Transcript Bacillus brevis - York College of Pennsylvania

The Effect of Space Exposure on the Antibiotic Susceptibility and Betalactamase Production of Bacillus brevis
Rebecca Tafoya
Department of Biological Sciences, York College of PA
Antibiotic
Methods (2)
Introduction
-Bacillus is one of the most commonly found bacterial species [1].
-Bacilli are one of the contaminants of laboratory and clinical workplaces, and
have also been found to cause infections such as endocarditis, sepsis,
meningitis and surgical wound infections [4].
-Bacteria are able to create dormant spores that are very resistant to damage
such as wet and dry heat, UV irradiation, and strong acidic and basic
chemicals [1,3].
-Bacillus brevis spores were isolated from tomato seeds that were contained
at 4 levels of space exposure on the LDEF led by NASA [7].
-Kirby-Bauer Disk Diffusion Method was used to administer the
antibiotics on each plate containing bacteria.
-Inhibition zone diameters were measured the next day using
electronic calipers, and interpreted as Sensitive, Intermediate, or
Resistant to the antibiotic
-Beta-Lactamase detection sticks from Oxoid were used to
detect beta-lactamase production by B. brevis isolates
-Beta-Lactamase stick was inserted into a colony, and
colorimetric results were read after 1 minute.
-Red : Positive for beta-lactamse
-No change: Negative for beta-lactamase
Methods
-Bacterial samples taken from LDEF levels A, B, C, D, Earth
Bacillus brevis. Taken from:
www.probioticpharma.com
Space D: Tetracycline 30µg. Inhibition
zone diameter.
Results: Antibiotic Disk Testing
40
Inhibition Zone (mm)
Penicillin
s
35
Streptomycin
30
ns
25
s
20
ns
s
s
s
ns
s
s
Chloramphenicol
Ampicillin
ns
10
5
R
R
R
R
S
I, S
S
I
S
S
S
S
I, S
S
S
S
S
S
S
Ampicillin
Erythromycin
Kanamycin
Tetracycline
Chloramphenicol
Streptomycin
Vancomycin
-Space level A and B isolates were negative for
beta-lactamase, yet they were resistant to
Penicillin, suggesting a mutation may occur which
confers Penicillin resistance without use of the
protective enzyme.
-Increases in exposure to space UV-radiation while
in spore form causes increased accumulation of
irreparable DNA and protein damage, making the
bacteria more susceptible to antibiotics than
colonies kept on Earth.
D
Sp
ac
e
C
Sp
ac
e
B
Sp
ac
e
A
Sp
ac
e
th
0
Level of Space E xposure
Figure 1. Graph showing mean inhibition zone diameter of
isolates. All isolates were Sensitive to the antibiotics. “NS”= not
significant, “S” = significant (p<0.05).
-Successfully thawed and revived B. brevis samples
-Bacteria were streak plated and characterized using colony and spore
morphology, and biochemical tests to ensure bacteria were B. brevis
R
-This study suggests that contamination by
extraterrestrial bacteria is minimal, as bacteria from
space have higher sensitivities to antibiotics than
bacterial species on Earth.
s
s
15
Ea
r
-We also checked for Beta-lactamase production to see if the enzyme’s
absence correlated to the antibiotic sensitivity, and level of space exposure
received.
Earth
-Increased levels of exposure increased the
antibiotic sensitivity of the isolates, although for
several antibiotics this could be due to chance
alone (p>0.05).
-DNA mutations may make bacteria more or less susceptible to common
antibiotics used to treat infections, which would call for new medication
protocol to be developed to treat illnesses caused by the altered bacteria.
-We retested the antibiotic susceptibility of several previously used antibiotics
and also a few different antibiotics to see if long-term space exposure would
affect the antibiotic sensitivity of the bacteria.
D
Discussion
-This space environment can cause damage to the spore’s proteins, nucleic
acids and mutagenic or lethal damage to its DNA [3].
Objectives of Study
C
Table 1. Table showing interpreted inhibition zone diameter results
from Space levels A, B, C, D, and Earth. S: Sensitive, I:
Intermediate, R: Resistant. Note how one replicate from Level D
and one from Level B produced two different results during
antibiotic testing.
-Spores were exposed to solar UV radiation, extreme hot and cold
temperatures, evaporation, and low pressures [3].
-Previous research has shown that B. brevis demonstrates an increased
sensitivity to the antibiotics Erythromycin, Penicillin, and Chloramphenicol at
exposure levels A and B [7,6]
B
Penicillin
-Levels were A (most exposure), B, C, and D (least exposure)[7].
-To defend against antibiotics, bacteria produce beta-lactamase, an enzyme
that renders some antibiotics useless [5].
A
Results: Beta-Lactamase Detection
-Earth, Space C, and Space D isolates were positive for betalactamase production.
-Space A and B were negative for beta-lactamase production
Literature Cited
1. Newcombe, D.A. et al. Survival of Spacecraft-Associated Microorganisms under
Simulated Martian UV Irradiation. Applied and Environmental Microbiology 2005; 71(12): 81478156.
2. Reva, O.N. et al. Antibiotic Susceptibility as a Taxonomic Characteristic of the Genus
Bacillus. International Journal of Systemic Bacteriology 1995; 45(2): 409-411.
3. Nicholson, W.L. et al. Resistance of Bacillus Endospores to Extreme Terrestrial and
Extraterrestrial Environments. Microbiology and Molecular Biology Reviews 2000; 64(3): 548572.
4. Weber, D.J. et al. In Vitro Susceptibility of Bacillus spp. to Selected Antimicrobial
Agents. Antimicrobial Agents and Chemotherapy 1988; 32(5): 642-645.
5. Chen, K.C., Knapp, J.S., and Holmes, K.K. Rapid, Inexpensive Method for Specific
Detection of Microbial β-Lactamases by Detection of Fluorescent End Products. Journal of
Clinical Microbiology 1984; 19(6): 818-825.
6. Rosenberg, E., and Mathur, C. Comparison of Bacillus brevis Isolates From Earthborne vs. SpaceExposed Tomato Seeds. 1990.
7. Kreuzer, A.M., and Mathur, C. A Comparative Study of Antibiotic Sensitivities of a Microorganism,
Bacillus brevis, Isolated From Space Exposed and Earth Based Lycopersicon lycopersicum
Seeds. 1992.
Acknowledgements
I would like to thank Dr. Mathur for her knowledge and guidance throughout this
project, and Dr. Rehnberg for his help in preparation of these graphs.