Antimicrobial Resistance in Serratia marcescens

2019-06-11T17:48:55Z (GMT) by Danielle Susan Sopovski
With the increase of antibiotic resistant bacteria strains, the need to determine the mechanisms of antimicrobial resistance is similarly rising. Serratia marcescens, 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 S. marcescens as one of the few bacteria resistant to antimicrobial compounds produced by Stemphylium vesicarium, an isolated fungal spinach endophyte. To identify the mechanism of antimicrobial resistance to the unknown Stemphylium 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 Stemphylium 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 S. marcescens antimicrobial resistance mechanisms, but it aids in our understanding of the mechanisms of antimicrobial resistance development in other human pathogens.