Analysis of Bacterial Resistance Henry R. Walther Grade 11 Pittsburgh Central Catholic High School.

Analysis of Bacterial ResistanceHenry R. Walther

Grade 11Pittsburgh Central Catholic High School

Bacteria Resistance Disease-causing microbes that

have become resistant to drug therapy

Tuberculosis, gonorrhea, malaria, and childhood ear infections are just a few of the diseases

Causes of Bacteria Resistance

horizontal gene transferunlinked point mutationsenvironmental pressurepass this trait to their offspring

Common Antibacterials DirithromycinErythromycin TroleandomycinCefiximeCefotaximeCefpodoximeCeftazidimeTriclosan

TriclosanA potent antibacterial

and antifungal agent. Increasing in US

consumer products (soaps, textiles, toys, kitchen utensils)

Binds to ENR enzyme in cell, which increases affinity for NAD+, a stable conformation of the 3 chemicals which inhibits fatty acid synthesis ( inculding synthesis of cAMP)

Relationship The August 6, 1998 issue of Nature warned that

triclosan's overuse could cause resistant strains of bacteria to develop, in much the same way that antibiotic-resistant bacterial strains are emerging, based on speculation that triclosan behaved like an antibiotic.

The 2003 Sunday Herald newspaper reported that some UK supermarkets and other retailers were considering phasing out products containing triclosan.

E. coli

• One of the most common forms of bacteria, free living, symbiotic or pathogenic.

• Has been utilized as the most studied prokaryote.

• There are many of different strains of E. coli, most of which are non-pathogenic. However, there are strains which can produce fatal disease.

Purposeto examine the potential resistance of E.coli to the antibiotic ingredient triclosan

Null Hypothesis Triclosan’s effect on the survivorship

of exposed, assumed resistant, bacteria and the unexposed bacteria will not differ outside of chance.

Materials Triclosan 60 sterile LB dishes E. Coli DH5α Micro Pipette Sterile Micro tubes Spreader bar Ethanol Scale Incubator (37°C) Sterile 250mL

Sidearm Flask

Fine tip permanent marker

Klett Spectrophotometer

Weigh Boat Vortex Tube Rack SDF media LB mediaLB MediaTryptone (1%)Yeast Extract (0.5%)NaCl (1%)NaOH

SDF Media100mM KH2PO4

100mM K2HPO4

10mM MgSO4

100mM NaCL (5%)

Procedure 1. A culture of E Coli DH5α was grown overnight in a sterile 250mL

sidearm flask in LB media at 37°C.

2. The culture was left to grow until it reached an absorbance reading of about 50 Kletts, which represents a cell density of about 108 cells/mL.

3. The culture was serially diluted in SDF to a concentration of approximately 103 cells/mL.

4. The triclosan solution was prepared. 0.075 grams of triclosan were weighed out and poured it into 5mls of ethanol. This created a 0.15% concentration of triclosan.

5. The following ingredients were mixed to attain the proper concentrations;

  0.15% 0.015% 0%

E. coli 0.01 ml 0.01 ml 0.01 ml

Triclosan 0.1 ml 0.01 ml 0 ml

SDF Media 0.89 ml 0.98 ml 0.99 ml

Procedure (cont.)6. Two plates were spread from each different concentration and let

grow over night in an incubator.

7. One bacteria colony was cultured from one of the 0% plates and one colony was cultured from each variable plate (assumed resistance).

8. Repeated step 1.

9. For each batch of bacteria repeat steps 2 through 5, but used 6 plates for each different concentration.

10. Record results.

0.000% 0.015% 0.150%0

50

100

150

200

250

Effect of Triclosan on E. Coli Survivorship

UnexposedExposed (1)Exposed (2)

[ ] Triclosan

Co

lon

y C

ou

nt

P value=0.075904

P value = 0.84567

ConclusionsThe null hypothesis can be

accepted because the p-value in the ANOVA is above the confidence cut off of .05. Exposing E. coli to triclosan did not appear increase the survivorship in 0.01X and 1X concentrations of triclosan. No evidence of resistance was found in this study.

Extensions/Improvements Use different species of bacteriaUse different antibacterial agentsGrow the bacteria with agent

over more generations Use different concentrations of

triclosanInfuse the agar with triclosan for

prolonged exposure time.

Referenceshttp://en.wikipedia.org/wiki/

Escherichia_colihttp://en.wikipedia.org/wiki/

Triclosanwww.beyondpesticides.org/pesticides/factsheets/Triclosan%20cited.pdf

en.wikipedia.org/wiki/Antibiotic_resistance

www.fda.gov/Fdac/features/795_antibio.html