>
		 </tr>
		 <tr>
			<td>KSV 2200 LB film balance</td>
			<td>KSV Chemicals, Finland</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Light microscope optical system </td>
			<td>CitoViva Technology Inc., Auburn, AL </td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>QCM-D</td>
			<td>Q-Sense AB, V&auml;stra Fr&ouml;lunda, Sweden </td>
			<td>E4</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scanning electron microscope (SEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL-7000F SEM </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Transmitting electron microscopy (TEM)</td>
			<td>JEOL USA Inc., Peabody, MA</td>
			<td>JEOL, JEM 2010 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Stericup, Presterilized</td>
			<td>Millipore Corporation, Billerica, MA</td>
			<td>SCGPU05RE</td>
			<td>0.22 &mu;m, GP Express PLUS membrane</td>
		 </tr>
		 <tr>
			<td>Bio-Assay dish</td>
			<td>NUNC A/S, Denmark</td>
			<td>240835</td>
			<td>Dimensions(mm), 245 x 245 x 25</td>
		 </tr>
		 <tr>
			<td>Pipettes</td>
			<td>Gilson, Pipetman, France</td>
			<td>P100, P200, P1000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>C24 Incubator Shaker</td>
			<td>New Brunswick Scientific, CT</td>
			<td>Classic C24</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gold-coated quartz pieces</td>
			<td>Auburn University, AL</td>
			<td></td>
			<td>Homemade</td>
		 </tr>
		 <tr>
			<td>Petri dishes</td>
			<td>Fisher Brand, USA</td>
			<td>0875713</td>
			<td>100 mmX15 mm </td>
		 </tr>
		 <tr>
			<td>SterilGard III Advance</td>
			<td>The Baker Company, ME</td>
			<td>SG403</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Culture Growing Flasks</td>
			<td>Corning Incorporated, NY</td>
			<td>4995</td>
			<td>PYREX 250 ml Erlenmeyer flasks </td>
		 </tr>
		 <tr>
			<td>Optical Spectrometer</td>
			<td>Genesys 20. Thermo Spectronic, USA.</td>
			<td>4001</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma, USA</td>
			<td>PDC-32G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Millipore water purification system</td>
			<td>Millipore</td>
			<td>Direct-Q</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Imaging Ellipsometer</td>
			<td>Accurion, USA</td>
			<td>nanofilm_ep3se
			</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Software </td>
			<td>Q-Sense AB, Sweden</td>
			<td>QSoft, QTools</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

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) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

The phage demonstrated lytic activity against all tested strains of S. aureus, including MRSA strains, as indicated by the phage spot test. Plaque sizes generally ranged from 5 to 15 mm. No activity was found against other test-cultures (Table 1).
A normal growth of S. aureus ATCC 12600 in NZY medium on shaker-incubator at 37 &deg;C is shown in Figure 1A (a curve labeled by empty circles). The number of bacteria increased from 3.2 x 106<...

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Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

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

biosensor-for-detection-antibiotic-resistant-staphylococcus

Research

JoVE Journal

Bioengineering

23.9K visualizações.  Auburn University. Biossensores fagos líticos e contas de anticorpos são capazes de discriminar entre resistente à meticilina (MRSA) e bactérias estafilococos sensíveis. Os fagos foram imobilizadas por um método de Langmuir-Blodgett sobre uma superfície de um sensor de microbalança de cristal de quartzo e trabalhado como de largo espectro de estafilococos sondas. Contas de anticorpos reconhecer MRSA.