Manuscript

"Susceptibility of Antimicrobial Agents on Pseudomonas aeruginosa"

Researched by: Andrew Williamson

Biology Department of Tennessee Technological University

Cookeville, TN 38501

Abstract

            Antimicrobial agents are used in laboratories to kill or hinder certain types of bacterium.  Bacteria can be resistant to many types of these agents, and will show no effects.  Sometimes combinations of these agents are needed in order to kill off a designated bacterium.  Pseudomonas aeruginosa will be used as a test subject for this experiment for the sole reason of its resistance to many different agents.  This subject is difficult to isolate and will more than likely require a combination of agents in order to be killed.

Keywords

Pseudomonas aeruginosa

Antimicrobial agents

Bacteria

Susceptibility

Resistance

Introduction

            In today’s society, many diseases arise from bacteria that grow resistant to the agents that are presented to them.  New research is done consistently to fight this constant upgrading of bacteria.  Through new research, scientists can develop new antimicrobial agents and antibiotics to ensure the elimination of a bacterium for a limited period of time.  Much research has been done on Pseudomonas aeruginosa.  Pseudomonas  aeruginosa is responsible for 16% of nosocomial pneumonia cases , 12% of hospital-acquired urinary tract infections , 8% of surgical wound infections , and 10% of bloodstream infections (Delden & Iglewski, Volume 4, Number 4).  One other very popular strain of Pseudomonas aeruginosa that is found in humans is in patients who have cystic fibrosis, in which the bacterium can almost never be treated.

It has been found that Pseudomonas aeruginosa develops plasmids that are that are able to transfer their genes through the processes of transduction and conjugation.  Transduction is the process of transferring genetic information from cell to cell through the mediation of a virus (phage) particle.   Conjugation is a one-way transfer of genetic material between cells by cell-to-cell contact.   By transferring their genetic material from cell to cell by this means, Pseudomonas aeruginosa is able to elude antimicrobial agents and antibiotics and survive.

The Null hypothesis will be disproved by proving that Pseudomonas aeruginosa is susceptible to certain antimicrobial agents.  Strains of Pseudomonas aeruginosa from a synthetic stock will be tested against individual tests commonly used in microbiology laboratories, and with common antimicrobial agents used in microbiology laboratories.  They will be tested individually, and if the results are not positive, then combinations of the chemicals will be tested.  All mediums will be collected and examined to determine if Pseudomonas aeruginosa is susceptible to these tests.  The objective of my research is to determine the susceptibility of Pseudomonas aeruginosa to certain antimicrobial agents found in common microbiology laboratories.

Methods and Materials

The sample used in this experiment came from a synthetic stock.

      Tests used include the following:

1)      Mueller Hinton / Pseudomonas Agar Disc Diffusion Assay Tests

      This technique allows you to test discs on top of the agar to see if they are resistant or susceptible.

2)      Minimum Inhibitory Concentrations (MIC’s)

      Determines the lowest concentration of a chemical that will inhibit growth.

3)      Tube Dilutions

      Used to determine the effective concentration of the chemical being used.

(Goss, 62-65)

Other materials that are needed during the experiment include:

• Petri dishes and specific agars

• Pseudomonas aeruginosa synthetic stock

• Glass tubes and racks for dilutions and MIC’s.

• Bleach (5% hypochlorite solution)

• Vesphene

• Isopropyl Alcohol

• 1 M Hydrochloric Acid

Using aseptic techniques, the experiment began with taking an inoculating loop, heating it, and obtaining a sample of the bacteria being used.  It is important to heat the loop in order to prevent any type of contamination of the bacteria from outside sources.  After heating the loop, the bacterium was then streaked onto an agar surface inside a petri dish.  As soon as this process was finished, the loop was heated again to kill off any excess bacteria that was left on the loop.  After this, the bacteria were exposed to discs dipped in each of the four chemicals.  The dishes were separated into quadrants for each of the four chemicals being used, so as to remember which chemical was which.

For the tube dilution assay, 32 small test tubes were obtained.  These were separated into groups of eight and labeled with roman numerals.  Each tube was filled with one mL of tryptic soy broth (TSB).  Dilutions of the chemical then occured as follows:  

1. Using a sterile pipet, one mL of the first chemical was placed into tube 2.

2. After this, the solution was pipetted up and down 2 to 3 times in order to mix the chemical and the       TSB.

3. Then, one mL of this solution is extracted and inserted into tube 3.  

4. The procedure is then followed as above for all tubes until tube 8.  

5. Extraction of 1 mL from tube 7 then occured and was discarded into the sink.

6. Tube 8 was then subjected to one mL of the chemical.

7. Repetition of this procedure was used for the other 3 chemicals.

8. After this is completed, subject tubes 1-7, but not 8, to 2 drops of the bacteria from the culture tube. 

      The petri dishes and tubes were then inoculated for 2-4 days.  After this time period, the petri dishes were then removed from the incubator and examination of the dishes is performed.

Results

           After careful analysis of the Pseudomonas aeruginosa plates and tube dilutions, it was determined that the following occurred:

          The disc diffusion assays on Mueller Hinton plates and Pseudomonas agar plates determined:

• Pseudomonas aeruginosa was resistant to isopropyl alcohol, 1 M hydrochloric acid, and vesphene.

• However, Pseudomonas aeruginosa was susceptible to a 5 % hypochlorite solution of bleach.

          MIC's proved that certain concentrations of each of the chemicals could inhibit the growth of Pseudomonas aeruginosa.

Discussion

This experiment was performed to see if any common antimicrobial agents could kill Pseudomonas aeruginosa, a very harmful, resistant bacteria.  After testing of the bacteria against individual chemicals, the results were conclusive enough to show that a 5% hypochlorite solution of bleach was useful in killing the bacteria.  This testing was very simple, and I hope that future testing, either by myself or by others, can lead to further discoveries in this field.  Just seeing this could show future researchers a way to use bleach in a different form in humans to kill the bacteria, but most importantly, it shows that if people are around a bacteria such as Pseudomonas aeruginosa, they can disinfect the workspace they used with bleach to kill the bacteria to prevent insertion of the bacteria into themselves, the host.

Performing this experiment again could see if combinations of these chemicals could kill this bacteria.  Changing the procedure by using combinations of chemicals could produce interesting results that could be very beneficial to the results that have already been obtained.  It could even lead to a new field in studying ways to reduce the amounts of diseases going on the in the world today.  For example, Pseudomonas  aeruginosa is responsible for 16% of nosocomial pneumonia cases , 12% of hospital-acquired urinary tract infections , 8% of surgical wound infections , and 10% of bloodstream infections (Delden & Iglewski, Volume 4, Number 4). As stated, if the right combinations of chemicals are found, it could produce a rapid change in the treatment of this bacteria.

Acknowlegdments

• Dr. Susan Goss, Biology Department of Tennessee Technological University

• William Andrew Todd, my cohort in this study.

Literature Cited

http://www.iit.edu/departments/csep/PublicWWW/codes/coe/ada-a.htm (Ethics Statement)

Goss, S.  2001-2002. Control of Microbial Growth.  Health Science Microbiology Laboratory Manual.  62-66

Delden, C.V. , Iglewski, B.H.  1998.  Cell-to-Cell Signaling and Pseudomonas aeruginosa Infections.  Emerging Infectious Diseases Volume 4, Number 4.  www.cdc.gov/ncidod/eid/vol4no4/vandelden.htm

Appendix

Andrew Williamson's Biology Web Page!