These are the preliminary results of 5 antibiotic-producers isolated as part of the Small World Initiative implementation at National University.

1. Isolation of soil microbes producing antibiotics
The Small World Initiative (SWI) 1,2, spear-headed
by Yale University, incorporates the
search for soil microbes producing antibiotics in
the undergraduate biology curriculum. At NU,
SWI has been implemented in Introductory
Microbiology Laboratory (BIO203A) courses3.
The major rationale behind SWI is the current
antibiotic crisis. The ESKAPE pathogens (see
table below) are responsible for a substantial
percentage of nosocomial infections in the
modern hospital and represent the vast majority
of antibiotic resistant isolates. Soil bacteria,
particularly from the genera Bacillus and
Pseudomonas, produce a large variety of
secondary metabolites with antibiotic activity
that not only protect them other microbes, but
also play an important part in quorum sensing,
biofilm formation, interactions with plants, and
sporulation 4,5,6–8. Besides their potential in
human medicine, antibiotics from the soil may
be used also to prevent plant diseases5,9. We
present the initial characterization of five
antibiotic-producing soil bacteria from CT
and CA.
RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
BACKGROUND
MATERIALS AND METHODS
1. A. M. Barral, H. Makhluf, P. Soneral, B. Gasper, FASEB J. 28, 618.41 (2014).
2. http://smallworldinitiative.org/.
3. A. M. Barral, H. Makhluf, in ASMCUE Microbrew Abstracts (Danvers, MA, 2014).
4. H. Chen et al., Lett. Appl. Microbiol. 47, 180–186 (2008).
5. J. M. Raaijmakers, I. de Bruijn, O. Nybroe, M. Ongena, Natural functions of lipopeptides
from Bacillus and Pseudomonas: More than surfactants and antibiotics. FEMS Microbiol.
Rev. 34 (2010), pp. 1037–1062.
6. I. Mora, J. Cabrefiga, E. Montesinos, Int. Microbiol. 14, 213–23 (2011).
7. T. Stein, Mol. Microbiol. 56, 845–57 (2005).
8. J. Shoji, H. Hinoo, Y. Wakisaka, K. Koizumi, M. Mayama, J. Antibiot. (Tokyo). 29, 366–374
(1976).
9. S. a Cochrane, J. C. Vederas, Med. Res. Rev., 1–28 (2014).
ACKNOWLEDGMENTS
Presenter contact: abarral@nu.edu @Bio_prof
Ana Maria Barral, Huda Makhluf, and Jacqueline Ruiz
Department of Mathematics & Natural Sciences, COLS
RESULTS
Spread/patch of SWI1, CFU4,
and R3 against S. cohnii.
CONCLUSIONS AND RECOMMENDATIONS
• Implementation of SWI, besides the obvious
educational benefits, also provides a
straightforward way of screening and
identification of antibiotic producing microbes.
• It is possible at NU to perform basic
characterization the isolates and preliminary
purification of the compounds.
• To identify both microbe and compound, more
advanced genetic (more targeted PCR,
whole genome sequencing) and chemical
methods are required.
• Other possibilities to explore include
screening for anti-fungal or anti-tumor effect.
• Soil samples were diluted and plated on different media (TSA, PDA).
• Colonies were tested for antibiotic activity against ESKAPE surrogates using spread/patch technique.
• Isolates with activity were further characterized by biochemical, morphological, and genetic (16s rRNA)
tests.
• Initial purification was achieved by ethyl acetate or 3-solvent extraction.
• Testing of soluble fractions was done using agar well diffusion, broth dilution, and other tests.
We describe five soil isolates with antibiotic
activity against ESKAPE surrogates that were
characterized by 16s rRNA sequencing and
traditional methods. The majority were from the
genus Bacillus, whose members are difficult to
discriminate. Initial purification using different
solvents showed activity in both organic (ethyl
acetate) and aqueous phases, pointing to the
presence of a variety of compounds of different
chemical nature.
ESKAPE PATHOGENS & THEIR SURROGATES
Phylogenetic analysis of the Bacillus isolates.
Dilution test of SWI ethyl acetate extracts on S. cohnii and E.coli
(#5 is K2.2, EA is ethyl acetate).
ESKAPE
Pathogens
ESKAPE
Surrogates
Gram Staining &
Morphology
Staphylococcus
aureus
Staphyloccocus
cohnii
Gram positive
Cocci in clusters
Enterobacter
species
Enterobacter
aerogenes
Gram negative
Rods
Klebsiella
pneumoniae
Escherichia coli
Gram negative
Rods
Pseudomonas
aeruginosa
Pseudomonas
putida
Gram negative
Rods
Enterococcus
faecium
Enterococcus
raffinosus
Gram positive
Cocci in chains
Acinetobacter
baumannii
Acinetobacter
bayley
Gram negative
Coccobacillus
Bacilllus subtilis
Gram positive
Rods
Soil isolate Genus Antibiotic effect
against
Origin & Coordinates
SWI1 Pseudomonas S.cohnii, S.aureus,
B.subtilis, A.bayley
Yale University yard
41.318534, -72.921868
SWI2 Bacillus P.putida, B.subtilis
Yale University yard
41.318534, -72.921868
CFU4 Bacillus S.cohnii, B.subtilis,
A.bayley
NU Costa Mesa campus
33.694816, -117.917937
K2.2 Bacillus S.cohnii, E.
raffinosus,
B.subtilis, A.bayley,
E.coli
Irvine, CA
33.667228, -117.827147
R5 Arthrobacter P.putida Lakewood, CA
33.853942, -118.100972
Agar well diffusion test of CFU4
colonies, supernatant, and
aqueous extract against S.
cohnii.
DISCUSSION
REFERENCES
Thanks to all the BIO203A SWI students, especially Bao Ly,
Kassia Valverde, and Robert Pfister (CFU4, K2.2, R5). Special
thanks to Tiffany Tsang from Yale who led SWI in 2013-14.
Summary of the five isolates, their origin & antibiotic activity.
Antibiotic producing soil isolates predominantly belonged to the genus Bacillus, with activity mainly
against Gram positive bacteria. Initial extraction was done both from solid and liquid cultures, with the
latter yielding more consistent results. Activity could be detected both in the ethyl acetate & aqueous
fractions.