Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

Summary

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.

Abstract

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.

Introduction

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 °C rather than 37 °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 (>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.

Protocol

1. Setting the Stage

1. 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.
2. Obtain PBP 2a antibody conjugated latex beads.
3. Prepare NZY medium as described ²¹.
4. Obtain phosphate buffered saline solution (PBS).
5. Prepare deionized water as subphase for Langmuir-Blodgett (LB) monolayer deposition.

2. Bacteriophage Propagation and Titration

1. Incubate 5 ml of overnight culture of S. aureus ATCC 12600 (3.6 x 10⁸ colony forming units (CFU)/ml) with 500 μl of phage (3 x 10⁹ PFU/ml) in 500 ml NZY medium in 2 L flack on shaker-incubator at 37 °C overnight.
2. Centrifuge culture at 4,424 x g for 10 min at 4 °C.
3. Re-centrifuged supernatant at 11,325 x g for 10 min at 4 °C.
4. Filter supernatant through a 0.22 μm Millipore filter.
5. Centrifuge filtrate at 75,395 x g for 1.5 hr.
6. Dissolve pellet in 100 μl of distilled water.
7. Prepare 10-fold dilutions of phage suspension in NZY medium.
8. Plate 1 ml of overnight (ON) culture of S. aureus ATCC 12600 onto each of 2 plates with NZY agar to make sure all surface is covered. Remove the excess of culture and let the surface dry for 30 min.
9. Spot a sample (10 μl) of appropriate phage dilution onto the surface of the plate and incubate at 37 ^oC for 18-24 hr.
10. Examine the formation of plaques and calculate the phage titer. Bacteriophage titers (T) are estimated on the basis of the number of plaques (N) formed per 10 μl volume (v) of phage suspension and the dilution factor (F). The titer was calculated by formula: T=N/(vxF). For example, for the number of plaques 75 at the dilution 10^-7 and the volume 10 μl (10 x 10^-3 ml), the titer is equal 75 / (10 x 10^-3 x 10^-7) = 7.5 x 10¹⁰ plaque forming units (PFU) per ml of suspension.

3. Gold-immobilized Phage

1. Clean gold-coated quartz pieces (60 mm²) by plasma etching in argon for 10 min and then sterilize for 6 hr under UV light in a sterile cabinet.
2. Add 50 microliters of 1 x 10⁹ PFU/ml phage suspension to the gold surface of each piece in a sterile Petri dish, and then incubate overnight in a humid chamber at room temperature.
3. Remove and titer the remaining phage suspension.
4. Wash pieces with bound phage 5 times with PBS to remove unbound phage.

4. Testing of Immobilized Phage Infectivity on a Dry Surface

1. Spread an O/N culture of S. aureus ATCC 12600 onto an NZY agar plate and allow drying.
2. Place a gold piece with immobilized phage onto the plate face down.
3. Observe a zone of lysis after 12 hr incubation at 37 °C that indicates infectivity.
4. Use gold covered pieces with no phage in control experiments.

5. Testing of Lytic Activity of Free and Bound Phage in Liquid

1. Add an ON culture of S. aureus ATCC 12600 to 10 ml of NZY in every of four 300 ml sidearm flasks (6 x 10⁶ CFU/ml in each flask).
2. Add In two flasks 2 x 10⁶ PFU/ml of free phage.
3. Use the other two flasks as controls.
4. Monitor the time course of S. aureus cell lysis for 570 min by optical density measurement (OD₆₀₀) at 30 min intervals for all flasks.
5. Take a final measurement at 24 hr.
6. Repeat steps 5.1-5.5, but use gold pieces with bound phages instead of free phages, and gold pieces with no phage in control flasks.

6. Loss of Bound Phages During Incubation

1. Add 50 μl of 1 x 10⁹ PFU/ml phage onto the surface of sterile gold piece in a Petri dish that is placed into a humid chamber overnight at room temperature.
2. Remove the suspension with unbound phage from the gold surface and titer.
3. Wash the gold piece with bound phage 5 times with PBS and place in 1 ml NZY solution in 15 ml tube in shaker-incubator at 37 °C for 8 hr.
4. Determine the titer of the detached phage as described in 2.

7. Phage Spheroids Preparation

1. Combining 400 μl of stock phage 12600 suspension (10¹¹ PFU/ml) with an equal volume of spectrophotometric grade chloroform in 1 ml vial at room temperature.
2. Gently vortex the phage-chloroform suspension 5-6 times (5 sec intervals) over one minute duration.
3. Allowed the suspension to stabilize for 30 sec and then pipette of the top phase containing spheroids was pipetted off for monolayer preparation.
4. Take a few microliters of the spheroids suspension for the transmission and scanning electron microscopy.
5. Use a remaining spheroid suspension for biosensor manufacturing.

8. Biosensor Preparation

1. Clean QCM-D sensors by plasma etching in argon for 10 min PDC-32G Harrick plasma cleaner.
2. Rinse sensor with hexane to remove any organic impurities.
3. Prepare and clean a trough of the film balance as described in ²².
4. Fill LB trough with a subphase solution and clean it with a trough barrier and stabilize the trough at 20±0.1 °C
5. Spread 300 μl aliquot of phage 12600 in aqueous suspension (10¹¹ PFU/ml) onto LB subphase.
6. Prepare phage monolayer onto LB subphase solution by allowing 300 μl aliquot of phage 12600 aqueous suspension (10¹¹ PFU/ml) to run down an inclined wettable glass rod that is partially submersed into the subphase ^22,23.
7. Stabilize the prepared monolayer for 10 min, and then compress it at a rate of 30 mm/min (45 cm²/min), until a constant pressure 19 N/m is attained.
8. Perform a vertical film deposition onto perpendicular positioned QCM-D sensor at a rate of 4.5 mm/min by successively dipping sensor in and out of the monolayer seven times. Electron microscopy of deposited phage monolayers before exposure to bacterial samples showed that the layers were continuous, homogeneous, and uniform (data not shown).
9. Repeat steps 4.5-4.8 with a phage spheroid suspension that is prepared in 3.
10. Use fabricated phage biosensors for MRSA detection, electron microscopy, and ellipsometry.

9. Biosensor Testing, Discrimination of Methicillin Resistant and Sensitive Staphylococcus Bacteria

1. In this protocol, biosensors with unmodified and modified phage and phage spheroids are prepared. A quartz crystal microbalance with four sensor flow chambers is used to monitor binding of bacteria to the phage immobilized on QCM sensor surface. PBP 2a antibody conjugated latex beads are utilized to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. All measurements are conducted in the flow mode (50 μl/min) at 5 MHz.
2. Establish a base line resonance frequency of the QCM sensor in water (~30 min).
3. Draw a suspension of live methicillin sensitive staphylococcus bacterial cells in water (10⁹ CFU/ml) through the test cell.
4. Continuously monitor changes in the sensors resonance frequency and energy dissipation for the first overtone, using the Q-Soft software.
5. When the changes in the sensors resonance frequency and dissipation reached saturation levels, add the suspension of PBP antibody conjugated latex beads (6.77 x10¹⁰ beads/ml) to the flow during the continuous monitoring of frequency and dissipation.
6. Repeat steps 9.2-9.5 for methicillin resistant staphylococcus bacterial cells (MRSA).
7. Define a frequency change through the mass change due to bacteria binding by the Sauerbrey equation ^24,25 Δf=-Δm xn/C, where C is the mass sensitivity constant (C=17.7 ng x cm^-2 x Hz^-1 at 5 MHz), m is mass and n is the overtone number (n = 1).
8. Define an energy dissipation change (ΔD) from the recording the exponential decay of oscillation (frequency and amplitude dampening, which allowed quantification of the energy dissipated and stored during one period of oscillation, E_dissipated and E_stored, respectively: ΔD = E_dissipated / 2πE_stored. ΔD is measured in dissipation units (DU), one DU = 10^-6 relative units).

Results

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 °C is shown in Figure 1A (a curve labeled by empty circles). The number of bacteria increased from 3.2 x 10⁶

Discussion

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...

Materials

List of materials used in this article

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