Pathological analysis and antimicrobial susceptibility of Chryseobacterium balustinum RTFCP 298 isolated from diseased rainbow trout, Oncorhynchus mykiss

In this study, six isolates of Chryseobacterium balustinum were characterized from diseased rainbow trout fingerlings. The virulence characteristics, pathogenicity, and antimicrobial susceptibility pattern of these isolates were investigated. The bacterium showed positive results for catalase, cytochrome oxidase, and aesculin hydrolysis, while negative results were obtained for DNase, gelatinase, methyl red, Voges-Proskauer's reaction, Simon citrate, Hydrogen sulphide, and starch hydrolysis. Amino acid metabolism analysis revealed the inability to metabolize arginine, lysine, and ornithine decarboxylase. Molecular characterization (16S rRNA) and phylogenetic analysis revealed the test isolates as C. balustinum, closely related to strain WLT (99.85% similarity) and C. balustinum P-27 (99.77%). Virulence assay indicated haemolytic activity and biofilm formation by the test bacterium. The challenge test confirmed moderate pathogenicity in rainbow trout and established Koch's postulates. The clinical manifestations of infection included fin erosion, eye and body surface haemorrhage, exophthalmia, and organ liquefaction. The minimum inhibitory concentrations of various antimicrobials ranged from 1 to > 256 µg mL−1. The novel synthetic antimicrobial peptides exhibited MICs of 8 to > 256 µg mL−1, suggesting a potential control method. These findings suggest that C. balustinum is an opportunistic pathogen with moderate pathogenicity in rainbow trout. Further research on the host–pathogen relationship is necessary to understand virulence characteristics and pathogenicity in aquaculture.

Isolation and growth conditions for Chryseobacterium balustinum.The remaining 10 live specimens were processed for the bacteriological investigation.Swab samples were collected from caudal peduncle regions of the infected trout specimens.For the collection of the tissue samples (liver and kidney), the trout juveniles were euthanized at 150 mg L −1 of Ethyl-3-aminobenzoate methanesulphonate (MS 222) to bring them to humane end points 35,36 .Then specimens were disinfected with 70% ethanol before collecting the tissue samples for isolation of pathogenic bacteria as etiological agents for the pathological conditions and infections recorded in the study.The tissue and swab samples were processed aseptically on different media; Shieh agar (supplemented with 0.5 µg mL −1 tobramycin; pH 7.2) and Hsu-Shotts agar (supplemented with 4.0 µg mL −1 neomycin sulphate; pH 7.2).The plates were incubated at 15 °C for 1 week and at 25 °C for 24-48 h.The plates incubated at 25 °C showed growth of yellow slimy pigmented colonies, whereas the plates incubated at 15 °C did not show the development of any yellow slimy pigmented colonies.The yellowish and slime-dominant colonies (n = 6) were selected randomly and screened for the 30% KOH test.The isolates were further sub-cultured on Tryptic Soya agar (TSA) at 25 °C for 24-48 h and preserved in 20% glycerol at − 20 °C for long-term storage.
Biochemical analysis.The biochemical identification of the isolates was carried out as per the standard methods published earlier [37][38][39] .Following the manufacturer's protocol, the Gramme reaction of bacterial isolates was determined using a Gram staining kit (HI MEDIA K001-1KT).The isolates were tested for the basic biochemical reactions such as cytochrome oxidase, catalase, Simon citrate, methyl-red (MR), Voges-Proskauer's reaction (VP), Hydrogen sulphide, starch and aesculin hydrolysis, DNase and gelatinase, urease, nitrate, indole, tyrosine, arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase.They were also tested for the acid production from sugars; mannitol, raffinose, mannose, sucrose xylose, salicin, trehalose, inositol, glucose, arabinose and lactose for biochemical analysis.In brief, a piece of sterile filter paper impregnated with freshly prepared oxidase reagent was placed in a petri dish.A streak of test bacterial culture was smeared across the filter paper with the help of a sterile platinum loop cytochrome oxidase test.For the catalase test, the colony of the test bacterium was smeared onto a clean glass slide.Then, a drop of 30% H 2 O 2 was placed on the smear.The ability of the test bacteria to utilize citrate as a sole source of carbon was tested by growing the culture on Simmons citrate agar slant for 24-48 h at 28 °C.For the MR test, a few drops of methyl red reagent were added to the 48-72 h grown culture in MR-VP broth.In the case of the VP test, VP reagents (solution A-0.6 mL and solution B-0.2 mL) were added to the 48-72 h grown bacterial culture in the MR-VP broth.Aseptically, a loopful of the bacterial culture was transferred to the Tryptic soy broth and incubated for 24-48 h at 28 °C.Once the bacterial growth was observed in the broth, ferrous sulfate solution as Hydrogen sulphide indicator was added to the broth grown with bacterial culture in Hydrogen sulphide test.Starch hydrolysis test was carried out by spot inoculating young test bacterial culture onto starch agar plate.After 18-24 h of incubation at 28 °C, the plates were flooded with Lugol's iodine solution.In the case of the aesculin test, bacterial cells were inoculated into aesculin broth and incubated at 28 °C for 24-48 h.A loop of bacterial culture was streaked as a single line on DNase agar plates.The plates were incubated at 28 °C for 2-4 days.After the incubation period, the plates were flooded with 1% HCl.For the gelatinase test, the gelatin plate was spot inoculated with bacteria culture and incubated at 28 °C for 24-48 h.For the urease test, the test bacteria was allowed to grow on urea agar slant at 28 °C for 6-24 h and observed for 6 days.The nitrate test was conducted by stab or streak inoculation of the test culture onto nitrate agar tubes followed by incubating the tubes at 28 °C for 24-48 h.Then nitrate solution was added to the tube.The indole test was conducted by adding heavy inoculum of test culture into tryptone broth in tubes.The tubes were incubated for a period of 48 h at 28 °C.Then 6-7 drops of Kovacs reagent were added to the tubes.In the tyrosine test, a loopful test culture was inoculated into tyrosine broth and the culture was incubated at 28 °C for 24-48 h.After observing the bacterial growth, a Durham tube was inserted into the tyrosine broth and allowed to incubate at 28 °C for an additional 24-48 h.The ability of test bacterial culture to produce arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase was tested by inoculating test culture in the medium containing these 3 amino acids in separate tubes.The tubes were overlaid with sterile liquid paraffin and incubated at 28 °C.Control tubes with the basal medium were also incubated for each test.The tubes were observed for a week.The bacterial abilities to produce acid from sugars; mannitol, raffinose, mannose, sucrose xylose, salicin, trehalose, inositol, glucose, arabinose and lactose were tested by supplementing the basal medium with individual sugar in separate tubes.Then tubes were inoculated with bacterial culture and incubated at 28 °C.

Molecular characterization (16S rRNA gene) and phylogenetic analysis.
Promega DNA isolation Kit was used to process the colonies that had grown on the agar plate in order to isolate the DNA and to analyse 16S rRNA gene by PCR.For PCR amplification universal primer UFF2 (5′-AGA GTT TGA TCC TGG CTC AG-3′) and URF2 (5′-ACG GGC GGT GTG TTC-3′) were used targeting the 16S rRNA gene.In the PCR reaction, the amplification of amplicons with a size of 1400 base pairs was achieved.The PCR product was sent for nucleotide sequencing by Sanger's sequencing method with the help of an ABI Big Dye Terminator Cycle Sequencing kit v3.1 and ABI 3730 XL (Applied Biosystems).The partial forward and reverse sequences of the 16S rRNA gene were assembled (CLC Genomics Workbench software, version 11.0.1) and the similarity search was conducted with the other Chryseobacterium spp., using BLASTn (NCBI, Bethesda, MD, USA).The 16S rRNA genes of all the isolates (n = 6) were found 100% identical.The test isolate was designated as laboratory strain no RTFCP 298 and submitted to NCBI as C. balustinum, RTFCP 298 (OP 604186) as a representative.The closely related sequences of the C. balustinum RTFCP 298 and other species under the genus were retrieved from the NCBI along with type strains and some Indian isolates.The multiple alignments were done by CLUSTAL W 40 .
The aligned sequences were used to infer evolutionary history according to the neighbour-joining algorithm 41 by Molecular Evolutionary Genetic Analysis (MEGA X) 42 .The Maximum Likelihood Method and Kimura 2-Parameter Model were used to infer the evolutionary history, and the tree with the highest log likelihood (− 6459.57) is presented, along with the percentage of associated taxa clustered together.To obtain the initial tree for the heuristic search, the Neighbour-Joining method was applied to a pair-wise distance matrix estimated using the Maximum Composite Likelihood (MCL) approach.The tree was drawn to scale, with branch lengths measured in substitutions per site.The analysis involved 22 nucleotide sequences, including 1st, 2nd, 3rd, and non-coding sites, with a total of 1548 positions in the final dataset.Acinetobacter calcoaceticus strain NCCB 22016 (NR042387) was included in the phylogenetic analysis as an out-group. 43and haemolytic activities were performed for phenotypic determination of the in vitro virulence characteristics of C. balustinum RTFCP 298.

Biofilm assay.
In 96-well flat bottom polystyrene microtiter plates, 30 µL of 10 8 CFU mL −1 C. balustinum RTFCP 298 was seeded with 200 µL of different growth media (HSU-SHOT, Cytophaga and SHIEH) with glucose and without glucose (0.45%) and incubated at 25 °C for 24-48 h.The planktonic cells were removed by washing twice with phosphate buffer saline (PBS) at pH 7.4.The staining was done by adding 150 µL of 0.1% crystal violet and kept for incubation for 1 h at 25 °C followed by washing with PBS (pH 7.4).Finally, the stain acquired by adherent bacteria was resolved by adding 200 µL of 95% ethanol and kept at 4 °C for 1 h.The biofilm formation was quantified by measuring optical density at 590 nm.
Haemolytic assay.The haemolytic assay was performed in 96 well U-bottom polystyrene microtitre plate, using defibrinated sheep blood 44 .The aseptically collected fresh defibrinated sheep blood was washed three times with PBS, centrifuged for 10 min at 1000×g and re-suspended at 10% (v/v) in PBS, containing 10 mM Dithiothreitol (DTT).The sheep erythrocytes were incubated with the freshly grown pure culture of C. balustinum RTFCP 298 in decreasing order of concentrations; 1.5 × 10 8 , 1.5 × 10 6 , 1.5 × 10 4 and 1.5 × 10 2 CFU mL −1 at 25 °C for 1 h.PBS (100 μL) and 0.2% Triton X-100 (100 μL) were used as a negative and positive control, respectively.The incubation of the plate was followed by centrifugation at 1000×g for 10 min and transferring of the supernatant to a flat bottom 96 well polystyrene microtitre plate.The absorbance of the plate was measured at 540 nm to estimate the lysis of the erythrocyte.
The percentage of haemolysis was calculated as: , where 0.2% Triton X-100 and PBS are positive control and Negative control respectively.

Resazurin assay for determination of minimum inhibitory concentration (MICs) of antibiotics and antimicrobial peptides (AMPs).
The minimum inhibitory concentrations (MICs) were measured in 96-well microtiter plates by broth dilution method 45 .The Muller Hinton broth medium (50 µL), containing decreasing concentrations (256-0.125 µg mL −1 ) of antibiotics (n = 5); erythromycin, florfenicol, neomycin, ampicillin and oxytetracycline and 15 AMPs, was inoculated with 10 5 CFU mL −1 of C. balustinum RTFCP 298 in 96-well microtiter plates, with each plate containing a positive control (only bacterial cells) and negative control (broth without bacteria).The MICs were determined after 18-20 h of incubation at 25 °C by the lowest concentration of antibiotics and AMP at which no visible growth occurred.The 10 μL of freshly prepared Resazurin (0.015%) diluted in PBS was added to all the wells of the MIC plate and incubated at 25 °C for 2 h.The colour of the entire well was recorded.A blue colour in the well was interpreted as no growth, whereas the development of pink colour scored as growth of the test bacterium.The MIC was defined as the lowest antibiotic/peptide concentration, which prevents a colour change from blue to pink.

Challenge assay.
To assess the pathogenicity of C. balustinum RTFCP 298, we conducted an experimental infection in rainbow trout fingerlings having an average weight of 25.0 ± 0.028 g and a length 12.5 ± 0.50 cm.The healthy fingerlings were collected from the state rainbow trout farm at Bairangna in district Chamoli, Uttarakhand for the challenge assay.Prior to the experimental infection in a flow-through system, the test fish (n = 30) were acclimated to the wet laboratory settings for 15 days in FRP tanks, each with a capacity of 500 L. Gill, liver and kidney and swab samples collected from randomly selected rainbow trout fingerlings (n = 6) were processed on SHIEH and Hsu-Shotts agar media as described previously for the screening of C. balustinum before the commencement of experimental infection.The fresh culture of the test bacteria was incubated in SHIEH-broth at 25 °C for 24 h.The cells were pelleted and washed twice with sterile 0.85% PBS.For the experimental infection, we maintained a constant cell density of 3.0 × 10 6 , 3.0 × 10 7 and 3.0 × 10 8 CFU mL −1 (DEN-1 McFarland densitometer, Grant-bio, England).To test Koch's hypotheses, 100 µL of 3.0 × 10 6 , 10 7 and 10 8 CFU of C. balustinum RTFCP 298 was administered to healthy rainbow trout fingerlings intraperitoneally in triplicate treatment groups and monitored for disease progression, abnormal behaviour, feeding and mortality.The control group was injected with 100 µL of 0.85% phosphate buffer saline (PBS).The water temperatures of experimental tanks were maintained at 18 °C.Following the post-challenge phase, the infected fingerlings (n = 6) randomly selected from each experimental group were again processed aseptically on SHIEH agar medium supplemented with 0.5 g mL −1 tobramycin, Hsu-Shotts agar supplemented with 4.0 µg mL −1 neomycin sulphate and TSA, as described previously.This was done to re-isolate and confirm the presence of C. balustinum in the organs of the challenged rainbow trout.The experimental trial was conducted for 90 days.
Histopathological analysis.The gill, liver, kidney, eye, spleen, intestine and muscle tissues from the experimentally infected rainbow trout were collected separately and fixed with Davidson's fixative for 12-16 h 46,47 .The fixed tissue samples were gradually dehydrated and blocks were prepared in embedding 'O' ring (HI Media, India).In brief, the tissue samples were dehydrated in the ascending grades of ethyl alcohol (50-100%) and then soaked in the clearing agent xylene twice to remove the dehydrating agent completely.The tissues were embedded in melted paraffin wax at 60-61 °C for 4-6 h, followed by the preparation of the blocks in embedding 'O' ring.Thin sections (4.0 µm) were cut (Micron HM 325, Thermo Scientific, USA) and adhered to the double frosted glass slides using egg albumin and glycerol mixed in a ratio of 1:1.After baking the slides at 37 °C for 1 h, the tissue sections were stained with 2% H & E stain 48 and further examined under the microscope (Leica DM 3000) for histopathological changes.

Results
Water quality parameters.During the collection of diseased rainbow trout samples, the average values of water quality parameters in the trout farms were as follows: water temperature of 18.5 °C, the dissolved oxygen concentration of 8.5 mg L −1 , pH of 7.5, total dissolved solids of 45 mg L −1 , and total ammonia nitrogen (TAN) level of 0.01 mg L −1 , indicating that the parameters were within the optimum range.
Clinical signs and bacterial isolation.The clinical signs, recorded in the study, were black pigmentation, haemorrhage in the dorsal body surface, fin rots and deep lesions at caudal peduncle regions.The microscopic examination of samples, prepared from diseased rainbow trout, did not reveal the presence of any parasitic infections.The PCR-based detection for aquatic herpesviruses, poxviruses, iridoviruses and adenoviruses using degenerated primers yielded negative results.The circular yellow colonies (n = 6) appeared on HSU-SHOT agar medium after 24-48 h of incubation at 25 °C.The pigmentation test (30% KOH reaction) detected the presence of flexirubin agent in all 6 isolates by turning yellow, slime bacterial colonies to brick red in colour., suggesting a close genetic relationship among these isolates.These strains were recovered alongside C. balustinum RTFCP 298 from the diseased rainbow trout samples as described in the "Materials and Methods" section.The phylogenetic tree involved analysis of 24 nucleotide sequences, and also established the identification of the present strain RTFCP 298 as C. balustinum in the study (Fig. 1).
Virulence characteristics.Biofilm.The biofilm formation test revealed that in the presence of glucose, C. balustinum RTFCP 298 formed significant biofilm (p < 0.01) as compared to glucose-limiting and medium control within the medium over the experimental period (24-48 h).When compared between glucose rich- www.nature.com/scientificreports/media; HSB, CB and SHIEH, the biofilm formation was significant in glucose rich SHIEH medium at 24 h and 48 h (p < 0.01) (Fig. 2A-B).
Challenge study.C. balustinum was found moderately pathogenic to rainbow trout fingerlings in the experimental infection to confirm Koch's postulate.The post-challenge period progressed with the development of initial pathological conditions and disease signs such as haemorrhage in eyes, black pigmentation, red patches and lethargy on the second day in all the experimental groups.During the entire course of experimental duration (90 days), the development of skin rashes and skin erosion at the base of the pectoral fins and the marked reduction in feed intake capacity among the challenged fish were recorded.Haemorrhage in the eyes of experimentally infected fish gradually turned into unilateral and bilateral exophthalmia conditions also.The necropsy Figure 1.Phylogenetic analysis was conducted using the maximum likelihood method with the kimura-2-parameter model, employing 500 bootstrap replications that included the 1st, 2nd, 3rd, and non-coding sites.The resulting tree with the highest log likelihood (− 6449.11) is presented, indicating the percentage of trees where associated taxa clustered together alongside the branches.Initial trees for the heuristic search were generated by applying the Neighbour-Joining method to a pair-wise distance matrix estimated using the Maximum Composite Likelihood (MCL) approach.The tree is scaled, with branch lengths measured in substitutions per site.This analysis involved 24 nucleotide sequences, and the final dataset consisted of 1541 positions.All evolutionary analyses were performed using MEGA X. www.nature.com/scientificreports/ of defunct or recently dead experimental fish revealed liquefaction in internal organs, haemorrhage in the liver and abnormal gall bladder (Fig. 4).

Mortality.
First mortality episodes of 12% and 15% were recorded in the fish group challenged with 3.0 × 10 7 and 3.0 × 10 8 CFU mL −1 of C. balustinum RTFCP 298, respectively post 4th day of the experimental infection.
The experimental group challenged with 3 × 10 6 CFU mL −1 of the test bacterium did not cause any mortality, but showed development of few signs of disease progression.On the 15th day of the post-challenge period, 4% mortality was recorded in the fish group infected with 3 × 10 6 CFU mL −1 of C. balustinum RTFCP 298.LD 50 value of C. balustinum RTFCP 298 was recorded at 10 8 CFU mL −1 , which led to the 50% mortality of the test fish on the 29th day.The fish in the control group of the experiment displayed no signs of disease progression, and no mortality was observed among the fish in this group.C. balustinum RTFCP 298 was re-isolated from the infected rainbow trout on SHIEH agar medium supplemented with 0.5 g mL −1 tobramycin and Hsu-Shotts medium sup-  www.nature.com/scientificreports/plemented with 4.0 g mL −1 neomycin sulphate, and phenotypic and partial 16S rRNA gene homology confirmed the characterization.A few isolates of Aeromonas group were also identified on TSA plate in the study.
Histopathology.Histopathological alterations in gill tissue included fusion of primary and secondary gill lamellae, hypertrophy, hyperplasia, vacuolation, epithelial lifting, telangiectasia in secondary lamellae, blood congestion and vasodilatation.The liver showed vacuolation, increasing sinusoidal space, hepatocyte nucleus and blood sinusoids, whereas haemorrhage and dilation of Bowman's space were recorded in the kidney.Spleen showed accumulation of hemosiderin, white and red pulp.The eye had increased space between pigmented epithelium and photoreceptor layer and cone-rod dystrophy.Necrosis, disruption of brush border and degeneration of lamina propria were observed in the intestine, whereas muscle showed necrosis and myocyte losses (Fig. 5).

Discussion
In the present study, C. balustinum RTFCP 298 isolates were recovered and characterised from the natural infection of rainbow trout in the Indian Himalayan Regions (IHR).The disease signs were similar to the cases of infection of Flavobacterium spp. in fish 49 .Since the first isolation in halibut, it has been recognised as a pathogenic bacterium rather than food spoilage bacterium, because it was isolated from the fin 4,26,50 .C. balustinum RTFCP 298 was identified based on the colony morphology, physiological and biochemical characteristics 20 .It was further confirmed by the PCR amplification of the 16S rRNA gene and phylogenetic analysis.The 16S rRNA gene is the most commonly used gene marker for identifying bacteria up to the species level.The 16S rRNA gene sequence alignment of C. balustinum, RTFCP 298 and phylogenetic analysis revealed that it shared maximum sequence similarity with C. balustinum strain WLT (MN317337) 20 .It is proposed that 16S rRNA gene identification can be useful in diagnosing C. balustinum infection in fish.Chryseobacterium WLT (MN 317337) from rainbow trout in the Republic of Korea revealed 99.24% similarity to other Chryseobacterium group 20 .Similarly, 16S rRNA gene of C. scophthalmum isolated from golden mahseer TPBLGL 18 (KM822770) has 99% similarity to the 16S rRNA gene of C. scophthalmum strain LMG 13028T (NR 025386) 24 .
Microdilution test utilizing resazurin revealed the lowest minimum inhibitory concentration against C. balustinum strain for oxytetracycline, florfenicol, and erythromycin.Due to their wide-ranging effectiveness and reduced harm to fish, the fish farmers in India frequently employ oxytetracycline, tetracycline, and ampicillin to combat bacterial diseases 51 .Consequently, the utilization of any of these three antibiotics could serve as a potential control measure during C. balustinum infection in trout.Previous studies have documented the natural resistance of Chryseobacterium spp. to a subset of antibiotics, including tetracyclines, erythromycin, linezolid, polymyxins, and chloramphenicol 52 .Furthermore, in our investigation, we assessed the antimicrobial efficacy of 15 synthetic novel antimicrobial peptides (AMPs) against C. balustinum.Remarkably, 5 out of the 15 AMPs exhibited notable antimicrobial activity (8-32 Μg mL −1 ) against the target bacterium.The susceptibility of peptides to bacterial interference may be attributed to their short length, which increases the likelihood of possessing unique sequences.As only a limited number of drugs and chemicals are approved for use in the treatment of diseases in aquaculture, an epizootic might cause significant losses in fish farming operations during the outbreak 53 .
The molecular virulence mechanism of non-model organisms such as C. balustinum can be better understood using information acquired from model species' virulence mechanisms.Few of the pathogenic determinants of C. balustinum have been investigated in vitro by comparative research on virulent colony types such as haemolytic test and biofilm formation in the current study.C. balustinum's haemolytic activity, like that of F. columnare, has been observed to have higher expression and detection against the potent haemolytic agent Triton X-100 (0.2%) 36 .Hemolysin of other organisms was identified by its ability to lyse RBCs or affect the biological functioning of other cells 54 .Proteases can play an important role in the formation of biofilm, which determines pathogenicity in bacteria 55,56 .The biofilm formation test revealed that C. balustinum was able to form biofilm with or without glucose in the medium.The biofilm generating bacteria are thought to be the primary factor for many opportunistic infections in fish, and they are extremely difficult to eliminate due to their much-enhanced resistance (1000 times) to several antimicrobials.As a result, greater concentrations of antimicrobial drugs must be used to kill or restrict the formation of pathogenic microbial consortiums established in biofilms 57 .cutaneous haemorrhage and ascites within the peritoneum, among other symptoms, have also been recorded in golden mahseer infected with Flavobacterium species 4,39,59,61,62 .In our study, C. balustinum RTFCP 298 was found to be moderately pathogenic to rainbow trout juveniles in the experimental infection trial to confirm the Koch's postulate test by revealing symptoms like dermal necrosis, liquefication, sub-peritoneal region-splenomegaly and diffuse petechiae, eye haemorrhage in the anterior chamber, internal organ haemorrhaging and ascites discharge from anus.Though the challenge test was conducted for 90 days, the pathogenicity of Chryseobacterium RTFCP 298 at experimental doses of 10 6 , 10 7 and 10 8 CFU mL −1 caused moderate mortality in rainbow trout.In general, Chryseobacterium pathogenicity in farmed fish is lower than that of the major bacterial pathogens (e.g., Flavobacterium spp and Lactococcus garvieae).Although the pathogenicity of Chryseobacterium is not much higher than that of other common pathogens in aquaculture, a 15-d experimental infection trial in rainbow trout weighing 20 g on average by Chryseobacterium strain WLT at 3 × 10 7 CFU of bacterium demonstrated 60% mortality and the LD 50 of the same strain recorded in between 10 6 to 10 7 CFU of the bacterium (Jung et al. 20 ).This Indian isolate, Chryseobacterium RTFCP 298 has shown its LD 50 at 10 8 CFU mL −1 .In the present study, the reproduction of clinical signs of disease progression and isolation of C. balustinum in the organs of the challenged rainbow trout established the test of Koch's hypotheses.However, detection of a few Aeromonas isolates on TSA plates may be correlated with the possible stress induction in such a long challenge test.
The present study investigated the histopathological changes associated with experimental infections of C. balustinum in rainbow trout.The gill tissue exhibited various alterations including necrotic deformities, fusion of primary and secondary gill lamellae, hypertrophy, hyperplasia, and vacuolation.Gill bacterial disease in fish is characterized by lamellar fusion and lamellar epithelial cell hyperplasia, as previously reported 63 .Similar histological changes in gill tissue have been observed in salmonid 4 , golden mahseer 24 and young turbot 64 infected with C. scophthalmum and other Chryseobacterium sp.Additionally, other organs such as the liver, eye, kidney, and spleen exhibited pathological changes such as vacuolation, increase of sinusoidal space, hepatocyte and hepatocyte nucleus alteration, blood sinusoid changes, increased space between pigmented epithelium and photoreceptor layer, cone-rod dystrophy, haemorrhage, dilation of Bowman's space, accumulation of hemosiderin, white pulp, and red pulp.Therefore, clinical signs and histopathological alterations suggested that the presence of disease in rainbow trout was due to the C. balustinum infection.

Conclusion
The present study reports the susceptibility of the test isolate, C. balustinum, to a range of FDA-approved antibiotics commonly used in aquaculture.Notably, this is the first report of the isolation of C. balustinum in the Indian context.The present findings underscore the potential of appropriate antibiotic treatment to effectively manage C. balustinum infection in farmed trout.Importantly, this research contributes to the identification and control of the disease in rainbow trout farming and highlights the necessity of implementing a regular monitoring and inspection regime.Therefore, analysis of infected samples from trout farms could aid in the prevention of C. balustinum and other diseases from spreading in aquaculture production units.

Figure 2 .
Figure 2. (A-B) Biofilm formation by C. balustinum RTFCP 298 in glucose-rich and glucose-limiting medium at (A) 24 h and (B) 48 h; HSB: HSU-Shot broth, SHIEH: Shieh broth, and CB: Cytophaga broth.Significant variation in biofilm formation recorded between glucose-rich and glucose limiting conditions with the medium (p < 0.01).The biofilm formation was also significant in glucose rich SHIEH medium at 24 h and 48 h (p < 0.01) as compared to other glucose-rich media.

Figure 4 .
Figure 4. Gross clinical and pathological signs in rainbow trout, O. mykiss experimentally challenged with C. balustinum.(a) Dermal necrosis or diffuse lateral scale-pocket.(b) Sub-peritoneal region-splenomegaly and diffuse petechiae.(c) Eye haemorrhage in the anterior chamber.(d) Exophthalmia, haemorrhages in the eye and dorsal discoloration are visible in the cranial region.(e) Eye haemorrhage in the anterior chamber (f) haemorrhaging and liquefaction in internal organ and (g) ascites discharge from anus.
Data on biofilm formation have been presented as mean ± standard error (Prism Version 5.01 GraphPad software).The significant differences (p < 0.01) in biofilm formation by C. balustinum RTFCPB 279 in different glucose-rich and glucose-limiting media were evaluated through One-way analysis of variance (ANOVA).To examine the significant variation (p < 0.01) in haemolytic activity between C. balustinum RTFCPB 279 concentration at 10 8 and 10 6 CFU mL −1 , the paired 't' test was performed.SPSS software version 19.0 (SPSS Inc, Chicago IL) was used for the statistical analysis in the present study.