Transcript Document

Isolation and Identification of Antibiotic-producing Actinomycetes from soil
samples in comparison with a known antibiotic producer, Streptomyces griseus
Amanda McCune
Department of Biological Sciences, York College
Streptomyces slide culture
Various antibiotic pills
Taken from:
http://en.wikipedia.org/wiki/Image:Streptomyces_sp_01.p
ng
Identification results (continued)
Taken from:
http://library.thinkquest.org/25462/uses.htm
l
Inclined coverslip slide
culture
Introduction
Antibiotic resistance is a major problem in the medical
industry due to the large increases in antibiotic resistant strains
of pathogenic bacteria, over the past 20 years. Bacteria are
constantly evolving into more efficient pathogens resulting in
their ability to resist antibiotics.
Many of the current, effective antibiotics in use, such as
streptomycin, vancomycin, chloramphenicol, and erythromycin,
are synthesized by a specific group of bacteria known as
actinomycetes (Strohl, 2003). Actinomycetes are aerobic, grampositive bacteria that form hyphae or branching filaments, and
are prolific producers of antibacterial chemicals (Bergey and Holt,
1994). Actinomycetes, typically present in soils, are found in
many different areas all over the world from the mountainous
regions of the Middle East to caves in Italy (Aghighi et al., 2004
and Laiz et al., 2000). Many researchers have suggested that
actinomycetes produce antibacterial chemicals to compete with
other microorganisms in the nutrient poor environments in which
they best survive (Aghighi et al., 2004). Therefore actinomycetes
are ideal candidates in the continuous search for new and better
antibiotics.
• Test bacteria streaked perpendicular to
the isolated bacteria
•Coverslip was placed at an angle
into an agar well
• Biochemical
• 0.1ml of test bacteria (E. coli, S. aureus,
and B. cereus) were spread over the entire
agar plate
S. griseus
•Distance of inhibition was measured from
edge of the disk to the edge of inhibition in
mm.
Isolate 2
disk
Isolate 1
disk
tests
Table 1: Biochemical test results for gram positive isolates and
Citrate
Mannitol Lactose
Isolate 1
+
-
-
-
Isolate 2
+
+
-
-
S. griseus
-
-
-
+
S. griseus
disk
Isolate 3
disk
•Differences in the antibiotic production testing
methods are due to the difference in the extraction
of the isolate synthesized chemicals.
Antibiotic production results
•Streak plate is direct contact with the organism
and its biosynthesized chemical
Streak plate results
•Disk diffusion plate uses the chemical extracted
from the actual organism
Table 2: Measurement of growth based on 0
(no growth) - +3 (complete growth) scale
- = orange, + = pink
Results
Isolated colonies from soil sample obtained along
the banks of Tyler Run
Isolate 1
Isolate 2
Isolate 3
Literature Cited
S. aureus
E. coli
B. cereus
Isolate 1
+3
0
+3
Isolate 2
+3
0
+3
Isolate 3
+3
+2
+3
S. griseus
+1
+2
+2
Identification
testing
Antibiotic
production testing
Gram stain
Streak plate method
Inclined coverslip
slide culture
Disk diffusion
method
Wet mount
Biochemical tests
•Laiz, L., Groth, I., Schumann, P., Zezza, F., Felske, A.,
Hermosin, B., and Saiz-Jimenez, C. 2000. Microbiology of
the stalactites from Grotta dei Cervi, Porto, Italy. International
Microbiology 3: 25-30.
Disk Diffusion results
Table 3: Zone of Inhibition (mm) ± SD
Identification results
• Gram staining
• gram (+) purple: isolate 1 and 2, S. griseus
• gram (-) pink: isolate 3
S. aureus
E. coli
B. cereus
0
0.33 ±0.38
0
Unknown 2
1.3±0.95
0.68 ±0.83
0
Unknown 3
0
0.18 ±0.35
0
S. griseus
0
0.83 ±0.95
0
Unknown 1
• Aghighi, S., Shahidi Bonjar, G.H., Rawashdeh, R.,
Batayneh, S., and Saadoun, I. 2004. First Report of
Antifungal spectra of Activity of Iranian Actinomycetes Strains
against Alternaria solani, Alternaria alternate, Fusarium
solani, Phytophthora megasperma, Verticillium dahliae, and
Saccharomyces cerevisiae. Asian Journal of Plant Sciences
3: 463-471.
• Bergey, D.H., and Holt, J.G. 1994. Bergey’s Manual of
Determinative Bacteriology Vol. 4. 9th ed. Baltimore, MD.
All plates were incubated and checked for growth of colonies
Colonies
isolated
•The antibiotic chemical being produced is most
effective on gram negative bacteria rather than the
gram positive, S. aureus or the spore forming gram
positive, B. cereus.
•Isolate 2 and S. griseus were most effective
against E. coli based on disk diffusion, isolates 1
and 2 were most effective against E. coli based on
streak plates.
Urea
+ = blue, - = green
Test bacteria
spread
Plated 10-4, 10-5, and 10-6 soil dilutions on
glycerol yeast extract agar (GYE)
Antibiotic production
• Soak each disk in supernatant obtained
from the broth culture of isolated bacteria 1-3
and S. griseus
Collected 5 Soil samples from various locations on campus
through
•Biochemical results point to Rhodococcus for
isolate 2, results were inconclusive for isolate 1.
Spiral chains of
conidia
Disk Diffusion method
Methods
Diluted soil samples from
S. griseus
B. cereus
Mannitol and Lactose
•Isolates 1 and 2 were found to be possible
actinomycetes from the gram positive results of the
gram stain. Isolate 3 was ruled out due to its gram
negative results.
•Through analysis of mycelium and sporulation
structures on the coverslip slide cultures and wet
mounts isolates 1 and 2 were narrowed down to
either Rhodococcus or Actinomadura genus levels
E. coli
Biochemical Tests
Urea
10-6
Branching
filaments
Short chains
of conidia
S. aureus
•Isolate and identify actinomycetes from soil samples
taken from York College campus
10-2
Identification
Isolate 2
Isolated bacteria 1 - 3 or S. griseus
Citrate
•Test for antibiotic production and compare to a known
antibiotic producing actinomycete, Streptomyces griseus
Isolate 1
•Growth of test bacteria was recorded as
0 for no growth or complete inhibition
through +3 for full growth or no inhibition
+ = yellow, - = red
Objectives
•Inclined coverslip slide
Streak plate method
•Inoculated edge of sterilized
coverslip by touching the bacterial
colony
Conclusions
• Strohl, William. 2003. Chapter 31: Antimicrobials. Pages
336-355 in Bull, Alan (ed.). Microbial Diversity and
Bioprospecting. ASM Press, Washington D.C.
Acknowledgments
I would like to thank Dr. Mathur for her advice and
support throughout the entire research process and
presentation. I would also like to extend my thanks the
biology staff for all of their interest and support through
the senior thesis process.