First Isolation, Characterisation of Chryseobacterium Shigense from Rainbow Trout

Background

Members the genus Chryseobacterium are widely distributed microorganisms that can be recovered from a wide variety of environments, such as fresh water, sewage and wastewater, soil or food sources, such as milk, poultry and meat and dairy products. Some species of Chryseobacterium have been involved in human infections, acting as sporadic but severe opportunistic nosocomial pathogens.

In veterinary medicine, chryseobacteria are not relevant pathogens for domestic animals, but they are widely distributed in aquatic environments and fish farms. Until recently members of the genus Chryseobacterium were not commonly associated with fish infections. However, there has been an increase in the frequency of clinical cases in which Chryseobacterium sp. strains have been isolated from different fish species. Thus, Chryseobacterium balustinum, Chryseobacterium scophtalmum and Chryseobacterium joostei have been isolated from diseased fish.

More recently, Chryseobacterium piscicola has been reported to produce mortalities in farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) in Chile and Finland, Chryseobacterium arothri was isolated from the kidneys of the pufferfish Arothron hispidus in Hawaii and Chryseobacterium chaponense from diseased farmed Atlantic salmon in Chile.

In fact, some Chryseobacterium species are considered potentially emerging pathogens in fish. However, many chryseobacteria isolated from diseased fish are usually identified only at the genus level due to the difficulty of their correct identification by phenotypically based laboratory methods alone, which limits the knowledge of the diversity of species associated with fish disease.

Results

All isolates gave shiny, round, yellow-pigmented colonies on Anacker and Ordal agar, a characteristic that lead to the presumptive diagnosis of infection by F. psychrophilum, but none of the isolates gave the expected amplicon product of 1,089 bp, specific of F. psychrophilum. Moreover, cells of trout isolates were straight short Gram negative rods after Gram staining. Comparative analysis of the 16S rRNA gene sequences revealed that the isolates shared 99.8-100 % sequence similarity between each other, thus demonstrating their high phylogenetic relatedness, 99.2-99.8 %, with the type strain of C. shigense and only 81.8-81.9 % with F. psychrophilum NCIMB 1947 T (GenBank accession nD12670).

The 16S rRNA gene sequences of the isolates included in this study have been deposited in GenBank under the accession numbers indicated in Figure 1.

Figure 1 Phylogenetic relationships of the clinically trout isolates and close related species in the genus Chryseobacterium inferred using the neighbor-joining method with 16S rRNA gene sequences. Bootstrap values (expressed as a percentage of 1000 replications) >50 % are given at the branching points. Leeuwenhoekiella marinoflava ATCC 19326 T (M58770) was used as outgroup.

Bar, 1 % sequence divergence Phenotypically all trout isolates were catalase and oxidase positive, non-motile, grew on nutrient agar with yellow and shiny colonies but not on MacConkey agar, produced flexirubin-type pigment, were able to grow at 530 C but not at 37 C, and hydrolysed starch, casein and gelatin. With the API 20NE system they exhibited homogeneous biochemical characteristics displaying the numerical profiles 3452205, while C. shigense CCUG 61059 T gave the numerical profile 2456204. With the APY ZYM strips, the trout isolates, as well as the type strain of C. shigense CCUG 61059 T, expressed activity for alkaline phosphatase, leucine arylamidase, trypsin, acid phosphatase and naphthol-AS-BIphophohydrolase but not for esterase C4, lipase C14, cystine arylamidase, a-chymotrypsin, a- galactosidase, -galactosidase, -glucuronidase, a-glucosidase, N-acetyl--glucosaminidase, a-mannosidase and a-fucosidase. Clinical isolates of C. shigense expressed activity for valine arilamidase and not for ester lipase C8 and -glucusidase, while C. shigense CCUG 61059 T gave opposite results for these tests.

After genetic characterization by random amplified polymorphic DNA, both oligonucleotides generated reproducible patterns, but an appropriate number on bands were produced with oligonucleotide P2. The seven C. shigense trout isolates showed undistinguishable RAPD fingerprints with amplifications bands ranging from 600 to 2500 bp, indicating genetic homogeneity among them. On the other hand, the strain CCUG 61059 T yielded a completely different fingerprint.

Conclusions

In this work we describe by first time the recovery of C. shigense from clinical specimens in trout, showing that it can also occur in a very different habitat to fresh lactic acid beverage where it was initially isolated.

Further Reading You can view the full report by clicking here.