In Vitro and In Vivo Efficacy of Florfenicol for Treatment of Francisella asiatica Infection in Tilapia

Abstract

Francisella asiatica is a recently described, Gram-negative, facultative intracellular fish pathogen, known to be the causative agent of francisellosis in warm-water fish. Francisellosis outbreaks have increased in frequency among commercial aquaculture operations and have caused severe economic losses in every case reported. The lack of effective treatments for piscine francisellosis led us to investigate the potential efficacy of florfenicol for inhibition of F. asiatica in vitro and as an oral therapeutic agent in vivo. The MIC of florfenicol for F. asiatica, as determined by the broth dilution method, was 2 g/ml, which indicates its potential efficacy as a therapeutic agent for treatment of francisellosis. The intracellular susceptibility of the bacterium to florfenicol in tilapia head kidney-derived macrophages (THKDM) was also investigated. Addition of florfenicol to the medium at 10 g/ml was sufficient to significantly reduce bacterial loads in the THKDM in vitro. Cytotoxicity assays done in infected THKDM also demonstrated drug efficacy in vivo, as determined by lactate dehydrogenase (LDH) release. Levels of LDH released from infected THKDM were significantly lower in macrophages treated with florfenicol (P < 0.001) than in untreated cells. In medicated-feed trials, fish were fed 15 mg of florfenicol/kg of fish body weight for 10 days, and the feeding was initiated at either 1, 3, or 6 days postchallenge. Immersion challenges resulted in 30% mean percent survival in nontreated fish, and fish receiving medicated feed administered at 1 and 3 days postinfection showed higher mean percent survival (100% and 86.7%, respectively). A significant decrease (P < 0.001) in bacterial numbers (number of CFU/g of spleen tissue) was observed in treated groups compared to nontreated infected fish at both 1 and 3 days postchallenge. There were no differences in bacterial burden in the spleens between fish treated 6 days postchallenge and untreated controls. In conclusion, if florfenicol is administered during early stages of infection, it has the potential for effectively treating piscine francisellosis, including the capacity for intracellular penetration and bacterial clearance.

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

“…A similar deactivation may be the reason for the lack of activity of flumequine seen in our study despite a favourable MIC and high intracellular concentration. Our findings that flumequine had no effect on intracellular replication are supported by the findings of Soto et al (2010Soto et al ( , 2013, who showed that treatment with florfenicol reduced mortality and prevented lethal disease caused by Francisella noatunensis subsp. orientalis in Nile tilapia Oreochromis niloticus L. but did not eradicate the bacteria.…”

“…Only a reduction in the number of bacteria in infected HK macrophages using 10 or 100 µg florfenicol per ml of cell culture medium were described. The methods used by Soto et al (2010Soto et al ( , 2013 differed from ours, and the intracellular concentrations in macrophages were determined using indirect methods. The advantage of the methods used in the present study is that there is a high accuracy in the measurements and the concentration of drugs and replication of the bacterium are performed directly on the target cells where F. noatunensis subsp.…”

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

“…However, according to Bowser et al (2009) resulted in plasma and tissue concentrations in tilapia similar to or higher than those found in cod by Samuelsen & Bergh (2004). Furthermore, a medication regime of 15 mg kg −1 daily for 10 consecutive days showed high efficacy in the treatment of a Francisella asiatica infection in tilapia if the drug was administered during the early stages of infection (1 to 3 d post challenge) (Soto et al 2010). The MIC value for the bacterial isolate was 2.0 µg ml ) in combination (Romet ® 30) showed plasma C max values of 22.1 µg ml −1 (sulphadimethoxine) and 1.6 µg ml −1 (ormethoprim) after 72 and 9 h, respectively.…”

Antibiotics in aquaculture: reducing their use and maintaining their efficacy

“…In fi nfi sh, salmonid rickettsial septicemia (SRS) caused by the intracellular bacterium Piscirickettsia salmonis is considered the main cause of mortality in farmed coho salmon in Chile (Bravo and Midtlyng, 2007). Francisella piscicida, has been reported as an emerging problem in cold water cod farming (Ottem et al, 2008) and Francisella asiatica has been associated with major epizootics in warm water fi nfi sh (Soto et al, 2010). In shrimp both necrotising hepatopancreatitis (NHP) a bacterial disease associated with infection by a rickettsial-like organism (Cuéllar-Anjel et al, 2010) and the emerging disease, streptococcosis (Hasson et al, 2009), both involve bacteria with an intracellular life stage.…”