CTX-M-15: Good News and Bad News Samantha Fanelli December 13th, 2010 One of the biggest challenges

facing global antibiotics is -lactam drug resistant, gram-negative bacteria. As resistance expands, the large variety of antibiotics that are available is slowly dwindling, leaving microbiologists and medical professionals struggling for a solution (Hanson, 2010). -lactam antibiotics, including penicillins, cephalosporins, carbapenams, and monobactams, prevent bacteria from synthesizing a cell wall, effectively lysing the cells. -lactamases, the enzymes present in these newly resistant bacteria, catalyze the hydrolysis of the -lactam ring that characterizes these antibiotics and renders them inactivated (Gunda, n.d.). These -lactamases are difficult to detect and control with current technology, especially extended-spectrum -lactamases (ESBLs) which have a wide capacity to inhibit -lactams, and this is an increasing concern because the encoding genes are carried on plasmids. Plasmids allow transfer of the resistant gene through simple bacterial conjugation, so this resistance has the ability to spread quickly and efficiently among isolates (Hanson, 2010). While scientists are working on ways to more effectively test antibiotic susceptibility and presence of the gene itself in bacteria, I spent the semester trying to determine how prevalent, if at all, this gene already is in America. Using polymerase chain reaction (PCR) and gel electrophoresis, I tested approximately fifty samples (see Table 1) from across the United States for presence of one of the most prevalent ESBLs, CTX-M-15, in E. coli bacteria. I was provided with a list of suggested bacteria samples to test for the CTXM-15 gene and grew them on tryptic soy agar plates to be isolated. After the plates incubated for a few days, I took a small sample of each bacterium to isolate the DNA by first heating the sample and then spinning it down. Once I isolated the DNA from each sample, I set to work running each sample through PCR, followed by gel electrophoresis.

Table 1. Samples Tested for Presence of CTX-M-15 from Penn States E. coli Reference Center
Sample Number 2 2970 2 3617 2 3620 2 3621 2 3622 2 3788 2 3789 2 3913 2 3914 2 3915 2 3916 2 4147 Result (+/-) Sample Number 2 4148 3 0136 3 0211 3 0212 3 0679 3 0680 3 0902 3 0914 3 0915 3 0916 3 0917 3 0918 Result (+/-) Sample Number 3 0921 3 0933 3 0934 3 1057 3 1058 3 2479 3 2659 3 2660 3 2661 3 2662 3 2663 3 3757 Result (+/-) Sample Number 3 3758 3 3759 3 3760 3 3761 3 3762 3 3763 3 3765 3 3766 3 3769 3 3770 3 3771 3 3772 Result (+/-) -

PCR uses a small mixture of DNA, primers (small pieces of DNA sequenced to match the segment being studied), DNA polymerase, and nucleotides to create billions of copies of a specific segment of DNA. After being mixed with these components, the sample is superheated in a thermocycler to separate the two strands of the template DNA and then cooled back down so the primers can attach at either end of the strand to mark a place for the DNA polymerase to start copying. The DNA polymerase attaches near the end of the primer and uses the added nucleotides to complete the rest of the strand. The mixture is constantly heated and cooled to repeat this process until a sizeable amount of DNA has been synthesized and can be used in the next analytical step, gel electrophoresis (PCR Virtual Lab, 2010). Beth provided me with two PCR mixtures to use, the first of which was unsuccessful. The combination of water, three primers, a master mix, and the isolated DNA for each sample produced murky results when the completed gel was photographed under ultraviolet light and was without a clear distinction of the two bands indicative of the CTX-M-15 gene (see Figure 1). Be it by human error or the test itself, we decided to try a different mixture which was far more successful (see Figure 2). The 2

mixture of a 2xMultiplex PCR mix, a mix of four CTX primers, and RNase-free water produced clear results when photographed. I began consistently using this PCR mix to prepare the DNA for gel electrophoresis throughout the rest of the experiment.

Lane: 9 8 7 6 5 4 3 2 1 Figure 1. Gel electrophoresis results of various E. coli samples with first PCR mixture. Lane 1 PCR DNA Ladder, Lane 2 CMX Positive Control, Lane 3 CMX Negative Control, Lane 4 Sample 2 4148, Lane 5 Sample 3 0136, Lane 6 Sample 3 0211, Lane 7 3 0212, Lane 8 3 0679, Lane 9 3 0680

Lane: 9 8 7 6 5 4 3 2 1 Figure 2. Gel electrophoresis results of the same E. coli samples with second PCR mixture. Lane 1 PCR DNA Ladder, Lane 2 CMX Positive Control, Lane 3 CMX Negative Control, Lane 4 Sample 2 4148, Lane 5 Sample 3 0136, Lane 6 Sample 3 0211, Lane 7 3 0212, Lane 8 3 0679, Lane 9 3 0680

Gel electrophoresis uses an electric current run through a stationary phase (in this case a 1% agarose gel) to separate different strands of DNA by size. The negative charge on DNA caused by its phosphates allows it to move through the gel, and increasingly smaller fragments move farther into the gel as the current is run through. A DNA ladder that serves as a molecular weight marker is placed in the 3

first well of the gel as a comparison for estimating the approximate size of the fragments (Hughes, n.d.). The next two lanes are a sample known to be positive for the CTX-M-15 gene for comparing to the other samples and one known to be negative as a control for the experiment. Dye is injected into these samples and the rest of those being run (all prepared during PCR) to allow for visibility of the moving samples and to weigh down the samples so they sink to the bottom of the wells (Hughes, n.d.). After the samples were run through a current at 200V for 25 minutes, the gel was photographed under ultraviolet light. The ethidium bromide added to the gel binds to the DNA and fluoresces under the light so the location of the DNA can be noted and compared to other samples. Be it fortunate or unfortunate, after testing all of the samples, none of them were positive for the CTX-M-15 gene. Though it did not make for a particularly exciting experiment, it is definitely good news to see the gene is not extremely prevalent yet in America. A high occurrence of this gene could mean a big problem for microbiologists, epidemiologists, physicians, and more importantly their patients. Health officials estimate approximately 70,000 cases of E. coli infection every year, and the emergence of extremely antibiotic resistant bacteria could cause an epidemic if not addressed quickly (Young, 2010). I was lucky in finding such optimistic results, but that certainly does not make the issue any less of a reality or a concern to health care officials and scientists more capable of solving this issue than an undergraduate like me. I can do little more than test for this gene and hope not to find it; it is up to the real scientists to figure out a way to keep that gene from finding its own way into my results.

References: Gunda, Tamas E. "On the Beta-Lactam Antibiotics in a Nutshell." KLTE. Web. 11 Dec. 2010. <http://www.cic.klte.hu/~gundat/betalaca.htm>. Hanson, Nancy D. "Molecular Diagnostics Could Help in Coping with Hidden Beta-Lactamases." Microbe Aug. 2010: 333-39. Print. Hughes, K. "Gel Electrophoresis." UTK. University of Tennessee - Knoxville. Web. 11 Dec. 2010. <http://web.utk.edu/~khughes/GEL/sld001.htm>. "PCR Virtual Lab." Genetic Science Learning Center. The University of Utah, 2010. Web. 11 Dec. 2010. <http://learn.genetics.utah.edu/content/labs/pcr/>. Young, Saundra. "E. Coli Cases down in 2009, CDC Says." CNN Health. CNN, 15 Apr. 2010. Web. 11 Dec. 2010. <http://articles.cnn.com/2010-04-15/health/foodborne.illness.cdc_1_foodnet-cases-of-ecoli-hemolytic-uremic-syndrome?_s=PM:HEALTH>.