Yersinia ruckeri is a pathogen that has an impact on aquaculture worldwide. The disease caused by this bacterial species, yersiniosis or redmouth disease, generates substantial economic losses due to the associated mortality and veterinary costs. For predicting outbreaks and improving control strategies, it is important to characterize the population structure of the bacteria. The phenotypic and genetic homogeneities described previously indicate a clonal population structure as observed in other fish bacteria. In this study, the pulsed-field gel electrophoresis (PFGE) and multi locus sequence typing (MLST) methods were used to describe a population of isolates from outbreaks on French fish farms. For the PFGE analysis, two enzymes (NotI and AscI) were used separately and together. Results from combining the enzymes showed the great homogeneity of the outbreak population with a similarity > 80.0% but a high variability within the cluster (cut-off value = 80.0%) with a total of 43 pulsotypes described and an index of diversity = 0.93. The dominant pulsotypes described with NotI (PtN4 and PtN7) have already been described in other European countries (Finland, Germany, Denmark, Spain and Italy). The MLST approach showed two dominant sequence types (ST31 and ST36), an epidemic structure of the French Y. ruckeri population and a preferentially clonal evolution for rainbow trout isolates. Our results point to multiple types of selection pressure on the Y. ruckeri population attributable to geographical origin, ecological niche specialization and movements of farmed fish.

This study presents the occurrence and abundance of Aeromonas antibiotic-resistant bacteria (ARB) and genes (ARGs) isolated from water, biofilm and fish in two commercial trout farms before and one week after flumequine treatment. Wild (WT) and non-wild (NWT) strains were determined for quinolones (flumequine, oxolinic acid and enrofloxacin), oxytetracycline (OXY), florfenicol (FFN), trimethoprim-sulfamethoxazole (TMP) and colistin (COL), and pMAR (presumptive multi-resistant) strains were classified. Forty-four ARGs for the mentioned antibiotics, β-lactams and multi-resistance were quantified for 211 isolates. BlaSHV-01, mexF and tetE were the dominant ARGs. A greater occurrence and abundance of tetA2, sul3, floR1, blaSHV-01 and mexF were observed for NWT compared to WT. The occurrence of pMAR and NWT Aeromonas for quinolones, OXY, FFN, TMP, COL and ARGs depended on the Aeromonas origin, antibiotic use and the presence of upstream activities. Our results revealed the impact of a flumequine treatment on Aeromonas present on a fish farm through an increase in NWT and pMAR strains. The link between fish and their environment was shown by the detection of identical ARB and ARGs in the two types of samples. There appears to be a high risk of resistance genes developing and spreading in aquatic environments.

Flavobacterium psychrophilum affects salmonid health worldwide and causes important economic losses. The genetic diversity of the pathogen must be considered to develop control methods. However, previous studies have reported both high and low levels of genetic diversity. The present longitudinal study aimed at assessing the genetic diversity of F. psychrophilum at a small temporal and geographic scale. Four farms located on the same watershed in France were studied. Rainbow trout (Oncorhynchus mykiss) batches were monitored and apparently healthy individuals were sampled over one year. A total of 288 isolates were recovered from fish organs (gills, spleen) and eggs. Pulse Field Gel Electrophoresis revealed high genetic diversity. Multi Locus Sequence Typing performed on a selection of 31 isolates provided congruent results: 18 STs were found, 13 of which were novel. The mean gene diversity (H=0.8413) was much higher than previously reported for this host species, although the sampling was restricted to a single watershed and one year. Seven isolates out of 31 were assigned to CC-ST10, which is the predominant clonal complex in the main salmonid production areas. A split decomposition tree reflected a panmictic population. This finding is important for aquaculture veterinarians in their diagnostic procedure, as the choice of the adequate antibiotic treatment is conditioned by the correct identification of the causative agent. Furthermore, this study expands our knowledge on genetic diversity required for the development of an effective vaccine against F. psychrophilum. Importance The bacterium Flavobacterium psychrophilum is a serious pathogen in many fish species, especially salmonids, that is responsible for considerable economic losses worldwide. In order to treat infections and to develop vaccines, the genetic diversity of this bacterium needs to be known. We assessed the genetic diversity of F. psychrophilum isolated from apparently healthy rainbow trout raised in several fish farms in the same watershed in France. Two different genotyping methods revealed high diversity. The majority of isolates were unrelated to CC-ST10, the clonal complex that is predominant worldwide and associated with disease in rainbow trout. In addition, we found 13 novel sequence types. These results suggest that a diverse subpopulation of F. psychrophilum may be harboured by rainbow trout.

The use of high-throughput methods allows a better characterization of food-related bacterial communities. However, such methods require large amounts of high quality bacterial DNA, which may be a challenge when dealing with a complex matrix that has a low concentration of bacteria like fresh fish fillets. Therefore, the choice of method used to recover bacteria from a food matrix in a cost-effective way is critical, yet little information is available on the performance of commonly used methods. We assessed the recovery capacity of two such methods: stomaching and mechanical rinsing. The efficiency of the methods was evaluated through the quantitative recovery and compatibility with end-point qPCR. Fresh rainbow trout ( Oncorhynchus mykiss ) fillets were inoculated with a bacterial marker, Brochothrix thermosphacta , at different concentrations (7.52 to 1.52 log CFU/g). The fillets were processed by one of the two methods and the recovery of the marker in the suspensions was assessed by plate counting and qPCR targeting B. thermosphacta - rpoC . The same analyses were performed on 6 non-inoculated fresh fillets. Stomaching and mechanical rinsing allowed an efficient and repeatable recovery of the bacterial communities from the 42 inoculated fillets. No significant differences of Recovery Ratios were observed between the marker enumerated in the inoculation suspensions and in the corresponding recovery suspensions after rinsing and stomaching. However, the stomaching method allowed too many particles to pass through the filters bag, making necessary a limiting supplementary filtration step. As a consequence, only the rinsing recovery method allowed a proper PCR quantification of the inoculated B. thermosphacta. The mean recovered bacterial level of the fillets was around 3 log CFU/g. It seems more relevant and cost-effective to recover the endogenous bacterial microbiota of a fish fillet structure using the rinsing method rather than the stomaching method.