Date of Completion

Spring 6-9-2020

Thesis Advisor(s)

Joerg Graf

Honors Major

Molecular and Cell Biology

Disciplines

Bacterial Infections and Mycoses | Bioinformatics | Medicine and Health Sciences | Microbiology | Pathogenic Microbiology

Abstract

In recent years, ciprofloxacin resistant (CpR) Aeromonas veronii and A. hydrophila strains have been isolated from the wounds of patients receiving leech therapy. Genome comparisons of these CpR isolates revealed the presence of chromosomal mutations in gyrA and parC as well as the gain of qnrS2 on either a large, 34 kb, conjugatable, low-copy plasmid, pAv42, or on a small, 6.8 kb, high-copy plasmid, pAh1471. The minimum inhibitory concentration, MIC, for Cp of these clinical isolates ranged from 1 to ≥32 µg/mL and some harbored a qnrS2 containing plasmid. We wanted to assess the contributions of these factors in an isogenic background. To this end, gyrA S83I and parC E91K mutations were introduced individually and in combination into a Cp-sensitive leech isolate, A. veronii Hm21. The pAh1471 was modified to encode a kanamycin resistance cassette. The resulting plasmid, pEL1, was electroporated into these four genetic backgrounds. pAv42 was conjugated into the four recipient strains by selecting for elevated Cp resistance. The genomes of all 12 strains were sequenced to screen for any secondary mutations, and Cp resistance was evaluated by E-tests. The results showed that the wild-type, gyrA, parC, and gyrA/parC mutants had a Cp MIC of 0.003, 0.094, 0.003, and 0.25 µg/mL, respectively. Furthermore, the presence of pEL1 or pAv42 increased the resistance by 0.032-3.0 µg/mL. These indicates that the presence of qnrS2 had synergistic effect on the MIC and that copy number did not affect MIC. In addition, no strain was CpR, which was observed in a number of the clinical isolates, suggesting that additional mutations contribute to Cp resistance. Ten lineages of Hm21RS gyrA parC pAv42 were grown under Cp selective pressure. Those grown in 2.0 μg/mL Cp acquired additional mutations and increased MICs. Key mutations discovered after sequencing populations include pncB T77P and phoE Δ367. Future investigations will clone these mutations into Hm21RS to assess their impact on Cp resistance.

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