Local epidemiological cutoff values and antimicrobial susceptibility profile for Brazilian Francisella orientalis isolates

Francisellosis is an emerging disease caused by Francisella orientalis and is responsible for major economic losses in tilapia farms worldwide. The method of choice for francisellosis outbreak control is oral antibiotic administration, as no commercial vaccines or autogenous effective vaccines are available. Although there is a standard method recommended by the Clinical and Laboratory Standards Institute (CLSI) for susceptibility testing of fish pathogenic Francisella spp., basic information on culture and growth conditions for this pathogen is scarce. In this study, the antibiotic susceptibility profile of Francisella orientalis was determined using the disk diffusion test. For that, 89 Francisella orientalis samples obtained from disease outbreaks in tilapia farms in 2012–2019 in various Brazilian states were selected for testing against different antibiotics. Because F. orientalis cannot grow on the standard Mueller-Hinton (MH) agar medium, two different culture media were selected for testing: modified heart cystine agar (CHAH) and modified cation-adjusted Mueller-Hinton agar (MMHA). Different incubation periods were also tested for more appropriate and accurate measurement data. CHAH was more suitable for performing the diffusion disk tests because MMHA required longer incubation periods and formed overestimated inhibition zones. Incubation time affected the antibiogram results mainly for β-lactam antibiotics, with 48 h being the ideal period for measuring inhibition zones. Epidemiological cutoff values of Francisella orientalis isolates using the normalized resistance interpretation (NRI) methodology were successfully calculated. All 89 isolates (100%) were considered wild type (WT) for florfenicol, oxytetracycline, erythromycin, and doxycycline, whereas 86 isolates (96.6%) were considered WT for amoxicillin, ampicillin, enrofloxacin, and norfloxacin. Three isolates (3.4%) were considered non-wild type (NWT) for enrofloxacin, norfloxacin, amoxicillin, and ampicillin. This is the first report of Francisella orientalis isolates resistant to amoxicillin and fluoroquinolones. This study provided important data on Francisella orientalis antibiotic susceptibility. Moreover, it showed that Brazil is already suffering the effects of the inadvertent use of licensed drugs and of off-label drugs, as the aforementioned drug classes are illegally used for farmed fish in the country.