Attack of The Enemy Mutants!
Since their discovery in the early 20th century, antibiotics have been considered the wonder
drugs of the era. Antibiotics are used to treat a wide variety of bacterial infections. Therapeutic use of
antibiotics increased from the 1950s until the mid 1980s1; it was around this time that the first
antibiotic-resistant bacteria were recognised2, and their harmful consequences were becoming
apparent. Why is the development of antibiotic resistance in bacteria of concern? Firstly, infection
caused by such bacteria may be very difficult, if not impossible (in some circumstances), to treat.
Secondly, the cost of treating people with such infections has substantially increased3.
The purpose of Attack of The Enemy Mutants was to study the rates at which a
Staphylococcus aureus isolate acquired resistance to nine antibiotics commonly used to treat such
infection, to study the rate at which this acquired antibiotic resistance recedes, and to determine
possible methods which could be used to slow down the rate at which antibiotic resistance to a given
My first hypothesis was that a Staphylococcus aureus isolate (from my nasal membrane)
would become resistant to one or more of the following antibiotics with repeated exposure:
gentamicin, clindamycin, enrofloxacin, erythromycin, amoxicillin/clavulanic acid, cephalexin,
April 19, 2004 Perryn Kruth - Attack of The Enemy Mutants page 1 of 5
tetracycline, methicillin and sulfamethoxazole/trimethoprim.
My second hypothesis was that the number of exposures needed for this S. aureus isolate to
achieve resistance would vary between different antibiotics. I also hypothesized that treating with
multiple antibiotics, either used concurrently, or by alternating antibiotics used, would delay the onset
of resistance. My final hypothesis was that bacteria would lose resistance if they were not continually
exposed to the antibiotic that they had acquired resistance to.
I swabbed my nasal membrane and cultured this swab on blood agar at 37o C for 24 hours. The
catalase test, Gram stains, and the observation of â-hemolytic colonies were used to identify a
Staphylococcus aureus culture4. This culture was sent to a commercial bacteriology lab to confirm
that pure Staphylococcus aureus was isolated, and to identify which antibiotics this isolate was
Based on this sensitivity testing, nine antibiotics commonly used to treat Staphylococcus aureus infections were chosen5. The S. aureus isolate was separately exposed to each of these nine
antibiotics in the form of sterile paper disks, impregnated with the drug (Antimicrobial Susceptibility
Test Disks, OXOID Ltd., Hampshire, England). Susceptibility was determined using the Kirby Bauer
method of measuring the zone of inhibition (the area around the antibiotic disk in which bacteria did
not grow, which relates to antibiotic sensitivity)6.Sixteen subsequent antibiotic exposures (dose
simulations) were made using a swab of bacterial growth from the inner margin of the previous zone
of inhibition, presumptively selecting for antibiotic resistance.
To determine the repeatability of this system, the original isolate was tested with one of the
antibiotics (gentamicin) on five identical agar plates, all cultured at the same time. Zones were
April 19, 2004 Perryn Kruth - Attack of The Enemy Mutants page 2 of 5
measured and compared using standard deviation.
To determine if the number of exposures needed for my isolate to acquire antibiotic resistance
could be increased by using different antibiotics concurrently, a modification of the original procedure
was used. Enrofloxacin and cephalexin (two of the antibiotics which induced a minimum of
intermediate sensitivity in the initial part of my study) antibiotic disks were used together in two
separate fashions. In the first test, the two antibiotic disks were placed side-by-side on the agar plate.
For the second test, these two antibiotics were used alternately for successive cultures (dose
simulations). To determine whether the acquired resistance to an antibiotic was lost, I continued to
subculture a cephalexin-resistant organism without the antibiotic while periodically monitoring zone
size. All results were analyzed using a zone of inhibition interpretation sheet unique to the brand of
antibiotic disks used in experimentation (BBLTM) to determine sensitivity to each antibiotic at each
Following the initial exposure period of sixteen days, six of the nine antibiotics used continued
to have an acceptable zone of inhibition (i.e., no development of resistance). These antibiotics were:
gentamicin, clindamicyn, erythromycin, clavulanic acid/amoxicilin, tetracycline,
sulfamethoxazole/trimethoprim. Three antibiotics had only an intermediate effect after multiple
exposures. These were: enrofloxacin at exposure 6; cephalexin at exposure 8; methicillin at exposure
13. Only two antibiotics lost effectiveness to the point of antibiotic resistance: cephalexin at exposure
The first conclusion that I am able to make is that the system I used for experimentation
April 19, 2004 Perryn Kruth - Attack of The Enemy Mutants Page 3 of 5
worked well and was reproducible. My first hypothesis, that my bacterial isolate will become resistant
to antibiotics commonly used to treat it, was partially correct. My S. aureus isolate achieved resistance
to gentamicin and cephalexin, and achieved intermediate sensitivity to enrofloxacin and methicillin; no
change in the level of resistance to the other antibiotics used was noted. The average duration of
antibiotic therapy is usually less than 14 days7. This suggests that, of the antibiotics used, cephalexin
would be the only one to potentially cause difficulties in initial treatment periods. My second
hypothesis, that the number of exposures needed for my Staphylococcus aureus isolate to achieve
resistance to different antibiotics will vary, was thus proven true. This is because antibiotics have
differing effects on bacteria; a certain mechanism of resistance that bacteria use against one form of
antibiotic may take longer to develop than another used against a different antibiotic. To
this point, at antibiotic exposure 13, when cephalexin and enrofloxacin were used alternately, my
isolate had not yet become consistently resistant. When these two antibiotics were used concurrently,
resistance development was delayed by six exposures. Thus, my third hypothesis, that treating with
multiple antibiotics will delay the onset of resistance appears to be true.
My final hypothesis, that bacteria will lose resistance if they are not continually exposed to the
antibiotic to which they have acquired resistance is yet to be fully proved. To date, I have determined
that antibiotic resistance is not lost within the first 12 days of no antibiotic exposure, but that
resistance became stronger during the first four days, before decreasing slightly. I still feel that my
hypothesis that resistance will be lost if the presence of antibiotics is lost for lengthened periods of
time may be correct, however this may take months to occur.
April 19, 2004 Perryn Kruth - Attack of The Enemy Mutants page 4 of 5
I would like to thank these people for their help and support over the course of my project: Mrs.
Hinan, Dr. Scott Weese, Joyce Rousseaux, and especially my parents and sisters.
Appendices
Amábile-Cuevas, C.F. New Antibiotics and New Resistance, American Scientist 91:138 - 148,
Brooks G.F., Janet S.B., Stephen A.M. Medical Microbiology, Twenty-First Edition, LANGE
Salyers A.A, White D.D. Bacterial Pathogenesis - A Molecular Approach, Second Edition, ASM
1. Salyers A.A, White D.D. Bacterial Pathogenesis - A Molecular Approach, Second Edition, ASM Press Washington, DC., 2002.
3. Calculations based upon quote from Pharmacist, Shoppers Drug Mart, Bullfrog Mall, Guelph Ontario
4. Brooks G.F., Janet S.B., Stephen A.M. Medical Microbiology, Twenty-First Edition, LANGE Medical Book, Toronto 1998.
5. University of Guelph, Laboratory Services, Animal Health Laboratory, (01/07/04)
6. BD BDLTM Antimicrobial Susceptibility Testing Disks p.1-4 2001/06
7. Pharmacist, Shoppers Drug Mart, Bullfrog Mall, Guelph Ontario
THINGS I WANT SOCIAL WORKERS TO KNOW Hi. My name is Anique. I am sixteen years old and in tenth grade at school. I was diagnosed with FAS for the first time when I was eleven years old. I was diagnosed again at Sunny Hill Hospital two years ago. I was also diagnosed at that time with Fetal Dilantin Syndrome. I also went to medical genetics because my birth mom has
Post-traumatic stress disorder and life events among recently resettled refugees Post-traumatic stress disorder and life events among recentlyDepartment of Public Health Sciences, Division of PsychosocialThis work is dedicated to absent friends:Hartmut Apitzsch for his clinical sensitivity and dedication, generous sharing of wisdom, andSten W. Jakobsson for his compassion, inspir