Time effect of experimental infection on Rainbow trout (Oncorhynchus mykiss) by immersion with Aeromonas salmonicida subsp. salmonicida.

Furunculosis caused by Aeromonas salmonicida subsp salmonicida (Ass) is a medically and economically important bacterial disease in salmonid farms that requires therapeutic measures to prevent and control the disease. Evaluation of the effectiveness of traditional measures such as antibiotics or vaccines usually requires infecting fish experimentally. The objective of this study is to develop a method of infectious challenge of large (250-g) Rainbow trout by immersion close to natural infection conditions. We compare mortality, morbidity and anti-Ass antibody production of Rainbow trout following different bathing times (2, 4, 8 and 24 h) at a final bacterial concentration of 106 CFU/mL. One hundred sixty fish divided in five groups corresponding to the 4 bathing times and the non-challenged group were studied. The 24 h contact duration resulted in the infection of all fish, with a mortality rate of 53.25%. The challenged fish developed acute infection with symptoms and lesions (inappetance, altering of swimming behaviour, presence of boils) similar to those observed in furunculosis, and produced antibodies against the bacterium at 4 weeks after challenging, in contrast with the non-challenged group.

No evidence for a relationship between farm or transformation process locations and antibiotic resistance patterns of Pseudomonas population associated with rainbow trout (Oncorhynchus mykiss).

AIMS: Study the relationship between antibiotic resistance patterns of Pseudomonas isolated from farmed rainbow trout fillets and farm or transformation process locations. METHODS AND RESULTS: Pseudomonas strains were isolated from rainbow trout sampled in two differently located farms and filleted in laboratory or in a processing factory. One hundred and twenty-five isolates were confirmed as belonging to Pseudomonas using CFC selective media, Gram staining, oxidase test and quantitative polymerase chain reaction methods. Fifty-one isolates from separate fish fillets were further identified using MALDI-TOF mass spectrometry, and the minimal inhibitory concentrations (MIC) of 11 antibiotics were also determined by microdilution method. Most of the isolates belonged to the Pseudomonas fluorescens group (94.1%), and no relationship was established between antibiotic resistance patterns and sampling locations (farms or filleting areas). Multiple resistance isolates with high MIC values (from 64 µg ml-1 to more than 1024 µg ml-1 ) were identified. CONCLUSIONS: Antibiotic resistance patterns found in Pseudomonas isolates were not influenced by farms or transformation process locations. Seven isolates were found highly resistant to four different antibiotic classes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study does not provide evidence of a relationship between farm or transformation process locations on antibiotic resistance patterns of Pseudomonas population.

Antimicrobial Susceptibility Profiles and Resistance Genes in Genus Aeromonas spp. Isolated from the Environment and Rainbow Trout of Two Fish Farms in France.

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, ß-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.

Evaluation of the efficacy of enrofloxacin in rainbow trout (Oncorhynchus mykiss) following experimental challenge with Yersinia ruckeri.

BACKGROUND: Use of enrofloxacin in trout farms is reported, especially for the treatment of yersiniosis, albeit various dosing regimens have been used. Therefore, optimal doses should be investigated. METHODS: Five groups of 15 fish were challenged with Y. ruckeri. Two days later, three groups received feed containing enrofloxacin (ENR) at 1, 2.5 and 5 mg/kg fish respectively, during 7 days; one group received a single intraperitoneal injection of ENR at 10 mg/kg; and one group was left untreated. On day 15, surviving fish were euthanized. RESULTS: All fish survived in the group treated by injection, compared to 53%, 60% and 40% of the fish treated with 1, 2.5 and 5 mg/kg oral ENR, respectively, and 53% in the infected untreated group. CONCLUSION: A single intraperitoneal injection of ENR at 10 mg/kg seems more relevant than repeated oral administrations. The ENR oral doses used in trout farms should be revised.

High Genetic Diversity in Flavobacterium psychrophilum Isolates from Healthy Rainbow Trout (Oncorhynchus mykiss) Farmed in the Same Watershed, Revealed by Two Typing Methods.

Flavobacterium psychrophilum affects salmonid health worldwide and causes 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 1 year. A total of 288 isolates were recovered from fish organs (gills and spleen) and eggs. Pulsed field gel electrophoresis revealed high genetic diversity. Multilocus sequence typing performed on a selection of 31 isolates provided congruent results, as follows: 18 sequence types (STs) were found, of which 13 were novel. The mean gene diversity (H = 0.8413) was much higher than that previously reported for this host species, although the sampling was restricted to a single watershed and 1 year. Seven isolates out of 31 were assigned to clonal complex ST10 (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 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. psychrophilumIMPORTANCE 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 isolates 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 clonal complex sequence type 10 (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 harbored by rainbow trout.

Antibiotic Resistance Genes and Bacterial Communities of Farmed Rainbow Trout Fillets (Oncorhynchus mykiss).

The rise of antibiotic resistance is not only a challenge for human and animal health treatments, but is also posing the risk of spreading among bacterial populations in foodstuffs. Farmed fish-related foodstuffs, the food of animal origin most consumed worldwide, are suspected to be a reservoir of antibiotic resistance genes and resistant bacterial hazards. However, scant research has been devoted to the possible sources of diversity in fresh fillet bacterial ecosystems (farm environment including rivers and practices, and factory environment). In this study bacterial communities and the antibiotic resistance genes of fresh rainbow trout fillet were described using amplicon sequencing of the V3-V4 region of the 16S rRNA gene and high-throughput qPCR assay. The antibiotic residues were quantified using liquid chromatography/mass spectrometry methods. A total of 56 fillets (composed of muscle and skin tissue) from fish raised on two farms on the same river were collected and processed under either factory or laboratory sterile filleting conditions. We observed a core-bacterial community profile on the fresh rainbow trout fillets, but the processing conditions of the fillets has a great influence on their mean bacterial load (3.38 ± 1.01 log CFU/g vs 2.29 ± 0.72 log CFU/g) and on the inter-individual diversity of the bacterial community. The bacterial communities were dominated by Gamma- and Alpha-proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The most prevalent genera were Pseudomonas, Escherichia-Shigella, Chryseobacterium, and Carnobacterium. Of the 73 antibiotic residues searched, only oxytetracycline residues were detected in 13/56 fillets, all below the European Union maximum residue limit (6.40-40.20 µg/kg). Of the 248 antibiotic resistance genes searched, 11 were found to be present in at least 20% of the fish population (tetracycline resistance genes tetM and tetV, ß-lactam resistance genes bla DHA and bla ACC, macrolide resistance gene mphA, vancomycin resistance genes vanTG and vanWG and multidrug-resistance genes mdtE, mexF, vgaB and msrA) at relatively low abundances calculated proportionally to the 16S rRNA gene.

Comparison of Stomaching versus Rinsing for Recovering Bacterial Communities from Rainbow Trout (Oncorhynchus mykiss) Fillets.

ABSTRACT: 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, such as 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 quantitative recovery and compatibility with end-point quantitative PCR (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 six noninoculated fresh fillets. Stomaching and mechanical rinsing allowed efficient and repeatable recovery of the bacterial communities from the 42 inoculated fillets. No significant differences in 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 proper PCR quantification of the inoculated B. thermosphacta. The mean recovered bacterial level of the fillets was approximately 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.

Mutations involved in the emergence of Yersinia ruckeri biotype 2 in France.

Yersina ruckeri is an enterobacteria responsible for Enteric redmouth disease (ERM), which causes significant economic losses in the aquaculture industry worldwide. Two biotypes have been described within Y. ruckeri: biotype 1 (BT1) and biotype 2 (BT2). Unlike BT1, BT2 is negative for motility and lipase secretion. The emergence of BT2 Y. ruckeri has been associated with disease outbreaks in vaccinated fish in several countries, notably France in the early 2000s. In this study, 15 BT2 strains (14 BT2 strains isolated in France and the BT2 reference strain EX5) were studied to compare the phenotypic characters of the BT1 and BT2 strains and to determine the genetic origin of the emergence of BT2 in France. BT1 bacteria are significantly longer in size than BT2 bacteria (a difference of 0.222 µm). The loss of motility of some French BT2 strains could be due to the loss of their ability to produce flagella caused by three mutations within the fliG, flhC and flgA genes. In the light of these results, the emergence of BT2 Yersinia ruckeri in France is discussed.

Genomic Characterization of Flavobacterium psychrophilum Serotypes and Development of a Multiplex PCR-Based Serotyping Scheme.

Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonids reared in freshwater worldwide. So far, serological diversity between isolates has been described but the underlying molecular factors remain unknown. By combining complete genome sequence analysis and the serotyping method proposed by Lorenzen and Olesen (1997) for a set of 34 strains, we identified key molecular determinants of the serotypes. This knowledge allowed us to develop a robust multiplex PCR-based serotyping scheme, which was applied to 244 bacterial isolates. The results revealed a striking association between PCR-serotype and fish host species and illustrate the use of this approach as a simple and cost-effective method for the determination of F. psychrophilum serogroups. PCR-based serotyping could be a useful tool in a range of applications such as disease surveillance, selection of salmonids for bacterial coldwater disease resistance and future vaccine formulation.

Pulsed-field gel electrophoresis and multi locus sequence typing for characterizing genotype variability of Yersinia ruckeri isolated from farmed fish in France.

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.

Yersinia ruckeri Biotypes 1 and 2 in France: presence and antibiotic susceptibility.

Yersinia ruckeri is the causative agent of yersiniosis, a disease reported in a number of fish species, especially rainbow trout. This study was undertaken to describe the phenotypes of Y. ruckeri on French rainbow trout farms. More than 100 isolates, collected during recent outbreaks on trout farms, were characterized by phenotypic tests, namely using biochemical tests of the API 20E system, serotyping, biotyping (tests for motility and lipase activity) and by describing the pattern of susceptibility to several antibiotics. The isolates showed a low phenotypic diversity with a prevalent serotype (O1) and API 20E profile 5 1(3)07 100. As in other European countries, Biotype 2 (BT2), which lacks both motility and secreted lipase activity, was found to be present in France. The emergence of 'French' BT2 was different than that observed for other European countries (Finland, Spain, Denmark and the UK). The antibiotic pattern was uniform for all isolates, regardless of the geographical area studied. The results indicate that no resistance has yet emerged, and the efficacy of the antibiotic generally used against yersiniosis in France, trimethoprim/sulfamethoxasol, is not compromised (minimum inhibitory concentrations [MIC] of between 0.016 and 0.128 µg ml-1). Enrofloxacin and doxycycline, not used as a first-line treatment in fish diseases, have reasonably good efficacies (with MICs ≤0.128 and 0.256, respectively).

Relative expression of genes involved in the resistance/sensitivity of Enterococcus faecalis JH2-2 to recombinant divercin RV41.

The relative expression of mptABCD operon, glpQ, pde, rpoN, mptR and gap-1 was studied by reverse transcription combined with the real time polymerase chain reaction to understand the role of each gene in resistance/sensitivity of Enterococcus faecalis to class IIa bacteriocins such as recombinant divercin V41 (DvnRV41). Comparative critical threshold methods in presence or absence of DvnRV41 were then used to determine the level of expression of each gene cited above. In the presence of DvnRV41, the rpoN and glpQ genes were down-regulated, mptR, mptC, gap-1 and pde genes were up-regulated, whilst expression of mptB and mptD genes remained unmodified.

Production of recombinant bacteriocin divercin V41 by high cell density Escherichia coli batch and fed-batch cultures.

To increase the yield of heterologous production of the class II bacteriocin DvnRV41 with Escherichia coli Origami (DE3) (pLysS/pCR03), induction of bacteriocin gene expression was optimized by varying the inducer isopropyl beta-D-thiogalactopyranoside (IPTG) concentration (0-2 mM), and controlled batch and fed-batch cultures were tested on a 2-L scale. A concentration of 0.5 mM IPTG was found to be optimal for cell growth and bacteriocin production. Shake flask cultivation of E. coli Origami (DE3) (pLysS/pCR03) gave biomass and bacteriocin yields of 1.54 +/- 0.06 g cdw/l and 18 +/- 1 mg DvnRV41/l, respectively. Biomass (2.70 +/- 0.06 and 6.8 +/- 0.6 g cdw/l, respectively) and bacteriocin yields (30 and 74 mg DvnRV41 per liter, respectively) were both increased with batch and fed-batch compared to shake flask cultures. Bacteriocin yields reported in this study are among the highest published for other heterologous expression systems in shake flasks.

Identification of new genes associated with intermediate resistance of Enterococcus faecalis to divercin V41, a pediocin-like bacteriocin.

It has been suggested that resistance to class IIa bacteriocins occurs at either a low or a high level. In listerial strains, low-level resistance (2-4-fold) to class IIa bacteriocins is attributed to alterations in membrane lipid composition. In Listeria monocytogenes and Enterococcus faecalis, high-level resistance (1000-fold) correlates with inactivation of the mptACD operon, which encodes the EII(Man)(t) mannose permease of the phosphotransferase system (PTS). Previous studies reported that in L. monocytogenes, high-level resistance involved the sigma(54) factor and the ManR activator. In this investigation, three genes associated with the resistance of Ent. faecalis JH2-2 to divercin V41, a pediocin-like bacteriocin from Carnobacterium divergens V41, were clearly identified by screening an insertional mutant library of Ent. faecalis JH2-2. These genes correspond to the well-known rpoN gene, which encodes sigma(54) factor, and to genes encoding a glycerophosphoryl diester phosphodiesterase (GlpQ) and a protein with a putative phosphodiesterase function (PDE). Resistance of the three mutants defective in the aforementioned genes appeared to be graduated: the rpoN mutant was more resistant than the glpQ mutant, which was more resistant than the pde mutant. Moreover, this resistance was specific to class IIa bacteriocins.