The overall goal of the following experiment is to discriminate methicillin resistant or MRSA and methicillin sensitive, or MSSA strains of staphylococcus aureus by especially selected anti modified lytic bacteria phage. When bacteria are attached to the long flexible tails of the lytic phage, the distance between the attached cells and the sensor surface is larger than the penetration of the QC m's acoustic wave. Therefore, the signal of the binding event is not generated.
Replacing the intact phage with spheroid results in fully functional phage with short tails, which bind the bacteria within the penetration depth of the QC m's acoustic wave, therefore contributing to the frequency and dissipation changes when phage steroids are deposited on A-Q-C-M-D crystal and exposed to the mixed bacterial suspension, they bind both the MRSA and MSSA strains of staphylococcus aureus, but not other bacteria separating the staphylococcus aureus from the other bacteria on the crystal surface. Finally, when Mr.RSA specific PPP two A antibody bound beads are added, a signal is generated in the presence of Mr.RSA strains of staphylococcus aureus. The main advantages of this technique over existing method such as PCR other that this method doesn't require DNA extraction and is not sensitive to empirics.
Though this method can provide insight into the screening and disinfection of staphylococcus orus, it can also be applied to other antibiotic resistant bacteria. Visual demonstration of this method is critical as the fabrication of biosensors requires precision, accuracy, and patience. Begin by cleaning gold coded quartz pieces by plasma etching in Argonne for 10 minutes, and then sterilize the pieces for six hours under UV light in a sterile cabinet after sterilization at a 50 microliters of phage suspension to the gold surface of each piece in a sterile Petri dish, and then incubate the pieces overnight in a humid chamber at room temperature the next morning, remove the remaining phage suspension for tittering, then wash each quartz piece with bound phage five times with PBS to remove any unbound phage.
To test the immobilized phage infectivity on a dry surface first spread an overnight culture of staphylococcus aureus onto an NZY eRate and allow the suspension to dry. Then place a gold coated quartz piece with immobilized phage face down onto the plate. After 12 hours at 37 degrees Celsius, a zone of lysis indicating infectivity can be observed to test the lytic activity of free phage in liquid.
First, add an overnight bacterial culture of staphylococcus aureus to 10 milliliters of NZY in every of four 300 milliliter sidearm flasks. Then add two times 10 to the six PFU per milliliter of free phage to two of the flasks. Now, monitor the time course of staphylococcus cell lysis for 570 minutes by optical density measurement at 30 minute intervals for each flask taking a final measurement at 24 hours.
To test the lytic activity of bound phage in liquid, repeat the just demonstrated steps, but add gold pieces with bound phage to the experimental flasks instead of free phage. To prepare phage steroids first combine 400 microliters of stock phage 1 2600 suspension with an equal volume of spectra photometric grade chloroform into a one milliliter vial at room temperature. Next, gently vortex the phage chloroform suspension five to six times at five second intervals over a one minute duration.
After allowing the suspension to stabilize for 30 seconds, collect the top fraction containing the steroid suspension. Transfer a few microliters of the steroid suspension into a 500 microliter vial for transmission and scanning electron microscopy. Save the remaining steroid suspension in a one milliliter vial for biosensor manufacturing.
To prepare the biosensor first, clean the QCMD sensors by plasma etching in argon for 10 minutes in a plasma cleaner. Then rinse the sensors with hexane to remove any organic impurities. Next, clean a trough of the film balance as previously described by Olson Etal in the Journal of Microbiological methods.
By filling an LB trough with a sub phase solution and cleaning it with a trough barrier. Then stabilize the trough at 20 degrees Celsius for 10 minutes. Now create a phage monolayer on the LB sub phase solution by carefully dripping a 300 microliter Eloqua of phage 1 2600 aqueous suspension done an inclined edible glass rod that is partially submersed in the SOPH phase After allowing the monolayer to stabilize for 10 minutes, compress the monolayer at a rate of 30 millimeters per minute until a constant pressure of 19 newton's per meter attained.
The vertical film deposition is the trickiest part of this procedure. To ensure success, one need to make sure all the components of this procedure are clean and also monolayer deposition parameters should be optimized. Now dip the sensor in and out of the monolayer seven times in succession at a rate of 4.5 millimeters per minute to perform a vertical film deposition.
Onto the perpendicularly positioned QCMD sensor. Vertical film deposition binds the intact phage to the biosensor surface. Finally, use fabricated phage biosensors for MRSA detection electron microscopy and el optometry of both the free and bound phage.
Begin this step by establishing baselines, resonance frequency and energy dissipation of the QCM sensor in water using qof T software. After about 30 minutes, draw a suspension of live methicillin sensitive staphylococcus aureus or MSSA bacterial cells in water through the test cell. The blue line displays the frequency value while the red line demonstrates the level of energy dissipation.
The horizontal lines show the establishment of the baseline. The number two in the middle of the screen indicates that events are related to the sensor. Number two out of four continuously monitor the changes in the resonance frequency and energy dissipation of the sensors for the first overtone shown.
Here are the changes after bacteria pass the test cell. Note that it only takes a few seconds for the frequency to decrease and the energy dissipation to increase when the changes in the sensors resonance frequency and inner energy dissipation reach saturation levels at the suspension of PVP antibody conjugated latex speeds to the flow and continue the continuous monitoring of the frequency dissipation. Shown here is the whole detection process showing the establishment of baselines, the few second change in frequency and energy dissipation after adding bacteria, the saturation, and finally, the changes after addition of beads.
This table shows the demonstrated phage lytic activity against all the tested strains of S reus, including MRSA strains as indicated by the phage spot test. The plaque sizes generally ranged from five to 15 millimeters. No activity was found against other test cultures.
Here, the normal growth of staphylococcus Urus A TCC 1 2600 in NZ Y medium on a shaker incubator at 37 degrees Celsius is demonstrated with the number of bacteria increasing from 3.2 times 10 to the six to 4.0 times 10 to the eight CFU per milliliter. In about 24 hours, phage immobilized on a gold surface demonstrated a similar lytic activity to the activity of the phage in suspension. The immobilized phage remained infective in a fresh bacterial infection even after use in a 24 hour growing experiment, several washes and storage in PBS for six days at four degrees Celsius.
In this image, the lysosome around the gold piece can be observed indicating that the immobilized phages are capable of lysing bacterial cells. Therefore, as illustrated here, the effective decrease of bacterial growth found at the co-culture of bacteria and demobilized phage is a result of the primary interaction of water suspended bacteria and bound phage. Here, transmission and scanning electron micrographs of the intact lytic phage 1 2600 on a gold substrate surface are shown when the phage suspension is subjected to chloroform treatment, the tail of the phage contracts in length and thickens and the polygonal head becomes rounded or steroid.
In spite of the significant structural changes. The lytic activity of the steroid as measured by plaque numbers did not change as a result of the chloroform treatment. QCM sensors with immobilized lytic phages showed no significant changes in the resonance frequency or energy dissipation when they were exposed to MRSA indicating the MRSA phage interaction resulted in a nom mass change.
According to the QCM, however, the electron micrographs of post assay biosensors revealed significant bacterial binding at the sensor surface. When MRSA suspensions were injected into the flow cell with phage steroid biosensors, a substantial decrease in the frequency and an increase in the dissipation were observed. Following phage sphero bacterial MRSA binding interactions, the assay sensors were exposed to BBP two A antibody conjugated latex speed suspensions.
These MRSA assay biosensors responded to the BPP two A antibody conjugated latex speed suspensions as a further decrease in the frequency and an increase in the dissipation were observed. The binding of the MSA to phage probes and PPP two A antibody conjugated latex speeds was confirmed using scanning electron microscopy investigations. When the phage steroid biosensor was exposed to MSSA, A substantial decrease in the frequency and an increase in the dissipation were observed following phage steroid MSSA binding interactions, the assay sensors were challenged with PPP two A antibody conjugated latex speed suspensions.
Initially, the MSSA assay biosensor showed short transience of increases in frequency and decreases in dissipation after a few minutes of MSSA and PPP two A antibody conjugated latex speeds interactions. However, the frequency return to post PVP two A antibody introduction levels, but the deci energy increased. The binding of MSSA to phage probes was then analyzed by scanning electron microscopy, but no binding between MSSA and PPP two A antibody conjugated latex speeds was observed here, an ellips symmetric thickness profile and a 3D thickness map of lytic phage are shown.
A line across the thickness map was drawn to generate the thickness profile. These last images show representative CCD optical camera images of MRSA captured by phages bound to the gold surface of A-Q-C-M-D sensor and 3D intensity profiles of MRSA on phage immobilized glass substrates at a concentration of 10 to the eight CFU per milliliter and 10 to the nine CFU per milliliter. Each round circle represents a single bacterium.
It is clear that the increase of bacteria concentration in liquid suspension from 10 to the eight CFU per milliliter to 10 to the nine CFU per milliliter results in the significant rise of captured bacteria in the 3D intensity profiles. The number of white peaks on the top layer is proportional to the number of captured bacteria, and as expected, the density of the peaks increases significantly when the bacterial exposure is increased from 10 to the eight to 10 to the nine CFUs per milliliter. The bottom dark blue layer corresponds to the phage layer contacting with a gold substrate.
As more bacteria are captured, the phage optical properties change as they bind bacteria resulting in a darker more intense blue color. Following this procedure, we can define bacterial capture efficiency in order to understand phage bacteria interactions After development. This technique shows the way for researchers in the field of bacteria phage therapy to explore pathogen inhibition in animals and humans Don't forget that working with organic solvents in pathogenic bacteria can be extremely hazardous, and following the strict regulation is very important.
