Date of Completion

5-1-2022

Thesis Advisor(s)

Joerg Graf

Honors Major

Molecular and Cell Biology

Disciplines

Bacteria | Infectious Disease | Medical Microbiology

Abstract

Infections from ciprofloxacin (Cp) resistant (CpR) Aeromonas veronii have been reported in increasing numbers among patients receiving leech therapy in recent years. Mutations in DNA gyrase, gyrA S83I, and DNA topoisomerase IV, parC E91K, along with acquisition of the qnrS2 gene located on the pAv42 plasmid, have been identified as genetic factors contributing to CpR in A. veronii. A strain of A. veronii harboring all known resistance factors was previously constructed and has a Cp minimum inhibitory concentration (MIC) of 3 µg/ml, which remains below the resistance threshold of 4 µg/ml and well below the high levels of resistance seen in isolates from infected patients, which showed MICs ranging from 1 to >32 µg/ml. This project aimed to identify additional ciprofloxacin resistance factors through experimental evolution of A. veronii in its natural host, the medicinal leech (Hirudo verbana), with exposure to low levels of Cp, which provides a selection pressure for the gain of mutations that increase CpR. E-testing was performed at multiple timepoints and revealed increased MIC values to >12 µg/ml in lineages exposed to the highest levels of Cp in the leech. Genomic sequencing analysis revealed several mutations which rose to 100% frequency in the population and remained fixed across several timepoints, indicating a potential role in CpR. Point mutations were identified in an MFS family multidrug efflux pump along with 10bp deletions in an adjacent ArsR transcriptional regulator. Future research will aim to more deeply investigate the contribution of these mutations to the CpR phenotype.

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