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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.

BACKGROUNDMATERIALS AND METHODS

ACKNOWLEDGMENTS

Presenter contact: abarral@nu.edu @Bio_prof

Ana Maria Barral, Huda Makhluf, and Jacqueline RuizDepartment of Mathematics & Natural Sciences, COLS

Isolation of soil microbes producing antibiotics

RESULTSSpread/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).

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.

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).

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.