Characterisation of atypical Aeromonas salmonicida isolates from Cleaner Fish in the United Kingdom

Abstract

Cleaner fish such as ballan wrasse (Labrus bergylta) and lumpfish (Cyclopterus lumpus) are currently used as a biological approach to controlling sea lice (Lepeophtheirus salmonis) in Atlantic salmon (Salmo salar) in the United Kingdom and elsewhere. However, these newly domesticated fish species are susceptible to infection by various bacterial pathogens, including atypical strains of A. salmonicida (aAs) that causes atypical furunculosis. Little is known of the diversity of aAs strains causing problems and no commercially licensed vaccine is presently available. Instead, multivalent autogenous vaccines (which include aAs strains) are used to control disease incidences, though outbreaks do still occur and these must be treated with antibiotics. For vaccines to be effective, the formulation must include isolates that confer protection against the prevalent circulating strains. Monitoring of the susceptibility of isolates to antibiotics is critical for these drugs to remain efficacious. Therefore, the aim of this thesis was to investigate the diversity of aAs strains causing disease problems in the UK and determine their antibiotic susceptibility. Seventy-seven aAs isolates collected from six different cleaner fish sites in the UK were characterised by phenotypic and genotypic methods. Two strain lineages were identified within the UK isolates by pulsed-field gel electrophoresis (PFGE), with a predominant group composed of ballan wrasse isolates and a minor cluster of mainly lumpsucker isolates. One pulsotype (B2) was detected at five UK sites and this accounted for 73% of isolates. The existence of two genetic groups was further supported by whole-genome sequencing (WGS) and virulence array protein gene typing of the A-layer (vapA). PFGE group B correlated with vapA type V isolates from ballan wrasse and PFGE group A with genotype vapA type VI typically from lumpsuckers. Additionally, the substrate, B-methyl-D-Glucoside from biolog assay differentiated between vapA type V and VI of aAs. Immuno-proteomic profiling of the aAs was performed by 1D and 2D SDS-PAGE to determine the protein profiles and the antigenic variation between the isolates. Six protein profiles (P1, P3-P6) were identified in the 77 aAs UK isolates. An association was noted between the isolate genotypes and the protein profiles, with five different protein profiles derived from pulsotype group B and vapA type V originating from wrasse. The remaining three related protein profiles originated from both cleaner fish species and the minor pulsotype group A and vapA type VI. Two key immuno-reactive proteins, enolase and vapA, were identified by MALDI-TOF-MS in SAIC-24 isolate. This will be useful in determining the function of aAs immune-related proteins and potential antigens for vaccine development. Antibiotic susceptibility of the aAs isolates was determined by disc diffusion assay and minimum inhibitory concentration (MIC) testing. The disc diffusion assay was of limited use in determining reduced susceptibility due to slow growth of the isolates and large zone sizes of growth inhibition. Conversely, provisional MIC cut-off values were successfully generated for seven antibiotics; florfenicol (FLO), oxytetracycline (OTC), enrofloxacin (ENR), flumequine (FLUQ), oxolinic acid (OXO), ormetoprim/sulphadimethoxine (ORS) and trimethoprim/sulphamethoxazole (TRS) by normalised resistance interpretation (NRI) analysis. The provisional MIC cut-off values of four antibiotics (FLO, OTC, OXO and ORS) were within the acceptable limits of precision and allowed for the setting of epidemiological cut-off values (ECV) of aAs strains and typical strains. There was evidence for reduced susceptibility to the antibiotics commonly used in the UK to treat atypical furunculosis, with 28 isolates (34%) having reduced susceptibility to multiple antibiotics. The prevalence of reduced susceptibility to the commonly used antibiotics, FLO, OTC and OXO increased between 2013 - 2019 Non-wild type (NWT) phenotypes were observed at all the UK farm sites, suggesting widespread presence of NWT aAs isolates within the UK. Gel-based plasmid profiling separated the aAs isolates into two main plasmid profiles that correlated with the vapA and PFGE genotype and host origin. Plasmid sequencing confirmed homology to typical A. salmonicida (tAs) plasmids associated with the type three secretion system (T3SS). A correlation was also found between acquired plasmids associated with reduced antibiotic susceptibility to OTC (pKp589-231, 2.9 kb) and extended spectrum beta lactamases (pAm08WL, 3.1 kb). Additionally, homology to a large conjugative plasmid pEIB202 (43.7 kb) encoding multidrug resistance determinants consistent with NWT phenotype of six aAs isolates was observed. Furthermore, WGS revealed chromosomal genes encoding resistance to erythromycin (ERY) and ampicillin (AMP) in all the 13 aAs genomes studied. In conclusion, this present study has provided insight into the diversity of aAs strains affecting cleaner fish in the UK and their susceptibility to antibiotics. These findings will assist in the prevention of atypical furunculosis and control strategies of cleaner fish management. Indeed, some of the isolates characterised herein were used in the development of a commercial whole-cell autogenous vaccine for use in Scotland that has shown to be successful in controlling outbreaks. In the absence of a licensed vaccine and selection of conserved antigenic targets, the antibiotic susceptibility patterns characterised in this present study will help inform on effective treatment regimes for aAs and assist to monitor the development of antibiotic resistance in UK aquaculture.