Cantaurus, Vol. 12, 7-9, May 2004 McPherson College Division of Science and Technology Pseudomonas aeruginosa resistance to tetracycline and triclosan

Abida A. Hamud-Socoro

P. aeruginosa is a gram-negative rod bacterium which is widespread in nature and causes dangerous infections in
humans. Tetracycline is a common antibiotic which is sometimes used to combat these infections. Triclosan is a
chemical widely used in consumer products as an antibacterial agent. Studies have shown P. aeruginosa to be
highly resistant to triclosan. The purpose of this study was to determine whether the bacterium would develop more
resistance to triclosan and tetracycline after exposure to these biocides. P. aeruginosa was exposed to different
concentrations of the two biocides, then bacteria growing in the highest concentrations were transferred to the next
highest concentration to determine whether growth would be observed. In the tetracycline experiment, bacteria in
the 50.0ug/ml concentration seemed to have developed resistance, because when they were moved to new sterile
plates containing 5.0ug/ml solution, they grew, although bacteria had not grown there originally. The limit solubility
for triclosan is 0.01g/L of water and tetracycline is 1g in 10ml of water.
Keywords: triclosan, Pseudomonas aeruginosa, tetracycline, resistance, FabI
Pseudomonas aeruginosa is a gram-negative rod-
shaped bacterium. These pathogens are widespread in P. aeruginosa has been isolated from soil and water, nature, inhabiting soil, water, plants, and animals and seems to cause disease in humans. Among including humans. They are known to cause human pathogens, P. aeruginosa is known for its nosocomial infections such as pneumonia, urinary tract multidrug resistance. Because of the efflux pump, P. infections, respiratory system infections and infections aeruginosa can be resistant to antibiotics such as of severe burns (Geyik, et al., 2003). This study used penicillin, cephalosporin, tetracycline and more, even Pseudomonas because it has been used in related without the R plasmid that is usually responsible for research projects dealing with antibiotic and triclosan antibiotic resistance among bacteria. Resistance in P. aeruginosa is caused by the outer membrane of the Only 4 structures of at least 12 resistance nodulation bacterium, because it is not very permeable (Livermore type efflux systems of the P. aeruginosa genome have et al., 1994). The efflux pump is located in the cell been characterized. Example of structural genes are membrane. The pump transports the antibiotics to the MexAB-OprM. MexCD-OprJ, MexEF-OprN and MexXY. outer membrane of the bacterial cell. P. aeruginosa is (Karkhoff-haeizer, 2000). With or without this efflux known for possessing metabolic versatility. These pump P. aeruginosa will still be resistant because of bacteria are chemoorganotrophic (Madigan et al., the strains, that maybe the same as other bacteria that 2002); they are able to grow up to 43 degree celcius has some resistant to triclosan and tetracycline. During and in neutral pH. P. aeruginosa does show a recent biochemical genetic studies it has been shown prediposition for growth in moist environments (Kwon the triclosan acts on defined bacterial targets and nonspecific one as previously thought as it pertains It can be possible that antibiotic resistant bacteria fatty acid biosynthetic pathway, ACP reductase. occur not only in humans but also in animals. Possessing both triclosan-sensitive and resistant Antibiotics are mostly used in animals to enhance enzymes, P. aeruginosa is unique. growth (O' Donnell, 2003). It seems that animals get Antibiotic resistance of bacteria is acquired by two more antibiotics than humans in a year. This has genetic processes. One process is mutation. become a major problem because these antibiotics are Sometimes bacterial DNA will spontaneously mutate in used to treat animals for infections. So when treated such a way that the efflux pump will expel antibiotics with antibiotics, the bacteria have often become (Poole, 2002). When a bacterial colony is spread with resistant to antibiotics because they are used in the antibiotic, most of the bacteria will be destroyed, but feed. We fertilize our crops with animal manure which bacteria that survive have a mutation that allowed them may contain already resistant bacteria; then the to resist the drug (Poole, 2002).These resistant bacteria might get in our soil and water and when the bacteria then multiply and create a resistant colony animals eat the plants and we eat the animals, the (O'Donnell). In the other process, acquired resistance bacteria might get in our food, which might infect occurs by the exchange of genes between bacterial humans with resistant bacteria (O'Donnell, 2003). strains through floating pieces of DNA known as This study will examine the effect of tetracycline and plasmids. These plasmids carry information from one triclosan on P. aeruginosa. Triclosan (2, 4, 4-tricloro- 2-hydroxydiphenyl ether) is a chemical widely used as from colonies growing on low concentration plates were antibacterial agent. Triclosan was introduced in 1972 transferred to new sterile plates whose [500.0ug/ml], in for hospital use (Jones et al., 2000). It is used in order to determine if any growth would be observed. consumer products such as antibacterial soap, This procedure was repeated, moving bacteria to plates detergent soaps, household cleaners and other hygiene of higher concentration, to determine at what products. Triclosan inhibits an enoyl-ACP reductase of concentration growth would be prevented. bacterial fatty acid biosynthesis. Recent studies showed P. aeruginosa contains two enoyl-ACP reductases known as FabI and FabK which are both resistant to triclosan. The enzyme Fabk is resistant to Maximum concentrations of triclosan that still allowed inhibitors that are designed to attack FabI (Heath and growth on plates of P. aeruginosa were in all plates. There was an error in each plate because of the Tetracycline is a bacteriostatic antibiotic and used to amount of triclosan that was in each plates was not select mutants of multidrug resistance (Ana et al., 1999) P. aeruginosa is resistant to tetracycline due to For tetracycline, the highest concentration that had low permeability of the outer membrane of the bacteria. P. aeruginosa growth contained 50.0ug/ml of When cell is contacted by tetracycline, the strains of tetracycline. The minimum inhibitory concentration, at the bacteria pump the antibiotic out of the cell which no bacteria were able to grow, was 500.0 ug/ml (Livermore, 1994). Overexpression or high mutation stress the strains of bacteria makes it multidrug Table 1. Table of Tetracycline of different
The objective of this study is to see if exposing an concentrations, Growth/No Growth, ug/ml of organism to tetracycline and to triclosan could select Tetracycline in the plate of P. aeruginosa. for resistance to tetracycline and triclosan. I will determine which different concentrations of tetracycline and triclosan can select for resistant strains of the bacteria, the minimum inhibitory concentration (MIC) and how quickly the resistance would develop. MATERIALS AND METHODS
Triclosan used in the experiment was purchased from Sigma-Aldrich. First 1g of 97% triclosan was dissolved in 17.5ml of ethanol and 82.5ml of distilled water, and filter-sterilized (0.2um). Equal amounts (2.8ml) of each dilution were pipetted into each test tube containing 25ml of tryptic soy agar. Six different concentrations of Table 2. Table of Triclosan of different concentrations,
triclosan were used ([1000.0],[100.0],[10.0], Growth/No Growth and ug/ml of tetracycline in the [1.0],[0.10],[0.010] ug/ml). A vortex machine was used to mix the contents. Each test tube was poured into a plate to be solidified. P. aeruginosa was purchased from Ward's Biology & Chemistry as freeze dried culture. Inoculation was used to culture bacteria, using 9ml of nutrient broth and a loop of the P. aeruginosa, which was then incubated for 24 hours. The spread method was used on plates by adding 0.3ml of P. aeruginosa in nutrient broth and the plates were incubated for 72 hours at 37 degrees. A powder of tetracycline was purchased from Sigma. Then 0.5g was dissolved in 10ml distilled water and filter-sterilized (0.2um). Equal amounts (2.8ml) of each DISCUSSION
dilution were pipetted into each test tube containing 25ml of tryptic soy agar. Another 7 dilutions ([5000.0], P. aeruginosa was more resistant to triclosan than to [500.0], [50.0], [5.0], [0.5], [0.05], [0.005]ug/ml) were tetracycline. The bacterium shows high resistance to prepared, using the antibiotic tetracycline. A vortex triclosan because P. aeruginosa has both the FabI and machine was used to mix the contents. The spread FabK gene. The lowest concentration of tetracycline method was used on plates by adding 0.3ml of a P. that could destroy the bacteria was 500.0ug/ml. The aeruginosa on the plates and the plates were incubated experimental findings may have been affected by errors in dissolving the solutions of triclosan and tetracycline The transferring method was performed. Bacteria P. aeruginosa Resistance to Tetracycline and Triclosan-Hamud-Socoro in water. The limits solubility of triclosan in water is resistant bacterial enzyme. Nature 406:145-146. 0.01g in 1000ml. The actual concentration for triclosan was 1000.0ug/ml, this make sense because much of Jones, R.D., H.B. Jampani, J.L. Newman, and A.S. triclosan was dissolved into a small amount of distilled Lee. 2000. Triclosan: A review of effectiveness water. In tetracycline the actual concentration was and safety in health care settings. American 5000.0ug/ml and limits solubility of tetracycline in water Journal of Infection Control 28: 184-196. is 1g in 10ml of distilled water. In the tetracycline Kwon, N.H., S.H. Kim, J.Y. Kim, J.Y. Lim, J.M. Kim, experiment, bacteria in the 50.0ug/ml solution seemed W.K. Jung, K.T. Park, W.K. Bae, K.M.Noh, J.W. to have developed resistance, because when they were Choi, J Hur, Y.H. Park 2003. Antimicrobial moved to new sterile plates that contain 50.0ug/ml performance of alkaline ionic fluid (GC-100X) and concentration of tetracycline, from the 5.0ug/ml plate, its ability to remove Escherichia coli O157:H7 from they grew, although bacteria had not grown there the surface of tomatoes. Journal of Food originally. In other studies compare with this research, the MIC of triclosan was much higher and MIC of tetracycline, which 11 strains had lower concentration Livermore, D.M., X.Z. Li, H. Nikaido. 1994. Role of and 7 other strains has higher concentration than what efflux pumps in intrinsic resistance of P. was found in the result. In between 2.56ug/ml and 0.256ug/ml there maybe MIC could be found but this chloramphenicol, and norfloxacin. Antimicrobial experiment was not taken farther to find the exact Agents and Chemotherapy. 38: (8) 1732-1741. number of MIC in between these two concentrations. In other studies cross-resistance between triclosan and Madigan, M.T., M. M. John, P.Jack. 2002. 10th tetracycline in P. aeruginosa and found amino acid edition. Brock Biology of Microorganisms. 368-70 changes due to exposure to triclosan makes it cross- resistant to other antimicrobial agents (Karkhoff- O'Donnell, W.M. 2003. Inducing ampicillin resistance This study could be refined in future studies by using in Escherichia coli. Transaction of Kansas limited concentration of triclosan and tetracycline in order to dissolve in water. Furthermore, mutation could be encouraged in the bacteria by exposing them to Poole, K. 2002. Mechanisms of bacterial biocide and ultraviolet light. More genetic mutations in the antibiotic resistance. Journal of Applied bacterium might lead to a greater possibility of its

Dr. Frye, Dr. Kim Stanley and the entire science

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R. Karkhoff-Schweizer and Herbert P. S.2001. Cross- Resistance Between Triclosan and Antibiotics in Pseudomonas aeruginosa Is Mediated by Multidrug Efflux Pumps: Exposure of a Susceptible Mutant strains to Triclosan Selects nfxB Mutants Overexpressing MexCD-OprJ. Vol. 45, 428-432. Geyik, M.F., M. Aldemir, S. Hosoglu, H.I. Tacyildiz. 2003. Epidemiology of burn unit infections in children. American Journal Infected Control 6: 342-6. Heath, R.J and O.C. Rock. 1995. A triclosan-

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