The overall goal of this procedure is to identify and assess antibiotic susceptibility of blood culture isolates in less time than with conventional methods. This is accomplished by first identifying the blood culture isolate. Using a novel 16 s ribosomal, DNA MultiPro PCR assay.
Then the isolate is incubated with a panel of antibiotics for six hours. The next step is to determine growth or growth in inhibition of the isolate in the presence of the antibiotic using a quantitative 16 s ribosomal D-N-A-P-C-R assay. Ultimately, the results show that identification and antibiotic susceptibility testing of a blood culture isolate can be obtained more rapidly through this combination of culture and PCR.
The main advantage of this technique over existing methods such as those using automated phoenix or IIC systems, is that no prior culturing status required, and that results from identification and antibiotic susceptibility testing can be available within the same day. Demonstrating the procedure will be Vandy Hanson and GID ing PhD students from our laboratory Dilute 100 microliters of blood culture positive for microbial growth in a reaction tube containing 900 microliters of 0.9%sodium chloride. Then centrifuge the sample at 13, 400 times G for five minutes to pellet the bacteria following the centrifugation.
Reese, suspend the pellet in 100 microliters of sterile demineralized water. Install the sample at four degrees Celsius until further use. Once all the samples are ready, prepare the real-time PCR reaction mixes to amplify specific 16 s ribosomal DNA from the bacteria of interest.
Use demineralized water to adjust the volumes to 20 microliters per reaction. Aliquot 20 microliters of PCR mix per well in a 96 well PCR plate. Then add five microliters of sample to each well and seal the PCR plate.
Next, run the PCR on the A BI Prism 7, 900 HT real-time PCR system to analyze the results once the PCR is completed, first use the analysis settings tab to adjust the CT analysis to 0.1. Then narrow the baseline configurations to start cycle six and end cycle 15. Record the CT value for each sample.
The cutoff value to consider a PCR result as positive can generally be set to a CT value of 35. In this example, the identification profile of an e coli infected blood culture is shown in two reaction mixes. Universal 16 s ribosomal DNA primers were included and generated two amplification curves of CT 25.2 and 25.95.
The third amplification curve is derived from the probe specific for Coli. Other specific probes did not generate detectable signals. Once the pathogen has been identified, antibiotic testing can be performed.
Begin by transferring five milliliters of broth from a positive blood culture into a serum separator tube. Centrifuge the sample at 2000 times G for 10 minutes following the centrifugation, discard the supan agent from the tube. Then using a sterile cotton swab, transfer the bacteria from the gel layer of the tube into 0.9%sodium chloride until a solution with a turbidity of a 0.5 McFarland standard is obtained.
This corresponds to about 1.5 times 10 to the eight colony forming units per milliliter. Next, dilute the suspension in double concentration Muller Hinton. Two broth to obtain a concentration of approximately five times 10 to the five colony forming units per milliliter.
Aliquot 50 microliters per well of the suspension in a microtiter plate already containing 50 microliters per well of a selection of antibiotics. Be sure to include positive growth controls for each sample using wells with water instead of antibiotics. Cover the plate when finished.
Then incubate the plate at 37 degrees Celsius for six hours to allow for bacterial growth. Be sure to store an aliquot of the bacterial suspension at four degrees Celsius as a negative growth control. After the six hour incubation, transfer the content of each well and the negative control sample into separate sterile tubes.
Following a five minute centrifugation at 16, 000 times. G, remove 80 microliters of supernatant and re suspend the pellets in 80 microliters of sterile demineralized water. Dilute the samples at one to 10 in water before using them for 16 s ribosomal, DNA real-time PCR.
Prepare the PCR mix using the IQ cyber green super mix and 16 s ribosomal DNA primers and aliquot. 20 microliters per well in a 96 well PCR plate. Then add five microliters of each sample to the wells and seal the plate.
Next, run the PCR to analyze the results. First, calculate the cutoff CT value. In general, the cutoff CT equals the CT of the positive growth control plus 0.5 times the CT of the negative growth control minus the CT of the growth control.
However, for piperacillin piperacillin with tazobactam and CEF dazadi in gram-negative rods as well as for amoxicillin, oxacillin, and trimethoprim with sulfamethoxazole in staphylococcus aureus or from amoxicillin. In enterococcus SPP, the multiplication factor of 0.5 should be reduced to 0.25. The susceptibility or resistance of the strains in each sample can be determined by comparing the CT of the sample to the cutoff ct.
A CT value above the cutoff indicates susceptibility while a CT below the cutoff indicates resistance. Here is an example of an antibiotic susceptibility amplification plot. The e coli strain identified earlier was tested for sensitivity to several antibiotics each represented by a separate curve.
Samples with a low CT value are samples in which growth has occurred in the presence of an antibiotic indicating resistance to the tested antibiotic. On the contrary, a high CT value represents a sample in which no growth has occurred indicating susceptibility to the tested antibiotic Once mastered. This technique can be done in nine hours if it's performed properly.
