1. Searching for antibiotic producing soil isolates
Aaron Rosenbloom Snow, Scott Chimileski, Karen N. Pelletreau
University of Connecticut, Department of Molecular and Cell Biology, Storrs, Connecticut
Purpose:
As bacterial resistance increases and antibiotic discovery slows, there is
an increasing need for new antibiotics. The purpose of this experiment
was to isolate antibiotic producing soil species to potentially discover
new classes of antibiotics and test its efficacy against todays most
relevant pathogens.
Funding:
ASM
Helmsley
HHMI
UCONN
Pilot:
SWI
Yale
UCONN
Characteristics of Lysobacter antibioticus:
Collected January 28th 2014 Storrs, CT from 1 to 3 cm deep mud under a
thawed section of a stream. Lysobacter antibioticus was isolated from a
plate of 10% TSA after plating the soil sample. Tests were performed to
characterize the isolate.
Sequence data determined that the species was Lysobacter antibioticus with
a 99% match in the database from NCBI’s 16S rRNA and the Ribosomal
Database Project.
Left: Ethyl acetate
Extract vs B. subtilis
Right: Lysobacter
Antibioticus colonies
Antibiotic activity of Lysobacter antibioticus:
Lysobacter antibioticus produced zones of inhibition against
five of eight safe ESKAPE pathogens .All zones of inhibition
completely prevented growth, rather than slowing it.
Lysobacter antibioticus inhibited both Gram-positive and
Gram-negative bacteria.
5/8 (62.5%) safe ESKAPE species were inhibited by the
antibiotics being produced.
Antibiotic tests :
An ethyl acetate extraction was done on Lysobacter
antibioticus to isolate the antibiotic compound [4]. This
extract was plated under a soft agar plate (liquid agar
overlay of ESKAPE relative) containing B. subtilis to test if
the extract contained the antibiotic. The Eukaryotic specie,
Candita albicans, was grown in the presence of this extract
to test its toxicity [5].
Isolation and identification:
Soil was diluted onto 10% Tryptic soy agar (TSA) to calculate CFU/g (3.3x 105)
[1]. Isolates were picked from these dilutions and patched onto spread plates
of the safe ESKAPEs. Plates were incubated for 2 days at 27°C and checked for
zones of inhibition [2]. Lysobacter antibioticus was determined to produce the
most ZOIs and was put through polymerase chain reaction (PCR). A 1392R
primer of the 16S gene was used for the PCR reaction. Electrophoresis was run
on the PCR products to determine quality of products, and they were sent for
sequencing. Sequences were compared to BLAST sequence data from the NCBI
and RDP databases to determine genus and species [3].
Lysobacter antibioticus is known to
produce β – lactams, macrocyclic
lactams, and katanosins. These inhibit
both Gram-positive and Gram-
negative bacteria.
Results:
Candita albicans was not inhibited by Lysobacter
antibioticus or the ethyl acetate extract.
Acknowledgements: University of Connecticut Department of Molecular and Cell Biology, Yale Center for Scientific Teaching, Helmsley Charitable Trust,
Davis Educational Foundation, HHMI
Bacillus subtilis
Methods:
[1] [2] [3] [4] [5]
C. albicans
vs Lysobacter
antibioticus
C. Albicans vs
Ethyl acetate
extract