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Research Project: Integrated Research to Improve On-Farm Animal Health in Salmonid Aquaculture

Location: Cool and Cold Water Aquaculture Research

Title: Detection of the florfenicol resistance gene floR in Chryseobacterium isolates from rainbow trout. Exception to the general rule?

Author
item Verner-jeffreys, David - Centre For Environment, Fisheries And Aquaculture Science (CEFAS)
item Brazier, Thomas - University Of Exeter
item Perez, Ramon - University Of Stirling
item Ryder, David - Centre For Environment, Fisheries And Aquaculture Science (CEFAS)
item Hoare, Rowena - University Of Stirling
item Welch, Timothy - Tim
item Ngao, Thao - University Of Stirling
item Card, Roderick - Animal & Plant Health Agency Apha
item Mclaren, Nikki - Animal & Plant Health Agency Apha
item Ellis, Richard - Animal & Plant Health Agency Apha
item Rowe, William - University Of Cambridge
item Bartle, Kerry - University Of Stirling

Submitted to: FEMS Microbiology Ecology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/8/2017
Publication Date: 2/11/2017
Citation: Verner-Jeffreys, D., Brazier, T., Perez, R.Y., Ryder, D., Hoare, R., Welch, T.J., Ngao, T., Card, R., Mclaren, N., Ellis, R., Rowe, W., Bartle, K. 2017. Detection of the florfenicol resistance gene floR in Chryseobacterium isolates from rainbow trout. Exception to the general rule? FEMS Microbiology Ecology. 93(4):fix015. doi:10.1093/femsec/fix015.

Interpretive Summary: Bacteria of the family Flavobacteriaceae are important members of freshwater aquatic ecosystems and include several pathogens. For the fish pathogenic members of this family, antimicrobial therapy is critical for controlling disease and reducing losses yet little is known regarding the capacity of these bacteria to obtain and express antimicrobial resistance genes. In this study, a large collection of Flavobacteriaceae isolated from fish were analyzed for antimicrobial resistance and antimicrobial resistance genes. We demonstrate the presence of an integrative Conjugative Element (ICE) containing the resistance gene floR in two highly florfenicol resistant Chryseobacterium isolates, as well as a range of other putative resistance genes. To our knowledge, this is the first demonstration of the presence of an ICE containing clinically important resistance genes in a pathogen from this major group of aquatic bacteria. These results establish the potential for the acquisition and phenotypic expression of clinically important antimicrobial resistance genes among pathogenic members of the Flavobacteriaceae.

Technical Abstract: Bacteria from the family Flavobacteriaceae include a number of pathogens of fish and other animals. An examination of 78 diverse Flavobacteriaceae isolates recovered from diseased salmonid fish revealed that many of these organisms were resistant to a range of antimicrobials. However, there is limited information on the actual disease risks such organisms pose, or on their antibiotic resistance mechanisms. Thirty-five Flavobacteriaceae isolates that could grow in greater than 2 mg/l florfenicol in vitro were examined for the presence of resistance genes by microarray. With the exception of two Chryseobacterium spp. isolates that were positive for floR, no other well-characterized clinical resistance genes were identified by microarray in any of the isolates. Whole genome sequence analysis revealed the presence, in both the floR positive isolates, of a likely Integrative Conjugative Element (ICE) that encoded for a number of putative AMR genes. These included floR, a tetX tetracycline resistance gene, a streptothricin resistance protein and a chloramphenicol acetyl GC transferase. Other resistance genes were also identified, including a putative Extended Spectrum Beta lactamase and multidrug efflux pump-encoding genes. Follow up in silico analysis of 377 published Flavobacteriaceae isolates from a range of sources, confirmed that well-characterized resistance gene cassettes could not be readily identified in bacteria from this group. The limited exception was floR, that was also identified in a plasmid associated with an avian pathogen, Riemerella anatipestifer. Efflux pump-mediated increased tolerance to a range of antimicrobials, including oxolinic acid, chloramphenicol and enrofloxacin, was confirmed in both isolates using an efflux pump inhibitor PAßN disc diffusion assay. The two isolates characterized in detail possessed a number of putative virulence factors, including the ability to grow in iron restricted environments, production of siderophores, hemolysins and other exoproteases. Mortalities were induced in rainbow trout injected with high doses of the two Chryseobacterium spp. isolates but not in Atlantic salmon. In general, results support the suggestion that, despite the discovery of an ICE carrying floR, resistance to antimicrobials in this group (Flavobacteriaceae) is mostly mediated via other mechanisms than the horizontally acquired resistance genes more normally associated with Gram negative bacterial pathogens.