Enrofloxacin, Effective Treatment of Pseudomonas aeruginosa and Enterococcus faecalis Infection in Oreochromis niloticus

Enrofloxacin is a broad-spectrum synthetic antimicrobial drug widely used in veterinary medicine. The present study aimed to determine the effective enrofloxacin dose for treating Pseudomonas aeruginosa and Enterococcus faecalis infection in Oreochromis niloticus. P. aeruginosa and E. faecalis isolates were verified using selective differential media and biochemically using the Vitek 2 test. Bacterial isolates were virulent for O. niloticus with LD50 equal to 2.03 × 106 and 2.22 × 107 CFU fish−1 for P. aeruginosa and E. faecalis, respectively. Infected fish suffered from decreased feed intake followed by off-food, tail erosion, darkening of the external body surface, exophthalmia, ascites, and loss of escape reflex. Internally, congested hemorrhagic hepatopancreas with engorged distended gall bladder were dominant. The posterior kidney was congested with enlarged spleen, and empty elementary tract. Pathologically, severe degenerative changes were dominant in the hepatopancreas, posterior kidney, spleen, stomach, and gills of infected fish. Antimicrobial sensitivity test indicated the high susceptibility of P. aeruginosa and E. faecalis to enrofloxacin with MIC estimated at 1 and 0.0625 µg/mL, respectively. Enrofloxacin effectively protected O. niloticus against E. faecalis and P. aeruginosa infection when used with medicated feed at doses of 10 and 20 mg kg−1 body weight.


Introduction
In Egypt, Oreochromis niloticus is the first economically important fish; cultured tilapia production exceeded 1.1 million tons in 2020 [1].Summer mortality syndrome represents the most severe challenge facing tilapia culture in Egypt over the past few years [2].Disease outbreaks hit most tilapia farms in Egypt [3]; A. veronii, A. hydrophila, A. caviae, A. sobria, Pseudomonas sp., and Streptococcus sp.isolated from the affected farms [4].
Bacterial infections are the most abundant diseases affecting cultured fish [5].Globally, fish diseases are estimated to contribute to more than 30% of the overall production loss, and bacterial diseases represent a serious challenge for tilapia culture worldwide [6].Gram-negative bacteria related to the genus Aeromonas, Pseudomonas, Vibrio, and Flavobacterium are responsible for high mortality rates and severe economic losses in cultured fish and shrimp [7,8].Pseudomonas aeruginosa is one of the most virulent Gram-negative pathogens affecting many cultured fresh and marine fish species, including O. niloticus, Clarias gariepinus, Dicentrarchus labrax, Oncorhynchus mykiss and Sparus aurata [9][10][11][12].E. faecalis is a newly emerged Gram-positive fish pathogen responsible for high mortality rates in the aquaculture of various fish species worldwide.E. faecalis infections were recorded in cultured O. niloticus, Barbonymus gonionotus, Cyprinus carpio, Oncorhynchus sp., Pterogymnus laniarius, Scophthalmus maximus, stinging catfish and walking catfish [13][14][15][16][17].
Enrofloxacin is a synthetic antibacterial drug related to fluoroquinolones and remains extensively used in veterinary medicine [18].Enrofloxacin acts by inhibiting the DNA gyrase enzyme (topoisomerase II); DNA gyrase is responsible for the standard coiling of DNA within the nucleus [19].Enrofloxacin has a potent broad-spectrum bactericidal

Infectivity Test
The infectivity test was performed to determine the virulence of bacterial isolates against O. niloticus (to satisfy Koch postulates) and to calculate lethal dose fifty (LD 50 ).

Bacterial Isolates
P. aeruginosa and E. faecalis were previously isolated from a diseased O. niloticus farm during a summer mortality outbreak.

Verification of Bacterial Isolates
Bacterial isolates were preliminarily identified on the selective media, Pseudomonas selective agar supplemented with cephalothin, fucidin, and cetrimide for P. aeruginosa and M-Enterococcus agar base media for E. faecalis.After that, isolates were reconfirmed using the Vitek 2 automatic biochemical identification system following the method described by Ali [25].One bacterial colony (from a fresh bacterial culture) was suspended in 5 mL of 0.5% sodium chloride solution; after that was adjusted to 0.6 McFarland standards.Identification cards were inoculated with bacterial suspensions in the Vitek 2 system, and the biochemical profile was recorded.

Bacterial Inoculum Preparation for the Infectivity Test
A single bacterial colony was picked up from the selective agar and then inoculated on brain heart infusion broth, and incubated at 35 • C for 12 h.Bacterial growth was harvested by centrifugation at 5000 rpm for 3 min.The bacterial pellet was suspended in 0.1% peptone water and adjusted with a spectrophotometer to 0.451 absorbances at 600 nm (equivalent to the second McFarland standard 6 × 10 8 CFU mL −1 ).One ml of sterile phosphate buffer saline was added to five ml of bacterial suspension to achieve a final concentration of 5 × 10 8 CFU mL −1 .Tenfold serial dilutions were prepared four consecutive times to obtain the following concentrations (5 × 10 7 , 5 × 10 6 , 5 × 10 5 , and 5 × 10 4 ) CFU mL −1 .

Experimental Design
Two hundred and eighty-eight fish were randomly divided into twelve groups as in Table 1; each group consists of 24 fish in three triplicates (8 fish per replicate).Fish in groups (1-5) were intraperitoneally inoculated with 0.2 mL of P. aeruginosa bacterial suspension containing (5 × 10 4 , 5 × 10 5 , 5 × 10 6 , 5 × 10 7 and 5 × 10 8 ) CFU mL −1 equivalent to (10 4 , 10 5 , 10 6 , 10 7 and 10 8 ) CFU fish −1 .Fish in the last group were inoculated with 0.9% saline and served as a control negative.The same experimental design was performed for groups (6-10) using E. faecalis bacterial suspension.Each replicate was maintained in a 100 L glass aquarium; water temperature was thermostatically maintained at 28 ± 1 • C, and aquaria water was changed at a continuous rate (5 Liter per hour).Feeding was restricted for 24 h before the challenge and then resumed 12 h after infection.All fish groups were observed for seven days to record the clinical signs, postmortem lesions, and mortality rates.Dead fish were considered only after the re-isolation of challenging bacteria, and LD 50 was calculated according to Reed and Muench [26].

Clinical Picture
Fish were observed daily throughout the experimental period to record any abnormal signs and behavioral changes, as described by Austin and Austin [27].Dead fish were immediately dissected under aseptic conditions for bacterial re-isolation.After that, the gross internal lesions were recorded, and tissues were sampled for the histopathological examination.

Histopathological Investigation
The histopathological examination was performed according to Suvarna et al. [28].Small pieces from the hepatopancreas, gills, spleen, stomach, and posterior kidney of moribund fish were fixed in 10% buffered formalin.Fixed tissues were dehydrated in ascending-grade ethyl alcohol and then cleared in xylene.Cleared samples were impeded in soft then hard paraffin wax and sectioned to 5 µm thickness using Leica RM2235 microtome (Lecia, Germany).Thin sections were mounted over labeled glass slides and finally stained with hematoxylin and eosin.Stained slides were examined and photographed using a microscope equipped with a digital camera.
2.6.Antimicrobial Susceptibility Tests 2.6.1.Agar Disc Diffusion Test Susceptibility of P. aeruginosa and E. faecalis to enrofloxacin was assayed.Overnightseeded broth was adjusted to 1.5 × 10 8 CFU mL −1 , and then 2 mL was spread on the Mueller-Hinton agar (Oxoid, UK) plate surface with a rotation movement.The plate was allowed to stand in an inverted position on the refrigerator for 20 min to absorb the excess moisture.The sensitivity disc (ENR 5 µg), Oxoid, UK, was gently fixed into the agar surface using sterile forceps.The agar plate was incubated at 35 • C for 24 h, and Escherichia coli ATCC 25,922 was used as a control.The inhibition zone was measured to the nearest mm using a digital caliper and interpreted as susceptible (21 mm or more), intermediate susceptible (16-20 mm), and resistant (less than 15 mm) according to breakpoints mentioned by CLSI [29].

Broth Dilution Test
The minimum inhibitory concentration (MIC) of enrofloxacin was determined for the tested isolates using the broth dilution test, as indicated by Ali et al. [23].Briefly, 256 µL of Enrofloxacin 10% was added to 1744 µL sterile distilled water.Double-fold serial dilution was performed 15 successive times.The overnight cultured tryptic soy broth was adjusted to 0.5 McFarland standard (absorbance of 0.063 at 600 nm).One ml of TSB was added to 199 mL of sterile Mueller-Hinton broth.After that, tetrazolium chloride (20 mg) was added as an indicator for bacterial growth.Each screw-capped test tube was loaded with 4.9 mL of seeded Mueller-Hinton broth (a final volume of 5 mL).After that, 100 µL from previously prepared enrofloxacin standard solution was added to the corresponding test tubes to achieve a final concentration of 265, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 and 0.015625 µg mL −1 , respectively.The last tube was left antibiotic-free as a control; tubes were incubated at 35 • C for 24 h.MIC was determined as the lowest antibiotic concentration, preventing bacterial growth.Red-colored broth indicated bacterial growth.Results interpreted as susceptible when MIC equals (1 µg/mL or less), intermediately susceptible (2-4 µg/mL), and resistant (more than 4 µg/mL) as mentioned by CLSI [29].

Protective Effect of Enrofloxacin against P. aeruginosa and E. faecalis Challenge
The protective effect of enrofloxacin against P. aeruginosa and E. faecalis infection in O. niloticus was assayed as the following: Two hundred and ten fingerlings were randomly divided into eight groups, as shown in Table 2. Groups (11 and 12 and +ve control) were experimentally infected through intraperitoneal injection with P. aeruginosa 2.03 × 10 6 CFU fish −1 , while groups (13 & 14 & +ve control) received E. faecalis 2.22 × 10 7 CFU fish −1 , control -ve groups received 0.2 mL of normal saline.Groups (11 and 13) received medicated feed containing enrofloxacin 10 mg kg −1 body weight equivalent to 340 mg kg −1 fish ration at (3%) feeding rate of fish weight.Groups (12 and 14) received 20 mg kg −1 (equivalent to 680 mg kg −1 ration).Enrofloxacin powder was mixed with 10 mL of fish oil, and the mixture was evenly distributed to one kg of fish feed.Medicated feed was left for one day at room temperature to absorb the drug and then preserved at 8 • C. The experimental infection was performed after consumption of the medicated feed by all the treated groups; treatment continued for seven successive days; the mortality rate was daily recorded for ten days.

Verification of Bacterial Isolates
P. aeruginosa was grown as yellowish-green colonies against a greenish background on Pseudomonas selective agar, while E. faecalis raised as dark red colonies on M-Enterococcus agar base media Figure 1a,b.Tested isolates were confirmed as P. aeruginosa and E. faecalis with 99% probability using the VITEK 2 automated biochemical identification system.The biochemical characteristics of both pathogens are recorded in Table 3

Verification of Bacterial Isolates
P. aeruginosa was grown as yellowish-green colonies against a greenish background on Pseudomonas selective agar, while E. faecalis raised as dark red colonies on M-Enterococcus agar base media Figure 1 a, b.Tested isolates were confirmed as P. aeruginosa and E. faecalis with 99% probability using the VITEK 2 automated biochemical identification system.The biochemical characteristics of both pathogens are recorded in Table 3.    α = Alpha, β = beta, γ = Gamma.

Antimicrobial Susceptibility
The agar disc diffusion test indicated that both bacterial isolates were susceptible to enrofloxacin with 21.5-and 56.5-mm zone diameters for P. aeruginosa and E. faecalis, as represented in Figure 1c,d.Broth dilution test confirmed the high susceptibility of P. aeruginosa and E. faecalis to enrofloxacin with MIC equals 1 and 0.0625 µg/mL, respectively Figure 1e,f.

Infectivity Test Result
The LD 50 of P. aeruginosa in challenged Nile tilapia was 2.03 × 10 6 CFU.Fish −1 , and it was 2.22 × 10 7 CFU.Fish −1 for E. faecalis, the mortality rate is demonstrated in Table 1.
Fish number in each group = 24.

Clinical Picture
Infected fish with P. aeruginosa or E. faecalis showed similar clinical signs in which fish suffered from decreased feed intake followed by off-food, with disease progression tail erosions and darkening of the external body surface taking place.Some infected fish showed exophthalmia and ascites, Figure 2a,b; fish swam near or at the water surface and lost escape reflex shortly before death.

Antimicrobial Susceptibility
The agar disc diffusion test indicated that both bacterial isolates were susceptible to enrofloxacin with 21.5-and 56.5-mm zone diameters for P. aeruginosa and E. faecalis, as represented in Figure 1 c, d.Broth dilution test confirmed the high susceptibility of P. aeruginosa and E. faecalis to enrofloxacin with MIC equals 1 and 0.0625 µg/mL, respectively Figure 1e,f.

Infectivity Test Result
The LD50 of P. aeruginosa in challenged Nile tilapia was 2.03 × 10 6 CFU.Fish −1 , and it was 2.22 × 10 7 CFU.Fish −1 for E. faecalis, the mortality rate is demonstrated in Table 1.
Fish number in each group = 24.

Clinical Picture
Infected fish with P. aeruginosa or E. faecalis showed similar clinical signs in which fish suffered from decreased feed intake followed by off-food, with disease progression tail erosions and darkening of the external body surface taking place.Some infected fish showed exophthalmia and ascites, Figure 2 a, b; fish swam near or at the water surface and lost escape reflex shortly before death.Pale hepatopancreas tinged with petechial hemorrhages or even large hemorrhagic spots was the most prominent gross internal finding observed during the dissection of the infected fish.Enlarged distended gall bladder, congested posterior kidney, enlarged spleen, and empty intestine were also reported, as represented in Figure 2c-f.Pale hepatopancreas tinged with petechial hemorrhages or even large hemorrhagic spots was the most prominent gross internal finding observed during the dissection of the infected fish.Enlarged distended gall bladder, congested posterior kidney, enlarged spleen, and empty intestine were also reported, as represented in Figure 2c-f.

Histopathological Examination
Infected O. niloticus with P. aeruginosa or E. faecalis showed severe degenerative changes in all tissue samples.Hepatopancreas of diseased fish showed diffused hepatocellular vacuolation, severe inflammation, mononuclear inflammatory cell infiltration, and the presence of necrotic foci with appendant melanomacrophage centers as represented in Figure 3a,b.The posterior kidney was also severely affected; renal corpuscles showed shanked glomeruli with dilated Bowmans's space, the presence of interstitial hemorrhage, mononuclear cell infiltration, degenerated proximal and distal convoluted tubules, detached tubular epithelium, hyaline droplet degeneration, and tubular obliteration as observed in Figure 3c,d.The affected fish spleen demonstrated diffused lymphocytic proliferation clusters, cuboidalshaped endothelial cells, and melanomacrophage centers Figure 4a,b.The stomach of experimentally infected fish showed destruction and detachment of mucosal lining, coagulative necrosis of some gastric glands with abundant lymphocytic infiltration in the lumen of gastric folds Figure 4c,d.The gill tissue of affected fish showed degeneration and fusion of secondary gill lamellae with sloughing of necrotic cells and epithelial lifting Figure 4e,f.

Histopathological Examination
Infected O. niloticus with P. aeruginosa or E. faecalis showed severe degenerative changes in all tissue samples.Hepatopancreas of diseased fish showed diffused hepatocellular vacuolation, severe inflammation, mononuclear inflammatory cell infiltration , and the presence of necrotic foci with appendant melanomacrophage centers as represented in Figure 3a,b.The posterior kidney was also severely affected; renal corpuscles showed shanked glomeruli with dilated Bowmans's space, the presence of interstitial hemorrhage, mononuclear cell infiltration, degenerated proximal and distal convoluted tubules, detached tubular epithelium, hyaline droplet degeneration , and tubular obliteration as observed in Figure 3c,d.The affected fish spleen demonstrated diffused lymphocytic proliferation clusters, cuboidal-shaped endothelial cells , and melanomacrophage centers Figure 4a,b.The stomach of experimentally infected fish showed destruction and detachment of mucosal lining, coagulative necrosis of some gastric glands with abundant lymphocytic infiltration in the lumen of gastric folds Figure 4c,d.The gill tissue of affected fish showed degeneration and fusion of secondary gill lamellae with sloughing of necrotic cells and epithelial lifting Figure 4e,f.

Result of the Treatment Trial
Enrofloxacin showed an excellent protective effect for the challenged O. niloticus against P. aeruginosa and E. faecalis infection.Enrofloxacin at a dose of 10 mg.kg −1 completely protected the challenged fish against E. faecalis infection by decreasing the mortality rate from 54.16 in the infected non-treated group to 0%, while P. aeruginosa infection required a much higher dose (20 mg.kg −1 ) to reduce mortality from 66.7 to 8.3 %, as represented in Table 2.

Discussion
Bacterial fish diseases are responsible for a huge annual loss estimated at USD 6 billion in 2014 [30]; this figure has increased to 9.58 in 2020 [31].P. aeruginosa and E. faecalis are among the most common bacterial pathogens affecting cultured fishes [10,32]; the present study aimed to treat such serious infections using an effective antimicrobial drug such as enrofloxacin.

Result of the Treatment Trial
Enrofloxacin showed an excellent protective effect for the challenged O. niloticus against P. aeruginosa and E. faecalis infection.Enrofloxacin at a dose of 10 mg kg −1 completely protected the challenged fish against E. faecalis infection by decreasing the mortality rate from 54.16 in the infected non-treated group to 0%, while P. aeruginosa infection required a much higher dose (20 mg kg −1 ) to reduce mortality from 66.7 to 8.3 %, as represented in Table 2.

Discussion
Bacterial fish diseases are responsible for a huge annual loss estimated at USD 6 billion in 2014 [30]; this figure has increased to 9.58 in 2020 [31].P. aeruginosa and E. faecalis are among the most common bacterial pathogens affecting cultured fishes [10,32]; the present study aimed to treat such serious infections using an effective antimicrobial drug such as enrofloxacin.
Selective media is a preliminary procedure used in microbial identification [33].In the present study, P. aeruginosa grew as greenish colonies on Pseudomonas selective agar due to the secretion of pyocyanin pigment; other bacteria growth was inhibited by CFC supplement [34].On the other hand, E. faecalis raised as dark red colonies on M-Enterococcus agar due to the uptake of triphenyl tetrazolium chloride and sodium azide preventing the growth of other microorganisms [35].
P. aeruginosa and E. faecalis were further verified by their specific biochemical profile with 99% probability using the Vitek 2 ® automatic microbial biochemical identification system.Vitek 2 ® system is among the most recent reliable techniques for identifying pathogenic bacteria [36].Vitek 2 ® system is the gold standard for P. aeruginosa identification with 100% accuracy, as described by Moehario et al. [37].
In the present work, LD 50 of E. faecalis was 2.22 × 10 7 CFU fish −1 , so it is less virulent when compared to P. aeruginosa (2.03 × 10 6 CFU fish −1 ); this could be due to many potent virulence factors P. aeruginosa has.
Rizkiantino et al. [38] found that the LD50 of E. faecalis in tilapia was 0.79 × 10 8 CFU mL −1 which was slightly higher than that reported in the present work; this variation could be attributed to the difference in challenged fish size as well as the diversity of used strain.The calculated LD50 of P. aeruginosa was nearly like that reported by Thomas et al. [39] in tilapia which was 4.5 × 10 6 CFU/fish.
Pyocyanin is the major virulence factor responsible for P. aeruginosa's pathogenicity [40].Dead fish showed the characteristic clinical and postmortem lesions of Pseudomonas septicemia, including exophthalmia, ascites, and hemorrhages over the external body surface, with congested hepatopancreas and posterior kidney.Similar results were observed by [10,14].Furthermore, histopathological examination indicated the presence of congestion, inflammation, and degeneration of the hepatopancreas, spleen, and posterior kidney; Refs.[23,41,42] reported similar results.Virulent P. aeruginosa induced high mortality rate, serious clinical signs, and postmortem lesions with severe pathological tissue changes because of virulence factors that the pathogen has such as pyocyanin.Pyocyanin is essential for Pseudomonas pathogenicity; it has toxic effects responsible for cellular death and interferes with many cellular functions by inducing oxidative stress, altering the expression and release of many cytokines [43][44][45].Outer membrane porin F, biofilm formation, exotoxin A, adhesins, and tissue-digesting enzymes as proteases are also responsible for the virulence of P. aeruginosa [46][47][48][49].
Enterococci are normal inhabitant gut microbes of human and other animal species [50].Enterococci emerged as a potential bacterial pathogen inducing severe localized or septicemic life-threatening infections in humans and animals [51].Infected O. niloticus expressed the general signs of septicemia, hemorrhagic batches on the skin, scale desquamation, and tail erosions.Internally, enlarged hemorrhagic hepatopancreas with distended gall bladder and congested spleen were clear on dissected fish.Refs.[52,53] recorded the same clinical signs from infected fish.The pathological changes involved the stomach and gills with impairment of their normal physiological functions; Elgohary et al. [16] and Abdelsalam et al. [54] reported similar descriptions.E. faecalis expresses many virulence factors directly responsible for disease progression in the affected animal.Enterococcal surface protein, surface aggregating protein, and large surface protein are responsible for biofilm formation, which subsequently helps in cell adherence, colonization, and evasion of the host immune system.E. faecalis produces extracellular metalloprotease (gelatinase) that hydrolyzes gelatin, collagen, and hemoglobin; it also produces serine protease and cytolysin A [55][56][57], these enzymes are directly responsible for disease pathogenesis.
Enrofloxacin is the most widely used fluoroquinolone in veterinary medicine; it has a broad spectrum of activity against many Gram-positive and Gram-negative bacterial pathogens affecting animals and fish [58,59].The result of antimicrobial sensitivity tests indicated high susceptibility of P. aeruginosa and E. faecalis to enrofloxacin.
In harmony with the present research findings [10,60], they reported that 100% of P. aeruginosa isolates retrieved from diseased O. niloticus were highly sensitive to ciprofloxacin.Also, Anifowose et al. [61] found that most of the E. faecalis isolates recovered from Clarias gariepinus Juveniles were sensitive to enrofloxacin.
The treatment trial showed remarkable efficacy in protecting O. niloticus against challenged bacterial pathogens.Enrofloxacin effectively protects the challenged fish against E. faecalis at a dose of 10 mg kg −1 , but the double dose was protective against P. aeruginosa.The difference in therapeutic dose could be due to the difference in MIC of both pathogens.E. faecalis was more susceptible to enrofloxacin than P. aeruginosa by four folds (MIC was 0.0625 and 1 µg mL −1 ), respectively.Enrofloxacin is an effective antibacterial agent when administrated with fish feed; it has excellent activity against sensitive fish pathogens.Moreover, it is a non-water-soluble powder, so the given dose is almost delivered to fish even if the feed remains for some time in the water.
In recent research, enrofloxacin is still used in many regions in the world for prophylaxis and treatment of cultured fish diseases; Amable et al. [62] used the subtherapeutic doses of enrofloxacin as a growth promotor and prophylactic for Piaractus mesopotamicus fish (the most cultured fish in Argentina), the drug was administrated in feed twice daily for 120 days.No significant difference was observed in drug resistance between the treated and control groups in the intestinal microbiota up to 90 days of the feeding trial.The antibiotic residues in meat samples showed no differences between controls and treatment.Concha et al. [63] reported that quinolones are still used in Chilean salmon farming and are currently approved for use in this industry.Among the 65 bacterial isolates from fish farms, only 4.6% showed resistance to enrofloxacin.
Oxytetracycline, oxolinic acid, flumequine, sarafloxacin, enrofloxacin, amoxicillin, erythromycin, sulfadimethoxine, ormetoprim, and florfenicol are the most used antibiotics in aquaculture worldwide [64].Fluoroquinolones are the most common quinolones used in veterinary medicine; they are the most used class of antibiotics in aquaculture worldwide [65,66].The residual limit of enrofloxacin is 30 µg/kg in the United States and the European Union.FAO and the WHO stipulate the allowable daily intake (ADI) of ENR as 2 µg/kg [67].The withdrawal time of enrofloxacin should be considered before use in fish treatment; some studies estimated it at 45 days [68].Ferri et al. [69] reported that the acceptable maximum residue limit (MRL) of enrofloxacin in finfish is 100 µg/kg.For the sustainability of the accelerated aquaculture growth as an important source of animal protein, this growth was accompanied by increased relay on antimicrobials to maintain fish health and fight diseases so, there is an urgent need for stewardship on antimicrobials use and monitoring the withdrawal time and drug residues [68].

Conclusions
P. aeruginosa and E. faecalis were highly pathogenic for O. niloticus, experimental infection-induced typical disease signs, high mortality rate, and severe pathological lesions.Enrofloxacin effectively protected O. niloticus against susceptible P. aeruginosa and E. faecalis infection when used with medicated feed at doses of 20 and 10 mg kg −1 body weight, respectively.Institutional Review Board Statement: Naturally infected and experimental seabass were handled, transported, examined, and euthanized following the National Advisory Committee for Laboratory Animals Research [70,71] guidelines regarding the care and use of fish in teaching and research.The Institutional Care of Aquatic Organisms and Experimental Animals Committee, National Institute of Oceanography and Fisheries has approved this work under certificate number (NIOF-AQ2-F-21-P-002).

Figure 1 .
Figure 1.(a) Dark red characteristic colonies of E. faecalis on Enterococcus agar base media.(b) Yellowish green characteristic colonies of P. aeruginosa against a greenish background due to pyocyanin secretion on Pseudomonas selective.(c) Wide inhibition zone induced by enrofloxacin (5 µg) disc indicated high sensitivity of E. faecalis to tested antibacterial.(d) Inhibition zone induced by enrofloxacin (5 µg) disc indicated sensitivity of P. aeruginosa to tested antibacterial.(e) MIC of enrofloxacin (0.0625 µg mL −1 ) completely inhibits E. faecalis growth while the left tube that showed bacterial growth.(f) MIC of enrofloxacin is 1 µg mL −1 which completely inhibits P. aeruginosa growth in contrast with the left tube sowed bacterial growth.

Figure 1 .
Figure 1.(a) Dark red characteristic colonies of E. faecalis on Enterococcus agar base media.(b) Yellowish green characteristic colonies of P. aeruginosa against a greenish background due to pyocyanin secretion on Pseudomonas selective.(c) Wide inhibition zone induced by enrofloxacin (5 µg) disc indicated high sensitivity of E. faecalis to tested antibacterial.(d) Inhibition zone induced by enrofloxacin (5 µg) disc indicated sensitivity of P. aeruginosa to tested antibacterial.(e) MIC of enrofloxacin (0.0625 µg mL −1 ) completely inhibits E. faecalis growth while the left tube that showed bacterial growth.(f) MIC of enrofloxacin is 1 µg mL −1 which completely inhibits P. aeruginosa growth in contrast with the left tube sowed bacterial growth.

Figure 4 .
Figure 4. (a, b) Spleen of S. faecalis infected fish demonstrated diffused clusters of lymphocytic proliferation (L), cuboidal-shaped endothelial cells (black arrow), Splenic capsule-trabecula systems (red arrow) and melanomacrophage centers (M and brown arrow), H & E, X = 400 (a) & 100 (b).(c, d) Stomach of S. faecalis experimentally infected fish showed destruction and detachment of mucosal lining (blue arrow), coagulative necrosis of some gastric glands (C) with abundant lymphocytic infiltration between the gastric glands (I) and in the lumen of gastric folds (L), H & E, X = 100 (c) and 400 (d).(e, f) Gills of P. aeruginosa infected fish with degeneration and fusion of secondary gill lamellae (F) with sloughing of necrotic cells (N), and epithelial lifting (grey arrow), H & E, X = 100 (a) and 400 (b).

Figure 4 .
Figure 4. (a,b) Spleen of S. faecalis infected fish demonstrated diffused clusters of lymphocytic proliferation (L), cuboidal-shaped endothelial cells (black arrow), Splenic capsule-trabecula systems (red arrow) and melanomacrophage centers (M and brown arrow), H & E, X = 400 (a) & 100 (b).(c,d) Stomach of S. faecalis experimentally infected fish showed destruction and detachment of mucosal lining (blue arrow), coagulative necrosis of some gastric glands (C) with abundant lymphocytic infiltration between the gastric glands (I) and in the lumen of gastric folds (L), H & E, X = 100 (c) and 400 (d).(e,f) Gills of P. aeruginosa infected fish with degeneration and fusion of secondary gill lamellae (F) with sloughing of necrotic cells (N), and epithelial lifting (grey arrow), H & E, X = 100 (a) and 400 (b).

Author Contributions:
Conceptualization, N.G.A.; methodology, N.G.A. and I.A.; validation, N.G.A.; formal analysis, N.G.A. and I.A.; investigation, N.G.A. and I.A.; resources, N.G.A.; data curation, N.G.A. and I.A.; writing-original draft preparation, N.G.A. and I.A.; writing-review and editing, N.G.A.; visualization, N.G.A. and I.A.All authors have read and agreed to the published version of the manuscript.Funding: This research received no external funding.

Table 1 .
Experimental design and mortality rate of O. niloticus fingerlings challenged with P. aeruginosa and E. faecalis.

Table 2 .
Protective effect of enrofloxacin for O. niloticus fingerlings challenged with P. aeruginosa and E. faecalis. P.

Table 3 .
The biochemical characteristics of P. aeruginosa and E. faecalis.