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].

-Levels were A (most exposure), B, C, and D (least exposure)[7].

-Spores were exposed to solar UV radiation, extreme hot and cold temperatures, evaporation, and low pressures [3].

-This space environment can cause damage to the spore’s proteins, nucleic acids and mutagenic or lethal damage to its DNA [3].

-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.

-To defend against antibiotics, bacteria produce beta-lactamase, an enzyme that renders some antibiotics useless [5].

-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]

Objectives of Study

-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.

-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.

Methods

-Bacterial samples taken from LDEF levels A, B, C, D, Earth

-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

Literature Cited1. Newcombe, D.A. et al. Survival of Spacecraft-Associated Microorganisms under

Simulated Martian UV Irradiation. Applied and Environmental Microbiology 2005; 71(12): 8147- 8156.2. 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): 548-

572.4. 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. Space- Exposed 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.

AcknowledgementsI 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.

The Effect of Space Exposure on the Antibiotic Susceptibility and Beta-lactamase Production of Bacillus brevis

Rebecca Tafoya

Department of Biological Sciences, York College of PA

Methods (2)-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

Space D: Tetracycline 30µg. Inhibition zone diameter.

Antibiotic A B C D Earth

Penicillin

R R R R R

Ampicillin

S I, S S I S

Erythromycin

S S S I, S S

Kanamycin

Tetracycline

Chloramphenicol

Streptomycin

Vancomycin

S S S S S

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.

Results: Antibiotic Disk Testing

Results: Beta-Lactamase Detection

-Earth, Space C, and Space D isolates were positive for beta-lactamase production.

-Space A and B were negative for beta-lactamase production

Discussion

-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).

-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.

-This study suggests that contamination by extraterrestrial bacteria is minimal, as bacteria from space have higher sensitivities to antibiotics than bacterial species on Earth.

Earth

Space

A

Space

B

Space

C

Space

D0

5

10

15

20

25

30

35

40Penicillin

Streptomycin

ChloramphenicolAmpicillin

ns

ss

s

s

s

s

s

ns

Level of Space E xposure

Inh

ibit

ion

Zo

ne

(m

m) s

sns

ns

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).

Bacillus brevis. Taken from: www.probioticpharma.com