Antimicrobial Susceptibility of Flavobacterium psychrophilum from Chilean Salmon Farms and Their Epidemiological Cut-Off Values Using Agar Dilution and Disk Diffusion Methods

Abstract

Flavobacterium psychrophilum is the most important bacterial pathogen for freshwater farmed salmonids in Chile. The aims of this study were to determine the susceptibility to antimicrobials used in fish farming of Chilean isolates and to calculate their epidemiological cut-off (COWT) values. A number of 125 Chilean isolates of F. psychrophilum were isolated from reared salmonids presenting clinical symptoms indicative of flavobacteriosis and their identities were confirmed by 16S rRNA polymerase chain reaction. Susceptibility to antibacterials was tested on diluted Mueller-Hinton by using an agar dilution MIC method and a disk diffusion method. The COWT values calculated by Normalized Resistance Interpretation (NRI) analysis allow isolates to be categorized either as wild-type fully susceptible (WT) or as manifesting reduced susceptibility (NWT). When MIC data was used, NRI analysis calculated a COWT of ≤0.125, ≤2, and ≤0.5 μg mL-1 for amoxicillin, florfenicol, and oxytetracycline, respectively. For the quinolones, the COWT were ≤1, ≤0.5, and ≤0.125 μg mL-1 for oxolinic acid, flumequine, and enrofloxacin, respectively. The disk diffusion data sets obtained in this work were extremely diverse and were spread over a wide range. For the quinolones there was a close agreement between the frequencies of NWT isolates calculated using MIC and disk data. For oxolinic acid, flumequine, and enrofloxacin the frequencies were 45, 39, and 38% using MIC data, and 42, 41, and 44%, when disk data were used. There was less agreement with the other antimicrobials, because NWT frequencies obtained using MIC and disk data, respectively, were 24 and 10% for amoxicillin, 8 and 2% for florfenicol, and 70 and 64% for oxytetracycline. Considering that the MIC data was more precise than the disk diffusion data, MIC determination would be the preferred method for susceptibility testing for this species and the NWT frequencies derived from the MIC data sets should be considered as the more authoritative. Despite the high frequency of isolates showing full susceptibility to florfenicol, the significant frequencies of isolates exhibiting reduced susceptibility to oxytetracycline and quinolones may result in treatment failures when these agents are used.

Characterization of Mechanisms Lowering Susceptibility to Flumequine among Bacteria Isolated from Chilean Salmonid Farms

“…The Chilean salmon farming industry is commonly affected by an important number of bacterial diseases causing high mortalities, consequently prompting the necessity of using high amounts of antibiotics to ensure salmon production [1,2]. However, it is well known that intensive use of antibiotics is responsible for therapy failures, probably due to a reduction in the susceptibility to antibacterials of the bacterial pathogens associated with this industry [2][3][4][5][6][7].…”

Antimicrobial susceptibility of Flavobacterium psychrophilum isolates from the United Kingdom

“…The global situation with respect to FFN susceptibility of F. psychrophilum can be assessed from a number of studies that have been published. Studies that have employed standard MIC testing protocols and that generated data of adequate precision have been reported from Denmark and the UK (Smith et al., ), Chile (Miranda et al., ) and USA (Van Vliet et al., ). Studies of the antimicrobial susceptibility of Danish (Bruun et al., ) and French (Michel et al., ) F. psychrophilum isolates that used non‐standardized agar dilution protocols have also been published.…”

“…The global situation with respect to FFN susceptibility of F. psychrophilum can be assessed from a number of studies that have been published. Studies that have employed standard MIC testing protocols and that generated data of adequate precision have been reported from Denmark and the UK , Chile (Miranda et al, 2016) and USA (Van Vliet et al, 2017). Studies of the antimicrobial susceptibility of Danish and French (Michel et al, 2003) F. psychrophilum isolates that used non-standar- These data would indicate, rather surprisingly, that the selective pressure that must have resulted from the use of this agent during the 25 or more years since the introduction of the antibiotic to aquaculture (Smith, 2008) has not yet resulted in any significant emergence of strains of F. psychrophilum with reduced susceptibility to this agent.…”

“…are licensed for use in aquaculture by the UK Veterinary Medicines Directorate (VMD; http://www.vmd.defra.gov.uk/). Several studies have examined the antimicrobial susceptibility of F. psychrophilum isolated from the USA (Pacha, 1968;Soule, Lafrentz, Cain, Lapatra, & Call, 2005;Van Vliet, Loch, Smith, & Faisal, 2017), the UK (Rangdale, Richards, & Alderman, 1997;Smith et al, 2016;Verner-Jeffreys & Taylor, 2015), Denmark (Bruun, Madsen, & Dalsgaard, 2003;Bruun, Schmidt, Madsen, & Dalsgaard, 2000;Lorenzen, Dalsgaards, & Bernardet, 1997;Schmidt, Bruun, Dalsgaard, Pedersen, & Larsen, 2000;Smith et al, 2016), France (Michel, Kerouault, & Martin, 2003), Japan (Izumi & Aranishi, 2004), Turkey (Boyacioglu & Akar, 2012;Boyacio glu et al, 2015;Durmaz, Onuk, & C ß iftci, 2012;Kum, Kirkan, Sekkin, Akar, & Boyacioglu, 2008), Canada (Hesami & Parkman, 2010), Spain (Del Cerro, M arquez, & Prieto, 2010), Norway (Nilsen et al, 2011), Chile (Henr ıquez-N uñez, Evrard, Kronvall, & Avendaño-Herrera, 2012;Miranda, Smith, Rojas, Contreras-Lynch, & Alonso Vega, 2016) and Finland (Sundell, Heinikainen, & Wiklund, 2013). However, differences in the medium and growth conditions used in these studies and variations in the interpretive criteria used make comparisons difficult.…”

Current Status of the Use of Antibiotics and the Antimicrobial Resistance in the Chilean Salmon Farms

“…Furthermore, 39 of the 40 isolates carried a single plasmid or combinations of two plasmids, but a relationship between plasmid and resistance could not be established. In a recent study, Miranda et al (2016) determined the susceptibility of 125 F. psychrophilum Chilean isolates to antimicrobials used in fish farming and calculated their CO WT values by using an agar dilution MIC method and a disk diffusion method. The data generated by the disk diffusion protocol used in this work were shown to have low precision, in agreement with Henríquez-Núñez et al (2012) , confirming that MIC determination would be the preferred method for susceptibility testing for this species.…”