Antimicrobial susceptibility of Francisella noatunensis subsp. noatunensis strains isolated from Atlantic cod Gadus morhua in Norway

Abstract

A total of 30 isolates of Francisella noatunensis subsp. noatunensis isolated from Atlantic cod Gadus morhua L. were tested for susceptibility, in the form of minimal inhibitory concentration (MIC) values, against the following antibacterial agents: flumequine, oxolinic acid, ciprofloxacin, florfenicol, oxytetracycline, erythromycin, streptomycin sulphate, trimetoprim/ sulphadiazine and rifampin. All the isolates had a low susceptibility to oxytetracycline, trimetoprim/sulphadiazine (Tribrissen ® ), erythromycin, ciprofloxacin and streptomycin with MIC values of 64, 64 to 128, 16, 8 and 32 to 128 µg ml Dis Aquat Org 98: [57][58][59][60][61][62] 2012 markedly more active against oxolinic-acid-resistant isolates of Aeromonas salmonicida (Lewin & Hastings 1990). F. noatunensis subsp. noatunensis is a slowgrowing bacterium, and rifampin, erythromycin and streptomycin sulphate are all antibacterial agents that are used in human medicine in the treatment of Mycobacterium tuberculosis, another slow-growing bacterium. Erythromycin is also occasionally used against bacterial kidney disease (BKD) in salmonids.The aim of the present study was to test the susceptibility, in the form of MIC values, to a number of isolates of Francisella noatunensis subsp. noatunensis against the selected group of antibacterial agents. MATERIALS AND METHODS Bacterial strainsA total of 30 isolates of Francisella noatunensis subsp. noatunensis from Atlantic cod Gadus morhua L. were analysed. Of these, 28 isolates were provided by the Norwegian Veterinary Institute (Norway) and 2 isolates, GM2212/LMG 24256 (Nylund et al. 2006, Ottem et al. 2007) and EK-4b, were provided by the Institute of Marine Research (Norway). All isolates originate from farmed cod, except EK-4b, which was isolated from a wild cod. The strains were previously identified as F. noatunensis subsp. noatunensis using the methods described by Ottem et al. (2008). ChemicalsFlumequine, oxolinic acid, oxytetracycline, florfenicol, Tribrissen (trimethoprim/sulfadiazine), erythromycin, ciprofloxacin, streptomycin sulphate and rifampin were all obtained from Norwegian Medical Depot (Bergen, Norway). Stock solutions of antibacterial agents were prepared at a concentration of 1.0 mg ml −1 in methanol (florfenicol, Tribrissen, rifampin), in water (oxytetracycline, erythromycin, streptomycin sulphate), 0.03 M NaOH (flumequine, oxolic acid) and 0.03 M NaOH/methanol (1:1) (ciprofloxacin). MIC determinationIn order to meet the demands of Francisella noatunensis subsp. noatunensis for specific growth factors, the method of choice in the present work was a modified agar dilution test for the determinations of MIC values (Alderman & Smith 2001). Briefly, F. noatunensis subsp. noatunensis isolates were grown on modified Mueller-Hinton agar (MMHA) supplemented with 3% foetal bovine serum (FBS) (PAA Laboratories), 0.8% glucose (Merck), 0.4% L-cystein (Sigma) and 4% Yeastolate Ultrafiltrate (Gibco) and containing 2-fold dilutions of the antibacterial agents tested. All supplements and a...

Broth microdilution protocol for minimum inhibitory concentration (MIC) determinations of the intracellular salmonid pathogen Piscirickettsia salmonis to florfenicol and oxytetracycline

“…Susceptibility presented in the form of minimum inhibitory concentration (MIC) values is an alternative way to measure antibiotic resistance as proposed by Isachsen et al . () for Francisella noatunensis subsp. noatunensis , also an intracellular pathogen.…”

Antibiotic uptake by cultured Atlantic cod leucocytes and effect on intracellular Francisella noatunensis subsp. noatunensis replication

“…In this study, an HK/plasma ratio of 1.16 for flumequine and 1.40 for oxolinic acid were found 48 h following in vivo administration. If accepting the assumption that the HK/plasma ratio is reasonably constant over time, our results indicate that a higher drug concentration may be present in HKLs compared to plasma, and therefore, based on the theoretical assessments by Isachsen et al (2012), therapeutic effects using daily doses of at least 15 and 25 mg kg −1 for flumequine and oxolinic acid, respectively, should be likely. However, when the effect of 5 µg ml −1 flumequine on survival and replication activity of Francisella noatunensis subsp.…”

“…noatunensis (Isachsen et al 2012). During incubation, the wells were covered by sealing tape as described above and wrapped in tinfoil to avoid photo-degradation of the antibacterials.…”

“…Isachsen et al (2012) found that 30 Norwegian isolates of Francisella noa tunensis subsp. noatunensis were susceptible to only a few of the antibacterials tested, including florfenicol with an in vitro MIC of 0.5 µg ml −1 and flumequine and oxolinic acid, both with an in vitro MIC of 0.25 µg ml −1 .…”

“…AliAbadi & Lees (2000) suggested an AUC 24 /MIC value greater than 100 and in some cases greater than 250. Isachsen et al (2012) estimated that an oral dose of 25 mg kg −1 of oxolinic acid would give an AUC 24 / MIC value of 100, indicating efficacy, whereas for oral doses of 15 and 25 mg kg −1 of flumequine, AUC 24 / MIC values of 262 and 276, respectively, were calculated. Therefore, due to the superior AUC 24 / MIC value of flumequine, this drug was chosen for the effect study.…”