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
ABSTRACT 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 INTRODUCTION 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
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
LITERATURE CITED Alonso, A., E. Campanario and J.S Martinez. 1999. Emergence of multidrug-resistant mutants is Increased under antibiotic selective pressure in Pseudomonas aeruginosa. Microbiology 145, 2857-2862. Chuanchuen, R., K. Beinlich, T.T. Hoang, A. Becher, R.
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-
SUMMARY OF THE PRODUCT CHARACTERISTICS NAME OF THE VETERINARY MEDICINAL PRODUCT Bimectin vet. 10 mg/ml, solution for injection QUALITATIVE AND QUANTITATIVE COMPOSITION Active substance(s) List of excipients PHARMACEUTICAL FORM Solution for injection. Clear colourless to slightly yellow coloured solution. PHARMACOLOGICAL PROPERTIES Pharmacodynamic properties Iverme
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