Esblb Strain Lab Report

K. pneumoniae is a gram negative, facultative anaerobic, gamma-proteobacteria that can cause pneumonia, urinary tract infections, cholecystitis, and septicemia. (Strand & Shulman 1988)
As shown in Table 1., many organisms have developed resistances to commonly used antibiotics. The difference between the standard strain of Klebsiella pneumoniae and the extended spectrum betalactemase (ESBL) strain, for example, is striking. 12 antibiotics had greater than or equal to 95 percent effectiveness against the common K. pneumoniae strain, while only 2 antibiotics were as effective against the ESLB strain. P. aeruginosa and K. pneumoniae are only two of the many clinically relevant gram negative species that have recently become resistant to various
antibiotics. Many efforts have been made to prevent organisms from developing new resistances; however it is difficult to combat organisms that are so proficient at adaptation. (Mahon, Lehman, & Manuselis 2011)

Goals and Potential Products.
With this research, I hope to increase understanding of the severity of the MDR problem that the healthcare field is facing. I hope to lay down the ground work for future research. A future project might be the study of bacteria in the field, with the goal of tracking how far and fast MDR wild type bacteria are spreading. I would like to discuss the results of this research with others in the field, with the hope that they may further develop this line of study.

Experiment Methods
I propose an experiment to determine the readiness and rate in which different species of medically relevant bacteria are able to transfer genes for resistance.
In this experiment we will inoculate individual vials of nutrient broth with various organisms that are known to be resistant to multiple antibiotics. We will also inoculate various organisms that are as close to the wild type as is feasible. The vials will be allowed to incubate at 37 degrees Celsius for 24 hours. The number of bacteria in a given cultured sample is too great to be counted directly. The samples will be serially diluted by a factor of 10 in sterile saline, to be repeated 7 times. A 10 microliter aliquot from each dilution will be placed on a 5% blood agar plate or a MacConkey plate, depending on the type of organisms plated. These plates are selected because of the ease of differentiation between organisms. The original vial and all diluted aliquots will then be refrigerated to restrict growth until the plates can be processed and evaluated. The plates will be streaked in the standard format for colony count. The plates will be incubated at 37 degrees Celsius for 24 hours. The plates will then be examined for purity of culture and for colony count. The number of colonies will be counted for each dilution. The plates that have a colony count between 300 and 30 will be used to estimate the number of viable cells in that dilution. This range is chosen because small dilution errors could have a
drastic effect on a plate with less than 30 organisms, and plates with more than 300 organisms would show poor isolation. Some organisms, especially Staphylococcus and Streptococcus spp., form multiple cell arrangements. A colony may consist of groups of bacteria rather than a single organism. For accuracy, we will refer to these as colony forming units (CFUs). We will determine the average number of CFUs per 10 microliters of sample by finding the number of colonies on all suitable plates, multiplying it by the dilution factor for each plate, then taking the average of these numbers. (Linne & Ringsrud 1992)
The dilution will allow us preform the next step with more accuracy. We will choose one organism that exhibits antibiotic resistance and one that does not. We will take the chosen organisms’ dilution aliquots that have a similar number of CFU’s and combine 20 microliters of each aliquot into a new vial of nutrient broth. The broth will be incubated for 24 hours. The sample will be serially diluted by a factor of 10, to be repeated 4 times. 20 microliters will then be plated to either 5% sheep blood agar or MacConkey agar. The plates will be streaked to isolate using the standard method. After 24 hours of incubation at 37 degrees Celsius, pure colonies will be picked from the plate that shows the most even distribution of organisms and the best separation of colonies. These colonies will be inoculated to new agar and streaked out. In the area of heavy inoculation on each plate, an antibiotic disk will be placed away from the edges of the plate, but in an area that is completely inoculated with bacteria. The plates will be incubated as all previous cultures have, and then examined to determine the “zone of no growth” around the disc. The zone of no growth will be measured and compared to the standards indicated in the material sheet for the disc. A result of sensitive, intermediate, or resistant will be recorded in a data table. This process is known as the “disc diffusion method of sensitivity testing.” The stated process will be repeated for each combination of organisms tested. The control for this test will be the organisms that are known to be susceptible to the antibiotics tested. (Forbes, Sahm, & Weissfeld 2007)
The timeline of this experiment would be between 1 to 4 months.
Required materials include stock cultures of organisms to be tested. Methicillin resistant S. aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Proteus mirabilis, and ESBL K. pneumoniae would be excellent choices because of their characteristic morphologies. (Gladwin & Trattler 2011) Multiple Cefepime and Ceftriaxone antibiotic discs will be needed to preform sensitivities. 5% sheep blood agar plates and MacConkey agar plates in sufficient quantities to subculture each test sample regularly. Nutrient broth tubes, or alternatively a nutrient broth bottle with sterile test tubes, will allow for mixing samples and incubation. Parafilm wax will be used seal tubes and plates. Sterile saline will be needed to dilute the nutrient broth. Prepacked sterile inoculating loops or a metal loop with portable incinerator will be used to streak out the plates. 37 degree Celsius Incubator will be needed. A Micropipeter (10 microliter) with tips are required to prepare aliquots and
dilutions.

Impact on students’ career/education
I currently work as a medical laboratory technician. Upon receiving my bachelor's degree in biology, I will be able to sit for the national board examination to become a medical laboratory scientist. I currently specialize in the microbiology and blood banking departments within the lab. Microbiology and blood banking give me a unique perspective on the interaction with micro organisms and their effect on humans, especially when patients develop septicemia. By completing this project, I will be one step closer to completing the requirements needed to graduate. This research will also help me to prepare for the presentation of policies and procedures that is required in my field of work.

Timeline
The project should be completed during the summer semester. Most of the first week will be spent setting up the stock cultures and performing preliminary quality control. The next week should be repetition of procedure and the reading of results. I expect that before the end of the third week, at least some of the tested organisms will begin to develop resistances. The experiment will be carried out throughout the summer, or until all non-resistant strains become resistant, whichever comes first. Research could be extended into the fall semester, if time and resources allowed.