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2011 OMIG Abstract 14

Identification of a bacteriocin that can kill biofilm bacteria
R.M.Q. Shanks1, A. Dashiff2, J.S. Alster2, D.E. Kadouri2
1The Charles T. Campbell Laboratory, UPMC Eye Center, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA,
2Department of Oral Biology, University of Medicine and Dentistry of New Jersey, Newark, NJ

Purpose: To isolate novel antimicrobials and test them for anti-biofilm activity.

Methods: A screen was performed in which >100 bacterial strains were tested for the ability to inhibit the growth of or kill other bacterial species.  The pBT20 transposon delivery vector was used for mutagenesis.  Arbitrary PCR and sequencing were used for mapping mutations. Susceptibility assays were performed using lawns of bacteria and spotting on antimicrobial containing crude lysates or affinity purified ColA-43864.  The ColA-43864 gene was cloned with a poly-histidine tag for affinity purification.  Biofilms were prepared on polyvinyl chloride and challenged with crude lysates or recombinant ColA-43864 before being analyzed by serial dilution and colony counts or fluorescent microscopy using live/dead staining.

Results: A strain of Citrobacter freundii was found that was able to prevent the growth of Escherichia coli and several other gram-negative bacteria. Transposon mutagenesis was used to isolate mutants unable to produce the antimicrobial compound.  Mutations were mapped using arbitrary PCR and sequencing, and were found to be in a plasmid-borne bacteriocin gene, ColA-43864.  The colA-43864 was cloned with a poly-histidine tag for affinity purification.  Crude lysates of E. coli bearing the ColA-43864 plasmid and purified recombinant ColA-43864 were able to kill bacteria in biofilms.

Conclusions: This research supports the concept that bacteriocins may be effective in killing surface-attached bacteria, which are notoriously difficult to treat with antibiotics.

Support:  Research to Prevent Blindness, NIH EY08098

Disclosure: P

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