With
the increase of antibiotic resistant bacteria strains, the need to determine
the mechanisms of antimicrobial resistance is similarly rising. <i>Serratia marcescens</i>, a ubiquitous,
Gram-negative opportunistic pathogen is known to have strong, natural
resistance to diverse antimicrobial agents including antibiotics and
antimicrobial peptides. Recently, we identified <i>S. marcescens</i> as one of the few bacteria resistant to antimicrobial
compounds produced by <i>Stemphylium
vesicarium</i>, an isolated fungal spinach endophyte. To identify the mechanism
of antimicrobial resistance to the unknown <i>Stemphylium</i>
antimicrobial compounds, we designed a transposon mutant screen identifying
mutants sensitive to antimicrobial inhibition of bacterial growth. A transposon
mutant library was constructed using the Tn5 EZ-Transposome (Epicentre) system and
contains 1,824 individual mutants with 127 being identified as having a
decreased resistance to the <i>Stemphylium</i>
antimicrobial compounds. The transposon growth inhibition screen initially evaluates
the mutants for reduced growth in the presence of 25% fungal metabolite over 24
hours. The growth phenotype is then confirmed
in triplicate in a 12-hour time course growth experiment. Identification of the
genomic insertion site of the Tn5 transposon utilized a multi-step modified
nested-PCR protocol, termed TAIL-PCR. Following PCR purification, nanodrop
spectroscopy and gel electrophoresis were performed to ensure the amplification
purity of the extracted DNA and was subsequently sequenced via WideSeq analysis.
BLAST identified insertions in genes necessary for membrane biogenesis, drug
transport, pili formation, and iron metabolism. Future work is aimed at
confirming these results and understanding the role of iron sequestration. Not
only will this research contribute to our understanding of <i>S. marcescens</i> antimicrobial resistance mechanisms, but it aids in
our understanding of the mechanisms of antimicrobial resistance development in
other human pathogens.
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/8059304 |
| Date | 11 June 2019 |
| Creators | Danielle Susan Sopovski (6650222) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/Antimicrobial_Resistance_in_Serratia_marcescens/8059304 |
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