Determination of Bacteria obtained from the

Science Library Men’s Restroom Door

Handle and a Household Restroom Knob for
resistance to Ampicillin, Chloramphenicol,
Ciprofloxacin, Kanamycin, Lomefloxacin,
Nalidixic Acid, Streptomycin, and
Tetracycline.
HASSAN ALYASSIN AND AUDREY NGUYEN

The purpose of this experiment was to determine whether isolated

bacteria from two different restroom handles would possess resistance to

any of eight antibiotics. We used the Kirby-Bauer disk diffusion method.

The eight antibiotics were Ampicillin, Chloramphenicol, Ciprofloxacin,

Kanamycin, Lomefloxacin, Nalidixic Acid (NAL), Streptomycin, and

Tetracycline. The unknown isolates were obtained from the inside handle

of the men’s restroom on the second floor of the Science Library

(yellowish, Gram-positive, irregular-rod shaped) and from the knob of a

household restroom (whitish, Gram-positive, cocci). Escherichia coli

strain ATCC 25922 was used as a control because it is susceptible to all

eight of the antibiotics that we are testing and is relatively easy to handle.

Interestingly, both unknown isolates were resistant to Nalidixic Acid and

were susceptible to all of the other antibiotics. A simple substitution

 mutation in Shigella sonnei of a Ser83->Leu (TCG->TTG) is all that is

needed to confer resistance to NAL (1). In order to analyze the results, we

utilized a quality control test where we used the ATCC 25922 strain of E.

coli to determine the effectiveness of the antibiotics. If the antibiotics

inhibited the growth of bacteria within an experimentally accepted radius

then the results were accepted.

Materials and Methods

Three strains of bacteria were obtained. Two unknown isolates along with E. coli

strain ATCC 25922. To allow for comparative analysis, we diluted all of our cultures to a

density of about 1.0 5 108 cell/mL. We used sterile saline as a diluent. To determine

density we used a spectrometer set at 625nm. We plated the three organisms on Mueller-

Hinton Agar plates (one organism per plate). Each of the three agar plates received eight

discs. Every disc contained 10 µ L of one of the following antibiotics: Ampicillin,

Chloramphenicol, Ciprofloxacin, Kanamycin, Lomefloxacin, Nalidixic Acid (NAL),

Streptomycin, or Tetracycline. The antibiotics were allowed to soak into the agar for 10

minutes before being inverted and incubated at 37°C overnight. We measured, in mm,

the size of the zone of inhibition of the plate containing the E. coli ATCC 25922 strain to

determine if our results were acceptable. There was a zone of inhibition between

Kanamycin and Lomefloxacin on the plate containing the bacteria from the household

doorknob. We cross-referenced our results with the “Quality Control Log for BBL

™Sensi-Disc™ Antimicrobial Disc chart. For the results indicated that the antibiotic was
working properly we interpreted the susceptibility of the antibiotic on our unknown

isolates. For the results that indicated that the antibiotic was not working properly we

noted that the results were inconclusive.

Results

Table 1: Radii of the Zones of Inhibition from 10 µ L of Antibiotic

Antibiotic E. coli ATCC Science Household
25922 Library Isolate Isolate
Ampicillin 9mm 14mm 8mm
Chloramphenicol 12mm 19mm 14mm
Ciprofloxacin 18mm 10mm 12mm
Kanamycin 12mm 12mm 11mm
Lomefloxacin 18mm 10mm 12mm
Nalidixic Acid 14mm 0mm 0mm
Streptomycin 13mm 9mm 7mm
Tetracycline 14mm 18mm 13mm

Table 2: Susceptibility to Antibiotics

Antibiotics Amount on E. coli ATCC Science Library Household
Disk 25922 Isolate Isolate
Ampicillin 10μL Susceptible Susceptible Susceptible
Chloramphenicol 10μL Susceptible Susceptible Susceptible
Ciprofloxacin 10μL Susceptible Intermediate Susceptible
Kanamycin 10μL Susceptible Susceptible Susceptible
Lomefloxacin 10μL Inconclusive Not Valid Not Valid
Nalidixic Acid 10μL Susceptible RESISTANT RESISTANT
Streptomycin 10μL Inconclusive Not Valid Not Valid
Tetracycline 10μL Inconclusive Not Valid Not Valid
 Lomefloxacin, Streptomycin, and Tetracycline all gave inconclusive results. This

means that the zone of inhibition was out of the normal range. The accepted range

for Lomefloxacin’s zone of inhibition is 27 – 33 mm but our experimental value

was determined to be 36 +/- 2 mm. The accepted range for Tetracycline’s zone of

inhibition is 18 - 25 mm but our experimental value was determined to be 28 +/- 2

mm. These values are very close to the accepted values but not they don’t fall

within them. Streptomycin, on the other hand, has an accepted zone of inhibition

of 12 – 20 mm. Our experimental value was 26 +/- 2 mm, which is very far from

accepted values. This means that we could not accept the results of how these

antibiotics reacted to our unknown isolates. Some reasons why an antibiotic would

fail include: age of antibiotic, temperature, and pH of media.

Discussion

Three of the eight tests were inconclusive. Lomefloxacin and tetracycline were

within one mm of the accepted values. We re-measured three different times to make

sure our measurements were accurate and each time we confirmed the same number.

One problem that was obvious was that the borders were difficult to define because they

were fuzzy. The borders of these two antibiotics were probably fuzzy because the

swabbing techniques created little grooves on the agar in which the antibiotic fluid was

able to flow. The results were that the zones of inhibition were not clearly defined.

Streptomycin was the only antibiotic that was noticeably far from its expected values.

The other five antibiotics tested resulted in conclusive results. Both unknown isolates
were resistant to Nalidixic Acid but were susceptible at varying degrees to Ampicillin,

Chloramphenicol, Ciprofloxacin, and Kanamycin.

From the unknown isolate obtained from the inside handle of the men’s restroom

on the second floor of the Science Library, I found that some colonies developed

resistance and began to grow in the presence of the antibiotic. There are now colonies

growing on the Chloramphenicol and Kanamycin.

From the unknown isolate obtained from the household doorknob, no new resistant

colonies have arisen. However, there appears to be inhibitory activity between

Kanamycin and Lomefloxacin because there is a clear but “foggy” zone where the two

zones of inhibition overlap. Under the microscope the size of the bacteria derived from

the household doorknob was 4.8 5 10-1 µ m. The size of the bacteria derived from the

men’s restroom in the Science library was 2 µ m in length.

A good follow-up to this experiment would be to create different arrangements of

the antibiotics to see if different over-lapping zones result in synergistic or inhibitory

activity. Also it would be fun to sequence these bacteria to see what mutation these

bacteria underwent to confer them this type of resistance. I would begin my search with

the nalA gene (2).

LITERATURE CITED

1. Jeong, Y. S., J. C. Lee, H. Y. Kang, H. S. Yu, E. Y. Lee, C. H. Choi, S. H. Tae,

Y. C. Lee, D. T. Cho, and S. Y. Seol. 2003. Epidemiology of nalidixic acid
resistance and TEM-1- and TEM-52-mediated ampicillin resistance of Shigella
sonnei isolates obtained in Korea between 1980 and 2000. Antimicrob. Agents
Chemother. 47:3719-3723.
2. Rella, M., and D. Haas. 1982. Resistance of Pseudomonas aeruginosa PAO to
nalidixic acid and low levels of beta-lactam antibiotics: mapping of chromosomal
genes. Antimicrob. Agents Chemother. 22:242-249.