Improved Broth Microdilution Method for Antimicrobial Susceptibility Testing of Francisella Noatunensis Orientalis

Abstract

In this project we optimized a minimal inhibitory concentration testing protocol for Francisella noatunensis orientalis. Thirty-three F. noatunensis orientalis isolates recovered from different fish species and locations were tested, and Escherichia coli ATCC 25922 was used as a quality control reference strain. A modified cation-adjusted Mueller Hinton broth supplemented with 2% IsoVitalex and 0.1% glucose (MMH) was tested at a pH of 6.4 ± 0.1, 7.1 ± 0.1, and 7.3 ± 0.1. Growth curves generated for F. noatunensis orientalis indicated that MMH at a pH of 6.4 ± 0.1 provided optimal growth. There were no significant differences in the growth curves obtained from isolates recovered from different fish species or from fresh or marine water. The pH of 6.4 ± 0.1 in the MMH media interfered with the inhibitory properties of the potentiated sulfonamides (ormetoprim-sulfadimethoxine and trimethoprim-sulfamethoxazole) when using the E. coli ATCC reference strain. Minimal inhibitory concentrations of eight antimicrobials (gentamicin, enrofloxacin, ampicillin, oxytetracycline, erythromycin, florfenicol, flumequine, and oxolinic acid) were similar for all F. noatunensis orientalis isolates. The in vitro susceptibility data provided here can provide a baseline for monitoring the development of antimicrobial resistance among F. noatunensis orientalis isolates, as well as provide valuable data in the development of potential therapeutics.

Received October 27, 2015; accepted April 13, 2016 Published online August 2, 2016

Acknowledgments

We gratefully thank Merck Animal Health, Madison, New Jersey, for financial support of this study. We also thank Dr. John Hansen from the Interdisciplinary Program in Pathobiology, University of Washington, Seattle, and Dr. Duncan J. Colquhoun at the Section for Bacteriology, Norwegian Veterinary Institute, Oslo, for sharing some of the F. noatunensis orientalis isolates used in the study. Finally, we thank Dr. Patricia Gaunt from Mississippi State University, College of Veterinary Medicine, Stoneville, and Dr. Ron Miller from the Food and Drug Administration’s Center for Veterinary Medicine, Office of New Animal Drug Evaluation, Rockville, Maryland, for a critical review of this paper.